Synthesis and in vitro cytotoxicity of cross-conjugated prostaglandin A and J series and their hydroxy derivatives

Article abstract of DOI:10.1039/c5ob00550g

The synthesis of two cross-conjugated prostaglandin analogues of known neurotrophic activity and their new hydroxy derivatives was accomplished starting from the diastereoisomeric (+)-camphor protected 3-[(dimethoxyphosphoryl)methyl]-4,5-dihydroxycyclopent-2-enones. The cytotoxicity of these compounds was determined against HeLa, K562, HL-60 human cancer cell lines and normal human cells (HUVEC). We found that NEPP11 and its C7-hydroxy derivative demonstrated high anticancer activity against the HeLa and HL-60 human cancer cell lines at concentrations ranging from 1 to 2 μM. Moreover, the C7-hydroxy derivative of NEPP11 displayed high cytotoxic selectivity between cancer cell lines and normal human cells. On the other hand, the J-type prostaglandin analogue of NEPP11 and its C13-hydroxy derivatives were much less toxic or nontoxic against the cancer and normal cells at concentrations up to 1 mM.

Full text of DOI:10.1039/c5ob00550g

Organic &
Biomolecular Chemistry
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PAPER
Synthesis and in vitro cytotoxicity of cross-
conjugated prostaglandin A and J series and
their hydroxy derivatives†
Cite this: Org. Biomol. Chem., 2015,
3, 7000
1
a
a
b
b
Remigiusz Żurawiński,* Marian Mikołajczyk,* Marcin Cieślak, Karolina Królewska
b
and Julia Kaźmierczak-Barańska
The synthesis of two cross-conjugated prostaglandin analogues of known neurotrophic activity and their
new hydroxy derivatives was accomplished starting from the diastereoisomeric (+)-camphor protected
3
-[(dimethoxyphosphoryl)methyl]-4,5-dihydroxycyclopent-2-enones. The cytotoxicity of these com-
pounds was determined against HeLa, K562, HL-60 human cancer cell lines and normal human cells
HUVEC). We found that NEPP11 and its C7-hydroxy derivative demonstrated high anticancer activity
(
against the HeLa and HL-60 human cancer cell lines at concentrations ranging from 1 to 2 μM. Moreover,
the C7-hydroxy derivative of NEPP11 displayed high cytotoxic selectivity between cancer cell lines and
normal human cells. On the other hand, the J-type prostaglandin analogue of NEPP11 and its
C13-hydroxy derivatives were much less toxic or nontoxic against the cancer and normal cells at concen-
trations up to 1 mM.
Received 18th March 2015,
Accepted 15th May 2015
DOI: 10.1039/c5ob00550g
www.rsc.org/obc
nucleophiles, such as the sulfhydryl group of cysteine residues.
While the formation of covalent adducts of cyPGs with gluta-
Introduction
Prostaglandins (PGs) are part of a family of naturally occurring thione (GSH) leads to the loss of their antitumor activity,
biologically active lipid mediators derived from the oxidation binding with intracellular proteins impairs the function of
of polyunsaturated twenty-carbon fatty acids. These labile, but these proteins, thus affecting cellular viability. Among the
highly potent molecules, regulate a broad range of physiologi- known targets for cyPGs there are transcription factors such as
9
10
11
cal processes in animals and humans, including blood circula- NF-κB and AP-1, nuclear receptor PPARγ, and proteins
1
2,13
tion, the contraction and relaxation of smooth muscle tissue, that are involved in the regulation of cellular redox status
renal physiology, the cytoprotection of gastric mucosa, diges- and cytoskeletal proteins.
1
4,15
The cellular mechanism of anti-
1
,2
tion and reproduction.
pathophysiological processes associated with inflammation
and cancer.
They are also involved in many tumor activity of cyPGs is complex, multiple and depends
among other factors on the cell type, cyPG structure and the
On the other hand, various members of the concentration used. The antitumor activity of cyPGs mainly
3
4
,5
cyclopentenone PG family (cyPG), which are characterized by manifests by inducing apoptosis or suppressing the tumor
the presence of the α,β-unsaturated carbonyl moiety, exhibit growth by influence on the expression of genes involved in cell
6
–8
anti-neoplastic, anti-inflammatory and antiviral activities. In growth and cell proliferation, as well as, on stress-induced
6
,8
contrast to other PGs, which exert their effects by binding to genes. Cell cycle arrest is correlated with modulation of cell
1
6
17
G-protein coupled receptors, the cyPGs interact with other cel- cycle regulatory proteins, such as cyclins D1 and B1, cyclin-
1
8
19
lular targets, including signaling molecules and transcription dependent kinases, or cyclin-dependent kinase inhibitors.
factors. These interactions are mainly attributed to the pres- The antitumor activity of cyPGs is inherently connected
ence of the α,β-unsaturated carbonyl group which makes these with the presence of the cyclopentenone backbone and
PGs susceptible to the Michael addition reaction with soft depends on the degree of the olefin–ketone conjugation as
well as on accessibility and the nucleophilic reactivity of the
endocyclic β-carbon atom of the α,β-unsaturated carbonyl
a
moiety. PGs with a cross-conjugated cyclopentadienone system
Department of Heteroorganic Chemistry, Centre of Molecular and Macromolecular
7
12
12,14
Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Łódź, Poland.
like Δ -PGA , Δ -PGJ and Δ
-PGJ (Fig. 1) have consider-
2
1
2
E-mail: remzur@cbmm.lodz.pl, marmikol@cbmm.lodz.pl
ably a higher antitumor activity in comparison with PGs con-
b
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular
20
taining a simple enone unit and substantially lower than
Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Łódź, Poland
†
cross-conjugated dienone punaglandins containing an
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob00550g
electron-withdrawing chlorine atom attached to the α-carbon
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Fig. 3 Structures of the (+)-camphor protected diastereoisomeric
3-[(dimethoxyphosphoryl)methyl]-4,5-dihydroxycyclopent-2-enones 1.
Fig. 1 Structures of cross-conjugated PGs mentioned in the text.
synthesis of bioactive cyclopentenones and cyclopentanones
2
8
using phosphorus reagents, the synthesis of both enantio-
mers of TEI-9826 has been presented based on the diastereo-
isomeric camphor protected 3-[(dimethoxyphosphoryl)methyl]-
2
9
4,5-dihydroxycyclopent-2-enones 1 (Fig. 3).
These chiral cyclopentenone building blocks are easily
available in a two-step reaction sequence, which involves com-
plete desymmetrization of meso-tartaric acid during the acid-
catalyzed reaction with (+)-camphor and methyl orthoformate
and the transformation of the camphor protected dimethyl tar-
trate formed to a separable mixture of the diastereoisomeric 1a
Fig. 2 Order of anticancer activity of PGs and their derivatives.
3
0
and 1b upon treatment with an α-phosphonate carbanion.
Continuing our interest in this field, we report herein a new
approach to the synthesis of the two cross-conjugated
analogues of the prostaglandin A and J series, 2 and 3, respecti-
vely, and their stereo-defined hydroxy derivatives anti-4, syn-5b
and anti-5b, using chiral building blocks 1 as substrates
atom of the α,β-unsaturated carbonyl moiety and the hydroxyl
group at C12 (Fig. 2).
21
The evidence that the cyclopentenone ring with the conju-
gated enone moiety is responsible for the anticancer pro-
perties of cyPGs is given by cyclopent-2-enone itself. This
compound mimics most of the biological activities of
(
Fig. 4).
It was demonstrated that 2 (designated NEPP11) and its
3
1–33
2
2,23
J-type analogue 3 exhibited neurotrophic activities.
They
cyPGs.
It was also shown that it was cytotoxic and pro-
protect HT22 neuronal cells against oxidative glutamate tox-
icity, promote neurite outgrowth from PC12 cells induced by
nerve growth factor, and suppress the manganese-induced
apoptosis of PC12 cells. In spite of a great attention toward the
neuroprotective and neuroregenerative properties of 2 and 3,
no experimental details on their synthesis and information on
the expected anticancer activities have been reported. There-
fore, in this paper we also report the cytotoxicity of these com-
pounds as well as of their new hydroxy derivatives anti-4, syn-
5b and anti-5b, determined against human cervical carcinoma
apoptotic in cancer cells derived from human malignant
2
4
tumors. The structure–activity relationship studies on cyPGs
also revealed that the hydroxyl group at C15 was not required
for a high antitumor activity with both the PGA and PGJ series
2
0
compounds and in the PGA series, inversion of the configur-
ation on the isomerase-sensitive C12 stereocenter from the
natural S to the unnatural R configuration significantly
increases biological stability while maintaining antitumor
2
5
potency. The above observations led to the synthesis of
7
1
3,14-dihydro-15-deoxy-Δ -PGA methyl ester (TEI-9826) which
1
(
HeLa), chronic leukemia (K562) and human acute leukemia
exhibited a highly improved stability in serum in comparison
7
(HL-60) cancer cell lines and normal human umbilical vein
endothelial cells (HUVEC). We have identified compounds that
were toxic against the HeLa, K562 and HL-60 cells and
induced apoptosis. We have also demonstrated that at sub-
lethal doses some of these cyPG analogues induce the for-
mation of aneuploid HeLa cells and arrest cells in the G1 and
G2/M phase of the cell cycle.
1
to Δ -PGA . Lipid microsphere-integrated TEI-9826 has been
shown to be active against human ovarian carcinoma cells in
nude mice and to retain in vivo activity against cisplatin-resist-
2
6
ant tumors. Moreover, this compound displayed a unique
antitumor activity profile by using the COMPARE program
2
7
with 38 tumor cell lines in vitro. Although the use of cyPGs
and their analogues as anticancer drugs is still a matter of the
future, their multiple mechanisms of action makes them
attractive and promising antitumor agents in human chemo-
therapy, especially from the viewpoint of overcoming multi-
drug chemoresistance.
Results and discussion
Synthesis of the target compounds
Recently, in the frame of our research program aimed at Because our strategy devised for the synthesis of the title
the invention and development of general methods for the compounds assumed installation of a suitable ω-chain in the
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Fig. 4 Structures of the target compounds.
