Skeletal Optimization of Cytotoxic Lipidic Dialkynylcarbinols

Article abstract of DOI:10.1002/cmdc.201800118

In line with a recent study of the pharmacological potential of bioinspired synthetic acetylenic lipids, after identification of the terminal dialkynylcarbinol (DAC) and butadiynyl alkynylcarbinol (BAC) moieties as functional antitumor pharmacophoric units, this work specifically addresses the issue of carbon backbone length. A systematic variation of the aliphatic chain length was thus carried out in both the DAC and BAC series. The critical impact of the length of the lipidic skeleton was first confirmed in the racemic series, with the highest cytotoxic activity observed for C₁₇ to C₁₈ backbones. Enantiomerically enriched samples were prepared by asymmetric synthesis of the optimal C₁₈ DAC and C₁₇ BAC derivatives. Samples with upgraded enantiomeric purity were alternatively produced by enzymatic kinetic resolution. Eutomers possessing the S configuration displayed cytotoxicity IC₅₀ values as low as 15 nm against HCT116 cancer cells, the highest level of activity reached to date in this series.

Full text of DOI:10.1002/cmdc.201800118

Accepted Article
Title: Skeletal optimization of antitumor lipidic dialkynylcarbinols
Authors: Maroua Bourkhis, Hafida Gaspard, Pauline Rullière, Diana
K. C. de Almeida, Dymytrii Listunov, Etienne Joly, Raoudha
Abderrahim, Marcos C. de Mattos, Maria C. F. de Oliveira,
Valérie Maraval, Remi Chauvin, and Yves Genisson
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To be cited as: ChemMedChem 10.1002/cmdc.201800118
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10.1002/cmdc.201800118
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Skeletal optimization of cytotoxic lipidic dialkynylcarbinols
Maroua Bourkhis,[a,b] Hafida Gaspard,[a] Pauline Rullière,[a] Diana K. C. de Almeida, [c] Dymytrii
Listunov,[a,d] Etienne Joly,[e] Raoudha Abderrahim,[b] Marcos C. de Mattos,[c] Maria C. F. de Oliveira,[c]
Valérie Maraval,[d] Remi Chauvin*[d] and Yves Génisson*[a]
[a]
Dr M. Bourkhis, Dr H. Gaspard, Dr P. Rullière, Dr D. Listunov, Dr. Y. Génisson
SPCMIB, UMR-CNRS 5068,
Université Paul Sabatier-Toulouse III
118 route de Narbonne, 31062 Toulouse Cedex 9, FRANCE
E-mail: genisson@chimie.ups-tlse.fr
[b]
[c]
Dr M. Bourkhis, Prof. R. Abderrahim
05/UR/13-01, LPMLNMH, Carthage University, Faculty of Sciences of Bizerte, 7021
Jarzouna, Tunisia
Dr. M. C. F. de Oliveira, Dr. M. C. de Mattos, D. K. C. de Almeida
Laboratory of Biotechnology and Organic Synthesis
Federal University of Ceará
Campus do Pici, Fortaleza, Ceará, BRAZIL
[d]
[e]
Dr D. Listunov, Dr V. Maraval, Prof. R. Chauvin
Department LCC, UPR-CNRS 8241
Université Paul Sabatier-Toulouse III
2205 route de Narbonne, 31077 Toulouse Cedex 4, FRANCE
E-mail: chauvin@lcc-toulouse.fr
Dr E. Joly
Institute of Pharmacology and Structural Biology
Department of Structural Biology and Biophysics
205 route de Narbonne, 31077 Toulouse Cedex 4, FRANCE
Supporting information for this article is given via a link at the end of the document.
Abstract: In line with a recent study of the pharmacological potential
of bio-inspired synthetic acetylenic lipids, after identification of the
terminal dialkynylcarbinol (DAC) and butadiynyl alkynylcarbinol
(BAC) moieties as functional antitumor pharmacophoric units, this
work specifically addresses the issue of the carbon backbone length.
A systematic variation of the aliphatic chain was thus carried out in
both the DAC and BAC series. The critical impact of the length of the
lipidic skeleton was first confirmed in the racemic series, with the
highest cytotoxic activity for C17 to C18 backbones.
Enantiomerically enriched samples were prepared by asymmetric
synthesis of the optimal C18 DAC and C17 BAC derivatives.
Samples with upgraded enantiomeric purity were alternatively
produced by enzymatic kinetic resolution. Eutomers possessing the
(S) configuration displayed IC50 of cytotoxicity against HCT 116
cancer cells down to 15 nM, the highest level of activity reached to
date in this series.
fully synthetic derivative (S)-2 was increased more than 100
times as compared to 1 (Fig. 1).
