Cytotoxic heterocyclic triterpenoids derived from betulin and betulinic acid

Article abstract of DOI:10.1016/j.bmc.2012.03.066

The aim of this work was to synthesize a set of heterocyclic derivatives of lupane, lup-20(29)-ene, and 18α-oleanane, and to investigate their cytotoxic activities. Some of those heterocycles were previously known in the oleanane (allobetulin) group; however, to our knowledge the syntheses and biological activities of lupane heterocycles have not been reported before. Starting from betulin (1) and betulinic acid (2), we prepared 3-oxo compounds and 2-bromo-3-oxo compounds 3-10, 2-hydroxymethylene-3-oxo compounds 11-13 and β-oxo esters 14-16. Condensation of these intermediates with hydrazine, phenylhydrazine, hydroxylamine, or thiourea yielded the pyrazole and phenylpyrazole derivatives 17-22, pyrazolones 23-25, isoxazoles 26 and 27, and thiazoles 28-31. Fifteen compounds (14-16, 18-25, and 29-32) have not been reported before. The cytotoxicity was measured using panel of seven cancer cell lines with/without MDR phenotype and non tumor MRC-5 and BJ fibroblasts. The preferential cytotoxicity to cancer cell lines, particularly to hematological tumors was observed, the bromo acids 5, 6 showed highest activity and selectivity against tumor cells.

Full text of DOI:10.1016/j.bmc.2012.03.066

Bioorganic & Medicinal Chemistry 20 (2012) 3666–3674
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cytotoxic heterocyclic triterpenoids derived from betulin and betulinic acid
Milan Urban a,d, Martin Vlk b, Petr Dzubak c, Marian Hajduch c, Jan Sarek d,
⇑
a Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA
b Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Brehova 7, Prague 1, Czech Republic
c Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry,
Palacky University and University Hospital in Olomouc, Puskinova 6, 775 20 Olomouc, Czech Republic
d Department of Organic Chemistry, Faculty of Science, and Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, 17.
listopadu 1192/12, 771 46 Olomouc, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this work was to synthesize a set of heterocyclic derivatives of lupane, lup-20(29)-ene, and
Received 6 December 2011
Revised 5 March 2012
Accepted 9 March 2012
Available online 11 April 2012
18a-oleanane, and to investigate their cytotoxic activities. Some of those heterocycles were previously
known in the oleanane (allobetulin) group; however, to our knowledge the syntheses and biological
activities of lupane heterocycles have not been reported before. Starting from betulin (1) and betulinic
acid (2), we prepared 3-oxo compounds and 2-bromo-3-oxo compounds 3–10, 2-hydroxymethylene-3-
oxo compounds 11–13 and b-oxo esters 14–16. Condensation of these intermediates with hydrazine,
phenylhydrazine, hydroxylamine, or thiourea yielded the pyrazole and phenylpyrazole derivatives 17–
22, pyrazolones 23–25, isoxazoles 26 and 27, and thiazoles 28–31. Fifteen compounds (14–16, 18–25,
and 29–32) have not been reported before. The cytotoxicity was measured using panel of seven cancer
cell lines with/without MDR phenotype and non tumor MRC-5 and BJ fibroblasts. The preferential cyto-
toxicity to cancer cell lines, particularly to hematological tumors was observed, the bromo acids 5, 6
showed highest activity and selectivity against tumor cells.
Keywords:
Lupane
Oleanane
Heterocycles
Cytotoxicity
Diazotization
Pyrazoles
Isoxazoles
Thiazoles
Ó 2012 Elsevier Ltd. All rights reserved.
Triterpenes
Bromination
1. Introduction
the A-ring or the E-ring of a variety of lupane derivatives. Within
this set of pyrazines, three compounds were significantly cytotoxic
Triterpenoids are a large group of natural compounds that are
found in numerous living organisms, and are particularly prevalent
in plants. They often have a variety of biological activities.1,2
Betulinic acid (2) has strong anti-HIV3–5 and anti-cancer6–8 activi-
ties and belongs to a group of the most interesting lupane deriva-
tives. Zuco et al. were the first to prove9 that the cytotoxic activity
of 2 is selective against cancer cells, without affecting normal cells.
Since then, many research groups have tried to find new triterpe-
noids with improved pharmacological properties that would make
them useful candidates for HIV and cancer treatment. These
studies have either explored new plant species or modified the
structures of known active compounds. Among hundreds of new
compounds, we have synthesized several derivatives of lupane that
are highly cytotoxic in a variety of cancer cell lines in vitro, includ-
ing those that are resistant to current chemotherapeutic
agents.10–17 Previously, we described the partial synthesis of trite-
rpenic heterocycles,14 in which a pyrazine ring was connected to
in vitro to warrant the extension of the in vitro studies to in vivo
testing in mice. All of the active compounds have a single pyrazine
ring that is connected to the A-ring of 2, whereas, the other deriv-
atives, in which the pyrazine cycle is connected to a different ring
or those derivatives with a quinoxaline system instead of pyrazine,
were inactive. These findings suggest that a simple prediction of
the structure–activity relationship is difficult, especially with com-
pounds that contain more complicated structures, such as triter-
penes. There are many examples in the literature in which a
small modification of a barely active triterpene tremendously in-
creases the biological activity, and there are also instances where
highly active compounds are rendered less active by a small mod-
ification. Examples with anti-HIV activity have been previously re-
ported.3,18,19 The cytotoxic activity of some triterpenic compounds
was also successfully increased by using heterocyclic modifica-
tions.20–22
The promising cytotoxicity of triterpenic pyrazines,14 nonterpe-
nic heterocycles,23,24 and pyrrole and indole derivatives of 225
encouraged us to synthesize several different types of heterocycles.
Except for a few cases,25 most efforts to modify 2 have focused on
⇑
Corresponding author. Tel.: +420 606 234 503; fax: +420 226 015 452.
E-mail address: jan.sarek@gmail.com (J. Sarek).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.066

M. Urban et al. / Bioorg. Med. Chem. 20 (2012) 3666–3674
3667
heterocycles, wherein, a heterocycle is condensed with either the
acid (4), while several 18
a
-oleanane heterocycles (such as pyrraz-
carboxylic group at C-28 or to the C-30 position.20,21,26,27 To our
knowledge, pyrrazoles, imidazoles, pyrazolons, or thiazoles have
not been condensed with the A-ring of acid 2 or dihydrobetulonic
ole, methylpyrrazole, oxazole, N-methylpyrrole, and aminothia-
zole) were synthesized by Kim et al.28–31 along with a number of
5-membered heterocycles that are connected to glycyrrhetic acid.
29
29
21
30
x
x
21
22
19
30
12
20
20
18
19
O
22 O
12
26
E
17
18
17
13
13
11
9
11
9
28
25
26
25
R1
CO2H
e
1
28
14
14
EtO2C
HO
EtO2C
HO
16
16
1
4
2
(A)
2 (A)
10
8
7
10
8
7
15
15
e
A
a
27
27
(B)
3
(B)
3
6
6
5
5
4
14
23
24
23
24
A
x
R1
A
B
x
15 DB
16 SB
B
1 CH2OH CH2 C(H, β-OH) DB
7
8
9
CH2
CH2
C(H, β-OH)
C=O
b
2 CO2H
3 CO2H
4 CO2H
CH2 C(H, β-OH) DB
CH2 C=O
CH2 C=O
b
c
c
*CHBr C=O
DB
SB
SB
SB
c, 2 equiv.
