Synthesis and evaluation of biological activity of the auaternary ammonium salts of lupane-, oleanane-, and ursane-type acids

Article abstract of DOI:10.1055/s-0030-1258293

The anticancer properties of the derivatives of natural pentacyclic triterpenoids have received much attention recently. Here we present the preparation and the evaluation of the anticancer activity of a novel group of derivatives: quaternary ammonium esters. The esters were synthesized by quaternization of the respective 2-bromoethyl esters of betulinic, dihydrobetulinic, platanic, oleanolic, ursolic, and 3-acetoxy-21-oxolup-18-en- 28-oic acids with common low-molecular-weight tertiary amines, namely trimethylamine, triethylamine, pyridine, and triethanolamine. However, the desired quaternary salts were obtained only from trimethylamine, triethylamine, and pyridine. In case of the reaction with triethanol?amine the elimination of one of the 2-hydroxyethyl groups occurred and only tertiary amines were formed. Most of the prepared compounds showed significant in vitro cytotoxic activity on the CEM T-lymfoblastic leukaemia cell line. Three of the six selected compounds {2-(triethylammonio)ethyl 3-hydroxylup-20(29)-en-28-oate bromide, 2-(triethylammonio)ethyl 3b-hydroxylupan-28-oate bromide and 2-[bis(2-hydroxyethyl)amino]ethyl 3-hydroxylupan-28-oate] exhibited strong cytotoxic activity on a panel of ten cell lines, including drug resistant. Georg Thieme Verlag Stuttgart · New York.

Full text of DOI:10.1055/s-0030-1258293

PAPER
3839
Synthesis and Evaluation of Biological Activity of the Quaternary Ammonium
Salts of Lupane-, Oleanane-, and Ursane-Type Acids
pe Acids iedermann, Barbara Eignerova, Marian Hajduch, Jan Sarek*e
a
b,c
d
Q
D
uaternary Ammo
a
nium Salts
v
of
L
upane-,
i
O
leana
d
ne-,
a
nd Ursane-T
yB
a
b
c
d
e
Institute of Microbiology, Academy of Sciences of the Czech Republic, Centre of Biocatalysis and Biotransformations, Vídeňská 1083,
42 20 Prague, Czech Republic
1
Department of Organic and Nuclear Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague 2,
Czech Republic
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6,
Czech Republic
Laboratory of Experimental Medicine, Department of Pediatrics, Faculty of Medicine, Palacky University in Olomouc, Puškinova 6,
775 20 Olomouc, Czech Republic
Department of Organic Chemistry, Faculty of Science, Palacky University in Olomouc, 17. Listopadu 1192/12, 771 46 Olomouc,
Czech Republic
Fax +420(226)015452; E-mail: jan.sarek@gmail.com
Received 6 May 2010; revised 20 August 2010
This paper is dedicated to Prof. Jiří Klinot and Dr. Eva Klinotová.
yl esters of triterpenic acids) and low-molecular-weight
tertiary amines is described. Our group has previously
Abstract: The anticancer properties of the derivatives of natural
pentacyclic triterpenoids have received much attention recently.
Here we present the preparation and the evaluation of the anticancer
activity of a novel group of derivatives: quaternary ammonium es-
published various syntheses of different cytotoxic triter-
penoids.1
0–12
The synthesis of hydrophobic derivatives
ters. The esters were synthesized by quaternization of the respective (fluoro derivatives) and the synthesis of moderately hy-
2
-bromoethyl esters of betulinic, dihydrobetulinic, platanic, olean- drophilic derivatives (oxo derivatives) has been accom-
olic, ursolic, and 3b-acetoxy-21-oxolup-18-en-28-oic acids with
common low-molecular-weight tertiary amines, namely trimethy-
lamine, triethylamine, pyridine, and triethanolamine. However, the
desired quaternary salts were obtained only from trimethylamine,
triethylamine, and pyridine. In case of the reaction with triethanol-
12,13
plished,
and the biological activity of the resulting
derivatives was studied. It was shown that the cytotoxic
activity strongly correlated with the hydrophilicity.
Therefore, we have focused on the synthesis of the previ-
amine the elimination of one of the 2-hydroxyethyl groups occurred ously unknown highly polar quaternary salts of triterpe-
and only tertiary amines were formed. Most of the prepared com- noids.
pounds showed significant in vitro cytotoxic activity on the CEM T-
The starting materials, betulin (1a) and the pentacyclic
lymfoblastic leukaemia cell line. Three of the six selected com-
triterpenic acids 2a, 3a, 4a, 5a, 6a, and 7a are shown in
pounds {2-(triethylammonio)ethyl 3b-hydroxylup-20(29)-en-28-
1
4
oate bromide, 2-(triethylammonio)ethyl 3β-hydroxylupan-28-oate Scheme 1. Acid 2a described by us previously is avail-
15
bromide and 2-[bis(2-hydroxyethyl)amino]ethyl 3b-hydroxylupan- able from betulin (1a) in five steps in 20% total yield.
16
2
8-oate] exhibited strong cytotoxic activity on a panel of ten cell
Betulin (1a) is by far the most abundant natural pentacy-
clic triterpene and, therefore, acid 2a is the most readily
lines, including drug resistant.
Key words: amines, esters, terpenoids, natural products, cytotoxic- available triterpenic acid (in multigram quantities) despite
ity screening
the multistep synthesis. Betulinic acid (3a) was isolated
from London Plane tree bark (Platanus × hispanica) by
1
7
extraction with methanol, platanic acid (4a) was pre-
Triterpenoids belong to a large family of natural com- pared from 3a by simple ozonolysis,18 and dihydrobetu-
1
,2
pounds exhibiting a wide range of biological activity.
linic acid (5a) was synthesized by the hydrogenation of
3
19
Pentacyclic triterpenoids show anticancer, chemopreven- 3a. Oleanolic (6a) and ursolic acids (7a) were obtained
4
5
6
7,8
tive, imunosuppresive, anti-inflammatory, anti-HIV,
from commercial suppliers.
9
6
antitrypanosomal, and antimicrobial properties. This
promising anticancer activity led to increased research ac-
tivity in this field. According to the Web of Science data-
base in the year 2000, four articles regarding the
antineoplastic activity of triterpenoids were published;
this number increased to thirty articles in 2009.
Preliminary experiments demonstrated reasonable acces-
sibility of 2-bromoethyl esters of triterpenic acids, a typi-
cal reaction protocol involved suspension of the starting
material and potassium carbonate in N,N-dimethylform-
amide and subsequent addition of 1,2-dibromoethane.
However, dimeric byproducts were formed during these
In this paper, the preparation of quaternary ammonium reactions (results not shown). This problem was overcome
salts using triterpenic bromides (particularly 2-bromoeth- by the addition of a large excess of 1,2-dibromoethane (10
equiv). Quaternization was achieved by the reaction of the
2
-bromoethyl ester with common low-molecular-weight
SYNTHESIS 2010, No. 22, pp 3839–3848
x
x.
x
x
.
2
0
1
0
Advanced online publication: 08.10.2010
DOI: 10.1055/s-0030-1258293; Art ID: T10110SS
Georg Thieme Verlag Stuttgart · New York
tertiary amines; trimethylamine, triethylamine, pyridine,
or triethanolamine; only trimethylamine, triethylamine,
©

3
840
D. Biedermann et al.
PAPER
2
9
Table 1 Typical Reaction Times of Triterpenic 2-Bromoethyl Es-
ters with Low-Molecular-Weight Tertiary Amines
2
0
O
3
0
21
1
9
1
8
22
Reagent
Reaction time (h)
2–3
2
8
5
steps
H
COOH
Me N
OH
3
3
HO
AcO
Et N
48–72
3
1
a
pyridine
60–96
2a
O
triethanolamine
<168
1
. O3, CH2Cl2,
80 °C
. Me2S
–
O
H
COOH
H
COOH
2a
2
O
O
N
Br
BrCH2CH2Br
K2CO3
HO
HO
MeCN
81%
2c
DMF, 50 °C
3
a
4a
O
61%
O
O
O
N
Br
C
H
COOH
O
N, DMF, 60 °
Et
Br
2d
O
HO
O
2
b
5a
Br
O
O
N
2
e
1
2
H
COOH
28
H
COOH
O
OH
3
HO
HO
O
N
6
a
7a
O
HO
Scheme 1
2
f
and pyridine afforded the desired quaternary salts. In the
case of triethanolamine, elimination of one of the 2-hy-
droxyethyl groups occurred and a tertiary amine was
formed. Other amines (pyrazine, imidazole, pyrrole, 2-
chloroethyl esters) did not afford satisfactory results and
complicated mixtures of products were formed or no reac-
tion occurred.
Scheme 2
could crystallize directly from the concentrated reaction
mixtures to yield very pure products. However, in the case
of the tertiary triterpenic amines 2f–7f and the quaternary
salts 2c, 2d, 3d, 4c, and 4e the product failed to crystallize
and the reaction mixture had to be purified by column
All reactions were carried out under an argon atmosphere chromatography.
in sealed glass vessel. The reactions proceeded very slow-
Initially, small groups of quaternary ammonium salts
were synthesized from a lupane-type 2-bromoethyl esters
ly, with the exception of the reactions with trimethyl-
amine (Table 1) even at higher temperatures (50–70 °C).
Reactions were generally very selective and differences in
the yields are mainly due to different solubilities of com-
pounds in N,N-dimethylformamide and difficulties in the
workup procedure.
2
2
b–5b. From 2-bromoethyl ester 2b, quaternary salts 2c,
d, 2e, and tertiary amine 2f were prepared (Scheme 2).
