Design and synthesis of pyrrolidine-containing sphingomimetics

Article abstract of DOI:10.1039/c1ob05324h

Based on the structures of natural sphingolipids, we designed heterocyclic sphingoid base mimetics in which the conformational restriction is introduced by incorporation of a pyrrolidine moiety between the 2-amino group and the C-4 carbon atom of the sphi

Full text of DOI:10.1039/c1ob05324h

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PAPER
Design and synthesis of pyrrolidine-containing sphingomimetics†
Seokwoo Lee, Sukjin Lee, Hyen Joo Park, Sang Kook Lee and Sanghee Kim*
Received 1st March 2011, Accepted 1st April 2011
DOI: 10.1039/c1ob05324h
Based on the structures of natural sphingolipids, we designed heterocyclic sphingoid base mimetics in
which the conformational restriction is introduced by incorporation of a pyrrolidine moiety between
the 2-amino group and the C-4 carbon atom of the sphingoid base. Our synthesis features a
regioselective nucleophilic ring opening of a cyclic sulfate with cyanide and subsequent manipulation of
the cyanide group. During the course of synthesis, Staudinger-type reductive cyclization of 1,3-azido
carboxylic acid and 1,4-azido alcohol offers a direct route to the five-membered pyrrolidone and
pyrrolidine products. The preliminary biological evaluation indicates that the designed pyrrolidine
analog is biologically active and its cytotoxic effect is associated with the induction of apoptosis.
Introduction
Sphingoid bases are long-chain aliphatic compounds typically
possessing a 2-amino-1,3-diol functionality.1 These compounds
serve a fundamental backbone of more complex metabolic
derivatives such as ceramide and sphingosine-1-phosphate. These
and all sphingoid base-containing compounds are referred to
as sphingolipids. Because sphingolipids play important roles in
membrane structure and in cell regulation as second messengers,2
they have been attractive targets for synthetic and medicinal
chemists over the last few decades.3
The increased appreciation and understanding of the biological
roles of sphingolipids raise the promising possibility that sphin-
golipid metabolites are biologically validated starting points for
the development of novel therapeutics.2a However, the physico-
chemical properties of sphingolipids themselves are not entirely
suitable for drug development. Thus, chemical modification of the
structure of sphingolipids, especially the sphingoid base moiety, is
deemed reasonable and necessary to confer drug-like properties.
The typical sphingoid bases are D-ribo-phytosphingosine (1,
Fig. 1) and D-erythro-sphingosine (2). In principle, sphingoid bases
can adopt multiple conformations depending on the environment,
because they are acyclic molecules and their amino and hydroxyl
groups can be engaged in several inter-/intramolecular hydrogen
bonds.4 Interestingly, there are several sphingoid base-like natural
products that have a cyclic moiety in their polar region.1b,5 The
representatives of this class are pramanicin (3),6 pachastrissamine
(4),7 and penaresidines (5).8 These heterocyclic compounds are
conformationally more restricted than the prototype sphingoid
bases 1 and 2 and exhibit various interesting biological activities.9
Fig. 1 Chemical structures of compounds 1–7.
There are also synthetic sphingoid base-like compounds that
have been designed and synthesized to provide conformational
restriction, especially in the polar region of the sphingoid base.10
Because the conformationally restricted analogs of sphingoid
bases might be useful in the investigation of the biological
functions of sphingolipids and might serve as valuable starting
points for the development of novel therapeutics, we have been
engaged in the design and synthesis of heterocyclic sphingoid
base-like compounds. In this regard, we have previously reported
sphingoid base mimetics, wherein the conformational restriction
is introduced by incorporation of a piperidine moiety between the
College of Pharmacy, Seoul National University, San 56-1, Shilim, Kwanak,
Seoul 151-742, Korea. E-mail: pennkim@snu.ac.kr; Fax: +82-2-888-0649;
Tel: +82-2-880-2487
† Electronic supplementary information (ESI) available: Copies of 1H
NMR and 13C NMR spectra of compounds 6–7 and 9–15. See DOI:
10.1039/c1ob05324h
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2-amino group and the C-4 carbon atom of the sphingoid base.11
These mimetics are much more effective than acyclic sphingoid
bases 1 and 2 at inhibiting cancer cell growth. This preliminary
biological result indicated that the polar region of the sphingoid
bases is amenable to conformational restriction by incorporation
into a heterocyclic ring, providing a basis for further investigation
on the structural modification.
As part of our ongoing sphingolipid research focused on the
design and preparation of conformationally constrained sphin-
gomimetics, we designed the heterocyclic compounds 6 and 7
(Fig. 1). These compounds can be considered cyclized mimetics
of sphingoid bases that bind the amine at C-2 and the carbon
at C-4 with the carbonyl or methylene linkages. The structure of
natural pramanicin (3) and penaresidines (5) led us to the above
design. The basic skeleton, stereochemistry, and chain length of
pramanicin (3) were retained, but its oxidized functionalities on
C-4 to C-9 were removed, including a quaternary hydroxyl group
and an epoxide group. This manoeuvre yielded the structure of
substituted pyrrolidone.6 Further removal of the carbonyl oxygen
of 6 resulted in the pyrrolidine compound 7.12 Pyrrolidine 7 can be
also considered a five-membered ring homologue of penaresidines
(5) with a simplified side chain. Herein, we report new sphingoid
base mimetics, their efficient synthetic route, and preliminary
activity studies.
Results and discussion
We envisioned that the designed heterocyclic compounds 6 and
7 could be accessible from the acyclic compound 9 through the
manipulation of its azido and nitrile groups (Scheme 1). The
requisite azido nitrile 9 might, in turn, arise from the regioselective
nucleophilic ring opening of cyclic sulfate 8 with cyanide. This
prediction was based on our previous observation that the ring
opening of cyclic sulfate 8 with iodide occurred exclusively at C-4
with clean inversion.13
Scheme 2 (a) i) NaCN, DMF, 1 h, ii) H2SO4/H2O/THF (1 : 1.5 : 50),
1 h; (b) TBSOTf, 2,6-lutidine, CH2Cl2, 0 ◦C to rt, 1 h; (c) DIBAL-H,
toluene, -78 ◦C, 1 h; (d) NaClO2, NaH2PO4·2H2O, 2-methyl-2-butene,
t-BuOH/H2O (1 : 1), 1 h; (e) NaBH4, MeOH/CH2Cl2 (2 : 1), 0 ◦C, 30 min;
(f) PPh3, toluene, reflux, 3 h; (g) PPh3, THF/H2O (100 : 1), reflux, 48 h;
(h) Bu4NF, THF, 0 ◦C to rt, 2 h; TBSOTf = tert-butyldimethylsilyl
trifluoromethanesulfonate; DIBAL-H = diisobutylaluminium hydride.
characteristics between the C-3 and C-4 carbons. Our computa-
tional studies on the truncated system of 8, shown in Fig. 2,
revealed that the electron densities on the cyclic sulfate carbon
atoms are different. The C-4 carbon displays a lower electron
density and less steric hindrance than the C-3 carbon. Thus, the
Scheme 1 Retrosynthetic plan for heterocyclic compounds 6 and 7.
