3,5-Bis(arylidene)piperid-4-ones containing 1,3,2-oxazaphosphorinane moieties: Synthesis and antitumor activity

Article abstract of DOI:10.1002/hc.21082

Two novel series of phosphorus-substituted 3,5-bis(arylidene)piperid-4-ones bearing 1,3,2-oxazaphosphorinane cycle either directly attached to the piperidone core through the P-N bond (diamidophosphates 4) or connected with it via thiocarbamoyl linker (th

Full text of DOI:10.1002/hc.21082

Heteroatom Chemistry
Volume 00, Number 0, 2013
3,5-Bis(arylidene)piperid-4-ones Containing
1,3,2-Oxazaphosphorinane Moieties: Synthesis
and Antitumor Activity
Anatolii E. Shipov,1 Mikhail V. Makarov,1 Pavel V. Petrovskii,1
Ekaterina Yu. Rybalkina,2 Yulia V. Nelyubina,1 and Irina L. Odinets1
1A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
Moscow, 119991, Russian Federation
2Institute of Carcinogenesis, N. N. Blokhin Russian Cancer Research Center, Russian Academy of
Medical Sciences, Moscow 115478, Russian Federation
Received 10 July 2012; revised 12 January 2013
INTRODUCTION
ABSTRACT: Two novel series of phosphorus-
substituted 3,5-bis(arylidene)piperid-4-ones bearing
1,3,2-oxazaphosphorinane cycle either directly at-
tached to the piperidone core through the P N bond
(diamidophosphates 4) or connected with it via thio-
carbamoyl linker (thioureas 5) were obtained by the
phosphorylation of NH precursors with 2-oxo-1,3,2-
oxazaphosphorinane chloride or the reaction of the
former ones with the corresponding cyclic isothio-
cyanate. According to the results of cytotoxicity screen-
ing against human carcinoma cell lines (A549, CaOv3,
KB), thioureas 5 were more active than the diami-
dophosphates 4 bearing the same arylidene rings, with
compounds with electron-withdrawing side groups
displaying IC50 in the micromolar range of 1.2–7 μM.
The discovery of antitumor properties of 1,3,2-
oxazaphosphorinanes (also referred to as oxaza-
phosphinanes) by Brock [1] promoted numerous
investigations in this area of organophosphorus
chemistry, and nowadays, among thousands of
drug candidates screened up to date, at least two
chemotherapeuticals—cyclophosphamide and ifos-
famide (Fig. 1)—found a wide range of applica-
tions in clinical practice. These related compounds
being not cytotoxic themselves are converted to
the active alkylating mustard via the hepatic cy-
tochrome P450 catalyzed 4-hydroxylation in the liver
[2]. These compounds can be considered as pro-
drugs in which 1,3,2-oxazaphosphorinane cycles fa-
cilitate transportation of the active cytostatics to
cancer cells and allow for a considerable reduction of
the general toxicity of plain mustard. Furthermore,
1,3,2-oxazaphosphorinan-2-thiones, especially those
bearing p-nitrophenoxy and butylthio (XR = 4-O2N-
C6H4, BuS) groups (Fig. 1), exhibit high nematocide
activity with low mammalian toxicity and have a
strong synergistic effect on pyrethroid insecticides
[3–5].
ꢀC
2013 Wiley Periodicals, Inc. Heteroatom Chem
00:1–9, 2013; View this article online at wileyon-
linelibrary.com. DOI 10.1002/hc.21082
Taking into account notable transport prop-
erties and metabolic peculiarities of the 1,3,2-
oxazaphosphorinane cycle, we focused on the
application of this phosphorus moiety for the mod-
ification of some pharmacophores to impart them
Correspondence to: Mikhail V. Makarov; e-mail: mmak78@
yandex.ru.
Contract grant sponsor: Chemical and Materials Sciences Divi-
sion of Russian Academy of Sciences.
The authors declare no conflict of interest.
ꢀC
2013 Wiley Periodicals, Inc.
1

2
Shipov et al.
CH2CH2Cl
N
in the case of 4-dimethylamino and 3-pyridyl deriva-
tives 4a and 4d, respectively. Obviously, worse re-
sults for the above compounds 4a,d can be explained
by the presence of residual water in the starting com-
pounds 1a,d, which could not be removed even by
prolonged drying over phosphorus pentoxide.
In the other approach, the desired compounds,
the main skeleton of which was connected with
the oxazaphosphorinane moiety via a thiocarbamoyl
linker, were readily obtained by the reaction of NH-
precursors 1a–c with isothiocyanate 3 derived, in
turn, from the chloride 2 according to the known
procedure [15]. The reaction proceeds smoothly
in CHCl3 at room temperature to give the crude
thioureas 5a–c in high yields. The isolated yields
of the derivatives 5b,c were still more than 85%
whereas the yield of 3-pyridyl derivative 5a de-
creased up to 25% over the chromatographic purifi-
cation.
H
N
H
N
N(CH2CH2Cl)2
NHCH2CH2Cl
XR
S
P
P
P
O
O
O
O
O
R = Alk, Ar
Ifosfamide
Cyclophosphamide
FIGURE 1 Biologically active 1,3,2-oxazaphosphorinanes.
better bioavailability or provide the compounds pos-
sessing higher or even other types of bioactivity. 3,5-
Bis(arylidene)piperid-4-ones possessing free radical
scavenging [6], α-glucosidase inhibitory [7], antimy-
cobacterial [8], and antitumor properties [9] due
to the presence of a 1,5-diaryl-3-oxo-1,4-pentadienyl
(dienone) pharmacophore, were among the basic
skeletons of interest. Antitumor properties of these
compounds can be adjusted either by the variation
of aromatic substituents or the introduction of dif-
ferent groups at the piperidone nitrogen atom. In-
deed, we have recently shown that incorporation
of a range of phosphorus-containing moieties, such
as phosphonate and methylenebisphonate groups,
as well as phosphoric acid residues either directly
bound with the heterocyclic nitrogen atom or con-
nected with it via an alkylene linker, into the struc-
ture of 3,5-bis(arylidene)piperid-4-ones, resulted in
a pronounced increase of their in vitro cytotoxicity
[10–13].