Lower yields of the salt 2d accounts for its failure to crys-
tallize and its difficult purification. From 2-bromoethyl
esters of betulinic and platanic acids 3b and 4b, analogous
The workup and purification of the polar compounds quaternary salts 3c–4e and tertiary amines 3f and 4f were
proved to be difficult, the prepared quaternary compounds prepared (Scheme 3). In the case of the quaternary deriv-
and tertiary amines are partially soluble in water and their atives of dihydrobetulinic acid 5c–5e, prepared from 5b,
solubility in chloroform-like solvents is very low. We higher yields are probably due to the lower solubility of
have tested several chromatographic methods to obtain the products in N,N-dimethylformamide (Scheme 4). The
pure quaternary salts; gradient method with elution with amine 5f can be easily purified by crystallization from
pure chloroform and then gradient of methanol up to 20% ethanol.
proved satisfactory. We also have observed that pure salts
Synthesis 2010, No. 22, 3839–3848 © Thieme Stuttgart · New York

PAPER
Quaternary Ammonium Salts of Lupane-, Oleanane-, and Ursane-Type Acids
3841
R
R2
R1
O
N
Br
H
COOH
R2
H
4
a
O
R1
3c: R = CH2
4c: R = O
HO
Br
N
BrCH2CH2Br
K2CO3
R
O
38%
MeCN, DMF
6a: R1 = H; R2 = Me
5
0 °C
a: R = Me; R2 = H
1
O
7
O
N
Br
BrCH CH Br
6c: R1 = H; R2 = Me
7
R
C
2
2
c: R1 = Me; R2 = H
6
b: 52% K2CO3
N, DMF, 60 °
O
Et
7b: 41% MeCN
DMF, 50 °C
R2
R1
R
3d: R = CH2
d: R = O
O
4
Br
Br
R2
H
C
R1
O
O
N
Br
N
N, DMF, 60 °
H
Et
O
O
3
4
b: R = CH2
b: R = O
O
O
Br
O
6
7
d: R1 = H; R2 = Me
d: R = Me; R2 = H
1
3
e: R = CH2
4e: R = O
BrCH2CH2Br
K2CO3
MeCN, DMF
0 °C
R2
R
b: R1 = H; R2 = Me
6
7
R1
6
4%
b: R = Me; R2 = H
1
5
HO
Br
O
N
O
H
3
a
N
6e
:
57%;
O
O
7e
OH
f: R = CH2
f: R = O
: 67%
e: R = H; R2 = Me
1
6
3
4
7e: R1 = Me; R2 = H
R2
Scheme 3
R1
OH
O
N
H
O
HO
O
N
Br
6f: R1 = H; R2 = Me
7
5
a
f: R = Me; R2 = H
1
O
BrCH2CH2Br
K2CO3
5c
Scheme 5
8
0%
MeCN
DMF, 50 °C
good. The tertiary amines 6f and 7f were formed also in
satisfactory yields.
70%
O
N
Br
C
O
Finally, the biological activity of the synthesized com-
pounds was investigated. Although anti-inflammatory
N, DMF, 60 °
Et
5
d
O
O
Br
(
suppression of production of NO in mice macrophages)
activity of all tested compounds was negligible, antican-
cer activity of these compounds was significant. All new-
ly prepared compounds were tested for their in vitro
cytotoxic activity against CEM human T-lymphoblastic
leukaemia cell line using the standard MTT test
Br
O
5
b
N
O
5
e
(
Table 2).20
HO
The results show that all quaternary ammonium salts pos-
sess higher cytotoxic activity than the starting 2-bromoet-
hyl esters. The most effective substances are lupane
derivative 5d and the ursane derivative 7d that exhibit
particularly good cytotoxic activity against the CEM line
O
N
O
OH
5
f
21
(
IC <5 mmol/L).
Scheme 4
50
In this work we have prepared 20 new alicyclic deriva-
Similarly, oleanane and ursane 2-bromoethyl esters 6b tives of triterpenic acids, 15 of them were quaternary am-
and 7b were used to prepare quaternary salts 6c–e and monium esters. 2-Bromoethyl esters were prepared from
7
c–e (Scheme 5). All of the oleanane- and ursane-type starting triterpenic acids and quaternized with tertiary
salts crystallized and their purity and the yields were amines (Me N, Et N, pyridine, and triethanolamine).
3 3
Quaternizations with trimethylamine, triethylamine, and
Synthesis 2010, No. 22, 3839–3848 © Thieme Stuttgart · New York

3
842
D. Biedermann et al.
PAPER
Table 2 Cytotoxic Activities of the Triterpenic Compounds Against
CEM T-Lymfoblastic Leukemia
246). Elemental analyses were recorded at the Institute of Organic
Chemistry Academy of Sciences of the Czech Republic on the PE
a
2
400 Series II CHNS/O Analyzer (Perkin Elmer, USA). Betulin
Compound
IC50 [mmol/L]
58.2 ± 10.9
134.8 ± 6.8
10.1 ± 1.4
19.0 ± 4.6
14.1 ± 3.4
141.0 ± 15.6
10.7 ± 1.5
75.0 ± 13.7
7.8 ± 1.7
Compound IC50 [mmol/L]
(1a) was isolated by extraction (EtOH) of the outer layers of birch
bark (Betula pendula). 21-Oxo acid 2a was prepared by a five-step
process from betulin (1a). Betulinic acid (3a) was isolated from
1
6
2
2
2
2
2
4
3
4
3
4
3
4
3
b
c
d
e
f
5c
5d
5e
5f
44.7 ± 17.6
1.5 ± 0.9
14
1
7
the London plane tree bark (Platanus × hispanica). Platanic acid
1
8
(
4a) was prepared by ozonolysis of betulinic acid (3a). Dihydro-
betulinic acid (5a) was prepared by hydrogenation of betulinic acid
9.2 ± 1.8
1
9
(
3a) as reported in the literature. Oleanolic acid (6a) and ursolic
7.9 ± 1.9
acid (7a) were obtained from Kaden Biochemicals GmbH,
Hamburg, Germany. All reagents and solvents were purchased from
Sigma-Aldrich.
6b
6c
6d
6e
6f
131.9 ± 9.2
119.4 ± 16.9
29.4 ± 8.3
10.7 ± 1.3
37.9 ± 9.5
28.9 ± 2.9
16.7 ± 2.3
4.1 ± 1.7
b
c
c
d
d
e
e
f
2
1
-Bromoethyl Esters 2b–7b; General Procedure 1
,2-Dibromoethane (10 equiv) was added to a stirred suspension of
a triterpenic acid in DMF (10 mL/g), MeCN (1 mL/g), and K CO
(1 equiv) and mixture was kept at 50 °C for 3 h [TLC monitoring
2
3
(
(
toluene–Et O, 5:1)]. The mixture was then poured into cold H O
10 × volume) and extracted with CHCl (2 × 15 mL each). The
2
2
3
combined CHCl layers were washed with H O (100 mL) and evap-
3
2
24.2 ± 2.8
9.6 ± 2.1
7b
7c
7d
7e
7f
orated. The crude residue was co-evaporated with H O (100 mL) to
2
remove residual DMF. The residue was dissolved in toluene, filtra-
tion over a short column (silica gel, toluene) afforded the product,
which was crystallized (i-PrOH).
89.0 ± 5.6
11.5 ± 0.8
12.2 ± 1.2
22.6 ± 3.7
13.4 ± 2.2
Quaternary Salts 2c–7e and 2-[Bis(2-hydroxyethyl)amino]ethyl
Derivatives 2f–7f; General Procedure 2
The reaction was performed under argon in a glass vessel gas tightly
closed with a screw cap (Supelco 27150-U). A soln of the starting
bromide 2a–7a at 70 °C in DMF (1 mL/1 g) was cooled to r.t. and
the respective amine (Me N, Et N, pyridine, or triethanolamine) (5
4f
a
The MTT assays were performed in duplicate.
3
3
pyridine afforded the desired quaternary amines in good
yields, reactions with triethanolamine, however, afforded heated [Me N and triethanolamine (50 °C), Et N (60 °C), and pyri-
equiv) was added. The glass vessel was closed and mixture was
3
3
exclusively substituted diethanolamines.
dine (70 °C)] until the reaction was completed (TLC, CHCl ). The
3
mixture was then worked up in two possible ways. Workup A: when
the product crystallized from the mixture, the mixture was filtered
through a frit (porosity 140–160 mm) and crystals were washed
Most of the quaternary esters showed significant in vitro
cytotoxic activity against CEM cancer cell line. During
tests against a panel of 10 cancer lines of different histo- (CH Cl ). The filtrate was dried under reduced pressure. Workup B:
2
2
genetic origin, three highly effective compounds 3d, 5d, when the product did not crystallize from the mixture, it was diluted
with H O (10 × volume) and extracted with CHCl (2 × twice the
and 5f were found (see Supporting Information). These
compounds are now the subject of further research (solu-
bility in water-based solvent systems, in vivo experiments
on B16F mice model and mechanistic studies).
2
3
volume of mixture). The combined CHCl layers were washed with
3
H O (100 mL) and evaporated. The crude residue was co-evaporat-
2
ed with H O to remove residual DMF. The dry residue was dis-
2
solved in MeOH and filtered over a short column (silica gel,
CHCl –MeOH, 4:1). The solvent was evaporated and the residue
3
was again dissolved in the minimal amount of MeOH and chro-
matographed (silica gel, gradient 0–20% MeOH–CHCl3).
Melting points were determined using a Büchi B-545 apparatus and
are uncorrected. Optical rotations were measured on an Autopol III
(
Rudolph Research, Flanders, USA) polarimeter in MeOH at 25 °C
–
1
2
–1
1
13
2-Bromoethyl 3b-Acetoxy-21-oxolup-18-en-28-oate (2b)
Following general procedure 1 using 2a (3.3 g, 6.6 mmol) gave 2b
(3.22 g, 81%) as a white powder; mp 209 °C (i-PrOH).
unless otherwise stated and are in [10 deg cm g ]. H and
NMR spectra were recorded on Varian
for H) or Varian
ing CDCl or CD OD as a solvent (25 °C). H NMR used TMS as
an internal standard (CDCl ) or were referenced to the residual sig-
nal of the solvent (CD OD). C NMR spectra were referenced to
CDCl (d = 77.00) or CD OD (d = 49.15). Mass spectra (EI) were
recorded on an Incos 50 (Finnigan MAT) mass spectrometer. ESI
spectra were recorded on Bruker Esquire 3000 in MeOH soln. IR
spectra were recorded on a Nicolet Avatar 370 FTIR; DRIFT = Dif-
fuse Reflectance Infrared Fourier Transform. TLC was performed
on Kieselgel 60 F254 (Merck) plates; the spots were detected by UV
fluorescence and charring with 10% H SO . The HPLC system con-
C
UNITY
Inova 400 (400 MHz
Inova 300 (300 MHz for H) instruments, us-
1
UNITY
1
1
25
[a]D –14.6 (c 0.44, MeOH).
3
3
3
–1
IR (CHCl ): 1255, 1610, 1698, 1729 cm .
1
3
3
3
1
H NMR (399.95 MHz, CDCl ): d = 0.84 (s, 3 H), 0.85 (s, 3 H), 0.91
3
3
3
(
s, 3 H), 0.94 (s, 3 H), 1.03 (s, 3 H), 1.22 (d, J = 7.2 Hz, 6 H, 2 CH3),
2
.05 (s, 3 H, OAc), 2.13 (dd, J = 18.6, 0.9 Hz, 1 H, H22a), 2.48
ddd, J = 13.3, 4.1, 2.5 Hz, 1 H, H16b), 2.52 (d, J = 18.6 Hz, 1 H,
H22b), 2.73 (dd, J = 12.8, 3.4 Hz, 1 H, H13b), 3.22 (septet, J = 6.9
(
Hz, 1 H, H20), 3.52 (m, 2 H, CH Br), 4.30 (m, 1 H, H3a), 4.52 (m,
2
2
H, CO CH ).