Cyclic sulfate 8 can be easily obtained from phytosphingosine
(1) in high overall yield, as previously reported by our group.13
The reaction of cyclic sulfate 8 with NaCN in DMF at room
temperature, after acidic hydrolysis of the intermediate O-sulfate,
afforded the C-4 cyanide compound 9 in 72% yield (Scheme 2).
As in previous ring opening reaction of 8 with iodide,13 the C-
1
4 substituted product 9 was the only regioisomer detected in H
NMR spectra of the crude product. The excellent regioselectivity
might be attributed to the differences in the steric and electronic
Fig. 2 The electrostatic potential map of the truncated system of 8
calculated at the PBE/DNP level.
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Table 1 Anti-proliferative effects of sphingolipids on cancer cells
C-4 carbon would be more susceptible to nucleophilic attack than
the C-3 carbon.
e
IC50 (mM)
For the efficient transformation of the cyano group, it was
desirable to protect the free hydroxyl group of 9. Thus, the hydroxyl
group was protected as its silyl ether using TBSOTf to give 10 in
90% yield (Scheme 2). The cyano group of 10 was reduced to the
corresponding aldehyde 11 (75%) using DIBAL-H, and then the
aldehyde group of 11 was oxidized with NaClO2 to give carboxylic
acid 12 (86%). In a parallel reaction, aldehyde 11 was reduced with
NaBH4 to give azido alcohol 13 (84%).
Cell line
1
2
6
7
B16 Melanomaa
A549b
30.4
29.8
13.7
17.4
29.6
38.6
27.9
21.9
59.6
>75
>75
>75
6.4
6.0
8.1
4.0
SK-HEP1c
HCT116d
a Mouse B16 melanoma cells. b Human lung carcinoma cells. c Human hep-
atocarcinoma cells. d Human colorectal carcinoma cells. e 50% inhibition
concentration.
For the Staudinger reduction of the azide functionality,14 the
1,3-azido carboxylic acid 12 was treated with PPh3 in refluxing
anhydrous toluene. This treatment resulted in concomitant in situ
cyclization to afford g-lactam 14 in 55% yield.15,16 Similarly, the
azido alcohol 13 also gave the cyclized product 15 in 84% yield
under the same reaction conditions. Removal of the silyl protecting
groups in compounds 14 and 15 with Bu4NF in THF afforded
the desired target compounds pyrrolidone 6 and pyrrolidine 7 in
85% and 77% yields, respectively. The structures of 6 and 7 were
supported by spectral and analytical data.
In our synthetic process, Staudinger-type transformations offer
a direct route to the five-membered pyrrolidone and pyrrolidine
products. Despite its synthetic potential, the Staudinger-type
reductive cyclization of 1,3-azido carboxylic acids and 1,4-azido
alcohols has not been widely reported. To our knowledge, the in
situ conversion of a 1,3-azido carboxylic acid to a g-lactam via the
Staudinger reaction has not been described previously, although
ring closure through the activation of the carboxy group has
been reported extensively.17,18 Only one case of the direct reductive
cyclization of a 1,4-azido alcohol to a pyrrolidine ring has been
previously reported,19 although the formation of aziridine from
1,2-azido alcohol has been widely explored.20,21
One plausible mechanism leading to 14 and 15 from 12 and
13 might be that the initially formed iminophosphoranes (16a
and 16b, Fig. 3) react with an intramolecular acid or hydroxyl
group to form the seven-membered [1,3,2]oxazaphosphepanone
or [1,3,2]oxazaphosphepane. However, this mechanism is ques-
tionable because there have been no reports on the existence of
seven-membered [1,3,2]oxazaphosphepane ring systems. To obtain
clues on the mechanism of this unusual Staudinger cyclization, the
reactions of 12 and 13 with PPh3 were carried out in the presence
of H2O in refluxing THF. Under these reaction conditions, 1,3-
azido carboxylic acid 12 was cleanly converted to the cyclized
product 14 in 75% yield. However, 1,4-azido alcohol 13 gave none
of the cyclized product 15, but instead gave a 78% yield of the
corresponding primary amine 17. From these results, the reaction
mechanisms might be assumed to be as follows. The formation
of 14 might include the initial hydrolysis of iminophosphorane
16a followed by subsequent condensation of the resulting amine
with the acid group, although the prior nucleophilic attack of the
iminophosphorane nitrogen atom on the carbonyl carbon could
not be excluded. For the formation of 15 from iminophosphorane
16b, the direct nucleophilic attack of the imino nitrogen atom
at the carbon atom bearing a hydroxyl group might be one
possible pathway. Nevertheless, more systematic experimental and
theoretical studies are needed to clarify the mechanisms.
The typical biological activities of sphingoid bases include the
modulation of multiple kinases, G protein-coupled receptors, and
channels.22 As a consequence of these actions, sphingoid bases
exhibit antiproliferative activity and induce apoptosis in cancer
cells.23 Based on this information, we first examined the cytotoxic
activities of sphingomimetics 6 and 7 as part of their preliminary
evaluation. As shown in Table 1, pyrrolidine 7 was found to be a
more effective initiator of cell death than the prototype sphingoid
bases 1 and 2, whereas pyrrolidone 6 showed less potent cytotoxic
activity than 1 and 2. For example, the IC50 values of 6 and 7
against B16 melanoma cells were 59.6 and 6.4 mM, while those
of sphingoid bases 1 and 2 were 30.4 and 29.6 mM, respectively.
The only structural difference between 6 and 7 is the presence of
a carbonyl oxygen group in the heterocyclic ring in 6. This simple
difference influences the biological activity significantly.
To determine whether the cytotoxic effects of 7 involve cellular
apoptotic pathways, caspase-3 activation was investigated because
induction of caspase-3 proteolytic activity is one of the most
important events in apoptosis.24 As illustrated in Fig. 4, the
treatment of B16 melanoma cells with 7 increased the caspase-
3-like activity in a concentration-dependent manner. These data
Fig. 4 Effects of 7 on the caspase-3-like activity of mouse melanoma
cells. B16 melanoma cells were treated with 7 for 18 h. Caspase-3-like
activity was determined by measuring the cleavage of the luminogenic
tetrapeptide substrate Ac-DEVD-pNA. Data are expressed as the mean
S.D. *Statistically significant compared to the control (*P < 0.05).