The structures of the phosphorylated products
1
4a–e, 5a–c were elucidated on the basis of H, 31P,
13C NMR, and IR spectral data along with a single
crystal X-ray diffraction analysis performed for di-
amidophosphate 4e (Fig. 2). According to the ana-
lytical data, some of the compounds obtained are
inclined to form either hydrates (4a,d and 5a) or
strong solvates with chloroform (5c). The 31P NMR
spectra of the corresponding products displayed sin-
glet signals at ca. 10 ppm for diamidophosphates
4a–e and at ca. –2 ppm for thioureas 5a–c. These
values of chemical shifts are typical for such phos-
In this paper, we report on the synthesis and cy-
totoxicity of novel 3,5-bis(arylidene)piperid-4-ones
modified with 1,3,2-oxazaphosphorinane cycle to
impart them better bioavailability or provide higher
antitumor properties.
1
phorus atom surrounding. In the H NMR spectra
of all compounds, the singlet resonances assigned
to vinyl hydrogen atoms are observed at ca. 7.7–
8.0 ppm, which is usual for this type of compounds.
On the whole, the spectral data are consistent with
E,E-geometry of the dienone moiety and are in good
agreement with the literature data for the known
analogs. Note that some compounds synthesized
possess poor solubility in common organic solvents,
thus preventing recording of 13C NMR spectra of
good quality.
RESULTS AND DISCUSSION
Synthesis
To attach 1,3,2-oxazaphosphorinane cycle to the 3,5-
bis(arylidene)piperid-4-one framework, we used two
different approaches. The first one was based on
the direct phosphorylation of the parent NH-3,5-
bis(arylidene)piperid-4-ones 1a–e with cyclic phos-
phorus acid chloride 2 [14] and provided compounds
4a–e bearing the direct P N bond, which may be
considered as prodrugs of 3,5-bis(arylidene)piperid-
4-ones, releasing the cytotoxic dienone system over
the hydrolytic cleavage (Scheme 1). Amides 4a–e
were synthesized using a slight excess (1.1 mol equiv)
of acid chloride 2 relative to the corresponding start-
ing NH-precursors 1a–e in CHCl3 in the presence of
4-dimethylaminopyridine (DMAP) as a base at room
temperature. Under these optimized conditions, the
phosphorylation afforded compounds 4b,c,e in good
yields (60–85%), which decreased to 43% and 38%
According to the X-ray investigation of the
compound 4e (as a crystallosolvate with one
CH2Cl2 molecule), the geometrical parameters of
the phosphorus-bound piperidone core are very
˚
close (within 0.01 A) to those in its 3,5-substituted
analogs [16, 17]. In particular, the piperidone moi-
ety adopts a chair conformation with the deviation
˚
of the nitrogen atom by 0.68(1) A, the bond P
N
is in the axial position and the olefin fragments in
its bulky substituents (that are rotated around the
C
Ph bonds) have E configuration.
The overall geometry of the phosphorus-
containing heterocycle in 4e is also typical for the
Heteroatom Chemistry DOI 10.1002/hc

Synthesis and Cytotoxicity of Novel 3,5-Bis(arylidene)piperid-4-ones with 1,3,2-Oxazaphosphorinanes Moieties
3
NH
P
O
Cl
Ar
O
O NH
2
O
O
N P
DMAP, CHCl3, r.t.
O
Ar
Ar
Ar
Ar
4a–e
O
NH
NH O
P
NCS
O
S
C
3
O NH
NH P
1a–e
N
CHCl3, r.t.
O
5a–c
Ar
Ar = 3-Pyridyl (a), 4-F-C6H4 (b), 4-O2N-C6H4 (c), 4-Me2N-C6H4 (d), 4-HO-3,5-t
SCHEME 1 Synthesis of 3,5-bis(arylidene)piperid-4-ones 4, 5 modified by 1,3,2-oxazaphosphorinane moieties.
FIGURE 2 General view of the compound 4e in representation of atoms via thermal ellipsoids at 40% probability level.
˚
Hydrogens at carbon atoms are not shown. Selected bond lengths in the oxazaphosphorinane moiety (A): P(1)–O(1) 1.4743(19),
P(1)O(2) 1.588(2), P(1)–N(1) 1.619(2), P(1)–N(2) 1.639(2), O(2)-C(1) 1.458(4), N(1)–C(3) 1.484(4), C(1)–C(2) 1.501(6), C(2)–
C(3) 1.510(6).
oxazaphosphorinane compounds with a P-NC2 moi-
ety (see, e.g., [18–21]), the major difference being
the position of the hydrogen atom at N(1)—axial
in 4e and equatorial in others. This caused the
expected changes in the bond lengths of the hetero-
cycle, in particular relative to 2-chloro-2-oxo-1,3,2λ5-
oxazaphosphinane 2, a rather air- and moisture-
stable compound, whose structure was also
elucidated by X-ray diffraction (Fig. 3).
However, replacement of the chlorine atom by the
bulky substituted piperidone causes a slight elon-
˚
gation of P(1)–O(2) bond from 1.5647(11) A in 2
˚
to 1.588(2) A in 4e. This effect can be explained
by the above differences in the position of the hy-
drogen atom at N(1) that allow for the formation
of the anomeric interaction lp(N)→σ*(P O) in 4e;
the pseudotorsion angle lpN(1)P(1)O(2) is 153.4(1)◦.
There is also a variation in the C3H6 fragment of the
oxazaphosphinane moiety, which can be attributed
to its strong liberation in a crystal of 4e.
