Fluorination of betulinines and other triterpenoids with DAST

Article abstract of DOI:10.1055/s-2005-861861

Betulinines are lupane, des-E-lupane, 18-lupene, 20(29)-lupene and 18α-oleanane derivatives with antitumor activity. We examined fluorination of these derivatives using diethylaminosulfur trifluoride (DAST) as fluorinating agent. We prepared 19β,28-epoxy-2,2-difluoro-18α- oleanan-3-one (3c), 19β,28-epoxy-2,2-difluoro-18α-oleanan-3β-ol (4a), methyl 3β-acetoxy-30-fluorolup-20(29)-ene-28-oate (6b), 3β,28-diacetoxy-22-oxo-21,21-difluorolup-18-ene (8b) and several other fluorinated betulinines for in vitro cytotoxicity tests which failed to demonstrate significant anticancer activity so far.

Full text of DOI:10.1055/s-2005-861861

PAPER
1157
Fluorination of Betulinines and Other Triterpenoids with DAST
a
a
a
b
b
F
D
luorinationof Tri
a
terpenoids
v
w
ith
D
A
ST id Biedermann, Jan Sarek,* Jiri Klinot, Marian Hajduch, Petr Dzubak
a
Department of Organic and Nuclear Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague 2,
Czech Republic
Fax +42(22)1951332; E-mail: jan.sarek@volny.cz
b
Laboratory of Experimental Medicine, Department of Pediatrics, Faculty of Medicine, Palacky University in Olomouc, Puskinova 6,
75 20 Olomouc, Czech Republic
7
Received 1 September 2004; revised 5 January 2005
Fluorination of Alcohols
Abstract: Betulinines are lupane, des-E-lupane, 18-lupene, 20(29)-
lupene and 18a-oleanane derivatives with antitumor activity. We
examined fluorination of these derivatives using diethylaminosulfur
trifluoride (DAST) as fluorinating agent. We prepared 19b,28-ep-
As a starting material we used betulin (1a), readily acces-
sible from birch bark. This compound undergoes an acid-
oxy-2,2-difluoro-18a-oleanan-3-one (3c), 19b,28-epoxy-2,2-di- catalyzed rearrangement with ring E expansion to allobet-
fluoro-18a-oleanan-3b-ol (4a), methyl 3b-acetoxy-30-fluorolup-
0(29)-ene-28-oate (6b), 3b,28-diacetoxy-22-oxo-21,21-difluoro-
uline (2a). For this reaction we used a ‘solid acid’ Mont-
2
morillonite K10. The procedure, as described in the
lup-18-ene (8b) and several other fluorinated betulinines for in vitro
cytotoxicity tests which failed to demonstrate significant anticancer
activity so far.
12
literature, did not work properly in our hands. We had to
increase the amount of Montmorillonite K 10 fivefold and
prolong the reaction time to 72 hours to obtain a good con-
version of the starting material. Treatment of allobetuline
Key words: antitumor agents, fluorine, rearrangements, steric hin-
drance, terpenoids
(
2a) with DAST under very mild conditions resulted in
the formation of an elimination product, olefin 2e, as we
expected. Elimination occurred probably due to heavy
steric hindrance of the alcohol group in position 3 by skel-
etal methyl groups at positions 23 and 24. We therefore
Introduction
Diethylaminosulfur trifluoride (DAST) is a widely used decided to prepare alcohol 2d, which we expected to be
1
–8
fluorinating reagent, very effective for converting alco- less sterically hindered. First of all we oxidized the hy-
hols, ketones, aldehydes and carboxylic acids into their droxy group in position 3 of the allobetuline (2a) with a
corresponding fluoro derivatives. Unlike other fluorinat- slightly modified procedure13 to obtain ketone 2b. We
ing agents, e.g. Olah reagent or SF , reactions with DAST then converted this compound into isomeric ketone 2c by
4
can be performed in glass equipments. The problem in refluxing with sulfur in morpholine.14 Reduction of the
performing fluorination lies in the possibility of rear- ketone 2c with NaBH resulted in the formation of desired
4
rangement or elimination instead of the desired reaction.
alcohol 2d. Reaction of alcohol 2d with DAST gave olefin
12
2
e as the single product (Scheme 1).
Recently, we have examined the structure-activity rela-
9
tionships in certain Betulinines (lupane, des-E-lupane, Olefin 2e with C-2 double bond was the only product of
1
8-lupene, 20(29)-lupene and 18a-oleanane derivatives), reactions of 2a and 2d with DAST. We therefore used hy-
9
1
5
and found significant antitumor activity. This effort was droxy ketones 3a and 3b, accessible from ketone 2b, to
directed towards highly polar compounds, while less polar avoid such elimination. Both hydroxy ketones 3a and 3b
compounds were not sufficiently examined. With a few reacted with DAST to form a single product, the difluoro
exceptions, no lupane or 18a-oleanane fluoro deriva- ketone 3c. Optimization of the reaction conditions led to
tives have been prepared until now. High antitumor activ- 38% yield.
1
0
ity of fluorinated compounds analogous to triterpenoids,
e.g. steroid Fluoxymesteron, is well known.
We next examined the effectiveness of DAST towards
1
1
2
,2-difluoro alcohol 4a, prepared from difluoro ketone 3c
Herein we report on the construction of new fluoro com- by reduction with NaBH . Treatment of 2,2-difluoro alco-
4
pounds, derived from lupane, des-E-lupane, 18-lupene, hol 4a with DAST afforded the difluoro olefin 4b, and the
2
0(29)-lupene and 18a-oleanane alcohols, oxo derivatives trifluoro derivative 4c as a result of rearrangement
and carboxylic acids using DAST as a source of fluorine. (Scheme 2).
The above-mentioned unexpected reactions, rearrange-
ments and low yields may be caused by the significant
steric hindrance and rigid skeleton. We confirmed this hy-
pothesis using unhindered enol ketone 5a and alcohol 6a
as substrates for fluorination (Scheme 3). These reactions
gave the desired fluoro derivatives 5b and 6b in high
yields (77–80%).
SYNTHESIS 2005, No. 7, pp 1157–1163
x
x
.
x
x
.2
0
0
5
Advanced online publication: 10.03.2005
DOI: 10.1055/s-2005-861861; Art ID: T10304SS
Georg Thieme Verlag Stuttgart · New York
©

1
158
D. Biedermann et al.
PAPER
2
9
3
0
2
O
1
9
21
F
2
3
0
O
1
18
22
28
1
a
1
0
1
a
2
5
26
17
HO
1
4
14
77%
2
3
16
O
9
6
OH
1
8
15
O
27
7
5
5a
5b
HO
1a
HO
2a
2
3
24
e
HO
F
b
5
8%
a
CO2Me
CO2Me
8
0%
O
O
AcO
2
e
6
a
6b
2
b
Scheme 3 Reagents and conditions: a. DAST, CH Cl , –78 °C to
r.t.
c
2
2
6
1%
e
HO
d
Fluorination of Oxo Derivatives
2
d
2
c
Utilization of DAST for the conversion of ketones and al-
dehydes into their corresponding geminal difluoro deriva-
tives demands harder condition than the substitution of a
Scheme 1 Reagents and conditions: a. Montmorillonite K10,
CHCl , reflux; b. PCC, Al O , CHCl ; c. S, morpholine, reflux; d.
NaBH , THF, MeOH; e. DAST, CH Cl , –78 °C to r.t.