Fig. 3 Chemical structures of iminophosphoranes 16 and amine 17.
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suggest that the cytotoxic effect of compound 7 is associated with
the induction of apoptosis, as is the case for sphingoid bases,
pramanicin (3),9a,c and penaresidines (5).8,9g
Previously, Yves Ge´nisson et al. have reported the synthesis of
pyrrolidine compound 18 (Fig. 5), which is structurally similar
to our pyrrolidine 7 but has a different stereochemistry at C-
4 and a different chain length.12 This C-4 epimeric compound
displayed a strong cytotoxicity in B16 melanoma cells, with an
IC50 of ca. 5 mM, which is very similar to that of pyrrolidine
7.
Conclusion
We have designed and synthesized conformationally constrained
sphingomimetics in which a pyrrolidine moiety was incorporated
between the 2-amino group and the C-4 carbon atom of the
sphingoid base. For the synthesis of the designed pyrrolidine-
containing compounds from phytosphingosine, we employed a
regioselective nucleophilic ring opening of a cyclic sulfate with
cyanide. The C-4 cyanide group thereby obtained was elaborated
further to pyrrolidone and pyrrolidine. In this process, a very
unusual Staudinger-type reductive cyclization was utilized. The
synthesized pyrrolidine 7 turned out to be much more effective
than the prototype sphingoid bases at inhibiting cancer cell
growth. Our study suggested that this cytotoxic effect is associated
with the induction of apoptosis. In addition, the pyrrolidine 7
down-regulated GCS gene expression. The observed biological
results indicate that the polar region of the sphingoid bases
is amenable to conformational restriction by incorporation of
a pyrrolidine ring. We believe that the presented pyrrolidine
analogues could be of value in the investigation of the biological
functions of sphingolipids as a chemical tool and in the develop-
ment of novel therapeutics as a lead scaffold.
Fig. 5 Structural comparison of compounds 7 and 18.
Because pyrrolidine 18 was reported to have an inhibitory
effect on the activity of glucosylceramide synthase (GCS), we
examined whether compound 7 affects GCS gene expression
in B16 melanoma cells. As shown in Fig. 6, semi-quantitative
PCR (qPCR) analysis of GCS mRNA expression demonstrated
that treatment of 7 resulted in the down-regulation of the GCS
mRNA expression in a concentration-dependent manner. GCS
is an enzyme that catalyzes the transfer of glucose to ceramide
in glucosylceramide biosynthesis.25 In addition to conferring
multidrug resistance to tumour cells, GCS also plays an impor-
tant role in many vital biological functions in cell growth and
apoptosis.26 Accumulating evidence suggests a reduced level of
GCS expression resulted in significant effects on the sphingolipid
pattern, with proapoptotic ceramide levels being elevated.27 Thus,
although further studies are required to conclusively demonstrate
the mechanism of action, the apoptotic cytotoxicity of pyrrolidine
7 might be a result of increased ceramide levels from a down-
regulation of GCS expression.
Experimental section
A: Preparation of the compounds
General. All chemicals were reagent grade and used as pur-
chased. All reactions were performed under an inert atmosphere
of dry argon or nitrogen using dry solvents. Reactions were
monitored with TLC analysis using Merck silica gel 60 F-254
thin layer plates. Flash column chromatography was carried out
on silica gel (230–400 mesh). H NMR and 13C NMR spectra
1
were recorded in d units relative to deuterated solvent as internal
reference at 300/400 and 75/100 MHz, respectively. IR spectra
were measured on a Fourier Transform Infrared spectrometer.
Mass spectra (MS) were recorded using fast atom bombardment
(FAB), chemical ionization (CI). High resolution mass spectra
(HRMS) were recorded using FAB or CI.
(R)-2-((1R,2S)-2-Azido-3-(tert-butyldiphenylsilyloxy-1-hydro-
xypropyl)hexadecanenitrile (9). To a solution of cyclic sulfate 8
(1.61 g, 2.50 mmol) in DMF (25 mL) was added NaCN (184 mg,
3.75 mmol). This reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was concentrated in vacuo to
remove the DMF, and then THF (10 mL), H2O (300 mL), and
conc. H2SO4 (200 mL) were added to the concentrated residue.
The mixture was stirred for 1 h at room temperature, diluted with
EtOAc and then washed with sat. NaHCO3 and brine. The organic
layer was dried over MgSO4 and concentrated in vacuo. The crude
product was purified using silica gel column chromatography
(hexane–EtOAc 10 : 1) to give nitrile alcohol 9 (1.06 g, 72%) as
a colorless oil. [a]D20 +37.6 (c 1.0, CHCl3); 1H NMR (CDCl3, 300
MHz) d 0.89 (t, J = 6.6 Hz, 3H), 1.07–1.09 (s, 9H), 1.27 (s, 24
H), 1.48–1.62 (m, 2H), 1.80–1.90 (m, 1H), 2.94 (ddd, J = 2.3, 5.3,
9.8 Hz, 1H), 3.54–3.65 (m, 2H), 3.99 (ddd, J = 4.5, 11.0, 20.9 Hz,
2H), 7.38–7.51 (m, 6H), 7.67–7.73 (m, 4H); 13C NMR (CDCl3, 75
MHz) d 14.1, 19.1, 22.7, 26.5, 26.7, 27.2, 29.0, 29.2, 29.3, 29.5,
29.57, 29.61, 29.64, 29.65, 31.9, 35.9, 64.0, 64.2, 70.9, 119.3, 127.7,
127.95, 127.99, 129.6, 130.13, 130.17, 132.1, 132.2, 134.8, 135.5; IR
Fig. 6 Down-regulation of GCS mRNA expression in mouse melanoma
cells. B16 melanoma cells were treated with the indicated concentrations
of 7 (10, 25, and 50 mM) for 24 h. The mRNA levels of GCS were
determined by quantitative real-time reverse transcriptase-polymerase
chain reaction (RT-PCR). Using the 2-DDCT method as described in the
Experimental section, the data are presented as the fold change in gene
expression normalized to a housekeeping gene, b-actin, and relative to
the untreated control (first bar). Data shown are the means SEM of
four determinations. *Statistically significant compared with the control
cultures (*P < 0.05, **P < 0.01).
