Substituted dienes prepared from betulinic acid – Synthesis, cytotoxicity, mechanism of action, and pharmacological parameters

Article abstract of DOI:10.1016/j.ejmech.2021.113706

A set of new substituted dienes were synthesized from betulinic acid by its oxidation to 30-oxobetulinic acid followed by the Wittig reaction. Cytotoxicity of all compounds was tested in vitro in eight cancer cell lines and two noncancer fibroblasts. Almost all dienes were more cytotoxic than betulinic acid. Compounds 4.22, 4.30, 4.33, 4.39 had IC₅₀ below 5 μmol/L; 4.22 and 4.39 were selected for studies of the mechanism of action. Cell cycle analysis revealed an increase in the number of apoptotic cells at 5 × IC₅₀ concentration, where activation of irreversible changes leading to cell death can be expected. Both 4.22 and 4.39 led to the accumulation of cells in the G0/G1 phase with partial inhibition of DNA/RNA synthesis at 1 × IC₅₀ and almost complete inhibition at 5 × IC₅₀. Interestingly, compound 4.39 at 5 × IC₅₀ caused the accumulation of cells in the S phase. Higher concentrations of tested drugs probably inhibit more off-targets than lower concentrations. Mechanisms disrupting cellular metabolism can induce the accumulation of cells in the S phase. Both compounds 4.22 and 4.39 trigger selective apoptosis in cancer cells via intrinsic pathway, which we have demonstrated by changes in the expression of the crucial apoptosis-related protein. Pharmacological parameters of derivative 4.22 were superior to 4.39, therefore 4.22 was the finally selected candidate for the development of anticancer drug.

Full text of DOI:10.1016/j.ejmech.2021.113706

European Journal of Medicinal Chemistry 224 (2021) 113706
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Substituted dienes prepared from betulinic acid e Synthesis,
cytotoxicity, mechanism of action, and pharmacological parameters
a
b
ꢀ a
ꢁ
ꢀ b
ꢁ
ꢁ
ꢀ b
Jan Pokorný , Denisa Olejníkova , Ivo Frydrych , Barbora Liskova , Sona Gurska ,
a
b
b
ꢀ a
ꢀ b
ꢀ
ꢀ
ꢁ
Sandra Benicka , Jan Sarek , Jana Kotulova , Marian Hajdúch , Petr Dzubak ,
Milan Urban b
,
*
a Department of Organic Chemistry, Faculty of Science, Palacký University Olomouc, 17. Listopadu 1192/12, 771 46, Olomouc, Czech Republic
b
ꢁ
ꢀ
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hnevotínska 1333/5, 779 00, Olomouc,
Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of new substituted dienes were synthesized from betulinic acid by its oxidation to 30-oxobetulinic
acid followed by the Wittig reaction. Cytotoxicity of all compounds was tested in vitro in eight cancer cell
lines and two noncancer fibroblasts. Almost all dienes were more cytotoxic than betulinic acid. Com-
Received 7 June 2021
Received in revised form
12 July 2021
Accepted 13 July 2021
Available online 15 July 2021
pounds 4.22, 4.30, 4.33, 4.39 had IC50 below 5 mmol/L; 4.22 and 4.39 were selected for studies of the
mechanism of action. Cell cycle analysis revealed an increase in the number of apoptotic cells at 5 ꢀ IC50
concentration, where activation of irreversible changes leading to cell death can be expected. Both 4.22
and 4.39 led to the accumulation of cells in the G0/G1 phase with partial inhibition of DNA/RNA synthesis
at 1 ꢀ IC50 and almost complete inhibition at 5 ꢀ IC50. Interestingly, compound 4.39 at 5 ꢀ IC50 caused
the accumulation of cells in the S phase. Higher concentrations of tested drugs probably inhibit more off-
targets than lower concentrations. Mechanisms disrupting cellular metabolism can induce the accu-
mulation of cells in the S phase. Both compounds 4.22 and 4.39 trigger selective apoptosis in cancer cells
via intrinsic pathway, which we have demonstrated by changes in the expression of the crucial
apoptosis-related protein. Pharmacological parameters of derivative 4.22 were superior to 4.39, therefore
4.22 was the finally selected candidate for the development of anticancer drug.
Keywords:
Triterpene
Betulinic acid
Wittig reaction
Cytotoxicity
Cell cycle
Mechanism of action
Apoptosis
Selectivity
Cancer
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
The main drawback of compound 2 is the low selectivity, which is
certainly caused by the presence of an electron-deficient double
Pentacyclic triterpenes are natural compounds with a broad
spectrum of biological activities [1e7]. Among them, betulinic acid
(1) is one of the molecules disposing of cytotoxic, antiviral (espe-
cially anti-HIV), or antiprotozoal activity [8e17], although its IC50 is
not sufficiently low for the treatment. Many scientists have been
focused on the research of derivatives of acid 1 with much better
cytotoxic activity that are promising candidates for the develop-
ment of anticancer drugs [18e25]. Similarly, derivatives of betulinic
acid (1) are often being used to investigate its mechanism of action
[26e30] or to improve its solubility and bioavailability [31e34].
One part of our research on cytotoxic derivatives of acid 1 has been
focused on the compounds modified in their position C-30 (Fig. 1).
Oxidation of acid 1 with SeO2 gives 30-oxobetulinic acid 2, which
has significant cytotoxic activity in multiple cancer cell lines [35].
bond 20(29) (Michaels acceptor), which binds non-specifically to
proteins by forming covalent bonds. One of the proposed solutions
to diminish this drawback was to replace the oxo-group with an
isosteric moiety to weaken the Michaels acceptor potency. In 2018,
we published a study about a new set of triterpenoid azines in
which the oxygen was replaced with a nitrogen-containing azine
group (Fig. 1, general formula 3), and the set showed a significant
improvement in selectivity [18]. Azines 3 were cytotoxic against the
CCRF-CEM lymphoblastic leukemia cell line (IC50 around 3 mmol/L),
while their cytotoxicity in other cancer cell lines and noncancer
fibroblasts was in the high micromolar range or below the detec-
tion limit. Despite this significant improvement of the therapeutic
index, the azines 3 were less appropriate for potential drug
development because of insufficient stability in an aqueous envi-
ronment [18]. Besides, some of the synthetic procedures gave low
yields when side-products formed instead of the quantitative
amounts of the desired products. Here we decided on another
* Corresponding author.