Horner olefination reaction (A-type PGs 2 and anti-3) or in the
Having aldehyde 6a in hand, we focused on the synthesis of
aldol condensation (J-type PG derivatives 3, syn-5, anti-5) with NEPP11 (2). The synthetic pathway to this compound is out-
appropriate aldehydes, 5-(4-methylphenyl)pentanal (6a) and lined in Scheme 2. The Horner olefination reaction of phos-
6
-(4-methylphenyl)hexanal (6b) were prepared in a five step phonate 1a and aldehyde 6a carried out in the presence of
reaction sequence starting from 2,3-dihydrofuran (7a) and 3,4- DBU and LiClO gave dienone 12. Hydrogenation of the latter
4
dihydro-2H-pyran (7b), respectively (Scheme 1). Thus, the acid catalyzed by 10%-Pd/C proceeded with complete diastereo-
catalyzed acetalization of 7a and 7b with 2,2-dimethyl-1,3- selectivity under stereocontrol of a chiral diol moiety affording
propanediol, followed by bromination of the hydroxyacetals 13 as a single diastereoisomer with the S configuration at the
formed with phosphorus tribromide in one pot afforded the newly formed stereogenic center. Reductive deacetalization of
3
4
corresponding bromoacetals 8a and 8b. The reaction of 13 with aluminum amalgam in aqueous solution followed by
Grignard reagents generated from with 4-methyl- water elimination from 3-hydroxycyclopentanone 14 produced
8
benzaldehyde gave benzyl alcohols 9 which subsequently were the enantiopure cyclopentenone 15. The aldol condensation of
subjected to the reductive deoxygenation with hydrogen in the 15 with methyl 6-formylhexanoate gave a mixture of anti- and
presence of a 10%-Pd/C catalyst to yield the appropriate satu- syn-aldols 4 in a ratio 5.4 : 1. They were separated by column
rated acetals 10. Because direct acidic deprotection of 10 to the chromatography and the absolute configuration at the C7
corresponding aldehydes did not afford the desired products carbon atom of anti- and syn-aldols 4 was assigned based on
in a satisfactory yield and with high purity, this transformation the value of the coupling constants between protons at C7 and
was accomplished in two steps involving transacetalization of C8, which were found to be 8.4 and 2.8 Hz, respectively. The
1
0 with methanol followed by deacetalization of dimethyl formation of aldol anti-4 as a major isomer can be rationalized
acetals 11 formed with hydrated iron(III) chloride. This pro- by the steric interaction in the chair-like cyclic transition state
cedure allowed us to prepare the desired aldehydes 6a and 6b involving lithium enolate and the aldehyde, and is in agree-
3
5
in very good yields and of satisfactory purity. Therefore, they ment with the observations described in the literature. Both
were used in the next synthetic step without purification.
anti- and syn-aldols 4 were subjected to mesylation with metha-
Scheme 1 Synthesis of aldehydes 6a and 6b.
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Scheme 2 Synthesis of NEPP11 (2).
nesulfonyl chloride and the corresponding anti- and syn-mesy- graphy only for the purpose of their characterization and cyto-
lates 16 formed were treated with neutral aluminum oxide to toxicity determination). The latter were converted in the
afford NEPP11 (2). The identical stereochemical outcome of reaction with methanesulfonyl chloride to the corresponding
the elimination of the mesyloxy group from anti- and syn-mesy- mesylates which upon treatment with neutral aluminum oxide
lates 16 can be explained in terms of the syn elimination gave the J-type PG analogue 3.
(
E1cB) and anti elimination (E2), respectively, as proposed by
3
5
Biological results
Kobayashi.
The J-type PG derivatives 3, syn-5 and anti-5 were syn-
Cytotoxicity. The cytotoxicity of the synthesized cyPG deriva-
thesized using a very similar protocol to that described above tives has been determined against human cancer cell lines
in which the order of installation of side-chains was reversed (HL-60, K562 and HeLa) and human normal cells (HUVEC).
(
Scheme 3). Thus, the introduction of the appropriate substitu- The viability of the cells was determined by the MTT assay at
−
2
ent at the C8 was achieved via the Horner olefination reaction four different prostaglandin concentrations: 1 mM, 1 × 10
of phosphonate 1b with methyl 5-formylpentanoate and the mM, 1 × 10− mM and 1 × 10 mM and IC50 values were calcu-
4
−6
subsequent stereocontrolled reduction of the carbon–carbon lated. We have previously reported that IC50 values obtained
double bond in the dienone 17 formed. The transformation of from 4-point dose–response curves can be reliable and success-
the protected diol moiety into an olefinic bond was accom- ful for the initial screening of cytotoxic compounds.3
6,37
As the
plished in a two-step reaction sequence involving reductive control cells with 100% viability, HeLa cells treated with 1%
deacetalization of 18 with an aluminum amalgam followed by DMSO (vehicle control) were used. Under our experimental
the acid catalyzed dehydration of the resulting hydroxycyclo- conditions, cross-conjugated derivatives of PGA and PGJ series
pentanone 19. The aldol condensation of 20 with 6-(4-methyl- NEPP11 (2) and 3, respectively, displayed comparable or higher
phenyl)hexanal (6b) gave a mixture of diastereoisomeric anti- in vitro cytotoxicity against HL-60 and HeLa cells (IC50 of
and syn-aldols 5 (they were separated by column chromato- 1–8 μM) than the enantiomers of TEI-9826 (IC50 of 5–200 μM),
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Scheme 3 Synthesis of PGJ derivatives 3 and 5.
Table 1 The IC50 values calculated from the dose–response curves
HL-60
K562
HeLa
HUVEC
Compound
IC50 24 h
IC50 48 h
IC50 24 h
IC50 48 h
IC50 24 h
IC50 48 h
IC50 24 h
IC50 48 h
a
(
(
R)-TEI-9826
S)-TEI-9826
80 µM
80 µM
40 µM
>1 mM
>1 mM
>1 mM
>1 mM
5 µM
5 µM
1 µM
nd
nd
nd
1 µM
nd
4 µM
nd
300 µM
80 µM
3 µM
>1 mM
5 µM
>1 mM
>1 mM
200 µM
10 µM
2 µM
8 µM
2 µM
>1 mM
>1 mM
nd
nd
100 µM
nd
>1 mM
nd
100 µM
200 µM
100 µM
>1 mM
>1 mM
>1 mM
>1 mM
nd
4 µM
nd
>1 mM
nd
nd
NEP11 (2)
3
anti-4
syn-5
anti-5
5 µM
>1 mM
>1 mM
>1 mM
nd
nd
a
nd – not determined.
which were used here as references (Table 1). At the same negative effect on the viability of the normal HUVEC cells at
time, 3 was much less toxic towards the normal HUVEC cells, concentrations up to 1 mM.
while cytotoxicity of NEPP11 (2) was comparable to that of
Determination of apoptosis and effects on cell cycle pro-
(R)-TEI-9826. In contrast to a cross-conjugated J-type PG analogue gression. cyPG derivatives 2, 3, anti-4, and (S)-TEI-9828
3
, the single enone derivatives of PGJ series with a hydroxy showing the highest cytotoxicity towards HeLa cells were inves-
substituent at C13 anti-5 and syn-5 were not toxic for any cell tigated for their ability to induce apoptosis. Because increased
line used. On the other hand, a single enone C7-hydroxy activity of caspases is a hallmark of apoptotic processes, the
derivative of PGA series anti-4 efficiently killed K562 and HeLa effector caspases 3 and 7 enzymatic cleavage assay that uses
cells, while being practically non-toxic for HL-60 cells up to a fluorescently labeled synthetic peptide as
a
substrate
concentration of 1 mM. After 48 h incubation in the cell (Z-DEVD-R110) was applied. The amount of fluorescent
culture medium anti-4 displayed high cytotoxicity against product generated is proportional to caspase-3/7 cleavage
HeLa cells with IC50 of 2 μM, which was equal to the cytotoxi- activity and can be quantified by the measurement of fluo-
city of a cross-conjugated dienone 2 and was 5-fold higher rescence of an analyzed sample using a spectrofluorometer.
than that observed for (S)-TEI-9826. Moreover, besides selective Cells exhibiting higher fluorescence values undergo apoptosis
toxicity against the cancer cell lines, anti-4 did not show any to a higher extent. HeLa cells grown in the presence of 1%
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Table 3 Influence of cyPG derivatives on the cell cycle distribution of
diploid HeLa cells. Mean values ± SD from 3 experiments are shown.
cyPG concentration in a cell culture is given in parentheses
%
of diploid
% of diploid
in S phase
% of diploid
in G2/M phase
in G1 phase
HeLa cells
DMSO (0.1%)
52.8 ± 0.6
52.9 ± 6.7
53.9 ± 4.5
51.9 ± 4.8
71.0 ± 7.5
48.7 ± 9.1
57.3 ± 0.9
53.5 ± 0.3
39.2 ± 0.6
36.1 ± 1.7
17.5 ± 6.3
37.0 ± 3.5
5.5 ± 8.8
40.9 ± 8.3
34.7 ± 0.9
38.5 ± 0.3
8.0 ± 0
8.0 ± 0
28.6 ± 1.8
11.1 ± 1.9
23.5 ± 1.4
10.4 ± 0.7
8.0 ± 0
(
R)-TEI-9826 (5 µM)
NEPP11 (2) (0.5 µM)
(5 µM)
3
Fig. 5 Activity of caspase 3/7 in HeLa cells treated with test compounds
for 18 h. Compound concentration: staurosporin 1 µM, NEPP11 (2)
anti-4 (0.5 µM)
syn-5 (5 µM)
anti-5 (5 µM)
1
5 µM, anti-4 25 µM, 3 40 µM, (S)-TEI-9826 50 µM. Mean values ± SD
8.0 ± 0
from 3 experiments are shown.
cycle distribution as compared to the control or DMSO-treated
cells. On the other hand, cross-conjugated PGJ derivative 3
induced accumulation of HeLa cells in the G1 and G2/M phase
of the cell cycle. In the presence of (R)-TEI-9826, we observed
significantly increased accumulation of HeLa cells specifically
in the G2/M phase with no distribution changes in the G1
phase. NEPP11 (2) and its hydroxy derivative anti-4 with the
highest cytotoxicity as determined by the MTT assay, did not
induce significant changes in HeLa cells distribution in both
G1 and G2/M phases of the cell cycle.
DMSO (vehicle) served as a negative control and cells treated
with staurosporin, a strong inducer of programmed cell death,
were used as a positive control. Cells were also incubated with
a given cyPG at the concentration of 5 × IC50 for 18 h. As
shown in Fig. 5, the highest fluorescence values were detected
in the cells which were treated with staurosporin (positive
control) and NEPP11 (2). This observation suggests that deriva-
tive 2 can activate caspases 3 and 7, which in consequence
leads to apoptosis in HeLa cells. On the other hand, we did
not observe any increase in fluorescence in cells treated with 3,
anti-4 or (S)-TEI-9826, which may imply that toxicity of these
compounds towards HeLa cells may result from necrosis.