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(CHCl3) nmax 3444, 2926, 2855, 2101, 1112 (cm-1); HRMS (FAB)
silica gel column chromatography (hexane–EtOAc 15 : 1) to give
calcd for C35H55N4O2Si 591.4094 ([M + H]+), found 591.4091.
azido carboxylic acid 12 (125 mg, 86%) as a colorless oil. [a]D25
1
-2.0 (c 0.77, CHCl3); H NMR (CDCl3, 400 MHz) d -0.06 (s,
(R) - 2 - ((5R,6S) - 6 - Azido - 3,3 - diethyl - 10,10 - dimethyl - 9,9-
diphenyl-4,8-dioxa-3,9-disilaundecan-5-yl)hexadecanenitrile (10).
To a solution of nitrile alcohol 9 (887 mg, 1.50 mmol) in CH2Cl2
(7.5 mL) were added 2,6-lutidine (700 mL, 6.00 mmol) and TBSOTf
(690 mL, 3.00 mmol) at 0 ◦C. This reaction mixture was stirred
for 1 h at room temperature, quenched with MeOH, poured into
sat. NaHCO3, and extracted twice with EtOAc. The combined
organic layers were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was purified using silica
gel column chromatography (hexane–EtOAc 15 : 1) to give nitrile
3H), 0.03 (s, 3H), 0.77 (s, 9H), 0.86 (t, J = 6.6 Hz, 3H), 1.06 (s,
9H), 1.24 (s, 24H), 1.36–1.39 (m, 1H), 1.64 (td, J = 2.3, 9.6 Hz,
1H), 2.45–2.49 (m, 1H), 3.63–3.72 (m, 2H), 3.80 (dd, J = 3.2, 9.3
Hz, 1H), 3.90 (t, J = 4.6 Hz, 1H), 7.35–7.43 (m, 6H), 7.65 (t, J =
6.4 Hz, 4H); 13C NMR (CDCl3, 75 MHz) d -4.9, -4.5, 14.1, 17.9,
19.1, 22.7, 25.7, 26.7, 27.7, 28.0, 29.35, 29.43, 29.6, 29.65, 29.69,
31.9, 49.0, 64.4, 66.3, 73.5, 127.8, 129.86, 129.90, 132.8, 132.9,
135.5; IR (CHCl3) nmax 2927, 2856, 2102, 1712, 1464, 1428, 1362,
1258, 1113, 837, 778, 740, 702 (cm-1); HRMS (CI) C41H70N3O4Si2
calcd for 724.4905 ([M + H]+), found 724.4905.
1
10 (953 mg, 90%) as a colorless oil. [a]2D5 -1.8 (c 0.7, CHCl3); H
NMR (CDCl3, 400 MHz) d -0.16 (s, 3H), 0.03 (s, 3H), 0.81 (s, 9H),
0.87 (t, J = 6.7 Hz, 3H), 1.08 (s, 9H), 1.26 (s, 22H), 1.38–1.45 (m,
1H), 1.52–1.54 (m, 1H), 1.66–1.71 (m, 1H), 2.80 (td, J = 2.6, 7.7
Hz, 1H), 3.61 (app dd, J = 2.2, 5.9 Hz, 1H), 3.65 (app d, J = 10.0
Hz, 1H), 3.73 (q, J = 6.1 Hz, 1H), 3.81 (dd, J = 4.4, 10.1 Hz, 1H),
7.36–7.46 (m, 6H), 7.65 (t, J = 6.1 Hz, 4H); 13C NMR (CDCl3,
75 MHz) d -4.4, -4.2, 14.1, 18.0, 19.1, 22.7, 25.6, 26.7, 27.4, 29.0,
29.3, 29.4, 29.55, 29.62, 29.64, 29.66, 29.8, 31.9, 36.4, 64.7, 66.3,
72.1, 119.4, 127.8, 127.88, 127.92, 129.97, 132.5, 132.6, 135.52,
135.54; IR (CHCl3) nmax 2927, 2856, 2104, 1470, 1428, 1258, 1114,
838, 778, 741, 702 (cm-1); HRMS (FAB) calcd for C41H68N4O2Si2
705.4959 ([M + H]+), found 705.4981.
(R) - 2 - ((5R,6S) - 6 - Azido - 3,3 - diethyl - 10,10 - dimethyl - 9,9-
diphenyl-4,8-dioxa-3,9-disilaundecan-5-yl)hexadecane-1-ol (13).
To a solution of aldehyde 11 (140 mg, 0.20 mmol) in CH2Cl2
(1 mL) and MeOH (2 mL) was added NaBH4 (11 mg, 0.29 mmol)
at 0 ◦C. This reaction mixture was stirred for 30 min at 0 ◦C,
quenched with sat. NH4Cl and extracted with twice EtOAc.
The combined organic layers were washed with brine, dried
over Na2SO4, and concentrated in vacuo. The crude product was
purified using silica gel column chromatography (hexane–EtOAc
10 : 1) to give azido alcohol 13 (119 mg, 84%) as a colorless oil.
[a]2D5 +11.3 (c 0.97, CHCl3); 1H NMR (CDCl3, 400 MHz) d -0.08
(s, 3H), 0.02 (s, 3H), 0.78 (s, 9H), 0.86 (t, J = 6.7 Hz, 3H), 1.07 (s,
9H), 1.24 (s, 25H), 1.59 (s, 1H), 2.03 (s, 2H), 3.60–3.66 (m, 2H),
3.68–3.73 (m, 3H), 3.80 (dd, J = 3.8, 10.5 Hz, 1H), 7.35–7.43 (m,
6H), 7.65 (t, J = 6.6 Hz, 4H); 13C NMR (CDCl3, 75 MHz) d -4.5,
-4.3, 14.1, 17.9, 19.1, 22.7, 25.8, 26.7, 27.6, 29.4, 29.55, 29.60,
29.64, 29.7, 29.8, 31.9, 42.8, 62.7, 65.0, 67.6, 75.0, 127.8, 129.86,
129.89, 132.8, 133.0, 135.6; IR (CHCl3) nmax 2927, 2855, 2103,
1471, 1257, 1112, 837, 777, 702 (cm-1); HRMS (FAB) calcd for
C41H72N3O3Si2 710.5112 ([M + H]+), found 710.5112.
