Synthesis of fluorinated brassinosteroids based on alkene cross-metathesis and preliminary biological assessment

Article abstract of DOI:10.1021/jm900495f

Three types of brassinosteroid analogues with perfluoroalkylated side chains were synthesized by using alkene cross-metathesis of a brassinosteroid derivative bearing a terminal alkene moiety with different (perfluoroalkyl) propenes. The presence of the d

Full text of DOI:10.1021/jm900495f

J. Med. Chem. 2009, 52, 5753–5757 5753
DOI: 10.1021/jm900495f
Synthesis of Fluorinated Brassinosteroids Based on Alkene Cross-Metathesis and
Preliminary Biological Assessment
ꢀ †,‡
ꢀ ‡
ꢁ
kova, Milos Budesı
ꢁꢁ
ꢀ ‡
ꢁ
ꢀ ‡
´
nsky, Blanka Klepetarova, Eva St’astna, and
ꢀꢁ ꢀ ‡
ꢀ ‡
ꢁ
Barbara Eignerova, Barbora Slavı
´
´
nsky, Martin Dracı
Martin Kotora*,†,‡
†Department of Organic and Nuclear Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague 2, Czech Republic,
and ‡Institute of Organic Chemistry and Biochemistry AS CR, Flemingovo naꢀm. 2, 166 10 Prague 6, Czech Republic
Received April 20, 2009
Three types of brassinosteroid analogues with perfluoroalkylated side chains were synthesized by using
alkene cross-metathesis of a brassinosteroid derivative bearing a terminal alkene moiety with different
(perfluoroalkyl)propenes. The presence of the double bonds in the cross-metathesis products allowed a facile
one-step double dihydroxylation to provide intermediates that after Baeyer-Villiger oxidation afforded the
target compounds. Biological activity of the prepared analogues was tested in GABAA receptor, cytotoxic,
and brassinolide activity, which reached in some cases the same range as their nonfluorinated analogues.
Introduction
In this regard, we aimed at two goals: the synthesis of a
modified molecule and the development of a suitable synthetic
methodology. As for the former, the synthesis of brassinoster-
oids bearing fluorinated side chains such as 1a-1c (Figure 1)
was expected to afford a new type of brassinosteroid deriva-
tives resistant to the above-mentioned undesirable side reac-
tion with increased metabolic stability,4,12,13 and perhaps
better biological activity. As for the latter, to demonstrate
that cross-metathesis of terminal alkenes with perfluoroalkyl-
propenes is a convenient method for synthesis of brassinoster-
oids bearing perfluoroalkylated side chains.
Fluorine-containing compounds have been an ever increas-
ing target of the pharmaceutical and specialty chemicals
industry.1-3 This stems from the fact that the presence of
fluorine or fluorinated functional groups with their unique
properties gives the target molecules special (desirable) prop-
erties (e.g., metabolic stability, increased lipophilicity, etc.).4
Brassinosteroidsare animportantclass of plant hormone with
many potential applications in agrochemistry due to their
ability to stimulate growth of plants under undesirable con-
ditions.5,6 In addition, some exerted unexpected antiviral and
cancerostatic activity.7-10 However, they are easily inacti-
vated, among other reactions, by the conversion to more
hydroxylated derivatives.11 The site of such a hydroxylation
has been found in the side chain, e.g., in position 26. Several
papers describe metabolic accumulation of C-26-, C-28-hy-
droxylated, or further oxidized products. Because the meta-
bolic stability of the C-F bond is very high, fluorine-
containing brassinosteroids would have practical significance
because of a good stability of products. This suggestion was
confirmed by synthesis and metabolic stability studies of their
monofluoroderivatives.12 Similar results were obtained for
brassinosteroids bearing perfluoroalkylated ester side chains
instead of the classical sterol one. They were found to possess
the activity comparable to natural brassinosteroids.13
Results and Discussion
Synthesis. The underlying strategy was based on the
following assumption: because the brassinolide side chain
contains a 1,2-diol moiety, it could be conveniently prepared
by dihydroxylation of a suitable unsaturated intermediate,
which in turn could be prepared by cross-metathesis of a
terminal alkene with the appropriately substituted perfluor-
oalkylpropene (Scheme 1).