E-mail address: milan.urban@upol.cz (M. Urban).
https://doi.org/10.1016/j.ejmech.2021.113706
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

ꢀ
J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
Fig. 1. Isosteric replacements and the context of this work with previous research [18,35].
isosteric replacement of the active moiety: C]O or C]N group for
C]C moiety (Fig. 1). This should yield stable compounds that
interact with similar molecular targets as active compounds from
Refs. [18,35] but they should not bind covalently. We used the same
starting compound, 30-oxobetulinic acid (2), and modified it in the
position 30 by the Wittig reaction. We have prepared 49 triphe-
nylphosphonium salts and optimized their use in the Wittig reac-
tion with the aldehyde 2. All new compounds were subsequently
tested for cytotoxic activity, and the best of them underwent the
search for the mechanism of action.
The selection of the substituents was partly motivated by the
most cytotoxic azines from our earlier work [18] but the main
intention here was to explore their variability. Our main focus was
oriented towards variously substituted benzenes (in o, m, and p
positions or more substituted), heterocycles are represented by
pyridines, alkanes by methyl- and cyclohexyl-. This was expected to
show us the differences in both the chemical reactivity of their
phosphonium salts and the SAR in biological activity.
improving the solubility of the starting aldehyde 2 and the product.
During the optimization, we found out that the reaction tempera-
ture plays an essential role in the reaction stereochemistry
outcome. When the Wittig reaction of 2 was performed at 25 ꢁC,
the typical outcome was a formation of both E and Z isomers in
various ratios, depending on the nature of the ylide used. For our
selection of substituted benzyl-type ylides, we proposed a starting
reaction temperature at 0 ꢁC when the formation of the E isomers
was predominant, and the procedures gave high yields of this iso-
mer. For most of the derivatives, the reaction temperature of 0 ꢁC
during the addition of aldehyde into the ylide was an essential
requirement for the effective reduction of the amount of the Z
isomer originating in the reaction mixture. There were some ex-
ceptions to this. The temperature of 0 ꢁC was insufficient to com-
plete the reaction of o-nitro and p-nitro derivatives (4.18, 4.39); the
starting material remained mostly unreacted. As a result, increasing
the reaction temperature to 70 ꢁC helped to finish the reaction for
the p-substituted derivative 4.39. However, it was still insufficient
for the o-derivative in which the conversion reached only about
40% (visualized by the TLC). Surprisingly, the reaction of both o- and
p-nitro derivatives at 70 ꢁC yielded almost pure E isomer, unlike the
majority of the other ylides. In contrast to the o- and p-nitro
products, the m-nitro isomer (4.27) reacted smoothly at 0 ꢁC
yielding the pure E isomer. Another exception was observed in the
case of m-COOH derivative (4.29), in which we were not successful
to prepare only one isomer selectively. Both isomers always formed
in a ratio of approximately 2 : 1 (E/Z), and the mixture was insep-
arable using the standard column chromatography. The same
behavior was observed for the derivative 4.2 bearing cyclohexyl
group. Both isomers were always found in a ratio of approximately
2.5 : 1 (E/Z) as an inseparable mixture.
The stability of the final products is essential for biological
evaluation. Since double bonds may undergo isomerization, and
since we had a negative experience with previously synthesized
azines [18], we paid special attention to this potential problem. We
observed that most of the products were stable under standard
conditions, in solutions in organic and aqueous solvents, and at
room temperature for several weeks; however, there were a few
exceptions. Dimethoxy derivative 4.50 (Scheme 1) has always
decomposed during column chromatography or in solution after
several days at r. t., while the derivative 4.49 seems to be reasonably
stable. The next unstable derivative 4.10 was prepared with a
longer conjugated chain in a mixture of several isomers. We have
also tried to prepare the amino derivative 4.45. We proved the final
product mass by HRMS in a mixture, but we were not able to purify
it enough for characterization by NMR, probably due to its insta-
bility in silica gel. Similar behavior was partly observed in the case
of the derivative 4.29 during longer column chromatography. Tri-
phenylphosphonium salts bearing OH- groups on the benzene ring
2. Results and discussion
2.1. Chemistry
The starting material of the synthesis was betulinic acid (1),
which was oxidized in the position C-30 by selenium dioxide in 2-
methoxyethanol [35] to form the aldehyde (2, Scheme 1). In the
next step, a wide range of triphenylphosphonium salts was pre-
pared (or purchased for R ¼ 1 and 7, see Scheme 1) by a reaction of
the corresponding bromide or iodide with triphenylphosphine in
refluxing toluene or acetonitrile. This reaction was usually quanti-
tative and did not require special conditions. The last step in the
reaction route, the Wittig reaction, was performed under an argon
atmosphere using dry tetrahydrofuran (THF), potassium t-butoxide
(t-BuOK), vacuum line, and Schlenk flask to avoid degradation of
the ylide. Formation of the ylide in the presence of the base was
always characterized by the change of color (a palette from yellow
to deep violet), while the completion of the reaction with the
aldehyde 2 was usually accompanied by decolorization of the re-
action mixture in 2e18 h (overnight). The optimum amount of the
triphenylphosphonium salt was 3.0 equivalents accompanied by
4.5 equivalents of t-BuOK, which yielded almost 100% conversion of
the aldehyde 2 to the product (according to the TLC). There were
only two exceptions to this procedure; the eCOOH substituted
derivatives (4.29, 4.41) required a double amount of the base to
complete the reaction.
