Design, cytotoxic and fluorescent properties of novel N-phosphorylalkyl substituted E,E-3,5-bis(arylidene)piperid-4-ones

Article abstract of DOI:10.1016/j.ejmech.2008.10.019

A series of E,E-N-phosphorylalkylene-3,5-bis(arylidene)piperid-4-ones 7a-k was prepared via the condensation of aromatic aldehydes with ω-aminophosphonates 5a-c and 6a,b bearing piperidone or a protected piperidone moiety, respectively. The synthetic rout

Full text of DOI:10.1016/j.ejmech.2008.10.019

European Journal of Medicinal Chemistry 44 (2009) 2135–2144
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, cytotoxic and fluorescent properties of novel N-phosphorylalkyl
substituted E,E-3,5-bis(arylidene)piperid-4-ones
Michael V. Makarova, Ekaterina Yu. Rybalkina b, Gerd-Volker Ro¨schenthaler c, Kurt W. Short d,
,
Tatiana V. Timofeeva d, 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 Institute for Inorganic and Physical Chemistry, University of Bremen, Leobener Strasse, D-28334 Bremen, Germany
d 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:
A
series of E,E-N-phosphorylalkylene-3,5-bis(arylidene)piperid-4-ones 7a–k was prepared via the
Received 15 July 2008
Received in revised form
10 October 2008
Accepted 20 October 2008
Available online 26 October 2008
condensation of aromatic aldehydes with -aminophosphonates 5a–c and 6a,b bearing piperidone or
u
a protected piperidone moiety, respectively. The synthetic routes to the starting aminophosphonates 5a–
c and 6a,b varied depending on the number of methylene groups in the alkylene chain and comprised
the Kabachnik–Fields reaction (n ¼ 1), the aza-Michael reaction (n ¼ 2) or alkylation of 4-piperidone
hydrochloride with diethyl
u-bromoalkylphosphonates under phase transfer catalysis conditions
(n ¼ 3,4). Phosphoryl substituted 3,5-bis(arylidene)piperid-4-ones 7b,c,e,f,h,i,k bearing both nitro groups
and fluorine atoms in the para-position of the arene rings possess cytotoxicity toward human carcinoma
cell lines CaOv3, Scov3, PC3 and A549 in the low micromolar range while their analogues having para-
Keywords:
Aminophosphonates
N-phosphorylalkylene-piperid-4-ones
N-phosphorylalkylene-3,5-
bis(arylidene)piperid-4-ones
Synthesis
dimethylamino groups had IC50 values greater than 50 mM . In contrast, only Me2N-substituted phos-
phonates 7g,j (n ¼ 3 and 4) and the salts of Me2N-substituted phosphonic acids 10c,f (n ¼ 2 and 3) display
fluorescence.
Structure–cytotoxicity–fluorescence
relationship
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
supposed mechanism of action comprising interaction with cellular
thiols with little or no affinity for hydroxyl and amino groups in
For years
g
-piperidone derivatives have been effectively used as
nucleic acids [9,12]. Cytotoxic properties were found for the related
cycloalkan-1-ones 2 possessing a conjugated structure [13–15] and
for curcumin 3 (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-hepta-
dien-3,5-dione), a natural component of the rhizome of Curcuma
longa, which proved to be the powerful chemopreventive and
anticancer agent [16,17] having also anti-inflammatory [18], anti-
bacterial [19], antihepatoxic [20], and antioxidative [21] properties.
Curcumin and other enones are supposed to have multiple targets
and interacting macromolecules within the cell [6,16,17]. The 1,5-
diaryl-3-oxo-1,4-pentadienyl 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 on the heterocyclic nitrogen atom in the piperidone
derivatives could influence the cytotoxic properties of the
compounds. In other words, it can increase the cytotoxic properties
by facilitating approach of the cytotoxin to a specific binding site or
reduce them preventing this interaction.
a base for design of pharmacologically active compounds such as
psycholeptics (e.g. the butyrophenone derivatives), local anes-
thetics (diammokann – 1-(b-anilinoethyl)-4-phenyl-4-(b-dieth-
ylaminoethoxy)piperidine N-cyclohexylsulfamine salt), analgetics
such as desprodin, prodin, meprodin, promedol, phentranyl and its
analogues which are currently used in clinical practice.
Recently, 3,5-bis(arylidene)-4-piperidones 1 (Fig. 1) were found
to possess high cytotoxic and antitumor activity towards different
cell lines [1–9] while hydrochloride salt of 3,5-bis(benzylidene)-4-
piperidone was well tolerated in mice whereby five daily doses up
to 240 mg/kg did not cause mortalities [10]. Moreover, these
compounds (generally screened as free bases) are non-lethal and
mice tolerated doses up to and including 300 mg/kg [11]. 3,5-
Bis(arylidene)-4-piperidones display anticancer properties via the
Recent observations demonstrate fluorescent properties of some
* Corresponding author.
E-mail address: odinets@ineos.ac.ru (I.L. Odinets).
representatives of 3,5-bis(arylidene)-4-piperidones 1 [22–24].
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.019

2136
M.V. Makarov et al. / European Journal of Medicinal Chemistry 44 (2009) 2135–2144
O
O
R3
R3
n
n
Z
Z
N
Z
Z
n
R1
R2
R2
1, R1 = H, Alk; R2, R3=H, OH; n = 0, 1; Z = NMe2, NO2, F
2, Z = C(NH)NH2, n = 0-5
O
O
OH
O
HO
OH
HO
OH
OCH3
OCH3
OCH3
OCH3
3, Curcumin
Fig. 1.
Natural fluorescence in the case of cytotoxic materials might be very
helpful in tracking their target organelle in cell. In the case of
piperidinones and their derivatives the cytotoxic activity increases
with the increase of electron withdrawing properties of the phenyl
ring substituents, which at the same time decreases the fluorescent
activity. Combination of antitumor activity and fluorescent proper-
ties makes these types of compounds exceedingly attractive as drug
candidates.
As mentioned above, antitumor activity and fluorescent prop-
erties of 3,5-bis(arylidene)piperid-4-ones are predominantly
determined by the conjugated system while solubility and
bioavailability are largely governed by the nature of the groups at
It should be mentioned that organophosphorus compounds
exhibit different types of bioactivity, and therefore form an
important series of compounds in the search for new drugs. For
instance, high-level anticancer activity has been found in a number
of phosphorus compounds of quite different structural types,
among them discovered in 1958 cyclophosphamide [25], which still
remains in wide clinical use, N-phosphonoacetyl-L-aspartic acid
(PALA) [26], derivatives of choline, including ether derivatives of
Lysophosphatidyl choline [27], 6–8 membered 1,2-azaphosphacy-
clanes [28] etc. Moreover, phosphorylated compounds may be used
as prodrugs to improve drug delivery to particular targets and to
increase the solubility of drugs.
the nitrogen atom of the piperidone moiety. However, the
p
-values
Taking into account bioactivity of organophosphorus
compounds in general and high and diverse biological activity of
aminophosphonates or so-called ‘‘phosphorus analogues’’ of ami-
noacids in particular [29], it seems reasonable to synthesize and
evaluate the cytotoxic and fluorescent properties of 3,5-bis(ar-
of the aryl substituents can affect both these parameters. 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.
So, the modification of the structures of 3,5-bis(arylidene)piperid-
4-ones both by variation of substituents at aromatic rings and by
insertion of bioactive groups to the piperidone nitrogen atom is
a very challenging route to novel fluorescent cytostatics. The
attempts to improve the antitumor properties of 3,5-bis(ar-
ylidene)-4-piperidones and affect the capacity of their trans-
portation via the cellular membrane were mostly concentrated on
modification at the nitrogen atom as N-acylation [2,4] or N-phos-
phorylation [22].
ylidene)piperidones bearing the
alkylene chain.
u-phosphonate group in the N-
2. Results and discussion
2.1. Chemistry
For the synthesis of the desired N-(u-phosphorylalkyl)-3,5-
bis(arylidene)piperid-4-ones two possible synthetic routes can be
path A
O
(EtO)2P(O)(CH2)nBr
PTC
R
N
H
4
R
O
5
7
6
8
4
3'
2'
3
2
11
9
R
N
R
10
O
N
(CH2)nP(O)(OEt)2
O
O
or
7
R
C(O)H
N
base or acid
path B
(CH2)nP(O)(OEt)2
(CH2)nP(O)(OEt)2
6
5
R=NMe2, F, NO2; n=1-4
Scheme 1. Possible synthetic approaches to N-phosphorylalkylated 3,5-bis(arylidene)piperidones 7.

M.V. Makarov et al. / European Journal of Medicinal Chemistry 44 (2009) 2135–2144
2137
O
N
O
O
O
O
O
K2CO3 / CH3CN
H2O- MeOH
rt
5b (n = 3)
5c (n = 4)
(EtO)2P(O)(CH2)nBr
+
(EtO)2PHO + CH2O +
N
H
.
HCl
N
N
H
(CH2)nP(O)(OEt)2
CH2P(O)(OEt)2
6a
Scheme 4. Synthesis of
g-aminopropyl- and
d-aminobutylphosphonates under PTC
conditions.
Scheme 2. Synthesis of a-aminomethylphosphonate via the Kabachnik-Fields reaction.
Piperidones 5b,c having three and four methylene groups in the
alkylene chain were prepared in high yields by alkylation of
used namely either alkylation of parent NH-3,5-bis(ar-
ylidene)piperid-4-ones 4 by phosphorylated halogenoalkanes or
condensation of preformed N-phosphorylalkyl substituted piper-
idones 5 or their O-protected derivatives 6 with aromatic aldehydes
under basic or acid conditions as outlined on Scheme 1. However,
previously we demonstrated [30] that reaction of NH-3,5-bis(ar-
ylidene)piperid-4-ones 4 with short-chain halogenoalkanes pro-
ceeded predominately as quaternization to afford N,N-dialkylated
products. Therefore, to avoid such side reaction condensation of
4-piperidone hydrochloride with diethyl
u-bromoalkylphospho-
nates under phase transfer catalysis conditions (Scheme 4).