The starting substrate 3 was prepared by standard syn-
thetic methodology in six steps from commercially available
carboxylic acid 2 (Scheme 2).29 Ester 3 was then reduced by
LiAlH4 to alcohol 4 (88%), which was oxidized by using
Dess-Martin reagent to aldehyde 5 (76%),30 whose Wittig
olefination afforded alkene 6 (93%).31 Deprotection of its
carbonyl group under acidic conditions afforded the re-
quired alkene 7 in good 96% isolated yield. With the alkene
7 on hand, cross-metatheses with perfluorohexyl- 8a, per-
fluoropropyl- 8b, and perfluoroisopropylpropene 8c (2-fold
molar excess) were carried out. On the basis of previous
results, the reaction was catalyzed by Hoveyda-Grubbs
second-generation catalyst32,33 (10 mol %) in refluxing
dichloromethane for 4 h.28 In all cases the cross-metathesis
proceeded smoothly to give the corresponding perfluor-
oalkylated products 9a-9c in good 67, 71, and 59% isolated
yields, respectively. According to NMR analysis, com-
pounds 9a and 9b were obtained as pure trans double-bond
isomers; only in the case of 9c a 19/1 trans/cis mixture was
Despite considerable advances in perfluoroalkylation meth-
odology,14-16 the search for new more synthetically flexible pro-
cedures is highly desirable. One such attractive process is highly
selective alkene cross-metathesis under mild reaction condi-
tions17 that has been utilized in the synthesis of a number of fluo-
rinated compounds.18-27 Recently, we have also reported that
the perfluoroalkylation could be achieved by cross-metathesis
with perfluoroalkylpropenes28 that showed higher reactivity,
selectivity, and efficiency in comparison with other methods.
*To whom correspondence should be addressed. Phone: þ420 221
951 334. Fax: þ420 221 951 326. E-mail: kotora@natur.cuni.cz. Ad-
dress: Martin Kotora, Department of Organic and Nuclear Chemistry,
Faculty of Science, Charles University in Prague, Hlavova 8, 128 43
Prague 2, Czech Republic.
r
2009 American Chemical Society
Published on Web 08/31/2009
pubs.acs.org/jmc

5754 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
Eignerovaꢀ et al.
obtained. The structure of trans-9c was unequivocally con-
firmed by a single crystal X-ray analysis.
the cyclohexene ring, an oxidizing agent is approaching the
double bond from the less hindered side, i.e., from the
bottom side of the molecule, and in the case of the side chain
double bond it is controlled by the presence of the center of
chirality on C-20. Although the formation of other possible
diastereoisomers can not be excluded, they were not detected
in the reaction mixture.
Finally, the synthesis was completed by Baeyer-Villiger
oxidation of 11a-11c by trifluoroperacetic acid (prepared by
mixing trifluoroacetic anhydride and hydrogen peroxide in
dichloromethane) under ambient conditions. In each case,
the oxidation afforded a mixture of two regioisomeric lac-
tones in 4:1 ratio in favor of the desired regioisomers 1a-1c
with natural configuration of diol moiety in the side chain.
The desired brassinosteroids 1a-1c were isolated by pre-
parative HPLC in 62, 70, and 61% yields, respectively.
Biology. Because the brassinosteroids are known to possess
various biological activities, the newly attained compounds,
1a-1c and 11a-11c, constituted ideal substrates for testing in
various assays because of their new structural and previously
unexplored feature, a perfluoroalkylated side chain.