(S) - 2 - ((5R,6S) - 6 - Azido - 3,3 - diethyl - 10,10 - dimethyl - 9,9-
diphenyl-4,8-dioxa-3,9-disilaundecan-5-yl)hexadecanal (11). To a
solution of nitrile 10 (705 mg, 1.00 mmol) in toluene (10 mL) was
added DIBAL-H (2.50 mL, 2.50 mmol, 1.0 M solution in toluene)
in a dropwise manner at -78 ◦C. This reaction mixture was stirred
for 1 h at the same temperature and then diluted with Et2O (10 mL),
followed by the addition of H2O (2.5 mL). The mixture solution
was stirred at room temperature for 1 h and then filtered through
a pad of Celite. The filtrate was concentrated in vacuo. The crude
product was purified using silica gel column chromatography
(hexane–EtOAc 30 : 1) to give aldehyde 11 (526 mg, 75%) as a
(R) - 2 - ((5R,6S) - 6 - Azido - 3,3 - diethyl - 10,10 - dimethyl - 9,9-
diphenyl-4,8-dioxa-3,9-disilaundecan-5-yl)hexadecane-1-ol (14).
To a solution of azido carboxylic acid 12 (109 mg, 0.15 mmol) in
THF/H2O (100 : 1, 15 mL) was added PPh3 (118 mg, 0.45 mmol).
This reaction mixture was refluxed for 48 h. The reaction mixture
was cooled to room temperature, and then diluted with EtOAc.
The organic layer was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The crude product was purified using
silica gel column chromatography (hexane–EtOAc 5 : 1) to give
pyrrolidone 14 (77 mg, 75%) as a colorless oil. [a]2D5 -8.2 (c 0.57,
1
colorless oil. [a]2D3 +17.1 (c 0.29, CHCl3); H NMR (CDCl3, 400
MHz) d -0.10 (s, 3H), 0.02 (s, 3H), 0.77 (s, 9H), 0.86 (t, J = 6.6
Hz, 3H), 1.06 (s, 9H), 1.24 (s, 25H), 1.45–1.48 (m, 1H), 1.69–1.71
(m, 1H), 2.21–2.23 (m, 1H), 3.67 (dt, J = 2.3, 7.6 Hz, 1H), 3.77 (dt,
J = 2.3, 7.6 Hz, 1H), 3.91–3.92 (m, 1H), 7.35–7.43 (m, 6H), 7.63
(t, J = 6.0 Hz, 4H), 9.67 (d, J = 2.5 Hz, 1H); 13C NMR (CDCl3,
75 MHz) d -4.7, -4.6, 14.1, 17.8, 19.1, 22.7, 25.5, 25.6, 26.7, 27.8,
29.3, 29.4, 29.5, 29.65, 29.67, 31.9, 55.0, 64.8, 66.4, 73.1, 127.83,
129.89, 129.92, 132.7, 132.8, 135.5; IR (CHCl3) nmax 2927, 2855,
2108, 1721, 1469, 1428, 1362, 1318, 1258, 1113, 1007, 837, 778,
740, 702 (cm-1); HRMS (CI) C41H70N3O3Si2 calcd for 708.4956
([M + H]+), found 708.4954.
1
CHCl3); H NMR (CDCl3, 400 MHz) d -0.02 (s, 3H), 0.01 (s,
3H), 0.84 (s, 9H), 0.86 (app t, J = 6.4 Hz, 3H), 1.04 (s, 9H), 1.24
(s, 22H), 1.61 (m, 2H), 1.80 (s, 2H), 2.30 (td, J = 2.30, 5.67 Hz,
1H), 3.46–3.48 (m, 1H), 3.50–3.54 (m, 1H), 3.57–3.61 (m, 1H),
4.19 (d, J = 5.8 Hz, 1H), 5.74 (s, 1H, -NH), 7.35–7.44 (m, 6H),
7.60–7.62 (m, 4H); 13C NMR (CDCl3, 75 MHz) d -5.1, -4.5,
14.1, 17.9, 19.1, 22.7, 23.7, 25.6, 26.8, 27.7, 29.3, 29.57, 29.64,
29.68, 29.8, 31.9, 46.2, 63.1, 64.4, 71.0, 127.9, 130.0, 132.7, 132.8,
135.48, 135.54, 178.1; IR (CHCl3) nmax 2926, 2855, 1704, 1464,
1428, 1390, 1257, 1113, 837, 775, 740, 702 (cm-1); HRMS (FAB)
C41H70NO3Si2 calcd for 680.4894 ([M + H]+), found 680.4879.
(S)-2-((5R,6S)-6-Azido-2,2,3,3,10,10-hexamethyl-9,9-diphenyl-
4,8-dioxa-3,9-disilaundecan-5-yl)hexadecanoic acid (12). To a
solution of aldehyde 11 (140 mg, 0.20 mmol) in t-BuOH (1 mL)
and 2-methyl-2-butene (130 mL, 1.20 mmol) were added NaClO2
(90 mg, 0.80 mmol) and NaH2PO4·2H2O (187 mg, 1.20 mmol)
in H2O (1 mL). This reaction mixture was stirred for 1 h at
room temperature, diluted with EtOAc and washed with sat.
NaHCO3 and brine. The organic layer was dried over MgSO4
and concentrated in vacuo. The crude product was purified using
(2S,3R,4R)-2-((tert-Butyldiphenylsilyloxy)methyl)-4-tetradecyl-
3-(triethylsilyloxy)pyrrolidine (15). To a solution of hydroxy-
methyl azido alcohol 13 (106 mg, 0.15 mmol) in toluene (7.5 mL)
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was added PPh3 (99 mg, 0.38 mmol). This reaction mixture
was refluxed for 3 h. The reaction mixture was cooled to room
temperature, and then diluted with EtOAc. The organic layer
was washed with brine, dried over Na2SO4 and concentrated in
vacuo. The crude product was purified using silica gel column
chromatography (hexane–EtOAc 3 : 1) to give pyrrolidine 15
(84 mg, 84%) as a colorless oil. [a]2D5 -8.3 (c 0.8, CHCl3); 1H NMR
(CDCl3, 400 MHz) d -0.02 (s, 3H), 0.03 (s, 3H), 0.84 (s, 9H),
0.86 (app t, J = 6.0 Hz, 3H), 1.05 (s, 9H), 1.24 (s, 23H), 1.42–1.45
(m, 1H), 1.75–1.82 (m, 2H), 1.89 (br s, 2H), 2.68 (dd, J = 10.2,
10.2 Hz, 1H), 2.96 (dd, J = 7.2, 9.6 Hz, 1H), 3.12 (t, J = 5.2 Hz,
1H), 3.49–3.53 (m, 1H), 3.57–3.60 (m, 1H), 4.04 (d, J = 3.9 Hz,
1H), 7.33–7.42 (m, 6H), 7.61–7.64 (m, 4H); 13C NMR (CDCl3, 75
MHz) d -4.9, -4.3, 14.1, 18.0, 19.2, 22.7, 25.8, 26.8, 26.9, 28.5,
29.4, 29.66, 29.68, 30.0, 31.9, 44.4, 50.5, 65.1, 68.5, 75.0, 127.7,
129.7, 133.3, 135.6; IR (CHCl3) nmax 2926, 2855, 1739, 1469, 1428,
1365, 1253, 1217, 1113, 836, 775, 740, 702 (cm-1); HRMS (FAB)
C41H72NO2Si2 calcd for 666.5102 ([M + H]+), found 666.5097.