2 2
2
4
sisted of a high pressure pump (Gilson, model 361), inject valve
Rheodyne), a preparative column (25 × 250 mm) packed with Bio-
spher 7 mm, (Labio, CZ), a differential–refractometrical detector
Laboratorní přístroje, Praha, CR) connected with a PC (software
13
C NMR (100.57 MHz, CDCl ): d = 15.9, 16.5, 16.7, 16.8, 18.1,
3
(
1
3
1
9.9, 20.2, 21.2, 21.3, 23.6, 25.1, 27.6, 27.9, 29.0, 29.0, 33.4, 34.8,
7.1, 37.8, 38.6, 41.3, 45.1, 45.4, 47.5, 51.0, 53.0, 55.4, 64.3, 80.7,
45.9, 171.0, 171.4, 174.0, 207.0.
(
Chromulan) and an automatic fraction collector (Gilson, model
Synthesis 2010, No. 22, 3839–3848 © Thieme Stuttgart · New York

PAPER
Quaternary Ammonium Salts of Lupane-, Oleanane-, and Ursane-Type Acids
3843
+
MS (EI, 70 eV): m/z (%) = 620 (M , 13), 560 (7), 515 (12), 467 (2),
07 (2), 368 (6), 355 (59), 313 (5), 264 (9), 203 (21), 189 (29).
SJ = 16.5 Hz, 1 H, H3a), 4.58 (dt, J = 9.6, 4.8 Hz, 1 H), 4.64 (dt,
4
J = 9.6, 4.8 Hz, 1 H), 5.00 (t, J = 5.1 Hz, 2 H), 8.15 (dd, J = 7.6, 6.4
Hz, 2 H), 8.67 (t, J = 7.8 Hz, 1 H), 8.97 (d, J = 4.0 Hz, 2 H).
Anal. Calcd for C H BrO : C, 65.90; H, 8.30. Found: C, 65.86; H,
3
4
51
5
1
3
8
.19.
C NMR (100.58 MHz, CD OD): d = 16.5, 17.0, 17.2, 17.3, 19.0,
3
2
0.2, 20.8, 21.3, 22.1, 24.4, 26.1, 28.4, 28.6, 29.8, 34.3, 35.7, 38.1,
2
-(Trimethylammonio)ethyl 3b-Acetoxy-21-oxolup-18-en-28-
38.7, 39.5, 42.3, 46.3, 46.5, 52.0, 54.1, 56.5, 61.1, 64.3, 82.1, 129.5,
146.1, 147.0, 147.6, 149.0, 172.7, 173.3, 174.1, 209.0.
oate Bromide (2c)
Following general procedure 2 using 2b (117 mg, 0.19 mmol) and
+
–
MS (ESI): m/z (%) = 618 (2, [M – Br ]), 558 (2), 512 (3), 468 (34),
26 (6), 409 (15), 393 (11), 365 (13), 249 (31), 205 (100), 189 (75).
Me N with workup B gave bromide 2c (78 mg, 61%) as a fine white
3
4
powder; mp 90 °C (CHCl –MeOH, dec.).
3
Anal. Calcd for C H BrNO : C, 67.03; H, 8.08; N, 2.00. Found:
25
39 56
5
[
a]D +10 (c 0.19).
C, 67.02; H, 8.11; N, 2.20.
IR (DRIFT): 1628 (C=C), 1732 cm–1 (C=O).
1
2-[Bis(2-hydroxyethyl)amino]ethyl 3b-Acetoxy-21-oxolup-18-
en-28-oate (2f)
Following general procedure 2 using 2b (250 mg, 0.40 mmol) and
triethanolamine with workup B gave amine 2f (126 mg, 48%) as a
H NMR (399.95 MHz, CD OD): d = 0.87 (s, 3 H), 0.88 (s, 3 H),
3
0
1
1
.88 (s, 3 H), 0.97 (s, 3 H), 1.11 (s, 3 H), 1.18 (d, J = 6.8 Hz, 3 H),
.20 (d, J = 6.4 Hz, 3 H), 2.03 (s, 3 H, OAc), 2.21 (d, J = 18.8 Hz,
H), 2.65 (d, J = 18.8 Hz, 1 H), 3.19 [s, 9 H, N(CH ) ], 3.29 (septet,
3
3
fine white powder; mp 174 °C (CHCl –MeOH, dec.).
3
J = 7.0 Hz, 1 H, H-20), 3.74 (t, J = 4.9 Hz, 2 H, COOCH CH N),
4
COOCH CH N).
1
2
2
2
5
.47 (m, SJ = 16.5 Hz, 1 H, H3a), 4.61 (m SJ = 45.2 Hz, 2 H,
[a]D –20 (c 0.20).
2
2
IR (DRIFT): 1688 (C=C), 1700, 1731 (C=O), 3437 cm–1 (O–H).
3
C NMR (100.57 MHz, CD OD): d = 16.4, 17.0, 17.4, 17.4, 19.2,
1
3
H NMR (399.95 MHz, CD OD): d = 0.87 (s, 3 H), 0.88 (s, 3 H),
3
2
3
1
0.3, 20.7, 21.2, 22.4, 24.6, 26.4, 28.4, 28.9, 30.1, 34.5, 36.0, 38.3,
0
1
1
.96 (s, 3 H), 1.01 (s, 3 H), 1.09 (s, 3 H), 1.19 (d, J = 1.2 Hz, 3 H),
.21 (d, J = 1.6 Hz, 3 H), 2.03 (s, 3 H, OAc), 2.15 (d, J = 18.9 Hz,
H, H22a), 2.51 (d, J = 18.8 Hz, 1 H, H22b), 2.70 (t, J = 5.8 Hz, 4
8.9, 39.7, 42.6, 46.6, 46.9, 48.3, 52.3, 54.3, 54.5, 56.7, 59.8, 82.3,
47.2, 172.8, 173.6, 174.3, 209.3.
+
MS (ESI): m/z (%) = 599 (100, [M – Br]).
H, NCH CH OH), 2.85 (t, J = 5.7 Hz, 2 H, COOCH CH N), 3.27
2
2
2
2
(
septet, J = 6.9 Hz, 1 H, H20), 3.58 (t, J = 5.8 Hz, 4 H,
Anal. Calcd for C H BrNO : C, 65.47; H, 8.91; N, 2.06. Found: C,
37
60
5
NCH CH OH), 4.22 (m, SJ = 16.2 Hz, 2 H, COOCH CH N), 4.47
(
2
2
2
2
6
5.22; H, 9.01; N, 2.14.
m, SJ = 16.5 Hz, 1 H, H3a).
1
3
2
-(Triethylammonio)ethyl3b-Acetoxy-21-oxolup-18-en-28-oate
C NMR (100.58 MHz, CD OD): d = 16.5, 17.0, 17.3, 17.4, 19.2,
3
Bromide (2d)
20.3, 20.6, 21.2, 22.4, 24.6, 26.4, 28.4, 28.8, 30.2, 34.5, 36.0, 38.3,
38.9, 39.6, 42.6, 46.0, 46.5, 46.8, 52.3, 54.3, 54.5, 56.7, 57.9, 60.8,
64.7, 79.5, 146.8, 172.8, 174.4, 175.5, 209.7.
Following general procedure 2 using 2b (285 mg, 0.46 mmol) and
Et N with workup B gave bromide 2d (76 mg, 24%) as a fine white
3
powder; mp 205 °C (CHCl –MeOH, dec.).
+
3
MS (ESI): m/z (%) = 644 (100, [M + H]), 584 (27).
25
[
a]D +76 (c 0.18).
Anal. Calcd for C H NO : C, 70.88; H, 9.55; N, 2.18. Found: C,
3
8
61
7
IR (DRIFT): 1634 (C=C), 1722 cm–1 (C=O).
71.02; H, 9.41; N, 2.29.
1
H NMR (399.95 MHz, CD OD): d = 0.87 (s, 3 H), 0.88 (s, 3 H),
3
2
-Bromoethyl 3b-Hydroxylup-20(29)-en-28-oate (3b)
0
1
2
.97 (s, 3 H), 1.02 (s, 3 H), 1.10 (s, 3 H), 1.19 (s, 3 H), 1.22 (s, 3 H),
Following general procedure 1 using 3a (500 mg, 1.1 mmol) and
.31 [t, J = 7.2 Hz, 9 H, N(CH CH ) ], 2.20 (d, J = 18.9 Hz, 1 H),
2
3 3
1
1
,2-dibromoethane gave 3b (395 mg, 64%) as white crystals; mp
85 °C (MeOH).
.47 (d, J = 12.0 Hz, 1 H), 2.64 (d, J = 18.9 Hz, 1 H), 2.73 (dd,
J = 8.0, 8.0 Hz, 1 H), 3.30 (septet, J = 6.9 Hz, 1 H, H20), 3.39 [q,
J = 7.4 Hz, 6 H, N(CH CH ) ], 3.65 (t, J = 5.1 Hz, 2 H,
2
5
[a]D +7 (c 0.38).
2
3 3
COOCH CH N), 4.50 (m, SJ = 32.4 Hz, 2 H, COOCH CH N).
–1
2
2
2
2
IR (CHCl ): 1641, 1722, 3612 cm .
3
1
3
C NMR (100.58 MHz, CD OD): d = 7.9, 16.5, 17.0, 17.4, 19.2,
1
3
H NMR (399.95 MHz, CD OD): d = 0.75 (s, 3 H), 0.82 (s, 3 H),
3
2
3
1
0.4, 20.7, 21.2, 22.4, 24.6, 26.4, 28.4, 28.8, 30.1, 34.9, 36.0, 38.3,
8.8, 39.6, 42.6, 46.7, 46.8, 52.3, 54.4, 54.8, 56.2, 56.7, 59.4, 82.3,
74.0, 172.8, 173.9, 174.4, 209.3.
0
3
.92 (s, 3 H), 0.96 (s, 3 H), 0.97 (s, 3 H), 1.69 (dd, J = 1.6, 0.8 Hz,
H, H30), 2.19 (ddd, J = 14.4, 11.6, 3.6 Hz, 1 H, H16b), 2.28 (m,
SJ = 16.8 Hz, 1 H, H13b), 3.02 (td, J = 11.1, 5.5 Hz, 1 H, H19b),
3.18 (dd, J = 6.3, 5.0 Hz, 1 H, H3a), 3.54 (t, J = 5.9 Hz, 2 H,
CH Br), 4.40 (m, SJ = 39.5 Hz, 2 H, CO CH ), 4.61 (m, SJ = 7.8
+
–
MS (ESI): m/z (%) = 641 (100, [M – Br ]), 613 (53).
2
2
2
Anal. Calcd for C H BrNO : C, 66.65; H, 9.23; Br, 11.08; N, 1.94.