10 CBr2
C=O
5 CO2H *CHBr C=O
c, 2 equiv.
6 CO2H
CBr2 C=O
d
d
x
O
CO2H
HO
O
HO
O
x
11
12 DB
13 SB
Scheme 1. Preparation of starting material for the synthesis of heterocycles. Reagents and conditions: (a) Montmorillonite K10, CHCl3, reflux; (b) Na2Cr2O7, AcONa, AcOH,
dioxane, rt; (c) Br2, AcOH, AcONa; (d) HCO2Et, NaH, dioxane, reflux; (f) Et2CO3, NaH, dioxane, reflux.
HO
HN
HN
N
N
Type
17 18α-oleanane
18 20(29)-lupene
Type
23 18α-oleanane
24 20(29)-lupene
25 lupane
a
19 lupane
a
11, 12, and 13
14, 15, and 16
b
b
N
N
NO REACTION
c
O
11
N
+
Type
N
O
20 18α-oleanane
21 20(29)-lupene
22 lupane
27 18α-oleanane type
26 18α-oleanane type
S
N
S
e
d
5 and 9
H2N
Br
N
Type
Type
28 18α-oleanane
29 lupane
30 18α-oleanane
31 lupane
O
N
f, g, h
H+
HO
i
9
9
O
O
32 18α-oleanane type
33 18α-oleanane type
Scheme 2. Reagents and conditions: (a) N2H4ꢀH2O, dioxane, reflux; (b) phenylhydrazine, AcOH, reflux; (c) NH2OH.HCl, EtOH, pyridine, reflux; (d) thiourea, morfoline, reflux;
(e) isoamylnitrite, TBAB, CHCl3, reflux; (f) urea or (g) guanidine or (h) acetamide, all refluxed in morpholine; (i) urea, reflux in pyridine or DMF or DMSO or N-
methylpyrrolidone.

3668
M. Urban et al. / Bioorg. Med. Chem. 20 (2012) 3666–3674
C30
C20
C29
C19
O1
C21
C22
C18
C12
C14
C11
C33
C13
C27
O3
C34
C2
C17
C28
C16
C1
N1
C9
C8
C10
C32
C25
C23
C31
C26
C15
C4
O2
C3
C5
C7
C6
C24
Figure 1. Ortep representation of morpholino derivative 32 with the atom numbering scheme. Thermal ellipsoids are drawn at the 50% probability level.41
Table 1
number of compounds for biological testing, we also included sev-
Cytotoxic activity of compounds 1–33 against CEM leukemia cell line
eral known heterocycles (17, 26, and 27).
a
a
Compound
IC50
(l
mol/L)
Compound
IC50 (lmol/L)
CEM
CEM
2. Material and methods
2.1. Chemicals
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
250
30
20
4
1
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
—
3
3
250
31
45
33
Betulin (1), betulinic acid (2), and dihydrobetulonic acid (4)
were obtained from company Betulinines (www.betulinines.com).
All other chemicals and solvents were obtained from Sigma–
Aldrich.
1
250
250
250
250
250
4
22
123
250
250
85
3
150
11
226
25
—
2.2. Cell lines
61
250
161
142
210
The CCRF-CEM (CEM), A549, K562, BJ and MRC-5 cell lines were
purchased from the American Tissue Culture Collection (ATCC).
Resistant clones, CEM daunorubicine resistant (CEM-DNR) and
K562 paclitaxel resistant (K562-TAX), were prepared by increasing
concentration of cytotoxic compounds and then characterized.42
Isogenic colorectal cancer cells bearing wild type (HCT116) or
deleted p53 gene (HCT116p53ꢁ/ꢁ) were obtained from Horizon
Discovery Ltd (www.horizondiscovery.com). The cells were main-
tained in nunc/corning 80 cm2 plastic tissue culture flasks and cul-
tured in cell culture medium according to ATCC recommendations
(DMEM/RPMI 1640 with 5 g/L glucose, 2 mM glutamine, 100 U/mL
penicillin, 100 mg/mL streptomycin, 10% fetal calf serum, and
NaHCO3).
a
The lowest concentration that kills 50% of tumor cells. The standard deviation in
cytotoxicity assays is typically up to 10% of the average value.
Imidazoles 26 and 27 were previously studied.32,33 However, only
Kumar et al.25 have reported on anti-cancer properties of their
products.
In this study, we prepared triterpenes with various 5-mem-
bered heterocycles condensed to the A-ring of betulonic acid (3),
dihydrobetulonic acid 4, and allobetulone (8). To increase the
Table 2
Cytotoxic activity of selected compounds 4–6, 12, 18, 19 and 29 against seven tumor and two normal fibroblast cell lines
a
Compound
IC50
(lmol/L)
CEM
CEM-DNR
A549
K562
K562-TAX
HCT116
HCT116p53ꢁ/ꢁ
BJ
MRC-5
04
05
06
12
18
19
29
3.7
1.4
1.0
3.5
2.8
2.6
3.5
9.4
11.4
7.0
29.6
10.4
8.2
5.9
11.9
8.4
17.6
5.2
2.5
1.6
0.9
8.4
2.6
3.5
4.8
4.0
1.7
1.1
12.7
3.0
2.7
4.1
4.3
2.4
12.1
4.5
3.9
6.1
4.1
2.6
13.7
3.3
2.8
22.6
17.8
13.3
55.5
35.8
22.4
24.9
11.7
14.6
8.0
29.9
14.1
13.4
15.7
3.6
7.0
11.2
6.9
5.1
4.3
aThe lowest concentration that kills 50% of tumor cells. The standard deviation in cytotoxicity assays is typically up to 10% of the average value.

M. Urban et al. / Bioorg. Med. Chem. 20 (2012) 3666–3674
3669
2.3. Cytotoxic MTT assay
EtOH at reflux proceeded as described in the literature,32,33, whereas
an inseparable mixture was obtained from the same reaction of
aldehydoketones 12 and 13. This problem is probably a result of
the free C-28 carboxyl group being attached to position 17 in both
of these compounds. This moiety may undergo various side reac-
tions. The reaction of both 2-bromo ketones 5 and 9 with thiourea
gave the best yield of the corresponding aminothiazoles 28 and 29
in morpholine at reflux, whereas larger amounts of the side-product
diosphenol 33 were obtained when other solvents were used.
Amines 28 and 29 were further modified into the bromo derivatives
30 and 31 by converting them into the corresponding diazonium
salts (Scheme 2). We attempted to prepare various heterocycles
by reactions of bromo ketone 9 with urea, guanidine, or acetamide,
however these reactions were not successful. Running these reac-
tions in morpholine at reflux resulted in the morpholino derivative
32, which easily hydrolyzed to diosphenol 33. The reaction of 9 with
urea in pyridine, DMF, N-methylpyrrolidone, or DMSO at reflux
yielded only diosphenol 33 and urea. No reaction occurred in dig-
lyme, EtOH, AcOH, THF, toluene, dioxane, or xylene at reflux. The
structure of the 32 (Fig. 1) was proved by X-ray crystallography.41
Cell suspensions were prepared and diluted according to the
particular cell type and the expected target cell density (2500–
30,000 cells/well based on cell growth characteristics). Cells were
added by pipette (80 lL) into 96-well microtiter plates. Inoculates
were allowed to pre-incubate for a period of 24 h at 37 °C and 5%
CO2 for stabilization. Four-fold dilutions of the intended test con-
centration were added in 20 lL aliquots at time zero to the micro-
titer plate wells. All tested compounds were dissolved in 10%
DMSO and the experiments were performed in quadruplicate.
