Synthesis of phthalates of betulinic acid and betulin with cytotoxic activity

Article abstract of DOI:10.1016/j.bmc.2005.03.006

Synthesis of 3β-O-phthalic esters from betulinic acid and its esters and synthesis of phthalic esters from betulin and its monoacetates using classical acylation procedure with phthalic anhydride. The evaluation of cytotoxicity of the prepared compounds was using numbers of tumor cell lines in MTT test. It was discovered that hemiphthalic esters had better cytotoxicity than starting compounds as betulinic acid or quite inactive betulin.

Full text of DOI:10.1016/j.bmc.2005.03.006

Bioorganic & Medicinal Chemistry 13 (2005) 3447–3454
Synthesis of phthalates of betulinic acid and
betulin with cytotoxic activity
Miroslav Kvasnica,a Jan Sarek,a,* Eva Klinotova,a Petr Dzubakb and Marian Hajduchb
aDepartment of Organic and Nuclear Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8,
128 43 Prague 2, Czech Republic
bLaboratory of Experimental Medicine, Departments of Pediatrics and Oncology, Faculty of Medicine, Palacky University in Olomouc,
Puskinova 6, 775 20 Olomouc, Czech Republic
Received 23 November 2004; revised 23 February 2005; accepted 1 March 2005
Available online 30 March 2005
Abstract—Synthesis of 3b-O-phthalic esters from betulinic acid and its esters and synthesis of phthalic esters from betulin and its
monoacetates using classical acylation procedure with phthalic anhydride. The evaluation of cytotoxicity of the prepared com-
pounds was using numbers of tumor cell lines in MTT test. It was discovered that hemiphthalic esters had better cytotoxicity than
starting compounds as betulinic acid or quite inactive betulin.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
lipophylic groups (e.g., acetates) lead to decrease of bio-
logic activity,9 right choice of appropriate substitution is
critical for improvement of pharmacological properties
of new compounds. In this respect we have mainly paid
attention to the synthesis of hemiesters of lower diacids
using competent anhydride.10 This procedure can lead
to acquirement of large groups of new polar derivatives
with increased cytotoxic activity. Here we report our re-
sults on synthesis of betulinic acid and betulin phthalate
esters and the effect of this substitution on biological
activity of new compounds. Methyl- and ethyl-phtha-
lates were also prepared within the structure–activity
relationship study.
Betulinic acid (1) (3b-hydroxy-lup-20(29)-en-28-oic
acid), pentacyclic lupane triterpene, is a known natural
compound with various biological effects.1,2 Cytotoxic
activity against various malignant versus nonmalignant
cell lines belongs to most intensively studied3 effects of
these compounds. Betulinic acid (1) demonstrated selec-
tive anticancer activity against neuroectodermal tu-
mors,4 including human melanoma, neuroblastoma,
and Ewing sarcomas. Therefore a lot of effort is focused
on modification5,6 of betulinic acid in order to increase
and broader its biological activity against tumor cells
of various histogenetic origin despite the fact that its
mechanism of effect is still being studied7 and is not clear
enough. In contrast to betulinic acid (1), betulin (5) (lup-
20(29)-en-3b,28-diol) has no significant cytotoxic
activities. However, based on structural similarity with
betulinic acid (1), one could expect that appropriate
derivatization could increase its cytotoxic potency.
2. Chemistry
Betulinic acid (1) was isolated from bark of plane trees
Platanus acerifolius using extraction with MeOH
according to literature.5 Treatment of acid 1 with etheri-
cal solution of diazomethane, resp. diazoethane, gave
methyl 2, resp. ethyl ester 3, in quantitative yield. Reac-
tion of acid 1 with benzyl bromide in the presence of
DBU afforded benzyl ester 4 in the yield 85%.
In this paper we performed synthesis of new triterpenic
compounds, particularly those derivatized on hydroxy
groups (mainly 3b- and 28-hydroxy groups).8 Since
derivatization of hydroxy group of betulinc acid (1) with
Betulin (5) was isolated from the birch bark (Betula pen-
dula) using the known extraction procedure.11 Monoace-
tate 6 was prepared using mild basic hydrolysis12 of
betulin diacetate with calcium hydroxide (5 equiv) at
room temperature in yield 65%. The monoacetate 7
Keywords: Triterpenes; Betulin; Phthalate; Cytotoxicity.
*
Corresponding author. Tel./fax: +420 221 951 332; e-mail:
jan.sarek@volny.cz
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.006

3448
M. Kvasnica et al. / Bioorg. Med. Chem. 13 (2005) 3447–3454
was prepared using selective acetylation of primary hy-
droxy group of betulin (5) with acetic anhydride in the
presence of imidazole13 in yield 85%.
Synthesis of phthalates of betulinic acid (1) and its deriva-
tives is described in Scheme 1, synthesis of phthalates
of betulin (5) and its monoacetates in Scheme 2. The
acylation reaction was always carried out by reflux
about 24 h. It should be noted that treatment of triter-
penes with phthalic anhydride requires reagent in at
least 5–10 times molar excess for completion of reaction.
After usual working up the crude products were sepa-
rated by chromatography over silica gel (EtOAc in hex-
ane, 10% ! 75%) or chromatographed using HPLC.
Products were then crystallized from the solvents men-
tioned in experimental section, if not otherwise stated.
The yields after crystallization were usually about 75–
85%. All methyl- and ethyl-phthalates, except 1b and
1c, were prepared using esterification with diazome-
thane, resp. diazoethane in quantitative yields. Esters
Scheme 2. Reagents and conditions for betulin derivatives: (i) phthalic
anhydride/DMAP/Py; (ii) CH2N2 (resp. CH3CHN2)/Et2O/CHCl3.
Pht = hemiphthaloyl,
phthaloyl.
MePht = methyl-phthaloyl,
EtPht = ethyl-
1b and 1c were prepared from esters 4b and 4c by trans-
fer-hydrogenolysis14 with 1,4-cyclo-hexadiene on Pd/C,
as described in Scheme 1, also in good yields.
