Brassinosteroids: Synthesis and activity of some fluoro analogues

Article abstract of DOI:10.1021/jm800085p

Three types of 5α-androstane and ergostane analogues of brassinolide, containing a fluorine atom in either the 3α or the 5α positions or in 3α and 5α positions, were prepared using standard operations (reaction of 3β-alcohols with (diethylamino)sulfur trifluoride, cleavage of epoxide with HF in py or BF₃·Et₂O). The 5α-fluorine was found to affect chemical reactivity (e.g., electrophilic addition to the Δ²-double bond) as well as physical properties (e.g., NMR, chromatographic behavior) of the products. Cytotoxicity of the products was studied using human normal and cancer cell lines with 28-homocastasterone as positive control and their brassinolide type activity was established using the bean second-internode test with 24-epibrassinolide as standard. The equivalence of F and OH groups was observed in some of the active compounds. The anticancer and the brassinolide-type activity do not correlate with each other: ergostane derivatives were most active in the former test while androstane derivatives were best in the latter.

Full text of DOI:10.1021/jm800085p

J. Med. Chem. 2008, 51, 3979–3984
3979
Brassinosteroids: Synthesis and Activity of Some Fluoro Analogues
Barbora Slavikova,† Ladislav Kohout,† Milos Budesinsky,† Jana Swaczynova,‡ and Alexander Kasal*,†
Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic, Fleming Square 2,
166 10 Prague 6, Czech Republic, Laboratory of Growth Regulators, Palacky UniVersity & Institute of Experimental Botany ASCR,
Slechtitelu 11, 783 71 Olomouc, Czech Republic
ReceiVed January 29, 2008
Three types of 5R-androstane and ergostane analogues of brassinolide, containing a fluorine atom in either
the 3R or the 5R positions or in 3R and 5R positions, were prepared using standard operations (reaction of
3ꢀ-alcohols with (diethylamino)sulfur trifluoride, cleavage of epoxide with HF in py or BF3 · Et2O). The
5R-fluorine was found to affect chemical reactivity (e.g., electrophilic addition to the ∆2-double bond) as
well as physical properties (e.g., NMR, chromatographic behavior) of the products. Cytotoxicity of the
products was studied using human normal and cancer cell lines with 28-homocastasterone as positive control
and their brassinolide type activity was established using the bean second-internode test with 24-epibrassinolide
as standard. The equivalence of F and OH groups was observed in some of the active compounds. The
anticancer and the brassinolide-type activity do not correlate with each other: ergostane derivatives were
most active in the former test while androstane derivatives were best in the latter.
Introduction
Brassinosteroids are plant hormones with many potential
applications in agriculture due to their ability to stimulate growth
of plants even under unsuitable conditions (lack of irrigation,
nutrients, inadequate temperature).1,2 Steroids were several times
found to act on other than expected sites.3,4 Thus some brass-
inosteroids also exert surprising antiviral and cancerostatic
activities even in human cells.5,6 The effect of brassinolide on
androgen-independent human prostate cancer PC-3 cell was
established: brassinolide (1, see Figure 1) was found to induce
a time and concentration-dependent cytotoxicity in PC-3 cells.
The mode of cell death appeared to be predominately apoptosis.
Western blot studies indicated that treatment with brassinolide
triggered a time-dependent decrease in the expression of
antiapoptotic protein Bcl-2. Some other members of the brassi-
nolide family (24-epibrassinolide and 28-homocastasterone,
compounds 2 and 3) were found7 to inhibit the growth, at
micromolar concentrations, of several human cancer cell lines
without affecting the growth of normal cells. In addition,
antiviral activity against herpes simplex virus types I and II
Figure 1
(selectivity index against HSV-1 > 78.5) was reported for
brassinosteroid derivatives.8,9
Electronegative substituents12,13 (a hydroxyl group, fluorine)
were also introduced to position 5R, leading to the active com-
pounds such as 6 and 7. Because the fluorine atom often14
behaves as a hydroxyl isoster, and the metabolic stability of
the C-F bond is much higher than that of the C-OH bond,
analogues of brassinosteroids were sought in which the 3R
hydroxyl was replaced with the 3R-fluorine (compound 8). This
would have its practical significance because good stability of
products applied is invaluable and the brassinosteroid metabo-
lism15 is known to start with oxidation of the 3R hydroxyl,
leading to inactive 3-ketones.
In another development, many structural changes were made
to the side chain of brassinosteroids. Recently, compounds
lacking the classical sterol side chain, e.g., compound 9, which
basically is ester of a 5R-androstane derivative,16 were also
found to have an activity comparable to natural brassinosteroids.
In this paper, we describe the preparation and properties of
two types of epicastasterone analogues with the fluorine atom
in positions 3R or 5R or both. The first type contains a 17ꢀ-
The most active brassinosteroid, brassinolide (1), was isolated
and later synthesized10 in many complicated and hence uneco-
nomic ways. More easily accessible analogues have been there-
fore sought to replace brassinolide: often used and studied 24-
epibrassinolide (2) is easily available from ergosterol although
its activity is slightly lower than that of brassinolide. Equally
active 28-homobrassinolide (4) was produced from commercial
sitosterol; it even retains the important (S) configuration at C-24.
Besides many others, the brassinosteroid family11 also includes
active compounds that contain a 6-oxo group instead of the
lactone ring (e.g., castasterone, 5).
* To whom correspondence should be addressed. Phone: +420 220 183
314. Fax: +420 220 183 578. E-mail: kasal@uochb.cas.cz. Address: RNDr.
Alexander Kasal, DSc., Fleming Square 2, CZ 166 10 Prague 6, Czech
Republic.
†
Institute of Organic Chemistry and Biochemistry of the Academy of
Sciences of the Czech Republic.
‡
Laboratory of Growth Regulators, Palacky University & Institute of
Experimental Botany ASCR.
10.1021/jm800085p CCC: $40.75
2008 American Chemical Society
Published on Web 06/17/2008

3980 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
SlaVikoVa et al.
