Synthesis and cytotoxic evaluation of β-alkyl or β-aryl-δ-methyl-α-methylene-δ-lactones. Comparison with the corresponding γ-lactones

Article abstract of DOI:10.1016/j.ejmech.2009.11.018

We present a simple and general strategy for the synthesis of β,δ-disubstituted-α-methylene-δ-lactones starting from easily available tert-butyl 2-(diethoxyphosphoryl)alk-2-enoates. The elaborated synthetic protocol includes pyrrolidine-catalyzed Michael addition of acetone, diastereoselective reduction of the carbonyl group, lactonization and finally the Horner-Wadsworth-Emmons reaction with formaldehyde. All α-methylene-δ-lactones were evaluated in vitro against mouse leukemia cell line L-1210 and two human leukemia cell lines HL-60 and NALM-6. Comparison of cytotoxic activity with corresponding α-methylene-γ-lactones is also discussed.

Full text of DOI:10.1016/j.ejmech.2009.11.018

European Journal of Medicinal Chemistry 45 (2010) 710–718
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxic evaluation of
-aryl- -methyl- -methylene- -lactones. Comparison with the
corresponding -lactones
b-alkyl or
b
d
a
d
g
qukasz Albrecht a, Jakub Wojciechowski b, Anna Albrecht a, Wojciech M. Wolf b, Anna Janecka c,
c
d
d
a
a,
*
_
´
Kazimierz Studzian , Urszula Krajewska , Marek Rozalski , Tomasz Janecki , Henryk Krawczyk
a
_
´
Institute of Organic Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Ło´dz, Poland
Institute of General and Ecological Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Ło´dz, Poland
Department of Biomolecular Chemistry, Medical University of Lodz, Mazowiecka 6/8, 92-215 Ło´dz, Poland
b
c
_
´
´
d
´
´ ´
Department of Pharmaceutical Biochemistry, Medical University of Lodz, Muszynskiego 1, 90-151 Łodz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
We present a simple and general strategy for the synthesis of b,d-disubstituted-a-methylene-d-lactones
Received 5 June 2009
Received in revised form
17 July 2009
Accepted 6 November 2009
Available online 14 November 2009
starting from easily available tert-butyl 2-(diethoxyphosphoryl)alk-2-enoates. The elaborated synthetic
protocol includes pyrrolidine-catalyzed Michael addition of acetone, diastereoselective reduction of the
carbonyl group, lactonization and finally the Horner–Wadsworth–Emmons reaction with formaldehyde.
All
human leukemia cell lines HL-60 and NALM-6. Comparison of cytotoxic activity with corresponding
-methylene- -lactones is also discussed.
a-methylene-d-lactones were evaluated in vitro against mouse leukemia cell line L-1210 and two
a
g
Keywords:
Ó 2009 Elsevier Masson SAS. All rights reserved.
a
a
-Methylene-
g
-lactones
-Methylene-
d
-lactones
Horner–Wadsworth–Emmons reaction
Diastereoselective synthesis
Cytotoxic activity
1. Introduction
particular in the context of leukemia as well as other cancer cell lines
[7–14].
a
-Alkylidene-
a variety of natural and synthetic products possessing diverse
biological activity [1]. Representative examples of natural -alkyli-
dene- -lactones such as: tulipaline A (2) and tulipaline B (3) [2,3],
eupomatilone 2 (4) [4] or helenalin (5) [5] are shown in Fig. 1. The
-alkylidene- -lactone moiety 1 seems to play a crucial role in the
g
-lactones 1 are found, as structural subunits, in
On the other hand,
abundant in nature [15–23], are well known. Representative exam-
ples of naturally occurring -methylene- -lactones such as: verno-
lepin (7) [15], vernomenin (8) [15], teucriumlactone (9) [17], crassin
(10a) and its acetate 10b [21,22] or pentalenolactone E (11) [16] are
depicted in Fig. 1. Many methods of their preparation have been
a-methylene-d-lactones 6, although much less
a
a
d
g
a
g
diverse pharmacological properties of these products, such as
cytotoxic, allergenic, anti-inflammatory, phytotoxic or antimicrobial
activity. The ability of the unsaturated lactone functionality to act as
a highly reactive Michael acceptor has brought forth the realization
of its crucial role as a physiologically important building block.
Indeed, these compounds have been shown to be active thiol
alkylators and to react with bionucleophiles, especially cysteine
mercapto groups what is crucial for their, usually strong and specific,
cytotoxic activity [5,6]. Structure–activity relationships (SAR) in the
reported in the literature [24–36]. a-Methylene-d-lactones 2 also can
act as Michael acceptors, therefore it is really surprising that the data
about the cytotoxic activity of these compounds are scarce and no
systematic SAR investigations have been undertaken so far. To the
best of our knowledge, there are only two publications on this
subject describing cytotoxicities of several estrogen derivatives
containing a-methylene-d-lactone moiety as a part of the A or D-ring
(Fig. 1, compounds 12, 13) [37,38].
Recently, we have significantly developed the methodology for
a
-alkylidene-
g
-lactones series have been extensively studied, in
the synthesis of
Wadsworth–Emmons olefination reaction which can be efficiently
used for the introduction of the alkylidene moiety into the -lactone
[14,39,40] and -lactone [41–47] ring. In this communication we
present further application of our methodology in the synthesis of
a-alkylidene-g- and d-lactones based on the Horner–
g
d
* Corresponding author. Fax: þ48 42 6365530.
E-mail address: henkrawc@p.lodz.pl (H. Krawczyk).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.018

Ł. Albrecht et al. / European Journal of Medicinal Chemistry 45 (2010) 710–718
711
Fig. 1.
b
-alkyl or
b
-aryl-
d
-methyl-
a
-methylene-
d
-lactones 18 and cytotoxic
In the next step, chemoselective reduction of the carbonyl group
was performed. In our initial studies tert-butyl 2-(diethox-
yphosphoryl)-3-(4-nitrophenyl)-5-oxohexanoate 15a was chosen
as a model substrate and various methods for its conversion into
evaluation of these compounds against the mouse leukemia cell line
L-1210 and two human leukemia cell lines NALM-6 and HL-60.
Furthermore, we compare the activity of
with the activity of the corresponding
a
b
-methylene-d-lactones 18
-aryl- -methyl-
g
a-methy-
the a-diethoxyphosphoryl-d-lactone 17a were evaluated. The use of
lene-
g
-lactones 19, which were very recently synthesized in our
KBH4 as a reducing agent provided 5-hydroxyhexanoate 16a
accompanied by the corresponding lactone 17a. 5-Hydroxyhex-
anoate 16a was completely converted into the lactone 17a by
treatment of the obtained mixture with CF3CO2H in DCM. 31P NMR
data of the lactone 17a revealed the formation of two diastereo-
isomers in a ratio 0.67:1. In order to find an effective, alternative
procedure for the highly diastereoselective synthesis of the lactone
17a we examined the stereoselectivity of the reduction of the
oxoester 15a in the presence of selected metal salts. Model studies
revealed that the treatment of the oxoester 15a with KBH4/CaCl2,
KBH4/CeCl3, KBH4/BaCl2 systems followed by lactonization under
laboratory using the same methodology [40].
2. Chemistry
b,d-Disubstituted-a-methylene-d-lactones 18 evaluated in this
study were obtained from easily available tert-butyl 2-(diethox-
yphosphoryl)alk-2-enoates 14a–g [48,49] as outlined in Scheme 1.
Our synthesis of these compounds begins with Michael addition of
acetone to the alk-2-enoates 14a–g. After preliminary experiments
it was found that the use of acetone as the reagent and solvent, and
pyrrolidine (0.2 equiv) as the catalyst gave the best results in terms
of yield and purity of the products. The additions proceeded
efficiently at room temperature and were terminated within 3–14
days. Data presented in Table 1 shows that a reasonable reaction
rate is limited to the alk-2-enoates 14a,b bearing electron-with-
drawing substituents on the aromatic ring. All the phosphonates
15a–g obtained were formed as inseparable mixtures of
diastereoisomers.
standard conditions gave the expected
a-diethoxyphosphoryl-d-
lactone 17a, however no improvement of diastereselectivity was
observed. The problem of highly diastereoselective reduction of
5-oxohexanoate 15a was eventually solved by employing DIBAL-H
as a reducing agent. The reduction performed in THF at ꢀ78 ꢁC for
3 h followed by CF3CO2H promoted cyclization of the resulting
5-hydroxyhexanoate 16a provided the lactone 17a as a mixture of
two diastereoisomers in a 0.05:0.95 ratio.
Scheme 1. Reagents and conditions: (a) Acetone, pyrrolidine (0.2 equiv), rt. (b) DIBAL-H (2.2 equiv), THF, Ar atm., ꢀ70 ꢁC, 3 h (c) CF3CO2H–CH2Cl2 (1:2), rt, overnight. (d) tBuOK
(1.2 equiv), Et2O, rt, 0.5 h.; then (HCHO)n (5 equiv), Et2O, rt, 1 h.

712
Ł. Albrecht et al. / European Journal of Medicinal Chemistry 45 (2010) 710–718
Table 1
tert-Butyl 3-substituted-2-diethoxyphosphoryl-5-oxohexanoates prepared 15a–g.
R
Yield [%]
Reaction time[days]
dra
a
b
c
d
e
f
4-NO2–C6H4–
4-Br–C6H4–
4-CH3O–C6H4–
3,4-(OCH2O)–C6H3–
2-CH3O–C6H4–
iPr
93
87
88
91
84
89
91
3
3
7
30:70
31:69
30:70
29:71
32:68
43:57
43:57
7
14
11
5
g
c-Hex-
a
Determined by 31P NMR.
This protocol was successfully extended to other 2-diethox-
yphosphoryl-5-oxohexanoates 15b–g. The -diethoxyphosphoryl-
a
d-
lactones 17b–g were formed as mixtures of diastereoisomers. Their
ratios were determined by 31P NMR and are shown in Table 2.
Analysis of these data revealed that electronic effect of the substit-
uents on the aromatic ring had a profound impact on the diaster-
eoselectivity of the reduction.
a-Diethoxyphosphoryl-d-lactones
17a,b,f,g derived from 3-aryl-5-oxohexanoates 15a,b bearing elec-
tron-withdrawing group on the aromatic ring and 3-alkyl-5-oxo-
hexanoates 15f,g exhibited high diastereoselectivity. In contrary,
3-aryl-5-oxohexanoates 15c–e bearing electron-donating substitu-
ents on the phenyl ring gave the corresponding products 17c–e with
moderate diastereoselectivity.