3. Results and discussion
Our preliminary investigation showed that betulinic acid
(1) and betulin (5) derivatives are potential lead com-
pounds for new anti-tumor agents. Majority of hemiph-
thalates showed increased cytotoxic activity judged
against starting compounds. The lowest effect of hemi-
phthalates was found in the case of bishemiphthalate
5a, since activity of those substances increased only
slightly compared with starting betulin (5). On the other
side, derivative 6a was substantially better than the par-
ent compound. Based on our results we can summarize
that derivatization of hydroxy groups of triterpenes by
phthalic anhydride increases cytotoxic activity. Esterifi-
cation of hemiphthalates using diazomethane (resp.
diazoethane) did not lead to cytotoxic derivatives, since
nearly all methyl- and ethyl-phthalates were found quite
inactive (IC50 > 250 lmol/L). There were only two
exceptions from this rule. Methyl- 1b and ethyl-phtha-
lates 1c of betulinic acid (1) showed significant biological
activity. Compound 1b was also the most active deriva-
tive prepared in this work. It was also found that active
compounds showed cytotoxic activity against broader
spectrum of tumors from different histogenetic origin,
for example, mesenchymal (CEM, K562, K562-tax), epi-
thelial (HT29, PC-3), and neuroektodermal (SK-MEL2)
tumors. Interestingly, introduction of methyl-phthalate
1b into the structure of betulinic acid (1) conferred drug
resistance, since this compound showed much lower
Scheme 1. Reagents and conditions for betulinic acid derivatives: (i)
phthalic anhydride/DMAP/Py; (ii) CH2N2 (resp. CH3CHN2)/Et2O/
CHCl3; (iii) 1,4-cyclo-hexadiene/Pd–C/THF/EtOH.

M. Kvasnica et al. / Bioorg. Med. Chem. 13 (2005) 3447–3454
3449
Table 1. Cytotoxic activity of compounds 1–7 and 1a,b,c–7a,b,c against CEM, K562, HT 29, PC-3, and SK MEL2 cells
Compound
IC50 (lmol/La)
CEM
K562
K562-tax
HT29
PC-3
SK MEL2
23.2 ( 1.5)
1
27.5 ( 2.7)
54.4 ( 29.4)
5.7 ( 3.0)
54.8 ( 5.6)
29.4 ( 4.8)
8.8 ( 1.5)
108.2 ( 12.1)
26.8 ( 3.2)
84.5 ( 6.6)
29.9 ( 4.2)
7.5 ( 2.0)
94.1 ( 8.7)
32.5 ( 3.4)
18.9 ( 9.0)
98.5 ( 31.3)
1a
1b
1c
60.3 ( 17.4)
13.4 ( 4.9)
183.9 ( 43.8)
47.8 ( 19.1)
71.1 ( 28.5)
30.4 ( 18.9)
44.5 ( 12.0)
40.7 ( 10.3)
2
155.1 ( 17.1)
29.8 ( 5.2)
>250
68.5 ( 10.7)
30.7 ( 4.9)
>250
67.2 ( 9.9)
23.1 ( 4.6)
>250
65.5 ( 15.2)
18.1 ( 4.6)
>250
117.7 ( 11.9)
24.6 ( 7.8)
>250
186.3 ( 22.4)
42.1 ( 0.6)
>250
2a
2b
2c
>250
>250
>250
>250
>250
>250
3
132.4 ( 14.4)
13.0 ( 3.2)
>250
114.0 ( 20.2)
17.2 ( 6.8)
>250
122.6 ( 37.6)
8.8 ( 1.1)
>250
121.3 ( 28.1)
9.9 ( 1.8)
>250
>250
217.9 ( 51.7)
25.8 ( 10.3)
>250
3a
3b
3c
10.5 ( 1.0)
>250
>250
>250
>250
>250
>250
>250
4
242.4 ( 13.7)
10.0 ( 0.8)
>250
>250
191.7 ( 50.4)
6.2 ( 1.1)
>250
248.9 ( 2.0)
8.9 ( 1.3)
>250
>250
>250
4a
4b
4c
10.1 ( 1.1)
>250
>250
10.9 ( 1.3)
>250
>250
15.3 ( 3.7)
>250
>250
>250
>250
>250
5
>250
>250
>250
>250
>250
>250
5a
5b
5c
184.5 ( 18.9)
>250
>250
149.6 ( 8.5)
>250
>250
110.1 ( 35.2)
>250
>250
129.1 ( 18.4)
>250
>250
134.3 ( 10.4)
>250
>250
204.2 ( 24.5)
>250
>250
6
96.4 ( 36.4)
13.6 ( 5.8)
>250
147.4 ( 37.7)
18.1 ( 5.7)
>250
165.6 ( 38.3)
8.3 ( 4.6)
>250
154.3 ( 14.2)
13.6 ( 5.7)
>250
248.6 ( 3.5)
20.1 ( 8.7)
>250
247.0 ( 4.7)
24.9 ( 9.9)
>250
6a
6b
6c
>250
>250
>250
>250
>250
>250
7
110.1 ( 17.5)
42.4 ( 7.6)
>250
93.5 ( 31.1)
37.8 ( 2.2)
>250
72.4 ( 27.3)
34.2 ( 6.4)
247.3 ( 5.6)
>250
76.7 ( 18.6)
36.1 ( 2.7)
248.3 ( 4.3)
>250
150.1 ( 20.0)
35.2 ( 11.2)
>250
223.7 ( 23.7)
90.0 ( 46.7)
>250
7a
7b
7c
>250
>250
>250
>250
Value >250 lmol/L means that compound is not active.
a The lowest concentration that kills 50% of tumor cells.
activity on P-glycoprotein overexpressing cell line K562-
tax15 compared to sensitive K562 leukemia cells. The
lowest cytotoxicity of synthesized phthalates was ob-
served on human melanoma SK MEL2 cells as summa-
rized in Table 1.
phthalates. For this reason hemiphthalates of triterpe-
noids represent interesting class of compounds for further
studies and/or development.