Two key structural alterations were (i) the inversion of
configuration of the carbinol center and (ii) the replacement of
the original internal double bond by a second triple bond. This
led to the identification of the (S)-configured terminal
dialkynylcarbinol (so-called DAC) unit as non-natural bioinspired
pharmacophore for antitumor activity (this series is viewed as
(R)-like in reference to the natural compound 1, the formal
retention of the S configuration being due to a change in CIP
priority).4 The third variable structural feature considered in the
initial study was the length of the linear lipidic skeleton. Indeed,
truncating the C20 backbone of the reference natural product 1
by three carbon atoms led to a six-fold increase in cytotoxicity in
both optical series.4
Introduction
Acetylenic lipids represent an unusual class of metabolites
combining
a circumscribed structural diversity with a rich
spectrum of biological activities, including anticancer properties.1
More than 100 such natural products were notably isolated from
various species of marine sponges over the past two decades.2
Whilst the presence of a terminal alkynylcarbinol unit has
previously been correlated with the antitumor activity of natural
representatives, it is only recently that the structure-activity
relationships of acetylenic lipids started being addressed in
detail.3 A systematic study based on the prototypical natural
product (S,E)-icos-4-en-1-yn-3-ol (1) isolated from the sponge
Cribrochalina vasculum was recently reported.4,5 Not only were
basic structural requirements for the cytotoxicity delineated, but,
as a result of several structural modifications, the potency of the
Figure 1. Natural alkynylcarbinol 15 and previously described synthetic
analogues 24 and 3.6
1
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The C17 skeleton, originally selected for the sake of availability
of the tetradecyne starting material, was retained as a standard
backbone for further structural modifications.7 More recently, the
extension of the pharmacophore was envisaged by means of an
ethynylogation approach.6 Insertion of an additional acetylenic
unit at the internal position of 2 gave rise to the butadiynyl
alkynylcarbinol (so-called BAC) function in the C19-derivative
(S)-3 displaying a promising IC50 value of 40 nM on the human
colon carcinoma cell line HCT 116. This result motivated a
systematic study of the impact of the linear carbon chain length
on the cytotoxic activity, with the aim of addressing two key
questions: is C17 the optimal length of the linear skeleton in the
DAC series? Is it also the case for BAC derivatives? A first set of
results along this line is presented below.
Scheme 2. Synthesis of racemic DACs 2, 6a-c and 6e-h.
With this series of eight racemic samples in hand, the impact of
aliphatic chain length on antitumor activity was evaluated by
measuring their IC50 of cytotoxicity at 72 h in HCT 116 colon
cancer cells (Table 1). A clear influence of the lipidic carbon
skeleton on the cytotoxic behavior was observed. In comparison
to the C17 derivative of reference (rac)-2, typically displaying an
IC50 of 150 nM, the cytotoxicity was found to decrease
dramatically upon shortening of the aliphatic chain. Chopping of
two methylene units led to a 30-fold loss in cytotoxicity, and the
IC50 was superior to 5 µM for the C14 derivative (rac)-6a (Table
1). Increasing the length of the acetylenic lipid backbone of 2
gave rise to a milder effect. A ca. 3-fold decrease in activity was
observed upon addition of 3 to 4 methylene fragments. On the
other hand, the direct homologue of the (rac)-2 reference
compound, the C18 lipidic DAC (rac)-6e, was found to be slightly
more cytotoxic with an IC50 value of 100 nM, suggesting some
room for improvement in the corresponding enantioenriched
form. Overall, this first part of the study confirmed that a carbon
skeleton of C17-C18 was indeed optimal in the DAC acetylenic
lipid sequence.
Results and Discussion
In order to assess the influence of the aliphatic backbone on
cytotoxicity, it was decided to first run the study with readily
accessible racemic samples of DAC and BAC lipids.
A
straightforward synthetic route was based on the use of the ynal
4 as precursor of the DAC terminus (Scheme 1). This substrate
can indeed undergo smooth addition of a variety of alkynyl
metals carrying the target lipophilic appendage. Such an
approach thus offers a unified access to both DAC and BAC
series.
Enantioselective dialkynylcarbinols synthesis. In order to
fully assess the cytotoxic activity of the C18 DAC derivative 6e,
enantio-enriched samples were prepared in both the (R) and (S)
series. Asymmetric synthesis of the targeted DACs was
accomplished using the previously developed modified Carreira
procedure for enantioselective addition of terminal alkynes onto
ynals 4.4,10 Treatment of 4 with pentadecyne in the presence of
Zn(OTf)2, Et3N and (+)- or (-)-N-methyl-ephedrine (NME) in
CH2Cl2 led to (S)- or (R)-5e, respectively, with high yields
(Scheme 3). Absolute configurations were assigned by analogy
with previous results,4,5 in agreement with Carreira’s
precedents.11
Scheme 1. Lipidic DACs and BACs retrosynthetic route.