To determine the influence of the synthesized cyPGs on the
cell cycle, HeLa cells were incubated at sublethal concen-
Conclusion
trations of the tested cyPGs for 24 h and their DNA content In conclusion, a new approach to the synthesis of NEPP11 (2)
was analyzed by FACS. First, we examined whether these cyPGs and its J-type PG analogue 3 has been developed based on the
can induce changes in ploidy of the HeLa cells. We found that diastereoisomeric (+)-camphor protected 3-[(dimethoxyphos-
3
, (R)-TEI-9826, NEPP11 (2) and anti-4 caused significant phoryl)methyl]-4,5-dihydroxycyclopent-2-enones 1. The key
increase in the number of aneuploid HeLa cells (Table 2). steps encompass a fully diastereoselective hydrogenation of
Thus, for example, after 24 h incubation with 3 or (R)-TEI-9826 the endocyclic carbon–carbon double bond in the cyclopente-
we observed approximately 40% of diploid and 60% of aneu- none ring proceeding under control of a chiral diol moiety and
ploid HeLa cells. In contrast, in control or DMSO treated cells the conversion of the latter into a new cyclopentenone with a
we observed 90% of diploid and 10% of tetraploid cells but no transposed olefinic bond. The cytotoxicity of NEPP11 (2) and
aneuploid cells.
its J-type PG analogue 3, as well as their C7- and C13-hydroxy
We have also analyzed cell cycle distribution of the diploid precursors, respectively, were investigated against the HeLa,
HeLa cells treated with the tested cyPGs (Table 3). As expected, K562 and HL-60 human cancer cell lines and the normal
syn-5 and anti-5 did not induce significant changes in cell human cells (HUVEC). NEPP11 (2) and its C7-hydroxy deriva-
tive anti-4 displayed a high anticancer activity against the HeLa
and HL-60 human cancer cell lines at concentrations ranging
from 1 to 2 μM. It was also found that NEPP11 (2) induced
Table 2 Influence of cyPG derivatives on ploidy of HeLa cells. Mean
apoptosis in HeLa cells being relatively nontoxic for the
values ± SD from 3 experiments are shown. cyPG concentration in a cell
normal human cells. Interestingly, the C7-hydroxy derivative of
culture is given in parentheses
NEPP11 anti-4 showed high cytotoxic selectivity between the
%
Diploid
% Tetraploid
% Aneuploid cancer cell lines and the normal human cells, which makes
this compound especially attractive for further investigations.
HeLa cells
DMSO (0.1%)
90.4 ± 1.4
92.6 ± 0.9
39.9 ± 1.7
46.3 ± 1.5
39.8 ± 4.1
62.7 ± 2.5
95.1 ± 0.4
95.7 ± 0.8
9.6 ± 1.4
7.4 ± 0.9
0
0
0
0
0
On the other hand, the J-type prostaglandin analogue of
0
(
R)-TEI-9826 (5 µM)
NEPP11 (2) (0.5 µM)
(5 µM)
60.1 ± 1.7
53.7 ± 1.5
60.2 ± 4.1
37.3 ± 2.5
0
NEPP11 (2), i.e. 3 and its C13-hydroxy derivatives syn-5 and
anti-5 were much less toxic or nontoxic against the cancer and
normal cells at concentrations up to 1 mM. At sublethal doses
NEPP11 (2), 3 and anti-4 induced changes in ploidy of HeLa
cells which manifested in notable increase of aneuploid cells
3
anti-4 (0.5 µM)
syn-5 (5 µM)
anti-5 (5 µM)
4.9 ± 0.4
4.3 ± 0.8
0
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with respect to diploid ones. Additionally, in contrast to 2 H, CArH), 7.14 (d, J = 8.1 Hz, 2 H, CArH), 4.62 (t, J = 6.5 Hz,
NEPP11 (2) and anti-4, compound 3 suppressed the growth of 1 H, CHOH), 4.38 (t, J = 4.9 Hz, 1 H, O–CH–O), 3.58 (d, J =
HeLa cells at the G1 and G2/M phases of the cell cycle.
11.0 Hz, 2 H, OCH
A
H
B
), 3.39 (d, J = 10.8 Hz, 2 H, OCH
A
H
B
),
C),
.71 (s, 3 H, CH C); C NMR (50 MHz, CDCl ): δ 141.85 (C ),
2
0
1
.34 (s, 3 H, CH
3
C
Ar), 1.88–1.19 (m, 9 H), 1.17 (s, 3 H, CH
3
1
3
3
3
Ar
37.06 (CAr), 129.05 (2 C, CArH), 125.82 (2 C, CArH), 102.06
), 74.27 (CHOH), 38.82 (CH ),
Experimental
General remarks
(
O–CH–O), 77.16 (2 C, OCH
2
2
3
4.67 (CH ), 30.09 (C(CH ) ), 25.68 (CH ), 23.76 (CH ), 22.93
2
3 2
2
2
Unless stated otherwise, all air and water sensitive reactions
were carried out under an argon atmosphere using freshly dis-
tilled dry solvents. All glassware was dried prior to use by
heating under vacuum. Commercial grade reagents and sol-
vents were used without further purification except as indi-
cated below. THF and diethyl ether were distilled over Na/
benzophenone prior to use. Thin layer chromatography (TLC)
was conducted on Silica Gel 60 F254 TLC purchased from
Merck. Column chromatography was performed using Merck
silica gel (70–230 mesh). NMR spectra were recorded on
Bruker AV 200, Bruker DRX 500 or Bruker Avance III 600
(
C
CH
3
), 21.80 (CH
3
3
), 21.06 (CArCH ); HRMS (EI) calcd for
18 28 3 28 3
H O 292.2038, found 292.2041; Anal. calcd for C18H O :
C, 73.93; H, 9.65. Found: C, 73.97; H, 9.71.
General procedure for the preparation of acetals 10
A mixture of hydroxyacetal 9 (4.07 mmol) and 10%-Pd/C
0.35 g) in ethanol (40 mL) was vigorously stirred at room
(
temperature under a hydrogen atmosphere for 24 h. Filtration
through a pad of Celite and concentration by rotary evapor-
ation furnished, after column chromatography with 100 : 3
petroleum ether/acetone the corresponding 5,5-dimethyl-2-
substituted-1,3-dioxane.
1
13
31
spectrometers. H, C and P chemical shifts are reported
relative to the residual proton resonance in the deuterated
5,5-Dimethyl-2-((4-methylphenyl)butyl)-1,3-dioxane
(10a).
solvents or referred to an 85% aqueous solution of H PO ,
3
4
Yield 94%. Colorless liquid; Rf = 0.63 (petroleum ether/
1
respectively. All chemical shifts (δ) are given in ppm and the
coupling constants (J) in Hz. HRMS were recorded on
Finnigan MAT 95 apparatus. Optical rotations were measured
using a Perkin-Elmer MC 241 photopolarimeter. Melting and
boiling points are uncorrected.
acetone 40 : 3); H NMR (200 MHz, CDCl ): δ 7.09 (d, J =
3
8
4
.7 Hz, 2 H, CArH), 7.05 (d, J = 8.6 Hz, 2 H, CArH), 4.40 (t, J =
.9 Hz, 1 H, O–CH–O), 3.60 (d, J = 11.0 Hz, 2 H, OCH ), 3.41
A
H
B
(
d, J = 10.8 Hz, 2 H, OCH H ), 2.57 (t, J = 7.5 Hz, 2 H, C CH ),
A
B
Ar
2
2
0
1
.31 (s, 3 H, CArCH
.72 (s, 3 H, CH C); C NMR (50 MHz, CDCl ): δ 139.51 (CAr),
3 3
3
), 1.84–1.33 (m, 6 H), 1.19 (s, 3 H, CH
3
C),
1
3
General procedure for the preparation of hydroxyacetals 9
34.95 (C ), 128.89 (2 C, C H), 128.22 (2 C, C H), 102.13
Ar
Ar
Ar
A mixture of activated magnesium turnings (4.88 g, 201 mmol) (O–CH–O), 77.20 (2 C, OCH
and bromoacetal 8 (67.5 mmol) in THF (80 mL) was heated in (CH ), 30.11 (C(CH
an oil bath at 60 °C for 3 h. After cooling to 0 °C, 4-methyl- 20.94 (C CH ); HRMS (EI) calcd for C H O 262.1933, found
2
C), 35.41 (CH
2
), 34.74 (CH
2
), 31.55
2
) ), 23.73 (CH ), 22.95 (CH ), 21.82 (CH ),
3 2 2 3 3
Ar
3
17 26 2
benzaldehyde (7.68 g, 64 mmol) in THF (5 mL) was added. The 262.1931; Anal. calcd for C17
mixture was stirred for 0.5 h, poured into saturated aqueous C, 77.53; H, 10.12.
28 3
H O : C, 77.82; H, 9.99. Found:
ammonium chloride, extracted with chloroform (4 × 30 mL),
5,5-Dimethyl-2-(5-(4-methylphenyl)pentyl-1,3-dioxane (10b).
= 0.64 (petroleum ether/
of the solvents the crude material was purified by column acetone 40 : 3); H NMR (200 MHz, CDCl ): δ 7.07 (s, 4 H,
chromatography using petroleum ether/acetone 10 : 1 as an CArH), 4.40 (t, J = 4.9 Hz, 1 H, O–CH–O), 3.60 (d, J = 11.0 Hz,
eluent to yield the corresponding hydroxyacetals. 2 H, OCH ), 3.41 (d, J = 10.9 Hz, 2 H, OCH ), 2.56 (t, J =
-(5,5-Dimethyl-1,3-dioxan-2-yl)-1-(4-methylphenyl)butan-1- 7.7 Hz, 2 H, CArCH ), 2.31 (s, 3 H, CArCH ), 1.73–1.23 (m, 8 H),
ol (9a). Yield 87%. Colorless oil; R = 0.35 (petroleum ether/ 1.19 (s, 3 H, CH C), 0.72 (s, 3 H, CH C). C NMR (50 MHz,
and dried over anhydrous sodium sulfate. After evaporation Yield 98%. Colorless liquid; R
f
1
3
A
H
B
A B
H
4
2
3
1
3
f
3
3
1
acetone 5 : 1); H NMR (200 MHz, CDCl
H, CArH), 7.14 (d, J = 8.1 Hz, 2 H, CArH), 4.69–4.57 (m, 1 H, (2 C, CArH), 102.21 (O–CH–O), 77.22 (2 C, OCH
3
): δ 7.23 (d, J = 8.3 Hz, CDCl
3
): δ 139.66 (CAr), 134.92 (CAr), 128.88 (2 C, CArH), 128.24
C), 35.34
2
2
CHOH), 4.40 (t, J = 4.8 Hz, 1 H, O–CH–O), 3.58 (d, J = 11.0 Hz, (CH ), 34.80 (CH ), 31.43 (CH ), 30.13 (C(CH ) ), 29.13 (CH ),
2
2
2
3 2
2
2
H, OCH
A
H
B
), 3.39 (d, J = 10.9 Hz, 2 H, OCH
A
H
B
), 2.33 (s, 3 H, 23.79 (CH
2
), 22.97 (CH
3
), 21.83 (CH
[M − H] 275.2011, found 275.2012;
3
), 20.95 (CArCH
3
). HRMS
+
CH Ar), 1.92–1.32 (m, 7 H), 1.17 (s, 3 H, CH
CH C); C NMR (50 MHz, CDCl ): δ 141.81 (C ), 137.00 (C ), Anal. calcd for C H O : C, 78.21; H, 10.21. Found: C, 78.00;
3
C
3
C), 0.71 (s, 3 H, (EI) calcd for C18
H
27
O
2
1
3
3
3
Ar
Ar
18 28 2
1
29.02 (2 C, CArH), 125.78 (2 C, CArH), 102.01 (O–CH–O), 77.12 H, 10.41.