As mentioned above, condensation of phosphorylated piper-
idones 5a–c with substituted benzaldehydes affording the target N-
phosphorylalkyl substituted 3,5-bis(arylidene)piperidones 7 can be
performed both under basic or acidic conditions. In the case of
dioxalane protected derivatives 6a,b only acidic conditions were
applied in order to ensure hydrolytic removal of the dioxalane
protective group (Table 1 and Scheme 5). It should be mentioned
that under acidic conditions (bubbling of gaseous HCl into
a mixture of reactants in acetic acid) the condensations of
compounds 5 and 6 were accompanied by partial dealkylation of
one ester group at the phosphorus atom in the phosphonate moiety
of the final 3,5-bis(arylidene)piperidones 7a–k (Scheme 5). In some
cases the corresponding semiesters 8 were isolated in pure form
either as free acids 8b,e (R ¼ F, n ¼ 1 and 2, respectively) or as
sodium salts 8j,g (n ¼ 3, 4, R ¼ NMe2) after the work-up procedure
(see Supplementary information). Such partial hydrolysis reduced
the yields of the 3,5-bis(arylidene)piperidones 7 and made work-
up and purification procedures difficult, especially in the case of p-
dimethylaminoarylidene derivatives where stable emulsions of
crude products in water were formed. The condensation under
basic conditions was the reasonable alternative to the acid-medi-
ated process, however, using alcoholic NaOH lead to a significant
decrease in product yields (less than 15%). Better results were
obtained when the reaction was performed in the presence of
piperidine as a base in alcoholic solutions. Under such conditions
the final products 7d,f,g,i were isolated in the lower yields
compared to acid-catalyzed condensation but the isolation proce-
dure was more convenient (see Section 4).
u
-(4-oxopiperidin-1-yl)alkylphosphonates 5 or their protected
forms 6 with aromatic aldehydes was the method of choice.
The synthetic routes to the starting aminophosphonates
bearing piperidone moiety 5 or their protected derivatives 6 varied
depending on the number of methylene groups of the alkylene
chain. Thus, N-phosphorylmethyl substituted piperidone (n ¼ 1)
was obtained as the dioxolane protected form 6a via the three-
component Kabachnik–Fields reaction of diethylphosphite,
commercially available 1,4-dioxa-8-azaspiro[4.5]decane and
formaldehyde in aqueous media (Scheme 2). Under these condi-
tions the reaction proceeds smoothly to give 6a as the main
reaction product (87% according to the 31P spectra of the reaction
mixture). However, distillation of crude product is accompanied by
partial decomposition reducing the total yield of purified 6a to
55%.
For the synthesis of b-aminophosphonates 5a, 6b bearing the
piperidone or protected piperidone moiety, respectively, we used
the aza-Michael reaction (Scheme 3). According to our data [31],
water as a solvent significantly accelerates the addition of various
amines to diethoxyvinylphosphonate to yield
b-amino-
phosphonates without any catalyst compared to other known
procedures for this reaction. When 4-piperidone hydrochloride was
used as a starting amine b-aminophosphonate 5a was isolated in
62% yield after purification by chromatography. In the case of the
protected piperidone derivative, 1,4-dioxa-8-azaspiro[4.5]decane,
no side reactions were observed and modified piperidone 6b was
isolated in 98% yield with >98% purity after a simple liophilization
procedure.
Total hydrolysis of ester groups at the phosphorus atoms in
phosphonates 7b–g,i was performed via the reaction with trime-
thylsilylbromide in chloroform followed by the treatment with aq.
Table 1
Synthesis of phosphonates 7a–k via Scheme 5.
Compound
n
R
Conditions/starting
substrate
Yield, %
O
O
7a
7b
7c
7d
7d
7d
7e
7e
7f
7f
7f
7g
7g
7h
7i
1
1
1
2
2
2
2
2
2
2
2
3
3
3
3
3
4
4
NMe2
F
i/6a
i/6a
i/6a
i/5a
i/6b
ii/6b
i/5a
i/6b
i/5a
i/6b
ii/6b
i/5b
ii/5b
i/5b
i/5b
ii/5b
i/5c
i/5c
34
42
43
34
37
34
27
40
40
57
29
32
27
37
47
23
43
30
N
.
HCl
NO2
NMe2
NMe2
NMe2
F
H
5a
(CH2)2P(O)(OEt)2
N
K2CO3 / H2O, rt
O
O
(EtO)2(O)P
F
NO2
NO2
NO2
NMe2
NMe2
F
NO2
NO2
NMe2
NO2
N
H
O
O
6b
H2O, rt
N
7i
7j
7k
(CH2)2P(O)(OEt)2
Scheme 3. Synthesis of b-aminoethylphosphonates via the aza-Michael reaction.

2138
M.V. Makarov et al. / European Journal of Medicinal Chemistry 44 (2009) 2135–2144
O
O
O
N
2 ArC(O)H
+
R
N
R
R
N
R
.
OH
(CH2)nP(O)(OEt)2
(CH2)nP
(CH2)nP(O)(OEt)2
i
OEt
5a-c
O
7a-k
8a-k
ii
O
O
O
n=1: R=NMe2 (7a,8a); F (7b,8b); NO2 (7c,8c);
n=2: R=NMe2 (7d,8d); F (7e,8e); NO2 (7f,8f);
n=3: R=NMe2 (7g,8g); F (7h,8h); NO2 (7i,8i);
n=4: R=NMe2 (7j,8j); NO2 (7k,8k)
2 ArC(O)H
R
N
R
N
(CH2)nP(O)(OEt)2
7d,f,g,i
(CH2)nP(O)(OEt)2
6a,b
Scheme 5. Synthesis of phosphonates 7a–k. Reagents and conditions: (i) HCl(g)/AcOH then K2CO3/H2O; (ii) piperidine/EtOH.
O
O
NaOH/EtOH
R
1.Me3SiBr
2. MeOH
R
N
R
R
N
xHBr
(CH2)nP(O)(OEt)2
(CH2)nP(O)(OH)2
7b-g,i
9a-g
O
n=1, R=F (9a,10a), NO2 (9b,10b);
n=2, R= NMe2 (9c,10c), F (9d,10d), NO2 (9e,10e)
n=3, R=NMe2 (9f,10f), NO2 (9g,10g)
R
N
R
(CH2)nP(O)(ONa)2
10a-g
Scheme 6. Hydrolysis of phosphonates 7.
MeOH. The corresponding phosphonic acids 9a–g were isolated
either as hydrobromides or as hydrobromides-hydrates containing
fractional amounts of hydrogen bromide and water molecules per
one molecule of acid. For evaluation of antitumor activity, the acid
hydrobromides 9a–g were further converted to water-soluble
phosphonic acid sodium salts 10a–g by treatment with an alcoholic
solution of NaOH (Scheme 6).
phosphonates and phosphonic acid salts having fluorine or nitro-
substituents, while compounds 7g,j and 10c,f possess intense
fluorescence at ca. 565 nm (Figs. 2 and 3). Fluorescence quantum
yields were determined using the fluorescence standard 9,10-
diphenylanthracene, 1 ꢀ10ꢁ5 M, dissolved in degassed ethanol.
Values were determined by the procedure of Maciejewski and Steer
[32].
2.2. Fluorescence
2.3. Cytotoxic properties
Taking into account the fluorescent properties of some aryli-
denepiperidones [22–24,30] giving the possibility to estimate their
distribution in cancer cells [30], absorption and emission spectra of
some phosphonates 7g–k and salts 10a–g were studied. Absorption
and emission maxima of all compounds are listed in Table 2. It can
be seen that compounds 7h and 7i,k bearing fluorine and nitro
groups in arene rings correspondingly have one absorption peak
around 330 nm while those 7g,j bearing dimethylamino groups
have two absorption peaks, one around 270 nm and the second
larger one around 455 nm. The absorption spectra of phosphonates
7g–k in chloroform are shown in Fig. S1. Similarly, two absorption
peaks, one around 280 nm and the second one around 475 nm are
observed for absorption spectra of salts 10c,f (R ¼ NMe2) in water
(Fig. S2). Moreover, two absorption peaks (higher at 330 nm and
lower at 235 nm) were observed for salts 10a,d bearing p-F moie-
ties and only one peak for p-nitrosubstituted derivatives.
The cytotoxic activity of the compounds synthesized was tested
against four human cell lines, namely CaOv3 and Scov3 (ovarian
carcinoma), PC3 (prostate carcinoma) and A549 (lung carcinoma).
Table 2
Photophysical data for phosphonates 7g–k and salts 10a–g.
Compound
Absorption
maximum
(nm)
Fluorescence
maximum
(nm)
Estimated
fluorescence
quantum yielda
7g
7h
7i
7j
455
329
331
456
331
331
327
476
331
326
476
327
566
417
385
567
391
392
388
570
394
389
570
382
0.022
4.638E-5
1.962E-5
0.024
8.719E-6
4.102E-5
3.231E-5
0.011
4.565E-5
2.674E-5
0.011
7k
10a
10b
10c
10d
10e
10f
10g
To obtain fluorescent spectra, compounds were excited at
lex ¼ 400 nm (in the case of 7g,j and 10c,f). The fluorescence
emission was measured at the wavelength interval of 410–800 nm.
It was also found that higher intensity emission is observed when
solutions are excited near the maximum of a longer wavelength
absorption peak. Practically no fluorescence is observed both for
5.506E-5
a
Relative to the fluorescence standard 9,10-diphenylanthracene, 1 ꢀ10ꢁ5 M, in
ethanol.

M.V. Makarov et al. / European Journal of Medicinal Chemistry 44 (2009) 2135–2144
2139
Table 3
Cytotoxicity of
u-aminophosphonates 7a–k and parent NH-piperidones 4a–c
against human CaOv3, Scov3, PC3 and A549 cell lines.