Scheme 1a
mixture of acetates 33 and 34. Alternatively, the triol 31 was
prepared by dihydroxylation of alcohol 35 and separation of its
mixture with compound 36.
The structure of all products prepared was confirmed by IR
and 1H NMR spectra (see Experimental Section). The inversion
of configuration at the fluorination of 3ꢀ-alcohols to 3R-fluorides
is manifested by a multiplet pattern of the H-3 signal in their
1H NMR spectra: instead of a broad multiplet of axial H-3 in
3ꢀ-alcohol (W ∼ 35 Hz; a triplet of triplets with two large diaxial
and two small axial-equatorial couplings), the 3R-fluorides
display an equatorial H-3 proton as a doublet of multiplets at
∼4.90 ppm that arise from a large geminal coupling (J(F,H) ∼
48 Hz) and four small vicinal interactions (J(H,H) ) 2.5-4
Hz). The presence of fluorine at 5R-position in compounds
24-36 is manifested in 1H NMR spectra by the additional
splitting of H-7R and H-7ꢀ protons (∼2.60 and ∼2.25 ppm)
with fluorine (J(F,H-7R) ∼ 7.5 Hz and J(F,H-7ꢀ) ∼ 3.5 Hz).
NMR Analysis. The presence of fluorine can be observed
either directly in 19F NMR spectra and/or indirectly as the
additional splitting of signals in 1H and 13C NMR spectra. The
identification of fluorine-coupled hydrogens and carbon atoms
requires a complete structural assignment of 1H and 13C NMR
signals. For such a detailed NMR analysis, we have chosen 3R-
fluoro derivatives 10, 20, and 21, 5R-fluoro derivatives 25, 33,
and 34, and 3R,5R-difluoro derivative 27. The usual combination
of homo- and heteronuclear 2D-NMR spectra (H,H-COSY, H,H-
ROESY, H,C-HSQC, and H,C-HMBC) was used for structural
hydroxy or 17ꢀ-acyloxy group, the second has the epibrassi-
nolide side chain.
Results
1
assignment and the H and 13C NMR data are summarized in
Synthesis. The simplest fluoro analogue of this series, com-
pound 10 (see Scheme 1), was prepared from the known17
i-steroid 11: the treatment with sulfuric acid, followed by the
reaction of intermediate 3ꢀ-alcohol 12 with (diethylamino)sulfur
trifluoride (DAST)12 afforded the 3R-fluoro compound 13, and
on deprotection, the target compound 10. Alternatively, i-steroid
14 was first converted into 3ꢀ-alcohol and 3R -fluoride 15 and
16, respectively; the 17-oxo group was then selectively reduced
with sodium borohydride in pyridine, yielding the above 17ꢀ-
alcohol 10.
The same sequence was repeated with 3R,5-cyclo-5R-ergost-
22-en-6-one (17), which was converted into 3ꢀ-hydroxy ketone
18. DAST was again used to produce the corresponding fluoride
19. Osmium tetroxide catalyzed dihydroxylation of the ∆22-
double bond produced an unseparable mixture of (22S,23S)-
and (22R,23R)-diols 20 and 21, respectively. Surprisingly, both
isomers had identical chromatographic properties on silica gel
and a HPLC column. They could, however, be distinguished
and characterized by NMR spectra (vide infra).
Our synthetic approach to 5R-fluoro analogues was based on
Ramirez’s experience18 with a hydrogen fluoride-pyridine
complex opening of 5ꢀ,6ꢀ-epoxides: thus compound 22 yielded
fluoro alcohol 23 (ref 19, see Scheme 2); alternative reaction
with boron trifluoride etherate afforded alcohol 23 in the same
yield (91%).
The Jones oxidation of the fluorohydrin 23 yielded ketone
24 that was partially hydrolyzed with one equivalent of
potassium carbonate to give monoacetate 25 (76%, the corre-
sponding diol 26 was only isolated in 22%). The 3ꢀ-alcohol 25
was either treated with DAST to produce 3R-fluoride 27 or
tosylated and solvolyzed with sodium nitrite in HMPA to yield
the corresponding 3R-hydroxy derivative 29. Hydrolysis of the
protecting acetoxy group in compounds 27 and 29 led to
analogues 28 and 30.
Supporting Information Tables 1 and 2.
Fluorine signals were observed as multiplets in 19F proton
coupled NMR spectra, and their chemical shifts are given in
Supporting Information Table 3. There is a clear difference in
chemical shifts between fluorine at the secondary position 3
(-177 to -184 ppm) and the tertiary position 5 (-154 to -160
ppm).
While the J(F,C) couplings were obtained from the doublets
observed for fluorine-coupled carbons in a 13C NMR spectrum,
the analysis of J(F,H) interactions from proton multiplets in a
1
1D H NMR spectrum is difficult, particularly for protons in
the crowded upfield region. Therefore, we used 2D-hetero-
nuclear 19F-1H-COSY spectroscopy20 for identification of fluo-
rine coupled proton signals.
The typical J(F,H) observed in 3R-fluoro-, 5R-fluoro-, and
3R,5R-difluoro derivatives for the selected compounds 10, 25,
and 27 are shown in Figure 2. The largest values were found
for geminal 2J(F-3,H-3) ∼ 48 Hz and the vicinal diaxial
3J(Fax,Hax) ) 43.8-45.6 Hz, while vicinal axial-equatorial
3J(Fax,Heq) were in the range 7.5-14 Hz. Fluorine at position
5R showed additional long-range couplings 4J(F,H-7R) ∼ 8 Hz,
4
4J(F,H-7ꢀ) ∼ 3.5 Hz, and J(F,H-19) ∼1.5 Hz.
The J(F,C) values in the selected compounds 10, 25, and 27
are shown in Figure 2. The largest values were found for one-
bond interactions (1J(C,F) ∼ 165-180 Hz), which are followed
by geminal coupling values (2J(F,C) ) 19-27 Hz). Vicinal
coupling values were found much smaller (3J(F,C) ) 0-5.8
Hz).