Spectroscopic studies were not sufficient in determining the
stereochemistry of the
a-diethoxyphosphoryl-d-lactones 17a–g
obtained. X-Ray crystallographic analysis conducted on the major
diastereoisomer 17a revealed that in the crystal the d-lactone ring
adopts 4H5 chair conformation [50]. The Cremer and Pople puck-
ering parameters [51] for the endocyclic atom sequence O5/C2/C1/
Fig. 2. The crystal structure of a-diethoxyphosphoryl-d-lactone 17a. The terminal C16
C6/C5/C3 are Q ¼ 0.481(1) Å,
q
¼ 135.2(2)ꢁ,
4
¼ 22.2(2)ꢁ. The phos-
atom of the diethoxy substituent is disordered over two partially occupied positions.
The picture shows site for which the occupation factor was 0.53(1). The slightly less
occupied position has not been shown for clarity. Displacement ellipsoids are drawn at
the 50% probability level. H atoms are represented by circles of an arbitrary radius.
phoryl and aryl groups are placed axially and are in mutual trans
relationship. The methyl group occupies equatorial position and is
located trans in respect to the 4-nitrophenyl subsituent (Fig. 2). This
allowed us to assign the (3R*, 4R*, 6S*) relative stereochemistry to
the all major diastereoisomers 17a–g. In this context, it is also worth
diastereomeric a-methylene-d-lactones 18a–g reflect the degree of
diastereoselection which is attained in the reduction of oxoesters
15a–g.
3
noting that the values of coupling constants JH3H4 ¼1.9–2.6 Hz,
4
3JPCipso ¼ 12.9–14.0 Hz and JH3H5 ¼1.3–1.7 Hz observed for major
diasteroisomers 17a–g confirm the conformation with trans diaxial
C-3 and C-4 substituents arrangement. The relative configuration of
all minor diastereoisomers was assigned to be (3R*, 4S*, 6S*) on the
basis of 1H and 13C NMR data. The values of coupling constants
3. Pharmacological results
3
3JH3H4 ¼ 4.5–5.4 Hz and JPCipso ¼ 4.5–5.1 Hz clearly proved the cis
All a-methylene-d-lactones 18a–g were evaluated in vitro against
mouse leukemia cell line L-1210 and two human leukemia cell lines
HL-60 and NALM-6. Cytotoxic activity of these compounds is
expressed as the concentration (mM) required to inhibit tumor cells
proliferation by 50% after 48 h exposure of the cells to a tested
compound (IC50 values). Results are given in Table 3. We also eval-
arrangement of the phosphoryl and aryl groups.
The Horner–Wadsworth–Emmons reaction of
yphosphoryl- -lactones 17a–g with excess of paraformaldehyde
performed in THF in the presence of potassium tert-butoxide affor-
ded the -methylene- -lactones 18a–g in high yield as inseparable
a-diethox-
d
a
d
uated the cytotoxicities of trans- and cis-
ylene- -lactones 19a–e prepared recently in our laboratory [40] in
order to compare the activity of - and -lactones. Carboplatin was
used as a reference compound [52].
In general, the -methylene-
b-aryl-g-methyl-a-meth-
mixtures of diastereoisomers. It is alsoworth to note that the ratios of
g
g
d
Table 2
a-Diethoxyphosphoryl-d-lactones 17a–g and
a-methylene-d-lactones 18a–g prepared.
a
d-lactones 18a–g synthesized
possess significant cytotoxic activity against all three cell lines.
However, there is a pronounced difference in the cytotoxicities of
R
17
18
Yield[%]a drb(3R*,4R*,6S*): Yield [%] dr(trans:cis)c
(3R*,4S*,6S*)
b
-aryl and
active against all three cell lines (IC50 values in a range from 0.80 to
7.00 M) and can be considered highly potent, according to the
M) [53]. Moreover their activities
b-alkyl substituted lactones. The first are much more
a
b
c
d
e
f
4-NO2–C6H4–
4-Br–C6H4–
4-CH3O–C6H4–
3,4-(OCH2O)–C6H3- 60
2-CH3O–C6H4–
iPr
66
65
62
95:5
85
87
78
72
68
84
84
>95:5
89:11
63:37
76:24
63:37
95:5
89:11
63:37
76:24
63:37
95:5
m
Kupchan’s classification (IC50 ꢂ 15
m
against L-1210 cell line are generally higher than against HL-60 or
NALM-6 cell lines. This observation confirms our earlier findings in
48
73
74
the
substituent in the lactone ring increases cytotoxicity.
substituted lactones 18f,g are less potent, with IC50 values between
13.0 and 61.2 M.
a-methylene-g-lactones series, that the presence of the aryl
g
c-Hex-
94:6
94:6
b-Alkyl
a
b
c
Overall yield for 2 steps.
Determined by 31P NMR.
Determined by 1H NMR.
m

Ł. Albrecht et al. / European Journal of Medicinal Chemistry 45 (2010) 710–718
713
Table 3
Cytotoxic Activity of
19a–e.
corresponding substitution pattern. Therefore, it seems that the size
of the ring does not influences the cytotoxicity.
a-methylene-d-lactones, 18a–g and a-methylene-g-lactones
5. Experimental
5.1. Chemistry
5.1.1. General
NMR spectra were recorded on a Bruker DPX 250 instrument at
250.13 MHz for 1H and 62.9 MHz for 13C and 101.3 MHz for 31P
NMR, respectively using tetramethylsilane as internal and 85%
H3PO4 as external standard. The multiplicity of carbons were
determined by DEPT experiments. IR spectra were measured on
a Specord M80 (Zeiss) instrument. Elemental analyses were per-
formed on a Perkin–Elmer PE 2400 analyzer. Melting points were
determined in open capillaries and are uncorrected. tert-Butyl
2-(diethoxyphosphoryl)alk-2-enoates 14a–g [48,49] and
lene- -lactones 19a–e [40] were prepared according to the litera-
ture procedures.
a-methy-
g
Compound
Cytotoxicity IC50
L-1210
(
m
M)a
HL-60
5.1.2. General procedure for the preparation of tert-butyl 3-
substituted-2-diethoxyphosphoryl-5-oxohexanoates 15a–g
NALM-6
18a
18b
18c
18d
18e
18f
18g
cis-19a
trans-19b
cis-19b
trans-19c
cis-19c
trans-19d
cis-19d
trans-19e
cis-19e
Carboplatin
1.20 ꢄ 0.20
1.75 ꢄ 0.35
0.80 ꢄ 0.09
7.00 ꢄ 1.20
2.10 ꢄ 0.72
44.4 ꢄ 3.47
13.0 ꢄ 1.90
1.90 ꢄ 0.20
0.58 ꢄ 0.08
7.80 ꢄ 1.10
1.95 ꢄ 0.05
4.40 ꢄ 0.82
0.80 ꢄ 0.06
1.40 ꢄ 0.12
1.50 ꢄ 0.19
5.50 ꢄ 0.97
9.7 ꢄ 1.2
6.12 ꢄ 0.71
4.77 ꢄ 0.35
5.95 ꢄ 0.48
5.52 ꢄ 0.42
6.44 ꢄ 0.40
61.2 ꢄ 1.80
40.6 ꢄ 7.70
21.7 ꢄ 6.65
6.66 ꢄ 0.11
4.28 ꢄ 0.54
5.56 ꢄ 0.33
4.82 ꢄ 0.61
5.35 ꢄ 0.53
33.3 ꢄ 3.6
3.42 ꢄ 0.41
4.51 ꢄ 0.63
5.76 ꢄ 0.41
5.46 ꢄ 0.21
6.26 ꢄ 0.28
56.4 ꢄ 2.80
15.8 ꢄ 2.50
3.77 ꢄ 1.10
1.10 ꢄ 0.14
4.25 ꢄ 0.36
5.42 ꢄ 0.26
5.59 ꢄ 0.31
5.00 ꢄ 0.74
4.70 ꢄ 0.70
4.39 ꢄ 0.37
5.97 ꢄ 0.22
0.7 ꢄ 0.3
To
a solution of the corresponding tert-butyl 2-(diethox-
yphosphoryl)alk-2-enoate 14 (3 mmol) in acetone (12 mL) pyrroli-
dine (51 mg, 0.6 mmol) was added and the resulting mixture was
stirred at room temperature for appropriate time shown in Table 1.
After complete consumption of alk-2-enoate 14 (monitored by 31P
NMR spectroscopy) the solvent was evaporated under reduced
pressure. The residue was dissolved in DCM (20 mL) and washed
with 1 M HCl aq (10 mL). Water layer was extracted with DCM
(2 ꢃ 10 mL). Combined organic layers were dried over anhydrous
MgSO4, filtered and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel (ethyl
acetate/hexane 2:1) to afford 3-substituted-2-diethoxyphosphoryl-
5-oxohexanoates 15.
5.01 ꢄ 0.21
5.62 ꢄ 0.21
2.9 ꢄ 0.1
5.1.2.1. tert-Butyl 2-diethoxyphosphoryl-3-(4-nitrophenyl)-5-oxo-
a
IC50, 50% inhibitory concentration determined by MTT assay after 48 h exposition
of the cells to the drugs. IC50 values were calculated from concentration-survival
curves using non-linear estimation (quasi-Newton algorithm) method. Each data
represents the mean from dose response curves of at least three experiments.
hexanoate (15a). (93%); colorless oil. IR (film): 1712, 1592, 1468,
1388, 1256, 1148, 1020 cmꢀ1 31P NMR (CDCl3):
d
¼ 20.55 (30%), 20.98
(70%); 1H NMR (CDCl3):
d
¼ 1.14 (s, 9H, (CH3)3C, major and minor),
1.19–1.41 (m, 6H, (CH3CH2)2OP, major and minor), 2.02 (s, 3H, CH3,
major), 2.04 (s, 3H, CH3, minor), 2.89–3.46 (m, 2H, CH2, major and
minor), 3.95–3.99 (m, 2H, CHP, CHAr, major and minor), 4.01–4.24
Comparison of the cytotoxicities of
analogously substituted -aryl- -lactones 19a–e reveals no signifi-
cant differences in their activities. Almost all cytotoxicities of 19a–e
are in the same range (between 0.5 and 7.8 M) as the cytotoxicities
of 18a–e (between 0.8 and 7.0 M). Only activities of cis-19a and cis-
19d against HL-60 cell line are significantly lower. Such decrease in
the activity for cis isomers was noticed by us previously in -aryl-
methyl- -methylene- -lactone serie [14]. Unfortunately, cytotox-
icities of -lactones 18a–g were determined for the mixtures of trans
b-aryl-d-lactones 18a–e with
b
g
(m, 4H, 2 ꢃ CH2OP, major and minor), 7.47 (d, 2H, 3JHH ¼ 8.7 Hz, CHAr
,
major and minor), 8.13 (d, 2H, 3JHH ¼ 8.7 Hz, CHAr, major and minor);
m
13C NMR (CDCl3):
d
¼ 15.93 (d, JCP ¼ 8.7 Hz, CH3CH2OP, major and
3
m
3
minor), 16.14 (d, JCP ¼ 5.6 Hz, CH3CH2OP, major and minor), 27.16
((CH3)3C, major), 27.58 ((CH3)3C, minor), 30.02 (CH3, minor), 30.08
g
b-
2
(CH3, major), 38.94 (d, JCP ¼ 2.7 Hz, CHAr, minor), 39.12
a
d
g
2
3
(d, JCP ¼ 3.1 Hz, CHAr, major), 46.45 (d, JCP ¼ 10.7 Hz, CH2,
1
minor),47.41 (CH2, major), 51.07 (d, JCP ¼ 128.3 Hz, PCH, major),
and cis diastereomers and no comparison in this respect can be
made.