Compound
R
n
CaOv3
Scov3
IC50
PC3
IC50
A549
IC50
IC50
(
m
M)a
(
m
M)
(m
M)
(mM)
4a
4b
4c
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
NMe2
F
NO2
NMe2
F
NO2
NMe2
F
NO2
NMe2
F
–
–
–
1
1
1
2
2
2
3
3
3
4
4
–
NTa
NTa
14 ꢂ 4.5
6.5 ꢂ 1.5
2.5 ꢂ 1.0
15 ꢂ 5
6 ꢂ 3
1.4 ꢂ 0.4
0.80 ꢂ 0.07
0.4 ꢂ 0.1
NTa
8.05 ꢂ 0.09
1.0 ꢂ 0.2
NTa
1.02 ꢂ 0.13
1.00 ꢂ 0.05
NTa
9.07 ꢂ 0.32
7.12 ꢂ 0.41
NTa
1.03 ꢂ 0.09
1.54 ꢂ 0.35
30 ꢂ 3.21
3.0 ꢂ 0.6
1.40 ꢂ 0.23
NTa
NTa
NTa
10.20 ꢂ 0.78
5.03 ꢂ 0.57
NTa
2.1 ꢂ 0.31
1.52 ꢂ 0.23
NTa
1.64 ꢂ 0.15
10.04 ꢂ 1.12
4.01 ꢂ 0.23
1.32 ꢂ 0.21
NTa
1.82 ꢂ 0.44
0.71 ꢂ 0.08
NTa
5.05 ꢂ 0.68 2.01 ꢂ 0.22
NTa
NTa
2.8 ꢂ 0.3
0.9 ꢂ 0.3
NTa
8.50 ꢂ 0.03
NO2
NMe2
NO2
–
5.62 ꢂ 0.21
NTa
1.01 ꢂ 0.07
4.1 ꢂ 0.2
1.2 ꢂ 0.2
3.0 ꢂ 0.3
Melphalan
20 ꢂ 5
60 ꢂ 10
70 ꢂ 20
50 ꢂ 20
a
NT means that at concentration 50 mM 50% cell death was not observed, further
increase of the concentration usually resulted in precipitation of the compound.
Fig. 2. The fluorescence spectra of phosphonates 7g–k in chloroform.
fluorine atom (compounds 4b and 7b,e,h) generally decreases the
activity in comparisonwith their analogues with a nitro-substituent
and the same length of the linker (compounds 7). It should be noted
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%). 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 [1–9,22,30]. For comparison the
activity of parent NH-piperidones 4a–c bearing the same substit-
uents in the phenyl rings was estimated in the same assay.
that dimethylamino substituted N-u-phosphorylated compounds
7a,d,g,j do not demonstrate cytotoxicity, while compound 4a (lines
Scov3, PC3 and A549) and non-phosphorylated N-alkyl-3,5-bis(ar-
ylidene)piperid-4-ones and their quaternized salts possess cyto-
toxic activity even when they have para-NMe2 or NEt2 groups in
benzene rings [line A549, see Ref. [30]].
For compounds 7b,e,h bearing the para-F atom, the introduction
of phosphorylalkyl substituent resulted in the increase of cytotoxic
properties for CaOv3, Scov3, and PC3 cells in comparison with the
NH-piperidone 4b while the tendency changed for the opposite one
for A549 screen. Such clear tendency was not observed in the case
of phosphonates having the para-NO2 groups possessing either
higher or lower activities in comparison with those for 4c
depending on the alkylene chain length between the phosphorus
atom and the heterocyclic nitrogen one. Thus, for Scov3 phospho-
nates 7c,f,i are more active than 4c, for A549 the cytotoxic prop-
erties of 4c are higher than those of its analogues 7c,f,i,k, for CaOv3
The parent NH-piperidones 4a–c and their phosphorylated
analogues 7 excluding those bearing NMe2 substituents (namely, 4a
for CaOv3 cell line, 7a for CaOv3, PC3 and A549 screens and 7d,g,j for
all cell lines used in these assays) had lower IC50 figures than
melphalan. In general, the cytotoxic properties of both the NH-
piperidones 4a–c and the E,E-N-phosphorylalkylene-3,5-bis(ar-
ylidene)piperid-4-ones 7a–k fit well with the above mentioned
general tendency that cytotoxic activity increases with the increase
of electronwithdrawing properties of the substituents R in the para-
position of benzene rings. Thus, compounds 7c,f,i,k (n ¼ 1, 2, 3 and 4
respectively) having strong electron withdrawing nitro group in the
para-position of benzene rings are the most active in this series. The
presence of the both weak acceptor and weak electron-donating
cell line only activity of d-aminophosphonate 7k was similar to
those of 4c while for PC3 IC50 values of all phosphorylated
compounds 7c,f,i,k were less comparing with 4c.
Moreover, the influence of the alkylene chain length manifested
variously for different cell lines. Thus, other conditions being equal,
d-aminophosphonate 7k demonstrates higher activity against the
CaOv3 cell line than its analogues with a shorter linker and was just
as potent as 4c. For Scov3 the tendency is in the opposite direction
and the activity of 7i (n ¼ 3) was even higher than that for 4c. For
prostate carcinoma PC3 the activity is a little bit lesser than that for
4c, comparable for 7c, 7f (n ¼ 1 and 2 correspondingly), and a little
bit decreased with elongation of the alkylene chain (compounds
7h,k). At the same time for lung carcinoma cell line A549,
nophosphonates 7c,e, and f are the most active.
b-ami-
Pharmaceutically acceptable salts are, because their solubility in
water is greater than that of the initial compounds, particularly
suitable for medical applications. Surprisingly, we failed to obtain
50% inhibition of the cells growth using salts 10 even those having
nitro-substituents.
3. Conclusion
We have developed efficient synthetic approaches to
u-ami-
Fig. 3. The fluorescence spectra of phosphonic acid sodium salts 10a–g in water.
nophosphonates bearing piperidone or a protected piperidone

2140
M.V. Makarov et al. / European Journal of Medicinal Chemistry 44 (2009) 2135–2144
moiety and differing in alkylene chain length. These are useful as
starting substrates for the synthesis of E,E-N-phosphorylalkylene-3,
5-bis(arylidene)piperid-4-ones. Investigation of cytotoxic proper-
ties of the compounds has revealed that a number of phosphonates
in the 3,5-bis(arylidene)piperid-4-one series have IC50 values in the
low micromolecular range towards four human carcinoma cell
lines. The difference in potencies between the compounds is
attributed to the difference in electronic properties of substituents
R in the para-position of the benzene rings rather than alkylene
chain length. Namely, the cytotoxicity increases with the increase
of electron withdrawing properties of the substituents R. Donor
amino groups result in increased fluorescence emission. This study
has revealed the high potential of these molecules for further
development to improve potencies.
completion of the reaction (monitoring via 31P NMR spectra).
Insoluble substances were filtered off and washed with CH2Cl2
(20 ml). Organic solutions were combined, evaporated in vacuum to
dryness and the residue was extracted by boiling hexane (4 ꢀ 25 ml).
Hexane was removed in vacuum to give diethyl 3-(4-oxopiperidin-1-
yl)propylphosphonate (4.5 g, 84%, purity 94% according to the 31P
and 1H NMR spectra) as light-yellow liquid. The compound was used
in the next step without further purification. 1H (CDCl3),
d
: 1.22 (t, 6H,
3
POCH2CH3, JHH ¼ 7 Hz), 1.72 (m, 4H, NCH2CH2CH2P and NCH2
3
CH2CH2P), 2.34 (appeared t, 4H, cyclic N(CH2CH2)2, JHH ¼ 5.4 Hz),
3
2.42 (t, 2H, NCH2CH2CH2P, JHH ¼ 6 Hz), 2.64 (appeared t, 4H, cyclic
N(CH2)2, JHH ¼ 5.4 Hz), 4.00 (m, 4H, POCH2). 13C (CDCl3),
d: 15.93
3
(POCH2CH3, 3JCP ¼ 6.4 Hz), 19.89 (NCH2CH2CH2P, 2JCP ¼ 4.8 Hz), 22.69
(NCH2CH2CH2P, JCP ¼ 142.5 Hz), 40.59 (C–C(O)), 52.35 (cyclic N
3
(CH2)2), 54.64 (NCH2CH2CH2P, JCP ¼ 16.8 Hz), 60.88 (POCH2CH3,
4. Experimental protocols
2JCP ¼ 6.4 Hz), 208.19 (CO). 31P (CDCl3),
d: 32.08. Anal. calcd. for
C12H24NO4P (%): C 51.98, H 8.72, P 11.17, found (%): C 51.48, H 8.75, P
11.26.
4.1. General remarks
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 of deuterated solvent as an internal
4.4. Diethyl 3-(4-oxopiperidin-1-yl)butylphosphonate 5c
The compound was obtained according the above procedure
except that diethyl 4-bromobutylphosphonate was used instead of
diethyl 3-bromopropylphosphonate and the reaction time was
ca.14 days. Yield 82%, purity 95%. The compound was used in the
standard (1H, 13C), CFCl3 19F) and H3PO4 31P) as an external stan-
( (
dard. 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. Melting points are uncorrected.
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 typical intensive
absorption bands in the 1200–1250 cmꢁ1 regions which may be
unambiguously assigned to vibrations of the P]O moiety were not
observed in the IR spectra of the phosphonates 7 as they are mixed
with vibrations of the conjugated aromatic system.