Biological Part. Anticancer Activity. Anticancer activity was
determined by comparing human normal (fibroblast BJ) and
cancer cell lines (T-lymphoblastic leukemia CEM, breast carci-
noma MCF 7, and multiple myeloma RPMI 8226). Cells of all
of these lines were exposed to six serial 4-fold dilutions of each
drugs for 72 h, and the proportions of surviving cells were then
estimated and IC50 values were calculated. The results, obtained
The last analogue, 5-fluoro-2R,3R,17ꢀ-trihydroxy-5R-an-
drostan-6-one (31) was obtained using OsO4 dihydroxylation
of the ∆2-unsaturated acetate 32 and hydrolysis of the 2:3

Brassinosteroids: Some Fluoro Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3981
Figure 2. The observed J(F,H) and J(F,C) (indicated with arrows) are shown in partial structures of 3R-fluoro, 5R-fluoro, and 3R,5R-difluoro
derivatives 8, 23, and 25.
Table 1. Cytotoxic Effect (IC50) of Brassinosteroids Determined by
Calcein-AM Assays
from calcein-AM assays, are presented in Table 1. 28-Ho-
mocastasterone was used as a positive control.
cell linea
Brassinolide-Type Activity. Brassinolide-type activity was
measured by the bean second-internode bioassay21 modified by
us16 (see Table 2). Compounds causing maximal elongation
above 20 mm are considered promising.
compound
CEMb
MCF 7c
RPMI 8226d
BJe
(µM)
(µM)
(µM)
(µM)
3
13 ( 2.8
>50
>50
26 ( 1.4
>50
>50
10
12
13
16
18
19
20, 21
25
26
27
28
29
30
31
32
33
34
35
36
>50
>50
>50
>50
>50
>50
Discussion
>50
>50
26.4 ( 0.1
>50
13.8 ( 2.3
>50
>50
>50
>50
>50
27.0 ( 0.04
>50
>50
Chemistry. All the reaction steps were carried out in a
standard manner. Surprisingly, the presence of the strongly
electronegative fluorine atom in position 5 exerted its effect on
the reactivity of 3ꢀ-alcohols (e.g., 12) with DAST: the yield of
the 3R-fluorides sought (e.g., 10) was lower than that of products
of elimination, ∆2-olefins (e.g., 32), which lack the strong 1,3-
diaxial interaction between substituents in positions 3R and 5R.
Another effect of the 5R-fluorine substitution was observed
in the osmium catalyzed dihydroxylation of olefin 32: while in
nonfluorinated substrates the R-attack, producing 2R,3R-diols,
markedly prevails, in the fluoro derivative 32, the 2ꢀ,3ꢀ-diol,
again lacking the strong 1,3-diaxial interactions, is the major
product. A similar effect of an electronegative substituent (OH
group) in position 5 on the ratio of substitution/elimination was
already observed by Spanish authors.13
Biological Activity. The 3R-fluoro analogues of 24-epicastas-
terone (20, 21) exhibit the same or better antiproliferative acti-
vity against the four tested cell lines (IC50 13.8-45.9 mM, see
Table 1) as the 3R-hydroxyl containing 28-homocastasterone
(3) and other natural brassinosteroids.7 The 3R,5R-difluoride
27 is only slightly active against the leukemia cell line; other
cell lines were not significantly affected.
>50
>50
40.4 ( 3.9
>50
42 ( 2.3
>50
25.7 ( 4.3
>50
23.5 ( 3.5
>50
45.9 ( 0.3
>50
>50
>50
>50
42.9 ( 4.9
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
45.4 ( 0.08
>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. d Multiple myeloma
RPMI 8226. e Human fibroblast BJ.
dihydroxy grouping, however, is rather an analogue of ecdysone.
This fact relates to the finding of ecdysteroids in men with severe
pathological conditions, which was once suggested as a potential
clinical marker.22
The surprising finding of a lower activity in the 3ꢀ-hydroxy
analogue 18 and complete inactivity of the 3R-fluoro analogue
19 may be ascribed to low solubility of compounds with a long
and lipophilic side chain.
Similarly, the 5R-fluoro ketone 36 was only marginally active
against multiple myeloma; this compound with a typical 2ꢀ,3ꢀ-
The others tested compounds had extremely weak or no
detectable activity, even when tested in concentrations of up to
50 µM.
The Brassinolide-Type Activity. The above fluoro steroids
exhibited surprisingly high activity (see Table 2), which negates
the generally accepted essentiality of the 3R hydroxyl group

3982 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Table 2. Activity in the Bean Second-Internode Bioassay
SlaVikoVa et al.
the highest brassinolide-type activity among the androstane23
derivatives (30).
prolongation of the second
internode SD (mm) at
compound
concentration 0-10 mol·L-1
SD
Experimental Section
10
12
13
16
18
19
20, 21
25
26
27
28
29
30
31
32
33
34
35
36
5.9
3.8
0.8
14.9
9.4
9.9
13.2
17.1
16.9
19.8
18.4
21.4
43.4
19.5
21.8
12.3
21.4
13.6
21.8
(2.5
(1.9
(0.7
(1.2
(0.7
(4.3
(5.1
(0.9
(6.7
(2.5
(2.3
(2.3
(0.9
(4.9
(3.0
(2.4
(4.1
(2.1
(2.9
3r-Fluoro-17ꢀ-hydroxy-5r-androstan-6-one (10). (1) From
Benzoate 13. Compound 13 (500 mg, 1.21 mmol) was added to a
solution of potassium carbonate in ethanol (5%, 50 mL) and the
reaction mixture was heated to reflux for 2 h. The solution was
concentrated to a quarter of its original volume and diluted with
brine. The product was extracted with chloroform and worked up
as usual (see Supporting Information), yielding Compound 10 as
white solid (240 mg, 64%); mp 189-193 °C (acetone/heptane),
[R]D -15.6 (c 0.26). IR: 3614, 1066 (OH), 1706 (CO), 991 (C-F).