40
66
5
Hz, 2 H, H29 pro-E), 4.74 (br d, J = 5.3 Hz, 1 H, H29 pro-Z).
Found: C, 66.41; H, 9.38; Br, 13.78; N, 2.0.
1
3
C NMR (100.58 MHz, CD OD): d = 14.7, 15.3, 16.0, 16.1, 18.3,
3
2
-Pyridinioethyl 3b-Acetoxy-21-oxolup-18-en-28-oate Bromide
19.4, 20.9, 25.5, 27.4, 28.0, 29.2, 29.7, 30.6, 32.0, 34.3, 37.0, 37.2,
38.3, 38.7, 38.8, 40.7, 42.4, 46.9, 49.4, 50.5, 55.3, 56.7, 63.3, 79.0,
109.7, 150.4, 175.7.
(2e)
Following general procedure 2 using 2b (308 mg, 0.50 mmol) and
pyridine with workup A gave bromide 2e (213 mg, 61%) as white
crystals; mp 249 °C (DMF, dec.).
+
MS (ESI): m/z (%) = 564 (M , 35), 544 (5), 503 (4), 412 (22), 367
(2), 356 (34), 342 (12), 327 (6), 220 (22), 207 (53), 189 (98).
25
[
a]D –16 (c 0.27).
Anal. Calcd for C H BrO : C, 68.19, H, 9.12. Found: C, 68.26, H,
9.10.
3
2
51
3
IR (DRIFT): 1636 (C=C), 1697, 1733 cm–1 (C=O).
1
H NMR (399.95 MHz, CD OD): d = 0.86 (s, 3 H), 0.86 (s, 3 H),
3
2
-(Trimethylammonio)ethyl 3b-Hydroxylup-20(29)-en-28-oate
0
1
.93 (s, 3 H), 0.96 (s, 3 H), 0.98 (s, 3 H), 1.18 (d, J = 6.8 Hz, 3 H),
.19 (d, J = 6.8 Hz, 3 H), 2.03 (s, 3 H, OAc), 2.11 (d, J = 8.8 Hz, 1
Bromide (3c)
Following general procedure 2 using 3b (200 mg, 0.36 mmol) and
Me N with workup A gave bromide 3c (162 mg, 73%) as a white
crystals; mp 279 °C (DMF, dec.).
H), 2.30 (d, J = 18.8 Hz, 1 H), 2.39 (dd, J = 9.7, 2.7 Hz, 1 H), 2.54
m, SJ = 16.2 Hz, 1 H), 3.23 (septet, J = 7.0 Hz, H20), 4.45 (m,
3
(
Synthesis 2010, No. 22, 3839–3848 © Thieme Stuttgart · New York

3
844
D. Biedermann et al.
PAPER
2
5
[
a]D 0 (c 0.28).
2-[Bis(2-hydroxyethyl)amino]ethyl 3b-Hydroxylup-20(29)-en-
28-oate (3f)
Following general procedure 2 using 3b (291 mg, 0.52 mmol) and
triethanolamine with workup B gave amine 3f (134 mg, 44%) as a
fine white powder; mp 146 °C (DMF, dec.).
IR (DRIFT): 1641 (C=C), 1729 (C=O), 3353 cm–1 (O–H).
1
H NMR (399.95 MHz, CD OD): d = 0.75 (s, 3 H), 0.86 (s, 3 H),
3
0
.95 (s, 3 H), 0.95 (s, 3 H), 1.01 (s, 3 H), 1.69 (s, 3 H), 1.91 (m,
SJ = 41.5 Hz, 2 H), 2.22 (dt, J = 13.0, 3.1 Hz, 1 H), 2.29 (td,
J = 11.8, 3.3 Hz, 1 H), 2.63 (m, SJ = 14.5 Hz, 1 H), 3.13 (m,
SJ = 16.2 Hz, 1 H, H3a), 3.25 [s, 9 H, N(CH ) ], 3.76 (t, J = 5.0 Hz,
2
5
[
a]D +6 (c 0.18).
IR (DRIFT): 1640 (C=C), 1723 (C=O), 3068 (O–H), 3331 cm–1 (O–
H).
3
3
2
H, COOCH CH N), 4.56 (m, SJ = 39.5 Hz, 3 H), 4.72 (d, J = 2.0
Hz, 1 H, H20b).
2
2
1
H NMR (399.95 MHz, CD OD): d = 0.74 (s, 3 H), 0.85 (s, 3 H),
3
1
3
C NMR (100.58 MHz, CD OD): d = 15.1, 16.1, 16.6, 16.8, 19.3,
0.94 (s, 3 H), 0.94 (s, 3 H), 0.99 (s, 3 H), 1.69 (s, 3 H), 1.89 (m,
SJ = 34.6 Hz, 2 H), 2.25 (m, SJ = 24.1 Hz, 2 H), 2.73 (t, J = 5.8 Hz,
4 H, NCH CH OH), 2.85 (t, J = 6.1 Hz, 2 H, COOCH CH N), 3.01
3
1
3
7
9.5, 21.9, 26.6, 27.9, 28.6, 30.8, 31.3, 32.7, 35.4, 37.5, 38.2, 39.3,
9.8, 40.0, 41.8, 43.4, 50.5, 51.8, 54.5, 56.7, 57.7, 58.4, 66.0, 78.7,
9.1, 79.4, 79.5, 110.5, 128.1, 137.3, 151.3, 176.1.
2
2
2
2
(m, SJ = 22.0 Hz, 1 H), 3.12 (m, SJ = 16.0 Hz, 1 H, H3a), 3.61 (t,
J = 5.7 Hz, 4 H, NCH CH OH), 4.17 (t, J = 6.1 Hz, 2 H,
COOCH CH N), 4.59 (s, 1 H), 4.71 (s, 1 H).
+
–
2
2
MS (ESI): m/z (%) = 543 (100, [M – Br ]), 484 (4).
2
2
Anal. Calcd for C H BrNO : C, 67.50; H, 9.71; N, 2.25. Found: C,
7.64; H, 9.68; N, 2.31.
35
60
3
13
C NMR (100.58 MHz, CD OD): d = 15.2, 16.1, 16.7, 16.7, 19.3,
3
6
1
3
6
9.6, 21.9, 26.6, 27.9, 28.6, 30.7, 31.5, 33.0, 35.4, 37.8, 38.2, 39.4,
9.8, 39.9, 41.8, 43.4, 50.5, 51.8, 54.3, 56.6, 57.7, 57.9, 58.0, 60.5,
0.7, 61.0, 63.0, 79.5, 110.3, 151.5, 177.4.
+
2
-(Triethylammonio)ethyl 3b-Hydroxylup-20(29)-en-28-oate
Bromide (3d)
Following general procedure 2 using 3b (1 g, 1.78 mmol) and Et N
MS (ESI): m/z (%) = 632 (33), 610 (100), 588 (33, M ), 528 (16).
3
with workup B gave a greyish residue, which was further crystal-
lized (EtOH) to afford salt 3d (462 mg, 39%) as white crystals; mp
Anal. Calcd for C H NO : C, 73.55; H, 10.46; N, 2.38. Found: C,
3
6
61
5
7
3.72; H, 10.41; N, 2.51.
2
50 °C (EtOH, dec.).
2
5
[
a]D 0 (c 0.16).
2-Bromoethyl 3b-Hydroxy-20-oxo-29-norlupan-28-oate (4b)
Following general procedure 1 using 4a (1.0 g, 2.15 mmol) and 1,2-
dibromoethane gave bromoethyl ester 4b (468 mg, 38%) as white
crystals. Analytical samples were recrystallized (hexane); mp 226
IR (DRIFT): 1637 (C=C), 1732 (C=O), 3311 cm–1 (O–H).
1
H NMR (399.95 MHz, CD OD): d = 0.75 (s, 3 H), 0.86 (s, 3 H),
3
0
1
1
.95 (s, 3 H), 0.96 (s, 3 H), 1.02 (s, 3 H), 1.35 (t, J = 7.2 Hz, 9 H),
.70 (s, 3 H), 2.21 (d, J = 13.0 Hz, 1 H), 2.30 (td, J = 12.5, 3.1 Hz,
H), 3.00 (td, J = 7.6, 5.5 Hz, 1 H), 3.13 (m, SJ = 16.2 Hz, 1 H,
°C (hexane).
2
5
[
a]D –63 (c 0.22, CHCl3).
H3a), 3.43 (q, J = 7.2 Hz, 6 H), 3.63 (m, SJ = 20.0 Hz, 2 H), 4.49
m, SJ = 54.5 Hz, 2 H), 4.61 (s, 1 H), 4.72 (s, 1 H).
IR (DRIFT): 1702 (C=O), 1723 (C=O), 3535 cm–1 (O–H).
(
1
1
H NMR (399.95 MHz, CD OD): d = 0.75 (s, 3 H), 0.81 (s, 3 H),
3
3
C NMR (100.58 MHz, CD OD): d = 7.9, 15.1, 16.1, 16.7, 16.8,
0.90 (s, 3 H), 0.96 (s, 3 H), 1.00 (s, 3 H), 2.19 (s, 3 H), 2.31 ( m,
SJ = 14.7 Hz, 1 H), 3.18 (dd, J = 11.1, 5.1 Hz, 1 H, H3a), 3.27 (td,
J = 11.1, 4.4 Hz, 1 H), 3.55 (t, J = 5.8 Hz, 2 H, COOCH CH N),
3
1
3
7
9.4, 19.5, 22.1, 26.8, 28.0, 28.6, 30.9, 31.4, 32.8, 35.5, 37.6, 38.3,
9.5, 39.9, 40.0, 41.9, 43.5, 50.7, 51.9, 54.7, 56.1, 56.8, 57.9, 58.0,
9.5, 110.5, 151.5, 176.4.
2
2
4.41 (td, J = 5.6, 1.1 Hz, 2 H, COOCH CH N).
2
2
+
–
13
MS (ESI): m/z (%) = 585 (100, [M – Br ]).
C NMR (100.58 MHz, CD OD): d = 14.6, 15.3, 15.9, 16.0, 18.2,
3
2
3
7
0.8, 29.1, 27.2, 27.9, 28.2, 29.1, 29.6, 30.1, 31.3, 31.5, 34.1, 36.6,
7.1, 37.4, 38.6, 38.8, 40.6, 42.1, 49.3, 50.3, 51.1, 55.2, 56.5, 63.4,
8.7, 175.6, 212.5.
Anal. Calcd for C H BrNO : C, 68.65; H, 10.01; Br, 12.02; N,
.11. Found: C, 68.79; H, 10.12; Br, 12.76; N, 2.16.