2
3
2
3
3
hydroxy group. Originally we used the literature condi-
4
2
2
1
tions, which recommended reflux with excess of DAST
in CH Cl . We however found this procedure to be effec-
2
2
tive only towards aldehyde 7a. Moreover, we obtained the
corresponding difluoro derivative 7b only in low yield
(22%) after 48 hours of reaction time (Scheme 4).
O
HO
a
We therefore decided to utilize other experimental condi-
tions. We performed the reaction with excess of DAST
O
2
O
2
b
3a
(
which served also as a co-solvent) in a sealed cylindrical
c
flask and heated to 70 °C. Under these conditions difluoro
ketones 8b and 9b were prepared from diketones 8a and
b
3
8%
9
a in yields below 30%. No tetrafluorinated product was
F
F
O
observed. In the case of heptanorketone 10a we also suc-
ceeded and prepared the difluoro derivative 10b. In con-
trast, ketones 2b, 2c and 11a showed no conversion under
these conditions (Schemes 1, 4).
c
O
HO
3
1%
3
c
3b
7
2%
d
Fluorination of Carboxylic Acids
F
F
6
1%
DAST is a convenient reagent for the synthesis of acyl
fluorides from the corresponding carboxylic acids under
F
F
c
1
4
b
very mild conditions. The experimental procedure is
analogous to the procedure we used for fluorination of al-
cohols. Reaction yields are above average (60–70%) and
no by-products were observed.
HO
+
4
a
F
3
4%
Acetylbetulinic acid (12b) was fluorinated with DAST to
acyl fluoride 12c. We used the same procedure success-
fully in the reactions of the saturated derivative 13a and
F
4
c
F
2
1-oxo acid 14a, to obtain acyl fluorides 13b and 14b, re-
Scheme 2 Reagents and conditions: a. MCPBA, MeOH, H SO , spectively (Scheme 5).
2
4
CHCl ; b. H SO , dioxane, MeOH, reflux; c. DAST, CH Cl , –78 °C
3
2
4
2
2
to r.t., d. NaBH , THF, MeOH
4
Synthesis 2005, No. 7, 1157–1163 © Thieme Stuttgart · New York

PAPER
Fluorination of Triterpenoids with DAST
1159
O
F
F
CO2CH3
CO2H
a
CO2CH3
a
2
2%
COF
3
0%
AcO
AcO
12b
7
b
1
2c
7
a
O
F
F
CO2H
O
O
a
b
R
R
6
3%
COF
AcO
1
3a
AcO
8b: R = CH OAc, 26%
2
9
b: R = CO2Me, 29%
13b
8
a: R = CH OAc
O
2
9
a: R = CO Me
2
O
O
CO2H
a
F
F
6
5%
b
COF
3
0%
AcO
1
4a
AcO
1
0b
14b
Scheme 5 Reagents and conditions: a. DAST, CH Cl , –78 °C to
1
0a
2
2
O
r.t.
strate significant anticancer activity on CEM leukemia
cells (TCS50 above 250 mM). We demonstrated the de-
crease of cytotoxic activity by introduction of fluorine
into these molecules. This fact probably originates in the
high lipophilicity of those fluoro derivatives.
b
no reaction
OAc
AcO
1
1a
Scheme 4 Reagents and conditions: a. DAST, CHCl , reflux; b.
3
Melting points were determined on Kofler block and are uncorrect-
ed. Optical rotations were measured on an Autopol III (Rudolph Re-
DAST, CHCl , 70 °C
3
1
13
19
search, Flanders, NJ) polarimeter in CHCl . H, C and F NMR
3
UNITY
1
spectra were recorded on Varian
Inova 400 (400 MHz for H),
using CDCl as a solvent. Chemical shifts are expressed in ppm with
3
1
Conclusion
tetramethylsilane as an internal standard for H spectra and with
1
9
13
CClF as an internal standard for F NMR spectra. C NMR spec-
3
tra are referenced to CDCl (77.00 ppm). Mass spectra (EI) were
We have shown that DAST is a suitable reagent for the
transformation of natural compounds with complicated
structure (triterpenoids) to their corresponding fluoro de-
rivatives. Alcohols, ketones, aldehydes and carboxylic ac-
ids can be used as substrates. The limitations of these
3
measured on INCOS 50 (Finnigan MAT) mass spectrometer. IR
spectra were recorded on a Perkin-Elmer 684 IR spectrometer in
CHCl . TLC was performed on Kieselgel 60 F (Merck) sheets;
3
254
the spots were detected by UV fluorescensce or spraying with 10%
H SO and heating to 110–200 °C. HPLC system consisted of High
2
4
fluorinations are the rigid skeleton of these natural com- Pressure Pump Gilson (model 361), Inject Valve Rheodyne, Prepar-
pounds and the steric hindrance caused by skeletal methyl ative Column (25 × 250 mm) with silica gel filling (Biospher 7 mm;
Labio), Differential–Refractometrical Detector (Laboratorní
přístroje, Praha, CR) connected with PC (software Chromulan) and
Automatic Fraction Collector Gilson (model 246). Betulin (1a) was
obtained by extraction of birch bark from our patrner papermill,
groups. The rigid skeleton induces low reactivity. Steric
hindrance may cause observed rearrangements and elimi-
nations. Having the reaction centre placed out of the rigid
skeleton, the yield increased dramatically up to 80%. Con-
version of ketones to difluoro derivatives demands hard
conditions. In contrast, for conversion of carboxylic acids ature.
to acyl fluorides very mild conditions are sufficient.
21
Billerud, Gruvön Mill, Sweden, using literature procedure. Betu-
linic acid (12a) was obtained from natural source according to liter-
1
8
WARNING: DAST is a dangerous substance which can decom-
pose explosively on temperature over 55 °C.
All thirteen fluoro derivatives prepared in this work (3c,
4
a–c, 5b, 6b, 7b, 8b, 9b, 10b, 12c, 13b, 14b) were tested Fluorination of the oxo compounds was carried out in a 10 mL en-
for in vitro cytotoxic activity. Unfortunately, fluorinated closed cylindrical flask (from Kimble–Kontes, art. no.: 747500-
0
010). The following compounds were prepared using literature
triterpenoids exemplified in this paper failed to demon-
Synthesis 2005, No. 7, 1157–1163 © Thieme Stuttgart · New York

1
160
D. Biedermann et al.
PAPER
1
2
procedures: allobetuline (2a), 19b,28-epoxy-18a-oleanan-3-one
ganic layer was dried (MgSO ), evaporated, and the residue was
4
14
(
2b),13 19b,28-epoxy-18a-oleanan-2-one (2c), 19b,28-epoxy-
crystallized from butanone to give 4a (340 mg, 72%); mp 299–
1
6
25
1
3
8a-oleanan-2b-ol (2d), 19b,28-epoxy-2a-hydroxy-18a-oleanan-
301 °C; [a]D +48 (c = 0.39).
-one (3a),15 19b,28-epoxy-3b–hydroxy-18a-oleanan-2-one (3b),
15
–1
IR (CHCl ): 3608 cm (C–OH).
