Novel synthesis, cytotoxic evaluation, and structure-activity relationship studies of a series of α-alkylidene-γ-lactones and lactams

Article abstract of DOI:10.1021/jm048970a

5-Alkyl- and 5-arylalkyl-3-methylenedihydrofuran-2-ones 13a-e, 3-alkylidenedihydrofuran-2-ones 18a-c, and 3-methylenepyrrolidin-2-ones 16a-e were synthesized utilizing ethyl 2-diethoxyphosphoryl-4-nitroalkanoates 9a-e as common intermediates. All obtained

Full text of DOI:10.1021/jm048970a

3516
J. Med. Chem. 2005, 48, 3516-3521
Novel Synthesis, Cytotoxic Evaluation, and Structure-Activity Relationship
Studies of a Series of r-Alkylidene-γ-lactones and Lactams
Tomasz Janecki,*,# Edyta Błaszczyk,# Kazimierz Studzian,‡ Anna Janecka,‡ Urszula Krajewska,† and
Marek Ro´z˘ alski†
Institute of Organic Chemistry, Technical University of Ło´dz´, Z˙ eromskiego 116, 90-924 Ło´dz´, Poland, Department of Medicinal
Chemistry, Medical University of Ło´dz´, Mazowiecka 6/8, 92-215, Poland, and Department of Pharmaucetical Biochemistry,
Faculty of Pharmacy, Medical University of Ło´dz´, Muszyn´skiego 1, 90-151, Ło´dz´, Poland
Received December 21, 2004
5-Alkyl- and 5-arylalkyl-3-methylenedihydrofuran-2-ones 13a-e, 3-alkylidenedihydrofuran-
2-ones 18a-c, and 3-methylenepyrrolidin-2-ones 16a-e were synthesized utilizing ethyl
2-diethoxyphosphoryl-4-nitroalkanoates 9a-e as common intermediates. All obtained com-
pounds were tested against L-1210, HL-60, and NALM-6 leukemia cells. The highest cytotoxic
activity was observed for 3-methylenefuranones 13d,e bearing benzyl or 3,4-dimethoxyphe-
nylmethyl substituents at position 5, with IC50 values of 5.4 and 6.0 µM, respectively. Contrary
to the literature reports, no enhancement in activity due to the presence of a hydroxy group
was found when the cytotoxicity of furanones 13a,b,d and 5-(1′-hydroxyalkyl)-3-methylenedi-
hydrofuran-2-ones 6a,b,d was compared. The anticancer activity of pyrrolidinones 16a-e and
3-alkylidenefuranones 18a-c was much weaker than that of furanones 13a-e.
Introduction
The crucial role of the R-methylene-γ-lactone moiety
in biological and especially cytotoxic activity of the
sesquiterpene natural products prompted us to develop
several new synthetic methods leading to this structural
unit. Successful application of the Horner-Wadsworth-
Emmons olefination reaction for the construction of
alkylidene bond gave us access to a series of 3-alky-
lidene-5,5-dimethyldihydrofuran-2-ones 515 and 3-me-
thylidene-5-(1′-hydroxyalkyl)dihydrofuran-2-ones 6.16
The latter compounds were tested for their cytotoxic
activity, and several of them proved to be very potent.17
Sesquiterpene lactones, found almost exclusively in
the species of the Compositae (Asteraceae) family, are
known to possess significant biological activity.1 Cyto-
toxic, allergenic, anti-inflammatory, phytotoxic, and
antimicrobial properties make them a desired target for
many synthetic organic chemists.1-3 Characteristic
structural features of this class of compounds are
R-methylene-γ-lactone 1, butenolide 2, and/or cyclopen-
tenone 3 moieties. From the recent comparative studies
it appears that the most important structural require-
ment for the activity of this class of compounds is an
R-methylene-γ-lactone moiety.4-6 It was also shown that
compounds containing this moiety can act as Michael
acceptors in the reaction with thiol groups of bionucleo-
philes.7 Furthermore, an R-alkylidene-γ-lactone moiety
readily forms a 2 + 2 cycloadduct with the DNA base
thymine.5 A number of possible drug candidates bearing
this lactone motif have been synthesized and tested.7-11
Much less common in nature are products with the
R-methylene-γ-lactam framework 4. Examples of a few
naturally occurring compounds of this structure are
pukeleimid E, isolated from cyanobacteria Lyngbya
majuscula,12 and two imidazole alkaloids anantin and
isoanantin, found in the leaf tissue of Cynometra.13
However, the biological activity of this class of com-
pounds is scarcely recognized.14
Here, we present the full account18 of a novel synthe-
sis of 3-alkylidenedihydrofuran-2-ones 13a-e and 18a-c
as well as 3-methylenepyrrolidin-2-ones 16a-e bearing
an alkyl or arylmethyl substituent at position 5. Cyto-
toxic evaluation of the target compounds was performed
on the mouse leukemia cell line L-1210 and two human
leukemia cell lines NALM-6 and HL-60. Structure-
activity relationships of the newly obtained compounds
are discussed. Also, cytotoxicities of furanones 13 and
furanones 6 containing a hydroxy group are compared.
Chemistry
3-Alkylidenedihydrofuran-2-ones 13a-e and 18a-c
and 3-methylenepyrrolidin-2-ones 16a-e were all syn-
thesized starting from ethyl 2-diethoxyphosphoryl-4-nitro-
alkanoates 9a-e, which are the key intermediates in
our method (Scheme 1). Nitroalkanoates 9a-e were
conveniently prepared by addition of nitroalkanes 8a-e
to ethyl 2-diethoxyphosphorylacrylate (7) in the pres-
ence of NaH, using a 2-fold excess of nitroalkane (7/8/
NaH ) 1:2:1.1). Column chromatography afforded 9a-e
as mixtures of diastereoisomers with close to a 3:2 ratio.