Because compounds 9a-9c possess two double bonds
within the molecule, simultaneous dihydroxylation was at-
tempted. The hydroxylation of the double bonds was carried
out by catalytic amount of OsO4 (15 mol %) and excess
N-methyl morpholine N-oxide (3.5 fold excess).13 Initial
hydroxylation of 9a for 2 h led only to a 1/1.5 mixture of
10a and 11a in 50% isolated yield. The previous findings
clearly demonstrated that dihydroxylation takes place pre-
ferentially on the more electron-rich double bond in the
cyclohexane ring. To achieve full conversion, the hydroxyla-
tion time was prolonged to 16 h. Under these conditions,
9a-9c were fully converted to tetraols 11a-11c that were
isolated as single diastereoisomers in good isolated yields of
68, 50, and 46%, respectively. The observed dihydroxylation
diastereoselectivity could be explained as follows: in case of
GABAA Receptors Activity. Initially, the binding of the
compounds 1a-1c to GABAA receptors was tested in vitro
using membranes isolated from the brains of male Wistar
rats. The specific steroid binding was detected by the de-
crease in the binding of 2 nM [35S]-tert-butylbicyclo-[2.2.2]
phosphorothionate (TBPS) after application of the tested
compounds incubated for 1 h at 37 °C. The results (see
Table 1) could be summarized as follows: the heptafluoro
derivative 1c compares favorably to natural hormone allo-
pregnanolone 12 and its higher metabolic stability (with
respect to potential hydroxylation of the side chain)4,12,13
should more than compensate for its slightly lower GABA-
like activity. The results are in agreement with structural
similarity of 1c and 28-norbrassinolide 13 (Figure 2).13
Compound 1a, which does not contain the steroidal i-octyl
side chain, is active at a higher concentration only and
compound 1b is inactive.
Figure 1. Brassinosteroids 1a-1c withperfluoroalkylated sidechain.
Scheme 1. Construction of Perfluoroalkylated 28-Norbrassi-
nosteroid Side Chain
Scheme 2. Synthesis of Brassinosteroids with Perfluoroalkylated Side Chains

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5755
Table 1. Modulatory Effect on GABAA Receptors
35S]-TBPS (%)a
Table 2. Cytotoxic Activity (IC50) of Brassinosteroids Determined by
Calcein-AM Assaysa
compd
[
Imax(%)b
IC50 (nM)c
80
900
compd
CEMb (μM)
MCF 7c (μM)
allopregnanolone 12
56.2 ( 6.0*
47.2 ( 15.1*
95.1 ( 14.8
56.6 ( 14.6*
79.0
57.9
28-homocastasterone 14
1a
13 ( 2.8
>50
>50
>50
>50
1a
1b
1c
d
d
1b
1c
>50
>50
59.4
100
>50
>50
a The values of samples containing tested compounds at 100 nM
concentrations were related to these of control samples with the buffer
and expressed in %. Data are presented as means ( SD (obtained from
triplicates or quadruplicates). To estimate statistical significance, non-
parametric Kruskal-Wallis test for a global comparison (χ2(4)=13.95,
p=0.0074) and Mann-Whitney-Wilcoxon test for pairwise compar-
isons (it was calculated with respect to the control samples, *p < 0.050).
b The maximal suppression of the binding. c the steroid concentration
producing a half-maximal inhibition were estimated using 1 nM TO
10 μM concentrations (in duplicates) of compounds. d Not determined.
11a
11b
11c
>50
48.2 ( 0.6
>50
35.3 ( 1.6
>50
a The IC50 values are expressed as mean ( SD values of three
independent experiments performed in triplicate. b T-lymphoblastic
leukemia cell line CEM. c Breast carcinoma cell lines MCF-7.
Table 3. Activity in the Bean Second-Internode Bioassay
PSIa
compd
SD
24-epibrassinolide 15
1a
32.3
3.1
(5.7
(1.1
(3.7
(0.3
(4.1
(3.1
(4.9
1b
1c
11.0
0.9
11a
11b
11c
14.1
9.6
11.6
a PSI: Difference of prolongation of the Second Internode SD (mm) at
concentration 10-10 mol L-1 to control.