(
(
(
2 C, OCH ), 74.15 (COH), 38.79 (CH ), 34.48 (CH ), 30.06
2
2
2
General procedure for the preparation of dimethyl acetals 11
A solution of 5,5-dimethyl-2-(substituted)-1,3-dioxane 10
C(CH ) ), 22.92 (CH ), 21.77 (CH ), 21.04 (C CH ), 20.35
3
2
3
3
Ar
3
CH
78.1880; Anal. calcd for C17
C, 73.38; H, 9.50.
2
); HRMS (EI) calcd for
C
17
H
26
O
3
278.1882, found
2
H
26
O
3
: C, 73.34; H, 9.41. Found: (2.33 g, 8.88 mmol) and p-toluenesulfonic acid hydrate (25 mg,
0.133 mmol) in methanol (50 mL) was heated under reflux for
5
-(5,5-Dimethyl-1,3-dioxan-2-yl)-1-(4-methylphenyl)pentan-1- 6 h. After cooling to room temperature, potassium carbonate
ol (9b). Yield 83%. Colorless oil; R = 0.37 (petroleum ether/ was added (30 mg, 0.217 mmol) and methanol was removed by
acetone 5 : 1); H NMR (200 MHz, CDCl ): δ 7.22 (d, J = 8.0 Hz, rotary evaporation. To the residue, water (5 mL) was added and
f
1
3
7006 | Org. Biomol. Chem., 2015, 13, 7000–7012
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the mixture was extracted with petroleum ether (3 × 20 mL). (CHO), 139.19 (CAr), 135.01 (CAr), 128.88 (2 C, CArH), 128.15
The combined organic extracts were dried over Na SO and (2 C, C H), 43.72 (CH CHO), 35.13 (C CH ), 31.22 (CH ),
2
4
Ar
2
Ar
2
2
concentrated under reduced pressure to afford a residue. This 28.63 (CH
2
), 21.82 (CH
2
3
), 20.89 (CArCH ); HRMS (EI) calcd for
residue was additionally treated three times in the same way as
above to achieve full transacetalization. The final dimethyl
acetals obtained were used without further purification in the
next reaction.
13
C H18O 190.1358, found 190.1359.
Preparation of dienone 12
Phosphonate 1a (2.88 g, 7.76 mmol) and lithium perchlorate
1
-(5,5-Dimethoxypentyl)-4-methylbenzene (11a). Yield 97%. (0.83 g, 7.99 mmol) were dissolved in THF (11 mL) and cooled
1
Colorless liquid; H NMR (200 MHz, CDCl
3
): δ 7.10 (d, J = to 0 °C. 1,8-Diazabicyclo-[5.4.0]undec-7-ene (DBU, 1.22 g,
.5 Hz, 2 H, CArH), 7.05 (d, J = 8.7 Hz, 2 H, CArH), 4.36 (t, J = 7.99 mmol) was then added and the mixture was stirred for
.6 Hz, 1 H, O–CH–O), 3.31 (s, 6 H, OCH ), 2.58 (t, J = 7.6 Hz, 15 min. 5-(4-Methylphenyl)pentanal (1.52 g, 8.63 mmol) in
8
5
2
1
1
3
H, CArCH
2
), 2.31 (s, 3 H, CArCH
3
), 1.75–1.52 (m, 4 H), THF (2 mL) was added to the mixture and the resulting solu-
): δ 139.19 (CAr), tion was stirred at 0–5 °C for 3 h. After evaporation of the
1
3
.47–1.28 (m, 2 H); C NMR (50 MHz, CDCl
34.75 (C ), 128.75 (2 C, C H), 128.06 (2 C, C H), 104.23 solvent under reduced pressure, the residue was subjected to
3
Ar
Ar
Ar
(
3
O–CH–O), 52.29 (2 C, OCH
1.29 (CH ), 24.12 (CH ), 20.76 (CArCH
C H O : C, 75.63; H, 9.97. Found: C, 75.53; H, 9.88.
3
), 35.24 (CArCH
2
), 32.17 (CH
2
), column chromatography (petroleum ether/acetone 5 : 1)
2
2
); Anal. calcd for affording 12 (2.80 g, 86%) as a yellowish oil. R = 0.48
3 f
1
9
(petroleum ether/acetone 5 : 1); [α] −102.1 (c 1.5 in CH Cl );
1
4
22
2
D
2
2
1
9
1
1
-(6,6-Dimethoxyhexyl)-4-methylbenzene (11b). Yield 96%. [α]
D
−93.0 (c 1.6 in acetone); H NMR (500 MHz, CDCl
3
):
1
3
Colorless liquid; H NMR (500 MHz, CDCl ): δ 7.09 (d, J = δ 7.09 (d, J = 8.0 Hz, 2 H, CArH), 7.06 (d, J = 8.0 Hz, 2 H, CArH),
8
5
2
1
1
.2 Hz, 2 H, C H), 7.06 (d, J = 8.2 Hz, 2 H, C H), 4.35 (t, J = 6.61 (dt, J = 15.7 Hz, J = 7.2 Hz, 1 H, CHvCHCH ), 6.42 (d, J =
Ar
Ar
2
.7 Hz, 1 H, O–CH–O), 3.31 (s, 6 H, OCH
3
), 2.57 (t, J = 7.7 Hz, 15.7 Hz, 1 H, CHvCHCH
2
), 5.79 (s, 1 H, C(O)CHvC), 5.27 (d,
H, CArCH
2 3
), 2.32 (s, 3 H, CArCH ), 1.65–1.52 (m, 2 H), J = 5.8 Hz, 1 H, C(O)CH–O), 4.34 (d, J = 5.8 Hz, 1 H, C(O)–CH–
1
3
.43–1.28 (m, 2 H); C NMR (125 MHz, CDCl ): δ 139.60 (C ), CH–O), 2.59 (t, J = 7.6 Hz, 2 H, C CH ), 2.32 (s, 3 H, C CH ),
3
Ar
Ar
2
Ar
3
2
34.99 (CAr), 128.90 (2 C, CArH), 128.24 (2 C, CArH), 104.44 2.32–2.24 (m, 2 H, CHvCHCH ), 2.08 (ddd, J = 13.1 Hz, J =
(
(
O–CH–O), 52.56 (2 C, OCH
3
), 35.35 (CH ), 32.36 (CH ), 31.50 4.6 Hz, J = 3.0 Hz, 1 H), 1.93 (ddd, J = 13.2 Hz, J = 9.4 Hz, J =
2
2
CH ), 29.08 (CH ), 24.43 (CH ), 20.98 (C CH ); HRMS (EI) 4.0 Hz, 1 H), 1.75 (t, J = 4.5 Hz, 1 H), 1.72–1.59 (m, 3 H),
2
2
2
Ar
3
+
calcd for C15
H
23
O
2
[M − H] 235.1698, found 235.1696.
1.57–1.47 (m, 3 H), 1.33 (td, J = 12.2 Hz, J = 4.7 Hz, 1 H), 1.20
(
(
ddd, J = 14.3 Hz, J = 7.2 Hz, J = 3.4 Hz, 1 H), 0.95 (s, 3 H, CH
camphor)), 0.82 (s, 3 H, CH (camphor)), 0.61 (s, 3 H, CH3
3
General procedure for the preparation of aldehydes 6
3
1
3
To a solution of appropriate dimethyl acetal (8.54 mmol) in (camphor)); C NMR (126 MHz, CDCl
dichloromethane (120 mL) and acetone (12 mL) at room temp- 168.72 (CvCHC(O)), 145.75 (CHvCHCH
3
): δ 201.19 (CvO),
), 139.12 (CArCH ),
2
2
erature was added iron(III) chloride hexahydrate (8.08 g, 135.17 (C CH ), 128.96 (2 C, C H), 128.23 (2 C, C H), 126.67
Ar
3
Ar
Ar
2
9.9 mmol). The resulting mixture was stirred for 3 h and (CvCHC(O)), 124.76 (CHvCHCH
2
), 124.01 (O–C–O), 77.98
poured into 80 mL of saturated aqueous sodium bicarbonate (C(O)–CH–O), 76.17 (C(O)–CH–CH–O), 51.55 (4° C), 47.83
solution. The aqueous layer was extracted three times with (4° C), 45.14 (CH), 44.55 (CH ), 35.26 (C CH ), 33.45 (CH ),
2
Ar
2
2
chloroform, and the combined organic extracts were washed 31.02 (CH
with brine and dried over anhydrous magnesium sulfate. Con- (CArCH ), 20.22 (CH
centration under reduced pressure yielded the corresponding (CH3 (camphor)); HRMS (EI) calcd for C H O 420.2664,
2
), 29.31 (CH
2
), 28.12 (CH
2
), 26.77 (CH
2
), 20.97
3
3 3
(camphor)), 20.20 (CH (camphor)), 9.37
28
36 3
aldehyde which was used in the next reaction without further found 420.2664; Anal. calcd for C28
purification. Found: C, 79.90; H, 8.47.
-(4-Methylphenyl)pentanal (6a). Yield 100%. Colorless
36 3
H O : C, 79.96; H, 8.63.
5
1
Camphor protected 2,3-dihydroxy-4-(6-(4-methylphenyl)hexyl)-
cyclopentanone (13)
liquid; H NMR (200 MHz, CDCl
3
): δ 9.76 (d, J = 1.6 Hz, 1 H,
CHO), 7.10 (d, J = 9.0 Hz, 2 H, CArH), 7.05 (d, J = 8.9 Hz, 2 H,
C H), 2.60 (t, J = 6.8 Hz, 2 H, C CH ), 2.44 (dt, J = 6.8 Hz, J = A mixture of dienone 12 (3.01 g, 7.16 mmol) and 10%-Pd/C
Ar
Ar
2
1
.7 Hz, 2 H, CH
2
CHO), 2.32 (s, 3 H, CArCH
3
), 1.74–1.58 (m, 4 (0.95 g) in ethanol (50 mL) was vigorously stirred at room
1
3
H); C NMR (50 MHz, CDCl
3
): δ 202.24 (CHO), 138.67 (CAr), temperature under a hydrogen atmosphere for 3.5 h. Filtration
1
35.00 (C ), 128.84 (2 C, C H), 128.07 (2 C, C H), 43.52 through a pad of Celite and concentration by rotary evapor-
Ar
Ar
Ar
(
(
1
CH
2
CHO), 34.98 (CArCH
2
), 30.79 (CH
2
2
), 21.46 (CH ), 20.79 ation furnished, after column chromatography (petroleum
C
ArCH
3
); HRMS (EI) calcd for C12
76.1199.