The starting diethoxy(3-bromopropyl)phosphonate [33] and
diethoxy(4-bromobutyl)phosphonate [34] were obtained by the
Arbuzov reaction of triethylphosphite and 1,3-dibromopropane or
1,4-dibromobutane correspondingly according to the known
procedures. Other reactants were purchased from Aldrich and used
without further purification.
next step without further purification. 1H (CDCl3),
d
: 1.22 (t, 6H,
3
POCH2CH3, JHH ¼ 7.1 Hz), 1.41–1.61 (m, 6H, NCH2CH2CH2CH2P),
3
2.34 (appeared quintet, 6H, cyclic N(CH2CH2), JHH ¼ 6 Hz and
3
NCH2CH2CH2CH2P, JHH ¼ 6 Hz), 2.68 (appeared t, 4H, cyclic
3
N(CH2)2, JHH ¼ 6.1 Hz), 3.88 (m, 4H, POCH2CH3). 13C (CDCl3),
d:
15.99 (POCH2CH3, 3JCP ¼ 5.9 Hz), 19.91 (NCH2CH2CH2CH2P,
2JCP ¼ 5.1 Hz), 24.95 (NCH2CH2CH2CH2P, JCP ¼ 140.5 Hz), 27.65
(NCH2CH2CH2CH2P, 3JCP ¼ 16 Hz), 40.70 (–H2C–C(O)), 52.58
(N(CH2)2 cyclic), 56.19 (NCH2CH2CH2CH2P), 60.91 (POCH2CH3,
2JCP ¼ 6.6 Hz), 208.53 (CO). 31P (CDCl3),
d: 32.00. Anal. calcd. for
C13H26NO4P$1/3H2O (%): C 52.51, H 9.04, P 10.42; found (%): C
52.56, H 9.12, P 10.17.
4.5. Diethyl (1,4-dioxa-8-azaspiro[4.5]dec-8-yl)methyl
phosphonate 6a
1,4-Dioxa-8-azaspiro[4.5]decane (4.29 g, 0.03 mol) was added
portionwise to a solution of diethylphosphite (4.14 g, 0.03 mol) and
formalin (2.43 g of 37% solution in water–MeOH, 0.03 mol) in
deionised water (15 mL) at room temperature, followed by stirring
of the reaction mixture over three days at the same temperature.
Water was removed under vacuum and the residue was distilled at
reduced pressure to give 4.90 g (55%) of phosphonate 6a of 94%
purity. B.p. 140–150 ꢃC (0.3 Hg). Analytically pure sample was
obtained by column chromatography on silica gel (gradient elution
4.2. Diethyl 2-(4-oxopiperidin-1-yl)ethylphosphonate 5a
To a solution of 4-piperidone hydrochloride monohydrate
(4.62 g; 0.03 mol) in deionised water (15 mL) at 0 ꢃC a solution of
potassium carbonate (4.14 g, 0.03 mol) in water (10 mL) was added.
Then diethyl vinylphosphonate (4.92 g, 0.03 mol) was added, and
reaction mixture was stirred at room temperature for two days. The
reaction solutionwas extracted with CH2Cl2, the organic extract was
dried over Na2SO4, filtered and evaporated at reduced pressure to
give the crude product (6.27 g, 78%) which was purified by column
chromatography on SiO2 (gradient from 100% CHCl3 to CHCl3/
from EtOAc/cyclohexane ¼ 4:1 to 100% EtOAc). 1H (CDCl3),
d: 1.11 (t,
3
6H, OCH2CH3, JHH ¼ 7.0 Hz), 1.51 (appeared t, 4H, cyclic
N(CH2CH2)2, 3JHH ¼ 5.6 Hz), 2.66 (appeared t, 4H, cyclic N(CH2CH2)2,
EtOH ¼ 20:1) to give 4.9 g (62%) of the title compound.1H (CDCl3),
d:
3JHH ¼ 5.6 Hz), 2.73 (d, 2H, CH2P, JPH ¼ 11.6 Hz), 3.87 (s, 4H, OCH2
2
-
3
1.22 (t, 6H, OCH2CH3, JHH ¼ 7 Hz), 1.89 (dt, 2H, CH2CH2P,
CH2O), 4.08 (m, 4H, OCH2CH3). 13C (CDCl3),
d
: 15.95 (d, POCH2CH3,
2JPH ¼ 20 Hz, JHH ¼ 8 Hz), 2.34 (appeared t, 4H, N(CH2CH2)2 cyclic,
3JCP ¼ 5.6 Hz), 34.26 (cyclic NCH2CH2), 52.69 (d, cyclic NCH2,
3
3JHH ¼ 6 Hz), 2.66 (m, 6H, N(CH2CH2)2 cyclic and CH2CH2P), 4.00 (m,
3JCP ¼ 10.4 Hz), 53.00 (d, NCH2, JCP ¼ 163 Hz), 61.55 (d, POCH2CH3,
4H, OCH2CH3). 31P (CDCl3),
d: 31.24. Anal. calcd. for C11H22NO4P: C
2JCP ¼ 6.4 Hz), 63.63 (s, OCH2CH2O), 105.98 (O–C–O). 31P (CDCl3),
d:
50.18, H 8.42, N 5.32, P11.76%; found:C 49.93, H 8.58, N5.18, P11.44%.
25.77. HRMS calculated for C12H24NO5P (Mþ) 293.13921, found
293.13962.
4.3. Diethyl 3-(4-oxopiperidin-1-yl)propylphosphonate 5b
4.6. Diethyl [2-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)
A
mixture of diethyl 3-bromopropylphosphonate (5.0 g,
ethyl]phosphonate 6b
0.0193 mol), piperidone monohydrate hydrochloride (2.97 g,
0.0193 mol) and potassium carbonate (6.5 g, 0.047 mol) in CH3CN
(40 mL) was stirred for ten days at room temperature until
Diethyl vinylphosphonate (8.20 g, 0.05 mol) was added to
a stirred solution of protected piperidone (7.15 g, 0.05 mol) in water

M.V. Makarov et al. / European Journal of Medicinal Chemistry 44 (2009) 2135–2144
2141
2
(15 mL) at room temperature and the mixture was stirred at the
same temperature for ca 20 h. Water was evaporated under
reduced pressure to give 15.1 g (98%) of crude 6b (>98% purity) as
a viscous liquid. The product was used for the further reactions
CH2P, JPH ¼ 11.6 Hz), 3.94–4.08 (m, 8H, cyclic NCH2 þ OCH2CH3),
7.09 (t, 4H, 3JHH ¼ 9.0 Hz, JFH ¼ 9.0 Hz), 7.36 (dd, 4H, JHH ¼ 8.0 Hz,
3
3
4JFH ¼ 5.0 Hz), 7.76 (s, 2H, CH]). 13C (CDCl3),
d: 15.91 (d, POCH2CH3,
3JCP ¼ 5.9 Hz), 51.78 (d, NCH2P, JCP ¼ 162 Hz), 55.48 (d, N(CH2)2
2
2
without purification. 1H (CDCl3),
d
:
1.15 (t, 6H, OCH2CH3,
cyclic, JCP ¼ 9.5 Hz), 61.73 (d, POCH2CH3, JCP ¼ 7.0 Hz), 115.37 (d,
3JHH ¼ 7.0 Hz), 1.57 (appeared t, 4H, cyclic N(CH2CH2)2,
C(8), C(10), C(80), C(100), JCF ¼ 22.0 Hz), 130.74 (d, C(6) and C(60),
2
3JHH ¼ 5.6 Hz), 1.80 (dt, 2H, CH2CH2P, JPH ¼ 19 Hz, JHH ¼ 8.2 Hz),
2.37 (appeared t, 4H, cyclic N(CH2CH2)2, 3JHH ¼ 5.6 Hz), 2.52 (m, 2H,
CH2CH2P), 3.78 (s, 4H, OCH2CH2O),3.93 (m, 4H, OCH2CH3). 13C
4JCF ¼ 3.3 Hz), 131.87 (C(3) and C(30)), 132.01 (d, C(7), C(11), C(70),
2
3
3
C(110), JCF ¼ 8.4 Hz), 135.44 (C(5) and C(50)), 162.54 (d, C(9) and
C(90), JCF ¼ 251 Hz),185.98 (C(4)).19F (CDCl3),
d
: ꢁ110.99. 31P (CDCl3),
(CDCl3),
d
: 16.05 (d, POCH2CH3, 3JCP ¼ 6.0 Hz), 23.44 (d, NCH2CH2P,
d
: 24.72. HRMS calculated for C24H26F2NO4P (Mþ) 461.15675, found
JCP ¼ 138 Hz), 34.32 (s, cyclic NCH2CH2), 50.35 (cyclic NCH2), 50.73
461.15624.
2
(NCH2CH2P), 61.12 (d, POCH2CH3, JCP ¼ 6.4 Hz), 63.79 (s, OCH2
-
CH2O), 106.53 (O–C–O). 31P (CDCl3),
d
: 31.85. HRMS calculated for
4.7.3. Diethyl 3,5-bis(4-nitrobenzylidene)-4-oxopiperidin-1-yl-
methylphosphonate 7c
C13H26NO5P (Mþ) 307.15486, found 307.15540.
Gradient elution starting from CH2Cl2 to CH2Cl2/EtOH ¼ 100:3;
crystallization from CH2Cl2/pentane mixture. Light-yellow crystal
solid, m.p. 197–200 ꢃC. IR: 1679 (C]O), 1620, 1592, 1511, 1346, 1315,
1304, 1265, 1248, 1197, 1108, 1047 and 1023 (P–O–C), 997, 972, 927,
4.7. N-( -(Diethoxyphosphorylalkyl)-3,5-bis(arylidene)piperid-4-
ones) 7 (general procedure)
u
Method A: dry hydrochloric acid was bubbled through a solu-
tion or suspension of the corresponding aminophosphonate 5a–c,
6a,b (0.01 mol) and aldehyde (0.02 mol) in glacial acetic acid (ca.
20 mL; in case of well soluble 4-fluorobenzaldehyde, volume of
AcOH was reduced to 15 mL) over 2–4 h. The reaction mixture was
kept at room temperature for 4–5 days. The volatiles were removed
under reduced pressure. Diethyl ether (30 ml) was added to the
residue to remove unreacted 4-fluoro- or 4-nitrobenzaldehyde and
the precipitate formed was filtered off. To this solid CH2Cl2 and an
aqueous solution of potassium carbonate or sodium carbonate (pH
w 8–9) were added. 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 (for
details of purification see below) followed by crystallization (slow
diffusion method) from a mixture of acetone and CH2Cl2, or CHCl3
and pentane to give substituted phosphonates 7.