3
easy access to a new type of brassinosteroids with perfluor-
oalkylated side chains. The flexibility of our approach per-
mits, in principle, introduction of any kind of side chains not
only on the brassinosteroid but also other frameworks and
provides a powerful tool for an analogue development and for
an elucidation of biological functions.
The preliminary biological testing showed that some of the
prepared brassinosteroids with fluorinated side chain exhibit
the GABAA activity comparable with the endogeneous neuro-
steroid allopregnanolone. On the other hand, the anticancer
activity was insignificant. Furthermore, the results of the
brassinolide activity of the prepared perfluoroalkylated
compounds were in the range of previously tested nonfluori-
nated analogues. These studies thus open not only new
possibilities for the synthesis new functionalized brassinoster-
oid analogues but also could serve as a general strategy for the
preparation of other classes of compounds bearing perfluori-
nated side chain.
Figure 2. Structures of allopregnanolone 12, 28-norbrassinolide
13, 28-homocastasterone 14, and 24-epibrassinolide 15.
Anticancer Activity. Brassinosteroids are also known to
exhibit anticancer activity.7,8,10 The cytotoxic activity of
1a-1c was determined by comparing human normal
(fibroblast BJ) and cancer cell lines (T-lymphoblastic leuke-
mia CEM and breast carcinoma MCF 7). These were
exposed to six serial 4-fold dilutions of each drug for 72 h,
the proportions of surviving cells were then estimated, and
IC50 values were calculated (28-homocastasterone was used
as a positive control). Unfortunately, only 11b exhibited
slight activity against CEM cell line (IC50 = 35.3 μM)
(see Table 2). The other tested compounds such as 1a-1c,
11a, and 11c had extremely weak or no detectable activity
(IC50 > 50 μM). However, it is important to emphasize that
tested compounds are not toxic toward normal human cells
at all.
Experimental Section
General. All solvents were used as obtained unless otherwise
noted. THF was distilled from sodium and benzophenone.
Perfluoroalkylated propenes 8a, 8b, and 8c (spectral character-
istics were in agreement with the previously reported data)35-37
were prepared according to the previously reported procedure.38
All other reagents were obtained from commercial sources. The
NMR spectra were measured on Bruker AVANCE 500 and 600
instruments (1H at 500 or 600 MHz, 13C at 125.7 or 150.9 MHz,
and 19F NMR at 470.3 MHz) as solutions in CDCl3 at 27 °C
unless otherwise noted. Chemical shifts are given in δ scale (1H
NMR spectra were referenced to TMS as an internal standard,
13C NMR spectra to CDCl3 at δ 77.0, and 19F NMR to C6F6
δ -163.0), coupling constants J are given in Hz. Melting points
(uncorrected) were determined using a Kofler apparatus. Infra-
red spectra were recorded as CHCl3 solutions or as KBr tablets
and are reported in wave numbers (cm-1). The HPLC system
used consisted of a high pressure pump (model 361, Gilson),
Rheodyne valve, preparative column (10 mm ꢀ 250 mm) with
silica gel filling (Biosher PSI 200 7micro-m, Labio), preparative
ELSD detector (Gilson) connected with PC (software Trilution
Brassinolide-Type Activity. Last but not least, brassinolide
activity was measured by the bean second-internode bioas-
say.13,34 The length of the second internodes was measured 5
days after the application of tested compounds in lanoline
and the difference in length between treated and control
plants provided a measure of activity (see Table 3). It was
found that compound 1b exhibited expressive swelling of the
second bean internode at concentration 10-7 mol L-1
.
Moreover, compound 1c exhibited surprising activity at
3
lower and higher concentrations differing by 5 orders (10-7
and 10-12 mol L-1, þ15.9 and þ10.7 mm, respectively).