For 1H, 13C, and 19F NMR data, see Supporting Information Tables
1-3. Anal. (C19H29FO2) C, H.
(2) From Diketone 16. A solution of sodium borohydride in
pyridine (1 mL, 0.65 mmol) was added to a solution of diketone
16 (120 mg, 0.39 mmol) in pyridine (2 mL) at -10 °C. After
standing at rt for 72 h, the reaction mixture was poured into dilute
hydrochloric acid, the product was extracted with chloroform, and
then worked up as usual. PLC chromatography yielded the product
10 (26 mg, 21%), identical with the sample prepared above.
3ꢀ-Hydroxy-6-oxo-5r-androstan-17ꢀ-yl Benzoate (12). Fol-
lowing the method A (see Supporting Information), i-steroid 1117
(3.31 g, 8.46 mmol) yielded compound 12 as white crystals (3.10
g, 90%); mp 232-236 °C (acetone), [R]D +9.5 (c 0.23). IR: 3609,
1054 (OH), 1710 (CO), 1280, 978 (C-O), 1603, 1451 (arom.). 1H
NMR δ: 0.79 (s, 3H, H-18), 0.94 (s, 3H, H-19), 3.58 (m, 1H, W ∼
35 Hz, H-3), 4.90 (t, 1H, J ) 8.5 Hz, H-17), 7.48 (m, 3H, aromatic
protons), and (8.05 m, 2H, aromatic protons). Anal. (C26H34O4)
C, H.
Scheme 2a
3r-Fluoro-6-oxo-5r-androstan-17ꢀ-yl Benzoate (13). Alcohol
12 (100 mg, 0.24 mmol) was treated with DAST according to the
method B. A little of 6-oxo-androst-2-ene-17ꢀ-yl benzoate was
found in a NMR spectrum of the product, therefore, the mixture
was treated with 3-chloroperoxybenzoic acid in dichloromethane
and the admixture was oxidized to a more polar derivative. White
crystals of compound 13 (37 mg, 37%) were obtained by PLC on
three plates (toluene/ethyl acetate, 7:3); mp 196-198 °C (acetone/
heptane), [R]D +9.7 (c 0.30). IR: 1710 (CO), 1279 (C-O), 1602,
1452 (arom), 991 (C-F). 1H NMR δ: 0.77 (s, 3H, H-18), 0.94 (s,
3H, H-19), 4.90 (t, 1H, J ) 7.8 Hz, H-17), 4.92 (dm, 1H, J(F,H-3)
) 48 Hz, H-3), 7.48 (m, 3H, aromatic protons), and 8.05 (m, 2H,
aromatic protons). Anal. (C26H33FO3) C, H.
3ꢀ-Hydroxy-5r-androstane-6,17-dione (15). i-Steroid 1424,25
(3.6 g, 12.57 mmol) was converted into a white solid 15 (2.9 g,
76%) according to the method A; mp 176-178 °C (acetone/heptane,
ref 25, records the same value), [R]D +57.7 (c 0.5, ref 25, gives
+58). 1H NMR δ: 0.79 (s, 3H, H-18), 0.88 (s, 3H, H-19), 3.59 (tt,
1H, J ) 11.2 and 4.7 Hz, H-3).
3r-Fluoro-5r-androstane-6,17-dione (16). The 3ꢀ-alcohol 15
(100 mg, 0.3 mmol) was converted into 3R-fluoro steroid 16
according to the method B. The product was treated with 3-chlo-
roperoxybenzoic acid as above and purified by PLC (3 plates,
toluene/ethyl acetate, 7:3) to yield compound 16 as a white solid
(44 mg, 44%); mp 211-213 °C (methanol), [R]D +50.3 (c 0.11).
IR: 1735, 1710 (CO), 989 (C-F). 1H NMR δ: 0.77 (s, 3H, H-18),
0.88 (s, 3H, H-19), 4.92 (dm, 1H, J(F,H-3) ) 48 Hz, H-3). Anal.
(C19H27FO2) C, H.
for this type of activity.12 The most active compounds were
5R-fluoro ketones 30 and related compounds 27-32, the effect
of the additional 3R-fluorine being insignificant. The effect of
the sole fluorine in the 3R position was lower than that in the
5R position (10 < 30).
The above results prove the equivalence of 3R-F and 3R-
OH groups in this respect, which, combined with the known
perfect metabolic stability of the C-F bond, shows that 3R-
fluorinated analogues of brassinolide can lead to products useful
for practical application. Both the activities studied, however,
do not correlate with each other: the highest anticancer activity
was found in the ergostane derivatives (compounds 20 and 21),
(22E,24R)-3ꢀ-Hydroxy-5r-ergost-22-en-6-one (18). i-Steroid
1726 (200 mg, 0.5 mmol) was converted into 3ꢀ-alcohol 1827,28
according to the method A. A white precipitate of compound 18
(200 mg, 95%) crystallized from methanol; mp 186-187 °C, (refs
1
27, 28 records the same value), [R]D -33.1 (c 0.5). H NMR δ:
0.68 (s, 3H, H-18), 0.76 (s, 3H, H-19), 0.82 (d, 3H, J ) 6.5 Hz,
H-26), 0.83 (d, 3H, J ) 7.0 Hz, H-27), 0.91 (d, 3H, J ) 6.4 Hz,
H-21), 1.01 (d, 3H, J ) 6.2 Hz, H-28), 3.58 (m, 1H, W ∼ 35 Hz,
H-3), 5.18 (m, 2H, H-23 and H-24).