The cells were incubated with the tested compounds for 72 h at
37 °C, in a 5% CO2 atmosphere at 100% humidity. At the end of
the incubation period, the cells were assayed by using the MTT
test. Aliquots (10 lL) of the MTT stock solution were pipetted into
each well and incubated for additional 1–4 h. After this incubation
period, the produced formazan was dissolved by the addition of
10% aq SDS (100 lL/well, pH = 5.5), and the cells were incubated
at 37 °C overnight. The optical density (OD) was measured at
540 nm with a Labsystem iEMS Reader MF. Tumor cell survival
(TCS) was calculated by using the following equation: TCS = (OD-
drug-exposed well/mean ODcontrol wells) ꢂ 100%. The IC50 value, the drug
concentration that is lethal to 50% of the tumor cells, was calcu-
lated from the appropriate dose-response curves.
3.2. Cytotoxicity
All of the compounds prepared in the study were tested for
cytotoxicity in highly chemosensitive T-lymphoblastic leukemia
CEM cell line in vitro, by using the MTT test39 (Table 1). In agree-
3. Results and discussion
3.1. Chemistry
ment with previous results, all of 18
found to be inactive. Betulinic (2) and betulonic acid (3) were only
moderately active (IC50 = 30 and 20 mol/L, respectively), whereas
a-oleanane derivatives were
l
dihydrobetulonic (4), 2-bromo dihydrobetulonic (5), 2,2-dibromo
dihydrobetulonic acid (6) and aldehydo-ketone 12, as well as pyr-
azoles 18, 19, and thiazole 29 were an order of magnitude more ac-
tive (IC50 = 4–1 lmol/L) on CEM cell line. Two more compounds,
diosphenol 33 and pyrazolone 24, were also moderately active
Allobetulin (7), allobetulon (8), and their derivatives were pre-
pared by the acidic rearrangement of betulin (1) by using montmo-
rillonite K10 in chloroform and subsequent oxidation with sodium
dichromate.14,40 One equivalent of Br2 was used to introduce bro-
mine at position 2 to give bromo derivatives 5 and 9, or 2 equiv of
Br2 were used to obtain dibromo compounds 6 and 10 (Scheme 1).
All of these procedures were described earlier.11,14,32,34–37
on these cells (Table 1).
Therefore, we chose five most active compounds 6, 12, 18, 19,
29 together with two starting compounds 5, 6 for extended testing
in the panel of seven cancer cell lines including multidrug resistant
and two non-tumor cell lines MRC-5 and BJ fibroblasts (Table 2).
The extended testing surprisingly shows that the most active
A modified procedure38 was used to synthesize aldehydoke-
tones 11–13. 3-Oxo derivatives 3, 4, or 8 were treated with ethyl
formate in a presence of a large excess of NaH (10 equiv) in anhy-
drous dioxane at reflux. These aldehydoketones exist predomi-
nantly in their enol form. A similar procedure was used for the
preparation of b-keto esters 14–16, which also predominantly exist
in their enol form.
Table 3
Crystalographic data of compound 32
The 5-membered heterocycles were synthesized by condensa-
tion reactions. The major issue with all of these reactions was
the limited solubility of the starting compounds in most of the sol-
vents that are usually used for similar reactions, and no reactivity
in the solvents in which the starting materials were soluble. For
this reason, unusual solvents, such as morpholine or a mixture of
EtOH and pyridine, were used frequently.
Pyrrazoles 17–19 and pyrazolones 23–25 were prepared by the
reaction of b-dicarbonylic compounds 11–16 with anhydrous
hydrazine hydrate (Scheme 2). The optimum conditions for this
reaction was 2–3 h in dioxane at reflux, after which most of the
products crystallized from the reaction mixture upon cooling. The
use of EtOH resulted in a much longer time to achieve the full con-
version of the reactant into product. In contrast, the reactions of
compounds 11–13 with phenylhydrazine only proceeded in glacial
acetic acid and not in any other solvent (dioxane, DMF, diglyme,
pyridine, pyridine/EtOH, morpholine, EtOH). This is probably be-
cause of a lower reactivity of phenylhydrazine in these solvents.
No reaction occurred between b-keto esters 14–16 and phenylhydr-
azine at reflux in any of the solvents mentioned. The preparation of
both isoxazoles 26 and 27 by the reaction of aldehydoketone 11
with hydroxylamine hydrochloride in a mixture of pyridine and
Empirical formula
Formula mass
Temperature
Wavelength
Crystal system
Space group
C34H53NO3, CHCl3
643.14
150(2) K
0.71073 Å
Orthorhombic
P212121 (No. 19)
Unit cell dimensions
a = 9.6402(2) Å;
a = 90°
b = 11.4835(3) Å; b = 90°
c = 30.2137(8) Å;
3344.75(14) Å3
4
c = 90°
Volume
Z
Density
Absorption coefficient
F(000)
1.277 g cmꢁ3
0.309 mmꢁ1
1384
Crystal size
0.50 ꢂ 0.20 ꢂ 0.18 mm
H
Range for data collection
1.90–27.47°
Index ranges
Total reflections collected
Independent reflections
ꢁ12 ꢃ h ꢃ 12, ꢁ8 ꢃ k ꢃ 14,ꢁ38 ꢃ l ꢃ 39
7474
6266 [R(int) = 0.0483]
Completeness to
H
H = 27.47°; 97%
Refinement method
Data/restraints/parameters
Goodness-of-fit on F
Final R indices [I>2
R indices (all data)
Largest difference peak/hole
Full-matrix least-squares on F2
7474/0/386
1.023
r
(I)]
R1 = 0.0483, wR2 = 0.1061
R1 = 0.0626, wR2 = 0.1149
0.405/ꢁ0.407 e Åꢁ3

3670
M. Urban et al. / Bioorg. Med. Chem. 20 (2012) 3666–3674
compounds are bromo acids 5, 6 or aldehydoketone 12, which
reached favorable in vitro therapeutic index >10 (ratio of cytotox-
icity in cancer versus non-cancer cell lines). While pyrazoles 18, 19
or thiazole 29 showed potency comparable to or slightly worse
than dihydrobetulonic acid (4) (Table 2).
a heating rate of 10 mA/s. Direct infusion APCI–MS experiments
were performed on an LCQ Fleet ion-trap mass spectrometer; the
system was controlled by a personal computer with Xcalibur soft-
ware (all by Thermo Fisher Scientific, San Jose, CA, USA). The APCI
vaporizer and heated capillary temperatures were set to 400 and
Interestingly, the most active compounds were among the
starting materials, and generally, all heterocyclic modifications to
the A-ring decreased the cytotoxicity. The majority of the heterocy-
cles were almost insoluble in any solvent, which might have played
an important role in the cytotoxicity testing. Solubility would also
be significant problem for the in vivo tests.