2-yl)-2,5-diphenyltetrazoliumbromide (MTT) to formazan.28 The
cells were incubated with the tested compounds for 72 h, following
which MTT (0.5 mg mL-1 in PBS) solution was added to the media,
and then the cells were incubated for 4 h at 37 ◦C. The media were
discarded, and then DMSO was added to dissolve the formazan
dye. The absorbance was measured at 570 nm and was then used
to calculate the IC50 value in comparison to control cells.
Caspase-3 activation assay. Caspase-3 activation assays were
performed using a Caspase-GloTM3/7 assay kit (Promega,
Madison, WI, USA) according to the manufacturer’s instructions.
Briefly, B16 melanoma cells were seeded in 96-well plates at a
density of 1 ¥ 104 cells per well. After 24 h, cells were treated
with 10, 25, and 50 mM compound 7. Caspase-Glo 3/7 reagent
(100 mL) was then added to each well treated with compound 7
for 18 h. After an incubation of 1 h at room temperature, chemi-
luminescence was measured with a Centro LB960 luminometer
(Berthold Technologies, Vilvoorde, Belgium) and expressed in
relative light units per second (RLU s-1).
General procedure for removal of the silyl protecting groups on
compounds 14 and 15. To a solution of 14 or 15 (68 mg or
67 mg, 0.10 mmol) in THF (2 mL) was added Bu4NF (0.25 mL,
0.25 mmol, 1.0 M solution in THF) at 0 ◦C. After stirring for 2 h
at room temperature, the solvent was removed in vacuo. The crude
product was purified using silica gel column chromatography
(CH2Cl2–MeOH–NH4OH 100 : 10 : 1) to give 6 or 7.
Real-time RT-PCR. Real-time RT-PCR was employed to
determine the expression level of GCS in mouse B16 melanoma
cells. Briefly, total RNA was extracted using TRI reagent and was
then reverse transcribed at 42 ◦C for 60 min in 20 mL of Reverse
Transcription System (Promega, Madison, WI) with 0.5 mg of
oligo(dT)15 primer. The levels of GCS mRNA were determined
using a MiniOpticon system (Bio-Rad, Hercules, CA) using 5 mL
R
of reverse transcription product, iQTM SYBRꢀ Green Supermix
(3S,4R,5S)-4-Hydroxy-5-(hydroxymethyl)-3-tetradecylpyrroli-
din-2-one (6). As a white solid (28 mg, 85%). [a]2D5 +9.7 (c 0.26,
MeOH); 1H NMR (CD3OD, 400 MHz) d 0.89 (t, J = 6.7 Hz, 3H),
1.28 (s, 23H), 1.39–1.43 (m, 1H), 1.46–1.52 (m, 1H), 1.57–1.65 (m,
2H), 2.47 (td, J = 4.3, 11.0 Hz, 1H), 3.42 (dd, J = 4.9, 4.9 Hz,
1H), 3.55 (d, J = 5.1 Hz, 2H), 4.26 (d, J = 6.0 Hz, 1H); 13C NMR
(CD3OD, 100 MHz) d 15.23, 15.24, 24.5, 25.5, 29.7, 31.27, 31.29,
31.5, 31.6, 31.7, 33.9, 48.1, 64.2, 66.1, 72.2, 181.8; IR (MeOH)
nmax 3428, 3215, 2912, 2848, 1663, 1471, 1053, 713 (cm-1); HRMS
(FAB) calcd for C19H38NO3 328.2852 ([M + H]+), found 328.2860.
(Bio-Rad, Hercules, CA), and primers in a total volume of 20 mL.
The standard thermal cycle conditions were employed: 95 ◦C for
20 s before the first cycle; 95 ◦C for 20 s, 56 ◦C for 20 s, and 72 ◦C for
30 s repeated 40 times; followed by 95 ◦C for 1 min and 55 ◦C for
1 min. Specific GCS primers were designed using Roche Applied
System (Basel, Swiss) and were provided by Bioneer Corporation
(Seoul, Korea). The following sequences were used: GCS for-
ward, 5¢-GTTTCAATCCAGAATGATCAGGT-3¢; GCS reverse,
5¢-AAGCATTCTGAAATTGGCTCA-3¢; b-actin (housekeeping
gene) forward, 5¢-AGCACAATGAAGATCAAGAT-3¢; b-actin
reverse, 5¢-TGTAACGCAACTAAGTCATA-3¢. The threshold
cycle (CT), indicating the fractional cycle number at which
the amount of amplified target gene reaches a fixed threshold
from each well, was determined using by MJ Opticon Monitor
software. Relative quantification, representing the change in gene
expression from real-time quantitative PCR experiments between
the treated samples and the untreated controls, was calculated by
the comparative CT method as published previously.29 The data
were analyzed using the equation 2-DDCT, where DDCT = [CT of
target gene - CT of housekeeping gene] treated group - [CT of
target gene - CT of housekeeping gene] untreated control group.
For the treated samples, evaluation of 2-DDCT represents the fold
change in gene expression, normalized to a housekeeping gene
(b-actin) and relative to the untreated control.
(2S,3R,4R)-2-(Hydroxymethyl)-4-tetradecylpyrrolidin-3-ol (7).
1
As a white solid (24 mg, 77%). [a]2D5 -13.5 (c 0.15, MeOH); H
NMR (CD3OD, 400 MHz) d 0.89 (t, J = 6.7 Hz, 3H), 1.28 (s,
26H), 1.57–1.59 (m, 1H), 1.93–1.99 (m, 1H), 2.71 (t, J = 10.7 Hz,
1H), 3.10 (dd, J = 7.2, 10.5 Hz, 1H), 3.15 (td, J = 2.6, 5.9 Hz, 1H),
3.57 (ddd, J = 6.1, 11.5, 17.9 Hz, 2H), 4.00 (dd, J = 2.3, 5.4 Hz,
1H); 13C NMR (CD3OD, 100 MHz) d 14.4, 23.7, 26.9, 29.0, 30.5,
30.65, 30.74, 30.8, 30.9, 33.1, 44.4, 60.4, 70.9, 73.1; IR (MeOH)
nmax 3387, 2918, 2849 (cm-1); HRMS (CI) calcd for C19H40NO2
314.3059 ([M + H]+), found 314.3059.