5. Experimental
4. Conclusion
Melting points were determined using a Kofler block
and are uncorrected. Optical rotations were measured
using CHCl3 solutions (unless otherwise stated) on an
Autopol III (Rudolph Research, Flanders, NJ) polari-
meter, with an accuracy of 2ꢁ. NMR spectra were re-
corded on a Varian UNITY INOVA 400 instrument
(1H NMR spectra at 399.95 MHz) using CDCl3 solu-
tions (unless otherwise stated), with SiMe4 as an internal
standard. Skeletal signals in the region approx. 0–2 ppm
were not recorded, except methyl group. EIMS spectra
were recorded on an INCOS 50 (Finigan MAT) spec-
trometer at 70 eV and an ion source temperature of
150 ꢁC. The samples were introduced from a direct
exposure probe at a heating rate of 10 mA/s. Relative
abundances stated are related to the most abundant
ion in the region of m/z > 180. TLC was carried out
using silica gel 60 F254, detection was by spraying with
10% aq H2SO4 and heating to 150–200 ꢁC. Column
chromatography was performed using silica gel 60
(Merck 7734). Used HPLC system consisted of High
Within the worldwide research of betulinic acid (1) and
betulin (5) in the field of anti-tumor agents, number of
structural modifications, and derivatizations were stud-
ied. Our study demonstrates that derivatization of 3b-hy-
droxy group of these triterpenoids with lower diacids can
result in compounds with higher cytotoxic activity against
cell lines of different histogenetic origin, including drug
resistant tumors. Even derivatization of quite inactive
betulin (1) can lead to compounds with higher cytotoxi-
city as shown in case of 6a or 7a. Prepared hemiphthalates
are also highly polar compounds (stated according to the
solubility in polar solvents, e.g., alcohols), which is very
useful in MTT screening, where 10% DMSO in water is
used, or in in vivo screenings, where using of polar sol-
vents is necessary. Another advantage of phthalates is
also possible of using of many substituted phthalic anhy-
drides (e.g., by halogen, nitro group) for derivatization
reaction in order to modify the properties of prepared

3450
M. Kvasnica et al. / Bioorg. Med. Chem. 13 (2005) 3447–3454
Pressure Pump Gilson (model 361), Inject Valve Rheo-
dyne, Preparative Column (25 · 250 mm) with filling Si
tonitrile–water gave colorless needles of 3a (500 mg,
77%), mp 129–131 ꢁC; [a]D +26 (c 0.45). IR m (CHCl3)
cmꢀ1: 3618, 1713, 1640, 1600, 1580, 1290. 1H NMR
(400 MHz): d 0.84, 0.88, 0.92, 0.94, 0.97 (each 3H, s,
5 · CH3), 1.27 (3H, t, J = 7.2 Hz, CH3), 1.69 (3H, s,
CH3), 1.84–1.94 (3H, m), 2.18–2.26 (2H, m), 3.01 (1H,
td, J = 11.0, 4.7 Hz), 4.07–4.21 (2H, m), 4.61 (1H, m),
4.73–4.77 (2H, m), 7.54–7.61 (2H, m), 7.73 (1H, m),
7.90 (1H, m). MS m/z (rel. intensity): 632 (2, M+), 617
(1), 588 (1), 559 (2), 543 (1), 515 (1), 502 (3), 484 (4),
466 (31), 451 (13), 423 (6), 393 (8), 357 (4), 330 (3),
311 (3), 287 (2), 272 (22), 263 (14), 247 (10), 215 (14),
203 (32), 189 (100). Anal. Calcd for C40H56O6: C,
75.91; H, 8.92. Found: C, 75.80; H, 9.05.
gel (Biospher 7 lm), Differential-Refractometrical
´
´
ˇ
Detector (Laboratornı prıstroje, Praha, CZ) connected
with PC (software Chromulan) and Automatic Fraction
Collector Gilson (model 246). Mixture of ethyl acetate
and hexane was used as the mobile phase, its composi-
tion is in each experiment. TLC was carried out on Kie-
sel gel 60 F254 plates (Merck).
5.1. General procedure for 3-O-acyl derivatives of
hydroxy-compounds with phthalic anhydrides
Phthalic anhydride (4 equiv) and DMAP (4 equiv) was
added to a solution of hydroxy-compound (1 mmol) in
pyridine (10 mL). After being refluxed for 30 h, the reac-
tion mixture was diluted with ethyl acetate and washed
twice with aqueous solution of HCl and twice with
water. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The residue was
chromatographed with gradient on silica gel (hexane–
ethyl acetate, 9:1–5:1).
5.5. Hemiphthalate of benzyl-betulinate (4a)
Starting with compound 4 (700 mg, 1.28 mmol), chro-
matography and crystallization from the mixture of
acetonitrile–water gave colorless needles of 4a (702 mg,
79%), mp 195–197 ꢁC; [a]D +30 (c 0.37). IR m (CHCl3)
1
cmꢀ1: 3508, 1716 sh, 1641, 1600, 1580, 1290. H NMR
(400 MHz): d 0.76, 0.82, 0.87, 0.93, 0.95, 1.68 (each
3H, s, 6 · CH3), 1.81–1.93 (3H, m), 2.18 (1H, td,
J = 12.8, 3.7 Hz), 2.28 (1H, br d, J = 12.4 Hz), 3.02
(1H, td, J = 10.8, 4.6 Hz), 4.60 (1H, m), 4.72–4.76 (2H,
m), 5.09 (1H, d, J = 12.3 Hz), 5.15 (1H, d, J = 12.3 Hz),
7.30–7.38 (5H, m), 7.54–7.62 (2H, m), 7.73 (1H, m),
7.90 (1H, m). MS m/z (rel. intensity): 694 (2, M+), 679
(1), 666 (1), 603 (1), 575 (1), 559 (6), 546 (4), 528 (38),
514 (14), 485 (11), 455 (2), 437 (100), 423 (5), 409 (12),
395 (11), 377 (2), 338 (9), 325 (6), 301 (1), 279 (2), 257
(5), 247 (13), 233 (10), 215 (7), 203 (12), 189 (44). Anal.
Calcd for C45H58O6: C, 77.77; H, 8.41. Found: C, 77.72;
H, 8.52.
5.2. Hemiphthalate of betulinic acid (1a)
Starting with compound 1 (500 mg, 1.09 mmol), chro-
matography and crystallization from the mixture of
acetonitrile gave a white powder of 1a (490 mg, 74%),
mp 259–260 ꢁC; [a]D +17 (c 0.35, THF). IR m (CHCl3)
cmꢀ1: 3511, 1699 sh, 1641, 1600, 1580, 1291. H NMR
1
(400 MHz): d 0.88, 0.89, 0.95, 0.96, 1.00, 1.70, (each
3H, s, 6 · CH3), 1.91–2.00 (2H, m), 2.23–2.29 (2H, m),
3.02 (1H, td, J = 11.0, 4.6 Hz), 4.61 (1H, m), 4.69–4.74
(2H, m), 7.52–7.54 (2H, m), 7.68 (1H, m), 7.75 (1H,
m). MS m/z (rel. intensity): 604 (1, M+), 589 (1), 558
(1), 543 (1), 519 (1), 456 (11), 438 (36), 423 (15), 409
(6), 395 (22), 369 (6), 327 (5), 302 (5), 287 (4), 259
(10), 248 (25), 234 (11), 220 (12), 203 (29), 189 (100).
Anal. Calcd for C38H52O6: C, 75.46; H, 8.67. Found:
C, 75.70; H, 8.81.