According to the literature [36], we started an optimization at
higher temperatures using toluene as the solvent. After several
modifications of the procedure, including attempts with other
solvents, we have found THF to be the best choice for the reaction
2

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J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
Scheme 1. The general reaction scheme for the preparation of the conjugates 4.1e4.51. Reagents and conditions: a) SeO2, 2-methoxyethanol, reflux, 3 h; b) PPh3, toluene or
acetonitrile, reflux, overnight; X ¼ Br, I (for R ¼ 5, 6) or Cl (for R ¼ 7); c) t-BuOK, THF, 30 min; subsequently 0 ꢁC, terpene 2, then r. t., 2e18 h *Yields for compounds 4.2 and 4.29 are
given for a mixture of both E/Z isomers. **Compounds were either not prepared or they were unstable, see the chemistry section (last paragraph).
(final structures 4.31, 4.44, 4.51) were unfortunately unreactive;
this class of salts cannot form reactive ylide form probably due to a
resonance deactivation after deprotection of the OH- group in the
presence of the base. We have tried some stabilized (4.7, 4.8) and
nonstabilized ylides (4.5, 4.6, 4.9). In these few cases, we were not
successful in preparing the desired product, or we obtained a
3

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J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
mixture of inseparable isomers in very low yields. The total number
of fully characterized new dienes (pure E-isomers) was 40.
2.2.3. Evaluation of cell death by annexin V/propidium iodide
double labeling
For further investigation of the predominant mode of cell death
induced by the most active compounds 4.22 and 4.39, we have used
dual Annexin V/propidium iodide labeling with subsequent flow
cytometry analysis. Based on the results, both compounds induced
a significant, concentration-dependent increase in the number of
apoptotic cells compared to the untreated control (Fig. 3).
Following individual quadrant analyses, the early (right lower
quadrant) together with late (right upper quadrant) apoptotic ef-
fects of compounds 4.22 and 4.39 for 1 ꢀ IC50 doses on the CCRF-
CEM cell line were determined as 6.7% and 7.3%, respectively. For
5 ꢀ IC50 doses were evaluated apoptotic population 10.0% and
27.2%. Compound 4.39 at 5 ꢀ IC50 concentration proved the most
considerable cytotoxic effect leading to a more than nine-fold in-
crease of apoptotic cells number compared to the control. The total
number of apoptotic cells from the Annexin V/propidium iodide
assay nicely correlates with the sub-G1 apoptotic cell portion
resulting from cell cycle analysis (Table 2).
2.2. Biology
2.2.1. Cytotoxicity
All prepared compounds were tested in vitro for their cytotoxic
activity in eight cancer cell lines and two noncancer fibroblasts in
the same manner as in Ref. [21]. All compounds were stable in a
water-containing environment. The used cancer cell lines were
derived from T-lymphoblastic leukemia (CCRF-CEM), chronic my-
elogenous leukemia (K562), and their MDR counterparts (CEM-
DNR, K562-TAX) resistant against daunorubicin or paclitaxel, solid
tumors including lung (A549) and colon (HCT116, HCT116 p53ꢂ/ꢂ
)
carcinomas, osteosarcoma (U2OS), and for comparison, the syn-
thesized derivatives were tested on two human non-cancer fibro-
blast cell lines (BJ, MRC-5). CCRF-CEM cell line is the most
chemosensitive cell line on the panel, and therefore it was used for
the SARs assumptions similarly to Ref. [21]. Compounds 4.22, 4.30,
4.33, and 4.39 had high and selective cytotoxic activity (IC50 in the
2.2.4. Western blot
range of 3.2e4.9
m
mol/L) against the CCRF-CEM cell line. In
Based on the results from cell cycle analysis and Annexin V/
propidium iodide assay, we further concentrated on a more
detailed analysis of apoptosis-as well as cell cycle-associated pro-
teins expression. For that purpose, we used standard western blot
analysis of cell extracts prepared from CCRF-CEM cells treated with
4.22 and 4.39 at 1 ꢀ IC50 and 5 ꢀ IC50 concentrations for 24 h. It is
well-known that caspases activation represents the critical hall-
mark of apoptotic cell death. Thus, the expression of the main
apoptotic caspases 3, 8, and 9 was analyzed. We have found that
both compounds at 5 ꢀ IC50 concentration induced activation of
initiation caspase-9 and execution caspase-3, demonstrated by the
presence of caspase's active form (Fig. 4). On the other hand, no
activation of caspase-8 was observed. Such results strongly suggest
the involvement of the mitochondrial apoptotic pathway. Both
compounds at 5 ꢀ IC50 concentration also induced PARP cleavage,
which represents one of the downstream substrates of activated
caspase-3. We further examined the possible modulation effect of
active compounds on the expression of selected proteins from the
Bcl-2 family since they are essential regulators of mitochondria-
mediated apoptosis. As presented in Fig. 4, compound 4.39 at
5 ꢀ IC50 concentration induces downregulation of Bcl-2 protein
while BAX expression is slightly (20%) upregulated. Finally, we
observed upregulation of p21 and downregulation of Cyclin D1
following treatment with 4.22 and 4.39 at 5 ꢀ IC50 concentration as
additional evidence for their impact on cell cycle progression.
contrast, the other compounds had moderate to low activity in
most cancer cell lines and low activity in noncancer fibroblasts
(Table 1). All compounds were less active against the CEM-DNR
multiresistant cell line, which can indicate the involvement of
general mechanisms of resistance like the presence of MDR trans-
porters. Compounds 4.22 and 4.39 had the highest cytotoxic ac-
tivity and favorable selectivity index, and therefore they were
selected to evaluate their mechanism of action in more detailed
biological tests e analysis of cell cycle, synthesis of DNA and RNA,
and protein expression analysis in cells treated with these
compounds.
Importantly, dienes 4.22, 4.30, 4.33, and 4.39 are far more se-
lective than aldehyde 2, which makes them much more promising
candidates for drug development even though they are less active.
In comparison to azines 3 from the lit. [18], dienes 4.22, 4.30, 4.33,
and 4.39 have similar cytotoxicity and selectivity, but they are more
stable. In conclusion, the Wittig reaction afforded four candidates
(4.22, 4.30, 4.33, and 4.39) for the anticancer drug development
that are superior to our previously published derivatives.