Taking into account that all the oxazaphospho-
rinane compounds with a P-NC2 fragment X-rayed
to date (Cambridge Structural Database, release
2011) have the H(N) atom in the equatorial plane
In both cases, the oxazaphosphinane moiety oc-
cupies a chair conformation with the atoms P(1)
and C(2) deviated from the N(1)O(2)C(1)C(3) plane
˚
by 0.50(1) and –0.66(1) A in 4e and by 0.55(1) and
˚
–0.67(1) A in 2, and the bond lengths within it are
˚
practically the same (the variation is ca. 0.01 A).
Heteroatom Chemistry DOI 10.1002/hc

4
Shipov et al.
TABLE 1 Cytotoxicity of 1,3,2-Oxazaphosphorinane Deriva-
tives 4a–e and 5a–c Against Human Carcinoma Cell Lines
Caov3, A549, KB 3-1, and KB 8-5. (IC50 (μM))
Human Carcinoma Cell Lines
Compound
CaOv3
A549
KB 3-1
30
KB 8-5
4a
>20
>20
5
30
18
4
4
4b
7
3
0.4
0.3
4.8 0.4 6.0 0.4
1.5 0.1 1.5 0.2 5.2 0.5
>20
>30
4c
4d
>20
>30
35
>30
4
33
>30
5
4e
5a
4.8 0.3 3.5 0.3 4.5 0.3 7.0 0.5
2.5 0.2 2.7 0.2 1.5 0.1 6.5 0.5
1.2 0.1 1.2 0.1 6.4 0.4 6.2 0.3
50 10
-
5b
5c
FIGURE 3 General view of the compound 2 in representa-
tion of atoms via thermal ellipsoids at 50% probability level.
Selected bond lengths in the oxazaphosphorinane moiety
Melphalan
Doxorubicin
50 10
-
-
-
1.2 0.1 3.3 0.2
˚
(A): P(1)–O(1) 1.4703(11), P(1)–O(2) 1.5647(11), P(1)–N(1)
1.6147(14), O(2)–C(1) 1.4741(17), N(1)–C(3) 1.4841(19),
C(1)–C(2) 1.510(2), C(2)–C(3) 1.519(2).
Cytotoxic Properties
The cytotoxic activity of the compounds synthe-
sized was tested in vitro (MTT test; MTT stands for
3-(4,5-dimethyldiazolyl-2)-2,5-diphenyl tetrazolium-
bromide) against human cancer cell lines, namely
Caov3 (ovarian carcinoma), A549 (lung carcinoma),
KB 3-1 (human oral epidermoid carcinoma), and KB
8–5 (drug-resistant subclone of the latter one with
Pgp170 hyperexpression). The results are summa-
rized in Table 1, showing the corresponding IC50
values (IC50 is the concentration of a compound re-
quired to inhibit the growth of the cells by 50%). An
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 the literature
(see, e.g., [7, 8]). Doxorubicin was used as the sec-
ond positive control.
The cytotoxicity screening has revealed
that compounds obtained are more active than
Melphalan used as a positive control for Caov3
and A549 cell lines. Among bisamidophospates 4,
compounds bearing donor dimethylamino groups
(4d) or a combination of tert-butyl and hydroxy
substituents (4e) possess much lower cytotoxicity
compared with those having electron-withdrawing
substituents in the side aryl rings such as fluorine
atom (4b) and nitro group (4c) (IC50 in the micromo-
lar range of 1.5–7 μM). Compound 4c was the most
active from this series. A similar correlation of cyto-
toxic properties of 3,5-bis(arylidene)-4-piperidones,
both phosphorylated and nonphosphorylated, with
electronic properties of side aryl groups, i.e., an
increase in the cytotoxicity with an increase in
the electron-withdrawing power of these groups,
was previously mentioned in the literature. Un-
expectedly, in contrast to a range of the known
phosphorylated E,E-3,5-bis(3-pyridinylmethylidene)
similarly to 2, its axial position in 4e is apparently
a consequence of crystal packing as a result of the
presence of bulky arylidene substituents or extra
proton donors/acceptors.
Thus, the diamidophosphate molecules in the
crystal 4e form centrosymmetric dimers through
˚
N(1) H···O(1) hydrogen bonds (N···O 2.982(4) A,
NHO 172(1)◦) in such a way that the second molecule
is shifted along the crystallographic axis that is
somewhat perpendicular to its largest dimension—
the bis(arylidene)-4-piperidone line. At the same
time, the group P O is also involved in the inter-
molecular H-bond with one of the OH groups (O···O
◦
˚
2.702(3) A, OHO 153(1) ), resulting in the formation
of infinite H-bonded tapes. The second OH group is
˚
H-bonded to the atom O(3) (O···O 2.832(3) A, OHO
129(1)◦), assembling the above supramolecular as-
sociates into double layers. The formation of the
three-dimensional (3D) framework in a crystal is
completed by a number of weak interactions
C
C
H···O, C H···π, π···π, and H···H, including
H···Cl ones with dichloromethane molecules.
In the crystal 2, the hydrogen◦bonds N H···O
˚
(N···O 2.9179(17) A, NHO 159(1) ) also give rise
to centrosymmetric dimers, but this time the
mean planes of the resulting H-bonded cycle and
the oxazaphosphinane moieties of the neighboring
molecules 2 practically coincide. In addition, the
presence of the covalently bound chlorine atom
makes these dimers hold together through the Cl···O
˚
interactions (Cl···O 3.191(1) A, lp(O)→σ*(P-Cl)); the
latter with a number of C H···Cl and C H···O con-
tacts complete the formation of the 3D framework
of 2.