* To whom correspondence should be addressed. Phone: (48 42)
6313220. Fax: (48 42) 6365530. E-mail: tjanecki@p.lodz.pl.
# Technical University of Ło´dz´.
‡ Department of Medicinal Chemistry, Medical University of Ło´dz´.
† Department of Pharmaucetical Biochemistry, Faculty of Pharmacy,
Medical University of Ło´dz´.
10.1021/jm048970a CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/16/2005

R-Alkylidene-γ-lactones and Lactams
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3517
Scheme 1a
a Reagents and conditions: (a) NaH, THF, room temp, 24 h; (b) (1) MeONa/MeOH, room temp, 0.5 h, (2) conc H2SO4, MeOH, -60 °C,
2 h; (c) (1) NaBH4, MeOH, NaOH, H2O, room temp, 20 h, (2) 1 N HClaq; (d) K2CO3, 36% formalin, 0-5 °C, 15 min; (e) NH4HCO2, 10%
Pd/C, MeOH/THF, 0 °C to room temp, 24 h; (f) (1) NaH, THF, (2) (CH2O)n, reflux, 1 h.
Scheme 2a
Table 1. Cytotoxic Activity and Lipophilicity of Compounds
13a-e, 16a-e, 18a-c, and 6a-d
cytotoxicity IC50 (µM)a
lipophilicity
compd
13a
log(P)
L-1210
32.5 ( 4,8 77.4 ( 6.5
6.0 ( 1.6
HL-60
NALM-6
0.82
1.29
2.48
2.51
2.00
0.17
0.64
1.83
1.86
1.35
3.59
4.08
5.08
0.04
0.45
1.31
1.88
41.0 ( 1.8
13b
13c
13d
13e
16a
16b
16c
16d
16e
18a
18b
18c
6a
39.5 ( 17.3 51.6 ( 24.0
20.0 ( 3.2 99.4 ( 31.5 23.6 ( 12.9
15.5 ( 2.9 42.7 ( 11.1 5.4 ( 0.3
a Reagents and conditions: (a) (1) NaH, benzene, room temp,
0.5 h, (2) R2CHO, benzene, reflux, 4 h.
4.3 ( 0.8
20.0 ( 5.7 640 ( 84
>100 894 ( 83
46.3 ( 1.8
6.0 ( 1.4
658 ( 47
387 ( 71
82.7 ( 8.4
420 ( 48
507 ( 26
380 ( 24
48.7 ( 2.8
71.0 ( 4.4
Conversion of the nitro group in 9a-e into a carbonyl
group (Nef reaction) followed by reduction of the 4-oxo-
alkanoates 10a-e gave 4-hydroxyalkanoates 11a-e,
which lactonized spontaneously to 3-(diethoxyphospho-
ryl)dihydrofuran-2-ones 12a-e. These compounds were
obtained as mixtures of diastereoisomers with close to
a 1:1 ratio. Horner-Wadsworth-Emmons olefination
of formaldehyde using 12a-e and applying the Villieras
procedure19 (36% formaline, K2CO3, 0-5 °C, 15 min)
gave the expected 3-methylenedihydrofuran-2-ones
13a-e. Furanones 13a,20 13b,c,21 and 13d2 have previ-
ously been prepared using other synthetic methods.
Olefination of isobutyraldehyde, benzaldehyde, and
1-naphthylaldehyde was accomplished using the sodium
derivative of furanone 12d in boiling benzene (Scheme
2). 3-Alkylidenedihydrofuran-2-ones 18a,b were ob-
tained as mixtures of E and Z isomers in a 30:70 and
85:15 ratio, respectively, while furanone 18c was formed
as a single E isomer. Configurational assignments were
made using the diagnostic deshielding effect of the
carbonyl group exerted on the cis-oriented vinyl pro-
ton.22
Reduction of the nitro group in 9a-e gave, after
spontaneous lactamization, 3-(diethoxyphosphoryl)pyr-
rolidin-2-ones 15a-e as mixtures of diastereoisomers
with close to a 3:2 ratio (Scheme 1). Olefination of
formaldehyde using 15a-e proceeded smoothly when
sodium hydride with paraformaldehyde in boiling THF
was used. Under these conditions the expected 3-me-
thylenepyrrolidin-2-ones 16a-e were formed along with
various amounts (6-26%) of 1-hydroxymethyl-3-meth-
ylenepyrrolidin-2-ones 17a-e. Purification and separa-
tion of these mixtures by column chromatography
afforded pure pyrrolidinones 16a-e. Pyrrolidinones
16a23 and 16d24 have previously been prepared using
other synthetic methods.
59.0 ( 6.9 397 ( 87
93.0 ( 12.4 490 ( 62
79.0 ( 7.5 402 ( 31
90.0 ( 6.9 168 ( 57
63.3 ( 10.0 60.6 ( 2.7
46.1 ( 9.2 49.8 ( 3.1
27.2 ( 6.1b 72.4 ( 16.9b
19.3 ( 3.7b 47.3 ( 3.7b
16.9 ( 3.9b 52.6 ( 4.9b
6b
6c
6d
8.0 ( 2.1b
9.7 ( 1.2
40.2 ( 2.4b
2.9 ( 0.1
carboplatin
0.7 ( 0.3
a IC50, 50% inhibitory concentration represents the mean from
dose response curves of at least three experiments. b From ref 17.