Since the early reports of its preparation,8 numerous syntheses
of the ynal precursor 4 have been described.9 Aldehyde 4 can
be prepared by direct formylation of TMS-acetylene.8b,9a-h
A
robust alternative procedure, relying on the oxidation of a
propargylic alcohol precursor, was applied here.8a,9i-t
Enantiomeric excesses (e.e.) were measured by chiral
supercritical fluid chromatography (SFC) of the corresponding 1-
naphthyl carbamate derivatives. Final protodesilylation of the
enantio-enriched adducts (S)- and (R)-5e, was carried out
smoothly by the action of K2CO3 in MeOH. The sign of the
optical rotations of the DACs (R)-(-)- and (S)-(+)-6e was in
agreement with our previous work,4,5 while the absolute value
remained very low, as expected from the weak local chiral
character of the DAC center ([a]D < 10° for e.e. ≥ 80 %).
Racemic dialkynylcarbinols synthesis. Lipidic skeletons
varying by the progressive addition of one carbon from C14 to
C21 were first targeted in the DAC series in order to
systematically browse a large range of aliphatic chain lengths.
Synthesis of the DAC derivatives proceeded readily through a
two-step sequence starting from commercially available terminal
alkynes (see SI for experimental details). Lithiation with n-BuLi
followed by addition of the corresponding acetylides to ynal 4 led
to trimethylsilyl-protected DAC intermediates 5a-h in moderate
to high yields. These primary products were desilylated by
treatment with K2CO3 in MeOH to deliver the targets DAC
derivatives (Scheme 2).
Evaluation of the antitumor potency of the enantiomers (R)- and
(S)-6e confirmed the dramatic influence of the carbinol absolute
configuration on the cytotoxicity, revealing an eudismic ratio
(obtained by dividing the IC50 of the eutomer by that of the
distomer) of 0.06. In addition, the IC50 value of the eutomeric
C18 DAC (S)-6e reached 60 nM, comparing favorably with the
value of 90 nM observed in the same assay for (S)-2, the
eutomeric C17 reference compound.
2
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Table 1. Cytotoxic activity of racemic lipidic DACs in HCT 116 cancer cells.
Backbone
length
C14
(n = 8)
C15
(n = 9)
C16
(n = 10)
C17
(n = 11)
C18
(n = 12)
C19
(n = 13)
C20
(n = 14)
C21
(n = 15)
compound
IC50 (nM)[a]
(rac)-6a
(rac)-6b
(rac)-6c
(rac)-2
(rac)-6e
(rac)-6f
(rac)-6g
(rac)-6h
>5000
5000
500
150
100
200
500
400
[a] Cells were seeded in 96-well plates and were treated with concentrations ranging from 10 µM to 5 nM; after 72 h, cells were incubated with
the MTT reagent, and the absorbance was measured.
AgNO3-catalyzed bromination of the alkynes with NBS was
followed by Cu(I)-mediated cross coupling in Et2NH in the
presence of hydroxylamine. Deprotection of the terminal position
was finally ensured by elimination of acetone in alkaline
conditions. The expected 1,3-diynes 9a-f were thus obtained in
39-48% overall yields.
The stage was then set for the construction of the series of
racemic BAC lipids. Lithiation of the terminal 1,3-diynes 9a-f by
treatment with n-BuLi at low temperature and subsequent
addition of the resulting alkylacetylides onto the pivotal ynal 4
led to the C-silylated precursors 10a-f in 57-71 % yields
(Scheme 5). Use of the mild K2CO3/MeOH protodesilylation
conditions proved unexpectedly inadequate, giving rise to
extensive degradation in the BAC series. The sensitivity of the
reactant to basic medium can be attributed to the presence of
the 1,3-diyne moiety. On the other hand, alternative treatment
with AgNO3 in acetone/H2O smoothly delivered the desired
desilylated BACs 11a-d,f in 60-75 % yields (Scheme 5).
Scheme 3. Asymmetric synthesis of (R)- and (S)-6e.
Racemic butadiynyl alkynylcarbinols synthesis. The BAC
series, comprising the prototypical C19 derivative (S)-3
displaying a remarkable IC50 of 40 nM,6 was then considered. In
order to gain insights into the optimal carbon backbone length, a
series of racemic samples of varying chain lengths were, there
again, first secured according to the general synthetic route
described in scheme 1. Based on the observations made in the
DAC series, a smaller collection of six lipidic skeletons was
selected with a length ranging from C15 to C21, with a focus on
the C17-C18 region. The 1,3-diyne precursors 9a-f carrying the
target aliphatic appendage were readily obtained from the
corresponding terminal alkynes (Scheme 4) (see SI for
experimental details).