Heteroatom Chemistry DOI 10.1002/hc

Synthesis and Cytotoxicity of Novel 3,5-Bis(arylidene)piperid-4-ones with 1,3,2-Oxazaphosphorinanes Moieties
5
piperid-4-ones having an electron-withdrawing
3-pyridyl ring and possessing excellent antitumor
activity toward the same cell lines, the cytotoxicity
of compound 4a was rather low and comparable
with that of 4d and 4e bearing donor side groups.
Therefore, this compound 4a represents a rare
exception from the above tendency, i.e., an in-
crease in antitumor activity for compounds with
electron-withdrawing side groups.
The potency of the diamidophosphates 4 toward
KB human oral epidermoid carcinoma cell lines was
in general the same as was observed for ovarian and
lung carcinoma cells, i.e., 4c > 4b >> 4a ∼ 4d ∼ 4e.
Similar to doxorubicin, the cytotoxicity toward the
KB3-1 cell line was higher comparing with that for
drug-resistant sublone KB8-5.
Thioureas 5 bearing the same arylidene rings
were more active than the diamidophosphates 4, and
in this case the derivative 5a bearing 3-pyridyl rings
was only slightly less potent than those having 4-
fluoro- and 4-nitro-benzylidene groups. Moreover,
thiourea 5c demonstrated a similar activity toward
both clones of KB cell lines. These cell lines differ
from each other in the expression of the transmem-
brane transporting P-glycoprotein (permeability gly-
coprotein abbreviated as Pgp170) being responsi-
ble for the elimination of hydrophobic compounds,
including the cytostatics, from tumor cells and as-
sumed to be a reason for multidrug resistance in the
treatment of cancers [22]. A similar toxicity toward
these cell lines for compound 5c allowed suggesting
that it is not the substrate for Pgp170 and hence can
be of interest for the treatment of resistant tumors.
useful tool for their modification, providing in-
creased antitumor activity and better bioavailability.
EXPERIMENTAL
NMR spectra were recorded with a Bruker AMX-400
spectrometer (Germany) (1H, 400.13; 31P, 161.98;
13C, 100.61; and 19F 376.49) or Bruker Avance-
300 spectrometer (Germany) (1H, 300.13 and 31P,
121.49) using residual proton signals of a deuterated
solvent as an internal standard (1H, 13C) and H3PO4
(31P) as an external standard. The 13C NMR spec-
tra were registered using the JMODECHO mode;
the signals for the C atoms bearing odd and even
numbers of protons have opposite polarities. Melt-
ing points were determined using an EZ melt ap-
paratus (Stanford research systems, Sunnyvale, CA)
and are uncorrected. IR spectra were recorded in
KBr pellets on a Magna-IR750 (Nicolet, Madison,
WI) Fourier spectrometer, resolution 2 cm−1, 128
scans. The starting 3,5-bis(arylidene)piperid-4-ones
1a–e [23] and compounds 2 [14] and 3 [15] were
prepared according to the known procedures, and
their chemical–physical characteristics fit well with
the literature data. All reagents and solvents used
were obtained from Aldrich (St. Louis, MO) and used
without further purification.
N-(2-Oxo-1,3,2λ5-oxazaphosphinan-2-yl)-3,5-
bis(benzyliden)-4-piperidones 4a–e (General
Procedure)
A
mixture of the corresponding NH-3,5-
bis(aryliden)piperid-4-one 1a–e (1 equiv), 2-chloro-
2-oxo-1,3,2λ5-oxazaphosphinane 2 (1.1 equiv), and
DMAP (1.2 equiv) in CHCl3 (10 mL) was stirred at
room temperature for 7–8 h. The resulting mixture
was concentrated in vacuo (or filtered in the case
of compounds 4c), and the product was purified by
chromatography on SiO2 (CHCl3/MeOH 100/1) or
recrystallization.
CONCLUSIONS
To summarize the results presented, two conve-
nient procedures for the introduction of the 1,3,2-
oxazaphosphorinane ring into a backbone of 3,5-
bis((hetero)arylidene)piperid-4-ones have been sug-
gested. The cytotoxicity screening demonstrated that
the introduction of such a phosphorus heterocy-
cle resulted in compounds possessing high anti-
tumor properties toward human carcinoma cell
lines with IC50 values in a micromolecular range
for the most potent compounds bearing electron-
withdrawing side phenyl rings. The presence of a
thiocarbamoyl linker between two heterocycles, i.e.,
piperidone and 1,3,2-oxazaphosphorinane, led to the
pronounced increase in the activity, and thiourea 5c
was found to be the most potent and promising drug
candidate from the series. Thus, the introduction
of 1,3,2-oxazaphosphorinane moiety into the back-
bone of biologically active compounds seems to be a
N-(2-Oxo-1,3,2λ5-oxazaphosphinan-2-yl)-
(3E,5E)-3,5-bis(3-pyridinylmethylidene)-4-
piperidinone (4a)
A
reaction of 3,5-bis(3-pyridinylmethylidene)-4-
piperidinone 1a (0.28 g, 1.0 mmol) with 2-chloro-2-
oxo-1,3,2λ5-oxazaphosphinane 2 (0.18 g, 1.1 mmol)
in the presence of DMAP (0.15 g, 1.2 mmol) provided
0.17 g (43%) of the final product after purification
by column chromatography. Yellow solid, mp 151◦C
(decomp.). IR (KBr, ν, cm−1): 3398, 3209, 2969,
2889, 1668 (C O), 1611 (C C), 1582, 1561, 1478,
1412, 1360, 1327, 1267, 1246, 1204, 1195, 1176, 1131,
1096, 1083, 1063, 1045, 1022, 980, 964, 937, 893, 873,
Heteroatom Chemistry DOI 10.1002/hc

6
Shipov et al.