250 °C, respectively; the corona discharge current was 3.0 lA.
Nitrogen served as both the sheath and auxiliary gas at a flow rate
of 40 and 14 arbitrary units, respectively. The MS spectra of the
positively charged ions were recorded from 180 to 700 m/z. The
sample solution in acetonitrile/chloroform (99:1, flow rate of 5–
30 lL/min) was introduced into the APCI source. The concentra-
tions and time of data collections were chosen to obtain optimal
spectra. Column chromatography was performed on silica gel 60
(Merck 7734). The HPLC system consisted of a high-pressure pump
(Gilson model 361), a Rheodyne injection valve, and a preparative
3.3. X-Ray structure of morpholino derivative 32
Single crystals of 32 were grown from mixture of chloroform/
methanol. A colorless crystal was measured at Bruker Nonius Kap-
column (25 ꢂ 250 mm) filled with silica gel (Biospher 7
lm). The
paCCD diffractometer by monochromatized Mo
K
radiation
differential refractometer detector (Laboratorni Pristroje, Praha,
CZ) was connected to a PC (software Chromulan) and an automatic
fraction collector (Gilson model 246). A mixture of EtOAc and hex-
ane was used as the mobile phase, its composition specified in each
experiment. TLC was carried out on Kieselgel 60 F254 plates
(Merck) with toluene/Et2O (5:1) as eluent, and the results were
visualized by spraying the plate with 10% aqueous H2SO4 and heat-
ing to 150–200 °C. Work-up refers to pouring the reaction mixture
into H2O, extracting the product with an organic solvent, washing
the organic layer successively with H2O, dilute aqueous HCl, H2O,
saturated aqueous NaHCO3, and H2O again, and drying the organic
layer with MgSO4. This process was followed by filtration and
evaporation of the filtrate under reduced pressure. Analytical sam-
ples were dried over P2O5 under reduced pressure. 1, 2, and 4 were
obtained from Betulinines (www.betulinines.com). All other chem-
icals and solvents were obtained from Sigma–Aldrich.
a
(l = 0.71073 Å) at 150(2) K. The structure was refined by full-matrix
least squares based on F2 (SHELXL97). CCDC-80928841 (Table 3).
4. Conclusions
Starting from betulin (1) and betulinic acid (2), we prepared a
set of lupane, lup-20(29)-ene, and 18a-oleanane derivatives (3–
16) that were further converted into their heterocyclic analogues.
These analogues have a 5-membered heterocycle connected to
their A-ring in position 2 and 3. In spite of many problems (espe-
cially with solubility), we obtained a set of 15 heterocycles. All of
synthesised compounds were tested for cytotoxicity against the
sensitive acute lymphoblastic leukemia cell line (CEM). Five most
active compounds 6, 12, 18, 19, 29 and starting compounds 4, 5
were selected and tested against seven cancer cell lines derived
from various solid and hematological tumors including lung
(A549), colon (HCT116p53WT) and myeloid leukemia (K562) can-
cer cell lines in vitro; Importantly, the most active compounds are
bromo dihydrobetulonic acid (5), dibromo dihydrobetulonic acid
(6) and aldehydoketone 12 with preferential toxicity to leukemia
cancer cells and not to normal human fibroblasts (BJ or MRC-5),
demonstrating promising therapeutic index (>10) under in vitro
conditions. These compounds have also high cytotoxic activity
against multidrug resistant P-glycoprotein positive (K562-TAX)
and p53 inactivated (HCT116p53ꢁ/ꢁ) cancer cell lines, suggesting
therapeutic potential against these therapeutically problematic tu-
mors. However, the compounds failed to exhibit significant cyto-
toxic activity in lung resistant protein positive cells (CEM-DNR)
suggesting their limited use in these cancers.14 Poor solubility of
the heterocyclic derivatives in water-based media and other sol-
vents might be the main reason for their lower activity in compar-
ison with dihydrobetulonic acid (4).
5.2. Betulonic acid (3)
Betulonic acid (3) was prepared from betulinic acid (1) by oxi-
dation using sodium dichromate11
5.3. Bromination of 2-oxo compounds
A solution of bromine (2.3 mL, 45 mmol) and sodium acetate
(1 g) in acetic acid (80 mL) was added dropwise to a vigorously
stirred solution of the corresponding 3-oxo compound (20 g,
43.9 mmol) in CHCl3 (300 mL) and acetic acid (50 mL). The product
was isolated by pouring the reaction mixture into water, filtering
the precipitate, and drying. The product was purified by chroma-
tography on silica gel (200 g), elution with toluene/diethyl ether
(20:1) gave an epimeric mixture of 2a- and 2b-bromo ketones.
The same procedure was used to prepare dibromo derivatives 6
and 10 except 2 equiv of bromine were used.
5. Experimental
5.3.1. 2-Bromo dihydrobetulonic acid (5)
Yield: 18 g (77%), white microcrystals; mp 255–257 °C (CHCl3/
- and 2b-bromo ketones; ½a D25
ꢄ
5.1. General experimental procedures
MeOH) for epimeric mixture of 2
a
+14 (c 0.17, CHCl3) (lit.32 mp 125–127 °C for 2
a-bromo ketone
Melting points were determined with a Kofler block and are
uncorrected. Optical rotations were measured in CHCl3 solutions
(unless otherwise stated) on an Autopol III (Rudolph Research,
Flanders, NJ) polarimeter. Infrared spectra were recorded in CHCl3
solution or DRIFTS (KBr) on a NICOLET Impact 400D. Wave num-
bers are given in cmꢁ1. NMR spectra were recorded on a Varian
Unity INOVA 400 instrument (1H NMR spectra at 399.95 MHz) in
CDCl3 solutions (unless otherwise stated), with SiMe4 as an inter-
nal standard. EI-MS were recorded on an INCOS 50 (Finigan MAT)
spectrometer at 70 eV and an ion source temperature of 150 °C.
The samples were introduced through a direct exposure probe at
(CHCl3/MeOH).
5.3.2. 2,2-Dibromo dihydrobetulonic acid (6)
Yield: 22 g (85%), white microcrystals; mp 249–249 °C (CHCl3/
MeOH; decomp.); ½a D25
ꢄ
ꢁ14 (c 0.35, CHCl3); 1H NMR d 0.77 (d,
J(H-29, H-30) = 6.8 Hz), 0.87 (d, J(H-30, H-29) = 6.8 Hz), 0.92 (s), 0.97 (s),
0.97 (s), 1.22 (s), 1.54 (s) (21H, 7 ꢂ CH3), 1.90 (1H, dd, J1 = 12.5 Hz,
J2 = 7.2 Hz), 3.13 (1H, d, J(H-1a, H-1b) = 16.0 Hz, H-1a), 3.65 (1H, d,
J(H-1b, H-1a) = 16.0 Hz, H-1b); EI-MS m/z 614 [M+] (1), 599 (1), 569
(40), 534 (55), 489 (100), 455 (41), 409 (77); Anal. C 58.53, H 7.62,
Br 25.95, Calcd for C30H46Br2O3, C 58.64, H 7.55, Br 26.01.