5.6. Bis(hemiphthalate) of betulin (5a)
Starting with compound 5 (500 mg, 1.13 mmol), chro-
matography and crystallization from ethanol gave
a white powder of 5a (633 mg, 76%), mp 178–180 ꢁC;
[a]D +28 (c 0.55). IR m (CHCl3) cmꢀ1: 3509, 1716,
5.3. Hemiphthalate of methyl-betulinate (2a)
1
1641, 1600, 1581, 1291. H NMR (400 MHz): d 0.89,
Starting with compound 2 (500 mg, 1.06 mmol), chro-
matography and crystallization from the mixture of ace-
tonitrile–water gave colorless needles of 2a (505 mg,
77%), mp 223–225 ꢁC; [a]D +25 (c 0.42). IR m (CHCl3)
cmꢀ1: 3508, 1717, 1704, 1641, 1600, 1581, 1290. 1H
NMR (400 MHz): d 0.84, 0.88, 0.91, 0.94, 0.97, 1.69
(each 3H, s, 6 · CH3), 1.84–1.92 (2H, m), 2.16–2.25
(3H, m), 3.00 (1H, td, J = 10.6, 4.1 Hz), 3.67 (3H, s,
CH3), 4.61 (1H, m), 4.72–4.77 (2H, m), 7.54–7.61 (2H,
m), 7.72 (1H, m), 7.89 (1H, m). MS m/z (rel. intensity):
618 (5, M+), 603 (1), 558 (6), 543 (1), 515 (1), 470 (2), 452
(100), 437 (32), 409 (17), 393 (12), 377 (5), 355 (5), 316
(3), 283 (2), 273 (6), 262 (22), 257 (6), 248 (13), 233
(10), 215 (12), 203 (29), 189 (91). Anal. Calcd for
C39H54O6: C, 75.69; H, 8.80. Found: C, 75.62; H, 8.92.
0.90, 0.93, 1.02, 1.07, 1.70 (each 3H, s, 6 · CH3), 2.48
(1H, td, J = 11.0, 5.5 Hz), 4.08 (1H, d, J = 10.7 Hz),
4.56 (1H, d, J = 10.7 Hz), 4.60 (1H, m), 4.70 (1H, br
d, J = 2.3 Hz), 4.74 (1H, dd, J = 11.4, 4.7 Hz), 7.54–
7.63 (4H, m), 7.74 (1H, m), 7.77 (1H, m), 7.85–7.88
(2H, m). MS m/z (rel. intensity): 738 (1, M+), 590 (1),
572 (8), 559 (2), 529 (5), 513 (1), 485 (1), 457 (1), 438
(7), 424 (14), 411 (5), 393 (7), 381 (4), 313 (1), 299 (4),
288 (3), 271 (4), 257 (6), 245 (5), 234 (13), 216 (20),
203 (41), 189 (100). Anal. Calcd for C46H58O8: C,
74.77; H, 7.91. Found: C, 74.71; H, 7.98.
5.7. Hemiphthalate of 3b-O-acetyl-betulin (6a)
Starting with compound 6 (500 mg, 1.03 mmol), chro-
matography and crystallization from acetonitrile gave
a white powder of 6a (505 mg, 77%), mp 197–198 ꢁC;
[a]D +20 (c 0.50). IR m (CHCl3) cmꢀ1: 3512, 1720,
5.4. Hemiphthalate of ethyl-betulinate (3a)
1
Starting with compound 3 (500 mg, 1.03 mmol), chro-
matography and crystallization from the mixture of ace-
1641, 1600, 1581, 1291. H NMR (400 MHz): d 0.82
(6H, s, 2 · CH3), 0.83, 0.96, 1.05, 1.69, 2.05 (each 3H,

M. Kvasnica et al. / Bioorg. Med. Chem. 13 (2005) 3447–3454
3451
s, 5 · CH3), 2.50 (1H, td, J = 11.0, 5.6 Hz), 4.12 (1H, d,
J = 11.1 Hz), 4.46 (1H, dd, J = 10.0, 6.1 Hz), 4.51 (1H,
d, J = 11.1 Hz), 4.60 (1H, m), 4.70 (1H, br d,
J = 2.0 Hz), 7.55–7.64 (2H, m), 7.72 (1H, dd, J = 7.4,
1.7 Hz), 7.93 (1H, dd, J = 7.3, 1.5 Hz). MS m/z (rel.
intensity): 632 (1, M+), 617 (1), 589 (6), 572 (31), 557
(7), 546 (12), 514 (15), 466 (42), 406 (35), 381 (7),
313 (1), 299 (5), 288 (2), 271 (4), 257 (7), 245 (6),
234 (18), 216 (25), 203 (44), 189 (100). Anal. Calcd
for C40H56O6: C, 75.91; H, 8.92. Found: C, 75.87; H,
8.98.
3.00 (1H, td, J = 11.0, 4.3 Hz), 3.67 (3H, s, CH3),
4.32–4.40 (2H, m), 4.61 (1H, m), 4.72 (1H, dd,
J = 11.6, 4.6 Hz), 4.74 (1H, br s), 7.49–7.54 (2H, m),
7.67 (1H, m), 7.72 (1H, m). MS m/z (rel. intensity):
646 (6, M+), 631 (1), 586 (4), 466 (3), 452 (100), 437
(26), 409 (14), 393 (8), 377 (3), 316 (2), 283 (1), 273
(3), 262 (12), 257 (13), 248 (9), 233 (3), 215 (8), 203
(17), 189 (57). Anal. Calcd for C41H58O6: C, 76.12; H,
9.04. Found: C, 76.10; H, 9.11.
5.12. Methyl-phthalate of ethyl-betulinate (3b)
5.8. Hemiphthalate of 28-O-acetyl-betulin (7a)
HPLC (hexane–ethyl acetate; 6:1), mp 167–169 ꢁC
(methanol); [a]D +33 (c 0.36). IR m (CHCl3) cmꢀ1
:
1
Starting with compound 7 (500 mg, 1.03 mmol), chro-
matography and crystallization from the mixture aceto-
nitrile–water gave a white powder of 7a (530 mg,
80%), mp 192–194 ꢁC; [a]D +30 (c 0.44). IR
1715, 1639, 1600, 1580, 1289. H NMR (400 MHz): d
0.87, 0.88 (each 3H, s, 2 · CH3), 0.93 (6H, s, 2 · CH3),
0.98 (3H, s, CH3), 1.27 (3H, t, J = 7.2 Hz, CH3), 1.70
(3H, s, CH3), 1.82–1.94 (3H, m), 2.19–2.26 (2H, m),
3.02 (1H, td, J = 11.0, 4.8 Hz), 4.08–4.21 (2H, m), 4.61
(1H, m), 4.70–4.74 (2H, m), 7.50–7.54 (2H, m), 7.69
(1H, m), 7.72 (1H, m). MS m/z (rel. intensity): 646 (7,
M+), 631 (1), 618 (1), 573 (4), 466 (70), 451 (29), 438
(3), 423 (19), 409 (2), 393 (11), 371 (6), 330 (5), 309
(2), 287 (7), 276 (20), 263 (13), 215 (17), 203 (29), 189
(100). Anal. Calcd for C41H58O6: C, 76.12; H, 9.04.