17
18
9
19
3
enol ketone 5a, alcohol 6a, aldehyde 7a, diketone 8a, diketone
1
9
9
22
20
H NMR: d = 0.80 (s, 3 H), 0.89 (d, 3 H, J = 1.2 Hz), 0.92 (s, 3 H),
9
a, ketone 10a, ketone 11a, acetylbetulinic acid (12b), dihy-
1
9
9
0.93 (s, 3 H), 1.00 (s, 6 H), 1.08 (s, 3 H), 2.33 (dt, 1 H, J = 4.6, 14.0
Hz), 3.45 (d, 1 H, J = 7.8 Hz, H-28a), 3.52 (s, 1 H, H-19a), 3.77 (dd,
1
drobetulinic acid (13a), and 21-oxoacid 14a. DAST was pur-
chased from Sigma-Aldrich.
H, J = 7.8, 1.5 Hz, H-28b), 3.35 (dd, 1 H, J = 22.4, 7.3 Hz, H-3a).
1
9b,28-Epoxy-18a-oleanan-2-ene (2e)
From Alcohol 2a: To a stirred solution of alcohol 2a (200 mg, 0.45
mmol) in CH Cl (5 mL) cooled to –78 °C was added DAST
13
C NMR: d = 46.3 (t, J = 20 Hz, C-1), C-2 not found, 78.5 (t, J = 20
Hz, C-3), 39.6 (d, J = 6 Hz, C-4), 55.1 (C-5), 18.0 (C-6), 33.6 (C-7),
40.9 (C-8), 51.4 (C-9), 38.4 (d, J = 5 Hz, C-10), 21.4 (C-11), 26.3
2
2
(
200 mL, 1.04 mmol). The reaction mixture was then allowed to
(
C-12), 34.0 (C-13), 40.8 (C-14), 26.3 (C-15), 36.7 (C-16), 41.5 (C-
warm to r.t. and the reaction was quenched by addition of H O (1
2
17), 46.8 (C-18), 87.9 (C-19), 36.2 (C-20), 32.7 (C-21), 26.2 (C-22),
19.1 (C-23), 15.6 (C-24), 15.9 (d, J = 5 Hz, C-25), 15.3 (C-26),
13.4 (C-27), 71.2 (C-28), 24.5 (C-29), 28.8 (C-30).
mL). The separated organic layer was diluted with CHCl (50 mL),
3
washed with H O (2 × 40 mL), dried (MgSO ) and evaporated under
reduced pressure. The dark yellow-brown residue was filtered over
a short column of silica gel (3 g, toluene) and crystallized from
i-PrOH to give olefin 2e (129 mg, 58%); mp 240–243 °C (Lit. mp
2
2
4
1
9
F NMR: d = –90.71 (dq, J = 245.2, 5.4 Hz, F-2a), –110.20 (dddd,
2
1
J = 245.2, 34.7, 22.2, 13.3 Hz, F-2b).
25
1
+
44.5–245 °C); [a] +81 (c = 0.22). The H NMR spectrum is
MS: m/z (%) = 478 (M , 100), 458 (69), 447 (34), 438 (17), 427
(23), 407 (78), 342 (11), 243 (9), 220 (11), 203 (17), 191 (25).
D
1
12
identical with the H NMR spectrum of an authentic sample.
From Alcohol 2d: Alcohol 2d (110 mg, 0.248 mmol) reacted with
DAST in the same manner as alcohol 2a to give 2e (74 mg, 61%).
Anal. Calcd for C30
C, 75.62; H, 9.88; F, 7.88.
H F O : C, 75.27; H, 10.11; F, 7.94. Found:
48 2 2
1
9b,28-Epoxy-2,2-difluoro-18a-oleanan-3-one (3c)
Difluoro Olefin 4b and Trifluoro Derivative 4c
To a stirred solution of alcohol 4a (170 mg, 0.35 mmol) in CH Cl
From Hydroxy Ketone 3a: To a stirred solution of hydroxy ketone
2 2
3
1
a (2.1g, 4.46 mmol) in CH Cl (20 mL) was added DAST (2 mL,
0.35 mmol) slowly at r.t.. The reaction was quenched after 24 h by
(5 mL), cooled to –78 °C was added DAST (170 mL, 0.88 mmol).
The reaction mixture was then allowed to warm to r.t. and the reac-
2
2
addition of H O (10 mL). The separated organic layer was diluted
tion was quenched by addition of H
ic layer was diluted with CHCl (50 mL), washed with H
mL), dried (MgSO ) and evaporated under reduced pressure. The
2
O (1 mL). The separated organ-
2
with CHCl (100 mL), washed with H O (2 × 400 mL), dried
3
2
O (2 × 40
3
2
(
MgSO ) and the organic layer was evaporated under reduced pres-
4
4
sure. The pale brown residue was chromatographed on silica gel
toluene–Et O, 40:1) and crystallized from i-PrOH to give difluoro
ketone 3c (800 mg, 38%); mp 243–245 °C; [a]D +123 (c = 0.52).
brown residue was separated by HPLC (3.5% EtOAc in hexane) and
(
lyophilized (t-BuOH) to give difluoro olefin 4b (93 mg, 61%); mp
2
2
5
25
232–233 °C (subl.); [a]
+55 (c = 0.36) and trifluoro derivative 4c
D
2
5
(
56 mg, 34%); mp 235–236 °C; [a]D +79 (c = 0.38).
From Hydroxy Ketone 3b: Hydroxy ketone 3b (200 mg, 0.42 mmol)
reacted with DAST in the same manner as hydroxy ketone 3a to
give 3c (63 mg, 31%).
4
b
–
1
IR (CHCl ): 1683 cm (C=C).
3
IR (CHCl ): 1743 cm– (C=O).
1
3
1
H NMR: d = 0.80 (s, 3 H), 0.83 (t, 3 H, J = 2.0 Hz), 0.93 (s, 3 H),
1
H NMR: d = 0.81 (s, 3 H), 0.89 (s, 3 H), 0.94 (s, 3 H), 0.95 (s, 3 H),
0
.94 (s, 3 H), 1.00 (s, 3 H), 1.80 (dt, 3 H, J = 3.7, 2.5 Hz, CH C=C),
3
0
.99 (s, 3 H), 1.16 (s, 3 H), 1.23 (d, J = 1.6 Hz, 3 H), 2.08 (ddd, 1
1
3
.87 (q, J = 2.7 Hz, CH C=C), 2.10–2.18 (m, 3 H, S J = 31.7 Hz),
3
H, J = 30.1, 15.3, 6.1 Hz, H-1a), 2.26 (dt, J = 15.1, 19.8 Hz, 1 H,
.45 (d, 1 H, J = 7.8 Hz, H-28a), 3.52 (s, 1 H, H-19a), 3.77 (dd, 1
H-1b), 3.46 (d, J = 7.8 Hz, 1 H, H-28a), 3.53 (s, 1 H, H-19a), 3.77
H, J = 7.8, 1.8 Hz).
(
dd, J = 7.8, 1.7 Hz, 1 H, H-28b).