3
8
66
3
2
+
2
-Pyridinioethyl 3b-Hydroxylup-20(29)-en-28-oate Bromide
MS (EI): m/z (%) = 566 (10), 564 (10, M ), 548 (22), 546 (22), 533
(3e)
(4), 531 (4), 412 (4), 369 (5), 329 (8), 207 (50), 189 (100).
Following general procedure 2 using 3b (285 mg, 0.51 mmol) and
pyridine with workup A gave bromide 3e (227 mg, 76%) as white
crystals; mp 257 °C (DMF, dec.).
Anal. Calcd for C H BrO : C, 65.83; H, 8.73. Found: C, 65.81; H,
3
1
49
4
8
.69.
2
5
[
a]D 0 (c 0.18).
2-(Trimethylammonio)ethyl 3b-Hydroxy-20-oxo-29-norlupan-
28-oate Bromide (4c)
Following general procedure 2 using 4b (316 mg, 0.56 mmol) and
IR (DRIFT): 1635 (C=C), 1725 (C=O), 1731 (C=O), 3398 cm–1 (O–
H).
1
Me N with workup B gave bromide 4c (151 mg, 43%) as a white
3
H NMR (399.95 MHz, CD OD): d = 0.75 (s, 3 H), 0.75 (s, 3 H),
3
powder; mp 264 °C (MeOH, dec.).
0
.83 (s, 3 H), 0.94 (s, 3 H), 0.95 (s, 3 H), 2.07 (m, SJ = 42.0 Hz, 2
2
5
[
a]D –20 (c 0.16).
H), 2.86 (dt, J = 10.9, 4.9 Hz, 1 H), 3.11 (m, SJ = 16.3 Hz, 1 H),
4
.60 (m, SJ = 18.9 Hz, 2 H), 4.68 (br s, 1 H), 5.04 (t, J = 5.1 Hz, 2
IR (DRIFT): 1726 (C=O), 3345 cm–1 (O–H).
H), 8.23 (t, J = 7.2 Hz, 2 H), 8.70 (tt, J = 7.9, 1.2 Hz, 1 H), 9.19 (dd,
J = 6.6, 1.1 Hz, 2 H).
1
H NMR (299.91 MHz, CD OD): d = 0.75 (s, 3 H), 0.86 (s, 3 H),
3
0
.95 (s, 3 H), 0.95 (s, 3 H), 1.02 (s, 3 H), 2.19 (s, 3 H), 2.27 (d,
1
3
C NMR (100.58 MHz, CD OD): d = 15.1, 16.1, 16.7, 16.7, 19.4,
J = 12.9 Hz, 1 H), 3.13 (dd, J = 10.5, 5.4 Hz, 1 H, H3a), 3.25 [s, 9
H, N(CH ) ], 3.77 (t, J = 4.9 Hz, 2 H, COOCH CH N), 4.57 (m,
SJ = 48.3 Hz, 2 H, COOCH CH N).
13
3
1
3
1
9.4, 22.0, 26.6, 28.0, 28.6, 30.8, 31.4, 32.7, 35.5, 37.5, 38.3, 39.5,
9.9, 40.0, 41.8, 50.6, 51.8, 56.7, 57.9, 61.5, 63.6, 79.5, 110.5,
29.7, 146.6, 147.7, 151.3, 176.3.
3
3
2
2
2
2
C NMR (75.42 MHz, CD OD): d = 14.7, 15.1, 16.2, 16.6, 16.8,
3
+
–
MS (EI, 70 eV): m/z (%) = 564 (3, [M – Br ]), 546 (1), 456 (22),
38 (9), 410 (11), 356 (9), 248 (34), 207 (55), 189 (100).
19.4, 20.3, 22.1, 24.1, 28.0, 28.4, 28.6, 29.1, 29.9, 30.9, 32.3, 35.5,
37.4, 38.3, 38.6, 40.0, 40.1, 41.9, 43.4, 50.7, 51.9, 52.4, 54.5, 56.8,
4
5
7.6, 58.8, 64.2, 66.1, 79.5, 176.0, 214.6.
Anal. Calcd for C H BrNO : C, 69.14; H, 8.78; Br, 12.43; N, 2.18.
Found: C, 69.21; H, 8.62; Br, 11.57; N, 2.19.
37
56
3
+
–
MS (ESI): m/z (%) = 544 (100, [M – Br ]), 413 (41).
Synthesis 2010, No. 22, 3839–3848 © Thieme Stuttgart · New York

PAPER
Quaternary Ammonium Salts of Lupane-, Oleanane-, and Ursane-Type Acids
3845
Anal. Calcd for C H BrNO : C, 65.37; H, 9.36; N, 2.24. Found: C,
38.7, 39.9, 40.1, 41.9, 43.3, 50.7, 51.9, 52.7, 54.6, 56.8, 57.7, 58.1,
34
58
4
6
5.21; H, 9.24; N, 2.31.
60.9, 79.6, 177.3, 215.1.
MS (ESI): m/z (%) = 589 (100, M+).
2
2
-(Triethylammonio)ethyl 3b-Hydroxy-20-oxo-29-norlupan-
Anal. Calcd for C H NO : C, 71.27; H, 10.08; N, 2.37. Found:
8-oate Bromide (4d)
35 59
6
C, 71.32; H, 10.16; N, 2.29.
Following general procedure 2 using 4b (250 mg, 0.44 mmol) and
Et N with workup A gave a grey powder residue, which was recrys-
tallized (EtOH) to afford salt 4d (132 mg, 45%) as white petals; mp
3
2
-Bromoethyl 3b-Hydroxylupan-28-oate (5b)
Following general procedure 1 using 5a (500 mg, 1.1 mmol) and
1
1
2
48 °C (MeOH, dec.).
,2-dibromoethane gave 5b (494 mg, 80%) as white needles; mp
86 °C (MeOH).
25
[
a]D –19 (c 0.18).
IR (DRIFT): 1729 (C=O), 3359 cm–1 (O–H).
25
[
a]D –14.3 (c 0.42).
1
H NMR (399.95 MHz, CD OD): d = 0.75 (s, 3 H), 0.86 (s, 3 H),
IR (DRIFT): 1722, 3613 cm–1.
3
0
2
.95 (s, 3 H), 1.02 (s, 3 H), 1.36 [t, J = 7.2 Hz, 9 H, N(CH CH ) ],
2 3 3
1
H NMR (299.91 MHz, CDCl ): d = 0.75 (d, J = 7.2 Hz, 3 H), 0.76
.19 (s, 3 H, H30), 2.24 (dt, J = 13.1, 4.4 Hz, 1 H), 3.13 (dd,
3
(
(
(
4
s, 3 H), 0.83 (s, 3 H), 0.86 (d, J = 7.2 Hz, 3 H), 0.91 (s, 3 H), 0.97
s, 3 H), 1.82 (m, SJ = 46 Hz, 2 H), 2.24 (m, SJ = 52 Hz, 3 H), 3.19
J = 10.8, 5.2 Hz, 1 H, H3a), 3.26 (m, SJ = 22.3 Hz, 1 H), 3.44 [q,
J = 7.2 Hz, 6 H, N(CH CH ) ], 3.56 (m, SJ = 13.6 Hz, 2 H,
2
3 3
dd, J = 11.2, 4.9 Hz, 1 H, H3a), 3.54 (t, J = 5.9 Hz, 2 H, CH Br),
COOCH CH N), 4.50 (m, SJ = 55.5 Hz, 2 H, COOCH CH N).
1
2
2
2
2
2
.38 (m, SJ = 24 Hz, 2 H, CO CH ).
1
3
2
2
C NMR (100.58 MHz, CD OD): d = 7.9, 15.1, 16.1, 16.6, 16.7,
3
1
3
C NMR (75.42 MHz, CDCl ): d = 14.6, 14.7, 15.4, 16.0, 16.1,
1
3
7
9.4, 22.1, 28.0, 28.4, 28.6, 29.1, 29.8, 31.0, 32.2, 35.5, 37.4, 38.3,
8.6, 39.9, 40.0, 41.9, 43.3, 50.8, 51.9, 52.4, 54.8, 56.8, 57.7, 58.2,
9.5, 176.2, 214.7.
3
1
3
6
8.3, 20.9, 22.8, 23.0, 26.9, 27.4, 28.0, 29.2, 29.7, 29.7, 31.9, 34.4,
7.1, 37.3, 38.2, 38.7, 38.8, 40.7, 42.5, 44.2, 48.9, 50.3, 55.3, 57.1,
3.2, 79.0, 175.9.
+
–
MS (ESI): m/z (%) = 587 (100, [M – Br ]).
+
MS (EI, 70eV): m/z (%) = 566 (M , 16), 548 (12), 531 (4), 503 (4),
4
Anal. Calcd for C H BrNO : C, 66.41; H, 9.85; N, 1.94. Found: C,
37
64
4
13 (55), 395 (9), 369 (8), 342 (7), 327 (6), 259 (17), 207 (98), 189
6
6.64; H, 9.67; N, 2.10.
(
96).
Anal. Calcd for C H BrO : C, 67.95; H, 9.44; Br, 14.13. Found:
C, 68.06; H, 9.20; Br, 14.03.
2
-Pyridinioethyl 3b-Hydroxy-20-oxo-29-norlupan-28-oate Bro-
32 53
3
mide (4e)
Following general procedure 2 using 4b (300 mg, 0.53 mmol) and
pyridine with workup B and crystallization (EtOH) gave bromide 4e
2
-(Trimethylammonio)ethyl 3b-Hydroxylupan-28-oate Bro-
mide (5c)
(
[
177 mg, 52%) as white crystals; mp 239 °C (EtOH, dec.).
Following general procedure 2 using 5b (203 mg, 0.36 mmol) and
Me N with workup A gave bromide 5c (156 mg, 70%) as a white
powder; mp 265 °C (DMF, dec.).
25
a]D –14 (c 0.18).
3
IR (DRIFT): 1713 (C=O), 3388 cm–1 (O–H).
1
25
H NMR (299.91 MHz, CD OD): d = 0.73 (s, 3 H), 0.75 (s, 3 H),
[a]D –16 (c 0.16).
3
0
.83 (s, 3 H), 0.95 (s, 3 H), 0.96 (s, 3 H), 2.16 (s, 3 H), 3.09 (m,
IR (DRIFT): 1728 (C=O), 3329 cm–1 (O–H).
SJ = 26.7 Hz, 2 H), 4.62 (t, J = 4.9 Hz, 2 H, COOCH CH N), 5.05
(
(
1
2
2
1
H NMR (399.95 MHz, CD OD): d = 0.76 (s, 3 H), 0.79 (d, J = 6.8
Hz, 3 H), 0.88 (s, 3 H), 0.88 (d, J = 6.8 Hz, 3 H), 0.96 (s, 3 H), 0.96
t, J = 5.0 Hz, 2 H, COOCH CH N), 8.22 (t, J = 7.1 Hz, 2 H), 8.71
3
2
2
d, J = 7.8 Hz, 1 H), 9.14 (d, J = 6.0 Hz, 2 H).