2.2.2. Influence of compounds 4.22 and 4.39 on cell cycle and DNA/
RNA synthesis in CCRF-CEM cancer cells
To further understand the mechanism of action of the most
active compounds 4.22 and 4.39, their effect on the cell cycle and
DNA and RNA synthesis in cancer cells (CCRF-CEM) was studied
(Table 2, Fig. 2). It was found that after incubation with 1 ꢀ IC50
concentration, the cells were still viable, with only a slight increase
in the percentage of apoptotic cells with fragmented DNA. An in-
crease in the numbers of apoptotic cells was observed at a higher
5 ꢀ IC50 concentration, where we can already expect activation of
irreversible changes leading to cell death. Compound 4.22 led to the
accumulation of cells at the G0/G1 cell cycle phase with partial
inhibition of DNA and RNA synthesis at 1 ꢀ IC50 and almost com-
plete inhibition at 5 ꢀ IC50. Similarly, the cells’ accumulation in the
G0/G1 phase was observed after treatment with a 1 ꢀ IC50 con-
centration of compound 4.39. Interestingly, at 5 ꢀ IC50, the accu-
mulation of cells was observed in the S phase of the cell cycle. To
explain such a phenomenon, we can hypothesize that higher con-
centrations could inhibit more targets (off-targets) than lower and,
therefore, more specific cytotoxic concentrations of the tested
drugs. Thus, mechanisms disrupting cellular metabolism can lead
to the accumulation of cells in the synthetic phase of the cell cycle.
2.2.5. Pharmacological parameters
Two compounds with the highest cytotoxicity e 4.22 and 4.39 e
were selected to evaluate the fundamental pharmacokinetic pa-
rameters such as absorption, distribution, metabolism, and excretion
(ADME). Knowledge of the ADME properties of a new chemical en-
tity is critical during its evaluation to become a leading candidate in a
drug discovery program and/or its use as a functional research tool.
Both candidates 4.22 and 4.39 were incubated with human
plasma in vitro for fast determination if they are susceptible to
plasma degradation and to what extent. Compounds 4.22 and 4.39
slowly hydrolyzed in plasma (after incubation for 2 h, 70%e80% of
the original amount of each substance was remaining).
For the microsomal stability assay, human liver microsomes and
NADPH cofactor were used to assess phase I oxidation by cytochrome
P450 and flavin monooxygenases. The intrinsic clearance calculated
from the microsomal stability assay indicated a low or medium
category. This means that the studied compounds 4.22 and 4.39 were
not subject to rapid metabolism by liver microsome enzymes.
4

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J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
Table 1
Cytotoxic activities of the final compounds on eight tumor and two normal fibroblast cell lines.
IC50 (m
mol/L)a
CCRF-CEM
Comp.
CEM-DNR
HCT 116
HCT116 p53ꢂ/ꢂ
K562
K562-TAX
A549
U2OS
BJ
MRC-5
SIb
2c
0.8
11.1
8.0
3.8
1.9
1.9
3.4
1.3
>50
23.9
>50
41.8
32.9
26.8
29.7
29.4
28.6
29.7
>50
37.0
45.2
>50
36.5
28.7
29.2
33.1
38.7
>50
35.1
48.8
26.2
>50
40.1
>50
40.6
45.4
>50
>50
>50
41.4
14.3
46.0
>50
>50
>50
45.5
32.5
32.2
19.0
2.2
6.7
2.6
5.7
3.1
>6.3
>7.7
>3.9
1.7
2.4
2.0
2.4
>1.9
>2.2
>2.3
>2.1
>2.5
>4.9
3.1
>13.0
>5.7
>4.3
>4.1
>5.8
>3.8
>4.4
>2.5
>10.3
>8.5
8.5
4.1
25.1
45.7
30.2
47.0
22.8
23.2
42.1
49.1
>50
26.8
>50
30.6
27.7
21.4
23.1
25.5
35.9
>50
21.6
28.1
25.3
17.4
33.9
30.6
26.1
31.4
33.1
28.9
>50
27.0
40.9
22.9
20.5
32.0
>50
>50
27.8
>50
>50
13.7
24.1
15.2
35.5
30.2
29.7
24.5
26.6
27.2
29.6
36.6
33.0
34.8
>50
24.7
23.0
16.4
25.3
27.3
35.1
23.1
37.5
30.9
43.4
34.6
21.4
35.4
39.5
33.2
30.7
39.1
40.9
18.6
35.0
39.0
42.4
>50
21.8
33.5
32.2
17.3
24.3
>50
39.5
26.3
25.9
22.6
24.8
24.5
29.5
47.0
36.1
39.3
>50
22.3
21.0
15.2
26.8
29.5
29.4
24.8
35.5
32.8
43.1
39.6
18.2
41.0
38.5
31.8
33.3
43.3
31.9
15.7
30.6
42.5
48.9
>50
20.8
33.6
36.3
20.8
15.7
>50
40.0
>50
28.1
21.8
23.9
21.6
25.1
>50
48.7
>50
>50
24.9
21.6
27.7
23.6
25.2
>50
26.5
>50
>50
>50
41.4
>50
36.2
46.3
>50
>50
>50
40.5
11.5
43.4
>50
>50
>50
40.6
26.0
29.8
22.2
26.2
43.6
25.3
45.8
25.9
25.1
39.3
47.9
>50
24.8
>50
29.8
29.2
23.6
21.9
25.6
32.8
48.4
22.7
25.1
24.3
14.2
36.7
25.6
26.3
29.8
29.2
29.3
>50
27.2
45.6
23.4
20.6
39.2
48.6
>50
25.2
>50
>50
16.0
24.1
>50
24.2
27.2
28.0
27.7
28.1
27.8
28.5
40.4
29.6
33.1
>50
18.2
27.7
17.3
27.6
28.4
>50
24.6
29.3
30.9
>50
25.2
>50
23.9
25.8
27.0
32.2
44.1
28.2
19.5
29.7
49.0
48.6
45.1
26.5
29.7
30.2
22.3
44.6
>50
>50
>50
33.0
42.1
42.3
46.7
>50
>50
>50
>50
>50
>50
41.5
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
44.6
>50
>50
>50
>50
49.5
24.3
>50
>50
>50
>50
>50
43.0
>50
47.6
24.3
>50
>50
43.1
31.5
31.7
32.6
35.3
29.5
>50
>50
>50
>50
42.2
32.1
32.6
>50
>50
>50
42.9
45.1
>50
>50
44.1
>50
38.3
>50
35.2
41.2
49.7
42.4
19.2
>50
>50
>50
>50
32.4
33.7
43.2
29.2
4.2d
4.3
6.5
4.4
11.8
19.1
15.4
18.7
16.8
20.6
23.2
21.8
23.7
19.7
9.5
11.7
3.2
8.8
11.6
12.1
8.0
12.4
11.3
20.1
4.6
4.11
4.12
4.13
4.14
4.15
4.16
4.17
4.18
4.19
4.20
4.21
4.22
4.23
4.24
4.25
4.26
4.27
4.28
4.29d
4.30
4.32
4.33
4.34
4.35
4.36
4.37
4.38
4.39
4.40
4.41
4.42
4.43
4.46
4.47
4.48
4.49
5.9
4.9
5.4
6.5
16.0
23.8
21.5
3.6
>9.2
>6.6
>2.9
>2.1
2.1
6.1
5.9
>8.4
>2.9
>2.4
>1.1
>7.5
1.7
17.5
20.9
45.6
5.5
22.4
22.1
10.0
>2.1
3.8
a
The lowest concentration that kills 50% of cells. The maximum standard deviation in cytotoxicity assays is typically 15% of the average value. Compounds with
mol/L were considered inactive.