1
3
C NMR: d = 52.9 (t, J = 23 Hz, C-1), C-2 not found, 131.6 (t,
J = 25 Hz, C-3), 128.8 (C-4), 55.6 (C-5), 22.4 (C-6), 33.2 (C-7),
0.3 (C-8), 48.1 (C-9), 41.3 (C-10), 23.5 (C-11), 26.4 (C-12), 34.1
C-13), 40.8 (C-14), 26.2 (C-15), 36.8 (C-16), 41.5 (C-17), 46.9 (C-
8), 88.0 (C-19), 36.3 (C-20), 32.7 (C-21), 26.1 (C-22), 22.2 (d,
J = 5 Hz, C-23), 20.4 (C-24), 15.4 (C-25), 15.7 (C-26), 13.4 (C-27),
1
3
C NMR: d = 51.8 (dd, J = 23, 20 Hz, C-1), 115.8 (dd, J = 258, 245
Hz, C-2), 204.6 (dd, J = 25, 23 Hz, C-3), 54.8 (C-4), 50.9 (C-5),
4
(
1
1
9.6 (C-6), 32.3 (C-7), 40.8 (C-8), 50.1 (C-9), 37.0 (dd, J = 5, 2 Hz,
C-10), 21.8 (C-11), 26.2 (C-12), 34.3 (C-13), 40.4 (C-14), 26.3 (C-
1
3
5), 36.6 (C-16), 41.4 (C-17), 46.6 (C-18), 87.8 (C-19), 36.2 (C-20),
2.6 (C-21), 26.1 (C-22), 28.0 (d, J = 4 Hz, C-23), 21.0 (d, J = 1 Hz,
7
1.2 (C-28), 24.5 (C-29), 28.8 (C-30).
C-24), 18.5 (C-25), 15.1 (C-26), 13.4 (C-27), 71.2 (C-28), 24.5 (C-
1
9
F NMR: d = –80.80 (dt, J = 248.5, 19.0 Hz), –82.72 (dq,
2
9), 28.8 (C-30).
J = 248.5, 19.6 Hz).
1
9
F NMR: d = –100.10 (ddd, J = 262.1, 20.4, 6.1 Hz), –87.76 (dddt,
+
MS: m/z (%) = 460 (M , 9), 440 (100), 420 (8), 409 (2), 389 (1), 365
1), 316 (1), 205 (15), 191 (3).
J = 262, 30, 20, 2 Hz).
(
+
MS: m/z (%) = 476 (M , 100), 458 (8), 445 (20), 405 (80), 388 (1),
41 (1), 281 (1), 204 (16), 191 (23).
Anal. Calcd for C H F O: C, 78.21; H, 10.06; F, 8.25. Found:
3
0
46 2
3
C, 75.11; H, 10.01; F, 8.11.
Anal. Calcd for C H F O : C, 75.59; H, 9.73; F, 7.97. Found:
30
46
2
2
C, 75.33; H, 9.37; F, 7.13.
4
c
–
1
IR (CHCl ): 1241 cm (C–O–C).
3
1
9b,28-Epoxy-2,2-difluoro-18a-oleanan-3b-ol (4a)
1
H NMR: d = 0.80 (s, 3 H), 0.93 (s, 3 H), 0.94 (s, 3 H), 0.98 (s, 3 H),
To a solution of difluoro ketone 3c (475 mg, 0.99 mmol) in a mix-
1
2
.01 (s, 3 H), 1.49 (d, 3 H, J = 22.7 Hz), 1.51 (d, 3 H, J = 23.0 Hz),
.12 (dd,
ture of THF (12 mL) and MeOH (12 mL) cooled in an ice-bath was
1 H, J = 17.6, 13.4 Hz), 2.83 (dddd, 1 H,
added NaBH (200 mg, 5.29 mmol). After 2 h, the reaction mixture
4
J = 21.3, 17.8, 13.9, 12.1 Hz, H-3a), 3.45 (d, 1 H, J = 7.8 Hz, H-
2
was poured into 5% aq HCl (100 mL) and extracted with CHCl3
8a), 3.52 (s, 1 H, H-19a), 3.77 (dd, 1 H, J = 7.8, 1.6 Hz, H-28b).
(100 mL). The organic layer was washed with H O (3 ×). The or-
2
Synthesis 2005, No. 7, 1157–1163 © Thieme Stuttgart · New York

PAPER
Fluorination of Triterpenoids with DAST
1161
1
3
C NMR: d = 52.7 (J = 21 Hz, C-1), C-2 not found, 57.7 (C-3),
7.1 (d, J = 165 Hz, C-4), 53.5 (d, J = 7 Hz, C-5), 20.1 (C-6), 34.4
J = 12.2 Hz, C-20), 27.3 (C-21), 36.6 (C-22), 27.9 (C-23), 16.5 (C-
24), 16.2 (C-25), 15.9 (C-26), 14.6 (C-27), 176.5 (C-28), 110.9 (d,
J = 11.1 Hz, C-29), 85.0 (d, J = 169 Hz, C-30), 21.3 (3b-OCOCH3),
171.0 (3b-OCOCH ), 51.3 (CO CH ).
9
(
2
4
2
1
C-7), 40.9 (C-8), 50.4 (C-9), 43.3 (d, J = 7 Hz, C-10), 24.0 (C-11),
6.4 (C-12), 34.1 (C-13), 40.8 (C-14), 26.1 (C-15), 36.8 (C-16),
1.5 (C-17), 46.9 (C-18), 87.9 (C-19), 36.3 (C-20), 32.7 (C-21),
6.2 (C-22), 26.8 (C-23), 29.1 (C-24), 15.3 (C-25), 16.2 (C-26),
3.5 (C-27), 71.2 (C-28), 24.5 (C-29), 28.8 (C-30).
3
2
3
19
F NMR: d = –215.22 (td, J = 47.3, 2.3 Hz).
+
MS: m/z (%) = 530 (M , 63), 510 (20), 470 (100), 455 (42), 427
(
14), 411 (14), 291 (13), 219 (13), 207 (19), 189 (56).
1
9
F NMR: d = –79.42 (ddd, J = 240.2, 21.3, 14.7 Hz), –91.59 (dm,
Anal. Calcd for C H FO : C, 74.68; H, 9.69; F, 3.58. Found:
J = 240 Hz, S J¢ = 64.6 Hz), –126.77 (m, S J = 161.5 Hz).
33 51
4
C, 74.34; H, 9.72; F, 3.66.
+
MS: m/z (%) = 480 (M , 90), 460 (6), 449 (33), 440 (54), 425 (82),
4
09 (100), 389 (4), 371 (3), 341 (2), 272 (5), 258 (8), 225 (49), 205
Difluoro Olefin 7b
(
61), 191 (30).
To a stirred solution of aldehyde 7a (200 mg, 0.36 mmol) in CHCl3
(5 mL) was added DAST (500 mL, 2.6 mmol). The reaction mixture
was refluxed 24 h, diluted with CHCl (30 mL), and the CHCl layer
Anal. Calcd for C H F O: C, 74.96; H, 9.86; F, 11.86. Found:
C, 74.69; H, 10.12; F, 12.07s.
3
0
47 3
3
3
was washed carefully with 5% aq NaHCO solution (40 mL) and
3
Fluoro Ketone 5b
H O (40 mL). The organic layer was dried (MgSO ) and evaporated
under reduced pressure. The dark yellow-brown residue was filtered
2
4
To a stirred solution of enol ketone 5a (107 mg, 0.23 mmol) in
CH Cl (5 mL), cooled to –78 °C was added DAST (100 mL, 0.52
over a short column of silica gel (3 g, toluene–Et O, 10:1) and sep-
2
2
2
mmol). The reaction mixture was then allowed to warm to r.t. and
arated by HPLC (6% EtOAc in hexane) to give difluoro olefin 7b
2
5
then quenched by addition of H O (1 mL). The separated organic
layer was diluted with CHCl (50 mL), washed with H O (2 × 40
mL), dried (MgSO ) and evaporated under reduced pressure. The
dark yellow residue was separated by HPLC (6% EtOAc in hexane)
to give fluoro ketone 5b (81 mg, 77%) as white crystalline solid; mp
(46 mg, 22%); mp 246–248 °C (MeOH); [a]D +21 (c = 0.31).