Scheme 5. Synthesis of racemic BACs 3, 11a-d and 11f.
The series of racemic BACs derivatives was subjected to
cytotoxicity evaluation in HCT 116 cancer cells (Table 2). A
milder effect of the carbon chain length on antitumor potency
was observed as compared to the previous DAC series, the
shorter C15 and the longer C21 derivatives displaying equivalent
cytotoxicity with an IC50 in the 220-250 nM range. Truncating the
lipid backbone by one carbon at a time from C19 to C17 led to a
gradual enhancement of potency, reaching an IC50 value of 60
nM for (rac)-11c. This finding indicated that C17 was the optimal
length for the lipidic BAC skeleton.
Enantioselective butadiynyl alkynylcarbinols synthesis. The
preparation of 11c in enantio-enriched form was thus envisioned.
Its enantioselective preparation paralleled that of the DAC
analogue 6e. The first example of asymmetric addition of a 1,3-
diyne nucleophile onto ynal using the modified Carreira’s
procedure was previously reported.6 Thanks to this precedent,
both enantiomers of the homologue 10c could be obtained, (+)-
Scheme 4. Synthesis of 1,3-diynes 9a-f.
A
three-step reaction sequence relying on the Cadiot-
Chodkiewicz coupling with 2-methylbut-3-yn-2-ol, used as a
protected form of acetylene, was implemented (Scheme 4).
3
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Table 2. Cytotoxic activity of racemic BAC derivatives in HCT 116 cancer cells.
Backbone
length
C15
(n = 7)
C16
(n = 8)
C17
(n = 9)
C18
(n = 10)
C19
(n = 11)
C21
(n = 12)
(rac)-11f
250
compound
IC50 (nM)[a]
(rac)-11a
(rac)-11b
(rac)-11c
(rac)-11d
(rac)-3
220
150
60
90
100
[a] Cells were seeded in 96-well plates and were treated with concentrations ranging from 10 µM to 5 nM; after
72 h, cells were incubated with MTT reagent, and the absorbance was measured.
or (-)-NME inducing the formation of the silylated (S)- or (R)-
selective trans-acetylation catalyzed by the Novozym 435 lipase
thus proved useful for the early synthesis of (S)-docosa-
(4E,15Z)-dien-1-yn-3-ol.12 A similar approach employing Lipase
AK (Amano) was later used to access (S)-eicos-(4E)-en-1-yn-3-
ol (1),13 both enantiomers of the two-headed compounds
adociacetylene,14 and duryne,15 and (S,S)-dideoxypetrosynol.16
Lipase PS (Amano) was also reported to catalyze an (R)-
selective acetylation, exploited for the synthesis and absolute
configuration determination of a new two-headed cytotoxic
metabolite from the marine sponge Petrosia sp. (4E,24E)-14-
BAC, respectively (Scheme 6). Treatment with AgNO3 in an
acetone/H2O mixture delivered the desilylated BACs (R)- or (S)-
11c, respectively. The optical rotations measured for (R)-(-)-
and (S)-(+)-11c were again in agreement with precedent results,
corroborating the assignment of the absolute configuration from
literature standards.
methyloctacosa-4,24-diene-1,27-diyne-3,26-diol.17
attempt to resolve a racemic DAC moiety by an enzymatic
method was reported in pioneering publication of
The only
a
Chattopadhyay and coll. who made use of a lipase from Candida
rugosa in the preparation of an enantio-enriched early
intermediate en route to (S)-eicos-(4E)-en-1-yn-3-ol (1)18 and the
non-natural 15E-isomer of (R,R)-duryne.19
In the present context, the DAC 2 was selected as a reference to
implement such a chemoenzymatic approach. In a preliminary
round of experiments, a series of lipases were screened for the
kinetic resolution of the acetate (rac)-2-Ac. Only three enzymes
out of the 12 tested gave a significant level of resolution, namely
Amano lipase PS (Burkholderia cepacia) immobilized in
diatomaceous earth, Thermomyces lanuginosus lipase and
Candida antartica lipase B (CAL B). The latter gave the optimal
results, allowing isolation of (R)-2 in 39% yield and 92% e.e. (as
determined by chiral SCF analysis of the corresponding 1-
naphthyl carbamate) and (S)-2-Ac in 40% yield with e.e. >99%
(as determined by chiral GC analysis) (Scheme 7) (see SI for
experimental details). Further saponification of (S)-2-Ac
(K2CO3/MeOH, 88% yield) afforded (S)-2 with 98.5% e.e. (as
determined by chiral SCF analysis of the corresponding 1-
naphthyl carbamate). Cytotoxicity tests indicated a value of IC50
of 40 nM for the enzymatically resolved sample of (S)-2 (vs. 3
µM for the corresponding (R)-2). Upgrading the e.e. led to a
more pronounced eudismic ratio reaching 0.013. The
corresponding (S)-2-Ac displayed an IC50 of 100 nM possibly
due to (S)-2 itself resulting from partial (~40 %) in cellulo
hydrolysis of the labile acetate in (S)-2-Ac.