3
Conclusion
The synthetic route based on the cross-metathesis between
terminal alkenes and perfluoroalkylpropenes constitutes an

5756 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
Eignerovaꢀ et al.
LC, Gilson), and automatic fraction collector (model 346, Gilson).
Purity of the prepared compounds was determined by a combina-
tion of 1H NMR and by HLPC techniques and was >95%.
General Procedure for Cross-Metathesis of 7 with (Perfluoro-
alkyl)propenes 8. To a mixture of a terminal alkene 7 (1 mmol) and
(perfluoroalkyl)propenes 8a-8c (2mmol) inCH2Cl2 (8mL) under
Ar atmosphere was added Hoveyda-Grubbs second-generation
catalyst (63 mg, 0.1 mmol) and it was stirred at 42 °C for 4 h. Eva-
poration of the volatiles under reduced pressure followed by column
chromatography on silica gel afforded expected compounds.
(20S)-20-(40,40,50,50,60,60,70,70,80,80,90,90,90-Tridecafluoronon-
10-en-10-yl)-5r-pregn-2-en-6-one (9a). The reaction was carried
out with 7 (250 mg, 0.77 mmol) and 8a (551 mg, 1.53 mmol)
according to the general procedure. Column chromatography
on silica gel (50/1 hexane/EtOAc) and crystallization (MeOH)
yielded 338 mg (67%) of the title compound 9a as white
(20S,10R,20R)-2r,3r,10,20-Tetrahydroxy-20-(40,40,50,50,60,60,60-
heptafluorohex-10-yl)-5r-pregnan-6-one (11b). The reaction was
carried out with 9b (190 mg, 0.37 mmol). Column chromatogra-
phy on silica gel (1/2 hexane/EtOAc) afforded 107 mg (50%) of
the title compound 11b as white crystals: mp 176-178 °C
20
(acetone/heptane); [R]D -23.9 (c 0.13, CHCl3). 1H NMR
(600 MHz, CD3OD) δ 0.75 (s, 3H), 0.77 (s, 3H), 1.10 (d, J=
6.6 Hz, 3H), 2.40 (m, 2H), 2.74 (bdd, J=11.5, 3.4 Hz, 1H), 3.44
(dd, J=4.9, 1.5 Hz, 1H), 3.66 (ddd, J=11.8, 4.8, 3.0 Hz, 1H), 3.95
(bq, J=3.0, 1H), 4.22 (ddd, J=7.8, 3.9, 1.5 Hz, 1H). HR-MS
(ESI) calcd for C27H40O5F7 [Mþ þ 1] 577.2758, found 577.2759.
Rf (20/1 toluene/EtOAc) = 0.23.
(20S,22R,23R)-2r,3r,22,23-Tetrahydroxy-25,26,26,26,27,27,
27-heptafluoro-cholestan-6-one (11c). The reaction was carried
out with 9c (190 mg, 0.37 mmol). Column chromatography on
silica gel (1/2 hexane/EtOAc) afforded 98 mg (46%) of the title
compound 11c as white crystalsand 11 mg (5%) of the compound
cis-10c as a colorless oil. 11c: mp 153-154 °C (acetone/heptane);
20
needles: mp 101-102 °C (MeOH); [R]D þ6.4 (c 0.22, CHCl3).
1H NMR (600 MHz, CDCl3) δ 0.70 (s, 3H), 0.72 (s, 3H), 1.05
(d, J=6.6 Hz, 3H), 2.75 (m, 2H), 5.32 (dt, J=15.2, 7.2 Hz, 1H),
5.55 (ddt, J = 15.3, 8.8, 1.3 Hz, 1H), 5.57 (m, 1H), 5.69 (m, 1H).