1
2
51.47 (d, JCP ¼ 132.1 Hz, PCH, minor), 62.24 (d, JCP ¼ 6.9 Hz,
CH3CH2OP, minor), 62.48 (d, 2JCP ¼ 6.9 Hz, CH3CH2OP, minor), 62.73
2
2
(d, JCP ¼ 5.7 Hz, CH3CH2OP, major), 62.84 (d, JCP ¼ 8.1 Hz,
CH3CH2OP, major), 81.60 ((CH3)3C, major), 82.48 ((CH3)3C, minor),
4. Conclusions
123.05 (2 ꢃ CHAr, major), 123.12 (2 ꢃ CHAr, minor), 129.24 (2 ꢃ CHAr
minor), 129.30 (2 ꢃ CHAr, major), 146.52 (CAr major), 146.62
minor), 149.36
,
In conclusion, we have developed a simple and diastereoselective
,
3
method for the synthesis of
b,d
-disubstituted-
a
-methylene-
d-
(CAr3
,
minor), 148.83 (d, JCP ¼ 7.5 Hz, CAr
,
2
lactones from the corresponding tert-butyl 2-(diethoxyphosphor-
yl)alk-2-enoates. Our methodology benefits from easily available
starting materials, experimental simplicity and high yields. Cyto-
(d, JCP ¼ 16.9 Hz, CAr, major), 166.09 (d, JCP ¼ 4.4 Hz, COO, major),
2
166.68 (d, JCP ¼ 5.6 Hz, COO, minor), 204.94 (C(O), minor), 205.03
(C(O), major). Anal. calcd for C20H30NO8P: C, 54.17; H, 6.82. Found: C,
toxicities of all
b-aryl substituted
d-lactones 18a–e against L-1210
54.01; H, 6.62.
and HL-60 and NALM-6 leukemia cell lines were very high and their
activity was comparable with the activity of the previously prepared
5.1.2.2. tert-Butyl 3-(4-bromophenyl)-2-diethoxyphosphoryl-5-oxo-
in our laboratory
b-aryl substituted
g-lactones 19a–e with the
hexanoate (15b). (87%); colorless oil. IR (film): 1720, 1592, 1488,

714
Ł. Albrecht et al. / European Journal of Medicinal Chemistry 45 (2010) 710–718
1388, 1260, 1148, 1024 cmꢀ1 31P NMR (CDCl3):
(69%); 1H NMR (CDCl3):
1.19–1.48 (m, 6H, (CH3CH2)2OP, major and minor), 2.00 (s, 3H, CH3,
major), 2.04 (s, 3H, CH3, minor), 2.87–3.36 (m, 2H, CH2, major and
minor), 3.76–4.01 (m, 2H, CHP, CHAr, major and minor), 4.14–4.25 (m,
4H, 2 ꢃ CH2OP, major and minor), 7.12–7.18 (m, 2H, CHAr, major and
minor), 7.35–7.41 (m, 2H, CHAr, major and minor); 13C NMR (CDCl3):
d
¼ 21.10 (31%), 21.62
((d, 2JCP ¼ 3.3 Hz, CHAr, minor), 39.66 (d, 2JCP ¼ 3.5 Hz, CHAr, major),
3
d
¼ 1.15 (s, 9H, (CH3)3C, major and minor),
47.15 (d, JCP ¼ 10.2 Hz, CH2, minor), 48.37 (CH2, major), 52.46
1
1
(d, JCP ¼ 127.8 Hz, PCH, major), 52.60 (d, JCP ¼ 131.5 Hz, PCH,
2
minor), 62.20 (d, JCP ¼ 6.9 Hz, CH3CH2OP, minor), 62.51 (d,
2JCP ¼ 6.5 Hz, CH3CH2OP, minor), 62.69 (d, 2JCP ¼ 6.3 Hz, CH3CH2OP,
2
major), 62.87 (d, JCP ¼ 7.1 Hz, CH3CH2OP, major), 81.53 ((CH3)3C,
major), 82.25 ((CH3)3C, minor), 100.81 (OCH2O, major), 100.86
(OCH2O, minor), 107.89 (CHAr, major), 108.01 (CHAr, minor), 108.49
(CHAr, minor),), 108.69 (CHAr, major), 121.44 (CHAr, major), 121.59
d
¼ 15.81 (d, 3JCP ¼ 7.0 Hz, CH3CH2OP, minor), 15.96 (d, 3JCP ¼ 6.2 Hz,
CH3CH2OP, minor),16.05 (d, 3JCP ¼ 5.8 Hz, (CH3CH2)2OP, major), 27.07
((CH3)3C, major), 27.51 ((CH3)3C, minor), 30.03 (CH3, minor), 30.16
(CHAr, minor), 134.95 (CAr, minor), 135.22 (CAr, major), 146.31 (CAr
,
2
(CH3, major), 38.64 (d, JCP ¼ 3.3 Hz, CHAr, minor), 38.92
major), 146.43 (CAr, minor), 147.32 (CAr, major), 147.45 (CAr, minor),
(d, 2JCP ¼ 3.3 Hz, CHAr, major), 46.62 (d, 3JCP ¼ 10.5 Hz, CH2, minor),
166.55 (d, JCP ¼ 5.2 Hz, COO, major), 167.33 (d, JCP ¼ 3.9 Hz, COO,
minor), 206.17 (C(O), major and minor). Anal. calcd for C21H31O8P:
C, 57.01; H, 7.06. Found: C, 57.22; H, 6.93.
2
2
1
47.67 (CH2, major), 51.48 (d, JCP ¼ 128.5 Hz, PCH, major), 51.85
1
2
(d, JCP ¼ 131.8 Hz, PCH, minor), 61.98 (d, JCP ¼ 7.0 Hz, CH3CH2OP,
minor), 62.26 (d, 2JCP ¼ 6.5 Hz, CH3CH2OP, minor), 62.48
2
2
(d, JCP ¼ 6.4 Hz, CH3CH2OP, major), 62.66 (d, JCP ¼ 7.2 Hz,
CH3CH2OP, major), 81.42 ((CH3)3C, major), 82.11 ((CH3)3C, minor),
120.38 (2 ꢃ CHAr, major), 120.51 (2 ꢃ CHAr, minor), 129.81 (2 ꢃ CHAr,
5.1.2.5. tert-Butyl 2-diethoxyphosphoryl-3-(2-methoxyphenyl)-5-
oxohexanoate (15e). (84%); colorless oil. IR (film): 1720, 1612, 1512,
1448, 1392, 1368, 1256, 1156, 1024 cmꢀ1 31P NMR (CDCl3):
d
¼ 22.23
minor), 129.90 (2 ꢃ CHAr, major), 130.90 (CAr, major), 131.00 (CAr
,
(32%), 23.26 (68%); 1H NMR (CDCl3):
d
¼ 1.15 (s, 9H, (CH3)3C, major
minor), 139.85 (d, 3JCP ¼ 7.6 Hz, CAr, minor), 140.32 (d, 3JCP ¼ 17.1 Hz,
and minor),1.17–1.48 (m, 6H, (CH3CH2)2OP, major and minor), 2.00 (s,
3H, CH3, major), 2.04 (s, 3H, CH3, minor), 2.85–3.38 (m, 2H, CH2,
major and minor), 3.84 (CH3, major), 3.85 (CH3, minor), 3.74–3.96 (m,
2H, CHP, CHAr, major and minor), 4.15–4.24 (m, 4H, 2 ꢃ CH2OP, major
and minor), 6.79–6.86 (m, 2H, CHAr, major and minor), 7.15–7.22 (m,
2
CAr
,
major), 166.18 (d, JCP ¼ 5.3 Hz, COO, major), 166.86
2
(d, JCP ¼ 3.9 Hz, COO, minor), 205.40 (C(O), minor), 205.41 (C(O),
major). Anal. calcd for C20H30BrO6P: C, 50.32; H, 6.33. Found: C,
50.63; H, 6.36.