846, 821, 807, 762, 709, 689, 628, 543, 523, 475. 31P
in the presence of DMAP (0.15 g, 1.2 mmol)
provided 0.29 g (60%) of the final product after
recrystallization from pyridine with ether. Yel-
low solid, mp 240–242◦C (decomp.). 31P (121.49,
(121.49, CDCl3), δ: 10.27. 1H (400.13, CDCl3),
δ: 1.38–1.48, 1.62–1.72 (both m, 1H
+
1H,
CH2CH2CH2), 2.80–2.91, 2.07–3.18, 3.27–3.33,
(three m, 1H + 1H, NHCH2), 3.89–3.98, 4.12–4.17
(both m, 1H + 1H, CH2O), 4.48 (HA) and 4.56 (HB)
1
DMSO-d6), δ: 9.74. H (300.13, DMSO-d6), δ: 1.32–
1.37, 1.52–1.61 (both m, 1H + 1H, CH2CH2CH2),
2.67–2.78, 2.90–2.99 (both m, 1H + 1H, CH2NH),
3.82–3.93, 3.98–410 (both m, 1H + 1H, CH2O), 4.44
2
(ABX system, 4H, piperidone NCH2, JHH = 15.6
3
3
Hz, JPHA = 5.6, JPHB = 5.8 Hz), 7.28 (dd, 2H, HPy,
3
3JHH = 4.8 Hz, JHH = 7.8 Hz), 7.65 (d, 2H, HPy,
3
(d, 4H, piperidone NCH2, JHP = 9.5 Hz), 4.79 (dt,
3
2
3JHH = 8.1 Hz), 7.68 (s, 2H, CH ), 8.51 (dd, 2H,
1H, NH, JHH = 4.3 Hz, JHP = 6.5 Hz), 7.76 (s, 2H,
HC ), 7.80 (d, 2H, CHAr, 3JHH = 8.8 Hz) 8.33 (d, 2H,
CHAr, 3JHH = 8.8 Hz). Anal. calcd. for C22H21N4O7P:
C 54.55, H 4.37, N 11.57. Found: C 54.59, H 4.27, N
11.59.
3
4
HPy, JHH = 4.8 Hz, JHH = 1.4 Hz), 8.59 (d, 2H,
HPy, 4JHH = 1.7 Hz). 13C (100.61, CDCl3), δ: 25.64 (d,
CH2CH2CH2, 3JPC = 6.6 Hz), 40.96 (d, NHCH2, 2JPC
2
= 2.6 Hz), 45.72 (d, NCH2, JPC = 3.3 Hz), 68.03 (d,
2
POCH2, JPC = 7.2 Hz), 123.33 (CPy), 130.29 (CPy),
N-(2-oxo-1,3,2λ5-oxazaphosphinan-2-yl)-3,5-
bis(4-dimethylaminobenzyliden)-4-
piperidone (4d)
132.84 (CH ), 134.31 (C CH), 136.72 (CPy), 149.68
(CPy), 150.96 (CPy), 185.88 (C O). Anal. calcd. for
C20H20N4O3P·1/3H2O: C 59.85, H 5.19, N 13.96.
Found: C 59.84, H 5.55, N 13.91.
A reaction of 3,5-bis(4-dimethylaminobenzyliden)-4-
piperidone 1d (0.36 g, 1.0 mmol) with 2-chloro-2-
oxo-1,3,2λ5-oxazaphosphinane 2 (0.17 g, 1.1 mmol)
in the presence of DMAP (0.15 g, 1.2 mmol)
provided 0.17 g (38%) of the final product af-
ter chromatography purification. Red solid, mp
220◦C (decomp.). IR (KBr, ν, cm−1): 3427, 3276,
2898, 2816, 1653, 1582, 1524, 1445, 1369, 1325,
1310, 1273, 1245, 1231, 1182, 1171, 1129, 1082,
1066, 1045, 1005, 984, 945, 918, 867, 809, 765,
523. 31P (161.97, CDCl3) δ: 10.46. 1H (400.13,
CDCl3), δ: 1.43–1.47, 1.72–1.81 (both m, 1H +
N-(2-Oxo-1,3,2λ5-oxazaphosphinan-2-yl)-3,5-
bis(4-fluorobenzyliden)-4-piperidone (4b)
A
reaction
of
3,5-bis(4-fluorobenzyliden)-4-
piperidone 1b (0.31 g, 1.0 mmol) with 2-chloro-2-
oxo-1,3,2λ5-oxazaphosphinane 2 (0.17 g, 1.1 mmol)
in the presence of DMAP (0.15 g, 1.2 mmol) provided
0.27 g (63%) of the final product after chromato-
graphic purification followed by recrystallization
from an acetone–hexane mixture. Yellow solid,
mp 166–167◦C. 31P (121.49, CDCl3), δ: 10.28. 1H
(300.13, CDCl3), δ: 1.46–1.51, 1.72–1.79 (both m, 1H
+ 1H, CH2CH2CH2), 2.55 (br s, 1H, NH), 2.86–2.98,
3.12–3.25 (both m, 1H + 1H, CH2NH), 3.93–4.04,
2
1H, CH2CH2CH2), 2.65 (d, 1H, NH, JHP = 4.8 Hz),
2.85–2.93, 3.10–3.19 (both m, 1H + 1H, CH2NH),
3.02 (s, 12H, CH3), 3.96–4.02, 4.13–4.20 (both m,
1H + 1H, CH2O), 4.45 (HA) and 4.53 (HB)
4.16–4.28 (both m, 1H + 1H, CH2O), 4.42 (HA) and
2
(ABX system, 4H, piperidone NCH2, JHH
=
2
4.49 (ABX system, 4H, piperidone NCH2, JHH
=
3
3
15.6 Hz, JPHA = 5.6, JPHB = 5.8 Hz), 6.69 (d,
3
3
16.2 Hz, JPHA = 6.9, JPHB = 6.4 Hz), 7.12 (dd,
3
3
2H, CHAr, JHH = 8.8 Hz), 7.35 (d, 2H, CHAr, JHH
= 8.8 Hz), 7.74 (s, 2H, HC ). Anal. calcd. for
C26H33N4O3P·0.5H2O: C 63.79, H 7.00, N 11.44.