Scheme 6. Enantioselective synthesis of BACs (R)-(-)- and (S)-(+)-11c.
For the sake of comparison, cytotoxicity experiments with the
C17 BAC (R)- and (S)-11c were there again carried out on the
HCT 116 cancer cell line. The distomeric (R)-11c displayed an
IC50 of cytotoxicity of 300 nM. Remarkably, an unprecedented
IC50 of 15 nM was found for the S enantiomer, corresponding to
an eudismic ratio of 0.05. This potency was higher than that of
the C19 reference compound (S)-3, displaying an IC50 of
cytotoxicity of 40 nM in the same bioassay.
The unprecedented level of cytotoxicity reached for (S)-11c,
compared to its enantiomer and the reference lipidic BAC (S)-3,
stimulated efforts for accessing DAC and BAC samples with
upgraded enantiomeric purity. Despite its practicality, the
asymmetric synthesis approach indeed delivers alkynylcarbinols
with e.e. not exceeding 90%.
Enzymatic kinetic resolution. The enzymatic kinetic resolution
of racemic samples was thus explored as an alternative strategy.
Such a chemoenzymatic approach has previously been used in
the total synthesis of several marine acetylenic lipids bearing the
naturally occurring alkenyl alkynylcarbinol (AAC) moiety. An (S)-
4
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Conclusions
Initial studies had shown that several simple structural
modifications were sufficient to enhance by two orders of
magnitude the cytotoxicity of the natural lipidic AAC (S,E)-1.
Fine optimization of the chiral alkynylcarbinol unit having led to
original dialkynyl- and butadiynyl-carbinol (respectively DAC and
BAC) pharmacophores, the lipidic backbone was still deserving
consideration. This first account confirms that the length of the
carbon skeleton is indeed critical, showing that the C17 to C18
backbone is optimal. In particular, the eutomeric C17 BAC
derivative (S)-11c revealed to be the most potent synthetic
derivative to date in this sequence of lipidic dialkynylcarbinols.
Scheme 7. Kinetic enzymatic resolution of (rac)-2-Ac.
The chemoenzymatic approach was then applied to the
preparation of the most cytotoxic BAC, (S)-11c (Scheme 8).
Treatment of the racemic mixture of the acetate (rac)-11c-Ac
with CAL B delivered the carbinol (R)-11c (44% yield, 96% e.e.,
as determined by the chiral SFC) along with the unreacted (S)-
11c-Ac (47% yield, 95% e.e., as determined by chiral GC
analysis) (see SI for experimental details).
Experimental Section
Chemistry
General procedure A: Carreira asymmetric alkynylation modified for
ynals. A flame dried flask was charged with Zn(OTf)2 (4 equiv) and (-)-
or (+)-N-methylephedrine (4 equiv) and dried under vacuum for 30 min.
After purging with argon, CH2Cl2 and triethylamine (4 equiv) were added.
The resulting mixture was vigorously stirred for 2 h at rt before addition in
one portion of 1-pentadecyne or tetradeca-1,3-diyne 9c (4 equiv). After 1
h stirring, 3-(trimethylsilyl)-2-propynal 4 (1 equiv) in CH2Cl2 was added,
and the mixture was stirred at rt overnight under inert atmosphere. After
treatment with a saturated aqueous NH4Cl solution, the aqueous layer
was extracted with CH2Cl2. The organic phase was then washed with
brine, dried over MgSO4 and concentrated under reduced pressure. The
crude product was purified by flash chromatography on silica gel to give
the expected enantioenriched DACs (R)- or (S)-5e or BACs (R)- or (S)-
10c.
Scheme 8. Kinetic enzymatic resolution of (rac)-11c-Ac.
In contrast to the DAC 2-Ac, the BAC 11c-Ac turned out to be
highly sensitive to standard saponification conditions. Exposure
to one equivalent of K2CO3 in MeOH led to rapid decomposition
(Scheme 9). Changing the base for NaHCO3 slowed down the
transformation and allowed identification of the unstable ynone
12 (obtained selectively as a 1.2:1 mixture with the expected
carbinol 11c), likely resulting from a base-induced isomerization
in allendiyne ester20 followed by the terminal addition of a
molecule of MeOH. Other hydrolysis conditions proved to be
non-productive (MeCN/NH4OH, Et2O/dilute aq. AcOH).