2
3
2
–1
IR (CHCl ): 1255 cm (C–O–C).
3
4
1
H NMR: d = 0.83 (s, 3 H), 0.84 (s, 3 H), 0.84 (s, 3 H), 0.91 (s, 3 H),
0
.97 (s, 3 H), 2.04 (s, 3 H), 3.08 (td, 1 H, J = 11.3, 4.3 Hz), 3.68 (s,
2
5
1 H, CO CH ), 4.45 (m, 1 H, S J = 16.0 Hz, H-3a), 5.24 (s, 1 H, H-
2
31–233 °C (MeOH); [a]D +75 (c = 0.17).
2 3
3
0a), 5.30 (t, 1 H, J = 3.2 Hz, H-30b), 6.03 (t, 1 H, J = 55.9 Hz).
–
1
IR (CHCl ): 1616 (C=C), 1689 cm (C=O).
3
1
3
C NMR: d = 38.4 (C-1), 23.7 (C-2), 80.9 (C-3), 37.8 (C-4), 55.4
1
H NMR: d = 0.81 (s, 3 H), 0.84 (d, 3 H, J = 0.9 Hz), 0.94 (d, 3 H,
(
C-5), 18.2 (C-6), 34.3 (C-7), 40.7 (C-8), 50.4 (C-9), 37.1 (C-10),
J = 0.9 Hz), 0.94 (s, 3 H), 1.02 (s, 3 H), 1.09 (s, 3 H), 1.10 (s, 3 H),
2
3
1.0 (C-11), 27.1 (C-12), 38.1 (C-13), 42.3 (C-14), 29.6 (C-15),
2.0 (C-16), 56.3 (C-17), 34.3 (C-18), 38.2 (C-19), 148.7 (t,
2
2
7
.86 (dt, 1 H, J = 16.1, 2.1 Hz, H-1), 3.46 (d, 1 H, J = 7.9 Hz, H-
8a), 3.55 (s, 1 H, H-19a), 3.79 (dd, 1 H, J = 7.8, 1.4 Hz, H-28b),
.35 (ddd, 1 H, J = 82.6, 3.1, 1.5 Hz, H-31).
J = 18.7 Hz, C-20), 33.0 (C-21), 36.5 (C-22), 16.5 (C-23), 27.9 (C-
4), 16.1 (C-25), 15.9 (C-26), 14.6 (C-27), 176.3 (C-28), 114.4 (t,
2
1
3
C NMR: d = 38.5 (C-1), 119.7 (C-2), 207.0 (C-3), 45.5 (C-4), 53.1
J = 9.9 Hz, C-29), 116.6 (t, J = 238.5 Hz, C-30), 171.0 (3b-
(
C-5), 20.1 (C-6), 32.7 (C-7), 40.4 (C-8), 48.9 (C-9), 35.8 (C-10),
OCOCH ), 21.3 (3b-OCOCH ), 51.3 (CO CH ).
3
3
2
3
2
3
3
1
1
1.7 (C-11), 26.4 (C-12), 34.3 (C-13), 40.8 (C-14), 26.4 (C-15),
6.7 (C-16), 41.5 (C-17), 46.8 (C-18), 87.9 (C-19), 36.3 (C-20),
2.8 (C-21), 26.2 (C-22), 28.8 (C-23), 22.0 (C-24), 15.3 (C-25),
6.0 (C-26), 13.4 (C-27), 71.3 (C-28), 24.5 (C-29), 28.7 (C-30),
56.8 (d, J = 278 Hz, C-31).
19
F NMR: d = –115.72 (dd, J = 298, 56 Hz), –114.68 (dd,
J = 298.5, 56 Hz).
+
MS: m/z (%) = 550 (M , 13), 488 (28), 473 (13), 284 (15), 249 (13),
25 (29), 205 (32), 189 (100).
2
1
9
Anal. Calcd for C H F O : C, 72.23; H, 9.18; F, 6.92. Found:
33 50 2 4
C, 71.90; H, 9.08; F, 7.32.
F NMR: d = –121.14 (ddd, J = 82.4, 6.4 Hz, 4.1 Hz).
+
MS: m/z (%) = 470 (M , 100), 450 (15), 439 (23), 399 (69), 383 (1),
3
41 (1), 323 (1), 215 (14), 203 (6), 189 (7).
Difluoro Ketone 8b
Anal. Calcd for C H FO : C, 79.10; H, 10.06; F, 4.04. Found:
Diketone 8a (500 mg, 0.9 mmol) was reacted with DAST (2 mL,
3
1
47
2
C, 79.34; H, 9.76; F, 4.53.
10.4 mmol) in CHCl (1 mL) in a sealed flask. The sealed flask with
3
the reaction mixture was positioned in the fume hood behind protec-
tive shielding, and heated to 70 °C in an oil bath. After 24 h, the
mixture was cooled in an ice-bath and carefully opened before
Methyl 3b-Acetoxy-30-fluorolup-20(29)-en-28-oate (6b)
To a stirred solution of alcohol 6a (100 mg, 0.18 mmol) in CH Cl
2
2
(
5 mL), cooled to –78 °C was added DAST (100 mL, 0.52 mmol).
work-up. The mixture was diluted with CHCl (50 mL), and washed
3
The reaction mixture was then allowed to warm to r.t. and then
carefully with aq NaHCO solution (40 mL) and H O (40 mL). The
3
2
quenched by addition of H O (1 mL). The separated organic layer
organic layer was dried (MgSO ) and evaporated under reduced
2
4
was diluted with CHCl (50 mL), washed with H O (2 × 40 mL),
pressure. The dark brown residue was chromatographed on a col-
3
2
dried (MgSO ) and evaporated under reduced pressure. The dark
umn of silica gel (50 g). Elution with a mixture of toluene and Et O
4
2
yellow residue was separated by HPLC (6% EtOAc in hexane) and
(10:1) followed by crystallization from butanone gave difluoro ke-
tone 8b (135 mg, 26%); mp 188–190 °C; [a]D +67 (c = 0.23).
2
5
crystallized from MeOH to give 6b (80 mg, 80%); mp 219–221 °C;
25
[
a]D –6.0 (c = 0.37).
–1
IR (CHCl ): 1253 (C–O–C), 1608 (C=C), 1727 cm (C=O).
3
IR (CHCl ): 1256 (C–O–C), 1655 (C=C), 1721 cm– (C=O).
1
3
1
H NMR: d = 0.85 (s, 3 H), 0.86 (s, 3 H), 0.93 (s, 3 H), 0.96 (s, 3 H),
1
H NMR: d = 0.83 (s, 3 H), 0.84 (s, 3 H), 0.84 (s, 3 H), 0.91 (s, 3 H),
1.14 (s, 3 H), 1.20 (dd, 1 H, J = 7.2, 2.6 Hz), 1.25 (dd, 1 H,
J = 7.2, 2.5 Hz), 1.98 (s, 3 H, 28-OAc), 2.05 (s, 3 H, 3b-OCOCH3),
2.93 (m, 1 H, S J = 20.4 Hz, H-13b), 3.38 (septet, 1 H, J = 7.0 Hz,
H-20), 3.96 (d, 1 H, J = 11.1 Hz, H-28a), 4.49 (m, 1 H, S J = 16.3
Hz, H-3a), 4.77 (d, 1 H, J = 11.1 Hz, H-28b).