Selectivity index is calculated for IC50 of CCRF-CEM line vs. the average IC50 values of both fibroblasts (BJ and MRC-5).
Parent compound is used as a standard.
IC50 > 50
m
b
c
d
IC50 value was measured for a mixture of both E/Z isomers.
Table 2
Cell cycle analysis and influence on DNA and RNA synthesis in CCRF-CEM cancer cells treated with 1 ꢀ IC50 and 5 ꢀ IC50 of compounds 4.22 and 4.39.
Used conc. (
mmol/L)
Sub G1 (%)
G0/G1 (%)
S (%)
G2/M (%)
pH3Ser10 (%)
DNA synthesis (%)
RNA synthesis (%)
Control
4.22
0
2.77
5.66
9.13
6.54
25.04
40.20
42.87
49.79
48.40
29.57
38.17
42.75
37.93
28.44
57.61
21.62
14.39
12.28
23.16
12.81
1.72
1.50
1.52
2.90
0.93
41.56
35.56
0.40
43.17
3.06
36.54
18.97
11.37
34.98
4.07
1 ꢀ IC50
5 ꢀ IC50
1 ꢀ IC50
5 ꢀ IC50
3.19
15.95
3.57
17.85
4.39
The measurement of plasma protein binding was performed
using equilibrium dialysis, compounds 4.22 and 4.39 were bound
by 79% and 68%, respectively.
Both derivatives showed low ability (ꢂlog Papp > 6 cm/s) to
diffuse through an artificial cellular membrane in the Parallel
Artificial Membrane Permeability Assay (PAMPA).
intestinal [37] and blood-brain barriers, respectively [38]. Molecule
4.22 showed a moderate ability of intestinal absorption (Papp (AB) in
the range of 5 $ 10ꢂ6 cm/s e 20 $ 10ꢂ6 cm/s) and ability to cross
blood-brain barriers (Papp (AB) > 10 $ 10ꢂ6 cm/s; CNS positive), while
compound 4.39 manifested a low category of permeability in both
cell assays. We assessed rates of transport across MDCK-MDR1 and
Caco-2 monolayers in both directions (apical to basolateral [AeB],
and basolateral to apical [BeA]) across the cell monolayer, which
enables us to determine the efflux ratio and shows if the compound
undergoes active efflux. Studied derivatives 4.22 and 4.39 were
actively exported from the cells in both barrier models as indicated
Compound 4.22 shows
a small improvement in cellular
permeability (without category change) compared to compound
4.39 in our PAMPA model and permeability data from cell perme-
ability assay: Caco-2 and MDCK-MDR1 cell lines. The Caco-2 and
MDCK-MDR1 permeability assays are established models of
5

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J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
Fig. 2. Graphs and dot plots from flow cytometry analyses show the influence of the best compounds 4.22 and 4.39 on cell cycle and DNA/RNA synthesis in cancer cells monitored
by incorporation of BrdU/BrU into DNA/RNA.
by efflux ratios ER > 2. Still, the values of efflux ratio were very close
to the limit of active and passive efflux that the compounds cannot
be clearly identified as substrates of MDR1 efflux pump present in
both cell types. Results from all in vitro pharmacology testing are
summarized in Table 3.
6

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J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
Fig. 3. Representative dot plot diagrams of dual Annexin V/propidium iodide staining of CCRF-CEM cells treated with 4.22 and 4.39 at 1 ꢀ IC50 and 5 ꢀ IC50 concentrations for 24 h.
The fluorescence signal was measured at green (Annexin V) and red (propidium iodide) channels using FACSAria II flow cytometer. Debris identified by FSC/SSC dot plot was
excluded from the PI/FITC channels of the dot plot diagram. At least 10,000 cells were analyzed in each sample and analysis of the individual quadrant was performed. The portion
(%) of cell numbers in each quadrant is shown.