HR-MS (EI) calcd. for C30H35OF13 [Mþ] 658.2480, found
658.2485. Rf (20/1 hexane/EtOAc) = 0.24.
1
[R]D20 -12.0 (c 0.16, CHCl3). H NMR (600 MHz, CD3OD) δ
0.74 (s, 3H), 0.77 (s, 3H), 1.09 (d, J=6.6 Hz, 3H), 2.42 (m, 2H),
2.74 (ddd, J=12.5, 3.4, 1.0 Hz, 1H), 3.41 (dd, J=5.0, 1.5 Hz, 1H),
3.66(ddd, J=11.8, 4.8, 3.0 Hz, 1H), 3.95 (bq, J=3.0 Hz, 1H), 4.16
(bd, J=7.8 Hz, 1H). HR-MS (ESI) calcd. for C27H40O5F7 [Mþ þ
1] 577.2758, found 577.2759. Rf (20/1 toluene/EtOAc) = 0.23.
General Procedure for Baeyer-Villiger Oxidation.39 A solu-
tion of trifluoroperoxyacetic acid in dichloromethane (20 mL),
prepared from trifluoroacetic anhydride (3.23 g, 8.24 mmol) and
30% hydrogen peroxide (0.5 mL, 4.8 mmol), was added to a
solution of ketone 11 (2 mmol) in dichloromethane (16 mL) and
stirred for 4 h. Then the reaction mixture was poured into a 10%
KHCO3 solution (200 mL), extracted with CHCl3 (3 ꢀ 150 mL),
the combined organic extracts washed with water (200 mL), and
dried over anhydrous MgSO4. Evaporation of the volatiles fol-
lowed by column chromatography on silica gel (6/1 EtOAc/hexane)
afforded 4/1 mixture of regioisomeric lactones 11/110. Further
preparative HPLC (6/1 EtOAc/hexane) yielded target compounds.
(20S,10R,20R)-2r,3r,10,20-Tetrahydroxy-7-oxa-7a-homo-20-(40,
40,50,50,60,60,70,70,80,80,90,90,90-tridecafluoronon-10-yl)-5r-preg-nan-
6-one (1a). The reaction was carried out with 11a (95 mg, 0.13
mmol) and trifluoroperoxyacetic acid (2 mL). Column chroma-
tography followed by HPLC afforded 60 mg (62%) of the title
(20S)-20-(40,40,50,50,60,60,60-Heptafluorohex-10-en-10-yl)-5r-
pregn-2-en-6-one (9b). The reaction was carried out with 7
(360 mg, 1.1 mmol) and 8b (463 mg, 2.2 mmol) according to
the general procedure. Column chromatography on silica gel
(50/1 hexane/EtOAc) and crystallization (MeOH) yielded 397
mg (71%) of the title compound 9b as white crystals: mp
117-119 °C (MeOH); [R]D þ12.9 (c 0.20, CHCl3). 1H NMR
20
(600 MHz, CDCl3) δ 0.70 (s, 3H), 0.72 (s, 3H), 1.05 (d, J=6.6
Hz, 3H), 2.75 (m, 2H), 5.32 (dt, J=15.3, 7.1 Hz, 1H), 5.55 (ddt,
J=15.3, 8.8, 1.2 Hz, 1H), 5.57 (m, 1H), 5.69 (m, 1H). HR-MS
(ESI) calcd. for C27H36OF7 [Mþ þ 1] 509.2649, found
509.2650. Rf (20/1 hexane/EtOAc)=0.24.
22-(E)-(20S)-25,26,26,26,27,27,27-Heptafluoro-cholesta-2,22-
dien-6-one (9c). The reaction was carried out with 7 (340 mg, 1.04
mmol) and 8c (440 mg, 2.0 mmol) according to the general
procedure. Column chromatography on silica gel (50/1 hexane/
EtOAc) and crystallization (MeOH) yielded 315 mg (59%) of the
title compound 9c as white crystals: mp 125-126 °C (MeOH);
[R]D þ19.5 (c 0.17, CHCl3). 1H NMR (600 MHz, CDCl3)
20
20
compound 1a as a colorless oil: [R]D þ7.5 (c 0.11, CHCl3). 1H
δ 0.69 (s, 3H), 0.71 (s, 3H), 1.03 (d, J=6.7 Hz, 3H), 2.77 (bdd, J=
20.0, 7.0 Hz, 2H), 5.30 (bdt, J=15.1, 7.0 Hz, 1H), 5.52 (bdd, J=
15.1, 8.8 Hz, 1H), 5.57 (m, 1H), 5.69 (m, 1H). HR-MS (ESI)
calcd for C27H36OF7 [Mþ þ 1] 509.2649, found 509.2649. Rf (20/
1 hexane/EtOAc)=0.24.