-(4-Methylphenyl)hexanal (6b). Yield 100%. Colorless (c 1.8 in CH
f
H16O 176.1201, found ether/acetone gradient) 13 (2.38 g, 86%) as a white solid. R =
1
9
0.49 (petroleum ether/acetone 5 : 1); mp 47 °C; [α] +90.6
D
1
9
1
6
2
Cl ); [α]546 +114.6 (c 1.8 in CH
2
2 2
Cl ); H NMR
1
liquid; H NMR (500 MHz, CDCl
3
): δ 9.77 (t, J = 1.4 Hz, 1 H, (500 MHz, CDCl ): δ 7.09 (d, J = 8.1 Hz, 2 H, CArH), 7.07 (d, J =
3
CHO), 7.12 (d, J = 8.0 Hz, 2 H, C H), 7.09 (d, J = 8.0 Hz, 2 H, 8.1 Hz, 2 H, C H), 4.63 (t, J = 4.3 Hz, 1 H, O–CH–CH–C(O)),
Ar
Ar
C
ArH), 2.60 (t, J = 7.7 Hz, 2 H, CArCH
2
), 2.44 (td, J = 7.4 Hz, J = 4.03 (d, J = 5.1 Hz, 1 H, O–CH–C(O)), 2.57 (t, J = 7.8 Hz, 2 H,
1
4
.4 Hz, 2 H, CH
2
CHO), 2.35 (s, 3 H, CArCH
3
), 1.72–1.61 (m,
2 3
CArCH ), 2.32 (s, 3 H, CArCH ), 2.30–2.10 (m, 3 H), 2.00–1.90
1
3
H), 1.43–1.34 (m, 2 H); C NMR (126 MHz, CDCl ): δ 202.70 (m, 2 H), 1.74 (t, J = 4.5 Hz, 1 H), 1.71–1.50 (m, 5 H), 1.48–1.29
3
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1
3
(
A B
m, 8 H), 1.2 (ddd, J = 12.6 Hz, J = 9.5 Hz, J = 4.6 Hz, 1 H), 0.98 2.1 Hz, 1 H, CH H C(O)), 1.70–1.20 (m, 10 H); C NMR
(
3
s, 3 H, CH (camphor)), 0.84 (s, 3 H, CH (camphor)), 0.71 (s, (50 MHz, CDCl ): δ 209.88 (CvO), 168.52 (CHvCHC(O)),
3
3
3
1
3
H, CH
3
(camphor)); C NMR (126 MHz, CDCl
3
): δ 214.36 139.46 (CArCH
2
3
), 134.88 (CArCH ), 133.43 (CHvCHC(O)),
(
CvO), 139.65 (CArCH
2
), 134.99 (CArCH ), 128.90 (2 C, CArH), 128.83 (2 C, CArH), 128.14 (2 C, CArH), 41.34 (CHvCHCH),
3
1
7
28.24 (2 C, C H), 119.85 (O–C–O), 78.65 (O–CH–CH–C(O)), 40.91 (CH C(O)), 35.33 (C CH ), 34.60 (CH ), 31.39 (CH ),
Ar
2
Ar
2
2
2
8.03, (O–CHC(O)), 51.64 (4° C), 47.75 (4° C), 45.15 (CH), 43.62 29.35 (CH
2
), 28.98 (CH
2
), 27.41 (CH
2
), 20.88 (CArCH
3
); HRMS
(
2 2 2 2
CH ), 40.06 (CH ), 35.42 (2 C, CH and CH), 31.53 (CH ), (EI) calcd for C18H24O 256.1827, found 256.1828; Anal. calcd
3
0.10 (CH ), 29.53 (CH ), 29.46 (CH ), 29.11 (CH ), 27.37 for C H O: C, 84.32; H, 9.44. Found: C, 84.09; H, 9.72.
2
2
2
2
18 24
(
9
CH
2
), 26.93 (CH
2
), 20.98 (CArCH
3
), 20.24 (2 C, CH
3
(camphor)),
Preparation of aldols 4
3 40 3
.18 (CH (camphor)); HRMS (EI) calcd for C28H O 424.2977,
found 424.2962; Anal. calcd for C H O : C, 79.20; H, 9.50. To a stirred solution of i-Pr NH (122 mg, 1.21 mmol) in THF
2
8
40
3
2
Found: C, 79.08; H, 9.61.
(10 mL), n-BuLi (0.42 mL, 2.43 M in hexane, 1.02 mmol) was
added at −30 °C under an argon atmosphere. Stirring was con-
tinued for 15 min and the mixture was allowed to warm to
(
(
3R,4S)-3-Hydroxy-4-(6-(4-methylphenyl)hexyl)cyclopentanone
14)
0
°C. The resulting solution of LDA was cooled to −78 °C and
A freshly prepared aluminum amalgam from granular alumi- 15 (238 mg, 0.93 mmol) in THF (2 mL) was added. The
num (1.0 g, 37.1 mmol) and a saturated solution of mercuric mixture was stirred for 10 min and then methyl 6-formyl-
chloride (15 mL) was added to a solution of 13 (567 mg, hexanoate (176 mg, 1.11 mmol) was added. After 30 min at
2
.07 mmol) in 8 : 1 THF/H
2
O (6 ml). Additional portions of the −78 °C, the solution was poured into an ice-cold saturated
aluminum amalgam were added after 10 and 17 h. After being aqueous NH Cl solution. The organic layer was separated and
4
stirred for an additional 7 h, the reaction mixture was filtered the aqueous phase was extracted with CH
through a pad of Celite, concentrated under vacuum, and the combined organic extracts were dried over Na
2
Cl
2
(3 × 30 mL). The
SO , concen-
2
4
crude product was purified by column chromatography (pet- trated under vacuum, and the residue was subjected to flash
roleum ether/acetone gradient) to yield 14 (317 mg, 87%) as a column chromatography (petroleum ether/acetone gradient) to
f
colorless solid. R = 0.27 (petroleum ether/acetone 5 : 1); mp afford anti-4 (287 mg, 75%) and syn-4 (53 mg, 13%) as colorless
1
9
19
5
CH
C
2–53 °C; [α] +98.8 (c 1.7 in CH Cl ); [α]
+98.8 (c 1.7 in liquids. anti-4 (less polar): R = 0.25 (petroleum ether/acetone
D
2
2
546
f
1
22
D
22
2
Cl
2
); H NMR (200 MHz, CDCl
ArH), 7.06 (d, J = 8.7 Hz, 2 H, CArH), 4.47 (br s, J = 7.6 Hz, 1 H, CH
3
): δ 7.10 (d, J = 9.0 Hz, 2 H, 5 : 1); [α]
−88.5 (c 2.3 in CH
); H NMR (500 MHz, CDCl
2
Cl
2
); [α]546 −106.8 (c 2.3 in
): δ 7.68 (dd, J = 5.7 Hz, J =
1
2
Cl
2
3
HOCH), 2.57 (t, J = 7.5 Hz, 2 H, C CH ), 2.32 (s, 3 H, C CH ), 2.4 Hz, 1 H, CHvCHC(O)), 7.09 (d, J = 8.1 Hz, 2 H, C H), 7.06
Ar
2
Ar
3
Ar
1
3
2
.43–2.00 (m, 5 H), 1.72–1.20 (m, 11 H); C NMR (50 MHz, (d, J = 8.1 Hz, 2 H, CArH), 6.13 (dd, J = 5.7 Hz, J = 1.7 Hz, 1 H,
CDCl ): δ 217.66 (CvO), 139.59 (CAr), 134.96 (CAr), 128.88 (2 C, CHvCHC(O)), 3.87 (s, 1 H, OH), 3.72–3.63 (m, 1 H, CHOH),
3
C H), 128.20 (2 C, C H), 70.86 (CHOH), 48.72 (C(O)CH –CH– 3.66 (s, 3 H, OCH ), 2.65–2.59 (m, 1 H, CHvCHCH), 2.56 (t, J =
Ar
Ar
2
3
OH), 42.26 (HO–CH–CH–CH
2
C(O)), 40.88 (HO–CH–CH–CH
2
2 3
C- 7.7 Hz, 2 H, CArCH ), 2.31 (s, 3 H, CArCH ), 2.31 (t, J = 7.5 Hz,
(
2
2 2 2 2 2 2 3
O)), 35.41 (CArCH ), 31.48 (CH ), 29.60 (CH ), 29.38 (CH ), 2 H, CH CO CH ), 2.00 (dd, J = 8.4 Hz, J = 2.1 Hz, 1 H, CH–
1
3
9.11 (CH ), 27.60 (CH ), 20.93 (C CH ); HRMS (EI) calcd for CHC(O)), 1.72–1.27 (m, 18 H); C NMR (126 MHz, CDCl ): δ
2
2
Ar
3
3
C
18
H
26
O
2
274.1933, found 274.1937; Anal. calcd for C18
H
26
O
2
:
212.95 (CvO), 174.19 (CO
2
CH
3
), 168.60 (CHvCHC(O)), 139.49
C, 78.79; H, 9.55. Found: C, 78.87; H, 9.70.