Method B: a mixture of the corresponding 2-(4-oxopiperidin-1-
yl)alkylphosphonate 5a,b or its O-protected form 6b, (0.01 mol),
aldehyde (0.02 mol) and piperidine (1 g, 0.012 mol) in EtOH
(15 mL) were refluxed for 4 days. Volatiles were evaporated at
reduced pressure and the residue was purified by column chro-
matography followed by additional crystallization from CH2Cl2/
pentane mixture.
856, 758. 1H (CDCl3),
d
: 1.22 (t, 6H, OCH2CH3, 3JHH ¼ 7.0 Hz), 3.00 (d,
2H,
CH2P,
2JPH ¼ 11.6 Hz),
3.98–4.11
(m,
8H,
cyclic
NCH2 þ OCH2CH3), 7.54–7.58 (d, 4H, aromatic), 7.83 (s, 2H, CH]),
8.27–8.32 (d, 4H, aromatic). 13C (CDCl3),
d: 16.24 (d, POCH2CH3,
3JCP ¼ 6.0 Hz), 52.16 (NCH2P, 3JCP ¼ 160 Hz), 55.59 (d, N(CH2)2 cyclic,
2JCP ¼ 9.5 Hz), 62.13 (d, POCH2CH3, JCP ¼ 6.8 Hz), 123.71 (C(8),
2
C(10), C(80), C(100)), 130.74 (C(7), C(11), C(70), C(110)), 134.49 (C(5)
and C(50)), 135.14 (C(3) and C(30)), 140.92 (C(6) and C(60)), 147.47
(C(9) and C(90), 185.76 (C(O)). 31P (CDCl3),
d: 24.08. HRMS calculated
for C24H26N3O8P (Mþ) 515.14575, found 515.14390.
4.7.4. Diethyl 2-[3,5-bis(4-(dimethylamino)benzylidene)-4-
oxopiperidin-1-yl]ethylphosphonate 7d
Eluent EtOAc; crystallization from CH2Cl2/pentane mixture.
Orange-red crystal solid, m.p. 124–126 ꢃC. 1H (CDCl3),
d: 1.27 (t, 6H,
3
2
OCH2CH3, JHH ¼ 7.0 Hz), 1.97 (dt, 2H, CH2CH2P, JPH ¼ 18.0 Hz,
3JHH ¼ 8.0 Hz), 2.87 (m, 2H, CH2CH2P), 3.01 (s, 12H, NMe2), 3.84 (s,
4H, NCH2 cyclic), 4.04 (m, 4H, OCH2CH3), 6.68–6.72 (d, 4H,
aromatic), 7.29–7.34 (d, 4H, aromatic), 7.75 (s, 2H, CH]). 13C
3
(CDCl3),
d
: 16.20 (d, POCH2CH3, JCP ¼ 6.0 Hz), 24.15 (d, NCH2CH2P,
JCP ¼ 138 Hz), 39.86 (NMe2), 50.44 (NCH2CH2P), 54.49 (N(CH2)2
cyclic), 61.46 (d, POCH2CH3, 2JCP ¼ 6.4 Hz), 111.45 (C(8), C(10), C(80),
C(100)), 122.89 (C(6) and C(60)), 128.47 (C(3) and C(30)), 132.33 (C(7),
C(11), C(70), C(110)), 136.74 (C(5) and C(50)), 150.37 (C(9) and C(90),
4.7.1. Diethyl 3,5-bis[4-(dimethylamino)benzylidene]-4-
oxopiperidin-1-yl-methylphosphonate 7a
186.36 (C(O)). 31P (CDCl3),
d: 31.49. HRMS calculated for
C29H40N3O4P (Mþ) 525.27565, found 525.27664.
Eluent – EtOAc, crystallization from CHCl3/pentane mixture.
Orange-red crystal solid, m.p. 162–164 ꢃC. IR: 1659 (C]O), 1591,
1556, 1523, 1448, 1366, 1298, 1274, 1254, 1171, 1054 (P–O–C), 1022
4.7.5. Diethyl 2-[3,5-bis(4-fluorobenzylidene)-4-oxopiperidin-1-
yl]ethylphosphonate 7e
Eluent EtOAc; crystallization from CH2Cl2/pentane mixture. Light-
yellow crystal solid, m.p. 57–60 (first melting) and 90–93 ꢃC (second
(P–O–C), 995, 967, 943, 815, 772, 518. 1H (DMSO-d6),
d
: 1.13 (t, 6H,
3
OCH2CH3, JHH ¼ 7.0 Hz), 2.97 (s, 12H, NMe2), 3.08 (d, 2H, CH2P,
2JPH ¼ 12.0 Hz), 3.87–4.02 (m, 8H, cyclic NCH2 þ OCH2CH3), 6.73–
melting).1H (CDCl3),
d
:1.26(t, 6H, OCH2CH3, 3JHH ¼ 7.0 Hz), 1.90 (dt, 2H,
6.77 (d, 4H, aromatic), 7.31–7.36 (d, 4H, aromatic), 7.49 (s, 2H,
CH2CH2P, 2JPH ¼ 18.0 Hz, 3JHH ¼ 8.0 Hz), 2.84 (m, 2H, CH2CH2P), 3.79 (s,
CH]). 13C (CDCl3),
d
: 16.16 (d, POCH2CH3, JCP ¼ 6.0 Hz), 39.88
4H, NCH2 cyclic), 4.03 (m, 4H, OCH2CH3), 7.11 (t, 4H, JHH ¼ 8.0 Hz,
3
3
1
3
4
(NMe2), 51.89 (d, CH2P, JCP ¼ 163 Hz), 56.14 (d, cyclic N(CH2)2,
3JCP ¼ 9.4 Hz), 61.93 (d, POCH2CH3, 2JCP ¼ 6.6 Hz),111.49 (C(8), C(10),
C(80), C(100)), 122.97 (C(6) and C(60)), 128.33 (C(3) and C(30)), 132.40
(C(7), C(11), C(70), C(110)), 137.15 (C(5) and C(50)), 150.43 (C(9) and
3JFH ¼ 8.0 Hz), 7.36 (dd, 4H, JHH ¼ 8.0 Hz, JFH ¼ 6.0 Hz), 7.76 (s, 2H,
CH]). 13C (CDCl3),
d
: 16.20 (d, POCH2CH3, JCP ¼ 6.0 Hz), 24.12 (d,
3
NCH2CH2P, JCP ¼ 139 Hz), 50.47 (NCH2CH2P), 54.48 (N(CH2)2 cyclic),
61.57 (d, POCH2CH3, 2JCP ¼ 6.1 Hz), 115.67 (d, C(8), C(10), C(80), C(100),
2JCF ¼ 21.6 Hz),130.93 (d, C(6) and C(60), 4JCF ¼ 3.3 Hz),132.12 (C(3) and
C(30)), 132.20 (C(7), C(11), C(70), C(110)), 135.48 (C(5) and C(50)), 162.79
C(90)), 186.39 (C(O)). 31P (DMSO-d6),
d: 25.28. HRMS calculated for
C24H26N3O8P (Mþ) 511.26000, found 511.26020.
(d, C(9) and C(90), JCF ¼ 250 Hz),186.51 (C(4)).19F (CDCl3),
d
:ꢁ110.97. 31
P
4.7.2. Diethyl 3,5-bis(4-fluorobenzylidene)-4-oxopiperidin-1-yl-
methylphosphonate 7b
(CDCl3), d
: 30.95. HRMS calculated for C25H28F2NO4P (Mþ) 475.17240,
found 475.17241.
Eluent – EtOAc, crystallization from CH2Cl2/pentane mixture at
ꢁ10 ꢃC. Light-yellow crystal solid, m.p. 105–110 ꢃC (no sharp
melting). IR: 1675 (C]O), 1611, 1598, 1585, 1508, 1294, 1272, 1222,
1186,1162,1047 (P–O–C),1025 (P–O–C), 996, 959, 934, 828, 784, 528,
4.7.6. Diethyl 2-[3,5-bis(4-nitrobenzylidene)-4-oxopiperidin-1-
yl]ethylphosphonate 7f
Eluent EtOAc; crystallization from acetone/hexane mixture.
497. 1H (CDCl3),
d
: 1.17 (t, 6H, OCH2CH3, 3JHH ¼ 7.0 Hz), 2.95 (d, 2H,
Yellow crystal solid, m.p. 154–157 ꢃC. 1H (CDCl3),
d
: 1.25 (t, 6H,

2142
M.V. Makarov et al. / European Journal of Medicinal Chemistry 44 (2009) 2135–2144
3
2
OCH2CH3, JHH ¼ 7.0 Hz), 1.88 (dt, 2H, CH2CH2P, JPH ¼ 18.0 Hz,
3JHH ¼ 8.0 Hz), 2.86 (m, 2H, CH2CH2P), 3.82 (s, 4H, cyclic NCH2), 4.03
(m, 4H, OCH2CH3), 7.50–7.55 (d, 4H, aromatic), 7.80 (s, 2H, CH]),
NCH2CH2CH2P, JCP ¼ 142.3 Hz), 54.19 (N(CH2)2 cyclic), 56.84 (d,
3 2
NCH2CH2CH2P, JCP ¼ 17.2 Hz), 61.19 (d, POCH2CH3, JCP ¼ 6.2 Hz),
123.56 (C(8), C(10), C(80), C(100)), 130.60 (C(7), C(11), C(70), C(110)),
133.78 (C(5) and C(50)), 135.52 (C(3) and C(30)), 140.99 (C(6) and
8.26–8.30 (d, 4H, aromatic). 13C (CDCl3),
d: 16.23 (d, POCH2CH3,
3JCP ¼ 6.0 Hz), 24.08 (d, NCH2CH2P, JCP ¼ 139 Hz), 50.54
(NCH2CH2P), 53.98 (N(CH2)2 cyclic), 61.57 (d, POCH2CH3,
2JCP ¼ 6.4 Hz), 123.70 (C(8), C(10), C(80), C(100)), 130.67 (C(7), C(11),
C(70), C(110)), 134.17 (C(5) and C(50)), 135.18 (C(3) and C(30)), 140.93
C(60)), 147.28 (C(9) and C(90)), 186.10 (C(O)). 31P (CDCl3),
d: 31.59.