Results and Discussion
The cytotoxicity of all obtained compounds was as-
sessed in vitro against three leukemia cell lines (mouse
L-1210 and human HL-60 and NALM-6) and expressed
as IC50 values. IC50 is the concentration (µM) required
to inhibit tumor cell proliferation by 50% after 72 h of
exposure of the cells to a tested compound. The mea-
sured IC50 values for 5-alkyl-3-methylenedihydrofuran-
2-ones 13a-e, 5-alkyl-3-methylenepyrrolidin-2-ones 16a-
e, and 5-alkyl-3-alkylidenedihydrofuran-2-ones 18a-c
are summarized in Table 1. Carboplatin was used as a
reference compound.25 Cytotoxicities of 5-(1′-hydroxy-
alkyl)-3-methylenedihydrofuran-2-ones 6a-d, which are
structurally related to furanones 13a-e, are also given
in Table 1.
As can be seen from the presented data, cytotoxicity
of pyrrolidin-2-ones 16a-e is generally low. IC50 values
for these compounds against HL-60 and NALM-6 cell
lines are greater than 300 (with one exception only), and
they are 10-100 times less active than the correspond-
ing furan-2-ones 13a-e. Differences in activity between
these two series of compounds against the L-1210 cell
line are smaller but also apparent. These findings are

3518 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Janecki et al.
in agreement with the only other comparative study
describing the differences in activity between R-meth-
ylene-γ-lactones and R-methylene-γ-lactams.14 Replace-
ment of the oxygen by a nitrogen atom in the furanone
ring most likely reduces the efficacy of 3-methylenepyr-
rolidin-2-ones as Michael acceptors because of better
conjugation between the carbonyl group and the un-
shared electrons on the nitrogen atom compared to
unshared electrons on an oxygen atom. Comparison of
the cytotoxicities of 13d and 18a-c shows clearly that
substitution of the methylene group with the isopropyl
or aryl substituent decreases the activity. Steric hin-
drance introduced by the substituent, which makes the
double bond less vulnerable to the nucleophilic attack,
seems the most obvious explanation for this observation.
Furthermore, an isopropyl substituent is more deacti-
vating than aryl substituents. Once again, the steric
effect should be taken into account. Aryl substituents
are conjugated with an R,â-unsaturated system and
thus are coplanar with it, contrary to an isopropyl
substituent, which can rotate freely around the C1′-
C2′ bond. As a consequence, the double bond is less
hindered by an aryl than by an isopropyl substituent.
There is no clear relationship between the cytotoxicity
of furanones 13a-e against L-1210 or HL-60 cell lines
and the nature of the R1 substituents. However, cyto-
toxicities against the L-1210 cell line are generally
higher than against the HL-60 cell line. On the other
hand, cytotoxicities of furanones 13a-e against NALM-6
cells are clearly higher when R1 has an aromatic
character. Furanones 13d,e with benzyl and 3,4-
dimethoxyphenylmethyl substituents have remarkably
high cytotoxities against this cell line.
Recently we have synthesized and evaluated the
cytotoxic activity of a series of 5-(1′-hydroxyalkyl)-3-
methylenedihydrofuran-2-ones 6a-d against L-1210
and HL-60 cell lines.17 These investigations were en-
couraged by the reports26 that the presence of a stere-
ochemically defined carbinol unit may enhance the
biological properties of R-methylene-γ-lactones. The
present results gave the opportunity to verify this
hypothesis because the only structural difference be-
tween the furanones 13a,b,d described in this study and
the corresponding furanones 6a,b,d is the lack of the
hydroxy group in the former ones. Disappointingly, a
comparison of these two series of furanones shows no
significant differences in cytotoxicities. Evidently, the
presence of a hydroxy group in 6a,b,d does not enhance
their cytotoxic properties.
wise, higher cytotoxicities of 18b,c in comparison with
18a can be rationalized not only in terms of the steric
effect but also in terms of their more lipophilic charac-
ter.
In conclusion, we have developed a novel, general, and
straightforward route to 3-alkylidenefuran-2-ones 13a-e
and 18a-c and 3-methylenepyrrolidin-2-ones 16a-e
starting from easily available common intermediates,
ethyl 2-diethoxyphosphoryl-4-nitroalkanoates 9a-e. For
all obtained compounds, cytotoxic activity against the
L-1210, HL-60, and NALM-6 cell lines was determined
and lipophilicity was calculated. Two of the prepared
furanones 13d and 13e exhibited remarkable cytotox-
icity toward the NALM-6 cell line. Pyrrolidinones 16
proved to be generally much less active than furanones
13 and 18. Also, 3-alkylidenefuranones 18 had weaker
activity than 3-methylenefuranones 13. No clear cor-
relation between cytotoxicity and lipophilicity in these
series of compounds was found, and it seems that
observed differences in activity can be better rational-
ized in terms of steric and electronic effects. A compari-
son of the cytotoxicities of furanones 13a,b,d and 6a,b,d
does not support the literature reports that the presence
of a hydroxy group may enhance the activity of R-me-
thylene-γ-lactones.
Experimental Section
Organic solvents and reagents were purified by the ap-
propriate standard procedures. IR spectra were recorded on a
Specord M80 spectrometer. 1H NMR (250 MHz), 13C NMR (62.9
MHz), and 31P NMR (101 MHz) spectra were recorded on a
Bruker DPX-250 spectrometer with TMS as an internal
standard and 85% H3PO4 as an external standard. 31P NMR
spectra were recorded using broad-band proton decoupling.