(
s, 3 H), 1.00 (s, 3 H), 2.24 (m, SJ = 15.7 Hz, 1 H), 2.31 (td,
3
C NMR (75.42 MHz, CD OD): d = 15.0, 16.1, 16.6, 16.7, 19.4,
3
J = 11.8, 3.1 Hz, 1 H), 3.14 (m, SJ = 16.3 Hz, 1 H, H3a), 3.25 [s, 9
2
3
1
2.0, 28.0, 28.2, 28.6, 29.0, 29.7, 30.8, 32.2, 35.4, 37.4, 38.3, 38.6,
9.9, 41.7, 43.2, 50.7, 51.8, 52.5, 56.7, 57.8, 61.5, 63.8, 64.3, 79.5,
29.7, 146.6, 147.8, 176.1, 214.6.
H, N(CH ) ], 3.75 (t, J = 5.0 Hz, 2 H, COOCH CH N), 4.54 (m,
3
3
2
2
SJ = 58.4 Hz, 2 H, COOCH CH N).
2
2
1
3
C NMR (100.58 MHz, CD OD): d = 15.0, 15.0, 16.2, 16.7, 19.4,
2.2, 23.4, 23.6, 28.0, 28.2, 28.6, 30.9, 31.0, 32.8, 35.6, 38.0, 38.3,
+
–
3
MS (ESI): m/z (%) = 565 (100, [M – Br ]).
2
Anal. Calcd for C H BrNO : C, 67.07; H, 8.44; N, 2.17. Found: C,
39.3, 39.9, 40.1, 42.0, 43.7, 45.4, 50.2, 51.7, 54.5, 56.8, 58.3, 58.5,
66.1, 79.6, 176.4.
36
54
4
6
7.16; H, 8.36; N, 2.25.
MS (ESI): m/z (%) = 545 (100, M+).
2
-[Bis(2-hydroxyethyl)amino]ethyl 3b-Hydroxy-20-oxo-29-nor-
Anal. Calcd for C H BrNO : C, 67.28; H, 10.00; N, 2.24. Found:
lupan-28-oate (4f)
35 62
3
C, 67.12; H, 9.92; N, 2.11.
Following general procedure 2 using 4b (336 mg, 0.60 mmol) and
triethanolamine with workup B gave amine 4f (146 mg, 41%) as a
fine white powder; mp 172 °C (DMF, dec.).
2
-(Triethylammonio)ethyl 3b-Hydroxylupan-28-oate Bromide
(
5d)
25
[
a]D +11 (c 0.22).
Following general procedure 2 using 5b (305 mg, 0.54 mmol) and
Et N with workup A gave salt 5d (159 mg, 44%); mp 248 °C (DMF,
3
IR (DRIFT): 1648 (C=C), 1735 (C=O), 3297 cm–1 (O–H).
1
dec.).
H NMR (299.91 MHz, CD OD): d = 0.75 (s, 3 H), 0.86 (s, 3 H),
3
2
5
0
.94 (s, 3 H), 0.95 (s, 3 H), 1.01 (s, 3 H), 2.18 (s, 3 H), 2.73 [t,
[a]D –20 (c 0.23).
J = 5.9 Hz, 4 H, N(CH CH OH) ], 2.86 (t, J = 6.0 Hz, 2 H,
COOCH CH N), 3.13 (dd, J = 10.8, 5.4 Hz, 1 H, H3a), 3.61 [t,
J = 5.8 Hz, 4 H, N(CH CH OH) ], 4.18 (t, J = 6.0 Hz, 2 H,
COOCH CH N).
1
IR (DRIFT): 1735 (C=O), 3295 cm–1 (O–H).
2
2
2
2
2
1
H NMR (399.95 MHz, CD OD): d = 0.76 (s, 3 H), 0.88 (s, 3 H),
3
2
2
2
0
2
(
.96 (s, 3 H), 0.96 (s, 3 H), 1.00 (s, 3 H), 1.35 (t, J = 7.2 Hz, 9 H),
.21 (m, SJ = 15.9 Hz, 1 H), 2.31 (dt, J = 12.4, 3.5 Hz, 1 H), 3.14
2
2
3
C NMR (75.42 MHz, CD OD): d = 15.1, 16.1, 16.6, 16.7, 19.4,
3
m, SJ = 16.2 Hz, 1 H), 3.42 (q, J = 7.3 Hz, 6 H), 3.62 (m, SJ = 33.7
2
2.1, 25.2, 28.0, 28.4, 28.6, 29.3, 29.8, 30.9, 32.5, 35.5, 37.7, 38.3,
Hz, 2 H), 4.47 (m, SJ = 55.7 Hz, 2 H).
Synthesis 2010, No. 22, 3839–3848 © Thieme Stuttgart · New York

3
846
D. Biedermann et al.
PAPER
1
3
25
C NMR (100.58 MHz, CD OD): d = 7.9, 15.0, 15.0, 16.1, 16.7,
[a]D +46 (c 0.21).
3
1
3
5
9.4, 22.1, 23.4, 23.6, 28.0, 28.2, 28.6, 30.9, 31.0, 32.7, 35.6, 38.0,
8.3, 39.3, 39.9, 40.1, 42.0, 43.7, 45.4, 50.3, 51.7, 54.7, 56.8, 57.9,
8.4, 59.6, 79.6, 176.5.
IR (DRIFT): 1631 (C=C), 1725 (C=O), 3433 cm–1 (O–H).
1
H NMR (399.95 MHz, CD OD): d = 0.78 (s, 3 H), 0.78 (s, 3 H),
3
0
.92 (s, 3 H), 0.95 (s, 3 H), 0.95 (s, 3 H), 0.98 (s, 3 H), 1.18 (s, 3 H),
+
–
MS (ESI): m/z (%) = 587 (100, M – Br ).
1
.92 (dd, J = 8.8, 3.5 Hz, 2 H), 2.10 (td, J = 4.0 Hz, 1 H), 2.86 (dd,
Anal. Calcd for C H BrNO : C, 68.44; H, 10.28; Br, 11.98; N,
J = 13.0, 5.1 Hz, 1 H), 3.15 (dd, J = 11.1, 4.9 Hz, 1 H, H3a), 3.24
3
8
68
3
2
.10. Found: C, 68.11; H, 10.22; Br, 12.48; N, 2.03.
[s, 9 H, N(CH ) ], 3.73 (m, SJ = 50.1 Hz, 2 H, COOCH CH N),
3
3
2
2
4
.51 (m, SJ = 45.3 Hz, 2 H, COOCH CH N), 5.28 (t, J = 3.4 Hz, 1
2 2
2
-Pyridinioethyl 3b-Hydroxylupan-28-oate Bromide (5e)
H, H12).
Following general procedure 2 using 5b (277 mg, 0.49 mmol) and
pyridine with workup A gave bromide 5e (240 mg, 76%) as white
crystals; mp 272 °C (DMF, dec.).
13
C NMR (100.58 MHz, CD OD): d = 15.9, 16.3, 17.9, 19.5, 23.9,
3
2
4
4.1, 24.5, 26.4, 27.8, 28.7, 31.6, 33.4, 33.9, 34.7, 38.2, 39.8, 40.6,
2.7, 42.9, 47.0, 48.0, 54.5, 56.7, 59.0, 59.6, 79.6, 124.2, 144.8,
[
a] –16 (c 0.30).
D
178.1.
IR (DRIFT): 1634 (C=C), 1735 (C=O), 3289 cm–1 (O–H).
MS (ESI): m/z (%) = 543 (100, [M – Br]).
+
1
H NMR (399.95 MHz, CD OD): d = 0.74 (d, J = 6.5 Hz, 3 H), 0.75
Anal. Calcd for C H BrNO : C, 67.50; H, 9.71; N, 2.25. Found: C,
67.52; H, 9.68; N, 2.39.
3
35 60
3
(
(
s, 6 H), 0.83 (s, 6 H), 0.94 (d, J = 6.5 Hz, 3 H), 0.94 (s, 3 H), 1.77
septet, J = 6.5 Hz, 1 H, H20), 2.07 (m, SJ = 23.5 Hz, 2 H), 3.12 (m,
SJ = 16.0 Hz, 1 H, H3a), 4.59 (dt, J = 9.2, 5.0 Hz, 2 H), 5.02 (d,
J = 4.7 Hz, 2 H), 8.19 (t, J = 7.0 Hz, 2 H), 8.68 (t, J = 7.6 Hz, 1 H),
2-(Triethylammonio)ethyl 3b-Hydroxyolean-12-en-28-oate
Bromide (6d)
9
.10 (s, 2 H).
Following general procedure 2 using 6b (295 mg, 0.52 mmol) and
1
3
Et
3
N with workup A gave salt 6d (116 mg, 34%) as white micro-
C NMR (100.58 MHz, CD OD): d = 15.0, 15.0, 16.1, 16.7, 16.7,
3
crystals; mp 230 °C (DMF, dec.).
1
3
7
9.3, 22.0, 23.4, 23.5, 27.9, 28.0, 28.6, 30.7, 30.9, 32.6, 35.6, 38.0,
8.2, 39.3, 39.9, 40.0, 41.8, 43.5, 45.4, 51.5, 56.7, 58.3, 61.5, 63.5,
9.5, 129.6, 146.5, 147.7, 176.4.
2
5
[a]D +43 (c 0.19).
IR (DRIFT): 1661 (C=C), 1719 (C=O), 3301 cm–1 (O–H).
+
–
MS (ESI): m/z (%) = 564 (100, M – Br ).
1
H NMR (399.95 MHz, CD OD): d = 0.78 (s, 3 H), 0.78 (s, 3 H),
3
Anal. Calcd for C H BrNO : C, 68.92; H, 9.07; N, 2.17. Found: C,
0.93 (s, 3 H), 0.95 (s, 3 H), 0.95 (s, 3 H), 0.98 (s, 3 H), 1.18 (s, 3 H),
37
58
3
6
8.97; H, 8.91; N, 2.09.
1.35 [t, J = 7.2 Hz, 9 H, N(CH CH ) ], 2.84 (m, SJ = 16.2 Hz, 1 H),
2
3 3
3
.15 (dd, J = 11.2, 4.7 Hz, 1 H, H3a), 3.22 (q, J = 7.3 Hz, 1 H), 3.42
2
-[Bis(2-hydroxyethyl)amino]ethyl 3b-Hydroxylupan-28-oate
(q, J = 7.2 Hz, 6 H), 3.61 (m, SJ = 37.1 Hz, 3 H), 4.42 (t, J = 5.2 Hz,
2 H, COOCH CH N), 5.28 (br s, 1 H, H12).
(
5f)
2
2
Following general procedure 2 using 5b (258 mg, 0.46 mmol) and
triethanolamine with workup B and crystallization (EtOH) gave
bromide 5f (201 mg, 74%) as small white crystals; mp 182 °C
13
C NMR (100.58 MHz, CD OD): d = 7.9, 15.9, 16.3, 18.0, 19.5,
3
2
3
1
4.0, 24.1, 24.5, 26.5, 27.8, 28.7, 31.6, 33.4, 33.5, 33.9, 34.6, 38.2,
9.8, 39.8, 42.7, 42.9, 47.0, 54.8, 56.0, 56.7, 58.6, 59.6, 79.6, 124.2,
44.9, 178.3.