(
-
)-(3S)-1-(Trimethylsilyl)octadeca-1,4-diyn-3-ol
(S)-5e:
General
procedure A was applied to (+)-N-methylephedrine (179 mg, 1 mmol),
Zn(OTf)2 (363 mg, 1 mmol), 1-pentadecyne (0.25 mL, 1 mmol), Et3N
(0.13 mL, 1 mmol) and 3-(trimethylsilyl)-2-propynal 4 (32 mg, 0.25 mmol,
as an 80% solution in CH2Cl2). The crude product was purified by flash
chromatography on silica gel (petroleum ether/Et2O: 9/1; Rf 0.15) to give
(S)-5e (63 mg, 75% yield) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ
5.13 (t, J 2.1 Hz, 1H), 2.26 (td, J 7.1, 2.1 Hz, 2H), 2.13 (s, 1H), 1.61 –
1.50 (m, 2H), 1.46 – 1.26 (m, 20H), 0.90 (t, J 6.6 Hz, 3H), 0.22 (s,
9H).13C NMR (75 MHz, CDCl3) δ (C≡C) 102.57, (C≡C) 88.87, (C≡C)
85.89, (CH-OH) 52.82, (12 CH2) 31.95, 29.70, 29.69, 29.68, 29.66, 29.56,
29.38, 29.16, 28.89, 28.32, 22.72, 18.77, (CH3) 14.15, (3C-Si) -0.28.
-0.4 (c 1, CHCl3). HRMS-DCI (CH4) m/z [M+H]+
#$
"
Optical rotation
α
calcd for C21H39OSi: 335.2770, found: 335.2761. Enantiomeric excess
(ee 80%) was evaluated from the chiral SFC analysis of the
corresponding 1-naphtyl carbamate (S)-5’e (see SI).
Scheme 9. Attempt to saponify acetate (rac)-11c-Ac.
Finally, the enzymatic approach was attempted for the
hydrolysis of (S)-11c-Ac. Treatment of this acetate with CAL B
yielded the expected BAC (S)-11c in 77% yield and 94% e.e. (as
determined by chiral SCF analysis) (Scheme 8) (see SI for
experimental details). Enzymatically resolved BAC samples
were submitted to cell viability experiments. Unlike its DAC
congener (S)-2-Ac, the eutomeric BAC acetate (S)-11c-Ac
showed a moderate level of cytotoxicity (IC50 1000 nM) that may
be correlated to its observed chemical instability. The (S)-11c
however displayed an IC50 of 15 nM (vs. 1000 nM for the
distomeric (R)-11c, corresponding to an eudismic ration of
0.015) fully confirming the trends observed previously.
(+)-(3R)-1-(Trimethylsilyl)octadeca-1,4-diyn-3-ol
(R)-5e:
General
procedure A was applied to (-)-N-methylephedrine (358 mg, 2 mmol, 4
equiv), Zn(OTf)2 (727 mg, 2mmol), 1-pentadecyne (0.5 mL, 2 mmol),
Et3N (0.27 mL, 2mmol) and 3-(trimethylsilyl)-2-propynal 4 (63 mg, 0.5
mmol, as an 80% solution in CH2Cl2). The crude product was purified by
flash chromatography on silica gel (petroleum ether/Et2O: 9/1; Rf 0.15) to
1
give (R)-5e (66 mg, 80% yield) as a colorless oil. H NMR and 13C NMR
were identical to that of (S)-5e.
α
#$ +0.38 (c 1, CHCl3). Enantiomeric
"
excess (ee 90%) was evaluated from the chiral SFC analysis of the
corresponding 1-naphtyl carbamate (R)-5’e (see SI).
General procedure B: K2CO3-promoted protodesilylation in MeOH.
K2CO3 (1 equiv) was added to a solution of the TMS-protected DAC 5 (1
equiv) in MeOH (0.2 M). The solution was stirred at rt until complete
consumption of the starting material, as judged by TLC monitoring (about
5
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4 hours). After addition of brine, the aqueous layer was extracted with
CH2Cl2, dried over MgSO4 and concentrated under reduced pressure.
The crude product was purified by flash chromatography on silica gel to
give 6.
11c (40 mg, 59% yield) as an orange viscous product. 1H NMR (300 MHz,
CDCl3) δ 5.14 (s, 1H), 2.58 (d, J 2.3 Hz, 1H), 2.28 (t, J 6.4 Hz, 2H), 2.24
(br s, OH), 1.53 (m, 2H), 1.45 – 1.08 (m, 14H), 0.88 (t, J 6.7 Hz, 3H). 13
C
NMR (75 MHz, CDCl3) δ 83.42, 79.95, 73.23, 71.12, 70.13, 63.95, 52.38,
31.87, 29.53, 29.44, 29.28, 29.03, 28.79, 27.99, 22.66, 19.25, 14.10.