M. Urban et al. / Bioorg. Med. Chem. 20 (2012) 3666–3674
3671
5.3.3. 2-Bromo allobetulon (9)
(2H), 2.39 (1H, d, J(H-1a, H-1b) = 15.4 Hz, H-1a), 3.04 (1H, td,
J(H-19b, ) = 10.4 Hz, J(H-19b, ) = 10.4 Hz, J(H-19b,
7 was converted into allobetulon 8 as previously described,14,40
and subsequently brominated as described above. Yield (bromina-
tion): 17 g (72%), white microcrystals; mp 226–228 °C (CHCl3/
=
H-21b)
H-18
a
H-21
a
4.7 Hz, H-19b), 4.18 (2H, m, CH2–Et), 4.61 (1H, m, H-29 pro E),
4.75 (1H, bd, J = 1.9 Hz, H-29 pro Z), 12.59 (1H, s, 3-OH); EI-MS
m/z 526 [M+] (100), 511 (9), 480 (39), 465 (14), 452 (10), 434
(13), 419 (7), 391 (42), 317 (56), 189 (27); Anal. C 75.08, H 9.66,
Calcd for C33H50O5, C 75.25, H 9.57.
MeOH); ½a 2D5
ꢄ
+68 (c 0.55, CHCl3) (lit.36,37 mp 216–225 °C; ½a 2D5
ꢄ
+74.
5.3.4. 2,2-Dibromoallobetulon (10)
Yield: 19 g (71%), white microcrystals; mp 217–220 °C (CHCl3/
b-Keto ester 16 was purified by chromatography on silica gel
(200 g) with gradient elution from hexane to hexane/ethyl acetate
MeOH); ½a 2D5
ꢄ
ꢁ16 (c 0.3, CHCl3) (lit.36 mp 214–216 °C; ½a 2D5
ꢁ14.
ꢄ
(9:1).Yield: 46%; mp. 231–234 °C (EtOH); ½a D25
ꢄ
+12 (c 0.45, CHCl3);
5.4. General procedure for the preparation of aldehydoketones
11–13
IR (CHCl3) m
max 3517, 1733, 1693, 1647, 1612 cmꢁ1; 1H NMR d 0.76
(d, J(H-29, H-30) = 6.8 Hz), 0.83 (s), 0.86 (d, J(H-30, H-29) = 6.8 Hz), 0.97
(s), 0.97 (s) (15H, 5 ꢂ CH3), 1.30 (3H, t, J = 7.2 Hz, CH3–Et), 2.19–
2.31 (2H), 2.41 (1H, d, J(H-1a, H-1b) = 15.6 Hz, H-1a), 4.19 (2H, m,
CH2–Et), 11.00 (1H, br s, 28-COOH), 12.58 (1H, s, 3-OH); EI-MS
m/z 528 [M+] (100), 513 (7), 482 (29), 467 (12), 454 (13), 358
(44); Anal. C 75.11, H 9.84, Calcd for C33H52O5, C 74.96, H 9.91.
A modified procedure from the literature36 was used. Each of
the 3-oxo derivatives 3, 4, and 8 (20 g, 45 mmol) was dissolved
in dry dioxane (400 mL) and excess NaH (22 g) was added slowly
to the solution, under argon. The mixture was heated slowly to
boiling and then ethyl formate (6 mL) was slowly added over 3 h.
The mixture was cooled to room temperature and any remaining
NaH was quenched with 30 mL of EtOH in dioxane (100 mL). The
crude product was precipitated out of the solution by adding 10%
HCl (1 L) and was then extracted into chloroform. The product
was purified by chromatography on silica gel (200 g) in toluene
(for compound 11) or by using gradient from toluene to 10%
diethyl ether in toluene (for acids 12 and 13) and was subsequently
crystallized from CHCl3/MeOH to give the pure aldehyde.
5.6. General procedure for the preparation of pyrazoles 17–19
Hydrazinehydrate, 100%(1 mL, 20 mmol)wasadded dropwiseto
a solution of each aldehyde 11–13 (2.1 mmol) in refluxing dioxane
(20 mL) and the solution was heated for another 2 h. White crystals
precipitatedfromthereactionmixtureuponcooling toroomtemper-
ature. The dioxane was evaporated and the crystalline product was
washed with CHCl3 (3 mL, twice) to remove unreacted aldehyde.
5.4.1. 2-Hydroxymethylene-allobetulone 11
5.6.1. Pyrazole 17
Yield: 71%; mp 242–244 °C (CHCl3/MeOH); ½a D25
ꢄ
+84 (c 0.40,
+76).
Yield: 85%; mp. 342–343 °C (dioxane); ½a D25
ꢄ
+60 (c 0.22, CHCl3);
pyridine) (lit.38 mp 253–257 °C (benzene/EtOH); ½a 2D5
ꢄ
IR (CHCl3) m
max 3467, 3282vb cmꢁ1; 1H NMR d 0.81, 0.82, 0.95, 1.03,
1.19, 1.29 (21H, all s, 7 ꢂ CH3), 2.00 (1H, d, J(H-1a, H-1b) = 14.6 Hz, H-
5.4.2. 2-Hydroxymethylene-betulonic acid 12
1a), 2.68 (1H, d, J(H-1b, H-1a) = 14.8 Hz, H-1b), 3.46 (1H, d, J = 7.8 Hz,
Yield: 70%; mp 235–238 °C (CHCl3/MeOH); ½a D25
ꢄ
+41 (c 0.30,
H-28a), 3.55 (1H, s, H-19a), 3.79 (1H, d, J = 7.8 Hz, H-28b), 4.69
CHCl3); 1H NMR contains singlet peak of methylene at 8.60 ppm
as described in lit.6
(1H, br s, NH), 7.23 (1H, s, H-31); EI-MS m/z 464 [M+] (100), 450
(19), 433 (8), 393 (12). Physical constants were same as reported
in the lit.27
5.4.3. 2-Hydroxymethylene-dihydrobetulonic acid 13
Yield: 72%; mp 284–287 °C(CHCl3/MeOH); ½a D25
ꢄ
+12 (c 0.42,
5.6.2. Pyrazole 18
CHCl3); 1H NMR contains singlet peak of methylene at 8.60 ppm
as described in lit.6
Yield: 85%; mp 219–220 °C (dioxane); ½a D25
ꢄ
+30 (c 0.35, CHCl3); IR
(KBr) m
max 3209b, 1680 cmꢁ1; 1H NMR d 0.79, 105, 1.02, 1.03, 1.17,
1.28 (15H, all s, 5 ꢂ CH3), 1.71 (3H, s, 3 ꢂ H-30), 1.90–2.04 (2H),
1.98 (1H, d, J(H-1a, H-1b) = 14.5 Hz, H-1a), 2.27 (1H,dt, J1 = 12.6 Hz,
J2 = J3 = 3.16 Hz), 2.34 (1H, td, J1 = J2 = 12.6 Hz, J3 = 3.5 Hz, 2.63 (1H,
5.5. General procedure for a preparation of b-keto esters 14–16
Each of the 3-oxo derivatives 3, 4, and 8 (20 g, 45 mmol) was
dissolved in dioxane (400 mL) and excess NaH (30 g) was slowly
added to the solution, under argon. The mixture was slowly heated
up to its boiling point and then diethyl carbonate (25 mL) was
slowly added over 3 h. The work-up procedure was same as for
aldehydes 11–13.