3. Conclusions
The main goal of this work was to modify betulinic acid (1) at
the position C-30 in order to find compounds with selective cyto-
toxic activity in cancer cell lines and reveal their mechanism of
action. From the past work, we knew that the oxidation of 1 gives
30-oxobetulinic acid 2 with high cytotoxicity, which unfortunately
is not selective and kills both cancer and healthy cells and therefore
is not useable in drug development [18,41]. The first attempt to
reach this goal was to mask the reactive Michaels acceptor of
compound 2 as azines and as expected, the selectivity improved
significantly, the activity was still reasonable, but compounds suf-
fered from low stability in the water environment [18]. In this work,
we replaced the reactive part of azines ¼ Nꢂ with isosteric ¼ CHꢂ
and obtained a set of 40 compounds of which 4.22, 4.30, 4.33, and
4.39 had cytotoxicity below 5
mmol/L and all of them had high
selectivity. The goal was achieved. The other compounds had
moderate activities but they provide important data points to our
long-term SAR studies based on which we may predict the cyto-
toxicity of proposed new structures in the future and decide
whether to synthesize new compounds or not. To better under-
stand the mechanism of action, we studied the cell cycle and DNA/
RNA synthesis in CCRF-CEM cells treated with 4.22 and 4.39 and
found that both compounds interfere with the cell cycle and induce
G1 cell cycle phase block, which may be associated with p21
overexpression and cyclin D1 downregulation [42]. Inhibition of
DNA and RNA synthesis was also observed. Similarly, apoptosis was
confirmed by analysis of caspase activation and cleavage of their
substrates. Interestingly, Bcl-2 expression decreased strongly in
contrast to the BAX level, which increased after 4.39 treatment at
Fig. 4. Western blot analysis of selected proteins involved in apoptosis and cell cycle
regulation. Cell extracts were prepared from CCRF-CEM cells treated with 4.22 and
4.39 at 1 ꢀ IC50 and 5 ꢀ IC50 for 24 h. As a protein loading control,
b-actin was used.
7

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J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
5 ꢀ IC50 concentration. Such changes could correspond to a
disruption of mitochondrial permeability and lead to the release
of cytochrome c prior to activation of apoptosis [43]. As part of
preclinical development, we tested in vitro pharmacological pa-
rameters. Good stability and favorable binding to plasma proteins
were observed for both compounds. In vitro models predicting the
permeability of potential drugs across the intestinal and blood-
brain barrier showed a favorable profile of compound 4.22. De-
rivative 4.39 has worse pharmacological parameters, it does not
cross the PAMPA artificial membrane, and the Caco-2 permeability
assay indicates poor intestinal absorption. Due to the high cyto-
toxic activity 3.2 mmol/L against chemosensitive CCRF-CEM cancer
cell line, favorable selectivity >12 and advantageous pharmaco-
logical parameters, derivative 4.22 is a promising candidate for
further preclinical development.
4. Materials and methods
4.1. Chemistry
HRMS analysis was performed using LC chromatograph Dionex
UltiMate 3000 (Thermo Fischer Scientific) and mass spectrometer
Exactive Plus Orbitrap high-resolution (Thermo Fischer Scientific).
Source of ionization: electrospray or APCI. Spectra: positive and
negative mode in the range of 400e700 m/z. Chromatographic
separation: column Phenomenex Gemini (C18, 50 ꢀ 2 mm, 3
mm
particle), isocratic elution, mobile phases: 80% ACN and 20% buffer
(0.01 mol/L ammonium acetate) or 95% MeOH þ 5% water þ 0.1%
HCOOH. Sample preparation: dissolution in MeOH. Spectra were
processed in the Thermo Scientific Xcalibur 4.1.31.9 software. All
1H and 13C NMR experiments were recorded at 500 MHz (Jeol
JNM-ECX-500) or 400 MHz (Jeol JNM-ECA400II) for 1H NMR, and
126 MHz or 100 MHz for 13C NMR, respectively, at 20 ꢁC in CDCl3,
CD3OD, or DMSO‑d6. Chemical shifts (
the residual solvent peak (for CDCl3 dH
d
) are reported relative to
7.26 ppm,
¼
dC ¼ 77.16 ppm; for CD3OD dH ¼ 3.31 ppm, dC ¼ 49.00 ppm; for
DMSO‑d6 dH ¼ 2.50 ppm, dC ¼ 39.52 ppm). NMR spectra were
processed using MestReNova 6.0.2e5475 and JEOL Delta 5.0.5.1
software. Melting points were determined using a Stuart Melting
Point Apparatus SMP30 and are uncorrected. Specific rotations
were measured in THF solutions at 22 ꢁC using an Atago POL-1/2
instrument, sample concentrations (c) are given in grams per
100 mL. IR spectra were recorded on a Nicolet Avatar 370 FTIR and
processed in the OMNIC 9.8.372 software. DRIFT stands for Diffuse
Reflectance Infrared Fourier Transform. TLC was carried out on
Kieselgel 60 F254 plates (Merck) detected first by UV light
(254 nm) and then by spraying with 10% aqueous H2SO4 and
heating to 150e200 ꢁC. Purification was performed using column
chromatography on Silica gel 60 (Merck 7734). Betulinic acid was
obtained from the company Betulinines (www.betulinines.com).
All other chemicals and solvents were obtained from Sigma-
Aldrich, Fluorochem, or Across Chemicals in analytical quality,
and were used as purchased.
4.1.1. Synthesis of 30-oxobetulinic acid (2)
10 g (21.9 mmol) of betulinic acid (1) was dissolved in 150 mL
of 2-methoxyethanol, and 6.1 g (54.8 mmol) of SeO2 was added.
The reaction was stirred under reflux for 3 h and monitored by
TLC. After completion, the hot reaction was immediately filtered
over a paper filter into 1 L icy water, where the product precipi-
tated as a yellow solid. The precipitate was then filtered using a
Büchner funnel, and the filter cake was dried at r. t. for 3 days. The
product was purified by flash column chromatography (toluene/
diethyl ether 1 : 1), affording 6.3 g (61%) of white solid. All spectral
and physical data agreed with the literature [35].