0
3
2
.96 (s, 3 H), 2.04 (s, 3 H), 2.95 (td, 1 H, J = 11.1, 4.4 Hz, H-19b),
.67 (s, 3 H, CO CH ), 4.47 (m, 1 H, S J = 16.0 Hz, H-3a), 4.84 (d,
2
3
H, J = 47.5 Hz, H-30), 5.02 (m, 2 H, S J = 6.3 Hz, H-29).
1
3
C NMR: d = 38.4 (C-1), 23.7 (C-2), 80.9 (C-3), 37.8 (C-4), 55.4
1
3
(
C-5), 18.2 (C-6), 34.2 (C-7), 40.7 (C-8), 50.4 (C-9), 37.1 (C-10),
C NMR: d = 38.6 (C-1), 23.6 (C-2), 80.7 (C-3), 37.8 (C-4), 55.4
2
3
1.0 (C-11), 26.7 (C-12), 38.2 (C-13), 42.3 (C-14), 29.7 (C-15),
2.0 (C-16), 56.6 (C-17), 50.1 (C-18), 42.2 (C-19), 150.1 (d,
(C-5), 18.1 (C-6), 34.7 (C-7), 41.3 (C-8), 50.9 (C-9), 37.1 (C-10),
21.3 (C-11), 27.7 (C-12), 41.6 (C-13), 44.2 (C-14), 27.3 (C-15),
Synthesis 2005, No. 7, 1157–1163 © Thieme Stuttgart · New York

1
162
D. Biedermann et al.
PAPER
2
6.8 (C-16), 52.2 (C-17), 149.1 (t, J = 10.3 Hz, C-18), 138.4 (t,
Anal. Calcd for C H F O : C, 73.13; H, 9.82; F, 9.25. Found:
2
5
40
2
2
J = 19.5 Hz, C-19), 25.8 (C-20), 117.0 (dd, J = 252.6, 249.5 Hz, C-
C, 72.98; H, 9.52; F, 9.26.
2
2
1
2
1), 204.2 (t, J = 24.8 Hz, C-22), 27.9 (C-23), 16.5 (C-24), 16.8 (C-
5), 16.8 (C-26), 15.7 (C-27), 63.7 (C-28), 22.1 (C-29), 22.1 (C-30),
71.0 (3b-OCOCH ), 21.3 (3b-OCOCH ), 170.3 (28-OCOCH ),
Fluorination of Ketones 2b, 2c and 11a with DAST
The ketones (200 mg, 0.38 mmol) were reacted with DAST (300
3
3
3
0.4 (28-OCOCH3).
mL, 1.33 mmol) in CHCl (500 mL) in the same manner as describe
3
1
9
above. The brown residue was separated by HPLC (15% vol.
EtOAc in hexane). According to spectral data, only starting material
was recovered.
F NMR: d = –97.71 (ddq, J = 301.8, 7.9, 3.0 Hz), –103.70 (d,
J = 301.8 Hz).
+
MS: m/z (%) = 576 (M , 1), 557 (14), 517 (7), 497 (11), 474 (10),
94 (10), 259 (7), 234 (10), 217 (14), 203 (29), 189 (100).
2
Acyl Fluoride 12c
To a stirred solution of acid 12b (200 mg, 0.40 mmol) in CH Cl (5
mL) was added DAST (200 mL, 1.04 mmol) at –78 °C. The reaction
mixture was then allowed to warm to r.t. and then quenched by ad-
Anal. Calcd for C H F O : C, 70.80; H, 8.74; F, 6.59. Found:
C, 70.57; H, 8.70; F, 6.37.
2
2
34
50
2
5
dition of H O (1 mL). The separated organic layer was diluted with
Difluoro Ketone 9b
Diketone 9a (450 mg, 0.8 mmol) was treated with DAST (1 mL, 5.2
2
CHCl (50 mL), washed with H O (2 × 40 mL), dried (MgSO ) and
3
2
4
evaporated under reduced pressure. The yellow residue was separat-
ed by HPLC (3% EtOAc in hexane) and crystallized from MeOH to
mmol) in CHCl (1 mL) in the same manner as described above.
3
The dark brown residue was separated by HPLC (25% EtOAc in
hexane). Lyophilization (t-BuOH) gave difluoro ketone 9b as a
white solid (138 mg, 29%); mp 131–133 °C; [a]D +31 (c = 0.25).
2
5
give acyl fluoride 12c (130 mg, 65%); mp 211–213 °C; [a]D +26
2
5
(c = 0.33).
–
1
–
1
IR (CHCl ): 1256 (C–O–C), 1720 (C=O), 1823 cm .
IR (CHCl ): 1255 (C–O–C), 1615 (C=C), 1728 cm (C=O).
3
3
1
1
H NMR: d = 0.83 (s, 3 H), 0.84 (s, 3 H), 0.85 (s, 3 H), 0.95 (s, 3 H),
H NMR: d = 0.84 (s, 3 H), 0.85 (s, 3 H), 0.91 (s, 3 H), 0.96 (s, 3 H),
0
.97 (s, 3 H), 1.69 (s, 3 H), 2.04 (s, 3 H, OCOCH ), 2.90 (td, 1 H,
1
2
.03 (s, 3 H), 1.24 (dd, J = 4.4, 2.4 Hz), 2.05 (s, 3 H, 3b-OCOCH ),
3
3
J = 10.8, 4.9 Hz, H-19), 4.47 (m, 1 H, S J = 16.2 Hz, H-3a), 4.64 (t,
.44 (dq, J = 13.4, 2.4 Hz), 2.65 (t, J = 9.3 Hz), 3.39 (septet, 1 H,
1
H, J = 1.5 Hz, H-29a), 4.75 (d, 1 H, J = 2.1 Hz, H-29b).
J = 7.0 Hz, H-20), 3.74 (s, 3 H, CO CH ), 4.48 (m, 1 H, S J = 16.5
Hz, H-3a).
2
3
13
C NMR: d = 38.4 (C-1), 23.7 (C-2), 80.9 (C-3), 37.8 (C-4), 55.4
1
3
(C-5), 18.1 (C-6), 34.2 (C-7), 40.7 (C-8), 50.4 (C-9), 37.1 (C-10),
C NMR: d = 38.5 (C-1), 23.6 (C-2), 80.7 (C-3), 37.7 (C-4), 55.4
C-5), 18.0 (C-6), 34.6 (C-7), 41.1 (C-8), 50.7 (C-9), 37.1 (C-10),
2
3
1
1
1
0.8 (C-11), 25.3 (C-12), 38.5 (C-13), 42.4 (C-14), 29.7 (C-15),
0.9 (C-16), 57.0 (d, J = 39 Hz, C-17), 49.1 (C-18), 46.7 (C-19),
49.3 (C-20), 30.0 (C-21), 35.6 (C-22), 27.9 (C-23), 16.5 (C-24),
6.2 (C-25), 15.9 (C-26), 14.7 (C-27), 165.2 (d, J = 374 Hz, C-28),
(
2
2
1.3 (C-11), 27.7 (C-12), 43.7 (C-13), 43.8 (C-14), 27.9 (C-15),
8.4 (C-16), 53.3 (C-17), 148.7 (t, J = 10.3 Hz, C-18), 138.3 (t,
J = 19.1 Hz, C-19), 25.8 (C-20), 117.7 (t, J = 253.1 Hz, C-21),
10.4 (C-29), 19.3 (C-30), 21.3 (3b-OCOCH ), 172.0 (3b-
2
1
1
04.1 (t, J = 25.0 Hz, C-22), 27.7 (C-23), 16.5 (C-24), 16.5 (C-25),
6.8 (C-26), 15.8 (C-27), 168.3 (C-28), 21.1 (C-29), 21.2 (C-30),
3
OCOCH3).