Found: C, 76.00; H, 9.15.
m
(CHCl3) cmꢀ1: 3510, 1721, 1641, 1600, 1580, 1290. H
NMR (400 MHz): d 0.85, 0.88, 0.95, 0.99, 1.03, 1.69,
2.08 (each 3H, s, 7 · CH3), 2.48 (1H, td, J = 11.0,
6.0 Hz), 3.86 (1H, d, J = 11.3 Hz), 4.25 (1H, d,
J = 11.3 Hz), 4.60 (1H, m), 4.75 (1H, br d, J = 2.3 Hz),
4.46 (1H, dd, J = 11.4, 4.4 Hz), 7.54–7.61 (2H, m),
7.72 (1H, m), 7.89 (1H, m). MS m/z (rel. intensity):
632 (2, M+), 617 (1), 589 (4), 572 (24), 557 (9), 514
(11), 466 (57), 406 (32), 381 (4), 313 (1), 299 (4), 288
(1), 271 (6), 257 (5), 245 (5), 234 (15), 216 (23), 203
(44), 189 (100). Anal. Calcd for C40H56O6: C, 75.91;
H, 8.92. Found: C, 75.52; H, 9.05.
1
5.13. Ethyl-phthalate of ethyl-betulinate (3c)
HPLC (hexane–ethyl acetate; 6:1), mp 172–174 ꢁC (eth-
anol); [a]D +27 (c 0.41). IR m (CHCl3) cmꢀ1: 1715, 1639,
1
5.9. General procedure for esterification of hemiphthalate
1600, 1580, 1289. H NMR (400 MHz): d 0.87, 0.89,
0.93, 0.94, 0.98 (each 3H, s, 5 · CH3), 1.27 (3H, t,
J = 7.2 Hz, CH3), 1.36 (3H, t, J = 7.2 Hz, CH3), 1.70
(3H, s, CH3), 1.82–1.94 (3H, m), 2.19–2.26 (2H, m),
3.02 (1H, td, J = 11.0, 4.8 Hz), 4.08–4.22 (2H, m),
4.30–4.42 (2H, m), 4.61 (1H, m), 4.70–4.74 (2H, m),
7.49–7.54 (2H, m), 7.68 (1H, m), 7.72 (1H, m). MS
m/z (rel. intensity): 660 (7, M+), 645 (2), 632 (1), 587
(9), 572 (1), 466 (95), 451 (64), 438 (3), 423 (29), 410
(4), 393 (27), 385 (15), 330 (8), 309 (6), 287 (32), 276
(38), 263 (28), 229 (12), 215 (23), 203 (75), 189 (100).
Anal. Calcd for C42H60O6: C, 76.33; H, 9.15. Found:
C, 76.30; H, 9.22.
A solution of diazomethane (resp. diazoethane) in dieth-
ylether was added to a solution of hemiphthalate
(1 mmol) in chloroform (5 mL). After 30 min the
reaction mixture was evaporated and the residue was
chromatographed using preparative-scale HPLC (hex-
ane–ethyl acetate) to afford the product.
5.10. Methyl-phthalate of methyl-betulinate (2b)
HPLC (hexane–ethyl acetate; 9:1), mp 224–226 ꢁC
(methanol); [a]D +28 (c 0.41). IR m (CHCl3) cmꢀ1
:
1
1720, 1641, 1600, 1580, 1292. H NMR (400 MHz): d
0.87, 0.88, 0.93, 0.94, 0.98, 1.70 (each 3H, s, 6 · CH3),
2.17–2.25 (2H, m), 3.00 (1H, td, J = 11.0, 4.3 Hz),
3.67, 3.90 (each 3H, s, 3 · CH3), 4.61 (1H, m), 4.72
(1H, dd, J = 11.6, 4.6 Hz), 4.74 (1H, br s), 7.50–7.54
(2H, m), 7.68–7.73 (2H, m). MS m/z (rel. intensity):
632 (9, M+), 617 (1), 572 (4), 452 (100), 437 (25), 409
(14), 393 (8), 371 (5), 316 (3), 283 (1), 273 (3), 262
(16), 257 (2), 248 (9), 233 (3), 215 (7), 203 (17), 189
(61). Anal. Calcd for C40H56O6: C, 75.91; H, 8.92.
Found: C, 75.83; H, 9.01.
5.14. Methyl-phthalate of benzyl-betulinate (4b)
HPLC (hexane–ethyl acetate; 9:1), mp 110–112 ꢁC
(methanol–chloroform); [a]D +33 (c 0.38). IR m (CHCl3)
cmꢀ1
:
1720, 1640, 1600, 1580, 1292. 1H NMR
(400 MHz): d 0.77, 0.85, 0.88, 0.93, 0.95, 1.69 (each
3H, s, 6 · CH3), 1.82–1.94 (3H, m), 2.19 (1H, td,
J = 12.8, 3.6 Hz), 2.28 (1H, br d, J = 12.5 Hz), 3.02
(1H, td, J = 10.8, 4.4 Hz), 3.89 (3H, s, CH3), 4.60 (1H,
br s), 4.70–4.73 (2H, m), 5.09 (1H, d, J = 12.4 Hz),
5.15 (1H, d, J = 12.4 Hz), 7.30–7.38 (5H, m), 7.50–7.54
(2H, m), 7.68–7.73 (2H, m). MS m/z (rel. intensity):
708 (4, M+), 693 (1), 680 (1), 617 (2), 590 (1), 573 (5),
528 (55), 514 (28), 485 (9), 437 (100), 423 (4), 409 (7),
393 (8), 371 (7), 338 (8), 325 (5), 301 (1), 279 (2), 271
(11), 255 (3), 247 (10), 233 (9), 221 (3), 203 (25), 189
(40). Anal. Calcd for C46H60O6: C, 77.93; H, 8.53.
Found: C, 77.88; H, 8.55.