d, J(H-1b, H-1a) = 14.9 Hz, H-1b), 3.05 (1H, td, J(H-19b, H-18 ) = 10.9 Hz,
a
J(H-19b,
) = 10.9 Hz, J(H-19b, H-21b) = 4.7 Hz, H-19b), 4.61 (1H, m,
H-21a
H-29 pro-E), 4.74 (1H, bd, J = 2.0 Hz, H-29 pro Z), 7.17 (1H, s, H-
31); EI-MS m/z 478 [M+] (100), 463 (38), 450 (2), 434 (13), 419 (1),
396 (19), 269 (32); Anal. C 77.63, H 9.71, N 5.73 Calcd for
C31H46N2O2, C 77.78, H 9.69, N 5.85.
b-Keto ester 14 was purified by chromatography on silica gel
(200 g) with gradient elution from toluene to toluene/diethyl ether
5.6.3. Pyrazole 19
(9:1). Yield: 67% mp 137–140 °C (EtOH); ½a D25
ꢄ
+75 (c 0.37, CHCl3);
Yield: 91%; mp 237–239 °C (dioxane); ½a D25
ꢄ
+9 (c 0.35, CHCl3); IR
IR (CHCl3) mmax 3666, 1736 cmꢁ1
;
1H NMR d 0.80, 0.86, 0.93, 0.94,
(CHCl3) mmax 3692, 3466, 1691 cmꢁ1 1H NMR d 0.77 (3H, d,
;
1.01, 1.08, 1.17 (21H, all s, 7 ꢂ CH3), 1.30 (3H, t, J(CH3, CH2) = 7.2 Hz,
J = 6.8 Hz), 0.79 (3H, s), 0.87 (3H, d, J = 6.8 Hz), 0.99 (3H, s), 1.00
(3H, s), 1.19 (3H, s), 1.30 (3H, s, 7 ꢂ CH3), 2.18–2.39 (3H), 2.65
(1H, d, J(H-1a, H-1b) = 14.8 Hz, H-1a), 7.25 (1H, s, H-31); MS EI-MS
m/z 480 [M+] (41), 465 (12), 452 (3), 436 (100), 419 (16), 409 (2),
267 (38); Anal. C 77.57, H 10.02, N 5.78, Calcd for C31H48N2O2, C
77.45, H 10.06, N 5.83.
CH –Et), 2.43 (1H, d, J(H-1a, H-1b) = 15.4 Hz, H-1a), 3.45 (1H, d,
3
J = 7.9 Hz, H-28a), 3.55 (1H, s, H-19), 3.79 (1H, dd, J1 = 7.8 Hz,
J2 = 1.3 Hz, H-28b), 4.20 (2H, m, CH2–Et), 12.59 (1H, s, 3-OH); EI-
MS m/z 512 [M+] (17), 439 (22), 424 (100), 409 (35), 393 (11),
381 (52), 355 (53), 342 (21), 189 (57); Anal. C 77.17, H 10.28, Calcd
for C33H52O4, C 77.30, H 10.22.
b-Keto ester 15 was purified by chromatography on silica gel
(200 g) with gradient elution from toluene to toluene/diethyl ether
5.7. General procedure for the preparation of phenylpyrazoles
20–22
(9:1).Yield: 52%; mp 234–236 °C (EtOH); ½a D25
ꢄ
+38 (c 0.34, CHCl3);
IR (CHCl3) mmax 3516, 1736, 1694, 1646, 1612 cmꢁ1
;
1H NMR d
Phenylhydrazine (1 mL, 20 mmol) was added dropwise to a
solution of each aldehyde 11–13 (2.1 mmol) in acetic acid
(20 mL) at reflux, under argon. The solution was heated for another
0.83, 0.97, 0.99, 1.06, 1.16 (15H, all s, 5 ꢂ CH3), 1.29 (3H, t,
J = 7.2 Hz, CH3–Et), 1.70 (3H, m, H-30), 1.94–2.07 (2H), 2.18–2.34

3672
M. Urban et al. / Bioorg. Med. Chem. 20 (2012) 3666–3674
2 h and then poured into water. The product was extracted into
ethyl acetate, washed with aqueous solution of NaHCO3, and dried
with MgSO4. The ethyl acetate was removed under reduced pres-
sure and the crude product was purified by chromatography on sil-
ica gel (150 g).
479 (37), 467 (19), 438 (62), 410 (43), 239 (100); Anal. C 79.31,
H 9.25, N 5.79, Calcd for C31H46N2O3, C 75.26, H 9.37, N 5.66.
Pyrazolone 25 was eluted with CHCl3/MeOH (10:1) and crystal-
lized from CHCl3/MeOH. Yield: 64%; mp 218–221 °C (CHCl3/
MeOH); ½a 2D5
ꢄ
+5 (c 0.32, pyridine); IR (CHCl3) mmax 1733 cm-1 1H
;
Phenylpyrazole 20 was eluted with a gradient from toluene to
toluene/diethyl ether (10:1) and crystallized from MeOH. Yield:
NMR d 0.77 (3H, d, J = 6.8 Hz), 0.81 (3H, s), 0.87 (3H, d,
J = 6.8 Hz), 0.98 (3H, s), 0.99 (3H, s), 1.13 (3H, s), 1.23 (3H, s,
7 ꢂ CH3), 2.5 (1H, bd, J(H-1a, H-1b) = 15.0 Hz, H-1a), 8–9 (1H, NH);
EI-MS m/z 496 [M+] (9), 468 (22), 458 (57), 440 (100), 425 (47),
397 (65), 267 (94); Anal. C 74.82, H 9.87, N 5.72, Calcd for
56%; mp 269–271 °C (MeOH); ½a D25
ꢄ
+73 (c 0.32, CHCl3); IR (CHCl3)
mmax 1598, 1500, 1384 cmꢁ1 1H NMR d 0.81, 0.88, 0.94, 0.95,
;
1.01, 1.03, 1.05 (21H, all s, 7 ꢂ CH3), 2.09 (1H, d, J(H-1a,
=
H-1b)
15.0 Hz, H-1a), 2.73 (1H, d, J(H-1b, H-1a) = 15.0 Hz, H-1b), 3.46
(1H, d, J = 8.0 Hz, H-28a), 3.57 (1H, s, H-19 ), 3.80 (1H, d,
C31H48N2O3, C 74.96, H 9.74, N 5.64.
a
J = 7.6 Hz, H-28b), 7.37–7.48 (5H, m, 5 ꢂ H-Ph), 7.40 (1H, s, H-
31); MS EI-MS m/z 540 [M+] (100), 525 (43), 509 (6), 469 (12); Anal.
C 81.83, H 9.52, N 5.21, Calcd for C37H52N2O, C 82.17, H 9.69, N
5.18.