Found: C 63.84, H 6.98, N 10.97.
3
2H, CHAr, JHH
=
3JHF = 8.6 Hz), 7.40 (dd, 2H,
3
3
CHAr, JHH = 8.6, JHF = 5.4 Hz), 7.76 (s, 2H,
HC ). 13C (75.47, CDCl3), δ: 25.93 (d, CH2CH2CH2,
3JPC = 5.1 Hz), 41.17 (d, NHCH2, JPC = 2.3 Hz),
2
2
45.87 (NCH2), 68.17 (d, POCH2, JPC = 5.7 Hz),
N-(2-Oxo-1,3,2λ5-oxazaphosphinan-2-yl)-
(3E,5E)-3,5-bis(3,5-ditert-butyl-4-
hydroxybenzylidene)-4-piperidone (4e)
2
115.97 (d, CArH, JCF = 16.4 Hz), 128.00 (C CH),
4
130.78 (d, ipso-CAr CH , JCF = 2.8 Hz), 132.35
3
(d, CArH, JCF = 6.2 Hz), 135.43 (CH ), 163.00 (d,
ipso-CAr F, 1JCF = 188.7), 186.7 (C O). Anal. calcd.
for C22H21F2N2O3P: C 61.39, H 4.92, N 6.51. Found:
C 61.24, H 4.94, N 6.48.
A
reaction
hydroxybenzylidene)-4-piperidone 1e (0.21 g,
0.38 mmol) with
2-chloro-2-oxo-1,3,2λ5-
of
3,5-bis(3,5-ditert-butyl-4-
oxazaphosphinane 2 (0.06 g, 0.40 mmol) in the
presence of DMAP (0.05 g, 0.39 mmol) provided
0.21 g (85%) of the final product after purification
by column chromatography. Yellow solid, mp
216–251◦C (decomp.). IR (KBr, ν, cm−1): 3624,
3549, 3380, 3001, 2958, 2915, 2874, 1660, 1594,
1571, 1562, 1436, 1425, 1390, 1359, 1332, 1260,
N-(2-Oxo-1,3,2λ5-oxazaphosphinan-2-yl)-3,5-
bis(4-nitrobenzyliden)-4-piperidone (4c)
A
reaction
of
3,5-bis(4-nitrobenzyliden)-4-
piperidone 1c (0.37 g, 1.0 mmol) with 2-chloro-2-
oxo-1,3,2λ5-oxazaphosphinane 2 (0.17 g, 1.1 mmol)
Heteroatom Chemistry DOI 10.1002/hc

Synthesis and Cytotoxicity of Novel 3,5-Bis(arylidene)piperid-4-ones with 1,3,2-Oxazaphosphorinanes Moieties
7
1238, 1202, 1194, 1161, 1120, 1094, 1051, 1033,
in anhydrous CHCl3 (20 mL), a solution of 2-
isothiocyano-2-oxo-1,3,2λ5-oxazaphosphinane
988, 938, 874, 839, 772. 31P (121.49, CDCl3), δ: 9.89.
1H (300.13, CDCl3), δ: 1.45 (s, 36H, CH3), 1.52,
1.63–1.80 (both m, 1H + 1H, CH2CH2CH2), 2.42
(br s, 1H, NH), 2.88–3.04, 3.13–3.27 (both m, 1H +
1H, CH2NH), 3.96–4.08, 4.17–4.31 (both m, 1H+1H,
CH2O), 4.48 (HA) and 4.56 (HB) (ABX system, 4H,
3
(0.20 g, 1.1 mmol) in CHCl3 (10 mL) was added
at room temperature. Dissolution of starting com-
pound 1b was accompanied by the formation of
precipitate of the desired product. After 7 h of
stirring, the precipitate formed was filtered off, sus-
pended in CHCl3 (30 mL), stirred for 2 h, filtered off,
and dried in vacuum to give compound 5b (0.36 g,
82%) as a yellow solid, mp 172–173◦C. IR (KBr, ν,
cm−1): 3422, 3322, 3109, 2969, 2926, 2056, 1674,
1615, 1599, 1575, 1509, 1439, 1413, 1323, 1262,
1239, 1202, 1189, 1160, 1137, 1098, 1056, 1012,
994, 949, 889, 834, 761, 526, 507, 476, 442. 31P
(161.97, DMF-d7), δ: 1.79. 19F (376.49, DMF-d7),
2
3
piperidone NCH2, JHH = 15. 6 Hz, JPHA = 5.6,
3JPHB = 5.8 Hz), 5.52 (s, 2H, OH), 7.27 (s, 4H, Ar),
7.77 (s, 2H, HC ). 13C (75.47, CDCl3), δ: 25.95 (d,
3
CH2CH2CH2, JPC = 6.6 Hz), 30.06 (Me), 34.24
(CMe3), 41.19 (NHCH2), 45.97 (NCH2), 68.02 (d,
2
POCH2, JPC = 6.6 Hz), 126.37 (CAr CH ), 128.08
3
(CArH), 130.23 (d, C CH, JPC = 6.6 Hz), 136.03
(CAr CMe3), 137.41 (C CH), 155.07 (CArOH),
187.05 (C O). Anal. calcd. for C38H55N2O5P: C
70.13, H 8.52, N 4.30. Found: C 69.97, H 8.39, N
4.12.