2H, CHAr, major and minor); 13C NMR (CDCl3):
d
¼ 15.99 (d,
3
5.1.2.3. tert-Butyl 2-diethoxyphosphoryl-3-(4-methoxyphenyl)-5-
3JCP ¼ 6.6 Hz, CH3CH2OP, major and minor), 16.10 (d, JCP ¼ 6.3 Hz,
CH3CH2OP, major and minor), 27.03 ((CH3)3C, major), 27.54 ((CH3)3C,
minor), 29.77 (CH3, minor), 29.92 (CH3, major), 35.63 (CHAr, minor),
37.28 (CHAr, major), 44.96 (d, 3JCP ¼ 11.1 Hz, CH2, minor), 45.86 (CH2,
oxohexanoate (15c). (88%); colorless oil. IR (film): 1724, 1612, 1512,
1444, 1392, 1368, 1256, 1160, 1024 cmꢀ1 31P NMR (CDCl3):
d
¼ 21.56
(30%), 22.18 (70%); 1H NMR (CDCl3):
d
¼ 1.13 (s, 9H, (CH3)3C, major and
1
minor), 1.17–1.48 (m, 6H, (CH3CH2)2OP, major and minor), 1.96 (s, 3H,
CH3, major),1.98 (s, 3H, CH3, minor), 2.84–3.34 (m, 2H, CH2, major and
minor), 3.75 (CH3, major), 3.77 (CH3, minor), 3.78–3.99 (m, 2H, CHP,
CHAr, major and minor), 4.12–4.24 (m, 4H, 2 ꢃ CH2OP, major and
minor), 6.77–6.88 (m, 2H, CHAr, major and minor), 7.17–7.20 (m, 2H,
major), 49.26 (d, JCP ¼ 131.1 Hz, PCH, minor), 49.75 (d,
1JCP ¼ 129.1 Hz, PCH, major), 54.96 (CH3, major and minor), 61.61
2
2
(d, JCP ¼ 6.9 Hz, CH3CH2OP, minor), 62.09 (d, JCP ¼ 6.9 Hz,
2
CH3CH2OP, minor), 62.19 (d, JCP ¼ 6.6 Hz, CH3CH2OP, major), 62.48
2
(d, JCP ¼ 7.0 Hz, CH3CH2OP, major), 80.73 ((CH3)3C, major), 81.58
CHAr, major and minor); 13C NMR (CDCl3):
d
¼ 15.90 (d, 3JCP ¼ 6.3 Hz,
((CH3)3C, minor), 110.41 (CHAr, major), 110.65 (CHAr, minor), 119.97
(2 ꢃ CHAr, major and minor), 128.01 (CHAr, major), 128.26 (CHAr,
minor), 129.94 (CAr, minor), 131.48 (CAr, major), 157.19 (CAr, minor),
CH3CH2OP, major and minor),16.12 (d, 3JCP ¼ 6.9 Hz, CH3CH2OP, major
and minor), 27.13 ((CH3)3C, major), 27.57 ((CH3)3C, minor), 30.07 (CH3,
minor), 30.27 (CH3, major), 38.67 (d, 2JCP ¼ 3.1 Hz, CHAr, minor), 39.00
2
157.20 (CAr, major), 166.71 (d, JCP ¼ 5.0 Hz, COO, major), 167.39 (d,
2
3
(d, JCP ¼ 3.8 Hz, CHAr, major), 47.13 (d, JCP ¼ 11.3 Hz, CH2, minor),
2JCP ¼ 4.4 Hz, COO, minor), 206.35 (C(O), minor), 206.57 (C(O), major).
1
48.22 (CH2, major), 52.19 (d, JCP ¼ 127.7 Hz, PCH, major), 52.46
Anal. calcd for C21H33O7P: C, 58.87; H, 7.76. Found: C, 58.41; H, 7.53.
(d,1JCP ¼ 132.1 Hz, PCH, minor), 54.85(CH3, minor),54.88(CH3, major),
2
2
61.83 (d, JCP ¼ 6.9 Hz, CH3CH2OP, minor), 62.13 (d, JCP ¼ 6.9 Hz,
CH3CH2OP, minor), 62.37 (d, 2JCP ¼ 6.4 Hz, CH3CH2OP, major), 62.56 (d,
2JCP ¼ 7.5 Hz, CH3CH2OP, major), 81.11 ((CH3)3C, major), 81.59((CH3)3C,
minor), 113.29 (2 ꢃ CHAr, major), 113.39 (2 ꢃ CHAr, minor), 128.98
5.1.2.6. tert-Butyl 2-diethoxyphosphoryl-3-isopropyl-5-oxohex-
anoate (15f). (89%); colorless oil. IR (film): 1724, 1592, 1488, 1388,
1256, 1148, 1020 cmꢀ1 31P NMR (CDCl3):
d
¼ 23.55 (43%), 23.82
3
(57%); 1H NMR (CDCl3):
d
¼ 0.79 (t, 3H, JHH ¼ 7.2 Hz, CH3, minor),
3
(2 ꢃ CHAr, minor), 129.08 (2 ꢃ CHAr, major), 132.65 (d, JCP ¼ 16.9 Hz,
0.85 (t, 3H, 3JHH ¼ 6.7 Hz, CH3, minor), 0.86 (t, 3H, 3JHH ¼ 6.7 Hz, CH3,
3
3
CAr, minor), 133.10 (d, JCP ¼ 7.5 Hz, CAr, major), 158.22 (CAr, major),
major), 0.89 (t, 3H, JHH ¼ 6.7 Hz, CH3, major), 1.29–1.35 (m, 6H,
2
158.28 (CAr, minor), 166.44 (d, JCP ¼ 6.6 Hz, COO, major), 167.22 (d,
(CH3CH2)2OP, major and minor), 1.47 (s, 9H, (CH3)3C, major and
minor), 1.82–1.86 (m, 1H, (CH3)2CH), 2.16 (s, 3H, CH3, major), 2.18
(s, 3H, CH3, minor), 2.30–2.68 (m, 2H, CH2, major and minor),
2.80–3.32 (m, 2H, CHP, CH, major and minor), 4.08–4.16 (m, 4H,
2JCP ¼ 5.0 Hz, COO, minor), 205.97 (C(O), major and minor); Anal. calcd
for C21H33O7P: C, 58.87; H, 7.76. Found: C, 58.41; H, 7.53.
5.1.2.4. tert-Butyl 3-(benzo[d][1,3]dioxol-5-yl)-2-diethoxyphosphoryl-
5-oxohexanoate (15d). (91%); colorless oil. IR (film): 1724, 1620,
1512, 1448, 1396, 1368, 1256, 1156, 1020 cmꢀ1 31P NMR (CDCl3):
2 ꢃ CH2OP, major and minor); 13C NMR (CDCl3):
d
¼ 15.92
3
3
(d, JCP ¼ 5.6 Hz, CH3CH2OP, minor), 15.98 (d, JCP ¼ 6.3 Hz,
3
CH3CH2OP, major and minor), 16.11 (d, JCP ¼ 5.6 Hz, CH3CH2OP,
d
¼ 21.40 (29%), 21.97 (71%); 1H NMR (CDCl3):
d
¼ 1.19 (s, 9H,
major), 19.39 (CH3, major), 19.48 (CH3, minor), 27.43 ((CH3)3C,
major), 27.48 ((CH3)3C, minor), 28.90 ((CH3)2CH, minor), 29.34
((CH3)2CH, major), 29.52 (CH3, minor), 29.69 (CH3, major), 31.16
(CH3, minor), 31.38 (CH3, major), 36.37 (d, 2JCP ¼ 3.8 Hz, CH, minor),
(CH3)3C, major and minor), 1.23–1.53 (m, 6H, (CH3CH2)2OP,
major and minor), 2.00 (s, 3H, CH3, major), 2.05 (s, 3H, CH3, minor),
2.77–3.38 (m, 2H, CH2, major and minor), 3.72–3.85 (m, 1H, CHAr,
major and minor), 3.92–4.05 (m, 1H, CHP, major and minor), 4.11–
4.23 (m, 4H, 2 ꢃ CH2OP, major and minor), 5.89 (s, 2H, OCH2O,
major), 5.91 (s, 2H, OCH2O, minor), 6.70–6.74 (m, 3H, CHAr, major
2
3
36.85 (d, JCP ¼ 3.8 Hz, CH, major), 41.00 (d, JCP ¼ 2.5 Hz, CH2,
minor), 43.33 (d, 3JCP ¼ 4.4 Hz, CH2, major), 46.30 (d, 1JCP ¼ 132.7 Hz,
1
PCH, major), 49.81 (d, JCP ¼ 130.8 Hz, PCH, minor), 61.82
and minor); 13C NMR (CDCl3):
d
¼ 16.14 (d, 3JCP ¼ 6.1 Hz, CH3CH2OP,
(d, JCP ¼ 5.6 Hz, CH3CH2OP, minor), 61.93 (d, JCP ¼ 6.9 Hz,
CH3CH2OP, minor), 62.29 (d, 2JCP ¼ 6.9 Hz, CH3CH2OP, major), 62.34
2
2
3
minor), 16.24 (d, JCP ¼ 6.3 Hz, CH3CH2OP, major and minor), 16.33
3
2
(d, JCP ¼ 6.2 Hz, CH3CH2OP, major), 27.44 ((CH3)3C, major), 27.81
(d, JCP ¼ 6.9 Hz, CH3CH2OP, major), 81.24 ((CH3)3C, major), 81.45
2
((CH3)3C, minor), 30.34 (CH3, minor), 30.52 (CH3, major), 39.27
((CH3)3C, minor), 167.47 (d, JCP ¼ 3.1 Hz, COO, major), 167.65

Ł. Albrecht et al. / European Journal of Medicinal Chemistry 45 (2010) 710–718
715
2
2
2
(d, JCP ¼ 5.3 Hz, COO, minor), 206.35 (C(O), minor), 206.80 (C(O),
(d, JCP ¼ 4.8 Hz, CH3CH2OP), 63.70 (d, JCP ¼ 7.0 Hz, CH3CH2OP),
73.82 (CHO),123.83 (2 ꢃ CHAr),127.80 (2 ꢃ CHAr),146.79 (CAr),149.93
(d, 3JCP ¼ 12.9 Hz, CAr), 165.62 (d, 2JCP ¼ 5.4 Hz, COO). Anal. calcd for
C16H22NO7P: C, 51.75; H, 5.97. Found: C, 51.98; H, 5.61.
major). Anal. calcd for C17H33O6P: C, 56.03; H, 9.13. Found: C, 56.24;
H, 9.31.