Column chromatography was performed on Fluka silica gel
60 (230-400 mesh).
Nitroethane, 1-nitropropane, and 1-nitrohexane were pur-
chased from Fluka. 2-Phenylnitroethane,29 2-(3,4-dimethoxy-
phenyl)nitroethane,29 and ethyl 2-diethoxyphosphorylacrylate
(7)18 were prepared according to the literature procedures.
General Procedure for the Preparation of Ethyl 2-di-
ethoxyphosphoryl-4-nitroalkanoates 9. A solution of ni-
troalkane 8 (17.0 mmol) in THF (10 mL) was added to a stirred
suspension of NaH (0.213 g, 8.9 mmol) in THF (40 mL) under
argon atmosphere at 0-4 °C. The reaction mixture was stirred
for 40 min at room temperature and cooled to 0-4 °C, and a
solution of ethyl 2-diethoxyphosphorylacrylate (7) (2.0 g, 8.5
mmol) in THF (10 mL) was added. The mixture was then
stirred for 24 h at room temperauture, THF was evaporated
at room temperauture, and the residue was quenched with
water (15 mL) and extracted with CH2Cl2 (4 × 20 mL). The
organic extracts were dried (MgSO4) and evaporated at room
temperauture to give a crude product that was purified by
column chromatography (eluent, CHCl3/acetone ) 90:10 for
9a-c and CHCl3/acetone ) 95:5 for 9d,e).
Structure-cytotoxicity relationship studies of ses-
quiterpene lactones revealed also that increased lipo-
philicity is often accompanied by increased cytotoxici-
ty.27 We calculated the lipophilicity of the newly obtained
compounds,28 and the results, expressed as log P where
P is a partition coefficient in the 1-octanol/water system,
are given in Table 1. In the series of furanones 6a-d
we have found a straightforward correlation between
lipophilicity and cytotoxicity with the more lipophilic
compounds being more cytotoxic. However, the cytotox-
icity of compounds 13a-e and 18a-c does not show a
clear correlation with their lipophilicity. Nevertheless,
high cytotoxicities of 13d and 13e against the NALM-6
cell line can be, at least partially, attributed to the
strongly lipophilic character of these compounds. Like-
Ethyl 2-diethoxyphosphoryl-5-(3,4-dimethoxyphenyl)-
4-nitropentanoate (9e): ratio of diastereoisomers ) 65:35;
oil, 85% yield; IR (film) 1732, 1552, 1260 cm-1
;
1H NMR30
3
(CDCl3) δ 1.28 (t, JHH ) 7.0 Hz, 3H, major + minor), 1.31 (t,
3JHH ) 7.0 Hz, 3H, major), 1.32 (td, 3JHH ) 7.2 Hz, 4JPH ) 0.5
3
4
Hz, 3H, minor), 1.33 (td, JHH ) 7.5 Hz, JPH ) 0.5 Hz, 3H,
3
4
major), 1.34 (td, JHH ) 7.0 Hz, JPH ) 0.5 Hz, 3H, minor),
2.28-2.75 (m, 2H + 2H, major + minor), 2.84-3.04 (m, 1H +
1H, major + minor), 3.02 (dd, 2JHH ) 14.5 Hz, 3JHH ) 7.2 Hz,
1H, minor), 3.03 (dd, JHH ) 14.5, JHH ) 5.5 Hz, 1H, major),
3.22 (dd, 2JHH ) 14.5 Hz, 3JHH ) 8.8 Hz, 1H, major), 3.23 (dd,
2JHH ) 14.5 Hz, 3JHH ) 7.5 Hz, 1H, minor), 3.85 (s, 3H + 3H,
major + minor), 3.86 (s, 3H + 3H, major + minor), 4.02-4.28
(m, 6H + 6H, major + minor), 4.67-4.81 (m, 1H, major), 4.90-
5.03 (m, 1H, minor), 6.62-6.82 (m, 3H + 3H, major + minor);
2
3

R-Alkylidene-γ-lactones and Lactams
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3519
13C NMR30 (CDCl3) δ 13.82 (s), 16.07 (d, 3JPC ) 6.0 Hz), 30.04
(d, 2JPC ) 4.5 Hz), 30.26 (d, 2JPC ) 3.5 Hz), 39.46 (s), 39.60 (s),
3 mmol), and aqueous 36% formaldehyde solution (0.54 mL,
7.0 mmol) was stirred at 0-4 °C for 15 min. The mixture was
next extracted with Et2O (4 × 15 mL), dried (MgSO4), and
evaporated. Residue was purified by column chromatography
(eluent, CHCl3) to give pure 13.
1
1
41.77 (d, JPC ) 130.2 Hz), 42.06 (d, JPC ) 130.6 Hz), 55.67
(s), 55.70 (s), 61.76 (s), 61.82 (s), 62.99 (d, 2JPC ) 6.5 Hz), 87.39
(d, 3JPC ) 8.4 Hz), 87.67 (d, 3JPC ) 15.0 Hz), 111.26 (s), 111.73
(s), 111.81 (s), 120.88 (s), 121.02 (s), 127.10 (s), 148.28 (s),
148.93 (s), 167.58 (d, 2JPC ) 5.7 Hz), 167.73 (d, 2JPC ) 6.3 Hz);
31P NMR30 (CDCl3) δ 20.46 (major), 21.12 (minor). Anal.
(C19H30NO9P): C, H, N, P.