71.0 (3b-OCOCH ), 21.2 (3b-OCOCH ), 51.7 (CO CH ).
19
3
3
2
3
F NMR: d = 35.90 (d, J = 11.3 Hz).
1
9
F NMR: d = –95.35 (ddd, J = 299.8, 8.4, 3.0 Hz), –103.92 (d,
J = 299.8 Hz).
+
MS: m/z (%) = 500 (M , 76), 485 (4), 453 (7), 440 (28), 425 (23),
3
97 (8), 304 (11), 250 (15), 203 (15), 189 (100).
+
MS: m/z (%) = 543 (M – 19, 100), 482 (69), 467 (5), 459 (3), 439
8), 423 (9), 348 (2), 304 (4), 280 (31), 203 (31), 190 (74).
Anal. Calcd for C H FO : C, 76.76; H, 9.86; F, 3.79. Found:
3
2
49
3
(
C, 76.73; H, 10.11; F, 3.28.
Anal. Calcd for C H F O : C, 70.43; H, 8.60; F, 6.75. Found:
C, 70.14; H, 8.55; F, 6.33.
33
48
2
5
Acyl Fluoride 13b
Dihydro acid 13a (131 mg, 0.25 mmol) in CH Cl (5 mL) was fluo-
2
2
rinated with DAST (130 mL, 0.68 mmol) in the same manner as de-
Difluoro Derivative 10b
Heptanorketone 10a (300 mg, 0.33 mmol) was treated with DAST
scribed for acid 12b. The yellow residue was separated by HPLC
(
3% EtOAc in hexane) and crystallized from i-PrOH to give acyl
(
300 mL, 1.33 mmol) in CHCl (300 mL) in the same manner as de-
3
2
5
fluoride 13b (66 mg, 63%); mp 247–251 °C; [a]D +2 (c = 0.51).
scribe above. After 12 h, the reaction mixture was worked up. The
dark brown residue was separated by HPLC (13% EtOAc in hex-
ane). Lyophylization (t-BuOH) gave difluoro derivative 10b as a
–1
IR (CHCl ): 1256 (C–O–C), 1720 cm (C = O).
3
1
2
5
H NMR: d = 0.76 (d, J = 6.8 Hz), 0.84 (s, 3 H), 0.85 (s, 3 H), 0.86
d, J = 6.8 Hz), 0.86 (d, J = 0.8 Hz), 0.95 (s, 3 H), 0.95 (s, 3 H), 2.05
white solid (40 mg, 30%); mp 168–170 °C; [a]D +101 (c = 0.18).
(
–
1
IR (CHCl ): 1255 (C–O–C), 1721 cm (C=O).
3
(s, 3 H, OCOCH ), 1.84 (ds, 1 H, J = 6.7, 2.9 Hz, H-20), 4.47 (m, 1
3
1
H, S J = 16.5 Hz, H-3a).
13
H NMR: d = 0.84 (s, 3 H), 0.86 (s, 3 H), 0.88 (s, 3 H), 0.99 (s, 3 H),
.01 (d, J = 2.0 Hz), 2.05 (s, 3 H, OCOCH ), 4.49 (m, 1 H, S
1
3
C NMR: d = 38.4 (C-1), 23.7 (C-2), 80.9 (C-3), 37.8 (C-4), 55.4
C-5), 18.1 (C-6), 36.0 (C-7), 40.7 (C-8), 50.2 (C-9), 37.1 (C-10),
20.8 (C-11), 26.7 (C-12), 38.4 (C-13), 42.6 (C-14), 29.7 (C-15),
J = 16.5 Hz, H-3a).
(
1
3
C NMR: d = 38.5 (C-1), 23.6 (C-2), 80.7 (C-3), 37.7 (C-4), 55.4
C-5), 18.1 (C-6), 33.2 (C-7), 40.9 (C-8), 50.4 (C-9), 37.1 (C-10),
9.1 (C-11), 20.1 (C-12), 43.9 (dd, J = 20.9, 20.9 Hz, C-13), 42.0
3
2
1
2
0.8 (C-16), 57.5 (d, J = 38 Hz, C-17), 48.6 (C-18), 43.9 (C-19),
9.5 (C-20), 34.3 (C-21), 22.3 (C-22), 27.9 (C-23), 16.5 (C-24),
6.2 (C-25), 15.9 (C-26), 14.6 (C-27), 165.5 (d, J = 376.2 Hz, C-
(
1
(
(
d, J = 7.6 Hz, C-14), 18.6 (d, J = 10.3 Hz, C-15), 30.2 (C-16), 34.5
d, J = 26.0 Hz, C-17), 124.7 (d, J = 240.0 Hz, C-18), 16.4 (C-23),
8), 14.6 (C-29), 22.8 (C-30), 21.3 (3b-OCOCH ), 171.0 (3b-
3
2
7.9 (C-24), 16.5 (C-25), 15.8 (C-26), 14.5 (d, J = 7.2 Hz, C-27).
OCOCH3).
1
9
19F
F NMR: d = 35.48 (d, J = 10.5 Hz).
NMR: d = –90.20 (d, J = 236.5 Hz), –106.35 (ddt,
J = 235.8, 29.8, 12.2 Hz).
+
MS: m/z (%) = 502 (M , 58), 455 (92), 442 (38), 428 (43), 399 (28),
+
249 (22), 204 (14), 189 (100).
MS: m/z (%) = 410 (M , 17), 350 (51), 335 (21), 307 (6), 249 (15),
2
28 (5), 189 (100).
Anal. Calcd for C H FO : C, 76.45; H, 10.22; F, 3.78. Found:
3
2
51
3
C, 76.30; H, 9.99; F, 3.47.
Synthesis 2005, No. 7, 1157–1163 © Thieme Stuttgart · New York

PAPER
Fluorination of Triterpenoids with DAST
1163
Acyl Fluoride 14b
Oxo acid 14a (200 mg, 0.39 mmol) in CH Cl (5 mL) was fluorinat-
ed with DAST (200 mL, 1.05 mmol) in the same manner as de-
scribed for acid 12b. The yellow residue was separated by HPLC
equipments), Czech Science Foundation (203/00/1232) (materials
and chemicals) and MPO project (FT-TA/027) (HPLC chromato-
graphy). Biological testing was supported by the Czech Science
Foundation (301/03/1570). We are grateful to Iva Tislerova for
measurements of NMR spectra, Stanislav Hilgard and Miroslav
Kvasnica for measurements of IR spectra. Special thanks go to Kri-
stin Israelsson from Billerud Papermill, Sweden for providing the
birch bark and Martin Buchta from IVAX, Pharmaceuticals for in-
dustrial extraction of the birch bark. We also thank to Bohunka
Sperlichova for measurement of optical rotation.
2
2
(
10% EtOAc in hexane) and crystallized from MeOH to give acyl
2
5
fluoride 14b (160 mg, 65%); mp 247–251 °C; [a]D –46 (0.50).
–
1
IR (CHCl ): 1255 (C–O–C), 1613 (C=C), 1706 (C=O), 1831 cm .