1
δ:−110.49. H (400.13, DMF-d7), δ: 1.61–1.65, 1.94–
2.01 (both m, 1H + 1H, CH2CH2CH2), 2.96–2.99,
3.19–3.26 (both m, 1H + 1H, CH2NH), 3.84–3.90,
4.12–4.20 (both m, 1H + 1H, CH2O), 4.53 (s, 1H,
NHCS), 5.07 (s, 1H, HNP), 5.58(HA) and 5.67 (HB)
N-[(2-oxo-1,3,2λ5-oxazaphosphinan-2-
yl)thiocarbamoyl]-3,5-bis(3-
pyridinylmethiliden)-4-piperidone (5a)
2
(AB system, 4H, piperidone NCH2, JHH = 16.8 Hz),
3
7.55 (dd, 4H, CHAr, JHH
=
3JHF = 8.8 Hz), 7.91
3
4
(dd, 4H, CHAr, JHH = 8.8 Hz, JHF = 5.7 Hz), 7.99
(s, 2H, HC = ). 13C (75.47, DMSO-d6), δ: 22.57 (d,
To a solution of 3,5-bis(3-pyridinylmethiliden)-
4-piperidone 1a (0.22 g, 0.8 mmol) in anhy-
drous CHCl3 (20 mL), 2-isothiocyano-2-oxo-1,3,2λ5-
oxazaphosphinane 3 (0.18 g, 1.0 mmol) in CHCl3
(10 m) was added at room temperature, and the
mixture was stirred for 11 h. The precipitated prod-
uct was filtered off, washed with CHCl3, and dried
in vacuum to give 0.39 g of crude compound. Re-
crystallization of the crude product from i-PrOH
and washing with CHCl3 and ether afforded 0.09 g
(25%) of the desired compound as a solvate with
CHCl3 and H2O. Yellow solid, mp 165–167◦C (de-
comp.). 31P (161.97, DMSO-d6), δ: 1.90. 1H (400.13,
DMSO-d6), δ: 1.25–1.29, 1.55–1.69 (both m, 1H+1H,
CH2CH2CH2), 2.80–2.91 (m, 1H, NHCH2, the second
signal of the NHCH2 group is overlapped with that
of DMSO-d6), 3.75–3.92 (m, 1H, OCH2, the second
signal of the OCH2 group is overlapped with that
of DMSO-d6), 4.42 (s, 1H, NHCS), 5.01 (br s, 1H,
HN-P), 5.25(HA) and 5.33(HB) (piperidone NCH2,
3
CH2CH2CH2, JPC = 7.7 Hz), 44.36 (NCH2), 54.91
2
(NHCH2), 67.54 (d, POCH2, JPC = 7.7 Hz), 116.32
2
(d, CArH, JCF = 21.4 Hz), 127.97 (C CH), 130.59
(d, ipso-CAr CH , 4JCF = 14.2 Hz), 133.36 (d, CArH,
3JCF = 8.2 Hz), 138.34 (CH ), 163.02 (d, CArF, 1JCF
=
2
249 Hz), 170.68 (d, C S, JPC = 12.6 Hz), 184.82
(C O). Anal. calcd. for C23H22F2N3O3PS: C 56.44, H
4.53, N 8.58, P 6.33. Found: C 56.37, H 4.48, N 8.47,
P 6.08.
N-[(2-Oxo-1,3,2λ5-oxazaphosphinan-2-
yl)thiocarbamoyl]-3,5-bis(4-nitrobenzyliden)-4-
piperidone (5c)
This compound was obtained in
a
similar
fashion to compound 5a starting from 3,5-
bis(4-nitrobenzyliden)-4-piperidone 1c (0.33 g,
0.9 mmol) and of 2-isothiocyano-2-oxo-1,3,2λ5-
oxazaphosphinane 3 (0.20 g, 1.1 mmol) in CHCl3
(30 mL). Very fine precipitate was filtered off,
washed with CHCl3, and dried in vacuum to give
0.44 g (87%) of a desired product as a solvate with
CHCl3. Yellow solid, mp 190–192◦C (decomp.). IR
(KBr, ν, cm−1): 3379, 3109, 2970, 2855, 2051, 1682,
1618, 1598, 1594, 1518 (NO2), 1494, 1412, 1345
(NO2), 1315, 1254, 1203, 1192, 1109, 1048, 989,
939, 886, 854, 806, 754, 507. 31P (161.97, DMSO-
2
AB system, 4H, JHH = 15.5 Hz), 7.56 (dd, 2H, HPy,
3
3JHH = 7.8 Hz, JHH = 4.0 Hz), 7.77 (s, 2H, CH ),
3
8.00 (d, 2H, HPy, JHH = 7.8 Hz), 8.63 (d, 2H, HPy,
3JHH = 4.0 Hz), 8.79 (s, 2H, HPy). Anal. calcd. for
C21H22N5O3PS·CHCl3·0.5H2O: C 45.26, H 4.14, N
12.00. Found: C 45.25, H 3.98, N 12.12.
N-[(2-oxo-1,3,2λ5-oxazaphosphinan-2-
yl)thiocarbamoyl]-3,5-bis(4-fluorobenzyliden)-4-
piperidone (5b)
1
d6), δ: 1.95. H (300.13, DMSO-d6), δ: 1.23–1.40,
1.55–1.75 (both m, 1H + 1H, CH2CH2CH2), 2.70–
2.95 (m, 1H, NHCH2, the second signal of the
NHCH2 group is overlapped with that of DMSO-d6),
To
a
stirred suspension of 3,5-bis(4-fluoro-
benzyliden)-4-piperidone 1b (0.28 g, 0.9 mmol)
Heteroatom Chemistry DOI 10.1002/hc

8
Shipov et al.
3.80–3.87 (m, 1H, OCH2, the second signal of the
OCH2 group is overlapped with that of DMSO-d6),
4.35 (s, 1H, NHCS), 5.25(HA) and 5.35(HB) (piperi-
observed reflections with I > 2 σ(I)). All calculations
were performed using SHELXTL PLUS 5.0.