B: Bioassay
Cell cultures and drug treatments. Mouse B16 melanoma cells
were kindly provided by Skin Research Institute, Amore-Pacific
Co., Korea. The cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with antibiotics and 10% (v/v)
fetal bovine serum (FBS). The cells were incubated at 37 ◦C in a
humidified atmosphere of 10% CO2. Each experiment was carried
out at least three times.
C: Theorical calculation
Electrostatic potential calculation. Theoretical calculations on
the truncated system of 8 was performed using the density
functional theory (DFT) method as implemented in the DMol3
package,30 which is available as part of the Material Studio 5.5
package. In the DFT calculations, we employed the Perdew, Burke,
Measurements of cell viability. Cell viability was determined by
the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-
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and Ernzerhof (PBE) function31 for the exchange–correlation in-
teraction within a generalized gradient approximation (GGA) and
a double numerical basis set including d-polarization functions
(DNP) as implemented in the DMol3.
G. Charron, A. Billich and D. Guerini, Bioorg. Med. Chem. Lett., 2007,
17, 491–494; (f) R. Zhu, A. H. Snyder, Y. Kharel, L. Schaffter, Q. Sun,
P. C. Kennedy, K. R. Lynch and T. L. Macdonald, J. Med. Chem., 2007,
50, 6428–6435; (g) K. Fuhshuku, N. Hongo, T. Tashiro, Y. Masuda, R.
Nakagawa, K. Seino, M. Taniguchi and K. Mori, Bioorg. Med. Chem.,
2008, 16, 950–964.
11 J. Cho, Y. M. Lee, D. Kim and S. Kim, J. Org. Chem., 2009, 74, 3900–
Acknowledgements
3904.
12 (2S,3R)-3-Hydroxy-2-(hydroxymethyl)pyrrolidine bearing various C4
lipophilic residues has been synthesized and evaluated. See: A. Rives, Y.
Ge´nisson, V. Faugeroux, C. Zedde, C. Lepetit, R. Chauvin, N. Saffon,
N. Andrieu-Abadie, S. Colie´, T. Levade and M. Baltas, Eur. J. Org.
Chem., 2009, 2474–2489.
13 S. Kim, S. Lee, T. Lee, H. Ko and D. Kim, J. Org. Chem., 2006, 71,
8661–8664.
14 (a) H. Staudinger and J. Meyer, Helv. Chim. Acta, 1919, 2, 635–
646; (b) Y. G. Gololobov, I. N. Zhmurova and L. F. Kasukhin,
Tetrahedron, 1981, 37, 437–472; (c) Y. G. Gololobov and L. F.
Kasukhin, Tetrahedron, 1992, 48, 1353–1406.
This work was supported by the World Class University Program
(R32-2008-000-10098-0) and the MarineBio Research Program
(NRF-C1ABA001-2010-0020428) of the National Research Foun-
dation of Korea (NRF) grant funded by the Korea government
(MEST).
Notes and references
1 (a) A. H. Jr. Merrill and K. Sandhoff, in Sphingolipids: Metabolism and
Cell Signaling, in Biochemistry of Lipids, Lipoprotein, and Membranes,
ed. D. E. Vance and J. E. Vance, Elsevier, New York, 2002, pp 373–407;
(b) S. T. Pruett, A. Bushnev, K. Hagedorn, M. Adiga, C. A. Haynes,
M. C. Sullards, D. C. Liotta and A. H. Jr. Merrill, J. Lipid Res., 2008,
49, 1621–1639.
2 For a good review with citations, see: (a) M. Kester and R. Kolesnick,
Pharmacol. Res., 2003, 47, 365–371; (b) H. Rosen and J. Liao, Curr.
Opin. Chem. Biol., 2003, 7, 461–468.
3 For recent reviews, see: (a) J. Liao, J. Tao, G. Lin and D. Liu,
Tetrahedron, 2005, 61, 4715–4733; (b) A. Delgado, J. Casas, A. Llebaria,
J. L. Abad and G. Fabrias, Biochim. Biophys. Acta, Biomembr., 2006,
1758, 1957–1977; (c) A. Delgado, J. Casas, A. Llebaria, J. L. Abad and
G. Fabra´s, ChemMedChem, 2007, 2, 580–606; (d) J. Antonio Morales-
Serna, J. Llaveria, Y. Diaz, M. M. Isabel Matheu and S. Castillon, Curr.
Org. Chem., 2010, 14, 2483–2521, and references therein.
4 (a) D. B. DuPre´ and M. C. Yappert, THEOCHEM, 1999, 467, 115–
133; (b) L. Li, X. Tang, K. G. Taylor, D. B. DuPre´ and M. C. Yappert,
Biophys. J., 2002, 82, 2067–2080; (c) H. Sasaki, H. Arai, M. J. Cocco and
S. H. White, Biophys. J., 2009, 96, 2727–2733, and references therein.
5 (a) T. F. Molinski and C. M. Ireland, J. Org. Chem., 1988, 53, 2103–2105;
(b) A. M. Aguinaldo and R. W. Read, Phytochemistry, 1990, 29, 2309–
2313; (c) H. Tsuneki, Y. You, N. Toyooka, T. Sasaoka, H. Nemoto, J.
A. Dani and I. Kimura, Biol. Pharm. Bull., 2005, 28, 611–614.
6 R. E. Schwartz, G. L. Helms, E. A. Bolessa, K. E. Wilson, R. A.
Giacobbe, J. S. Tkacz, G. F. Bills, J. M. Liesch, D. L. Zink, J. E. Cumtto,
B. Pramanik and J. C. Onishi, Tetrahedron, 1994, 50, 1675–1686.
7 I. Kuroda, M. Musman, I. I. Ohtani, T. Ichiba, J. Tanaka, D. G.
Gravalos and T. Higa, J. Nat. Prod., 2002, 65, 1505–1506.
15 According to ref. 16, the Staudinger-type reductive cyclization of 1,3-
azido nitrile 10 seemed to be feasible to give g-lactam 14. However, our
attempts to effect this reaction were not successful.
16 H. Ducatel, A. N. V. Nhien, S. Pilard and D. Postel, Synlett, 2006,
1875–1878.
17 For previous examples, see: (a) D. R. Williams, J. P. Reddy and G. S.
Amato, Tetrahedron Lett., 1994, 35, 6417–6420; (b) Y.-S. Hon, Y.-W. Liu
and C.-H. Hsieh, Tetrahedron, 2004, 60, 4837–4860; (c) M. Kawasaki, T.