8

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J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
4.1.2. General procedure for the preparation of
bromomethylpyridines
1H, J ¼ 11.3 Hz), 0.94, 0.87, 0.86, 0.75, 0.65 (15H, all s, 5 ꢀ eCH3)
ppm; 13C NMR (126 MHz, DMSO‑d6)
d 177.16, 152.43, 139.20, 113.27,
2.8 mmol of the corresponding 2-, 3- or 4-(bromomethyl)pyri-
dine hydrobromide was dissolved in 10 mL of distilled water, then
15 mL of DCM was added, and the solution was placed to stir at 0 ꢁC
112.45, 76.74, 55.40, 54.85, 49.90, 49.75, 41.92, 40.23, 40.11, 38.46,
38.23, 37.62, 36.69, 36.09, 33.93, 32.41, 31.58, 29.28, 28.06, 27.12,
26.52, 20.56, 17.92, 15.91, 15.76, 15.74, 14.40 ppm; HRMS (ESIþ) m/z
calcd for C31H49O3 [MþH]þ 469.3676, found 469.3680; IR (DRIFT)
in an ice bath. While continuous stirring,
a solution of
3.0 mmol K2CO3 dissolved in 10 mL H2O was added dropwise. After
30 min, the reaction mixture was extracted with DCM, the organic
phases were combined, dried over anhydrous MgSO4, filtered, and
evaporated at r. t. The residue was immediately used in the
following procedure to prepare the corresponding triphenylphos-
phonium salt.
nmax 2390e3670, 2939, 2868, 1685, 1628, 1593, 1448 cmꢂ1
.
4.1.5.2. 30-(Cyclohexylmethylene)betulinic acid 4.2. The product 4.2
was obtained as a mixture of inseparable E and Z isomers in a ratio
approximately 2.5 : 1, white solid, 114 mg (65%) by the general
procedure (orange ylide color) while the reaction time was 2 h: m.
p. 174e177 ꢁC; ½a D22
ꢃ
þ36ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
4.1.3. General procedure for the preparation of
triphenylphosphonium salts
d
5.96 (d, J ¼ 15.8 Hz, 0.4H, H-30 of Z-isomer), 5.76e5.69 (m, 1.4H,
H-30 of E-isomer and H-31 of Z-isomer), 5.43e5.36 (m, 1H, H-31 of
E-isomer), 4.97 (d, J ¼ 2.2 Hz, 1H, H-29a of E-isomer), 4.84, 4.82
(2 ꢀ 0.4H, both s, 2 ꢀ H-29 of Z-isomer), 4.72 (s, 1H, H-29b of E-
2.1 mmol of triphenylphosphine was dissolved in 5 mL of
toluene or acetonitrile (for 4.2), and 2.0 mmol of the corresponding
bromide was added. The mixture was stirred overnight under
reflux until white crystals of the salt precipitated. The precipitate
was then filtered, washed with diethyl ether, and dried under a
vacuum to provide white crystalline salt in quantitative yields. For
4.2: The mixture was cooled down after reflux, the solvent was
almost evaporated, and the residue was precipitated by adding
diethyl ether to obtain white crystals, which were filtered and dried
under vacuum (56% yield). The salts were used without further
purification.
isomer), 3.23e3.16 (m, 1.4H, H-3
a of both isomers), 3.12e3.00 (m,
1.4H, H-19
b
of both isomers), 2.53e2.44 (m, 1H), 0.98 (s, 1.2H, CH3-
group of Z-isomer), 0.96 (s, 4.2H, CH3- group of both isomers), 0.93
(s, 3H, CH3- group of E-isomer), 0.93 (s, 4.2H, CH3- group of both
isomers), 0.82 (s, 3H, CH3- group of E-isomer), 0.82 (s, 1.2H, CH3-
group of Z-isomer), 0.75 (s, 4.2H, CH3- group of both isomers);
HRMS (ESIꢂ) m/z calcd for C37H57O3 [MꢂH]ꢂ 549.4302, found
549.4308; IR (DRIFT) nmax 3680, 3340, 2925, 2870, 2851, 2654,1685,
1449 cmꢂ1
.
4.1.4. General procedure for the preparation of
triphenylphosphonium salts bearing eOH group
4.1.5.3. 30-Benzylidenebetulinic acid 4.3. The product 4.3 was ob-
tained as white solid, 125 mg (72%) by the general procedure (or-
ange ylide color), while the reaction time was 18 h: m. p.
4.0 mmol of the corresponding hydroxybenzyl alcohol was
dissolved in 10 mL of acetonitrile, and 4.5 mmol of triphenyl-
phosphine hydrobromide was added. The mixture was stirred
overnight under reflux until white crystals of the salt precipitated.
The precipitate was then filtered, washed with diethyl ether, and
dried under a vacuum to provide white crystalline salt in quanti-
tative yield.
170e173 ꢁC; ½a 2D2
ꢃ
þ1ꢁ (c 0.29, THF); 1H NMR (400 MHz, CDCl3)
d
7.47e7.42 (m, 2H, arom. ring), 7.35e7.29 (m, 2H, arom. ring),
7.25e7.19 (m, 1H, arom. ring), 6.81 (d, J ¼ 16.2 Hz, 1H, H-31), 6.70 (d,
J ¼ 16.2 Hz, 1H, H-30), 5.12. 5.07 (2H, all s, 2 ꢀ H-29), 3.29 (td,
J1 ¼11.1, J2 ¼ 4.7 Hz, 1H, H-19 ), 3.20 (dd, J1 ¼11.1, J2 ¼ 4.9 Hz, 1H, H-
b
3
a
), 2.03 (dd, J1 ¼ 12.6, J2 ¼ 7.8 Hz, 1H), 1.92 (t, J ¼ 11.3 Hz, 1H), 1.02
(s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.81 (s, 3H), 0.75 (s, 3H) ppm; 13C
NMR (101 MHz, CDCl3) 182.32, 152.70, 137.69, 131.72, 128.70,
4.1.5. General procedure for the preparation of conjugates 4.1e4.51
0.96 mmol of the triphenylphosphonium salt was introduced in
a dry Schlenk flask on the vacuum line, followed by 165 mg
(1.43 mmol) of t-BuOK. After 30 min of drying under vacuum, 2 mL
of dry THF was added, and the reaction was stirred for 1 h to form
the ylide. Then, the flask was cooled down in an ice bath, and after
10 min, 150 mg (0.32 mmol) of the aldehyde (2) was added under
an argon atmosphere. The reaction was stirred at 0 ꢁC until the ice
bath melted and then at r. t. 2e18 h. Completion of the reaction was
monitored by TLC (toluene/diethyl ether 1 : 1) followed by
extraction (1% HCl/DCM). Organic phases were combined, dried
over MgSO4, and evaporated under reduced pressure. The residue
was purified by flash column chromatography (toluene/diethyl
ether 2 : 1), affording the final product in moderate to high yields.