HRMS-DCI (CH4) m/z [M+H]+ calcd for C17H25O: 245.1905, found:
( )-(3R)-Octadeca-1,4-diyn-3-ol (R)-6e: The general procedure B was
-
#$
"
245.1901. α -8.5 (c 1.0, CHCl3). Chiral SFC analysis (Chiralpak IA-3
applied to (S)-1-(trimethylsilyl)octadeca-1,4-diyn-3-ol (S)-5e (22 mg, 0.06
mmol) and K2CO3 (8.3 mg, 0.06 mmol). Purification by flash
chromatography on silica gel (petroleum ether/Et2O: 9/1; Rf 0.15) gave
(R)-6e (15 mg, 93% yield) as a white solid. 1H NMR (300 MHz, CDCl3) δ
5.10 (d, J 2.2 Hz, 1H), 2.54 (d, J 2.3 Hz, 1H), 2.22 (td, J 7.1, 2.1 Hz, 2H),
1.98 (br s, OH), 1.59 – 1.44 (m, 2H), 1.37 (m, 2H), 1.25 (s, 18H), 0.88 (t,
J 6.7 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (CꢀC) 86.20, (CꢀC) 81.47,
µm (4.6x100 mm), CO2/MeOH, 1.5 mL/min, PDA detector (l 242 nm), rt
(R)-11c 3.05 min and (S)-11c 3.19 min), ee 84%.
(+)-(3S)-heptadeca-1,4,6-triyn-3-ol (S)-11c: The general procedure C
was applied to (+)-(3R)-1-(trimethylsilyl)heptadeca-1,4,6-triyn-3-ol (R)-
10c (75 mg, 0.24 mmol) and AgNO3 (4 mg, 0.024 mmol). Purification by
flash chromatography on silica gel (pentane/Et2O: 8/2; Rf 0.15) gave (S)-
(CꢀC) 76.91, (CꢀC) 72.17, (CH-OH) 52.4, (12 CH2) 31.91, 29.67, 29.64,
29.60, 29.48, 29.34, 29.09, 28.85, 28.25, 22.68, 18.66, (CH3) 14.11. α
11c (30 mg, 51% yield) as an orange viscous product. 1H NMR and 13C
#$
"
- 3.4 (c 1.1, CHCl3). HRMS-DCI (CH4) m/z [M+H]+ calcd for C18H31O:
NMR were identical to that of heptadeca-1,4,6-triyn-3-ol (R)-11c. α
+
"
#$
263.2375, found: 263.2374.
6.9 (c 1.1, CHCl3). Chiral SFC analysis (Chiralpak IA-3 µm (4.6x100 mm),
CO2/MeOH, 1.5 mL/min, PDA detector (l 242 nm), rt (R)-11c 3.13 min
and (S)-11c 3.18 min), ee 84%.
(+)-(3S)-Octadeca-1,4-diyn-3-ol (S)-6e: The general procedure B was
applied to (R)-1-(trimethylsilyl)octadeca-1,4-diyn-3-ol (R)-5e (50 mg, 0.15
mmol) and K2CO3 (21 mg, 0.15 mmol). Purification by flash
chromatography on silica gel (petroleum ether/Et2O: 9/1; Rf 0.15) gave
(S)-6e (35 mg, 90% yield) as a white solid. 1H NMR and 13C NMR were
identical to that of (R)-6e. α +3.3 (c 1.2, CHCl3).
Biology
Cytotoxicity: The drugs' cytotoxicity was determined by standard MTT
tests on HCT116 cells. In brief, 5.000 HCT116 cells were distributed in
96 flat bottom well plates in 100 µL of DMEM 10% FCS and 200
microliters in the column destined to receive the drug’s highest
concentrations, i.e. 10 µM. 2 µL of DMSO containing the drugs at 1 mM
were then added to each well of that first column. For each drug,
duplicates of concentrations ranging from 10 µM to 5 nM were carried out
by means of 12 successive 2-fold dilutions using a multi-channel pipette
to pass 100 µl to the wells in the adjacent column and mixing thoroughly
by pipetting up and down each time. Assays were performed 3 times
independently, and controls always included dilutions of the reference
drug (S)-2 (IC50 80-100 nM). Plates were then placed in a CO2 tissue
culture incubator for 72 h before the MTT test was performed. This was
done by adding 10 µL of MTT stock solution (12 µM, 5 mg/ml in PBS,
Sigma) to each well and incubating the plate for 90 min at 37 °C. 100 µl
isopropanol, 0.1 M HCl were then added to each well, and the plates
were returned to 37 °C for 90 min before reading the OD at 570nM.