5.11. Ethyl-phthalate of methyl-betulinate (2c)
HPLC (hexane–ethyl acetate; 7:1), mp 211–213 ꢁC (eth-
anol); [a]D +27 (c 0.43). IR m (CHCl3) cmꢀ1: 1719, 1641,
1
1600, 1580, 1289. H NMR (400 MHz): d 0.87, 0.89,
0.93, 0.94, 0.98 (each 3H, s, 5 · CH3), 1.36 (3H, t,
J = 7.1, CH3), 1.70 (3H, s, CH3), 2.17–2.25 (2H, m),

3452
M. Kvasnica et al. / Bioorg. Med. Chem. 13 (2005) 3447–3454
5.15. Ethyl-phthalate of benzyl-betulinate (4c)
7.56 (2H, m), 7.70 (1H, m), 7.75 (1H, m). MS m/z (rel.
intensity): 646 (1, M+), 631 (1), 603 (5), 586 (35), 560
(10), 543 (15), 466 (40), 406 (35), 381 (10), 313 (1), 299
(3), 288 (1), 271 (4), 257 (6), 245 (6), 234 (20), 216
(25), 203 (48), 189 (100). Anal. Calcd for C41H58O6: C,
76.12; H, 9.04. Found: C, 76.07; H, 9.05.
HPLC (hexane–ethyl acetate; 9:1), mp 111–112 ꢁC (eth-
anol); [a]D +28 (c 0.39). IR m (CHCl3) cmꢀ1: 1720, 1640,
1
1600, 1580, 1293. H NMR (400 MHz): d 0.77, 0.85,
0.87, 0.93, 0.95 (each 3H, s, 5 · CH3), 1.35 (3H, t,
J = 7.2 Hz, CH3), 1.69 (3H, s, CH3), 1.82–1.94 (3H,
m), 2.19 (1H, td, J = 12.8, 3.6 Hz), 2.28 (1H, br d,
J = 12.4 Hz), 3.02 (1H, td, J = 10.8, 4.4 Hz), 4.32–4.40
(2H, m), 4.60 (1H, br s), 4.70–4.73 (2H, m), 5.09 (1H,
d, J = 12.4 Hz), 5.15 (1H, d, J = 12.4 Hz), 7.30–7.38
(5H, m), 7.49–7.54 (2H, m), 7.68 (1H, m), 7.72 (1H,
m). MS m/z (rel. intensity): 722 (5, M+), 707 (1), 679
(1), 631 (4), 604 (1), 587 (6), 528 (62), 514 (25), 485
(10), 437 (100), 423 (4), 409 (6), 393 (7), 371 (7), 338
(9), 325 (5), 301 (1), 279 (2), 271 (15), 255 (4), 247
(12), 233 (9), 221 (3), 203 (24), 189 (43). Anal. Calcd
for C47H62O6: C, 78.08; H, 8.64. Found: C, 77.92; H,
8.70.
5.19. Ethyl-phthalate of 3b-O-acetyl-betulin (6c)
HPLC (hexane–ethyl acetate; 9:1), mp 186–188 ꢁC, (eth-
anol–butanone); [a]D +20 (c 0.44). IR m (CHCl3) cmꢀ1
:
1720, 1640, 1600, 1580, 1292, 1265. 1H NMR
(400 MHz): d 0.84, 0.85, 0.86, 0.99, 1.07 (each 3H, s,
5 · CH3), 1.37 (3H, t, J = 7.2 Hz, CH3), 1.70, 2.05 (each
3H, s, 2 · CH3), 2.50 (1H, td, J = 11.1, 5.8 Hz), 4.11
(1H, d, J = 11.0 Hz), 4.37 (2H, q, J = 7.2 Hz); 4.45–
4.49 (2H, m), 4.60 (1H, m), 4.71 (1H, br d,
J = 2.3 Hz), 7.52–7.54 (2H, m), 7.70 (1H, m), 7.74 (1H,
m). MS m/z (rel. intensity): 660 (1, M+), 645 (1), 617
(5), 600 (32), 574 (12), 557 (12), 466 (47), 406 (33), 381
(8), 313 (1), 299 (3), 288 (1), 271 (3), 257 (7), 245 (7),
234 (20), 216 (25), 203 (50), 189 (100). Anal. Calcd for
C42H60O6: C, 76.33; H, 9.15. Found: C, 76.30; H, 9.15.
5.16. Bis(methyl-phthalate) of betulin (5b)
HPLC (hexane–ethyl acetate; 4:1), mp 122–124 ꢁC
(methanol); [a]D +30 (c 0.50). IR m (CHCl3) cmꢀ1
:
1
1725, 1640, 1600, 1580, 1291. H NMR (400 MHz): d
0.89 (6H, s, 2 · CH3), 0.94, 1.00, 1.08, 1.71 (each 3H, s,
4 · CH3), 2.03 (1H, m), 2.51 (1H, td, J = 11.0, 5.8 Hz),
3.90, 3.91 (each 3H, s, 2 · CH3), 4.11 (1H, d,
J = 11.2 Hz), 4.50 (1H, dd, J = 11.2, 1.2 Hz), 4.61 (1H,
m), 4.71–4.75 (2H, m), 7.50–7.56 (4H, m), 7.68–7.77
(4H, m). MS m/z (rel. intensity): 766 (1, M+), 586 (100),
571 (14), 543 (13), 530 (1), 518 (1), 504 (1), 450 (3), 423
(14), 406 (40), 391 (11), 383 (1), 363 (16), 337 (3), 323
(2), 295 (2), 270 (5), 255 (4), 241 (3), 229 (7), 216 (18),
203 (36), 189 (95). Anal. Calcd for C48H62O8: C, 75.16;
H, 8.15. Found: C, 75.17; H, 8.21.
5.20. Methyl-phthalate of 28-O-acetyl-betulin (7b)
HPLC (hexane–ethyl acetate; 9:1), mp 89–91 ꢁC (lyophi-
lization from tert-butanol); [a]D +27 (c 0.21). IR m
1
(CHCl3) cmꢀ1: 1720, 1640, 1600, 1580, 1290, 1263. H
NMR (400 MHz): d 0.88, 0.89, 0.94, 0.99, 1.04, 1.69,
2.07 (each 3H, s, 7 · CH3), 2.45 (1H, td, J = 11.4,
6.0 Hz), 3.86 (1H, d, J = 10.8 Hz), 3.90 (3H, s, CH3),
4.26 (1H, dd, J = 10.8, 1.2 Hz), 4.60 (1H, m), 4.70
(1H, br d, J = 2.4 Hz), 4.72 (1H, dd, J = 11.8, 5.2 Hz),
7.50–7.54 (2H, m), 7.67–7.74 (2H, m). MS m/z (rel.
intensity): 646 (1, M+), 631 (1), 603 (5), 586 (28), 571
(7), 528 (9), 466 (61), 406 (35), 381 (5), 313 (2), 299
(4), 288 (1), 271 (7), 257 (5), 245 (7), 234 (18), 216
(23), 203 (48), 189 (100). Anal. Calcd for C41H58O6: C,
76.12; H, 9.04. Found: C, 76.10; H, 9.13.