5.1.2.7. tert-Butyl 3-cyclohexyl-2-diethoxyphosphoryl-5-oxohex-
anoate (15g). (91%); colorless oil. IR (film): 1724, 1596, 1468, 1388,
5.1.3.2. 4-(4-Bromophenyl)-3-diethoxyphosphoryl-6-methyltetrahy-
dro-2H-piran-2-one (17b). (65%); colorless oil. IR (film): 1728, 1592,
1256, 1188, 1024 cmꢀ1 31P NMR (CDCl3):
d
¼ 23.80 (43%), 23.96
(57%); 1H NMR (CDCl3):
d
¼ 0.82–1.00 (m, 2H, CH2, major and
1488, 1388, 1260, 1152, 1028 cmꢀ1 31P NMR (CDCl3):
d
¼ 21.00 (89%),
minor), 1.12–1.24 (m, 3H, CH2, CH, major and minor), 1.25–1.36
(m, 6H, (CH3CH2)2OP, major and minor), 1.47 (s, 9H, (CH3)3C, major
and minor), 1.58–1.82 (m, 6H, CH2, major and minor), 2.10 (s, 3H,
CH3, major), 2.20 (s, 3H, CH3, minor), 2.44–2.65 (m, 2H, CH2, major
and minor), 2.75–3.32 (m, 2H, CHP, CH, major and minor), 4.12–4.19
21.67 (11%). (3R*,4R*,6S*)-17b: 1H NMR (CDCl3):
d
¼ 1.32 (t, 3H,
3JHH ¼ 7.1 Hz, CH3CH2OP), 1.35 (t, 3H, 3JHH ¼ 7.1 Hz, CH3CH2OP), 1.38
(d, 3H, 3JHH ¼ 6.3 Hz, CH3), 1.81–1.90 (m, 1H, CH2), 2.31–2.43 (m, 1H,
2
3
4
CH2), 3.36 (ddd, 1H, JHP ¼ 28.6 Hz, JHH ¼ 2.6 Hz, JHH ¼ 1.3 Hz,
CHP), 3.82–3.94 (m, 1H, CHAr), 4.12–4.27(m, 4H, 2 ꢃ CH2OP), 4.31–
(m, 4H, 2 ꢃ CH2OP, major and minor); 13C NMR (CDCl3):
d
¼ 16.00
4.4.41 (m, 1H, CHO), 7.05 (d, 2H, JHH ¼ 7.5 Hz, CHAr), 7.47 (d, 2H,
3
3
3
3
(d, JCP ¼ 4.1 Hz, CH3CH2OP, minor), 16.03 (d, JCP ¼ 5.0 Hz,
3JHH ¼ 7.5 Hz, CHAr); 13C NMR (CDCl3):
d
¼ 15.97 (d, JCP ¼ 7.1 Hz,
3
3
CH3CH2OP, minor), 16.08 (d, JCP ¼ 6.1 Hz, CH3CH2OP, major), 16.11
CH3CH2OP), 16.08 (d, JCP ¼ 6.8 Hz, CH3CH2OP), 20.92 (CH3), 34.80
3
3
2
(d, JCP ¼ 5.9 Hz, CH3CH2OP, major), 26.02 (CH2, minor), 26.15
(d, JCP ¼ 2.0 Hz, CH2), 36.61 (d, JCP ¼ 2.0 Hz, CHAr), 44.12
2
(2 ꢃ CH2, major and minor), 26.34 (CH2, minor), 26.89 (CH2, major),
27.55 ((CH3)3C, major), 27.62 ((CH3)3C, minor), 29.57 (CH2, major),
29.74 (CH2, minor), 29.96 (CH2, major), 30.02 (CH3, major), 31.21
(CH3, minor), 36.14 (d, 2JCP ¼ 3.8 Hz, CH, major), 36.38
(d, 1JCP ¼ 129.7 Hz, PCH), 62.75 (d, JCP ¼ 6.8 Hz, CH3CH2OP), 63.56
2
(d, JCP ¼ 7.1 Hz, CH3CH2OP), 73.84 (CHO), 120.84 (CAr), 128.41
3
(2 ꢃ CHAr), 131.74 (2 ꢃ CHAr), 141.12 (d, JCP ¼ 13.2 Hz, CAr), 166.00
(d, 2JCP ¼ 5.4 Hz, COO). Anal. calcd for C16H22BrO5P: C, 47.42; H, 5.47.
2
3
(d, JCP ¼ 4.3 Hz, CH, minor), 40.30 (d, JCP ¼ 12.9 Hz, CH, minor),
Found: C, 47.21; H, 5.87.
3
3
41.44 (d, JCP ¼ 13.3 Hz, CH, major), 42.22 (d, JCP ¼ 2.5 Hz, CH2,
minor), 43.69 (d, 3JCP ¼ 3.9 Hz, CH, major), 46.16 (d, 1JCP ¼ 133.6 Hz,
5.1.3.3. 3-Diethoxyphosphoryl-4-(4-methoxyphenyl)-6-methylte-
1
PCH, major), 49.13 (d, JCP ¼ 131.5 Hz, PCH, minor), 61.88
trahydro-2H-piran-2-one (17c). (62%); colorless oil. IR (film): 1716,
2
2
(d, JCP ¼ 6.3 Hz, CH3CH2OP, minor), 62.02 (d, JCP ¼ 6.9 Hz,
CH3CH2OP, minor), 62.34 (d, 2JCP ¼ 6.9 Hz, CH3CH2OP, major), 62.46
1592, 1486, 1388, 1256, 1152, 1012 cmꢀ1 31P NMR (CDCl3):
d
¼ 21.49
(63%), 22.05 (37%); 1H NMR (CDCl3):
d
¼ 1.23–1.40 (m, 9H,
2
(d, JCP ¼ 7.5 Hz, CH3CH2OP, major), 81.31 ((CH3)3C, major), 81.51
(CH3CH2)2OP, CH3, major and minor), 1.59–1.69 (m, 1H, CH2, major),
1.80–1.89 (m, 1H, CH2, minor), 2.14–2.22 (m, 1H, CH2, minor), 2.31–
2.42 (m, 1H, CH2, major), 3.30 (dd, 1H, 2JHP ¼ 28.6 Hz, 3JHH ¼ 5.5 Hz,
CHP, minor), 3.39 (ddd, 1H, 2JHP ¼ 28.7 Hz, 3JHH ¼ 2.2 Hz,
4JHH ¼ 1.6 Hz, CHP, major), 3.71 (s, 3H, CH3, major and minor), 3.83–
4.05 (m, 1H, CHAr, major and minor), 4.13–4.28 (m, 4H, 2 ꢃ CH2OP,
major and minor), 4.31–4.43 (m, 1H, CHO, major), 4.79–4.91 (m, 1H,
CHO, minor), 6.84–6.88 (m, 2H, CHAr, major and minor), 7.06–7.16
2
((CH3)3C, minor), 167.54 (d, JCP ¼ 2.5 Hz, COO, major), 167.84
2
(d, JCP ¼ 5.0 Hz, COO, minor), 206.54 (C(O), minor), 206.94 (C(O),
major). Anal. calcd for C20H37O6P: C, 59.39; H, 9.22. Found: C, 59.42;
H, 9.01.
5.1.3. General procedure for the preparation of 4-substituted-3-
diethoxyphosphoryl-6-methyltetrahydro-2H-piran-2-ones 17a–g
To
a
solution of the corresponding tert-butyl 2-diethox-
(m, 2H, CHAr, major and minor); 13C NMR (CDCl3):
d
¼ 15.86
yphosphoryl-5-oxohexanoate 15 (2 mmol) in THF (20 mL) at ꢀ70 ꢁC
1.5 M solution of DIBAL-H (2.93 mL, 4.4 mmol) was added dropwise.
The resulting mixture was stirred at this temperature for 3 h,
quenched with 3 M HCl aq. (10 mL) and allowed to warm to room
temperature. Solvent was evaported off under reduced pressure and
the residue extracted with DCM (3 ꢃ 15 mL). Combined organic
layers were dried over anhydrous MgSO4, filtered and the solvent
was removed under reduced pressure. The residue was dissolved in
DCM (5 mL), CF3CO2H (2.5 mL) was added and the resulting solution
left without stirring at room temperature overnight. The solvent was
evaporated under reduced pressure, the residue dissolved in CH2Cl2
(20 mL), washed with sat. NaHCO3 (10 mL), dried over MgSO4,
filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (ethyl acetate/
(d, 3JCP ¼ 4.9 Hz, CH3CH2OP, major and minor), 16.05
3
(d, JCP ¼ 6.5 Hz, CH3CH2OP, major and minor), 20.74 (CH3, minor),
3
20.97 (CH3, major), 34.95 (d, JCP ¼ 1.5 Hz, CH2, major), 36.19
3
2
(d, JCP ¼ 2.5 Hz, CH2, minor), 38.53 (d, JCP ¼ 3.4 Hz, CHAr, minor),
39.49 (d, 2JCP ¼ 3.4 Hz, CHAr, major), 44.37 (d, 1JCP ¼ 128.6 Hz, PCH,
major), 47.86 (d, 1JCP ¼ 125.5 Hz, PCH, minor), 55.00 (CH3, major and
2
minor), 62.55 (d, JCP ¼ 6.9 Hz, CH3CH2OP, minor), 62.56 (d,
2JCP ¼ 6.8 Hz, CH3CH2OP, major), 63.38 (d, JCP ¼ 7.1 Hz, CH3CH2OP,
2
2
major), 63.71 (d, JCP ¼ 6.9 Hz, CH3CH2OP, minor), 73.92 (CHO,
major), 75.02 (CHO, minor), 113.96 (2 ꢃ CHAr, major and minor),
127.59 (2 ꢃ CHAr
,
major), 127.74 (2 ꢃ CHAr
,
minor), 133.96
3
3
(d, JCP ¼ 14.0 Hz, CAr, major), 135.81 (d, JCP ¼ 4.5 Hz, CAr, minor),
158.38 (CAr, major and minor), 166.38 (d, 2JCP ¼ 5.4 Hz, COO, major),
2
167.00 (d, JCP ¼ 5.0 Hz, COO, minor). Anal. calcd for C17H25O6P: C,
hexane 4:1) to afford
a-diethoxyphosphoryl-d-lactone 17.
57.30; H, 7.07. Found: C, 57.14; H, 7.25.