#$
"
(
-
)-(3S)-1-(trimethylsilyl)heptadeca-1,4,6-triyn-3-ol
(S)-10c:
The
general procedure A was applied to (+)-N-methylephedrine (376 mg, 2.1
mmol, 4 equiv), Zn(OTf)2 (763 mg, 2.1 mmol), tetradeca-1,3-diyne 9c (0.4
g, 2.1 mmol), Et3N (0.3 mL, 2.1 mmol) and 3-(trimethylsilyl)-2-propynal 4
(133 mg, 0.525 mmol, 1 equiv, as a 50% solution in CH2Cl2). The crude
product was purified by flash chromatography on silica gel (petroleum
ether/Et2O: 95/5; Rf 0.1) to give (S)-10c (96 mg, 58% yield) as an orange
oil. 1H NMR (300 MHz, CDCl3) δ 5.17 (d, J 6.8 Hz, 1H), 2.38 – 2.22 (m,
3H), 1.65 – 1.49 (m, 2H), 1.47 – 1.29 (m, 14H), 0.97 – 0.86 (m, 3H), 0.22
(s, 9H).13C NMR (75 MHz, CDCl3) δ (C≡C) 100.73, (C≡C) 90.25, (C≡C)
83.18, (C≡C) 71.62, (C≡C) 69.84, (C≡C) 64.20, (CH-OH) 53.00, (9 CH2)
31.91, 29.57, 29.48, 29.32, 29.08, 28.85, 28.07, 22.70, 19.31,
(CH3)14.14, (3 C-Si) -0.36. α #$ -10.1 (c 1.0, CHCl3). HRMS-DCI (CH4)
"
m/z [M+H]+ calcd for C20H33OSi: 317.2301, found: 317.2296. The
enantiomeric excess (ee 83%) was evaluated by the chiral SCF analysis
of (R)-11c (vide infra).
Acknowledgements
(+)-(3R)-1-(trimethylsilyl)heptadeca-1,4,6-triyn-3-ol (R)-10c: General
procedure A was applied to (-)-N-methylephedrine (376 mg, 2.1 mmol, 4
eqiuv), Zn(OTf)2 (763 mg, 2.1 mmol), tetradeca-1,3-diyne 9c (0.4 g, 2.1
mmol), Et3N (0.3 mL, 2.1 mmol) and 3-(trimethylsilyl)-2-propynal 4 (133
mg, 0.525 mmol, 1 equiv, as a 50% in CH2Cl2). The crude product was
purified by flash chromatography on silica gel (petroleum ether/Et2O:
95/5; Rf 0.1) to give (R)-10c (79 mg, 48% yield) as a white oil. 1H NMR
and 13C NMR were identical to that of 1-(trimethylsilyl)heptadeca-1,4,6-
I. Fabing, T. Castellan and the Integrated Screening Platform of
Toulouse (PICT) are warmly thanked for the chiral SFC analyses.
We are thankful to the Institute of Chemistry of Toulouse (ICT–
FR CNRS 2599) for IR, mass spectrometry and NMR analyses.
The government of Tunisia is acknowledged for a mobility grant
to M.B. The French Embassy in Kiev, Ukraine, is acknowledged
for a contribution to the fellowship of D.L., his investigations
having been performed within the framework of the GDRI
(Groupement Franco-Ukrainien en Chimie Moléculaire) funded
by the CNRS. P.R. is grateful to ARC foundation for cancer
research for postdoctoral funding. M.C.F.O. and M.C.M. thank
the Brazilian Funding Agency CNPq for providing their research
fellowship.
triyn-3-ol (S)-10c.
α
#$ +10.2 (c 1.0, CHCl3). The enantiomeric excess
"
(ee 84%) was evaluated by the chiral SCF analysis of (S)-11c (vide infra).
General procedure C: AgNO3-catalyzed protodesilylation in acetone.
AgNO3 (0.1 equiv) was added to a solution of TMS-protected BAC 10 (1
equiv) in acetone (0.1 M) followed by one drop of water. The mixture was
stirred until TLC monitoring showed disappearance of the starting
material (ca. 3 h) and then quenched with a saturated aqueous NaCl
solution. The aqueous layer was extracted with diethyl ether and the
combined organic layers were dried over Na2SO4 and concentrated
under reduced pressure. The crude product was purified by flash
chromatography on silica gel to give the corresponding butadiynyl
alkynylcarbinols 11.
Keywords: alkynol • marine lipid • asymmetric synthesis •
cytotoxicity • polyacetylene • enzymatic kinetic resolution
References:
( )-(3R)-heptadeca-1,4,6-triyn-3-ol (R)-11c: The general procedure C
-
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10c (90 mg, 0.28 mmol) and AgNO3 (5 mg, 0.028 mmol). Purification by
flash chromatography on silica gel (pentane/Et2O: 8/2; Rf 0.15) gave (R)-
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