5.17. Bis(ethyl-phthalate) of betulin (5c)
HPLC (hexane–ethyl acetate; 4:1), mp 175–177 ꢁC (etha-
nol); [a]D +29 (c 0.43). IR m (CHCl3) cmꢀ1: 1725, 1640,
1
1600, 1580, 1291. H NMR (400 MHz): d 0.89, 0.90,
5.21. Ethyl-phthalate of 28-O-acetyl-betulin (7c)
0.95, 1.00, 1.08 (each 3H, s, 5 · CH3), 1.36, 1.38 (each
3H, t, J = 7.2 Hz, 2 · CH3), 1.71 (3H, s, CH3), 2.51
(1H, td, J = 11.1, 6.0 Hz), 4.11 (1H, d, J = 11.0 Hz),
4.32–4.41 (4H, m), 4.50 (1H, dd, J = 11.2, 1.2 Hz), 4.61
(1H, m), 4.71–4.75 (2H, m), 7.49–7.56 (4H, m), 7.67–
7.77 (4H, m). MS m/z (rel. intensity): 794 (1, M+), 601
(100), 586 (21), 558 (15), 545 (2), 533 (1), 519 (2), 465
(5), 438 (16), 406 (42), 391 (15), 383 (1), 363 (20), 337
(5), 323 (1), 295 (2), 270 (5), 255 (8), 241 (1), 229 (7),
216 (22), 203 (30), 189 (93). Anal. Calcd for C50H66O8:
C, 75.53; H, 8.37. Found: C, 75.28; H, 8.49.
HPLC (hexane–ethyl acetate; 9:1), mp 96–98 ꢁC (lyophi-
lization from tert-butanol); [a]D +28 (c 0.36). IR m
1
(CHCl3) cmꢀ1: 1720, 1640, 1600, 1580, 1290, 1263. H
NMR (400 MHz): d 0.88, 0.90, 0.94, 0.99, 1.05 (each
3H, s, 5 · CH3), 1.36 (3H, t, J = 7.2 Hz, CH3), 1.69,
2.07 (each 3H, s, 2 · CH3), 2.45 (1H, td, J = 11.4,
6.0 Hz), 3.86 (1H, d, J = 10.8 Hz), 4.26 (1H, dd,
J = 10.8, 1.2 Hz), 4.31–4.42 (2H, m), 4.60 (1H, m),
4.70 (1H, br d, J = 2.4 Hz), 4.72 (1H, dd, J = 11.6,
5.0 Hz), 7.49–7.55 (2H, m), 7.66–7.74 (2H, m). MS m/z
(rel. intensity): 660 (1, M+), 645 (1), 617 (3), 600 (33),
574 (13), 557 (12), 466 (51), 406 (29), 381 (10), 313 (1),
299 (2), 288 (1), 271 (2), 257 (6), 245 (6), 234 (20), 216
(22), 203 (48), 189 (100). Anal. Calcd for C42H60O6: C,
76.33; H, 9.15. Found: C, 76.28; H, 9.22.
5.18. Methyl-phthalate of 3b-O-acetyl-betulin (6b)
HPLC (hexane–ethyl acetate; 9:1), mp 182–184 ꢁC
(methanol–butanone); [a]D +21 (c 0.48). IR m (CHCl3)
cmꢀ1: 1724, 1640, 1600, 1580, 1292, 1260. 1H NMR
(400 MHz): d 0.84, 0.85, 0.86, 0.99, 1.07, 1.70, 2.05 (each
3H, s, 7 · CH3), 2.50 (1H, td, J = 10.8, 5.8 Hz), 3.90
(3H, s, CH3), 4.10 (1H, d, J = 11.0 Hz), 4.45–4.51 (2H,
m), 4.60 (1H, m), 4.71 (1H, br d, J = 2.0 Hz), 7.53–
5.22. Methyl-phthalate of betulinic acid (1b)
1,4-cyclo-Hexadiene (300 lL, 3.2 mol) and 10% palla-
dium on charcoal (300 mg) was added to a solution of

M. Kvasnica et al. / Bioorg. Med. Chem. 13 (2005) 3447–3454
3453
4b (300 mg, 0.42 mmol) in THF (20 mL) and anhydrous
EtOH (10 mL). After being stirred at 30 ꢁC for 10 h,
palladium was filtered off and solvents were evaporated.
The residue was crystallized from methanol to afford 1c
as a white powder (228 mg, 88%), mp 250–252 ꢁC;
[a]D +32 (c 0.41). IR m (CHCl3) cm1: 3508, 1710 sh,
a pre-incubation period of 24 h at 37 ꢁC and 5% CO2
for stabilization. Fourfold dilutions, in 20 lL aliquots,
of the intended test concentration were added at time
zero to the microtiter plate wells. All test compound
(dissolved in 10 lL of 10% DMSO) concentrations were
examined in duplicate. Incubation of the cells with the
test compounds lasted for 72 h at 37 ꢁC, in a 5% CO2
atmosphere at 100% using MTT. Aliquots (10 lL) of
the MTT stock humidity. At the end of the incubation
period, the cells were assayed solution were pipetted
into each well and incubated for a further 1–4 h.
After this incubation period formazan produced was
dissolved by the addition of 100 lL/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 = (ODdrug-exposed
well/mean ODcontrol wells) · 100%. The IC50 value,
the drug concentration lethal to 50% of the tumor
cells, was calculated from appropriate dose–response
curves.
1
1690, 1641, 1600, 1580, 1290. H NMR (400 MHz): d
0.88 (6H, s, 2 · CH3), 0.94, 0.95, 0.99, 1.71 (each 3H,
s, 4 · CH3), 1.85 (1H, m), 1.95–2.04 (2H, m), 2.19
(1H, td, J = 12.4, 5.8 Hz), 2.28 (1H, td, J = 12.4,
3.0 Hz), 3.02 (1H, td, J = 10.7, 4.5 Hz), 3.89 (3H, s,
CH3), 4.62 (1H, m), 4.71–4.76 (2H, m), 7.50–7.54
(2H, m), 7.67–7.74 (2H, m). MS m/z (rel. intensity):
618 (2, M+), 603 (1), 588 (1), 572 (1), 528 (1), 438
(58), 423 (22), 395 (37), 369 (4), 287 (1), 269 (2), 259
(4), 248 (5), 232 (4), 215 (2), 202 (9), 189 (31). Anal.