(CArCH ), 135.01 (CArCH ), 132.81 (CHvCHC(O)), 128.89 (2 C,
2
3
C H), 128.19 (2 C, C H), 72.06 (CHOH), 55.86 (CH–CHC(O)),
Ar
Ar
(S)-4-(6-(4-Methylphenyl))hexyl)cyclopent-2-enone (15)
51.44 (OCH
3
), 44.87 (CHvCHCH), 35.37 (CH
2
), 35.29 (CArCH
2
),
A solution of 14 (284 mg, 1.03 mmol) and p-toluenesulfonic 33.94 (CH ), 33.80 (CH ), 31.46 (CH ), 29.55 (CH ), 29.04
2 2 2 2
acid hydrate (29 mg, 0.16 mmol) in diethyl ether (10 mL) was (CH ), 29.00 (CH ), 26.80 (CH ), 25.05 (CH ), 24.84 (CH ),
2
2
2
2
2
stirred at room temperature for 24 h. The reaction mixture was 20.95 (CArCH
3
); HRMS (EI) calcd for C26
38 4
H O 414.2770, found
poured into diethyl ether and saturated aqueous NaHCO . The 414.2776; Anal. calcd for C26H O : C, 75.32; H, 9.24. Found:
3 38 4
layers were separated, and the aqueous phase was extracted C, 75.27; H, 9.31. syn-4 (more polar): R = 0.18 (petroleum
f
2
2
22
several times with CHCl
dried over anhydrous Na
pressure. Flash column chromatography using 50 : 1 petroleum 5.7 Hz, J = 2.4 Hz, 1 H, CHvCHC(O)), 7.09 (d, J = 8.1 Hz, 2 H,
ether/acetone as the eluent yielded 15 (241 mg, 91%) as a ArH), 7.06 (d, J = 8.1 Hz, 2 H, CArH), 6.13 (dd, J = 5.7 Hz, J =
colorless liquid. R = 0.37 (petroleum ether/acetone 20 : 3); 1.8 Hz, 1 H, CHvCHC(O)), 4.03 (br s, 1 H, CHOH), 3.66 (s,
3
. The combined organic extracts were ether/acetone 5 : 1); [α]
D
−81.0 (c 0.9 in CH
2
Cl
2
); [α]546 −95.0
1
2
SO
4
and concentrated under reduced (c 0.9 in CH
2
Cl
2
); H NMR (500 MHz, CDCl
3
): δ 7.69 (dd, J =
C
f
2
1
21
546
[
α] −100.2 (c 1.7 in CH Cl ); [α]
−121.0 (c 1.7 in CH Cl ); 3 H, OCH ), 2.88–2.81 (m, 1 H, CHvCHCH), 2.56 (t, J = 7.6 Hz,
D
2
2
2
2
3
1
H NMR (200 MHz, CDCl
3
): δ 7.62 (dd, J = 5.6 Hz, J = 2.5 Hz, 2 H, CArCH
H, CHvCHC(O)), 7.10 (d, J = 9.0 Hz, 2 H, CArH), 7.05 (d, J = CH CO CH
.7 Hz, 2 H, C H), 6.14 (dd, J = 5.6 Hz, J = 2.0 Hz, 1 H, CH-CHC(O)), 1.70–1.26 (m, 18 H). C NMR (126 MHz, CDCl3):
2
), 2.32 (s, 3 H, CArCH
3
), 2.31 (t, J = 7.4 Hz, 2 H,
1
8
2
2
3
), 2.30–2.25 (m, 1 H, OH), 2.13 (t, J = 2.8 Hz, 1 H,
1
3
Ar
CHvCHC(O)), 3.00–2.81 (m, 1 H, CHvCHCH), 2.57 (t, J = δ 211.99 (CvO), 174.15 (CO ), 169.07 (CHvCHC(O)),
2
CH
3
7
.5 Hz, 2 H, CArCH ), 2.47 (dd, J = 18.8 Hz, J = 6.3 Hz, 1 H, 139.51 (CArCH ), 134.99 (CArCH ), 133.37 (CHvCHC(O)),
2
2
3
CH H C(O)), 2.32 (s, 3 H, CH ), 1.98 (dd, J = 18.8 Hz, J = 128.89 (2 C, C H), 128.19 (2 C, C H), 71.18 (CHOH), 56.80
A
B
3
Ar
Ar
7008 | Org. Biomol. Chem., 2015, 13, 7000–7012
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Paper
(
(
(
3
CH–CHC(O)), 51.46 (OCH ), 42.95 (CHvCHCH), 35.38 General procedure for the preparation of 15-deoxy-13,14-
7
C CH ), 34.26 (CH ), 34.16 (CH ), 33.90 (CH CO CH ), 31.47 dihydro-19,20-dinor-12-iso-18-(4-methylphenyl)-Δ -PGA
Ar
2
2
2
2
2
3
1
CH
2
), 29.57 (CH
2
), 29.06 (CH
2
), 28.88 (CH
). HRMS (EI) calcd for
C H O 414.2770, found 414.2782; Anal. calcd for C H O :
2 2
), 27.13 (CH ), methyl ester (NEPP11) (2)
2
2 2 3
5.85 (CH ), 24.78 (CH ), 20.95 (CArCH
A suspension of a syn- or anti-mesylate 16 (47 mg, 0.095 mmol)
2
6
38
4
26 38 4
and neutral aluminum oxide (80 mg) in dichloromethane
C, 75.32; H, 9.24. Found: C, 75.20; H, 9.37.
(
2.5 mL) was stirred at room temperature for 24 h (additional
equal portions of Al O were added after 2 and 6 h). The reac-
2
3
tion mixture was filtered through a pad of Celite, concentrated,
and the residue was purified by column chromatography to
afford the corresponding alkene.
General procedure for the preparation of mesylates 16
To an ice-cold solution of a syn- or anti-aldol 4 (0.27 mmol)
and Et N (273 mg, 2.70 mmol) in CH Cl (5 mL) was added
3
2
2
From anti-16. Yield 93%. Colorless liquid; R
petroleum ether/acetone 5 : 1); [α]D −127.1 (c 1.6 in CH Cl );
f
= 0.31
methanesulfonyl chloride (217 mg, 1.89 mmol). Stirring was
continued for 2 h at the same temperature and the solution
was poured into saturated aqueous NaHCO3 solution. The
mixture was extracted with dichloromethane and the com-
bined organic layers were dried over anhydrous sodium
sulfate. After concentration in vacuo, the residue was subjected
to flash chromatography (petroleum ether/acetone 5 : 1) to
afford a corresponding mesylate.
22
(
2
2
1
H NMR (500 MHz, CDCl ): δ 7.51 (dd, J = 5.8 Hz, J = 2.2 Hz,
3
1
8
H, CHvCHCO), 7.08 (d, J = 8.0 Hz, 2 H, CArH), 7.05 (d, J =
.0 Hz, 2 H, C H), 6.52 (t, J = 7.6 Hz, 1 H, CvCHCH ), 6.32
Ar
2
(
dd, J = 6.0 Hz, J = 1.6 Hz, 1 H, CHvCHC(O)), 3.66 (s, 3 H,
OCH ), 3.49–3.41 (m, 1 H, CH), 2.54 (t, J = 7.7 Hz, 2 H,
C CH ), 2.31 (t, J = 7.4 Hz, 2 H, CH CO CH ), 2.31 (s, 3 H,
3
Ar
2
2
2
3
C CH ), 2.30–2.18 (m, 2 H, CvCHCH ), 1.83–1.73 (m, 1 H),
Ar
3
2
anti-16. Yield 97%. Colorless liquid; R = 0.20 (petroleum
13
f
1
.70–1.18 (m, 15 H); C NMR (126 MHz, CDCl
3
): δ 196.87
2
4
1
ether/acetone 5 : 1); [α]
D
−52.2 (c 1.4 in CH
2 2
Cl ); H NMR
(
CvO), 173.96 (CO CH ), 161.86 (CHvCHC(O)), 139.51
2
3
(
500 MHz, CDCl ): δ 7.67 (dd, J = 5.7 Hz, J = 2.4 Hz, 1 H,
3
(
C CH ), 138.01 (HCvCC(O)), 135.16 (CvCHCH ), 134.95
Ar
2
2
CHvCHC(O)), 7.07 (d, J = 8.2 Hz, 2 H, C H), 7.05 (d, J = 8.2
Ar
(
CArCH
3
), 134.75 (CHvCHC(O)), 128.86 (2 C, CArH), 128.18
Hz, 2 H, CArH), 6.10 (dd, J = 5.7 Hz, J = 1.8 Hz, 1 H, CHvCHC-
(
2 C, C H), 51.42 (OCH ), 43.22 (CHvCHCH), 35.37 (C CH ),
Ar 3 Ar 2
(O)), 4.98 (dt, J = 8.6 Hz, J = 4.1 Hz, 1 H, CHOMs), 3.65 (s, 3 H,
3
2
3.83 (CH CO CH ), 32.37 (CH ), 31.43 (CH ), 29.60 (CH ),
2
2
3
2
2
2
OCH ), 2.99 (s, 3 H, CH S), 2.97–2.92 (m, 1 H, CH–CHC(O)),
3
3
9.05 (CH
2
), 28.83 (CH
2
), 28.82 (CH
2
), 28.27 (CH
2
), 25.72
2
C
.58–2.51 (m, 3 H, CArCH
2
and CH–CHC(O)), 2.30 (s, 3 H,
CO CH ), 1.81–1.70 (m, 1
H), 1.66–1.17 (m, 17 H); C NMR (126 MHz, CDCl ): δ 206.88
(
CH ), 24.66 (CH ), 20.91 (C CH ); Anal. calcd for C H O :
2 2 Ar 3 26 36 3
ArCH ), 2.29 (t, J = 7.5 Hz, 2 H, CH
3
2
2
3
C, 78.75; H, 9.15. Found: C, 78.82; H, 9.30.
From syn-16. Yield 84%. Colorless liquid; [α]
in CH Cl ).