NMR (600 MHz, CDCl3) δ 0.73 (s, 3H), 0.92 (s, 3H), 1.05 (d, J =
7.0 Hz, 3H), 2.48 (m, 1H), 3.12 (dd, J=12.3, 4.5 Hz, 1H), 3.51 (dd,
J=4.7, 1.6 Hz, 1H), 3.72 (ddd, J=12.1, 4.7, 2.8 Hz,1H), 4.03 (bq,
J =3.0 Hz, 1H), 4.08 (m, 2H), 4.28 (ddd, J = 7.9, 3.9, 1.6Hz, 1H).
HR-MS (ESI) calcd for C30H38O6F13 [Mþ - 1] 741.2466, found
741.2446. Rf (6/1 EtOAc/hexane)=0.16.
General Procedure for Dihydroxylation.13 A solution of OsO4
(13 mg, 0.05 mmol) in 2-methyl-propan-2-ol (0.12 mL) was added
to a solution of olefin 9 (0.35 mmol) in acetone (8 mL) and
tetrahydrofuran (8 mL). Next, N-methylmorpholine N-oxide
(140 mg, 1.2 mmol) in water (0.2 mL) was added. The mixture
was stirred under Ar atmosphere for 16 h at room temperature. A
solution of sodium sulfite (5 mL, 10%) was then added and the
mixture was stirred for 30 min, poured into water, and extrac-
ted with chloroform. Column chromatography on silica gel (1/2
hexane/EtOAc) and crystallization (heptane/acetone) yielded
compounds 11a-11c as a white crystals.
(20S,10R,20R)-2r,3r,10,20-Tetrahydroxy-7-oxa-7a-homo-20-(40,
40,50,50,60,60,60-heptafluorohex-10-yl)-5r-pregnan-6-one (1b). The
reaction was carried out with 11b (70 mg, 0.12 mmol) and
trifluoroperoxyacetic acid (2 mL). Column chromatography fol-
lowed by HPLC afforded 51 mg (70%) of the title compound1b as
20
a colorless oil: [R]D þ7.0 (c 0.14, CHCl3). 1H NMR (600 MHz,
CDCl3) δ 0.73 (s, 3H), 0.92 (s, 3H), 1.05 (d, J=7.0 Hz, 3H), 2.48
(m, 1H), 3.12 (dd, J=12.3, 4.5 Hz, 1H), 3.51 (dd, J=4.7, 1.6 Hz,
1H), 3.72 (ddd, J=12.1, 4.7, 2.8 Hz, 1H), 4.03 (bq, J=3.0 Hz, 1H),
4.08 (m, 2H), 4.28 (ddd, J=7.9, 3.9, 1.6 Hz, 1H). HR-MS (ESI)
calcd for C27H38O6F7 [Mþ - 1] 591.2562, found 591.2550. Rf (6/1
EtOAc/hexane)=0.16.
(20S,10R,20R)-2r,3r,10,20-Tetrahydroxy-20-(40,40,50,50,60,60,70,
70,80,80,90,90,90-tridecafluoronon-10-yl)-5r-pregnan-6-one (11a).
The reaction was carried out with 9a (115 mg, 0.17 mmol).