(22E,24R)-3r-Fluoro-5r-ergost-22-en-6-one (19). The 3ꢀ-alco-
hol 18 (210 g, 0.5 mmol) was treated with DAST according to the

Brassinosteroids: Some Fluoro Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3983
method B. The product was purified using PLC to yield 3R-fluoride
19 (81 mg, 38%) as a white solid; mp 168-169 °C (methanol),
[R]D -24.7 (c 0.24). IR: 1705 (CO), 986 (C-F). 1H NMR δ: 0.68
(s, 3H, H-18), 0.74 (s, 3H, H-19), 0.82 (d, 3H, J ) 6.6 Hz, H-26),
0.83 (d, 3H, J ) 6.6 Hz, H-27), 0.91 (d, 3H, J ) 6.8 Hz, H-21),
1.01 (d, 3H, J ) 6.6 Hz, H-28), 4.91 (dm, 1H, J(F,H-3) ) 48 Hz,
H-3), 5.18 (m, 2H, H-23 and H-24). Anal. (C28H45FO) C, H. Olefin
(22E,24R)-5R-ergosta-2,22-dien-6-one was isolated as a more polar
side product.
5-Fluoro-3r-hydroxy-6-oxo-5r-androstan-17ꢀ-yl Acetate (29).
4-Toluenesulfonyl chloride (200 mg, 1.04 mmol) was added to a
solution of 3ꢀ-alcohol (25, 200 mg, 0.55 mmol) in pyridine (2 mL).
After 48 h, the mixture was diluted with brine, the white precipitate
was extracted with chloroform, the extract was washed with water,
dilute hydrochloric acid, then potassium hydrogen carbonate
solution, and then dried over anhydrous sodium sulfate. Evaporation
of the solvent in vacuo to dryness afforded tosylate, which was
solvolyzed with sodium nitrite (480 mg, 0.70 mmol) in HMPA (25
mL) at 90 °C for 2 h under nitrogen atmosphere. The mixture was
poured into water and the product was extracted with chloroform.
The extract was subsequently washed with water, dilute hydro-
chloric acid, water, and then potassium hydrogen carbonate solution
and water. Evaporation of the solvent and PLC (5 plates, benzene/
ethyl acetate, 7:3) yielded the 3R-alcohol 29 (83 mg, 41%) as white
crystals; mp 139-142 °C (acetone/heptane), [R]D -13.6 (c 0.21).
IR: 3617, 1017 (OH), 1725 (AcO). 1H NMR δ: 0.78 (s, 3H, H-18),
0.80 (s, 3H, H-19), 2.04 (s, 3H, CH3CO), 4.08 (m, 1H, W ∼ 16
Hz, H-3), 4.64 (t, 1H, J ) 8.5 Hz, H-17). Anal. (C21H31FO4) C, H.
5-Fluoro-3r,17ꢀ-dihydroxy-5r-androstan-6-one (30). Com-
pound 29 (70 mg, 0.19 mmol) was hydrolyzed according to the
method D to afford compound 30 (50 mg, 81%) as white crystals;
mp 260-262 °C (acetone/heptane), [R]D -12.4 (c 0.31). IR: 3617,
1070 (OH), 1725 (CO), 1020 (C-F). 1H NMR δ: 0.74 (s, 3H,
H-18), 0.80 (s, 3H, H-19), 3.70 (t, 1H, J ) 8.5 Hz, H-17), 4.08
(m, 1H, W ∼ 16 Hz, H-3). Anal. (C19H29FO3) C, H.
(22S,23S,24R)-3r-Fluoro-22,23-dihydroxy-5r-ergostan-6-
one (20) and (22R,23R,24R)-3r-Fluoro-22,23-dihydroxy-5r-
ergostan-6-one (21). The 3R-fluoro derivative (19) (170 mg, 0.41
mmol) was hydroxylated according to the method C1. Evaporation
of the solvent gave a white solid of a mixture (20 and 21, 115 mg,
63%), which was inseparable using silica gel chromatography
(column or HPLC); mp 168-176 °C (methanol). IR: 3628, 3551
(OH), 1382, 1369 (CH3), 1082 (C-OH), 1704 (CO), 987 (C-F).
For 1H, 13C, and 19F NMR data, see Supporting Information Tables
1-3. Anal. (C28H47FO3) C, H.
5-Fluoro-6-oxo-5r-androstane-3ꢀ,17ꢀ-diyl Acetate (24). Al-
cohol 2319 (230 mg, 0.56 mmol) was oxidized in acetone (4 mL)
with the Jones reagent at 0 °C. After 5 min, the excess of reagent
was reduced with solution sodium metabisulfite (5 mL), and the
product was extracted with chloroform. The extract was washed
with brine, dried, and concentrated in vacuo. Crystallization yielded
white crystals of ketone 24 (210 mg, 91%); mp 167.5-168 °C
(acetone/heptane), [R]D -21.9 (c 0.3), (ref 19 gives 185-186 °C
and [R]D -27). IR: 1727, 1367, 1260, 1247, 1048 (acetate), 1017
5-Fluoro-2r,3r,17ꢀ-trihydroxy-5r-androstan-6-one (31). (1)
By Hydrolysis. Acetate 33 (50 mg, 0.13 mmol) was hydrolyzed
according to the method D, yielding triol 31 as white crystals (32
mg, 72%); mp 187-189 °C (methanol), [R]D -5.7 (c 0.19). IR:
3608, 3561 (OH), 1726 (CO), 1074, 1046, 999 (C-OH), 1015
(C-F).1H NMR δ: 0.74 (s, 3H, H-18), 0.83 (s, 3H, H-19), 3.71
(dt, 1H, J(17,16R) ) 8.4 Hz, J(17,16ꢀ) ) 8.5 Hz and J(17,OH) )
6.0 Hz, H-17), 3.75 (m, 1H, W ) 33 Hz, H-2), 4.04 (m, 1H, W )
26 Hz, H-3). Anal. (C19H29FO4) C, H.
(2) By Dihydroxylation. The olefin 35 (61 mg, 0.20 mmol) was
hydroxylated according to the method C2. PLC chromatography
(2 plates, elution toluene/ethyl acetate, 1:1) afforded the above triol
31 (21 mg, 31%) as the more polar product.