5.1.3.1. 3-Diethoxyphosphoryl-6-methyl-4-(4-nitrophenyl)tetrahydro-
2H-piran-2-one (17a). (66%); white crystals, mp 136–138 ꢁC. IR
(film): 1720, 1592, 1488, 1388, 1260, 1148, 1024 cmꢀ1 31P NMR
5.1.3.4. 4-(Benzo[d][1,3]dioxol-5-yl)-3-diethoxyphosphoryl-6-methy
ltetrahydro-2H-piran-2-one (17d). (60%); colorless oil. IR (film):
1716,1504,1440,1388,1324,1244,1136,1016 cmꢀ1 31P NMR (CDCl3):
(CDCl3):
(CDCl3):
d
¼ 20.47 (95%), 21.23 (5%). (3R*,4R*,6S*)-17a: 1H NMR
d
¼ 21.31 (76%), 21.92 (24%); 1H NMR (CDCl3):
¼ 1.25–1.40 (m, 9H,
d
3
d
¼ 1.31 (t, 3H, JHH ¼ 6.5 Hz, CH3CH2OP), 1.36 (t, 3H,
(CH3CH2)2OP, CH3, major and minor), 1.60–1.91 (m, 1H, CH2, major
and minor), 2.29–2.41 (m, 1H, CH, major and minor), 3.28 (dd, 1H,
2JHP ¼ 28.6 Hz, 3JHH ¼ 5.4 Hz, CHP, minor), 3.36 (ddd, 1H,
3JHH ¼ 6.4 Hz, CH3CH2OP), 1.41 (d, 3H, 3JHH ¼ 6.3 Hz, CH3), 1.90–1.98
2
(m, 1H, CH2), 2.36–2.48 (m, 1H, CH2), 3.41 (ddd, 1H, JHP ¼ 28.6 Hz,
3JHH ¼ 2.6 Hz, JHH ¼ 1.4 Hz, CHP), 3.98–4.04 (m, 1H, CHAr), 4.08–
2JHP ¼ 28.8 Hz, JHH ¼ 2.3 Hz, JHH ¼ 1.6 Hz, CHP, major), 3.78–3.89
(m, 1H,CHAr, major and minor), 4.06–4.28 (m, 4H, 2 ꢃ CH2OP, major
and minor), 4.32–4.46 (m, 1H, CHO, major), 4.80–4.87 (m, 1H, CHO,
minor), 5.94 (s, 2H, OCH2O, minor), 5.95 (s, 2H, OCH2O, minor), 6.59–
4
3
4
4.27(m, 4H, 2 ꢃ CH2OP), 4.33–4.4.40 (m, 1H, CHO), 7.36 (d, 2H,
3JHH ¼ 8.6 Hz, CHAr), 8.22 (d, 2H, JHH ¼ 8.6 Hz, CHAr); 13C NMR
3
(CDCl3):
d
¼ 15.92 (d, 3JCP ¼ 7.0 Hz, CH3CH2OP),16.05 (d, 3JCP ¼ 6.9 Hz,
3
CH3CH2OP), 20.78 (CH3), 34.79 (d, JCP ¼ 3.4 Hz, CH2), 37.05
6.78 (m, 3H, CHAr, major and minor); 13C NMR (CDCl3):
d
¼ 15.89
2
1
(d, JCP ¼ 2.0 Hz, CHAr), 44.02 (d, JCP ¼ 131.0 Hz, PCH), 62.72
(d, 3JCP ¼ 6.6 Hz, CH3CH2OP, major and minor), 16.00

716
Ł. Albrecht et al. / European Journal of Medicinal Chemistry 45 (2010) 710–718
3
(d, JCP ¼ 6.4 Hz, CH3CH2OP, major and minor), 20.67 (CH3, minor),
(94%). (3R*,4R*,6S*)-17g: 1H NMR (CDCl3):
d
¼ 0.91–1.00 (m, 2H,
3
20.89 (CH3, major), 34.94 (d, JCP ¼ 1.7 Hz, CH2, major), 36.66
CH2),1.11–1.25 (m, 3H, CH2, CH),1.31–1.42 (m, 9H, (CH3CH2)2OP, CH3),
1.62–1.70 (m, 7H, 3xCH2, CH),1.75–1.86 (m,1H, CH),1.90–2.08 (m, 2H,
CH2), 3.20 (ddd, 1H, 2JHP ¼ 28.9 Hz, 3JHH ¼ 1.9 Hz, 4JHH ¼ 1.7 Hz, CHP),
4.12–4.25 (m, 4H, 2 ꢃ CH2OP), 4.48–4.60 (m, 1H, CHO). 13C NMR
2
3
(d, JCP ¼ 2.4 Hz, CHAr, minor), 39.01 (d, JCP ¼ 3.4 Hz, CH2, minor),
39.37 (d, 2JCP ¼ 5.5 Hz, CHAr, major), 44.38 (d, 1JCP ¼ 128.8 Hz, PCH,
major), 47.73 (d, 1JCP ¼ 125.5 Hz, PCH, minor), 62.53 (d, 2JCP ¼ 6.2 Hz,
CH3CH2OP, minor), 62.64 (d, 2JCP ¼ 6.6 Hz, CH3CH2OP, major), 63.37
(CDCl3):
d
¼ 15.92
(d,
3JCP ¼ 6.2 Hz,
CH3CH2OP),
15.95
2
2
3
(d, JCP ¼ 7.1 Hz, CH3CH2OP, major), 63.69 (d, JCP ¼ 6.9 Hz,
CH3CH2OP, minor), 73.85 (CHO, major), 74.87 (CHO, minor), 100.86
(OCH2O, minor),100.94 (OCH2O, major),106.95 (CHAr, minor),107.18
(d, JCP ¼ 6.2 Hz, CH3CH2OP), 21.49 (CH3), 25.66 (2 ꢃ CH2), 25.75
(CH2), 28.44 (d, 3JCP ¼ 1.9 Hz, CH2), 29.28 (CH2), 30.78 (CH2), 36.55 (d,
2JCP ¼ 3.7 Hz, CHc-Hex), 38.48 (d, JCP ¼ 13.2 Hz, CH), 42.92
3
(CHAr, major), 108.01 (CHAr, major), 108.10 (CHAr, minor), 119.33
(d,1JCP ¼ 128.9 Hz, PCH), 62.32 (d, 2JCP ¼ 6.8 Hz, CH3CH2OP), 63.03 (d,
2JCP ¼ 7.0 Hz, CH3CH2OP), 74.31 (CHO), 166.05 (d, 2JCP ¼ 5.2 Hz, COO).
Anal. calcd for C16H29O5P: C, 57.82; H, 8.79. Found: C, 57.94; H, 8.52.
3
(CHAr, major), 119.87 (CHAr, minor), 135.78 (d, JCP ¼ 13.9 Hz, CAr
,
3
major), 137.53 (d, JCP ¼ 4.7 Hz, CAr, minor), 146.36 (CAr, major and
minor), 147.75 (CAr
,
minor), 147.85 (CAr
,
major), 166.16
(d, 2JCP ¼ 5.4 Hz, COO, major), 166.79 (d, 2JCP ¼ 5.0 Hz, COO, minor).
5.1.4. General procedure for the preparation of 4-substituted-6-
methyl-3-methylenetetrahydro-2H-piran-2-ones 18a-g
Anal. calcd for C17H23O7P: C, 55.13; H, 6.26. Found: C, 55.23; H, 6.15.
To a solution of the corresponding
a-diethoxyphosphoryl-d-
5.1.3.5. 3-Diethoxyphosphoryl-4-(2-methoxyphenyl)-6-methylte-
lactone 17 (1.0 mmol) in Et2O (10 mL) potassium tert-butoxide
(134 mg, 1.2 mmol) was added and the resulting mixture was stirred
at room temperature for 30 min. Then paraformaldehyde (150 mg,
5 mmol) was added in one portion. After 1 h the reaction mixture
was quenched with brine (10 ml), layers separated and the water
layer was extracted with CH2Cl2 (2 ꢃ 10 mL). The combined organic
layers were dried over MgSO4 and evaporated. The crude product
was purified by column chromatography (eluent: ethyl acetate/
hexane 1:4).
trahydro-2H-piran-2-one (17e). (48%); colorless oil. IR (film): 1720,
1592, 1486, 1388, 1256, 1154, 1016 cmꢀ1 31P NMR (CDCl3):
d
¼ 22.29
(63%), 23.24 (37%); 1H NMR (CDCl3):
d
¼ 1.22–1.38 (m, 9H,
(CH3CH2)2OP, CH3, major and minor), 1.84–2.08 (m, 1H, CH2, major
and minor), 2.17–2.34 (m, 1H, CH2, major and minor), 3.38 (dd, 1H,
2JHP ¼ 28.1 Hz, 3JHH ¼ 4.4 Hz, CHP, minor), 3.44 (ddd, 1H,
2JHP ¼ 28.3 Hz, 3JHH ¼ 2.4 Hz, 4JHH ¼ 1.4 Hz, CHP, major), 3.81 (s, 3H,
CH3, minor), 3.83 (s, 3H, CH3, major), 3.87–4.03 (m, 1H, CHAr, major
and minor), 4.03–4.27 (m, 4H, 2 ꢃ CH2OP, major and minor), 4.31–
4.44 (m, 1H, CHO, major), 4.86–4.99 (m, 1H, CHO, minor), 6.86–6.96
5.1.4.1. (4R*,6S*)-6-methyl-3-methylene-4-(4-nitrophenyl)tetrahydro-
2H-piran-2-one (18a). (85%); colorless oil (dr >95:5). IR (film): 1712,
3
(m, 2H, CHAr, major and minor), 7.03 (dd, 1H, JHH ¼ 7.4 Hz,
4JHH ¼ 1.6 Hz, CHAr, major), 7.13 (dd, 1H, 3JHH ¼ 7.6 Hz, 4JHH ¼ 1.6 Hz,
1512, 1352, 1196, 1132, 1056 cmꢀ1 1H NMR (CDCl3):
d
¼ 1.39 (d, 3H,
CHAr, minor), 7.21–7.30 (m, 1H, CHAr, major and minor); 13C NMR
3JHH ¼ 6.4 Hz, CH3), 2.16 (t, 2H, 3JHH ¼ 5.4 Hz, CH2), 4.21–4.26 (m,1H,
CHAr), 4.39–4.47 (m,1H, CHO), 5.56 (d,1H, 2JHH ¼ 1.1 Hz, CH), 6.75 (d,
1H, 2JHH ¼ 1.1 Hz, CH), 7.37 (d, 2H, 3JHH ¼ 8.4 Hz, CHAr), 8.22 (d, 2H,
3
(CDCl3):
d
3
¼ 15.82 (d, JCP ¼ 6.1 Hz, CH3CH2OP, major and minor),
15.97 (d, JCP ¼ 6.0 Hz, CH3CH2OP, major and minor), 20.54 (CH3,
minor), 20.90 (CH3, major), 32.89 (CH2, major and minor), 35.64 (d,
3JHH ¼ 8.4 Hz, CHAr); 13C NMR (CDCl3):
¼ 20.92 (CH3), 36.77 (CH2),
d
2JCP ¼ 2.7 Hz, CHAr, minor), 36.79 (d, JCP ¼ 2.9 Hz, CHAr, major),
41.52 (CHAr), 72.55 (CHO), 123.20 (2 ꢃ CHAr), 128.55 (2 ꢃ CHAr),
131.64 (CH2),135.01 (C),146.57 (CAr),149.78 (CAr),164.63(COO). Anal.
calcd for C13H13NO4: C, 63.15; H, 5.30. Found: C, 63.30; H, 5.62.