Anal. calcd. for C26H30N3O8P: C 57.46, H 5.56, N 7.73%; found: C
57.46, H 5.52, N 7.57%.
(C(6) and C(60)), 147.42 (C(9) and C(90), 185.88 (C(O)). 31P (CDCl3),
d:
4.7.10. Diethyl 3-[3,5-bis(4-dimethylaminobenzylidene)-4-
oxopiperidin-1-yl]-butylphosphonate 7j
30.53. HRMS calculated for C25H28N3O8P (Mþ) 529.16140, found
529.16073; calculated for [M–OH]þ 512.15866, found 512.15818.
Gradient elution starting from CHCl3 to CHCl3/acetone ¼ 10:1;
crystallization from CH2Cl2/hexane mixture. Orange-red crystal
4.7.7. Diethyl 3-[3,5-bis(4-dimethylaminobenzylidene)-4-
oxopiperidin-1-yl]-propylphosphonate 7g
solid, m.p. 146–148 ꢃC. 1H (CDCl3),
d: 1.25 (t, 6H, POCH2CH3,
3JHH ¼ 7.1 Hz), 1.63 (m, 6H, NCH2CH2CH2CH2P), 2.55 (t, 2H,
3
Gradient elution starting from CH2Cl2 to CH2Cl2/acetone ¼ 5:1;
crystallization from CHCl3/hexane mixture. Orange-red crystal
solid, m.p.140–142 ꢃC. IR: 1657 (C]O), 1587,1524, 1444,1372,1308,
NCH2CH2CH2CH2P, JHH ¼ 7.1 Hz), 3.00 (12H, 2NMe2), 3.80 (s, 4H,
N(CH2)2 cyclic), 4.01 (m, 4H, POCH2CH3), 6.69 (4H aromatic), 7.32
(4H aromatic), 7.73 (s, 2H, CH]). 13C (CDCl3),
d: 16.03 (d, POCH2CH3,
1233, 1160, 1063 (P–O–C), 1025 (P–O–C), 988, 948, 814, 514. 1H
3JCP ¼ 6.3 Hz), 19.97 (d, NCH2CH2CH2CH2P, JCP ¼ 4.8 Hz), 25.01 (d,
2
3
1
(CDCl3),
d
: 1.20 (t, 6H, POCH2CH3, JHH ¼ 7.1 Hz), 1.75 (m, 4H,
NCH2CH2CH2CH2P, JCP ¼ 140.4 Hz), 27.79 (d, NCH2CH2CH2CH2P,
3
NCH2CH2CH2P), 2.62 (t, 2H, NCH2CH2CH2P, JHH ¼ 6.4 Hz), 3.01 (s,
12H, 2NMe2), 3.83 (s, 4H, cyclic N(CH2)2), 3.98 (m, 4H, POCH2CH3),
6.70 (d, 4H aromatic, 3JFH ¼ 8.9 Hz), 7.33 (d, 4H aromatic, JHH ¼ Hz),
3JCP ¼ 16.3 Hz), 39.68 (NMe2), 54.78 (N(CH2)2 cyclic), 56.32
(NCH2CH2CH2CH2P), 60.97 (d, POCH2CH3, 2JCP ¼ 6.3 Hz),111.30 (C(8),
C(10), C(80), C(100)), 123.04 (C(6) and C(60)), 128.98 (C(3) and C(30)),
132.09 (C(7), C(11), C(70), C(110)),136.10 (C(5) and C(50)),150.13 (C(9)
7.74 (s, 2H, CH]). 13C (CDCl3),
d
: 16.14 (d, POCH2CH3, 3JCP ¼ 5.9 Hz),
2
20.12 (d, NCH2CH2CH2P, JCP ¼ 4.4 Hz), 22.78 (d, NCH2CH2CH2P,
JCP ¼ 141.2 Hz), 39.84 (NMe2), 54.78 (cyclic N(CH2)2), 57.04 (d,
and C(90)), 186.63 (C(O)). 31P (CDCl3),
d: 32.08. Anal. calcd. for
C31H44N3O4P:C 67.25, H8.01, N7.59%; found: C67.29, H8.01, N7.64%.
3
2
NCH2CH2CH2P, JCP ¼ 17.6 Hz), 61.18 (d, POCH2CH3, JCP ¼ 6.6 Hz),
111.48 (C(8), C(10), C(80), C(100)), 123.10 (C(6) and C(60)), 129.03
(C(3) and C(30)), 132.27 (C(7), C(11), C(70), C(110)), 136.30 (C(5) and
4.7.11. Diethyl 3-[3,5-bis(4-nitrobenzylidene)-4-oxopiperidin-1-yl]-
butylphosphonate 7k
C(50)), 150.33 (C(9) and C(90)), 186.76 (C(O)). 31P (CDCl3),
d
: 32.20.
Gradient elution starting from CH2Cl2 to CH2Cl2/acetone ¼ 5:1.
Anal. calcd. for C30H42N3O4P: C 66.77, H 7.84, N 7.79%; found: C
66.64, H 7.97, N 7.81%.
Yellow crystal solid, m.p. 117–119 ꢃC. 1H (CDCl3),
d
: 1.24 (t, 6H,
3
POCH2CH3, JHH ¼ 7.1 Hz), 1.60 (m, 6H, NCH2CH2CH2CH2P), 2.54 (t,
3
2H, NCH2CH2CH2CH2P, JHH ¼ 6.6 Hz), 3.78 (s, 4H, cyclic N(CH2)2),
4.7.8. Diethyl 3-[3,5-bis(4-fluorobenzylidene)-4-oxopiperidin-1-
yl]propylphosphonate 7h
4.01 (m, 4H, POCH2CH3), 7.53 (4H aromatic), 7.79 (s, 2H, CH]), 8.28
(4H aromatic). 13C (CDCl3),
d
: 16.17 (d, POCH2CH3, JCP ¼ 5.1 Hz),
3
Gradient elution starting from CH2Cl2 to CH2Cl2/acetone ¼ 5:1.
Yellow crystal solid, m.p. 84–90 ꢃC (no sharp melting). IR: 1673
(C]O), 1614, 1599, 1577, 1508, 1293, 1266, 1227, 1183, 1161, 1051 (P–
19.95
(d,
NCH2CH2CH2CH2P,
2JCP ¼ 3.7 Hz),
25.05
(d,
1
NCH2CH2CH2CH2P, JCP ¼ 141.2 Hz), 27.51 (d, NCH2CH2CH2CH2P,
3JCP ¼ 16.1 Hz), 54.36 (N(CH2)2 cyclic), 56.44 (NCH2CH2CH2CH2P),
61.21 (d, POCH2CH3, 2JCP ¼ 6.2 Hz), 123.60 (C(8), C(10), C(80), C(100)),
130.62 (C(7), C(11), C(70), C(110)), 133.79 (C(5) and C(50)), 135.63
(C(3) and C(30)), 141.08 (C(6) and C(60)), 147.31 (C(9) and C(90)),
O–C), 1034 (P–O–C), 1021, 967, 948, 908, 803, 780, 569, 533, 497. 1H
3
(CDCl3),
d
: 1.18 (t, 6H, POCH2CH3, JHH ¼ 7 Hz), 1.69 (m, 4H,
NCH2CH2CH2P), 2.61 (m, 2H, NCH2CH2CH2P), 3.78 (s, 4H, cyclic
N(CH2)2), 3.96 (m, 4H, POCH2CH3), 7.09 (t, 4H, C(10)–H, C(8)–H,
186.19 (C(O)). 31P (CDCl3),
d: 31.76. Anal. calcd. for C27H32N3O8P: C
3
3
C(100)–H, C(80)–H, JFH ¼ 8.6 Hz, JHH ¼ 8.6 Hz), 7.35 (dd, C(7)–H,
58.17, H 5.79, N 7.54%; found: C 58.17, H 5.71, N 7.39%.
C(11)–H, C(70)–H, C(110)–H, 3JHH ¼ 8.6 Hz, 4JFH ¼ 5.4 Hz), 7.75 (s, 2H,
CH]). 13C (CDCl3),
d
: 16.30 (d, POCH2CH3, JCP ¼ 6.6 Hz), 20.18 (d,
4.8. 3-[3,5-Bis(substituted benzylidene)-4-oxopiperidin-1-yl]alkyl
phosphonic acids 9a–g (general procedure)
3
NCH2CH2CH2P,
2JCP ¼ 4.4 Hz),
22.94
(d,
NCH2CH2CH2P,
JCP ¼ 141.5 Hz), 54.48 (cyclic N(CH2)2), 57.00 (d, NCH2CH2CH2P,
3JCP ¼ 16.9 Hz), 61.41 (d, POCH2CH3, JCP ¼ 6.6 Hz), 115.75 (d, C(8),
To a stirred solution of the corresponding diethyl phosphonate
(2 mmol) 7 dissolved in dry CHCl3 (10 mL) a solution of bromo-
trimethylsilane (8 mmol) in dry CHCl3 (3 mL) was added at one
portion from syringe at room temperature. In the case of
compounds 7d,g bearing the dimethylamino group an excess of
bromotrimethylsilane was used (28 mmol). Reaction solution was
stirred over 24 h at room temperature. The acids 9c,f precipitated
from the reaction solution and in this case the stirring over 3 days
was necessary to complete the transformation. CHCl3 was removed
at reduced pressure and the solid remaining was treated with
a mixture of MeOH:H2O ¼ 10:1 (v/v). Insoluble product was filtered
off, washed with a small portion of MeOH, then Et2O and dried
under P2O5 at 1 mm Hg to afford analytical pure compound. In the
case of acids 8c,f which were soluble in MeOH, methanol was
removed to dryness to give the desired compound.