1
3
21
D
3
−126.5 (c 0.8
(
(
CvO), 173.93 (COOCH
3
), 168.18 (CHvCHC(O)), 139.48
2
2
CArCH
2
), 134.89 (CArCH ), 133.11 (CHvCHC(O)), 128.82 (2 C,
3
C H), 128.16 (2 C, C H), 82.18 (CHOMs), 54.45 (CH–CHC(O)),
Ar
Ar
5
1.41 (OCH
3
), 43.86 (CHvCHCH), 38.32 (CH
3
S), 35.30
(
+)-Camphor protected methyl 7-((4R,5R)-4,5-dihydroxy-3-
(CArCH ), 34.14 (CH ), 33.72 (CH ), 31.39 (CH ), 30.70 (CH ),
2 2 2 2 2
oxocyclopent-1-en-1-yl)hept-6-enoate (17)
2
9.41 (CH ), 28.95 (CH ), 28.50 (CH ), 27.04 (CH ), 25.42
2
2
2
2
(
CH
2
), 24.49 (CH
2
), 20.88 (CArCH
3
); HRMS (EI) calcd for According to the procedure described for 12 phosphonate 1b
27 40 6
C H O 492.2546, found 492.2533. (1.95 g, 5.26 mmol) was transformed into dienone 17 (1.71 g,
syn-16. Yield 92%. Colorless liquid; R = 0.13 (petroleum 84%). Pale yellow oil; R = 0.41 (petroleum ether/acetone 5 : 1);
f
f
2
2
1
24
D
1
ether/acetone 5 : 1); [α]
(
D
−37.9 (c 1.0 in CH
2
Cl
2
); H NMR [α]
+99.3 (c 1.9 in CH
2
Cl
2
); H NMR (500 MHz, CDCl
), 6.41 (d, J = 15.8
CHvCHC(O)), 7.08 (d, J = 8.1 Hz, 2 H, C H), 7.06 (d, J = 8.1 Hz, 1 H, CHvCHCH ), 5.77 (s, 1 H, CvCHC(O)), 5.10 (d, J =
3
): δ 6.59
3 2
500 MHz, CDCl ): δ 7.73 (dd, J = 5.7 Hz, J = 2.4 Hz, 1 H, (dt, J = 15.7 Hz, J = 7.0 Hz, 1 H, CHvCHCH
Ar
2
Hz, 2 H, CArH), 6.17 (dd, J = 5.7 Hz, J = 1.8 Hz, 1 H, CHvCHC- 5.8 Hz, 1 H, C(O)CH–O), 4.42 (d, J = 5.8 Hz, 1 H, C(O)CH–CH–
O)), 5.03 (dt, J = 7.3 Hz, J = 2.6 Hz, 1 H, CHOMs, 3.66 (s, 3 H, O), 3.62 (s, 3 H, OCH ), 2.30 (t, J = 7.4 Hz, 2 H, CH COOCH ),
(
3
2
3
CH O), 3.07–3.00 (m, 1 H, CHvCHCH), 2.85 (s, 3 H, CH S), 2.25 (q, J = 7.3 Hz, 2 H), 2.05 (ddd, J = 13.1 Hz, J = 4.3 Hz, J =
3
3
2
.56 (t, J = 7.7 Hz, 2 H, CArCH
2
), 2.31 (t, J = 7.5 Hz, 2 H, 3.3 Hz, 1 H), 1.79–1.58 (m, 5 H), 1.56–1.41 (m, 3 H), 1.27 (td,
CH
CH–CHC(O)), 2.05–1.95 (m, 1 H), 1.80–1.23 (m, 17 H);
NMR (126 MHz, CDCl ): δ 208.41 (CvO), 173.94 (CO CH
68.87 (CHvCHC(O)), 139.44 (CArCH ), 135.03 (CArCH
2 2 3 3
CO CH ), 2.31 (s, 3 H, CArCH ), 2.22 (t, J = 2.6 Hz, 1 H, J = 12.2 Hz, J = 4.7 Hz, 1 H), 1.17–1.11 (m, 1 H), 0.94 (s, 3 H,
1
3
13
C
CH ), 0.77 (s, 3 H, CH ), 0.58 (s, 3 H, CH ); C NMR (126 MHz,
3
3
3
3
2
3
3
), CDCl
), (O)), 144.88 (CHvCHCH
3
): δ 201.42 (CvO), 173.74 (CO
2
3
CH ), 168.16 (CvCHC-
1
1
8
2
2
), 126.96 (CvCHC(O)), 124.86
32.89 (CHvCHC(O)), 128.90 (2 C, C H), 128.19 (2 C, C H), (CHvCHCH ), 124.02 (O–C–O), 77.91 (C(O)CH–O), 75.93
Ar
Ar
2
2.21 (CHOMs), 53.50 (CH–CHC(O)), 51.49 (OCH
3
), 42.60 (C(O)CH–CH–O), 51.56 (4° C), 51.41 (OCH
), 33.78 (CH), 44.51 (CH ), 33.62 (CH ), 33.06 (CH
3
), 47.67 (4° C), 45.09
), 29.65 (CH ), 27.72
(
(
(
CHvCHCH), 38.00 (CH
3
S), 35.35 (CArCH
2
), 34.17 (CH
2
2
2
2
2
CH ), 33.59 (CH CO CH ), 31.43 (CH ), 29.52 (CH ), 29.03 (CH ), 26.79 (CH ), 24.29 (CH ), 20.37 (CH ), 20.13 (CH ), 9.23
2
2
2
3
2
2
2
2
2
3
3
CH
2
), 28.58 (CH
2
), 26.68 (CH
2
), 25.15 (CH
2
), 24.58 (CH
2
), (CH
3
); HRMS (EI) calcd for
C
23
H
32
O
5
388.2249, found
2
4
0.94 (CArCH
92.2549.
3
); HRMS (EI) calcd for C27
H
40
O
6
492.2546, found 388.2241; Anal. calcd for C23
C, 70.98; H, 8.47.
32 5
H O : C, 71.11; H, 8.30. Found:
This journal is © The Royal Society of Chemistry 2015
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Organic & Biomolecular Chemistry
(
4
+)-Camphor protected methyl 7-((1R,2R,3R)-2,3-dihydroxy-
-oxocyclopentyl)heptanoate (18)
Preparation of aldols 5
Aldol condensation of 20 (127 mg, 0.57 mmol) with 6-(4-
The hydrogenation reaction of dienone 17 (1.70 g, 4.38 mmol) methylphenyl)hexanal (6b) (124 mg, 0.65 mmol) was per-
was performed according to the procedure described for 13 formed according to the procedure described for 4 to afford
and gave saturated cyclopentanone 18 (1.59 g, 92%) as a color- anti-5 (82 mg, 35%) and syn-5 (14 mg, 6%) as colorless oils.
2
0
less oil. R
(
f
= 0.47 (petroleum ether/acetone 5 : 1); [α]
D
f
−115.4 anti-5 (less polar): R = 0.23 (petroleum ether/acetone 20 : 3);
1
24
1
c 1.6 in CH Cl ); H NMR (200 MHz, CDCl ): δ 4.47 (dd, J = [α]D +82.6 (c 1.1 in CH Cl ); H NMR (500 MHz, CDCl ): δ 7.68
2 2 3 2 2 3
4
.6 Hz, J = 3.4 Hz, 1 H, O–CH–CHC(O)), 4.16 (d, J = 5.1 Hz, 1 H, (dd, J = 5.7 Hz, J = 2.4 Hz, 1 H, CHvCHC(O)), 7.08 (d, J =
), 2.32 (t, J = 7.4 Hz, 2 H, 8.0 Hz, 2 H, CArH), 7.06 (d, J = 8.2 Hz, 2 H, CArH), 6.14 (dd, J =
CH CO CH ), 2.24–2.12 (m, 2 H), 2.04 (ddd, J = 13.0 Hz, J = 4.3 5.7 Hz, J = 1.7 Hz, 1 H, CHvCHC(O)), 3.82 (s, 1 H, OH), 3.67
CHC(O)), 3.67 (s, 3 H, OCH
3
2
2
3
Hz, J = 3.3 Hz, 1 H), 1.94–1.78 (m, 1 H), 1.78–1.52 (m, 7 H), (s, 3 H, OCH
3
), 3.69–3.61 (m, 1 H, CHOH), 2.66–2.60 (m, 1 H,
1
0
.39 (d, J = 12.4 Hz, 8 H), 1.25–1.06 (m, 1 H), 0.98 (s, 3 H, CH ), CHvCHCH), 2.57 (t, J = 7.6 Hz, 2 H, C CH ), 2.31 (s, 3 H,
3
Ar
2
1
3
.83 (s, 3 H, CH ), 0.70 (s, 3 H, CH ); C NMR (50 MHz, C CH ), 2.31 (t, J = 7.4 Hz, 2 H, CH CO CH ), 2.00 (dd, J =
3
3
Ar
3
2
2
3
CDCl
3
2 3
): δ 214.01 (CvO), 173.97 (CO CH ), 120.23 (O–C–O), 8.4 Hz, J = 2.1 Hz, 1 H, CH–CHC(O)), 1.70–1.26 (m, 18 H);
1
3
7
9.84 (O–CH–CHC(O)), 77.37 (CHC(O)), 51.54 (4° C), 51.24
C NMR (126 MHz, CDCl ): δ 212.92 (CvO), 174.17 (CO CH ),
3 2 3
(
OCH ), 47.70 (4° C), 45.01 (CH), 44.08 (CH ), 39.78 (CH ), 168.50 (CHvCHC(O)), 139.60 (C CH ), 134.96 (C CH ),
3
2
2
Ar
2
Ar
3
3
2
5.31 (CH), 33.82 (CH
2
), 29.94 (CH
2
), 29.60 (CH
2
), 29.11 (CH
2
), 132.89 (CHvCHC(O)), 128.89 (2 C, CArH), 128.23 (2 C, CArH),
8.76 (CH ), 27.21 (CH ), 26.61 (CH ), 24.65 (CH ), 20.28 72.16 (CHOH), 55.91 (CH–CHC(O)), 51.50 (OCH ), 44.86
2
2
2
2
3
(
CH ), 19.91 (CH ), 9.52 (CH ); HRMS (EI) calcd for C H O
(CHvCHCH), 35.44 (CH ), 35.37 (CH ), 33.93 (CH ), 33.75
3
3
3
23 36
5
2
2
2
3
92.2563, found 392.2560; Anal. calcd for C23
H
36
O
5
: C, 70.38; (CH
2
), 31.53 (CH
2
), 29.32 (CH
2
), 29.17 (CH
2
2
), 28.91 (CH ),
H, 9.24. Found: C, 70.36; H, 9.20.
26.69 (CH ), 25.31 (CH ), 24.75 (CH ), 20.96 (C CH ); Anal.
2 2 2 Ar 3
calcd for C H O : C, 75.32; H, 9.24. Found: C, 75.25; H, 9.15.
2
6
38 4
f
syn-5 (more polar): R = 0.16 (petroleum ether/acetone 20 : 3);
Methyl 7-((1R,2S)-2-hydroxy-4-oxocyclopentyl)heptanoate (19)
The reductive deacetalization reaction of 18 (258 mg,
2
4
1
[α] +71.5 (c 0.6 in CH Cl ); H NMR (500 MHz, CDCl ): δ 7.70
D 2 2 3
(
dd, J = 5.4 Hz, J = 2.1 Hz, 1 H, CHvCHC(O)), 7.08 (d, J =
0
.658 mmol) with the aluminum amalgam was carried out
7
5
3
.6 Hz, 2 H, CArH), 7.06 (d, J = 7.3 Hz, 2 H, CArH), 6.14 (dd, J =
.3 Hz, J = 1.1 Hz, 1 H, CHvCHC(O)), 4.03 (br s, 1 H, CHOH),
according to the procedure described for 14 to afford 19
f
(142 mg, 89%) as a colorless solid. R = 0.25 (petroleum ether/
.66 (s, 4 H, OH and OCH ), 2.84 (br s, 1 H, CHvCHCH), 2.56
3
2
9
1
acetone 5 : 1); mp 49–50 °C; [α]
D
2 2
−110.1 (c 1.4 in CH Cl ); H
(
t, J = 7.4 Hz, 2 H, CArCH
2 3
), 2.31 (s, 3 H, CArCH ), 2.30 (t, J =
NMR (200 MHz, CDCl ): δ 4.47 (s, 1 H, CHOH), 3.67 (s, 3 H,
3
7.4 Hz, 2 H, CH CO CH ), 2.13 (dd, J = 2.7 Hz, J = 2.0 Hz, 1 H,
2
2
3
1
3
CO
2 3 2 2 3
CH ), 2.37 (t, J = 4.0 Hz, 2 H, CH CO CH ), 2.29 (d, J = 7.5
CH–CHC(O)), 1.77–1.18 (m, 18 H); C NMR (126 MHz, CDCl3):
δ 212.02 (CvO), 174.18 (CO CH ), 168.98 (CHvCHC(O)),
139.51 (C CH ), 135.00 (C CH ), 133.45 (CHvCHC(O)),
Hz, 2 H), 2.23–2.03 (m, 2 H), 1.71–1.50 (m, 6 H), 1.44–1.26 (m,
2
3
1
3
6
H); C NMR (50 MHz, CDCl ): δ 217.66 (CvO), 174.31
3
Ar
2
Ar
3
(
CO
2
CH
3
), 70.76 (CHOH), 51.45 (OCH
3
), 48.77 (C(O)-
CHCH ),
1
28.90 (2 C, C H), 128.22 (2 C, C H), 71.33 (CHOH), 56.78
Ar
Ar
CH
2
CHOH), 42.31 (C(O)CH
2
CHCH ), 40.92 (C(O)CH
2
2
2
(
CH–CHC(O)), 51.49 (OCH
3
2
), 42.93 (CHvCHCH), 35.35 (CH ),
3
3.95 (CH ), 29.29 (2 C CH ), 28.89 (CH ), 27.39 (CH ), 24.71
2
2
2
2
34.35 (CH ), 34.21 (CH ), 33.95 (CH CO CH ), 31.50 (CH ),
2 2 2 2 3 2
(
CH
2
22 4
); Anal. calcd for C13H O : C, 64.44; H, 9.15. Found: C,
2
9.34 (CH ), 29.05 (CH ), 28.93 (CH ), 27.02 (CH ), 26.11
2
2
2
2
6
4.38; H, 9.28.