5-Fluoro-6-oxo-5r-androst-2-en-17ꢀ-yl Acetate (32). The ole-
fin 32 (170 mg, 60%) was isolated as the less polar product in the
PLC preparation of compound 27; mp 156-158 °C (acetone/
heptane), [R]D -3.0 (c 0.21). IR: 1724 (CO), 1661 (CC), 1257
(C-O), 1018 (C-F). 1H NMR δ: 0.74 (s, 3H, H-18), 0.79 (s, 3H,
H-19), 2.05 (s, 3H, COCH3),4.64 (t, 1H, J ) 8.0 Hz, H-17), 5.63
(m, 2H, H-2 and H-3). Anal. (C21H29FO3) C, H.
1
(C-F). H NMR δ: 0.78 (s, 3H, H-18), 0.84 (s, 3H, H-19), 2.03
(s, 3H, COCH3), 2.04 (s, 3H, COCH3), 2.25 (dt, 1H, J ) 12.5 and
3.7 Hz, H-7ꢀ), 2.60 (dt, 1H, J ) 12.5 and 7.7 Hz, H-7R), 4.64 (t,
1H, J ) 8.6 Hz, H-17), 5.00 (m, 1H, W ∼ 35 Hz, H-3).
5-Fluoro-3ꢀ-hydroxy-6-oxo-5r-androstan-17ꢀ-yl Acetate (25).
A solution of potassium carbonate (670 mg, 4.85 mmol) in water
(12 mL) and methanol (24 mL) was added to a solution of diacetate
24 (1.83 g, 4.48 mmol) in methanol (180 mL). After 1 h at rt, acetic
acid (0.6 mL) was added and the solution was concentrated in
vacuo. Brine precipitated a white solid, which was extracted with
ethyl acetate. The extract was washed with brine, dried, and
concentrated in vacuo. Chromatography of the remainder on a
column of silica gel (150 mL) yielded alcohol 25 (1.25 g, 76%);
mp 165-167 °C (1.18 g, acetone/heptane), [R]D -16.4 (c 0.18).
1
IR: 3500, 3439, 3611, 1048 (OH), 1725, 1257 (COCH3). For H,
13C, and 19F NMR data, see Supporting Information Tables 1-3.
Anal. (C21H31FO4) C, H.
5-Fluoro-3ꢀ,17ꢀ-dihydroxy-5r-androstan-6-one (26). The more
polar component of the above chromatography, diol 26 (317 mg,
22%) crystallized from chloroform; mp 217-219 °C, [R]D -23.2
(c 0.23). IR: 3610, 3444, 1058 (OH), 1726 (CO). 1H NMR δ: 0.73
(s, 3H, H-18), 0.82 (s, 3H, H-19), 2.23 (ddd, 1H, Jgem ) 12.8, J(7) )
3.4 and J(7,5RF) ) 4.2 Hz, H-7ꢀ), 2.59 (dt, 1H, Jgem ) 12.8, J(7,8)
) 12.2 and J(7R,F) ) 7.9 Hz, H-7R), 3.66 (t, 1H, J ) 8.7 Hz, H-17),
3.89 (m, 1H, W ∼ 35 Hz, H-3). Anal. (C19H29FO3) C, H.
5-Fluoro-2r,3r-dihydroxy-6-oxo-5r-androstan-17ꢀ-yl Ac-
etate (33). The olefin 32 (70 mg, 0.20 mmol) was hydroxylated
according to the method C2. PLC chromatography (3 plates,
toluene/ethyl acetate, 6:4) yielded diol 33 (25 mg, 33%) as a white
solid; mp 172-174 °C (methanol), [R]D -7.3 (c 0.22). IR: 3604,
3560 (OH), 1726 (CO), 1255, 1034 (C-O), 1047, 996 (C-OH),
1019 (C-F). For 1H, 13C, and 19F NMR data, see Supporting
Information Tables 1-3. Anal. (C21H31FO5) C, H.
5-Fluoro-2ꢀ,3ꢀ-dihydroxy-6-oxo-5r-androstan-17ꢀ-yl Acetate
(34). The lipophilic zone from the above chromatography afforded
diol 34 (36 mg, 47%) as a white solid; mp 237-239 °C (methanol),
[R]D -21.3 (c 0.25). IR: 3604, 3560 (OH), 1726 (CO), 1257, 1035
(C-O), 1046, 993 (C-OH), 1018 (C-F). For 1H, 13C, and 19F NMR
data, see Supporting Information Tables 1-3. Anal. (C21H31FO5)
C, H.
5-Fluoro-17ꢀ-hydroxy-5r-androst-2-en-6-one (35). The 17ꢀ-
acetoxy derivative 32 (330 mg, 0.95 mmol) was hydrolyzed according
to the method D, yielding the 17ꢀ alcohol 35 (206 mg, 71%) as a
white solid; mp 151-153 °C (acetone/heptane), [R]D -5.0 (c 0.27).
IR: 3614, 1060 (OH), 1724 (CO), 1661 (CC), 1023 (C-F). 1H NMR
δ: 0.74 (s, 3H, H-18), 0.75 (s, 3H, H-19), 3.70 (dt, 1 H, J(17,16R) )
8.5, J(17,16ꢀ) ) 8.5 and J(17,OH) ) 6.0 Hz, H-17), 5.64 (m, 2H, W
∼ 47 Hz, H-2 and H-3). Anal. (C19H27FO2) C, H.
3r,5-Difluoro-6-oxo-5r-androstan-17ꢀ-yl Acetate (27). Fol-
lowing the method B, alcohol 25 (300 g, 0.8 mmol) was treated
with DAST. The residue was separated by PLC (6 plates, a mixture
of light petroleum/ether, 7:3). The polar zone afforded a white solid
(47 mg, 16%) of 3R-fluoride 27; mp 168-170 °C (acetone/heptane),
[R]D -23.2 (c 0.12). IR: 1725 (CO), 1256 (C-O), 1018, 993 (C-F).
For 1H, 13C, and 19F NMR data, see Supporting Information Tables
1-3. Anal. (C21H30F2O3) C, H.