General Procedure for the Preparation of Ethyl 2-di-
ethoxyphosphoryl-4-oxoalkanoates 10. A solution of 2-di-
ethoxyphosphoryl-4-nitroalkanoate 9 (4.0 mmol) in MeOH (4
mL) was added to a solution of sodium methoxide prepared
from sodium (100 mg, 4.4 mmol) and MeOH (8 mL), and the
reaction mixture was stirred under argon atmosphere at room
temperauture for 0.5 h. Then it was cooled to -60 °C and a
cold (0 °C) solution of H2SO4 (2.4 mL) in MeOH (12 mL) was
added. Stirring was continued for 2 h at -60 °C, and water
(30 mL) was added at such a rate that the temperature did
not exceed 4 °C. The solvent was evaporated at room temper-
auture under reduced pressure, and the residue was extracted
with CH2Cl2 (4 × 20 mL). Combined extracts were dried
(MgSO4) and evaporated, and the crude products 10 were
purified by column chromatography (eluent, CHCl3/acetone )
95:5).
5-(3,4-Dimethoxyphenylmethyl)-3-methylene-4,5-dihy-
drofuran-2-one (13e): oil, 48% yield; IR (film) 1772, 1664
cm-1; 1H NMR (CDCl3) δ 2.59 (ddt, 2JHH ) 17.0 Hz, 3JHH ) 6.0
4
2
3
Hz, JHH ) 2.8 Hz, 1H), 2.81 (dd, JHH ) 14.3 Hz, JHH ) 6.0
Hz, 1H), 2.89 (ddt, 2JHH ) 17.0 Hz, 3JHH ) 7.8 Hz, 4JHH ) 2.8
Hz, 1H), 2.95 (dd, 2JHH ) 14.3 Hz, 3JHH ) 6.0 Hz, 1H), 3.79 (s,
3H), 3.80 (s, 3H), 4.69 (dq, 3JHH ) 7.8 Hz, 3JHH ) 6.0 Hz, 1H),
5.49 (t, 4JHH ) 2.8 Hz, 1H), 6.10 (t, 4JHH ) 2.8 Hz, 1H), 6,65-
6,78 (m, 3H); 13C NMR (CDCl3) δ 32.50 (s), 41.27 (s), 55.90 (s),
55.92 (s), 77.21 (s), 111.32 (s), 112.74 (s), 121.68 (s), 127.89
(s), 148.14 (s), 149.02 (s), 121.98 (s), 134.37 (s), 170.13 (s). Anal.
(C14H16O4) C, H.
General Procedure for the Preparation of 3-Diethoxy-
phosphorylpyrrolidyn-2-ones 15. A mixture of 2-diethoxy-
phosphoryl 4-nitroalkanoate 9 (2.5 mmol), 10% Pd/C (0.118
g), and HCOONH4 (0.843 g; 13.4 mmol) in MeOH (15 mL) and
THF (15 mL) was stirred at 0-4 °C for 2 h, warmed to room
temperature and stirred for an additional 22 h. The reaction
mixture was filtered through a Celite bed, filtrate was
evaporated, CHCl3 (40 mL) was added to the residue, and
chloroform solution was filtered through a Celite bed. Evapo-
ration of the chloroform gave crude product that was purified
by column chromatography (eluent, CHCl3/MeOH ) 97:3).
Ethyl 2-diethoxyphosphoryl-5-(3,4-dimethoxyphenyl)-
4-oxopentanoate (10e): oil, 62% yield; IR (film) 1732, 1260
cm-1; 1H NMR (CDCl3) δ 1.26 (t, 3JHH ) 7.0 Hz; 3H), 1.30 (td,
4
3
3JHH ) 7.0 Hz, JPH ) 0.5 Hz, 3H), 1.33 (td, JHH ) 7.0 Hz,
3-Diethoxyphosphoryl-5-(3,4-dimethoxyphenylmeth-
4JPH ) 0.5 Hz, 3H), 2.91 (ddd, JHH ) 17.8 Hz, 3JPH ) 8.8 Hz,
2
yl)pyrrolidyn-2-one (15e): ratio of diastereoisomers 60:40;
3JHH ) 2.5 Hz, 1H), 3.28 (ddd, JHH ) 17.8 Hz, JHH ) 11.0
2
3
oil, 70% yield; IR (film) 3120, 1712, 1232 cm-1
;
1H NMR30
Hz, 3JPH ) 6.0 Hz, 1H), 3.47 (ddd, 2JPH ) 23.8 Hz, 3JHH ) 11.0
3
3
(CDCl3) δ 1.29 (t, JHH ) 7.0 Hz, 6H, major), 1.30 (t, JHH
)
3
Hz, JHH ) 2.5 Hz, 1H), 3.68 (s, 2H), 3.86 (s, 6H), 4.03-4.24
3
7.0 Hz, 3H, minor), 1.32 (t, JHH ) 7.0 Hz, 3H, minor), 1.84-
(m, 6H), 6.67-6.85 (m, 5H); 13C NMR (CDCl3) δ 13.00 (s), 15.25
2
2.92 (m, 4H + 4H, major + minor), 3.01 (dd, JHH ) 13.5 Hz,
3
3
2
(d, JPC ) 6.0 Hz), 15.30 (d, JPC ) 5.7 Hz), 37.72 (d, JPC
)
2
3
3JHH ) 3.5 Hz, 1H, major), 3.28 (dd, JHH ) 13.5 Hz, JHH
)
1
2.0 Hz), 39.07 (d, JPC ) 131.8 Hz), 48.08 (s), 54.85 (s), 54.89
(s), 60.63 (s), 61.88 (d, 2JPC ) 6.2 Hz), 61.91 (d, 2JPC ) 6.8 Hz),
110.49 (s), 111.52 (s), 120.68 (s), 125.09 (s) 147.24 (s), 148.11
3.5 Hz, 1H, minor), 3.75-3.88 (m, 6H + 6H, major + minor),
3.95-4.23 (m, 5H + 5H, major + minor), 6.70-6.82 (m, 3H +
3H, major + minor), 9.50 (bs, 1H + 1H, major + minor); 13C
(s), 167.24 (d, JPC ) 5.7 Hz), 204.14 (d, JPC ) 15.1 Hz); 31P
2
3
NMR30 (CDCl3) δ 15.50-16.20 (m), 22.30 (d, JPC ) 3.6 Hz),
2
NMR (CDCl3) δ 22.69. Anal. (C19H29O8P) C, H, P.