(
CH
2
), 24.78 (CH
2
3 38 4
), 20.96 (CArCH ); Anal. calcd for C26H O :
C, 75.32; H, 9.24. Found: C, 75.21; H, 9.11.
Methyl (R)-7-(4-oxocyclopent-2-en-1-yl)heptanoate (20)
1
5-Deoxy-13,14-dihydro-19,20-dinor-18-(4-methylphenyl)-
The dehydration of 19 (132 mg, 0.54 mmol) was carried out in
accordance with the procedure applied for 15 to afford 20
1
2
Δ -PGJ methyl ester (3)
1
(
112 mg, 92%) as a colorless liquid. R = 0.38 (petroleum ether/ To an ice-cold solution of syn- and anti-aldol 5 (68 mg,
f
2
3
1
acetone 7 : 1); [α]
D
2 2 3 2 2
+111.8 (c 1.7 in CH Cl ); H NMR 0.164 mmol) and Et N (166 mg, 1.64 mmol) in CH Cl (2 mL)
(
200 MHz, CDCl
CHvCHC(O)), 6.14 (dd, J = 5.6 Hz, J = 2.0 Hz, 1 H, CHvCHC- Stirring was continued for 2 h at the same temperature and
O)), 3.67 (s, 3 H, OCH ), 2.98–2.84 (m, 1 H, CHCH ), 2.53 (dd, the solution was poured into saturated aqueous NaHCO solu-
J = 18.8 Hz, J = 6.3 Hz, 1 H, C(O)CH ), 2.31 (t, J = 7.4 Hz, 2 tion. The mixture was extracted with dichloromethane and the
H, CH CO Me), 1.99 (dd, J = 18.8 Hz, J = 2.1 Hz, 1 H, C(O)- combined organic layers were dried over anhydrous sodium
3
): δ 7.63 (dd, J = 5.6 Hz, J = 2.5 Hz, 1 H, was added methanesulfonyl chloride (131 mg, 1.15 mmol).
(
3
2
3
A
H
B
2
2
1
3
CH
2
A
H
B
), 1.70–1.25 (m, 10 H); C NMR (126 MHz, CDCl
3
): δ sulfate. After concentration in vacuo, the residue was dissolved
Cl (2 mL) and neutral Al (1.2 g) was added in three
10.10 (CvO), 174.15 (CO
2
CH ), 168.63 (CHvCHC(O)), 133.52 in CH
3
2
2
2 3
O
(
CHvCHC(O)), 51.43 (OCH ), 41.37 (CHCH ), 40.94 (CH– equal portions after 3 and 6 h. The reaction mixture was
3
2
CH
2
C(O)), 34.56 (CH
), 27.34 (CH
24.1412, found 224.1410.
2
), 33.91 (CH
2
CO
2
2
Me), 29.15 (CH ), 28.88 stirred for 24 h at room temperature, filtered through a pad of
(
CH
2
2
), 24.74 (CH
2
); HRMS (EI) calcd for C13
H
20
O
3
Celite, concentrated, and the residue was purified by column
2
chromatography to afford 3 (57 mg, 87%) as a colorless liquid.
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2
1
3
R
f
= 0.31 (petroleum ether/acetone 5 : 1); [α]
D
+119.7 (c 1.4 in were used. 20 × 10 cells were seeded in each well of a 96-well
1
CH Cl ); H NMR (600 MHz, CDCl ): δ 7.52 (ddd, J = 6.0 Hz, J = plate. After 24 h the cells were exposed to the cyPGs at conc. of
2
2
3
2
.6 Hz, J = 0.7 Hz, 1 H, CHvCHC(O)), 7.08 (d, J = 7.9 Hz, 2 H, 5 × IC50 for another 18 h. The cells were also exposed to 1%
ArH), 7.06 (d, J = 8.1 Hz, 2 H, CArH), 6.54 (t, J = 7.7 Hz, 1 H, DMSO (in order to check if the reagent used to dissolve the
CvCHCH ), 6.32 (dd, J = 6.0 Hz, J = 1.8 Hz, 1 H,CHC(O)), 3.66 test compounds has any effect on the induction of apoptosis)
C
2
(
C
s, 3 H, OCH
), 2.31 (s, 3 H, CArCH
CH CO CH ), 2.27–2.19 (m, 2 H, CvCHCH ), 1.84–1.76 (m, sis was analyzed by the Apo-ONE® Homogeneous Caspase-3/7
3
), 3.46–3.43 (m, 1 H, CH), 2.56 (t, J = 7.7 Hz, 2 H, and 1 μM staurosporin (Sigma, St. Louis, MO) which is a
ArCH
2
3
), 2.29 (t, J = 7.5 Hz, 2 H, strong inducer of cell apoptosis. The induction of cell apopto-
2
2
3
2
1
H), 1.65–1.57 (m, 5 H), 1.55–1.47 (m, 3 H), 1.41–1.21 (m, 7 Assay (Promega, Madison, WI, USA). After 18 h of incubation
13
H); C NMR (151 MHz, CDCl
3
): δ 196.93 (CvO), 174.13 with the test compounds, the cells were treated with the
(
(
(
(
(
(
CO CH ), 161.77 (CHvCHC(O)), 139.42 (C CH ), 137.84 caspase-3/7 reagent (according to manufacturer’s instructions)
HCvCC(O)), 135.63 (CvCHCH ), 135.07 (CArCH ), 134.86 and incubated for additional 1.5 hours at room temperature.
2 3
2
3
Ar
2
CHvCHC(O)), 128.95 (2 C, CAr–H), 128.23 (2 C, CArH), 51.45 The fluorescence of wells was measured at the excitation wave-
OCH3), 43.24 (CHvCHCH), 35.35 (C CH ), 33.97 length 485 nm and emission wavelength 520 nm using the
Ar
2
CH
CH
2
2
CO
2
CH
3
), 32.37 (CH
2
), 31.41 (CH
2
), 29.43 (CH
2
), 29.04 microplate reader FLUOStar Omega (BMG-Labtech, Germany).
),
), 29.03 (CH
2
), 28.97 (CH
2
), 28.55 (CH
2
), 25.67 (CH
2
2
4.80 (CH ), 20.96 (C CH ); Anal. calcd for C H O : C, 78.75;
2 Ar 3 26 36 3
Cell cycle analysis
H, 9.15. Found: C, 78.66; H, 9.10.
HeLa cells were seeded in a 6-well plate at a density of 3.2 ×
5
Cells and cytotoxicity assays
10 cells per well. 24 hours later the cells were treated with a
given PG derivative for additional 24 h. After treatment, the
Human umbilical vein endothelial cells (HUVEC) were isolated
2
+
2+
38
cells were washed with ice cold PBS (free of Ca and Mg ),
trypsinized, collected and fixed with 70% ethanol at −20 °C
overnight. Directly before the flow cytometry assay the cells
were washed again with the ice cold PBS and treated with
from freshly collected umbilical cords as previously described,
and cultured in plastic dishes coated with gelatin, in RPMI
640 medium supplemented with 20% FBS (fetal bovine
1
−1
−1
serum), 90 U ml heparin, 150 µg ml ECGF (Endothelial Cell
Growth Factor, Roche Diagnostics, Mannheim, Germany) and
antibiotics (100 µg ml streptomycin and 100 U ml penicil-
−
1
RNase A (50 µg ml ) for 1 h at 37 °C. Subsequently, the cells
were stained with propidium iodide (10 µg ml− ) for 30 min at
room temperature in the dark. Flow cytometry was performed
on a BD FACS Calibur Flow Cytometry System (Becton Dickin-
son) using an Ar-ion laser (488 nm). Fluorescence dot plots
and histograms were generated using CellQuest software. The
distribution of cells in a given cell cycle phase was analyzed
1
−1
−1
3
lin). 10 × 10 cells were seeded in each well of a 96-well plate
(
Nunc). The HeLa (human cervix carcinoma), K562 (chronic leu-
kemia) and HL-60 (acute leukemia) cells were cultured in RPMI
640 medium supplemented with antibiotics and 10% fetal calf
serum (HeLa, K562) or 20% fetal calf serum (HL-60), under a
1
3
using ModFit LT software, based on measurements of at least
5
2
% CO –95% air atmosphere. 7 × 10 cells were seeded in each
4
1
0 cells in each experiment.
well of a 96-well plate (Nunc). 24 h later the cells were exposed
to the test compounds. Stock solutions (100 mM) of test com-
pounds were freshly prepared in DMSO. The final concen-
trations of the compounds tested in the cell cultures were: Acknowledgements
1
mM, 1 × 10− mM, 1 × 10 mM and 1 × 10 mM. The con-
2
−4
−6
The authors gratefully acknowledge financial support from the
Ministry of Science and Higher Education of Poland (grant
number N N204 129737).
centration of DMSO in the cell culture medium was 1%. The
IC50 values (the concentration of test compound required to
reduce the cell survival fraction to 50% of the control) were cal-
culated from the dose–response curves and used as a measure
of cellular sensitivity to a given treatment. The cytotoxicity of all
compounds was determined by the MTT [3-(4,5-dimethylthiazol-
Notes and references
2
-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO]
39
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with drugs, the cells were treated with the MTT reagent and
incubation was continued for another 2 h. MTT-formazan crys-
tals were dissolved in 20% SDS and 50% DMF at pH 4.7 and the
absorbance was measured at 570 and 650 nm on an ELISA plate
reader (FLUOstar Omega). As a control (100% viability), we used
cells grown in the presence of the vehicle (1% DMSO) only.
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