Column chromatography on silica gel (1/2 hexane/EtOAc)
afforded 86 mg (68%) of the title compound 11a as white
(20S,22R,23R)-2r,3r,22,23-Tetrahydroxy-7-oxa-7a-homo-
25,26,26,26,27,27,27-heptafluoro-cholestan-6-one (1c). The re-
action was carried out with 11c (85 mg, 0.15 mmol) and
trifluoroperoxyacetic acid (2 mL). Column chromatography
followed by HPLC afforded 54 mg (62%) of the title com-
20
crystals: mp 220-221 °C (acetone/heptane); [R]D -1.9
(c 0.11, MeOH). 1H NMR (600 MHz, CD3OD) δ 0.70 (s, 3H),
0.76 (s, 3H), 1.06 (d, J=6.8 Hz, 3H), 2.48 (m, 1H), 2.69 (ddd,
J = 12.6, 3.4, 0.8 Hz, 1H), 3.52 (dd, J = 4.6, 1.7 Hz, 1H),
3.77 (ddd, J=11.8, 4.8, 3.2 Hz, 1H), 4.06 (q, J=3.3 Hz, 1H),
4.30 (ddd, J= 8.0, 3.8, 1.7 Hz, 1H). HR-MS (ESI) calcd for
C30H39O5F13Na [Mþ þ Na] 749.2482, found 749.2476. Rf (2/3
toluene/EtOAc)=0.23.
20
pound 1c as a colorless oil: [R]D þ29.4 (c 0.07, MeOH).
1H NMR (600 MHz, CDCl3) δ 0.73 (s, 3H), 0.92 (s, 3H),
1.04 (d, J = 7.0 Hz, 3H), 2.75 (m, 1H), 3.12 (dd, J = 12.3,
4.5 Hz, 1H), 3.46 (dd, J=4.7, 1.7 Hz, 1H), 3.72 (ddd, J=12.1,
4.7, 2.7 Hz, 1H), 4.03 (bq, J=3.0 Hz, 1H), 4.09 (m, 2H), 4.21

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5757
(m, 1H). HR-MS (ESI) calcd for C27H38O6F7 [Mþ - 1]
591.2562, found 591.2547. Rf (6/1 EtOAc/hexane)=0.16.
Biological Evaluation. GABAA receptor binding assay,40,41
calcein-AM cytotoxicity assay,10 and the Bean second-inter-
node bioassay13,34 were carried out according to previously
reported methods (for details, see Supporting Information).
of I(CF2)nI homologues (n = 1-3); perfluoroalkylation of arenas
by RFI or [RFCO2]2; RFI in the synthesis of RFSR and segmented
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2001, 108, 147–175.
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Total syntheses of conformationally locked difluorinated pento-
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Org. Chem. 2007, 72, 1575–1587.
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Smith, C. A. Synthesis of 4,4-difluoroglycosides using ring-closing
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(20) Thibaudeau, S.; Fuller, R.; Gouverneur, V. Stereoselective synthesis
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Tetrahedron Lett. 2004, 45, 57–60.
(24) De Matteis, V.; van Delft, F. L.; de Gelder, R.; Tiebes, J.; Rutjes, F.
P. J. T. Fluorinated (hetero)cycles via ring-closing metathesis of
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(25) For an early example, see Chatterjee, A. K.; Morgan, J. P.; Scholl,
M.; Grubbs, R. H. Synthesis of functionalized olefins by cross and
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Acknowledgment. This work was supported by the grant
project IAA400550609 and the research projects Z40550506,
LC06070, LC06077, and MSM0021620857 from the Ministry
of Education of the Czech Republic. The authors are indebted
to Mgr. Oklestkova-Swaczynova (Laboratory of Growth
Regulators, Palacky University Olomouc, Czech Republic)
and Dr. Kristofikova (Prague Psychiatric Centre, Prague,
Czech Republic) for testing of the prepared compounds.
Supporting Information Available: All experimental proce-
dures, detailed compound characterization data, biological
evaluation methods, copies of spectra, and crystallographic
information file. This material is available free of charge via
the Internet at http://pubs.acs.org.
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