2
43.49 (d, 1JCP ¼ 129.8 Hz, PCH, major), 45.97 (d, 1JCP ¼ 125.2 Hz, PCH,
minor), 54.58 (CH3, minor), 54.74 (CH3, major), 62.35 (d,
2JCP ¼ 6.6 Hz, CH3CH2OP, major and minor), 63.16 (d, JCP ¼ 6.9 Hz,
2
CH3CH2OP, major), 63.50 (d, 2JCP ¼ 6.8 Hz, CH3CH2OP, minor), 74.34
(CHO, major), 74.47 (CHO, minor), 110.54 (CHAr, major), 110.86
(CHAr, minor), 120.25 (CHAr, minor), 120.31 (CHAr, major), 127.64
5.1.4.2. 4-(4-Bromophenyl)-6-methyl-3-methylenetetrahydro-2H-
piran-2-one (18b). (87%); colorless oil (dr 89:11). IR (film): 1720,
1488, 1384, 1304, 1204, 1072 cmꢀ1 (4R*,6S*)-18b: 1H NMR (CDCl3):
(CHAr, major), 128.12 (CHAr, major), 128.35 (CHAr, minor), 129.00
d
¼ 1.37 (d, 3H, 3JHH ¼ 6.4 Hz, CH3), 2.04–2.12 (m, 2H, CH2), 4.06–4.09
3
(CHAr
,
minor), 129.88 (d, JCP ¼ 12.9 Hz, CAr
,
major), 130.86
(m, 1H, CHAr), 4.38–4.51 (m, 1H, CHO), 5.55 (t, 1H,
4JHH ¼ 2JHH ¼ 1.4 Hz, CH), 6.69 (t,1H, 4JHH ¼ 2JHH ¼ 1.4 Hz, CH), 7.06 (d,
3
(d, JCP ¼ 5.1 Hz, CAr, minor), 156.29 (CAr, major and minor), 166.87
(d, 2JCP ¼ 4.6 Hz, COO, major), 167.53 (d, 2JCP ¼ 3.3 Hz, COO, minor).
Anal. calcd for C17H25O6P: C, 57.30; H, 7.07. Found: C, 57.01; H, 7.32.
2H, JHH ¼ 8.6 Hz, CHAr), 7.47 (d, 2H, JHH ¼ 8.6 Hz, CHAr); 13C NMR
3
3
(CDCl3):
d
¼ 21.11 (CH3), 37.18 (CH2), 41.24 (CHAr), 72.65 (CHO),
120.80 (CAr),129.29 (2 ꢃ CHAr),131.18 (CH2),131.76 (2 ꢃ CHAr),135.72
(C),141.32 (CAr),165.20 (COO). (4R*,6R*)-18b (representative signals):
1.44 (d, 3H, 3JHH ¼ 6.3 Hz, CH3), 3.77–3.84 (m,1H, CHAr), 5.24 (dd,1H,
4JHH ¼ 2.7 Hz, 2JHH ¼ 1.2 Hz, CH). Anal. calcd for C13H13BrO2: C, 55.54;
H, 4.66. Found: C, 55.28; H, 4.31.
5.1.3.6. 3-Diethoxyphosphoryl-4-isopropyl-6-methyltetrahydro-2H-
piran-2-one (17f). (73%); colorless oil. IR (film): 1720, 1464, 1392,
1252, 1160, 1032 cmꢀ1 31P NMR (CDCl3):
d
¼ 20.11 (5%), 22.13 (95%).
3
(3R*,4R*,6S*)-17f: 1H NMR (CDCl3):
d
¼ 0.95 (d, 3H, JHH ¼ 6.6 Hz,
3
CH3), 0.99 (d, 3H, JHH ¼ 6.5 Hz, CH3), 1.25–1.47 (m, 9H,
(CH3CH2)2OP, CH3), 1.58–1.61 (m, 1H, CH), 1.75–1.88 (m, 1H, CHiPr),
5.1.4.3. 4-(4-Methoxyphenyl)-6-methyl-3-methylenetetrahydro-2H-
piran-2-one (18c). (78%); colorless oil (dr 63:37). IR (film): 1712,
2.02–2.21 (m, 2H, CH2), 3.16 (ddd, 1H, 2JHP ¼ 28.8 Hz, JHH ¼ 2.1 Hz,
3
4JHH ¼ 1.6 Hz, CHP), 4.13–4.26 (m, 4H, 2 ꢃ CH2OP), 4.51–4.65 (m, 1H,
1468, 1364, 1312, 1224, 1068 cmꢀ1 1H NMR (CDCl3):
d
¼ 1.36 (d, 3H,
CHO). 13C NMR (CDCl3):
d
¼ 15.52 (d, 3JCP ¼ 6.1 Hz, CH3CH2OP),15.57
3JHH ¼ 6.4 Hz, CH3, major), 1.44 (d, 3H, JHH ¼ 6.3 Hz, CH3, minor),
1.85–2.17 (m, 2H, CH2, major and minor), 3.71–3.76 (m, 1H, CHAr,
minor), 3.80 (m, 3H, CH3O, major), 3.81 (m, 3H, CH3O, minor), 4.06
3
3
(d, JCP ¼ 6.1 Hz, CH3CH2OP), 18.67 (CH3), 20.19 (CH3), 21.03 (CH3),
3
3
28.54 (d, JCP ¼ 2.2 Hz, CH2), 28.98 (d, JCP ¼ 13.2 Hz, (CH3)2CH),
37.52 (d, 2JCP ¼ 3.7 Hz, CH), 43.09 (d, 1JCP ¼ 129.2 Hz, PCH), 61.89 (d,
3
(t, 1H, JHH ¼ 4.7 Hz, CHAr, major), 4.43–4.50 (m, 1H, CHO, major),
2JCP ¼ 6.9 Hz, CH3CH2OP), 62.65 (d, JCP ¼ 7.0 Hz, CH3CH2OP), 73.73
4.51–4.65 (m, 1H, CHO, minor), 5.25 (dd, 1H, JHH ¼ 2.6 Hz,
2
4
2
4
(CHO), 165.43 (d, JCP ¼ 5.2 Hz, COO). Anal. calcd for C13H25O5P: C,
2JHH ¼ 1.3 Hz, CH, minor), 5.55 (t, 1H, JHH ¼ 2JHH ¼ 1.4 Hz, CH,
53.42; H, 8.62. Found: C, 53.61; H, 8.19.
major), 6.57 (ddd, 1H, 4JHH ¼ 2.6 Hz, 4JHH ¼ 0.7 Hz, 2JHH ¼ 1.3 Hz, CH,
4
minor), 6.66 (t, 1H, JHH ¼ 2JHH ¼ 1.4 Hz, CH, major), 6.85–6.90 (m,
5.1.3.7. 4-Cyclohexyl-3-diethoxyphosphoryl-6-methyltetrahydro-2H-
2H, CHAr, major and minor), 7.07–7.12 (m, 2H, CHAr, major and
piran-2-one (17g). (74%); colorless oil. IR (film): 1724, 1448, 1392,
minor); 13C NMR (CDCl3):
minor), 37.36 (CH2, major), 38.60 (CH2, minor), 40.82 (CHAr, major),
d
¼ 21.03 (CH3, major), 21.45 (CH3,
1248, 1176, 1128, 1032 cmꢀ1 31P NMR (CDCl3):
d
¼ 20.24 (6%), 22.28

Ł. Albrecht et al. / European Journal of Medicinal Chemistry 45 (2010) 710–718
717
43.64 (CHAr, minor), 55.01 (CH3O, major and minor), 72.66 (CHO,
major), 75.50 (CHO, minor), 113.89 (2 ꢃ CHAr major), 113.99
major), 128.72 (2 ꢃ CHAr
5.1.4.7. 4-Cyclohexyl-6-methyl-3-methylenetetrahydro-2H-piran-2-
one (18g). (84%); colorless oil (dr 96:4). IR (film): 1720, 1468, 1384,
,
(2 ꢃ CHAr
,
minor), 128.39 (2 ꢃ CHAr
,
,
1312,1224,1136,1012 cmꢀ1 (4R*,6S*)-18g: 1H NMR (CDCl3):
d
¼ 0.83–
3
minor), 129.70 (CH2, minor), 130.37 (CH2, major), 133.95 (C, minor),
134.11 (C, major), 136.50 (CAr, major), 138.93 (CAr, minor), 158.26
(CAr, major), 158.42 (CAr, minor), 165.50 (COO, major and minor).
Anal. calcd for C14H16O3: C, 72.39; H, 6.94. Found: C, 72.31; H, 6.88.
0.95 (m, 2H, CH2), 1.14–1.27 (m, 3H, CH2), 1.37 (d, 3H, JHH ¼ 6.3 Hz,
2
CH3), 1.63–1.83 (m, 7H, CH2, CH), 2.13 (ddd, 1H, JHH ¼ 14.4 Hz,
3JHH ¼ 3.6 Hz, CH2), 2.30–2.35 (m, 1H, CH), 4.57–4.71 (m, 1H, CHO),
4
2
5.42 (dd, 1H, JHH ¼ 1.6 Hz, JHH ¼ 1.0 Hz, CH), 6.39 (dd, 1H,
4JHH ¼ 1.6 Hz, JHH ¼ 1.0 Hz, CH); 13C NMR (CDCl3):
d
¼ 21.45 (CH3),
2
5.1.4.4. 4-(Benzo[d][1,3]dioxol-5-yl)-6-methyl-3-methylenetetrahy-
dro-2H-piran-2-one (18d). (72%); colorless oil (dr 76:24). IR (film):
25.81 (3xCH2), 29.23 (CH2), 30.41 (CH), 37.17 (CH2), 42.48 (CH), 72.87
(CHO), 128.88 (CH2), 136.77 (C), 165.06 (COO). (4R*,6R*)-18g (repre-
sentative signals): 4.19–4.29 (m,1H, CHO), 5.51 (dd,1H, 4JHH ¼ 1.9 Hz,
2JHH ¼ 1.3 Hz, CH), 6.30 (dd,1H, 4JHH ¼ 1.6 Hz, 2JHH ¼ 1.3 Hz, CH). Anal.
calcd for C13H20O2: C, 74.96; H, 9.68. Found: C, 75.12; H, 9.98.