Crystallographic data for 2 and 4e·CH2Cl2 con-
taining the supplementary crystallographic data
for this paper, have been deposited with the
Cambridge Crystallographic Data Center, CCDC
881463 and 881464, respectively. These data can be
obtained free of charge from the Director, CCDC,
12 Union Road, Cambridge CB2 1EZ, UK (Fax:
+44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk
or www.ccdc.cam.ac.uk).
2
done NCH2, AB system, 4H, JHH = 16.0 Hz), 7.82
3
(d, 4H, CHAr, JHH = 9.0), 7.85 (s, 2H, CH ), 8.34
3
(d, 4H, CHAr, JHH = 8.6). 13C (75.47, DMSO-d6), δ:
22.51 (d, CH2CH2CH2, 3JPC = 7.7 Hz), 44.60 (NCH2),
2
54.85 (NHCH2), 67.57 (d, POCH2, JPC = 7.7 Hz),
124.07 (CArH), 131.75 (CArH), 140.44 (CAr), 147.81
2
(O2N-CAr), 170.93 (d, C S, JPC = 13.1 Hz), 184.85
(C O). Anal. calcd. for C23H22N5O7PS·0.2CHCl3: C
49.11, H 3.94, N 12.34. Found: C 49.24, H 3.87, N
12.41.
Evaluation of Cytotoxic Properties
X-ray Structure Determination
Cell lines used for estimation of toxicity of com-
pounds were CaoV3 (human ovarian carcinoma),
A549 (human lung carcinoma), KB 3-1 (human
oral epidermoid carcinoma cells), and drug-resistant
subclone of the latter one, i.e., KB 8-5, with Pgp170
hyperexpression. 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 in-
dividual compounds was measured for each cell line
after 72 h of cultivation by the MTT colorimetric
assay. The test is based on the ability of mitochon-
drial dehydrogenase in viable cells to convert a MTT
reagent (ICN Biomedicals, Eschwege, Germany) into
a soluble blue formazan dye. Briefly, the different
cell lines were seeded into 96-well plates at a con-
centration of 1×104 cells/100 μL/well. The cells were
allowed to attach overnight at 37◦C in a humidi-
fied atmosphere containing 5% CO2. The tested com-
pounds were initially dissolved in dimethylsulfoxide
(DMSO; Sigma-Aldrich) and the working solutions
were added to FBS free culture medium. The com-
pounds were added to wells with increasing drug
concentrations. After 72 h of incubation, 20 μL of
MTT reagent (5 mg/mL) was added and cell cultures
were incubated for 3 h at 37◦C. After the removal
of the culture medium, formazan crystals were dis-
solved in DMSO to determine the amount of for-
mazan product. The optic density (OD) was deter-
mined by the multiwell plate reader (Uniplan, Picon,
Russia) at 590 nm. The results were expressed as a
percentage decrease of cell viability as compared to
untreated controls. Each concentration of the com-
pound tested was examined in triplicate, and the IC50
values were determined graphically. The concentra-
The crystal of the compound 4e (as a solvate with one
molecule of CH2Cl2) suitable for the X-ray diffrac-
tion study was grown by slow diffusion of pentane
into a solution of the compound in methylene chlo-
ride, acid chloride 2 crystallized spontaneously after
purification.
Crystals of 4e·CH2Cl2 (C39H57Cl2N2O5P, M =
735.74) are monoclinic, space group P21/c, at 100
˚
K: a = 14.167(2), b = 17.270(3), c = 17.163(3) A, β =
3
˚
110.638(3)
, V = 3929.6(10) A , Z = 4 (Z’ = 1), dcalc
=
1.244 gcm−3, μ(Mo Kα) = 2.50 сm−1, F (000) = 1576.
Intensities of 40,956 reflections were measured with
a Bruker Smart APEX2 CCD diffra◦ctometer [λ(Mo
˚
Kα) = 0.71072 A, ω-scans, 2θ < 56 ], and 9471 in-
dependent reflections [Rint = 0.0662] were used in
further refinement. Crystals of 2 (C3H7ClNO2P, M =
155.52) are orthorhombic, space group Pbca, at 100
˚
K: a = 9.5554(19), b = 10.899(2), c = 12.156(2) A, V =
1265.9(4) A , Z = 8 (Z’ = 1), dcalc = 1.632 g cm−3
,
3
˚
μ(Mo Kα) = 7.66 сm−1, F (000) = 640. Intensities of
9167 reflections were measured with a Bruker Smart
APEX2 CCD diffractometer [λ(Mo Kα) = 0.71072
◦
˚
A, ω-scans, 2θ < 58 ], and 1682 independent reflec-
tions [Rint = 0.0380] were used in further refinement.
The structures were solved by a direct method and
refined by the full-matrix least-squares technique
against F2 in the anisotropic–isotropic approxima-
tion. The hydrogen atoms of NH and OH groups
were found in difference Fourier synthesis. The po-
sitions of H(C) atoms were calculated. All hydrogen
atoms were refined in the isotropic approximation
in the riding model. For 4e·CH2Cl2, the refinement
converged to wR2 = 0.1885 and GOF = 1.137 for
all independent reflections (R1 = 0.0652 was calcu-
lated against F for 6733 observed reflections with I
>2 σ(I)). For 2, the refinement converged to wR2 =
0.0627 and GOF = 1.000 for all independent reflec-
tions (R1 = 0.0250 was calculated against F for 1352
tions of compounds used were 5×10−5, 10−5, 10−6
,
10−7 M. Commercially available Melphalan (Sar-
colysin) and Doxorubicin purchased from Arkelan-
Glaxo were used as a positive control in the assay.
Heteroatom Chemistry DOI 10.1002/hc

Synthesis and Cytotoxicity of Novel 3,5-Bis(arylidene)piperid-4-ones with 1,3,2-Oxazaphosphorinanes Moieties
9
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