(
CHCl –MeOH, dec.).
3
25
[
a]D –17 (c 0.42).
+
–
MS (ESI): m/z (%) = 585 (100, [M – Br ]), 556 (3).
IR (DRIFT): 1724 (C=O), 3358 cm–1 (O–H).
Anal. Calcd for C H BrNO : C, 68.65; H, 10.01; N, 2.11. Found:
C, 68.71; H, 9.89; N, 1.98.
3
8
66
3
1
H NMR (399.95 MHz, CDCl ): d = 0.74 (d, J = 6.8 Hz, 3 H), 0.76
s, 3 H), 0.82 (s, 3 H), 0.85 (d, J = 6.4 Hz, 3 H), 0.91 (s, 3 H), 0.94
s, 3 H), 0.97 (s, 3 H), 1.79 (m, SJ = 16.2 Hz, 4 H), 2.75 (t, J = 5.3
3
(
(
2-Pyridinioethyl 3b-Hydroxyolean-12-en-28-oate Bromide (6e)
Following general procedure 2 using 6b (272 mg, 0.48 mmol) and
pyridine with workup A gave bromide 5e (270 mg, 88%) as white
crystals; mp 260 °C (DMF, dec.).
Hz, 4 H, NCH CH OH), 2.85 (t, J = 5.8 Hz, 2 H, COOCH CH N),
2
2
2
2
3
.19 (m, SJ = 16.2 Hz, 1 H, H3a), 3.64 (t, J = 5.3 Hz, 4 H,
NCH CH OH), 4.18 (m, SJ = 51.6 Hz, 2 H, COOCH CH N).
2
2
2
2
1
3
25
C NMR (100.58 MHz, CDCl ): d = 14.6, 14.6, 15.4, 16.0, 16.1,
[a]D +39 (c 0.18).
3
1
3
5
8.2, 20.9, 22.7, 23.0, 26.9, 27.3, 27.9, 29.6, 29.7, 32.0, 34.3, 37.1,
7.3, 38.0, 38.7, 38.8, 40.7, 42.5, 44.1, 48.9, 50.2, 53.3, 55.2, 56.4,
7.0, 59.7, 61.5, 77.2, 78.9, 176.3.
IR (DRIFT): 1633 (C=C), 1716 (C=O), 3361 cm–1 (O–H).
1
H NMR (399.95 MHz, CD OD): d = 0.46 (s, 3 H), 0.75 (s, 3 H),
3
0
.88 (s, 3 H), 0.88 (s, 3 H), 0.89 (s, 3 H), 0.94 (s, 3 H), 1.08 (s, 3 H),
+
–
MS (ESI): m/z (%) = 612 (100), 588 (41, [M – Br ]), 530 (34).
1
.99 (m, SJ = 35.6 Hz, 1 H), 2.70 (dd, J = 13.7, 3.8 Hz, 1 H), 3.11
Anal. Calcd for C H NO : C, 73.30; H, 10.76; N, 2.37. Found: C,
7
(dd, J = 11.1, 4.8 Hz, 1 H, H3a), 4.51 (m, SJ = 14.5 Hz, 2 H), 5.00
(m, SJ = 12.8 Hz, 2 H), 5.09 (t, J = 3.4 Hz, 1 H, H20), 8.21 (t,
J = 7.2 Hz, 2 H), 8.70 (t, J = 7.8 Hz, 1 H), 9.10 (d, J = 5.8 Hz, 2 H).
13
36
63
5
3.43; H, 10.92; N, 2.32.
2
-Bromoethyl 3b-Hydroxyolean-12-en-28-oate (6b)
C NMR (100.58 MHz, CD OD): d = 15.9, 16.3, 17.7, 19.4, 23.9,
3
Following general procedure 1 using 6a (5.0 g, 10.94 mmol) and
2
3
1
4.0, 24.3, 26.4, 27.8, 28.7, 31.5, 33.4, 33.6, 33.8, 34.6, 38.1, 39.7,
9.8, 40.4, 42.6, 42.7, 46.9, 56.6, 61.5, 64.1, 79.5, 79.6, 123.9,
29.7, 144.7, 146.6, 147.7, 178.2.
1
,2-dibromoethane gave 6b (3.2 g, 52%) as white needles. The
2
2
analytical data were in accord with literature values; mp 195 °C
(
i-PrOH).
+
–
MS (ESI): m/z (%) = 562 (100, [M – Br ]), 483 (2), 432 (3).
25
[
a]D +14 (c 0.18, CHCl3).
Anal. Calcd for C H BrNO : C, 69.14; H, 8.78; N, 2.18. Found: C,
3
7
56
3
6
9.02; H, 8.89; N, 2.31.
2
-(Trimethylammonio)ethyl 3b-Hydroxyolean-12-en-28-oate
Bromide (6c)
Following general procedure 2 using 6b (225 mg, 0.40 mmol) and
Me N reacted with workup A gave bromide 5c (192 mg, 77%) as a
3
white powder; mp 277 °C (DMF, dec.).
Synthesis 2010, No. 22, 3839–3848 © Thieme Stuttgart · New York

PAPER
Quaternary Ammonium Salts of Lupane-, Oleanane-, and Ursane-Type Acids
3847
2
-[Bis(2-hydroxyethyl)amino]ethyl 3b-Hydroxyolean-12-en-28-
39.9, 40.3, 40.3, 40.8, 43.2, 54.3, 54.6, 56.7, 59.0, 66.1, 79.6, 127.4,
139.4, 177.9.
oate (6f)
Following general procedure 2 using 6b (193 mg, 0.34 mmol) and
triethanolamine with workup B and crystallization (EtOH) gave
bromide 6f (109 mg, 57%) as a white powder; mp 230 °C (CHCl3–
MeOH, dec.).
+
–
MS (ESI): m/z (%) = 543 (100, [M – Br ]).
Anal. Calcd for C H BrNO : C, 67.50; H, 9.71; N, 2.25. Found: C,
35 60
3
6
7.63; H, 9.78; N, 2.12.
25
[
a]D +22 (c 0.15).
2
-(Triethylammonio)ethyl 3b-Hydroxyurs-12-en-28-oate Bro-
IR (DRIFT): 1618 (C=C), 1732 (C=O), 3363 cm–1 (O–H).
mide (7d)
1
Following general procedure 2 using 7b (300 mg, 0.53 mmol) and
H NMR (399.95 MHz, CD OD): d = 0.77 (s, 3 H), 0.78 (s, 3 H),
3
Et N with workup A gave salt 7d (270 mg, 77%) as a white powder;
3
0
1
2
.91 (s, 3 H), 0.94 (s, 3 H), 0.95 (s, 3 H), 0.98 (s, 3 H), 1.16 (s, 3 H),
.91 (dd, J = 7.6, 3.5 Hz, 1 H), 2.04 (dd, J = 15.3, 11.3 Hz, 1 H),
.73 [t, J = 5.8 Hz, 4 H, N(CH CH OH) ], 2.85 (t, J = 6.1 Hz, 2 H,
mp 212 °C (DMF, dec.).
2
5
[a]D +38 (c 0.28).
2
2
2
COOCH CH N), 2.88 (dd, J = 15.3, 5.0 Hz, 1 H), 3.15 (dd,
IR (DRIFT): 1634 (C=C), 1724 (C=O), 3322 cm–1 (O–H).
2
2
J = 11.2, 4.8 Hz, 1 H, H3a), 3.60 [t, J = 6.0 Hz, 4 H,
1
H NMR (399.95 MHz, CD OD): d = 0.78 (s, 3 H), 0.78 (s, 3 H),
N(CH CH OH) ], 4.12 (m, SJ = 51.4 Hz, 2 H), 5.27 (t, J = 3.5 Hz,
3
2
2
2
0
0
1
.79 (d, J = 8.0 Hz, 3 H), 0.98 (d, J = 6.8 Hz, 3 H), 0.98 (s, 3 H),
.98 (s, 3 H), 1.14 (s, 3 H), 1.35 [t, J = 7.2 Hz, 9 H, N(CH CH ) ],
1
1
H, H20).
3
2
3 3
C NMR (100.58 MHz, CD OD): d = 15.9, 16.3, 17.9, 19.5, 24.0,
3
.82 (td, J = 13.6, 4.5 Hz, 1 H), 1.94 (m, SJ = 25.9 Hz, 2 H), 2.13
td, J = 13.6, 4.4 Hz, 1 H), 2.21 (d, J = 11.1 Hz, 1 H), 3.16 (dd,
2
4
7
4.1, 24.5, 26.0, 43.0, 27.9, 28.7, 31.6, 33.5, 33.7, 34.8, 38.2, 39.8,
0.7, 42.7, 42.8, 45.9, 47.1, 48.0, 54.3, 56.7, 58.0, 60.9, 63.8, 79.5,
9.7, 123.9, 145.0, 179.5.
(
J = 11.6, 4.3 Hz, 1 H, H3a), 3.42 [q, J = 7.3 Hz, 2 H, N(CH CH ) ],
2
3 3
3
.62 (m, SJ = 18.5 Hz, 2 H, COOCH CH N), 4.41 (m, SJ = 28.2
2 2
+
–
MS (ESI): m/z (%) = 610 (100), 588 (46, [M – Br ]), 550 (3), 528
41).
Hz, 2 H, COOCH CH N), 5.27 (t, J = 3.7 Hz, 1 H, H12).
2 2
(
13
C NMR (100.58 MHz, CD OD): d = 8.0, 9.4, 16.0, 16.4, 17.6,
3
Anal. Calcd for C H NO : C, 73.55; H, 10.46; N, 2.38. Found: C,
7
18.0, 19.4, 21.5, 24.1, 24.4, 25.3, 27.9, 28.8, 29.1, 31.5, 34.2, 37.8,
38.1, 39.8, 39.9, 40.3, 40.4, 40.8, 42.9, 43.2, 47.9, 54.4, 54.8, 56.0,
36
61
5
3.58; H, 10.32; N, 2.26.
5
6.7, 58.4, 59.6, 79.6, 127.3, 139.5, 178.0.
2
-Bromoethyl 3b-Hydroxyurs-12-en-28-oate (7b)
+
–
MS (ESI): m/z (%) = 585 (100, [M – Br ]).