3
1
H NMR: d = 0.85 (s, 3 H), 0.86 (s, 3 H), 0.92 (s, 3 H), 0.95 (d,
J = 0.6 Hz), 1.08 (s, 3 H), 1.21 (d, J = 6.9 Hz), 1.22 (d, J = 7.0 Hz),
2
2
1
.05 (s, 3 H, OCOCH ), 2.23 (dd, 1 H, J = 18.7, 0.9 Hz, H-22a),
.66 (d, 1 H, J = 18.7 Hz, H-22b), 2.7 (dd, 1 H, J = 12.6, 3.3 Hz, H-
3a), 2.44 (ddd, 1 H, J = 13.6, 4.2, 2.5 Hz, H-16b), 3.22 (sept, 1 H, References
3
J = 7.0 Hz), 4.49 (m, 1 H, S J = 16.6 Hz, H-3a).
(
(
1) Hudlicky, M. Org. React. 1988, 35, 513.
2) Slavikova, B.; Kasal, A.; Chodounska, H.; Kristofikova, Z.
1
3
C NMR: d = 38.6 (C-1), 23.6 (C-2), 80.6 (C-3), 37.8 (C-4), 55.4
C-5), 18.1 (C-6), 34.8 (C-7), 41.3 (C-8), 51.0 (C-9), 37.1 (C-10),
(
Collect. Czech. Chem. Commun. 2002, 67, 30.
3) Kirihara, M. Yakugaku Zasshi 2000, 120, 339; Chem. Abstr.
2
3
2
1
2
1.0 (C-11), 27.5 (C-12), 45.5 (C-13), 54.3 (C-14), 28.8 (C-15),
3.4 (C-16), 52.6 (d, J = 46 Hz, C-17), 168.4 (C-18), 146.9 (C-19),
5.2 (C-20), 205.0 (C-21), 45.5 (C-22), 27.9 (C-23), 16.5 (C-24),
6.8 (C-25), 16.6 (C-26), 15.8 (C-27), 163.8 (d, J = 368 Hz, C-28),
(
2
000, 132, 347199.
(
4) Xia, J.; Chen, Y.; Liberatore, K. M.; Selinsky, B. S.
Tetrahedron Lett. 2003, 44, 9295.
0.0 (C-29), 19.9 (C-30), 21.3 (3b-OCOCH ), 171.0 (3b-
3
(5) Dax, K.; Albert, M.; Hammond, D.; Illaszewicz, C.;
Purkarthofer, T.; Tscherner, M.; Weber, H. Monatsh. Chem.
2000, 133, 427.
OCOCH3).
1
9
F NMR: d = 30.0 (d, J = 6.8 Hz).
(
6) Pu, Y. M.; Torok, D. S.; Ziffer, H. J. Med. Chem. 1995, 38,
120.
7) Bartmann, E.; Krause, J. J. Fluorine Chem. 1993, 61, 117.
+
MS: m/z (%) = 500 (M , 37), 457 (6), 440 (40), 425 (37), 411 (4),
4
3
97 (13), 305 (8), 281 (29), 251 (38), 203 (29), 189 (100).
(
Anal. Calcd for C H FO : C, 74.67; H, 9.20; F, 3.69. Found:
C, 74.87; H, 9.31; F, 3.68.
(8) Thomas, M. G.; Suckling, J. C.; Pitt, A. R.; Suckling, K. E.
32
47
4
J. Chem. Soc., Perkin Trans. 1 1999, 3191.
(
9) Sarek, J.; Klinot, J.; Dzubak, P.; Klinotova, E.; Noskova, V.;
Krecek, V.; Korinkova, G.; Thomson, J. O.; Janostakova, A.;
Wang, S.; Parsons, S.; Fischer, P. M.; Zhelev, N. Z.;
Hajduch, M. J. Med. Chem. 2003, 46, 5402.
Cytotoxic MTT Assay
Screening of cytotoxic activity was performed on highly chemosen-
sitive T-lympoblastic leukaemia CEM cells using cytotoxic MTT
9
assay. The cells were prepared and diluted according to the expect-
(10) Richtr, V.; Klinot, J.; Kraitr, M. Chem. Listy 1998, 92, 963.
(11) Matsuzawa, A.; Yamamoto, T. Cancer Res. 1977, 37, 4408.
(12) Li, T.; Wang, J.; Zheng, X. J. Chem. Soc., Perkin Trans. 1
1998, 3957.
ed target cell density (5000 cells/well). The cells were added by pi-
pette (80 mL) into 96-well microtiter plates. Inoculates were allowed
a pre-incubation period of 24 h at 37 °C and 5% CO for stabiliza-
2
tion. Four-fold dilutions, in 20 mL aliquots, of the intended test con-
centration were added at time zero to the microtiter plate wells. All
tested compounds were dissolved in 10% DMSO and concentra-
tions were examined in duplicate. Incubation of the cells with the
(13) Klinot, J.; Vystrcil, A. Collect. Czech. Chem. Commun.
1966, 31, 1079.
(14) Sejbal, J.; Klinot, J.; Protiva, J.; Vystrcil, A. Collect. Czech.
Chem. Commun. 1986, 51, 118.
test compounds lasted for 72 h at 37 °C, in a 5% CO atmosphere at
(15) Klinot, J.; Sejbal, J.; Vystrcil, A. Collect. Czech. Chem.
2
1
00% humidity. At the end of the incubation period, the cells were
Commun. 1989, 54, 400.
assayed using MTT. Aliquots (10 mL) of the MTT stock solution
were pipetted into each well and incubated for a further 1–4 h. After
this incubation period, formazan produced was dissolved by the ad-
dition of 100 mL/well of 10% aq SDS (pH 5.5), followed by a fur-
ther incubation at 37 °C overnight. The optical density (OD) was
measured at 540 nm with a Labsystem iEMS Reader MF. Tumour
cell survival (TCS) was calculated using the following equation:
(16) Nair, M. R.; Hilgard, S.; Klinot, J.; Waisser, K.; Vystrcil, A.
Collect. Czech. Chem. Commun. 1976, 41, 770.
(17) Klinot, J.; Svetly, J.; Kudlackova, D.; Budesinsky, M.;
Vystrcil, A. Collect. Czech. Chem. Commun. 1979, 44, 211.
(18) Pradhan, P. B.; Chakraborty, S.; Sinha, P. R. Ind. J. Chem.
Sect. B: Org. Chem. Incl. Med. Chem. 1995, 34, 540.
(19) Hajduch, M.; Sarek, J. PCT Int. Patent Appl. WO 0190046,
2001; Chem. Abstr. 2002, 136, 6179.
TCS = (ODdrug-exposed well/mean ODcontrol wells) × 100%. The TCS
50
value, the drug concentration lethal to 50% of the tumour cells, was
calculated from appropriate dose-response curves.
(20) Urban, M.; Sarek, J.; Klinot, J.; Korinkova, G.; Hajduch, M.
J. Nat. Prod. 2004, 67, 1100.
(21) Klinot, J.; Vystrcil, A. Collect. Czech. Chem. Commun.
1
964, 29, 516.
Acknowledgment
(
22) Fischer, P. M.; Sarek, J.; Blaney, P. M.; Collier, P.;
Fergusson, J. R. PCT Int. Patent Appl. WO 03/045971,
This study was supported by the Ministry of Education of the Czech
Republic (MSM 113100001 and MSM 151100001) (instrumental
2003; Chem. Abstr. 2003, 139, 7043.
Synthesis 2005, No. 7, 1157–1163 © Thieme Stuttgart · New York