Calcd for C39H54O6: C, 75.69; H, 8.80. Found: C,
75.63; H, 8.90.
5.23. Ethyl-phthalate of betulinic acid (1c)
Compound 1c was prepared from 4c by a procedure
similar to that described for 1b. Crystallization from
ethanol gave a white powder of 1c (220 mg, 85%), mp
247–249 ꢁC; [a]D +25 (c 0.42). IR m (CHCl3) cm1: 3508,
1710 sh, 1691, 1641, 1600, 1580, 1289. 1H NMR
(400 MHz): d 0.88, 0.89, 0.94, 0.95, 0.99 (each 3H, s,
5 · CH3), 1.35 (3H, t, J = 7.2 Hz, CH3), 1.71 (3H, s,
CH3), 1.85 (1H, m), 1.95–2.04 (2H, m), 2.19 (1H, td,
J = 12.4, 5.8 Hz), 2.28 (1H, td, J = 12.4, 3.0 Hz), 3.02
(1H, td, J = 10.7, 4.5 Hz), 4.31–4.42 (2H, m), 4.62 (1H,
m), 4.71–4.76 (2H, m), 7.49–7.54 (2H, m), 7.66–7.74
(2H, m). MS m/z (rel. intensity): 632 (4, M+), 617 (1),
586 (3), 528 (1), 466 (2), 438 (53), 423 (16), 395 (34),
369 (2), 287 (1), 259 (4), 248 (5), 232 (4), 217 (2), 202
(9), 189 (31). Anal. Calcd for C40H56O6: C, 75.91; H,
8.92. Found: C, 75.85; H, 8.94.
Acknowledgements
This study was supported in part by the Ministry of
Education of the Czech Republic (MSM 113100001,
151100001), which paid for instrumental equipment,
by the Czech Science Foundation (203/03/D152), from
which the chemicals were paid and by MPO project
(FT-TA/027) from which HPLC column, silica gel and
all other material support were paid. Biological testing
was supported by the Czech Science Foundation (301/
03/1570). We are grateful to Iva Tislerova for measure-
ment of NMR spectra. Special thanks to Bohunka Sper-
lichova for measurement of optical rotatory power.
6. Materials and methods
6.1. Chemicals
References and notes
1. Pisha, E.; Chai, H.; Lee, I. S.; Chagwedera, T. E.;
Farnsworth, N. R.; Cordell, G. A.; Beecher, C. W. W.;
Fong, H. H. S.; Kinghorn, A. D.; Brown, D. M.; Want,
M. C.; Wall, M. E.; Hieken, T. J.; Gupta, T. K. D.;
Pezzuto, J. M. Nat. Med. 1995, 1, 1046–1051.
2. Cichewicz, R. H.; Kouzi, S. A. Med. Res. Rev. 2004, 24,
90–114.
3. Zuco, V.; Supino, R.; Righetti, S. C.; Cleris, L.; Marchesi,
E.; Gambacorti-Passerini, C.; Formelli, F. Cancer Lett.
2002, 175, 17–25.
4. Kim, D. S. H. L.; Pezzuto, J. M.; Pisha, E. Bioorg. Med.
Chem. Lett. 1998, 8, 1707–1712.
5. Urban, M.; Sarek, J.; Klinot, J.; Korinkova, G.; Hajduch,
M. J. Nat. Prod. 2004, 67, 1100–1105.
6. Mukherjee, R.; Jaggi, M.; Rajedran, P.; Siddiqui, M. J. A.;
Srivastara, S. K.; Vardhan, A.; Burman, A. C. Bioorg.
Med. Chem. Lett. 2004, 14, 2181–2184.
Phthalic anhydride, DMAP, MTT (3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyl tetrazolium bromide) and SDS
were purchased from Sigma–Aldrich, s.r.o., Czech
Republic.
6.2. Cell lines
Cell lines CEM, HT29, K562, PC-3, and SK MEL2 were
purchased from the American Tissue Culture Collection
(ATTC). Paclitaxel-resistant subline of K562 cells
(K562-tax) was prepared and characterized in our
laboratories.15
6.3. MTT cytotoxicity assay
7. Fulda, S.; Jeremias, I.; Debatin, K. M. Oncogene 2004, 23,
7611–7620.
8. Hajduch, M.; Sarek, J.: Triterpenoid derivatives. PCT Int.
Patent Appl. WO0190046, 23 May 2001.
9. Kim, J. Y.; Koo, H. M.; Kim, D. S. H. L. Bioorg. Med.
Chem. Lett. 2001, 11, 2405–2408.
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

3454
M. Kvasnica et al. / Bioorg. Med. Chem. 13 (2005) 3447–3454
10. Hashimoto, F.; Kashiwada, Y.; Cosentino, L. M.; Chen,
C.-H.; Garrett, P. E.; Lee, K.-H. Bioorg. Med. Chem.
1997, 5, 2133–2143.
13. Tietze, L. F.; Heinzen, H.; Moyna, P.; Rischer, M.;
Neunaber, H. Liebigs Ann. Chem. 1991, 1245–
1249.
14. Felix, A. M.; Heimer, E. P.; Lambros, T. J.; Tzougraki,
C.; Meienhofer, J. J. Org. Chem. 1978, 43, 4194–
4196.
15. Noskova, V.; Dzubak, P.; Kuzmina, G.; Ludkova, A.;
Stehlik, D.; Trojanec, R.; Janostakova, A.; Korinkova,
G.; Mihal, V.; Hajduch, M. Neoplasma 2002, 49, 418–
425.
ˇ
´
ˇ
´
´
11. Sarek, J.; Klinot, J.; Brazˇinova, S.; Dzubak, P.; Klinotova,
E.; Noskova, V.; Krecek, V.; Korınkova, G.; Thomson, J.
´
ˇ ˇ ˇ´
´
ˇ ´
´
O.; JanostÕakova, A.; Wang, S.; Parsons, S.; Fischer, P.
M.; Zhelev, N. Z.; Hajdu´ch, M. J. Med. Chem. 2003, 46,
5402–5415.
12. Li, T.-S.; Wang, J.-X.; Zheng, X.-J. J. Chem. Soc., Perkin
Trans. 1 1998, 3957–3965.