1720, 1468, 1368, 1312, 1224, 1024 cmꢀ1 1H NMR (CDCl3):
d
¼ 1.37
3
3
(d, 3H, JHH ¼ 6.4 Hz, CH3, major), 1.43 (d, 3H, JHH ¼ 6.3 Hz, CH3,
minor), 1.83–2.17 (m, 2H, CH2, major and minor), 3.69–3.78 (m, 1H,
CHAr, minor), 4.03 (t, 1H, 3JHH ¼ 4.8 Hz, CHAr, major), 4.41–4.64 (m,
1H, CHO, major and minor), 5.31 (dd,1H, 4JHH ¼ 2.6 Hz, 2JHH ¼ 1.3 Hz,
CH, minor), 5.56 (t, 1H, 4JHH ¼ 2JHH ¼ 1.4 Hz, CH, major), 5.95 (s, 2H,
OCH2O, major), 5.96 (s, 2H, OCH2O, minor), 6.58–6.80 (m, 4H, CH,
5.2. Pharmacology
5.2.1. Cells and cytotoxicity assays
Mouse leukemia L-1210 cells were cultured in RPMI 1640
medium (Sigma, St. Louis, MO) supplemented with 10% foetal calf
CHAr, major and minor); 13C NMR (CDCl3):
21.34 (CH3, minor), 37.27 (CH2, major), 38.52 (CH2, minor), 41.19
(CHAr, major), 44.05 (CHAr, minor), 72.59 (CHO, major), 75.32 (CHO,
d
¼ 20.93 (CH3, major),
serum (Gibco, Berlin, Germany), gentamycin (50 mg/mL) and 0.02 M
minor),100.80 (OCH2O, minor),100.86 (OCH2O, major),107.65 (CHAr
major and minor),107.97 (CHAr, major),108.07 (CHAr, minor),120.48
(CH2, major),121.02 (CH2, minor),129.76 (CHAr, minor),130.52 (CHAr
major),135.63 (CHAr, minor),135.88 (CHAr, major),136.27 (C, major),
138.58 (C, minor),146.21 (CAr, major),146.35 (CAr, minor),147.75 (CAr
,
HEPES buffer (Gibco). Cytostatic effects were assayed by measuring
inhibitory effects on L-1210 cell proliferation. In this assay, cells
were seeded in 2 mL aliquots onto a 24-well plate (NUNC,
Denmark) at a concentration 1.5 ꢃ103 cells/mL. After 24 h drug
solution were added and incubation was carried out for an addi-
tional 48 h. The cell number relative to control was determined by
a tetrazolium dye method [54].
,
,
major and minor), 165.23 (COO, major), 165.37 (COO, minor). Anal.
calcd for C14H14O4: C, 68.28; H, 5.73. Found: C, 67.97; H, 5.62.
Human leukemia promyelocytic HL-60 and lymphoblastic
NALM-6 cell lines were used. Leukemia cells were cultured in RPMI
1640 medium supplemented with 10% heat–inactivated foetal
5.1.4.5. 4-(2-Methoxyphenyl)-6-methyl-3-methylenetetrahydro-2H-
piran-2-one (18e). (68%); colorless oil (dr 63:37). IR (film): 1720,
1488, 1384, 1308, 1212, 1068 cmꢀ1 1H NMR (CDCl3):
d
¼ 1.33 (d, 3H,
bovine serum (Invitrogen, Paisley, UK) and antibiotics (100 mg/ml
3JHH ¼ 6.4 Hz, CH3, major), 1.42 (d, 3H, JHH ¼ 6.3 Hz, CH3, minor),
1.90–2.20 (m, 2H, CH2, major and minor), 3.80 (m, 3H, CH3O, major),
3.85 (m, 3H, CH3O, minor), 4.12–4.21 (m,1H, CHAr, minor), 4.36 (t,1H,
3JHH ¼ 4.7 Hz, CHAr, major and minor), 4.40–4.57 (m, 1H, CHO, major
and minor), 5.26 (dd,1H, 4JHH ¼ 2.5 Hz, 2JHH ¼ 1.3 Hz, CH, minor), 5.53
streptomycin and 100 U/ml penicillin). Cells were grown in 37 ꢁC in
a humidified atmosphere of 5% CO2 in air. Cytotoxic activity was
determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nylterazolium bromide, Sigma, St. Louis, USA] assay [55]. Expo-
nentially growing leukemia cells were seeded at 8 ꢃ 103/well on
96-well plate (Nunc, Roskilde, Denmark). Stock solutions of the
analyzed compounds were freshly prepared in DMSO and diluted
with complete culture medium to obtain the concentration range
from 10ꢀ7 to 10ꢀ3 M. Cells were exposed to the test compounds for
46 h, then MTT reagent was added and incubation was continued
for 2 h. After incubation, MTT–formazan crystals were dissolved in
20% SDS and 50% DMF at pH 4.7 and absorbance was read at 562
and 630 nm on an ELISA–plate reader (ELX 800, Bio-Tek, USA). As
a control, cultured cells were grown in the absence of drugs. The
values of IC50 (the concentration of the tested compound required
to reduce the cells survival fraction to 50% of the control) were
calculated from concentration-response curves and used as
a measure of cellular sensitivity to a given treatment. Data points
represent means of at least three experiments each made in 6
repeats ꢄ SD.
3
4
4
(t, 1H, JHH ¼ 2JHH ¼ 1.4 Hz, CH, major), 6.41 (dd, 1H, JHH ¼ 2.7 Hz,
2JHH ¼ 1.3 Hz, CH, minor), 6.61 (t, 1H, 4JHH ¼ 2JHH ¼ 1.4 Hz, CH, major),
6.88–6.96 (m, 2H, CHAr, major and minor), 6.99–7.29 (m, 2H, CHAr,
major and minor); 13C NMR (CDCl3):
d
¼ 21.07 (CH3, major), 21.28
(CH3, minor), 35.41 (CH2, major), 36.56 (CH2, minor), 37.17 (CHAr,
major), 38.97 (CHAr, minor), 55.01 (CH3O, major and minor), 73.18
(CHO, major), 75.38 (CHO, minor), 110.64 (CAr, major), 110.94 (CAr,
minor), 120.36 (CHAr, major), 120.69 (CHAr, minor), 127.75 (CHAr,
minor), 128.03 (CHAr, major), 128.16 (CHAr, minor), 128.52 (CH2,
minor),128.67 (CHAr, major),129.55 (CH2, major),131.00 (CHAr, major
and minor), 136.64 (C, major), 138.47 (C, minor), 156.39 (CAr, major),
156.56 (CAr, minor), 166.00 (COO, major), 166.64 (COO, minor). Anal.
calcd for C14H16O3: C, 72.39; H, 6.94. Found: C, 72.51; H, 6.82.
5.1.4.6. 4-Isopropyl-6-methyl-3-methylenetetrahydro-2H-piran-2-
one (18f). (84%); colorless oil (dr 95:5). IR (film): 1724, 1464, 1384,
1296, 1184, 1136, 1048 cmꢀ1 (4R*,6S*)-18f: 1H NMR (CDCl3):
5.3. X-ray single crystal analysis for 17a
d
¼ 0.94 (d, 3H, 3JHH ¼ 6.5 Hz, CH3), 0.96 (d, 3H, 3JHH ¼ 6.5 Hz, CH3),
3
1.38 (d, 3H, JHH ¼ 6.3 Hz, CH3), 1.67–1.85 (m, 2H, CH2, CH), 2.09
Crystal and X-ray data. Formula: C16H22NO7P, Mw ¼ 371.32,
2
3
3
(ddd, 1H, JHH ¼ 14.3 Hz, JHH ¼ 3.6 Hz, JHH ¼ 4.1 Hz, CH2), 2.28–
2.35 (m, 1H, (CH3)2CH), 4.62–4.69 (m, 1H, CHO), 5.47 (dd, 1H,
colourless
crystal
0.50 ꢃ 0.20 ꢃ 0.15 mm,
a ¼ 14.0936(8),
b ¼ 17.4422(10), c ¼ 8.0163(5) Å,
b
¼ 120.6630(10)ꢁ, V ¼ 1695.07
4JHH ¼ 1.6 Hz, JHH ¼ 1.1 Hz, CH), 6.41 (dd, 1H, JHH ¼ 1.6 Hz,
(17) Å3, rcalcd ¼ 1.455 g cmꢀ3
,
l
¼ 0.71073 Å,
m
¼ 2.00 cmꢀ1, semi-
2
4
2JHH ¼ 1.1 Hz, CH); 13C NMR (CDCl3):
d
¼ 16.77 (CH3), 19.11 (CH3),
empirical absorption correction based on multiple scanned equiv-
20.64 (CH3), 28.53 (CH), 32.04 (CH2), 43.79 (CH), 73.24 (CHO),
127.56 (CH2), 137.49 (C), 165.75 (COO). (4R*,6R*)-18f (representative
alent reflections 0,8686 < T < 0.9704, Z ¼ 4, crystal system: mono-
clinic, space group: Cc, T ¼ 90 K,
u scans,17749 reflections collected,
3
signals): 1.39 (d, 3H, JHH ¼ 6.3 Hz, CH3), 4.20–4.32 (m, 1H, CHO),
(ꢄh, ꢄk, ꢄl), 2qmax ¼ 60.0ꢁ, 2468 unique reflections (Rint ¼ 0.019)
4
2
5.55 (dd, 1H, JHH ¼ 2.0 Hz, JHH ¼ 1.2 Hz, CH), 6.35 (dd, 1H,
and 2454 observed reflections [I ꢅ 2
s (I)], 344 refined parameters,
4JHH ¼ 1.6 Hz, JHH ¼ 1.2 Hz, CH). Anal. calcd for C10H16O2: C, 71.39;
refinement on F2, Robs ¼ 0.0225, Rall ¼ 0.0224, wR (F2) ¼ 0.0621, max
2
H, 9.59. Found: C, 71.65; H, 9.64.
(min) residual electron density Drmax ¼ 0.34 (Drmin ¼ ꢀ0.17) eÅꢀ3
,

718
Ł. Albrecht et al. / European Journal of Medicinal Chemistry 45 (2010) 710–718
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X-ray data collected with Bruker SMART APEX2 CCD area detector
diffractometer. Computer programs used: data collection APEX2
[56], data reduction SAINT-PLUS [57], absorption correction SADABS
[58], structure solution, refinement, and molecular graphics SHELXTL
[59]. All hydrogen atoms werelocated on difference Fourier map and
their positional and isotropic displacement parameters were
allowed to refine without constraints. Disorder of methyl in one of
the ethoxyl groups were satisfactory refined using a two site model
with both site occupancies representing each of the disordered
atoms tied to sum to unity. Crystallographic data (excluding struc-
ture factors) for the structure reported herein, have been deposited
with the Cambridge Crystallographic Data Centre as supplementary
publication CCDC 721865. Copies of the data can be obtained free of
charge on application to The Director, CCDC, 12 Union Road, Cam-
bridge CB2 1EZ, UK. Any request should be accompanied by a full
literature citation.
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Acknowledgments
Financial support from the Ministry of Education and Science
(Project No. 3 T09A 075 28), augmented in part by Medical
University of Lodz (503-30-15-2 to M. R.) and Institute of General
and Ecological Chemistry (JW, WMW) is gratefully acknowledged.
Appendix. Supporting information
of
a-alkylidene-g-butyrolactones and a-alkylidene-g-butyrolactams. in:
O.A. Attanasi, D. Spinelli (Eds.), Targets in Heterocyclic Systems. Italian
Society of Chemistry, Rome, 2006, pp. 301–320.
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