2
1
22.85 (d, JPC ) 3.8 Hz), 36.36 (s), 38.0 (s), 36.79 (d, JPC
)
General Procedure for the Preparation of 3-Diethoxy-
phosphoryl-3,4-dihydro-2(5H)-furanones 12. A solution of
NaBH4 (38 mg, 1.0 mmol) and NaOH (4 mg, 0.1 mmol) in H2O
(0.5 mL) was added to a solution of 2-diethoxyphosphoryl
4-oxoalkanoate 10 (2.0 mmol) in MeOH (2.5 mL), and the
reaction mixture was stirred for 20 h at room temperauture.
The mixture was acidified to pH ∼ 1 using 1 M HCl, and
MeOH was evaporated under reduced pressure. The residue
was extracted with CHCl3 (3 × 15 mL), combined extracts were
washed with H2O (10 mL), dried (MgSO4), and evaporated.
Crude products were purified by column chromatography
(eluent, CHCl3/acetone ) 95:5) to give 12 as mixtures of
diastereoisomers.
139.8 Hz), 36.18 (d, 1JPC ) 149.5 Hz), 55.54 (s), 55.71 (s), 58.31
3
3
2
(d, JPC ) 1.2 Hz), 59.31 (d, JPC ) 7.3 Hz), 62.12 (d, JPC
)
6.5 Hz), 62.21 (d, 2JPC ) 6.7 Hz), 62.95 (d, 2JPC ) 6.5 Hz), 63.14
(d, 2JPC ) 6.5 Hz), 111.06 (s), 112.45 (s), 112.57 (s), 121.26 (s),
121.45 (s), 127.94 (s), 128.78 (s), 147.51, 147.64 (s), 148.68 (s),
163.63 (d, 2JPC ) 3.8 Hz), 165.53 (d, 2JPC ) 3.8 Hz); 31P NMR30
(CDCl3) δ 23.59 (minor) 24.45 (major). Anal. (C17H26NO6P) C,
H, N.
General Procedure for the Preparation of 3-Methyl-
enepyrrolidyn-2-ones 16. A solution of 3-diethoxyphospho-
rylpyrrolidyn-2-one (1.0 mmol) in THF (7 mL) was added to a
suspension of NaH (0.025 g, 1.05 mmol) in THF (3 mL), and
the reaction mixture was stirred at room temperature for 0.5
h. Next, paraformaldehyde (0.033 g, 1.1 mmol) was added in
one portion, and the mixture was refluxed for 1 h and cooled
to 0-4 °C. Then H2O (3 mL) was added, THF was evaporated
under reduced pressure, and the residue was extracted with
CH2Cl2 (3 × 15 mL). Combined organic extracts were washed
with H2O (5 mL), dried (MgSO4), and evaporated to give crude
15 that were purified by column chromatography (eluent,
CHCl3).
3-Diethoxyphosphoryl-5-(3,4-dimethoxyphenylmethyl)-
3,4-dihydro-2(5H)-furanone (12e): ratio of diastereoisomers
1
) 60:40; oil, 70% yield; IR (film) 1772, 1260 cm-1, H NMR30
3
(CDCl3) δ 1.32 (t, JHH ) 7.0 Hz, 6H + 6H, major + minor),
2.12-2.37 (m, 1H + 1H, major + minor), 2.42-2.69 (m, 1H +
1H, major + minor), 2.81-3.24 (m, 3H + 3H, major + minor),
3.87 (s, 6H + 6H, major + minor), 4.09-4.30 (m, 4H + 4H,
3
3
major + minor), 4.64 (dq, JHH ) 8.8 Hz, JHH ) 6.5 Hz, 1H,
3
3
minor), 4.91 (dq, JHH ) 7.8 Hz, JHH ) 6.9 Hz, 1H, major),
6.72-6.85 (m, 3H + 3H, major + minor); 13C NMR30 (CDCl3)
5-(3,4-Dimethoxyphenylmethyl)-3-methylenepyrroli-
dyn-2-one (16e): oil, 40% yield; IR (film) 3100, 1684, 1662
cm-1; 1H NMR (CDCl3) δ 2.45 (ddt, 2JHH ) 17.0 Hz, 3JHH ) 4.0
Hz, 4JHH ) 2.2 Hz, 1H), 2.73 (ddt, 2JHH ) 17.0 Hz, 3JHH ) 7.5
3
2
δ 16.35 (d, JPC ) 5.6 Hz), 29.45 (d, JPC ) 3.4 Hz), 29.64 (d,
1
1
2JPC ) 3.1 Hz), 39.71 (d, JPC ) 150.8 Hz), 39.89 (d, JPC
)
2
139.6 Hz), 40.53 (s), 40.61 (s), 55.9 (s), 62.73 (d, JPC ) 6.7
4
2
3
2
2
Hz, JHH ) 2.2 Hz, 1H), 2.82 (dd, JHH ) 13.8 Hz, JHH ) 7.6
Hz), 62.92 (d, JPC ) 6.7 Hz), 63.51 (d, JPC ) 6.7 Hz), 63.56
Hz, 1H), 3.15 (dd, 2JHH ) 13.8 Hz, 3JHH ) 3.4 Hz, 1H), 3.86 (s,
2
3
3
(d, JPC ) 6.5 Hz), 79.84 (d, JPC ) 3.2 Hz), 79.95 (d, JPC
)
4
3H), 3.87 (s, 3H), 4.02-4.20 (m, 1H), 5.18 (t, JHH ) 2.2 Hz,
10.5 Hz), 111.45 (s), 112.67 (s), 112.75 (s), 121.52 (s), 121.67
(s), 127.84 (s), 128.31 (s), 148.09 (s), 148.16 (s), 149.02 (s),
171.28 (s), 171.48 (d, 2JPC ) 4.4 Hz); 31P NMR30 (CDCl3) δ 20.82
(minor), 21.03 (major). Anal. (C17H25O7P) C, H, P.