Shinada, M. Hamada and Y. Ohfune, Org. Lett., 2005, 35, 4165–4167.
18 Intermolecular Staudinger ligation of an azide and a carboxylic acid to
form an amide group has been reported. See :(a) J. Garcia, F. Urpı and
´
J. Vilarrasa, Tetrahedron Lett., 1984, 25, 4841–4844; (b) F. Urpı and J.
´
Vilarrassa, Tetrahedron Lett., 1986, 27, 4623–4624.
19 A. Dureault, C. Greck and J.-C. Depezay, J. Carbohydr. Chem., 1990,
´
9, 121–123.
20 For selected examples, see: (a) C. Xiong, W. Wang and V. J. Hruby,
J. Org. Chem., 2002, 67, 3514–3517; (b) H. Konno, S. Aoyama, K.
Nosaka and K. Akaji, Synthesis, 2007, 23, 3666–3672; (c) F. Sarabia, S.
Chammaa, M. Garcia-Castro and F. Martin-Galvez, Chem. Commun.,
2009, 5763–5765.
21 We are aware of only two examples of the direct reductive cyclization
of 1,3-azido alcohol to azetidine. See: (a) I. Coldham, A. J. Collis, R. J.
Mould and R. E. Rathmell, J. Chem. Soc., Perkin Trans. 1, 1995, 2739–
2745; (b) S. Obika, J.-I. Andoh, M. Onoda, O. Nakagawa, A. Hiroto,
T. Sugimoto and T. Imanishi, Tetrahedron Lett., 2003, 44, 5267–5270.
22 For a review, see: W. Zheng, J. Kollmeyer, H. Symolon, A. Momin,
E. Munter, E. Wang, S. Kelly, J. C. Allegood, Y. Liu, Q. Peng, H.
Ramaraju, M. C. Sullards, M. Cabot and A. H. Jr. Merrill, Biochim.
Biophys. Acta, Biomembr., 2006, 1758, 1864–1884.
8 J. Kobayashi, J.-F. Cheng, M. Ishibashi, M. R. Wa¨lchli, S.
Yamamura and Y. Ohizumi, J. Chem. Soc., Perkin Trans. 1, 1991,
1135–1137.
23 (a) E. H. Ahn and J. Schroeder, J. Exp. Biol. Med., 2002, 227, 345–353;
(b) Y. Nagahara, T. Shinomiya, S. Kuroda, N. Kaneko, R. Nishio and
M. Ikekita, Cancer Sci., 2005, 96, 83–92.
9 (a) G. H. Senel and C. Karakaya, in BS-Biological Science and
Engineering, Sabanci Universitesi, 2006, pp 26–38; (b) C.-Y. Kwan,
P. H. M. Harrison, P. A. Duspara and E. E. Daniel, Jpn. J. Pharmacol.,
2001, 85, 234–240; (c) O. Kutuk, A. Pedrech, P. Harrison and H. Basaga,
Apoptosis, 2005, 10, 597–509; (d) V. Ledroit, C. Debitus, C. Lavaudb
and G. Massiota, Tetrahedron Lett., 2003, 44, 225–228; (e) D. Canals, D.
Mormeneo, G. Fabria`s, A. Llebaria, J. Casas and A. Delgado, Bioorg.
Med. Chem., 2009, 17, 235–241; (f) Y. Salma, E. Lafont, N. Therville,
S. Carpentier, M.-J. Bonnafe´, T. Levade, Y. Ge´nisson and N. Andrieu-
Abadie, Biochem. Pharmacol., 2009, 78, 477–485; (g) K. Ohshita, H.
Ishiyama, Y. Takahashi, J. Ito, Y. Mikamib and J. Kobayashia, Bioorg.
Med. Chem., 2007, 15, 4910–4916.
10 For representative examples, see: (a) M. Macchia, S. Barontini, S.
Bertini, V. D. Bussolo, S. Fogli, E. Giovannetti, E. Grossi, F. Minutolo
and R. Danesi, J. Med. Chem., 2001, 44, 3994–4000; (b) H.-J. Ha,
M. C. Hong, S. W. Ko, Y. W. Kim, W. K. Lee and J. Park, Bioorg.
Med. Chem. Lett., 2006, 16, 1880–1883; (c) A. M. Dougherty, F. E.
McDonald, D. C. Liotta, S. J. Moody, D. C. Pallas, C. D. Pack and A.
H. Merrill, Org. Lett., 2006, 8, 649–652; (d) V. Wascholowski and A.
Giannis, Angew. Chem., Int. Ed., 2006, 45, 827–830; (e) S. Hanessian,
24 (a) Y.-G. Ren, K. W. Wagner, D. A. Knee, P. Aza-Blanc, M. Nasoff and
Q. L. Deveraux, Mol. Biol. Cell, 2004, 15, 5064–5074; (b) S. Notebaert,
L. Duchateau and E. Meyer, Vet. Res., 2005, 36, 229–240.
25 T. D. Butters, R. A. Dwek and F. M. Platt, Curr. Top. Med. Chem.,
2003, 3, 561–574.
26 (a) Y. Y. Liu, T. Y. Han, A. E. Giuliano and M. C. Cabot, J. Biol.
Chem., 1999, 274, 1140–1146; (b) R. J. Bleicher and M. C. Cabot,
Biochim. Biophys. Acta, Mol. Cell Biol. Lipids, 2002, 1585, 172–178;
(c) C. P. Reynolds, B. J. Maurer and R. N. Kolesnick, Cancer Lett.,
2004, 206, 169–180; (d) V. Gouaze, Y. Y. Liu, C. S. Prickett, J. Y. Yu, A.
E. Giuliano and M. C. Cabot, Cancer Res., 2005, 65, 3861–3867.
27 F. Di Sano, S. Di Bartolomeo, B. Fazi, C. Fiorentini, P. Matarrese, A.
Spinedi and M. Piacentini, Cell Death Differ., 2002, 9, 693–695.
28 M. C. Alley, A. Dominic and R. H. Shoemaker, Cancer Res., 1988, 48,
589–601.
29 K. J. Livak and T. D. Schmittgen, Methods, 2001, 25, 402–408.
30 (a) B. J. Delley, Chem. Phys., 1990, 92, 508–517; (b) B. J. Delley, Chem.
Phys., 2000, 113, 7756–7764.
31 J. P. Perdew, K. Burke and M. Ernzerhof, Phys. Rev. Lett., 1996, 77,
3865–3868.
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