2
2
C(10), C(80), C(100), JCF ¼ 21.3 Hz), 131.26 (d, C(6) and C(60),
4JCF ¼ 3.7 Hz), 132.72 (C(3) and C(30)), 132.31 (d, C(7), C(11), C(70),
C(110), 3JCF ¼ 8 Hz), 135.26 (C(5) and C(50)), 162.90 (d, C(9) and C(90),
JCF ¼ 251 Hz), 186.91 (C(4)). 19F (CDCl3),
d
: ꢁ110.66. 31P (CDCl3),
d:
31.88. Anal. calcd. for C26H30F2NO4P: C 63.80, H 6.18, N 2.86%;
found: C 63.85, H 6.13, N 2.77%.
4.7.9. Diethyl 3-[3,5-bis(4-nitrobenzylidene)-4-oxopiperidin-1-yl]-
propylphosphonate 7i
Gradient elution starting from CH2Cl2 to CH2Cl2/acetone ¼ 5:1;
crystallization from CH2Cl2/Et2O mixture. Yellow crystal solid, m.p.
133–136 ꢃC. IR: 1678 (C]O), 1618, 1598, 1592, 1514, 1348, 1339,
1308, 1279, 1260, 1247, 1234, 1204, 1180, 1120, 1110, 1099, 1057 (P–
O–C), 1027 (P–O–C), 1008, 966, 947, 926, 859, 810, 759. 1H (CDCl3),
d
: 1.19 (t, 6H, POCH2CH3, 3JHH ¼ 7.1 Hz), 1.65 (m, 4H, NCH2CH2CH2P),
3
2.62 (t, 2H, NCH2CH2CH2P, JHH ¼ 6.7 Hz), 3.80 (s, 4H, cyclic
4.8.1. [(3E,5E)-3,5-Bis(4-fluorobenzylidene)-4-oxo-1-
piperidinyl]methylphosphonic acid 9a
N(CH2)2), 3.96 (m, 4H, POCH2CH3), 7.52 (4H aromatic), 7.79 (s, 2H,
CH]), 8.27 (4H aromatic). 13C (CDCl3),
d
2
: 16.09 (d, POCH2CH3,
Yield 89%, decompose above 235 ꢃC. Light-yellowcrystal solid.1H
3JCP ¼ 6.2 Hz), 19.87 (d, NCH2CH2CH2P, JCP ¼ 4.4 Hz), 22.69 (d,
(DMSO-d6),
d
: 2.84 (d, 2H, CH2P, JPH ¼ 12 Hz), 4.07 (broad s, 24H,
2

M.V. Makarov et al. / European Journal of Medicinal Chemistry 44 (2009) 2135–2144
2143
cyclic NCH2 þ H2O þ P–OH), 7.29 (t, 4H, 3JHH ¼ 8 Hz, 3JFH ¼ 8 Hz), 7.57
acid 9 (0.001 mol). The reaction mixture was stirred at room
temperature for 24 h. Then the solid product was filtered off,
washed several times with anhydrous EtOH, Et2O and air-dried.
(dd, 4H, 3JHH ¼ 8 Hz, 4JFH ¼ 5 Hz), 7.60 (s, 2H, CH]).19F (DMSO-d6),
d:
ꢁ111.50. 31P (DMSO-d6),
d: 19.26. Anal. calcd. for C20H18F2NO4P$1/
2H2O: C 57.98, H 4.62, N 3.38%; found: C 57.95, H 4.45, N 3.37%.
4.9.1. Disodium [3,5-bis(4-fluorobenzylidene)-4-oxopiperidin-1-
4.8.2. [(3E,5E)-3,5-Bis(4-nitrobenzylidene)-4-oxo-1-
piperidinyl]methylphosphonic acid 9b
yl]methylphosphonate 10a
Yield 79%. Light-yellow crystal solid. 1H (D2O),
d: 2.63 (d, 2H,
Yield 93%, decompose >200 ꢃC. Yellow crystal solid. 1H (DMSO-d6),
:2.85(d, 2H, CH2P, 2JPH ¼ 12 Hz), 4.12 (s, 4H, NCH2 cyclic), 6.15 (broad s,
CH2P, 2JPH ¼ 12 Hz), 3.94 (s, 4H, NCH2 cyclic), 7.10 (t, 4H, 3JHH ¼ 8 Hz,
3
4
d
3JFH ¼ 8 Hz), 7.41 (dd, 4H, JHH ¼ 8 Hz, JFH ¼ 5 Hz), 7.52 (s, 2H,
4H, 0.5H2O þ P–OH), 7.68–7.73 (m, 4H, aromatic), 7.77 (s, 2H, CH]),
CH]). 19F (D2O),
C20H16F2NNa2O4P$6H2O: C 43.10, H 5.06, N 2.51%; found: C 42.98, H
d
: ꢁ111.26. 31P (D2O),
d: 15.18. Anal. calcd. for
8.23–8.28 (m, 4H, aromatic). 31P (DMSO-d6),
d: 19.16. Anal. calcd. for
C20H18N3O8P: C 52.30, H 3.95, N 9.15%; found: C 51.96, H 3.85, N 9.11%.
4.81, N 2.39%.
4.8.3. 2-[(3E,5E)-3,5-Bis(4-(dimethylamino)benzylidene)-4-oxo-1-
piperidinyl]-ethylphosphonic acid hydrobromide 9c
4.9.2. Disodium [3,5-bis(4-nitrobenzylidene)-4-oxopiperidin-1-
yl]methylphosphonate 10b
Yield 72%. Dark-red crystal solid. 1H (DMSO-d6),
d: 2.02–2.20 (m,
Yield 63%, yellow crystal solid. 1H (D2O),
d: 2.67 (d, 2H, CH2P,
2H, CH2CH2P), 3.03 (s, 12H, NMe2), 3.50 (m, 2H, CH2CH2P), 4.66 (s,
4H, NCH2 cyclic), 6.54 (broad s,11H, 3H2O þ acid protons), 6.86–6.90
(m, 4H, aromatic), 7.44–7.48 (m, 4H, aromatic), 7.77 (s, 2H, CH]). 31P
2JPH ¼ 12 Hz), 4.03 (s, 4H, NCH2 cyclic), 7.49–7.53 (m, 6H, aroma-
tic þ CH]), 8.09–8.12 (m, 4H, aromatic). 31P (D2O),
d: 15.16. Anal.
calcd. for C20H16N3Na2O8P$2H2O: C 44.54, H 3.74, N 7.79%; found: C
(DMSO-d6),
d: 20.96. Anal. calcd. for C25H32N3O4P$2.9HBr$1/2H2O: C
44.67, H 3.58, N 7.67%.
42.10, H 5.08, Br 32.49, N 5.89%; found: C 42.28, H 4.65, N 2.91%.
4.9.3. Disodium 2-[3,5-bis(4-(dimethylamino)benzylidene)-4-
oxopiperidin-1-yl]ethylphosphonate 10c
4.8.4. 2-[(3E,5E)-3,5-Bis(4-fluorobenzylidene)-4-oxo-1-
piperidinyl]ethylphosphonic acid hydrobromide 9d
Yield 89%, decompose above 200 ꢃC. Dark-red crystal solid. 1H
Yield 43%, m.p. 213–216 ꢃC (decompose). Light-yellow crystal
(D2O),
3.62 (s, 4H, NCH2 cyclic), 6.72–6.76 (m, 4H, aromatic), 7.27–7.31 (m,
4H, aromatic), 7.50 (s, 2H, CH]). 13C (D2O/DMSO-d6),
: 27.87 (d,
d: 1.58 (m, 2H, CH2CH2P), 2.84 (m, 14H, CH2CH2P and NMe2),
solid. 1H (DMSO-d6),
d
: 1.82–1.95 (m, 2H, CH2CH2P), 3.08 (m, 2H,
CH2CH2P), 4.17 (s, 4H, NCH2 cyclic), 4.52 (broad s, 13H, 5H2O þ P–
d
3
3
3
OH), 7.33 (t, 4H, JHH ¼ 8 Hz, JFH ¼ 8 Hz), 7.63 (dd, 4H, JHH ¼ 8 Hz,
NCH2CH2P, JCP ¼ 125 Hz), 39.56 (NCH2CH2P), 41.21 (NMe2), 54.83
(N(CH2)2 cyclic), 114.24 (C(8), C(10), C(80), C(100)), 124.31 (C(6) and
C(60)), 129.29 (C(7), C(11), C(70), C(110)), 135.23 (C(5) and C(50)),
140.49 (C(3) and C(30)), 153.26 (C(9) and C(90)), 189.54 (C(4)). 31P
4JFH ¼ 5 Hz), 7.72 (s, 2H, CH]). 19F (DMSO-d6),
d
: ꢁ110.87. 31P
(DMSO-d6), d: 23.34. Anal. calcd. for C21H20F2NO4P$0.625HBr: C
53.61, H 4.41, N 2.99%; found: C 53.45, H 4.95, Br 32.47, N 5.66%.
(D2O), d: 20.03. Anal. calcd. for C25H30N3Na2O4P$1.5H2O: C 55.55, H
4.8.5. 2-[(3E,5E)-3,5-Bis(4-nitrobenzylidene)-4-oxo-1-
piperidinyl]ethylphosphonic acid hydrobromide 9e
6.15, N 7.77%; found: C 55.07, H 5.81, N 7.61%.