3r,5-Difluoro-17ꢀ-hydroxy-5r-androstan-6-one (28). Acetate
27 (70 mg, 0.19 mmol) was hydrolyzed according to the method
D. Chromatography of the remainder by PLC on two plates yielded
compound 28 as white crystals (46 mg, 74%); mp 180-182 °C
(acetone/heptane), [R]D -15.6 (c 0.24). IR: 3614 (OH), 1725 (CO),
1
1027, 993 (C-F). H NMR δ: 0.74 (s, 3H, H-18), 0.79 (s, 3H,
H-19), 3.70 (dt, 1H (J(17,16R) ) 8.5, J(17,16ꢀ) ) 8.5 and J(17,OH)
) 6.0 Hz, H-17), 4.92 (dm, 1H, J ) 47 Hz, H-3). Anal.
(C19H28F2O2) C, H.

3984 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
5-Fluoro-2ꢀ,3ꢀ,17ꢀ-trihydroxy-5r-androstan-6-one
SlaVikoVa et al.
(4) Pellicciari, R.; Sato, H.; Gioiello, A.; Costantino, G.; Macchiarulo,
A.; Sadeghpour, B. M.; Giorgi, G.; Schoonjans, K.; Auwerx, J.
Nongenomic Actions of Bile Acids. Synthesis and Preliminary
Characterization of 23- and 6,23-Alkyl-Substituted Bile Acid Deriva-
tives as Selective Modulators for the G-Protein Coupled Receptor
TGR5. J. Med. Chem. 2007, 50, 4265–4268.
(5) Swaczynova, J.; Sisa, M.; Hnilickova, J.; Kohout, L.; Strnad, M.
Synthesis, biological, immunological and anticancer properties of a
new brassinosteroid ligand. Pol. J. Chem. 2006, 80, 629–635.
(6) Wu, Y. D.; Lou, Y. J. Brassinolide, a plant sterol from pollen of
Brassica napus L., induces apoptosis in human prostate cancer PC-3
cells. Pharmazie 2007, 62, 392–395.
(7) Malikova, J.; Swaczynova, J.; Kolar, Z.; Strnad, M. Anticancer and
Antiproliferative Activity of Natural Brassinosteroids. Phytochemistry
2008, 69, 418–426.
(8) Talarico, L. B.; Ramirez, J. A.; Galagovsky, L. R.; Wachsman, M. B.
Structure-activity relationship studies in a set of new brassinosteroid
derivatives assayed against herpes simplex virus types 1 and 2 in cell
cultures. Med. Chem. Res. 2002, 11, 434–444.
(36).
(1) By Hydrolysis. Following the method D, the acetate 34 (50
mg, 0.13 mmol) was hydrolyzed to yield white crystals of the 17ꢀ-
alcohol 36 (33 mg, 75%); mp 213-215 °C (methanol), [R]D -15.0
(c 0.22). IR: 3625, 3612 (OH), 1726 (CO), 1074, 1045, 998 (C-
1
OH), 1016 (C-F). H NMR δ: 0.73 (s, 3H, H-18), 1.03 (s, 3H,
H-19), 3.64 (dt, 1H, J(17,16R) ) 8.5, J(17,16ꢀ) ) 8.5 and J(17,OH)
) 6.0 Hz, H-17), 3.85 (m, 1 H, W ∼ 24.5 Hz, H-3), 3.98 (m, 1H,
W ∼ 14.0 Hz, H-2). Anal. (C19H29FO4) C, H.
(2) By Dihydroxylation. A less polar zone of the PLC plates
used in the above preparation of diol 31 from olefin 35 was eluted
to yield diol 36 (31 mg, 46%) identical with the above sample.
Biological Evaluation. Calcein-AM Cytotoxicity Assay. The
cell lines (T-lymphoblastic leukemia cell line CEM, breast carcinoma
cell lines MCF-7, multiple myeloma RPMI 8226, and human fibroblast
BJ) were cultured in DMEM medium (Gibco BRL) supplemented with
10% of fetal calf serum, 4 mM glutamine, 100 U/mL penicillin, and
100 µg/mL streptomycin, at 37 °C in a fully humidified atmosphere
containing 5% CO2. The cell suspension of approximate density of
1.25 × 105 cells/mL was redistributed into 96-well microtiter plates
and after 3 h of stabilization the tested steroids were added in different
concentrations. Tested compounds were dissolved in DMSO before
addition to cultures. Control cultures were treated with DMSO alone.
The final concentration of DMSO in the reaction mixture never
exceeded 0.6%. Four-fold dilutions of the intended test concentration
were added at time zero in 20 µL aliquots to the microtiter plate wells.
Each tested compound was evaluated at six 4-fold dilutions. In routine
testing, the highest well concentration was 50 µM, but it can be the
matter of change dependent on the agent. After 72 h of cultivation,
the cells were incubated with Calcein AM solution (Molecular Probes)
for 1 h. Fluorescence of viable cells was quantified with Fluoroscan
Ascent (Microsystems). The percentage of surviving cells in each well
(9) Michelini, F. M.; Ramirez, J. A.; Berra, A.; Galagovsky, L. R.; Alche,
L. E. In vitro and in vivo antiherpetic activity of three new synthetic
brassinosteroid analogues. Steroids 2004, 69, 713–720.
(10) Khripach, V. A.; Zhabinskii, V. N.; de Groot, A. E. Brassinosteroids:
A New Class of Plant Hormones; Academic Press: San Diego, 1999.
(11) Zullo, M. A. T.; Kohout, L.; de Azevedo, M. B. M. Some Notes on
Terminology of Brassinosteroids. Plant Growth Regul. 2003, 39, 1–11.
(12) Galagovsky, L. R.; Gros, E. G.; Ramirez, J. A. Synthesis and
bioactivity of natural and C-3 fluorinated biosynthetic precursors of
28-homobrassinolide. Phytochemistry 2001, 58, 973–980.
(13) Brosa, C.; Soca, L.; Terricabras, E.; Ferrer, J. C.; Alsina, A. New
Synthetic Brassinosteroids: a 5R-Hydroxy-6-ketone Analog with Strong
Plant Growth Promoting Activity. Tetrahedron 1998, 54, 12337–
12348.