Following general procedure 1 using 7a (2.5 g, 5.48 mmol) and 1,2-
dibromoethane gave 7b (1.27 g, 41%) as white needles; mp 201 °C
Anal. Calcd for C H BrNO : C, 68.65; H, 10.01; N, 2.11. Found:
38 66
3
C, 68.66; H, 10.24; N, 2.26.
(
i-PrOH).
25
[
a]D +8 (c 0.32, CHCl3).
2
-Pyridinioethyl 3b-Hydroxyurs-12-en-28-oate Bromide (7e)
IR (DRIFT): 1618 (C=C), 1727 (C=O), 3360 cm–1 (O–H).
Following general procedure 2 using 7b (272 mg, 0.48 mmol) and
pyridine with workup A gave bromide 7e (270 mg, 88%) as white
crystals; mp 231 °C (DMF, dec.).
1
H NMR (399.95 MHz, CD OD): d = 0.77 (s, 3 H), 0.79 (s, 3 H),
3
0
1
.87 (d, J = 6.8 Hz, 3 H), 0.92 (d, J = 6.0 Hz, 3 H), 0.96 (s, 3 H),
.00 (s, 3 H), 1.09 (s, 3 H), 1.80 (td, J = 13.7, 4.6 Hz, 1 H), 1.92 (dd,
2
5
[a]D +22 (c 0.19).
J = 9.0, 3.7 Hz, 2 H), 2.02 (td, J = 13.0, 4.5 Hz, 1 H), 2.25 (d,
J = 10.4 Hz, 1 H), 3.22 (dd, J = 10.8, 4.9 Hz, 1 H, H3a), 3.49 (t,
J = 6.0 Hz, 2 H, COOCH CH Br), 4.32 (t, J = 6.0 Hz, 2 H,
IR (DRIFT): 1628 (C=C), 1731 (C=O), 3405 cm–1 (O–H).
1
H NMR (399.95 MHz, CD OD): d = 0.77 (s, 3 H), 0.85 (d, J = 6.4
3
2
2
Hz, 3 H), 0.90 (s, 3 H), 0.90 (d, J = 8.0 Hz, 3 H), 0.96 (s, 3 H), 0.96
COOCH CH Br), 5.28 (t, J = 3.7 Hz, 1 H).
2
2
(
s, 3 H), 1.05 (s, 3 H), 2.72 (dd, J = 13.7, 4.0 Hz, 1 H), 3.14 (dd,
1
3
C NMR (100.58 MHz, CD OD): d = 15.5, 15.6, 17.0, 17.1, 18.3,
3
J = 11.1, 4.9 Hz, 1 H), 4.52 (m, SJ = 25.6 Hz, 2 H, COOCH CH N),
2
2
2
3
1
1.1, 23.3, 23.5, 24.1, 27.2, 28.0, 28.1, 29.0, 30.6, 33.0, 36.6, 37.0,
8.6, 38.7, 38.8, 39.0, 39.6, 42.0, 47.5, 48.2, 52.8, 55.2, 63.6, 79.0,
25.8, 137.9, 177.1.
5
.02 (m, SJ = 13.1 Hz, 2 H, COOCH CH N), 5.10 (t, J = 3.5 Hz, 1
2
2
H, H12), 8.23 (t, J = 6.7 Hz, 2 H), 8.71 (t, J = 7.8 Hz, 1 H), 9.13 (d,
J = 6.6 Hz, 2 H).
+
MS (ESI): m/z (%) = 564 (100), 562 (M , 93).
13
C NMR (100.58 MHz, CD OD): d = 16.0, 16.4, 17.5, 17.7, 19.3,
3
Anal. Calcd for C H BrNO : C, 69.19; H, 9.12; Br, 14.18. Found:
21.5, 24.0, 24.1, 24.2, 25.1, 27.8, 28.7, 29.0, 31.5, 33.6, 34.0, 37.9,
38.0, 38.1, 39.8, 40.2, 40.4, 40.6, 43.0, 48.2, 49.5, 54.1, 56.6, 61.5,
3
2
51
3
C, 69.05; H, 8.98; Br, 15.32.
6
4.2, 64.2, 79.5, 127.0, 129.7, 139.3, 146.7, 147.7, 178.0.
2
-(Trimethylammonio)ethyl 3b-Hydroxyurs-12-en-28-oate
+
–
MS (ESI): m/z (%) = 562 (100, M – Br ).
Bromide (7c)
Anal. Calcd for C H BrNO : C, 69.14; H, 8.78; N, 2.18. Found: C,
Following general procedure 2 using 7b (200 mg, 0.36 mmol) and
Me N reacted with workup A gave bromide 5c (211 mg, 94%) as
37 56
3
6
8.99; H, 8.76; N, 2.14.
3
white small crystals; mp 208 °C (DMF, dec.).
2
-[Bis(2-hydroxyethyl)amino]ethyl 3b-Hydroxyurs-12-en-28-
25
[
a]D +44 (c 0.26).
oate (7f)
IR (DRIFT): 1642 (C=C), 1731 (C=O), 3363 cm–1 (O–H).
Following general procedure 2 using 6b (200 mg, 0.36 mmol) and
triethanolamine with workup B and crystallization (EtOH) gave
bromide 6f (142 mg, 67%) as a white powder; mp 242 °C (CHCl3–
MeOH, dec.).
1
H NMR (299.91 MHz, CD OD): d = 0.74 (d, J = 11.4 Hz, 1 H),
3
0
.78 (s, 3 H), 0.80 (s, 3 H), 0.90 (d, J = 8.8 Hz, 3 H), 0.96 (d, J = 7.2
Hz, 3 H), 0.98 (s, 3 H), 0.98 (s, 3 H), 3.16 (dd, J = 10.8, 5.1 Hz, 1
H, H3a), 3.25 [s, 3 H, N(CH ) ], 3.75 (m, SJ = 42.0 Hz, 2 H,
2
5
[a]D +28 (c 0.21).
IR (DRIFT): 1627 (C=C), 1738 (C=O), 3312 cm–1 (O–H).
3
3
COOCH CH N), 4.48 [br s, 2 H, (COOCH CH N)], 5.27 (t, J = 3.6
2
2
2
2
Hz, 1 H, H12).
1
H NMR (299.91 MHz, CD OD): d = 0.76 (s, 3 H), 0.81 (s, 3 H),
1
3
3
C NMR (75.42 MHz, CD OD): d = 16.0, 16.4, 17.6, 18.0, 19.4,
3
0
1
.85 (d, J = 6.2 Hz, 3 H), 0.92 (d, J = 6.2 Hz, 3 H), 0.94 (s, 3 H),
.02 (s, 3 H), 1.09 (s, 3 H), 2.12 (m, SJ = 18.2 Hz, 1 H), 2.32 (d,
2
1.5, 24.2, 24.3, 25.3, 27.9, 28.8, 29.1, 31.6, 34.2, 37.7, 38.1, 39.8,
Synthesis 2010, No. 22, 3839–3848 © Thieme Stuttgart · New York

3
848
D. Biedermann et al.
PAPER
J = 11.2 Hz, 1 H), 2.82 [t, J = 6.9 Hz, 4 H, N(CH CH OH) ], 2.89
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2
2
2
(
t, J = 6.0 Hz, 2 H, COOCH CH N), 3.22 (m, SJ = 16.4 Hz, 1 H,
2 2
H3a), 3.68 [t, J = 5.9 Hz, 4 H, N(CH CH OH) ], 4.22 (t, J = 6.0 Hz,
2
2
2
2
H, COOCH CH N), 5.46 (t, J = 2.2 Hz, 1 H).
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540.
C NMR (75.42 MHz, CD OD): d = 14.5, 15.2, 17.0, 17.0, 19.2,
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(
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2.1, 23.1, 30.0, 24.1, 27.2, 28.2, 28.9, 29.1, 32.6, 33.9, 36.7, 37.0,
8.6, 38.7, 38.8, 39.1, 39.5, 40.1, 44.5, 47.0, 48.5, 54.6, 54.7, 58.8,
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Anal. Calcd for C H NO : C, 73.55; H, 10.46; N, 2.38. Found: C,
36
61
5
7
3.21; H, 10.37; N, 2.31.
(
Cytotoxic MTT Assay
Screening of cytotoxic activity was performed on highly chemosen-
sitive T-lymphoblastic leukaemia CEM cells using cytotoxic MTT
assay.20 Cell lines were purchased from the American Tissue Cul-
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1360.
(
(
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insoluble pentacyclic and tetracyclic terpenoids, a soluble
formulation of a pentacyclic or tetracyclic terpenoid and a
pharmaceutical composition containing this soluble formul-
ation: Sarek, J.; Hajduch, M.; Svoboda, M.; Novakova, K.;
Spacilova, P.; Kubelka, T.; Biedermann, D. US
3
7 °C and 5% CO for stabilization. Fourfold dilutions, in 20-mL
2
aliquots, of the intended test concentration were added at time zero
to the microtiter plate wells. All tests (dissolved in 10 mL of 10%
DMSO) were performed in duplicate. Incubation of the cells with
the test compounds lasted for 72 h at 37 °C, in a 5% CO atmosphere
2
at 100% using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide]. At the end of the incubation period, solns were
pipetted into each well and incubated for a further 1–4 h. After this
incubation period the formazan produced was dissolved by the ad-
dition of 100 mL/well of 10% aq SDS (pH 5.5), followed by a further
incubation at 37 °C overnight. The optical density (OD) was mea-
sured at 540 nm with a Labsystem iEMS Reader MF. Inhibition of
tumor growth/survival (IC) was calculated using the following
equation: IC = (OD drug-exposed well/mean OD control
wells) × 100%. The IC value, the drug concentration lethal to 50%
2009325919, 2009.
(
(
(
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Křeček, V.; Kořínková, G.; Thomson, J. P.; Janošt'áková,
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5
0
(
(
of the tumor cells, was calculated from appropriate dose-response
curves.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.
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(
(
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Acknowledgment
This study was supported by A/CZ0046/1/0022 of FM EEA/Norska,
Czech Science Foundation (305/09/1216) and Foundation for
Cancer Research. Indirect costs were paid from the Czech Ministry
of Education (MSM 6198959216). We are grateful to Stanislav
Hilgard for measurement of IR spectra. We also thank Bohunka
Šperlichová for measurement of optical rotation.
(
20) Hajduch, M.; Mihal, V.; Minarik, J.; Faber, E.; Safarova, M.;
Weigl, E.; Antalek, P. Cytotechnology 1996, 19, 243.
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preparation thereof: Šarek, J.; Biedermann, D.; Hajdúch, M.
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(
(
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Synthesis 2010, No. 22, 3839–3848 © Thieme Stuttgart · New York