Yield 96%, decompose > 200 ꢃC. Yellow crystal solid. 1H (DMSO-
4.9.4. Disodium 2-[3,5-bis(4-fluorobenzylidene)-4-oxopiperidin-1-
yl]ethylphosphonate 10d
d6), d: 1.79–1.97 (m, 2H, CH2CH2P), 3.09 (m, 2H, CH2CH2P), 4.28
(broad s, 17H, NCH2 cyclic and acid protons), 7.78–7.82 (m, 6H,
Yield 100%, decompose above 200 ꢃC. Light-yellow crystal solid.
aromatic þ CH]), 8.26–8.31 (m, 4H, aromatic). 31P (DMSO-d6),
d:
1H (D2O),
d: 1.50–1.65 (m, 2H, CH2CH2P), 2.78 (m, 2H, CH2CH2P),
3
3
23.11. Anal. calcd. for C21H20N3O8P$2/3H2O$1/2HBr: C 47.97, H 4.19,
3.78 (s, 4H, NCH2 cyclic), 7.14 (t, 4H, JHH ¼ 8 Hz, JFH ¼ 8 Hz), 7.44
Br 7.60, N 7.99%; found: C 47.98, H 4.04, Br 7.64, N 7.84%.
(m, 4H, aromatic), 7.62 (s, 2H, CH]). 13C(D2O): 25.91 (d, NCH2CH2P,
2
JCP ¼ 124 Hz), 53.00 (NCH2CH2P and N(CH2)2 cyclic, JCP ¼ 11 Hz),
4.8.6. 3-[(3E,5E)-3,5-Bis[4-(dimethylamino)benzylidene]-4-oxo-1-
piperidinyl]propylphosphonic acid hydrobromide 9f
115.62 (d, C(8), C(10), C(80), C(100), 2JCF ¼ 21.9 Hz),130.27 (s, C(6) and
C(60)), 131.25 (C(3) and C(30)), 133.22 (d, C(7), C(11), C(70), C(110),
3JCF ¼ 8.4 Hz), 137.40 (C(5) and C(50)), 163.03 (d, C(9) and C(90),
Yield 80%. Dark-red crystal solid. 1H (DMSO-d6),
d: 1.61 (m, 2H,
CH2CH2CH2P), 1.90 (m, 2H, CH2CH2CH2P), 3.05 (s, 12H, NMe2), 3.45
JCF ¼ 250 Hz), 188.60 (C(4)). 19F (D2O),
d
: ꢁ111.12. 31P (D2O),
d: 20.01.
3
(t, 2H, CH2CH2CH2P, JHH ¼ 7.5 Hz), 4.65 (broad s overlapped with
Anal. calcd. for C21H18F2NNa2O4P$2.5H2O: C 49.62, H 4.56, N 2.76%;
protons of water, NCH2 cyclic), 6.90–6.92 (m, 4H, aromatic), 7.45–
found: C 49.64, H 3.81, N 2.81%.
7.47 (m, 4H, aromatic), 7.81 (s, 2H, CH]). 31P (DMSO-d6),
d: 24.98.
This compound was transformed into the corresponding sodium
salt without additional purification.
4.9.5. Disodium 2-[3,5-bis(4-nitrobenzylidene)-4-oxopiperidin-1-
yl]ethylphosphonate 10e
Yield 100%, decompose above 200 ꢃC. Yellow crystal solid. 1H
4.8.7. 3-[(3E,5E)-3,5-Bis(4-nitrobenzylidene)-4-oxo-1-
piperidinyl]propylphosphonic acid hydrobromide 9g
(D2O),
NCH2 cyclic), 7.43–7.46 (m, 4H, aromatic), 7.47 (s, 2H, CH]), 8.03–
8.08 (m, 4H, aromatic). 13C (D2O/DMSO-d6),
: 25.83 (d, NCH2CH2P,
d: 1.53 (m, 2H, CH2CH2P), 2.75 (m, 2H, CH2CH2P), 3.75 (s, 4H,
Yield 81%. Yellow crystal solid. 1H (DMSO-d6),
d
: 1.55 (m, 2H,
d
CH2CH2CH2P), 1.82 (m, 2H, CH2CH2CH2P), 3.28 (t, 2H, CH2CH2CH2P,
JCP ¼ 125 Hz), 37.92 (NCH2CH2P), 53.08 (N(CH2)2 cyclic), 123.71
(C(8), C(10), C(80), C(100)), 131.70 (C(7), C(11), C(70), C(110)), 134.66
(C(5) and C(50)), 135.57 (C(3) and C(30)), 140.45 (C(6) and C(60)),
3JHH ¼ 7.1 Hz), 4.58 (s, 4H, NCH2 cyclic), 7.83–7.85 (m, 4H, aromatic),
7.98 (s, 2H, CH]), 8.33–8.35 (m, 4H, aromatic). 31P (DMSO-d6),
d:
24.98.
Anal.
calcd.
for
C22H22N3O8P$1.83H2O$0.83H
147.17 (C(9) and C(90), 187.56 (C(O)). 31P (D2O),
d: 20.02. Anal. calcd.
Br: C 44.95, H 4.54, Br 11.33, N 7.15%; found: C 44.64, H 4.07, Br
11.45, N 7.51%.
for C21H18N3Na2O8P$H2O: C 47.11, H 3.77, N 7.85%; found: C 47.14, H
3.61, N 7.94%.
4.9. [3E,5E-Bis(benzylidene)-4-oxopiperidin-1-yl]alkylphosphonic
acid disodium salts 10 (general procedure)
4.9.6. Disodium 2-[3,5-bis(4-(dimethylamino)benzylidene)-4-
oxopiperidin-1-yl]propylphosphonate 10f
Yield 63% (based on starting ester 7g), decompose above 200 ꢃC.
A filtered solution of sodium hydroxide (0.16 g, 0.004 mol) in
EtOH (10 mL) was added to a stirred powder of the corresponding
Dark-red crystal solid. 1H (D2O),
d
: 1.23 (m, 2H, CH2CH2CH2P), 1.53
(m, 2H, CH2CH2CH2P), 2.46 (m, 2H, CH2CH2CH2P), 2.75 (s, 12H,

2144
M.V. Makarov et al. / European Journal of Medicinal Chemistry 44 (2009) 2135–2144
NMe2), 3.50 (s, 4H, NCH2 cyclic), 6.61–6.63 (m, 4H, aromatic), 7.16–
7.18 (m, 4H, aromatic), 7.43 (s, 2H, CH]). 13C: (D2O),
: 21.51
(No MK-2464.2008.3) and NIH via the RIMI program (grant
1P20MD001104-01).
d
(NCH2CH2CH2P), 27.21 (d, NCH2CH2CH2P, JCP ¼ 131 Hz), 39.54
(NMe2), 53.57 (N(CH2)2 cyclic), 58.4 (d, NCH2CH2CH2P,
JCP ¼ 20.5 Hz), 112.58 (C(8), C(10), C(80), C(100)), 122.68 (C(6) and
C(60)), 127.33 (C(7), C(11), C(70), C(110)), 133.49 (C(5) and C(50)),
139.13 (C(3) and C(30)), 151.61 (C(9) and C(90)), 187.88 (C(4)). 31P
Appendix. Supplementary information
Supplementary data associated with this article can be found in
the online version, at doi: 10.1016/j.ejmech.2008.10.019.
(D2O), d: 21.64. Anal. calcd. for C26H32N3Na2O4P$3.5H2O: C 52.88, H
6.66, N 7.12%; found: C 52.55, H 6.13, N 7.07%.
References
4.9.7. Disodium 2-[3,5-bis(4-nitrobenzylidene)-4-oxopiperidin-1-
yl]propylphosphonate 10g
[1] J.R. Dimmock, V.K. Arora, M.J. Duffy, R.S. Reid, T.M. Allen, G.Y. Kao, Drug Des.
Discov. 8 (1992) 291.
[2] J.R. Dimmock, V.K. Arora, M. Chen, T.M. Allen, G.Y. Kao, Drug Des. Discov. 12
(1994) 19.
[3] J.R. Dimmock, M.P. Padmanilayam, R.N. Puthucode, A.J. Nazarali,
N.L. Motaganahalli, G.A. Zello, J.W. Quail, E.O. Oloo, H.-B. Kraatz, J.S. Prisciak,
T.M. Allen, C.L. Santos, J. Balzarini, E. De Clercq, E.K. Manavathu, J. Med. Chem.
44 (2001) 586.
[4] J.R. Dimmock, A. Jha, G.A. Zello, R.K. Sharma, A. Shrivastav, P. Selvakumar,
T.M. Allen, C.L. Santos, J. Balzarini, E. De Clercq, E.K. Manavathu, J.P. Stables, J.
Enzyme Inhib. Med. Chem. 18 (2003) 325.
Yield 95%, decompose above 200 ꢃC. Yellow crystal solid. 1H
(D2O), d: 1.25 (m, 2H, CH2CH2CH2P), 1.57 (m, 2H, CH2CH2CH2P), 2.56
(t, 2H, CH2CH2CH2P, 3JHH ¼ 7 Hz), 3.76 (s, 4H, NCH2 cyclic), 7.44–7.47
(m, 6H, aromatic þ CH]), 8.06–8.07 (m, 4H, aromatic). 31P (D2O),
d:
21.69. IR: 3108, 1677, 1616, 1599, 1593, 1516, 1344, 1315, 1259, 1176,
1109, 1058 (strong, broad), 980, 855, 808, 757, 713, 688. Anal. calcd.
for C22H20N3Na2O8P$4H2O: C 43.79, H 4.68, N 6.96%; found: C
43.53, H 4.42, N 6.68%.
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Cell lines used for estimation of toxicity of compounds 7a–k
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PC3 (human prostate carcinoma) and A549 (human lung carci-
noma). 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 h
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
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a soluble blue formazan dye. Briefly, the different cell lines were
seeded into 96-well plates at a concentration of 1 ꢀ104 cells/
100
humidified atmosphere containing 5% CO2. The tested
m
L/well. The cells were allowed to attach overnight at 37 ꢃC in
a
compounds were initially dissolved in dimethylsulfoxide (DMSO)
and the working solutions were added to FBS free culture
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Acknowledgements
This work was supported by the Deutsche For-
schungsgemeinschaft (DFG no. 436 RUS 113/905/0-1; RO 362/35-1),
Russian Foundation of Basic Research (grant no. 06-03-04003),
program of President of Russian Federation ‘‘For Young PhD’’