(14) Todoroki, Y.; Hirai, N.; Koshimizu, K. Synthesis and biological activity
of 1′-deoxy-1′-fluoro- and 8′-fluoroabscissic acids. Phytochemistry
1995, 40, 633–641.
(15) Bajguz, A. Metabolism of brassinosteroids in plants. Plant Physiol.
Biochem. 2007, 45, 95–107.
was calculated from the equation IC50 ) (ODdrug
/mean
exposed well
(16) Sisa, M.; Hnilickova, J.; Swaczynova, J.; Kohout, L. Syntheses of
new androstane brassinosteroids with 17beta-ester group: butyrates,
heptafluorobutyrates, and laurates. Steroids 2005, 70, 755–762.
(17) Kohout, L.; Cerny, V.; Strnad, M. On Steroids, Part CCCXXX.
Alternative Syntheses of 2R,3R,17ꢀ-Trihydroxy-7-oxa-B-homo-5R-
androstan-6-one and Some Androstane Brassinolide Analogs. Collect.
Czech. Chem. Commun. 1987, 52, 1026–1042.
(18) Ramirez, J. A.; Gros, E. G.; Galagovsky, R. L. Effects on Bioactivity
due to C-5 Heteroatom Substitutents on Synthetic 28-Homobrassinolide
Analogs. Tetrahedron 2000, 56, 6171–6180.
(19) Boswell, G. A., Jr. Reaction of Nitrosyl Fluoride and Selected Steroid
Enes. A New Synthesis of ∆5-4-Keto Steroids. J. Org. Chem. 1968,
33, 3699–3713.
(20) Hughes, D. W.; Bain, A. D.; Robinson, V. J. NMR Analysis of
fluorinated corticosteroids related to fluocinonide: detection of long-
range H-1 F-19 couplings using the heteronuclear equivalent of the
COSY experiment. Magn. Reson. Chem. 1991, 29, 387–397.
(21) Mitchell, J. W.; Livingstone G. A. Bean second internode method. In
Agricultural Handbook, No. 336; U.S. Government Printing Office:
Washington, DC, 1968; pp 26-28..
ODcontrol wells) × 100%; each compound was tested in triplicate, and the
entire test was repeated at least three times. The IC50 value, the
concentration lethal to 50% of the cells, of each tested substance, was
calculated from the obtained dose response curves.7
The Bean Second-Internode Bioassay. Seeds of bean (Phaseolus
Vulgaris L., cv. Pinto) germinated for 2 days were selected for
uniformity from a large population of seedlings and then transferred
into pots containing perlite and 1/10 diluted Hoagland solution (half
concentration, pH 5.7).21 Seedlings were grown in a light-controlled
cultivation room (25-27 °C, light 48 W ·m-2, light/dark period 16/8
h). Seven-day-old plants with second internodes 2 mm long were
treated with different amounts of tested compounds in 5 µL fractionated
lanolin. The substances were applied in microdrops to the scar left
after the removal of bract from the base of the second internode. The
control plants were treated with lanolin alone. At least seven plants
were used for each experiment, and the assays were repeated at least
three times. The length of the second internodes was measured after
5 days, and the difference in length between treated and control plants
provided a measure of activity (see Table 2).
(22) Gharib, B.; Baswald, S.; Quilici, M.; De Reggi, M. Ecdysteroid-like
compounds in human urine: They can occur in the absence of any
parasitic infection. Clin. Chim. Acta 1991, 199, 159–166.
(23) Strnad, M.; Kohout, L. Simple brassinolide analogue 2R,3R-dihydroxy-17ꢀ-
(3-methylbutyryloxy)-7-oxa-B-homo-5R-androstan-6-one inducing the second
bean internode splitting. Plant Growth Regul. 2003, 40, 39–47.
Acknowledgment. This work was supported by the Grant
Agency of ASCR (grant no. IAA400550609) and the research
project Z4 055 0506. We thank Jarmila Balonova for her excellent
technical assistance.
¨
(24) Butendant, A.; Suranyi, L. A. Uber einige Derivate des i-Androstans.
Chem. Ber. 1942, 75, 591–597.
(25) Labler, L.; Slama, K.; Sorm, F. On Steroids. CXV. Toxic effect of
some androstane derivatives on Pyrrhocoris apterus L Larvae. Collect.
Czech. Chem. Commun. 1968, 33, 2226–2237.
(26) Thompson, M. J.; Cohen, C. F.; Lancaster, S. M. Brassicasterol and
22,23-Dihydrobrassicasterol from Ergosterol via i-Ergosterol. Steroids
1965, 5, 745–752.
Supporting Information Available: Supplementary data on
routine experimental procedures, combustion analysis of products,
and their NMR spectra are given. This material is available free of
charge via the Internet at http://pubs.acs.org.
(27) Ferrer, J. C.; Lalueza, R.; Saavedra, O.; Brosa, C. Short step synthesis of
(22E,24R)-5R-ergosta-2,22-dien-6-one, a key intermediate for the prepara-
tion of 24-epibrassinolide. Tetrahedron Lett. 1990, 31, 3941–3942.
(28) Thompson, M. J.; Mandava, N.; Flippen-Anderson, J. L.; Worley, J. F.;
Dutky, S. R.; Robbins, W. E.; Lusby, W. Synthesis of Brassinosteroids:
New Plant-Growth-Promoting Steroids. J. Org. Chem. 1979, 44,
5002–5004.
References
(1) Hayat, S.; Ahmad, A. Brassinosteroids. BioactiVity and Crop Pro-
ductiVity: Kluwer Academic Publishers: Dordrecht, 2003.
(2) Kagale, S.; Divi, U. K.; Krochko, J. E.; Keller, W. A.; Krishna, P.
Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica
napus to a range of abiotic stresses. Planta 2007, 225, 353–364.
(3) Mellon, S. H. Neurosteroid regulation of central nervous system
development. Pharmacol. Ther. 2007, 116, 107–124.
JM800085P