5.9. Isoxazoles 26 and 27
The procedure used were analogous to those that have been
reported in the literature.27,40 Hydroxylamine hydrochloride
(0.5 g, 7.3 mmol) was added to a solution of aldehyde 9 (0.5 g,
1.1 mmol) in a mixture of pyridine/EtOH (1:1, 25 mL) and the mix-
ture was heated to reflux for 7 h. The reaction mixture was then
poured into 8% aqueous HCl. The product was extracted with ethyl
acetate, washed with aqueous solution of NaHCO3, and dried with
MgSO4. The ethyl acetate was removed under reduced pressure
and the crude mixture of isoxazoles 26 and 27 was purified by
HPLC with 15% ethyl acetate in hexane as the eluent. The HPLC
separation was repeated to achieve the full separation of the
regioisomers.
Phenylpyrazole 21 was eluted with a gradient from toluene to
toluene/diethyl ether (3:1) and crystallized from CHCl3. Yield:
66%; mp 258–260 °C (CHCl3); ½a D25
ꢄ
+25 (c 0.41, pyridine); IR (CHCl3)
m
max 1734, 1694 cmꢁ1; 1H NMR (in CDCl3/CD3OD) d 0.86, 0.99, 1.00,
1.01, 1.03 (15H, all s, 5 ꢂ CH3), 1.72 (3H, s, H-30), 2.05 (1H, d,
J(H-1a, H-1b) = 14.7 Hz, H-1a), 2.68 (1H, d, J(H-1b, H-1a) = 14.9 Hz, H-
1b), 3.04 (1H, td, J(H-19b,
) = 10.4 Hz, J(H-19b,
) = 10.4 Hz,
H- 21a
H-18a
J(H-19b, H-21b) = 4.7 Hz, H-19b), 4.63 (1H, m, H-29 pro-E), 4.75 (1H,
bd, J = 2.4 Hz, H-29 pro Z), 7.37 (1H, s, H-31), 7.34–7.40 (2H, m,
Ph), 7.42–7.50 (3H, m, Ph); MS EI-MS m/z 554 [M+] (100), 539
(47), 510 (16), 495 (3), 485 (1), 467 (2), 441 (5), 198 (31), Anal. C
80.43, H 9.31, N 5.16, Calcd for C37H50N2O2, C 80.10, H 9.08, N 5.05.
Phenylpyrazole 22 was eluted with a gradient from toluene to
toluene/diethyl ether (3:1) and crystallized from CHCl3. Yield:
5.9.1. Isoxazole 26
Yield: 18%; mp 303–305 °C (hexane/EtOAc); ½a D25
ꢄ
+71 (c 0.26,
CHCl3); IR (CHCl3) mmax 1610, 1451 cmꢁ1 1H NMR d 0.80, 0.81,
;
70%; mp 298–300 °C (CHCl3); ½a D25
ꢄ
0 (c 0.28, pyridine); IR (KBr) mmax
0.94, 1.03, 1.29, 1.38 (21H, all s, 7 ꢂ CH3), 1.92 (1H, d, J(H-1a, H-1b)
=
2200–3450 vb, 1696 cmꢁ1
;
1H NMR d 0.78 (3H, d, J = 6.8 Hz), 0.87
14.9 Hz, H-1a), 2.79 (1H, d, J = 15.1, H-1b), 3.46 (1H, d, J = 7.8 Hz,
H-28a), 3.55 (1H, s, H-19), 3.79 (1H, dd, J1 = 7.2 Hz, J2 = 1.1 Hz,
H-28b), 8.04 (1H, d, J = 1.4 Hz, H-31); EI-MS m/z 465 [M+] (95),
450 (22), 437 (100), 422 (24), 394 (87), 384 (23).
(3H, s), 0.88 (3H, d, J = 6.8 Hz), 0.99 (3H, s), 1.00 (6H, s), 1.04 (3H,
s, 7 ꢂ CH3), 1.74 (1H), 1.85 (2H), 2.06 (1H, d, J(H-1a, H-1b) = 14.8 Hz,
H-1a), 2.20–2.40 (3H), 2.70 (1H, d, J(H-1b, H-1a) = 14.8 Hz, H-1b),
7.38 (1H, s, H-31), 7.35–7.40 (2H, m, Ph), 7.43–7.50 (3H, m, Ph);
MS EI-MS m/z 556 [M+] (100), 541 (67), 512 (7), 497 (3), 469 (4),
198 (52); Anal. C 79.74, H 9.16, N 5.03, Calcd for C37H52N2O2, C
79.81, H 9.41, N 5.03.
5.9.2. Isoxazole 27
Yield: 28%; mp 274–277 °C (hexane/EtOAc); ½a D25
ꢄ
+56 (c 0.21,
CHCl3); IR (CHCl3) mmax 1641, 1481, 1455 cmꢁ1
;
1H NMR d 0.81,
0.84, 0.94, 1.03, 1.20, 1.31 (21H, all s, 7 ꢂ CH3), 1.98 (1H, d,
J(H-1a, H-1b) = 15.1 Hz, H-1a), 2.52 (1H, d, J = 15.1, H-1b), 3.46 (1H,
d, J = 7.7 Hz, H-28a), 3.55 (1H, s, H-19), 3.79 (1H, dd, J1 = 7.8 Hz,
J2 = 1.1 Hz, H-28b), 7.99 (1H, s, H-31); EI-MS m/z 465 [M+] (100),
450 (10), 437 (56), 422 (53), 394 (2), 384 (47). The spectra of both
isoxazoles were compared with those of identical compounds in
the literature.33
5.8. General procedure for the preparation of pyrazolones 23–25
from b-keto esters 14–16
Hydrazine hydrate, 100% (1 mL, 20 mmol) was added dropwise
to a solution of each b-keto ester 14–16 (2.0 mmol) in dioxane
(20 mL) at reflux, under argon. The solution was heated for another
3 h and then poured into 8% aqueous HCl. The product was ex-
tracted into ethyl acetate, washed with an aqueous solution of
NaHCO3, and dried with MgSO4. The ethyl acetate was removed
under reduced pressure and the crude product was purified by
chromatography on silica gel (50 g).
5.10. Thiazoles 28–31
Thiourea (2 g, 25.6 mmol) was added to a solution of either 5 or
9 (1 g, 1.9 mmol, mixture of
a- and b-diastereomers) in morpho-
Pyrazolone 23 was eluted with toluene/isopropanol (10:1) and
line and the mixture was heated at reflux for 6 h, then poured into
water. The product was extracted into chloroform, washed with
aqueous solution of NaHCO3 and dried over MgSO4. The solvents
were removed under reduced pressure and the crude product
was purified by chromatography on silica gel (50 g).
crystallized from CHCl3/MeOH. Yield: 59%; mp over 300 °C
(CHCl3/MeOH); ½a D25
ꢄ
+68 (c 0.15, pyridine); IR (CHCl3) mmax 3487,
;
1H NMR d 0.76, 0.81, 0.92, 0.93, 0.99 (15H,
3159, 1615 vb cmꢁ1
all s, 5 ꢂ CH3), 2.5 (1H, bd, J(H-1a, H-1b) = 12.7 Hz, H-1a), 3.45 (1H,
d, J = 7.5 Hz, H-28a), 3.55 (1H, s, H-19
a), 3.79 (1H, d, J = 7.5 Hz,
Amino thiazole 28 was eluted with toluene/diethyl ether (10:1)
and crystallized from MeOH. Yield: 63%; mp 313–318 °C (MeOH);
H-28b), 8–9 (1H, br s, NH); MS EI-MS m/z 480 [M+] (100), 465
(9), 449, (8), 409 (13), 138 (92); Anal. C 77.26, H 9.94, N 5.71, Calcd
for C31H48N2O2, C 77.45, H 10.06, N 5.83.