General Procedure for the Preparation of 3-Methyl-
enedihydro-2-furanones 13. A mixture of 3-diethoxyphos-
phoryltetrahydro-2-furanone 12 (1.0 mmol), K2CO3 (0.415 g,
1H), 5.83 (t, 4JHH ) 2.2 Hz, 1H), 6.53-6.57 (m, 3H); 13C NMR
(CDCl3) δ 28.13 (s), 37.41 (s), 55.81 (s), 58.20 (s), 115.92 (s),
111.14 (s), 112.69 (s), 121.72 (s), 128.17 (s), 147.89 (s), 148.93
(s), 135.58 (s), 163.89 (s). Anal. (C14H17NO3) C, H, N.
General Procedure for the Preparation of 3-Alky-
lidene-5-benzyldihydrofuran-2-ones 18. A solution of 12d

3520 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Janecki et al.
(2) Ballini, R.; Bosica, G.; Livi, D. A New Synthesis of exo-Methylene
Butyrolactones from Nitroalkanes. Synthesis 2001, 1519-1522
and references therein.
(0.310 g, 1.0 mmol) in benzene (5 mL) was added to a
suspension of NaH (0.033 g, 1.1 mmol) in benzene (3 mL), and
the reaction mixture was stirred at room temperature for 0.5
h. Next, appropriate aldehyde (1.1 mmol) was added and the
mixture was refluxed for 4 h. After cooling to room tempera-
ture, water (5 mL) was added, layers were separated, and
water layer was washed with benzene (2 × 10 mL). Combined
organic extracts were dried (MgSO4) and evaporated to give
crude 18 that was purified by column chromatography (eluent,
CHCl3/acetone ) 95:5).
(3) GowriSankar, S.; Lee, Ch. G.; Kim, J. N. Facile Synthesis of
Lactones and Dihydronaphthalenes from Methyl 2-Isobutenyl
(or 2-Isopentenyl) Cinnamates as the Common Intermediates.
Tetrahedron Lett. 2004, 45, 6949-6953 and references therein.
(4) Dirsch, V. M.; Stuppner, H.; Ellmerer-Muˆller, E. P.; Vollmar,
A. M. Structural Requirementes of Sesquiterpene Lactones To
Inhibit LPS-Induced Nitric Oxide Synthesis in RAW 264. 7
Macrophages. Bioorg. Med. Chem. 2000, 8, 2747-2753.
(5) Heilmann, J.; Wasescha, M. R.; Schmidt, T. J. The influence of
Glutathione and Cysteine Levels on the Cytotoxicity of Helena-
nolide Type Sesquiterpene Lactones against KB Cells. Bioorg.
Med. Chem. 2001, 9, 2189-2194.
(6) Patel, B. P.; Waddel, T. G.; Pagni, R. M. Explaining Photoder-
matosis: Cyclopentenone vs R-Methylene-γ-lactone Natural
Products. Fitoterapia 2001, 72, 511-515.
(7) Gonza´lez, A. G.; Silva, M. H.; Padro´n, J. I.; Leo´n, F.; Reyes, E.;
AÄ lvarez-Mon, M.; Pivel, J. P.; Quintana, J.; Este´vez, F.; Bermejo,
J. Synthesis and Antiproliferative Activity of a New Compound
Containing an R-Methylene-γ-Lactone Group. J. Med. Chem.
2002, 45, 2358-2361 and references therein.
(8) Wang, T.-Ch.; Zhao, Y.-L.; Kuo, D.-H. Synthesis and Vasorelax-
ing Evaluation of R-Methylidene-γ-butyrolactone Bearing Quino-
lin-2(1H)-one and 3,4-Dihydroquinolin-2(1H)-one Derivatives.
Eur. J. Med. Chem. 2001, 36, 909-914.
(9) Lee, K.-H.; Huang, B.-R. Synthesis and Cytotoxic Evaluation of
R-Methylene-γ-butyrolactone Bearing Naphthalene and Naphthol-
[2,1-b]furan Derivatives. Eur. J. Med. Chem. 2002, 37, 333-
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(10) Paintner, F. F.; Allmendinger, L.; Bauschke, G.; Berns, C.;
Heisig, P. Synthesis and Antimicrobial Activity of Tetrodeca-
mycin Partial Structures. Bioorg. Med. Chem. 2003, 11, 2823-
2833.