½
a 2D5
ꢄ
+81 (c 0.30, CHCl3); IR (CHCl3) mmax 3488, 3391, 1603,
1525 cmꢁ1 1H NMR d 0.81, 0.92, 0.94, 1.03, 1.11, 1.21 (21H, all s,
;
Pyrazolone 24 was eluted with CHCl3/MeOH (10:1) and crystal-
lized from CHCl3/MeOH. Yield: 64%; mp 241–243 °C (CHCl3/
7 ꢂ CH3), 2.14 (1H, d, J(H-1a, H-1b) = 15.3 Hz, H-1a), 2.58 (1H, d,
J = 15.4, H-1b), 3.46 (1H, d, J = 7.8 Hz, H-28a), 3.55 (1H, s, H-19),
3.80 (1H, bd, J1 = 7.3 Hz, H-28b), 4.84 (2H, br s, NH2); EI-MS m/z
496 [M+] (100), 481 (23), 261 (7), 193 (12). FABMS m/z 497
[M++H], 481; Anal. C 74.68, H 9.59, N 5.80, S 6.30, Calcd for
MeOH);
½
a 2D5
ꢄ
+15 (c 0.31, pyridine); IR (CHCl3) mmax 1734,
1694 cmꢁ1
;
1H NMR d 1.00, 1.01, 1.16, 1.26, 1.70 (15H, all s,
5 ꢂ CH3), 1.96 (3H), 2.29 (3H), 3.00 (1H, m, H-19b), 4.61 (1H, m,
H-29 pro-E), 4.74 (1H, bd, H-29 pro Z); EI-MS m/z 494 [M+] (41),
C31H48N2OS, C 74.95, H 9.74, N 5.64, S 6.45.

M. Urban et al. / Bioorg. Med. Chem. 20 (2012) 3666–3674
3673
Amino thiazole 29 was eluted with toluene/diethyl ether (10:1)
and crystallized from MeOH. Yield: 57%; mp 310–315 °C (MeOH;
(21H, all s, 7 ꢂ CH3), 2.57 (2H, br s, CH2 morpholine), 2.89 (2H, br
s, CH2 morpholine), 3.46 (1H, d, J = 8.0 Hz, H-28a), 3.56 (1H, s, H-
19), 3.78 (1H, d, J = 7.6 Hz, H-28b), 3.83 (4H, m, 2 ꢂ CH2 morfoline),
6.01 (1H, br s, H-1); EI-MS m/z 524 [M++H] (100), 508 (84), 498
(67), 481 (7), 466 (14), 455 (2); Anal. C 78.12, H 10.02, N 2.51, Calcd
for C34H53NO3, C 77.96, H 10.20, N 2.67.
decomposition); ½a D25
ꢄ
+22 (c 0.37, CHCl3); IR (CHCl3) mmax 3510,
3480, 3385, 1738, 1606 cmꢁ1
;
1H NMR d 0.77 (3H, d, J = 6.8 Hz),
0.86 (3H, d, J = 6.8 Hz), 0.89 (3H, s), 0.98 (3H, s), 0.99 (3H, s), 1.10
(3H, s), 1.19 (3H, s, 7 ꢂ CH3), 2.10 (1H, d, J(H-1a, H-1b) = 15.4 Hz, H-
1a), 2.20–2.38 (3H), 2.53 (1H, d, J = 15.4, H-1b), 4.07 (2H, br s,
NH2); EI-MS m/z 512 [M+] (63), 497 (17), 451 (9), 423 (8), 313
(100); Anal. C 72.55, H 9.37, N 5.46, S 6.33, Calcd for C31H48N2O2S,
C 72.61, H 9.43, N 5.46, S 6.25.
Acknowledgements
This study was supported by the Czech Science Foundation
(305/09/1216 and 301/09/P433) and the Grant Agency of the CTU
in Prague, SGS11/133/OHK4/2T/14. Indirect costs were paid by
the internal grant of Palacky University (IGA UPOL 9011100251).
Infrastructural part of this project (Institute of Molecular and
Translational Medicine) was supported from the Operational Pro-
gram Research and Development for Innovations (Project
CZ.1.05/2.1.00/01.0030). Special thanks go to Ivana Cisarova (sup-
ported from MSM0021620857) for the X-ray analysis of compound
32. We are also grateful to Stanislav Hilgard for running IR spectra,
and to Dr. Kesner and Dr. Pasztor (NICOLET CZ) for special services
in IR instrumentation. We are also grateful to Bohunka Šperlichová
for measurement of optical rotation, and Richard Threlfall for edit-
ing the manuscript.
5.11. Bromo thiazoles 30 and 31
Each amino thiazole 28 (200 mg, 0.4 mmol) or 29 (200 mg,
0.4 mmol) was dissolved in chloroform (5 mL) and tetrabutylam-
monium bromide (300 mg, 1 mmol) and isopentyl nitrite (106 lL,
0.8 mmol) were added to the solution. The reaction mixture was
heated to 60 °C for 4 h and then poured into water. The product
was extracted with chloroform, washed with aqueous solution of
Na2SO3 and Na2S2O3 and dried with MgSO4. The solvents were re-
moved under reduced pressure and the crude products were puri-
fied by chromatography.
Bromo thiazole 30 was purified by preparative HPLC on silica gel
with hexane/ethyl acetate (95:5) as eluents, and then lyophilized
from t-BuOH. Yield: 120 mg (53%); mp 271–273 °C (t-BuOH);
Supplementary data
½
a 2D5 +68 (c 0.51, CHCl3); IR (CHCl3) mmax 1603, 1525 cmꢁ1 1H
;
ꢄ
Supplementary data (1H, 13C NMR spectra of new compounds
and X-ray crystallographic data for compound 32) associated with
this article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2012.03.066. These data include MOL files and
InChiKeys of the most important compounds described in this
article.
NMR d 0.81, 0.90, 0.94, 1.03, 1.18, 1.28 (21H, all s, 7 ꢂ CH3), 2.21
(1H, bd, J(H-1a, H-1b) = 15.0 Hz, H-1a), 2.77 (1H, bd, J(H-1b,
=
),
H-1a)
15.0 Hz, H-1b), 3.46 (1H, d, J = 6.0 Hz, H-28a), 3.55 (1H, s, H-19
a
3.79 (1H, d, J = 6.0 Hz, H-28b); MS APCI+: m/z 562 [M-81Br+H]+
(100), 560 [M-79Br+H]+ (97); Anal. C 66.22, H 8.41, Br 14.12, N
2.57, S 5.78, Calcd for C31H46BrNOS, C 66.41, H 8.27, Br 14.25, N
2.50, S 5.72.
Bromo thiazole 31 was purified by chromatography on silica gel
(50 g) with chloroform/acetone (100:1), and then lyophilized from
References and notes
t-BuOH. Yield: 73 mg (33%); mp 189–191 °C (t-BuOH); ½a D25
ꢄ
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