5-Benzyl-3-(2-methylpropylidene)-4,5-dihydrofuran-2-
one (18a): ratio of diastereoisomers E/Z ) 30:70, oil, 78% yield;
1
3
IR (film) 1752, 1672 cm-1; H NMR30 (CDCl3) δ 0.96 (d, JHH
) 6.8 Hz, 3H, major), 0,97 (d, 3JHH ) 6.8 Hz, 3H, major), 1.01
3
3
(d, JHH ) 6.8 Hz, 3H, minor), 1.02 (d, JHH ) 6.8 Hz, 3H,
minor), 2.34 (d sept, 3JHH ) 9.8 Hz, 3JHH ) 6.8 Hz, 1H, minor),
2
3
4
2.55 (ddd, JHH ) 16.5 Hz, JHH ) 6.5 Hz, JHH ) 3.0 Hz, 1H,
2
3
4
minor), 2.60 (ddd, JHH ) 17.8 Hz, JHH ) 6.5 Hz, JHH ) 2.2
Hz, 1H, major), 2.76-2.94 (m, 2H + 2H, major + minor), 3.08
(dd, 2JHH ) 14.0 Hz, 3JHH ) 6.5 Hz, 1H, major), 3.12 (dd, 2JHH
3
3
) 13.8 Hz, JHH ) 5.5 Hz, 1H, minor), 3.70 (d sept, JHH
)
3
3
10.0 Hz, JHH ) 6.8 Hz, 1H, major), 4.69 (dq, JHH ) 7.8 Hz,
3JHH ) 6.5 Hz, 1H, major), 4.67-4.83 (m, 1H, minor), 5.90 (dt,
3JHH ) 10.0 Hz, JHH ) 2.5 Hz, 1H, major), 6.51 (dt, JHH
)
4
3
4
9.8 Hz, JHH ) 3.0 Hz, 1H, minor), 7.20-7.37 (m, 5H + 5H,
major + minor); 13C NMR30 (CDCl3) δ 21.34 (s), 21.37 (s), 22.24
(s) 22.29 (s), 26.14 (s), 29.63 (s), 30.00 (s), 34.18 (s), 41.64 (s),
41.93(s), 77.10 (s), 77.37 (s), 122.03 (s), 123.71 (s), 126.75 (s),
126.81 (s), 128.45 (s), 128.50 (s), 129.40 (s), 135.57 (s), 135.72
(s), 146.18 (s), 150.45 (s), 169.33 (s), 171.01 (s). Anal. (C15H18O2)
C, H.
(11) Baraldi, P. G.; Nu´nez, M. C.; Tabrizi, M. A.; Clerq, E.; Balzarini,
J.; Bermejo, J.; Este´vez, F.; Romagnoli, R. Design, Synthesis and
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ylene-γ-butyrolactanes and Polypyrrole Minor Groove Binders.
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(15) Janecki, T.; Błaszczyk, E. A Convenient Synthesis of 3-Alky-
lidenetetrahydro-2-furanones from 3-Diethoxyphosphoryl-2,5-
dihydro-2-furanones. Synthesis 2001, 403-408.
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Hydroxyalkyl)-3-methylidenetetrahydro-2-furanones. Tetrahe-
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(18) For a preliminary communication, see the following: Janecki,
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Synthesis 1984, 406-408.
Cells and Cytotoxicity Assays. Mouse leukemia L-1210
cells were cultured in RPMI 1640 medium (Sigma) supple-
mented with heat-inactivated 10% fetal bovine serum (Gibco)
in a 5% CO2/95% air atmosphere. Cytotoxic effects were
assayed by measuring the inhibitory effects on L-1210 cell
proliferation. In this assay, cells were seeded in 2 mL aliquots
in 6 mL tissue culture tubes (Corning) at a concentration of 5
× 103 cells/mL and exposed to drugs for 72 h at 37 °C. The
cell number relative to control was then determined by the
colorimetric tetrazolium dye method.31
Human promyelocytic leukemia HL-60 cells and lympho-
blastic NALM-6 cells were grown in RPMI 1640 (Cambrex,
Belgium) supplemented with heat-inactivated 10% fetal bovine
serum (Cytogen, Germany), penicillin (100 U/mL), and strep-
tomycin (100 µg/mL) under a 5% CO2/95% air atmosphere.
Exponentially growing HL-60 and NALM-6 cells were seeded
at 0.2 × 106 cells per each well onto a 24-well plate (Nunc,
Denmark), and cells were then exposed to the tested com-
pounds or carboplatin for 72 h. The number of viable cells was
counted in Bu¨rker hemocytometer using the trypan-blue
exclusion assay. Concentration-response curves were deter-
mined. IC50 values (the concentration of the tested compounds
required to reduce a fraction of surviving cells to 50% of that
observed in the control cells) were calculated from dose-
response curves and used as an index of cellular sensitivity to
a given treatment. All the data were expressed as the mean
( SD.
Acknowledgment. This work has been partially
supported by the Polish State Committee for Scientific
Research (KBN, Project No. 4 T09A 135 24).
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(22) Macomber, R. S. A. Complete Introduction to Modern NMR
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Supporting Information Available: Elemental analysis
results, IR, and full 1H, 13C, and 31P NMR spectra for
compounds 9a-d, 10a-d, 12a-d, and 15a-d and IR and full
1H and 13C NMR spectra for compounds 13a-d, 16a-d, and
18b,c. This material is available free of charge via the Internet
at http://pubs.acs.org.
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JM048970A