Modification of the conditions is described in the respective
experiment if necessary.
d
128.15, 127.48, 126.63, 112.72, 79.21, 56.59, 55.47, 50.79, 50.63,
42.53, 40.85, 40.18, 38.97, 38.83, 38.67, 37.32, 37.07, 34.50, 33.26,
32.37, 29.98, 28.13, 27.40, 27.15, 21.17, 18.40, 16.25 (2C), 15.49,
14.91 ppm; HRMS (ESIþ) m/z calcd for C37H53O3 [MþH]þ 545.3989,
found 545.3988; IR (DRIFT) nmax 2450e3610, 2939, 2869, 1692,
1601, 1450 cmꢂ1
.
4.1.5.4. 30-(Naphth-3-ylmethylene)betulinic acid 4.4. The product
4.4 was obtained as white solid, 119 mg (63%) by the general pro-
cedure (red ylide color), while the reaction time was 2 h: m. p.
187e190 ꢁC; ½a 2D2
ꢃ
þ21ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
d
7.82e7.76 (m, 4H, arom. ring), 7.69e7.64 (m, 1H, arom. ring),
7.46e7.40 (m, 2H, arom. ring), 6.93 (d, J ¼ 16.2 Hz, 1H, H-31), 6.87
(d, J ¼ 16.2 Hz, 1H, H-30), 5.17, 5.11 (2H, all s, 2 ꢀ H-29), 3.35 (td,
J ¼ 11.1, 4.7 Hz, 1H, H-19
b
), 3.18 (dd, J ¼ 11.5, 4.6 Hz, 1H, H-3
a), 2.22
(td, J ¼ 12.3, 3.3 Hz, 1H), 2.05 (dd, J ¼ 12.8, 7.7 Hz, 1H), 1.95 (t,
4.1.5.1. 30-Methylenebetulinic acid 4.1. The product 4.1 was ob-
tained as white solid, 100 mg (67%) by the general procedure
(yellow ylide color) while the reaction time was 18 h: m. p.
J ¼ 11.4 Hz, 1H), 1.02 (s, 3H), 0.97 (s, 3H), 0.95 (s, 3H), 0.79 (s, 3H),
0.72 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
d 181.70, 152.82,
135.20, 133.88, 133.08, 132.13, 128.32, 128.27, 128.07, 127.79, 126.70,
126.38, 125.89, 123.75, 112.95, 79.17, 56.60, 55.49, 50.98, 50.65,
42.57, 40.89, 40.17, 38.99, 38.84, 38.76, 37.35, 37.10, 34.52, 33.39,
32.41, 30.00, 28.13, 27.50, 27.21, 21.21, 18.40, 16.29, 16.25, 15.45,
14.95 ppm; HRMS (ESIꢂ) m/z calcd for C41H53O3 [MꢂH]ꢂ 593.3989,
found 593.3994; IR (DRIFT) nmax 3629, 3450, 3186, 2931, 2867, 2612,
228e231 ꢁC; ½a 2D2
ꢃ
ꢂ5ꢁ (c 0.29, THF); 1H NMR (500 MHz, DMSO‑d6)
d
12.06 (s, 1H, eCOOH), 6.35 (dd, 1H, J1 ¼17.5 Hz, J2 ¼ 11.0 Hz, H-30),
5.25 (d, 1H, J ¼ 17.5 Hz, H-31 pro-E), 5.06 (d, 1H, J ¼ 10.9 Hz, H-31
pro-Z), 5.00, 4.98 (2H, all s, 2 ꢀ H-29), 4.24 (d, 1H, J ¼ 5.2 Hz, eOH),
3.07 (td, 1H, J1 ¼ 11.1 Hz, J2 ¼ 4.7 Hz, H-19
b), 3.01e2.92 (m, 1H, H-
3
a
), 2.02e1.95 (m, 1H), 1.80 (dd, 1H, J1 ¼12.3 Hz, J2 ¼ 7.9 Hz), 1.74 (t,
1948, 1686, 1598, 1450 cmꢂ1
.
9

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J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
4.1.5.5. 30-(2-Methylbenzylidene)betulinic acid 4.11. The product
4.11 was obtained as a white solid, 162 mg (91%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
J1 ¼11.2, J2 ¼ 4.8 Hz,1H, H-19 ), 3.18 (dd, J1 ¼11.4, J2 ¼ 4.8 Hz,1H, H-
b
3
a
), 2.37e2.32 (m, 1H), 2.22 (td, J1 ¼12.4, J2 ¼ 3.4 Hz, 1H), 2.03 (dd,
J1 ¼12.7, J2 ¼ 7.9 Hz, 1H), 1.95 (t, J ¼ 11.4 Hz, 1H), 1.01 (s, 3H), 0.96 (s,
3H), 0.95 (s, 3H), 0.81 (s, 3H), 0.75 (s, 3H) ppm; 13C NMR (126 MHz,
p. 167e170 ꢁC; ½a D22
ꢃ
ꢂ9ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
CDCl3) d 181.51, 152.39, 137.62, 134.05, 133.11, 128.66, 127.63, 127.58,
d
7.51e7.46 (m, 1H, arom. ring), 7.21e7.13 (m, 3H, arom. ring), 6.91
126.80, 124.34, 114.34, 79.19, 56.58, 55.50, 50.65, 50.41, 42.61, 41.38,
40.89, 39.01, 38.86, 38.71, 37.36, 37.06, 34.52, 32.71, 32.35, 29.94,
28.15, 27.53, 27.06, 21.16, 18.43, 16.27 (2C), 15.49, 14.95 ppm; HRMS
(ESIꢂ) m/z calcd for C37H50O3Br [MꢂH]ꢂ 621.2938, found 621.2947;
IR (DRIFT) nmax 3610, 3450, 3219, 2939, 2868, 2371, 1913, 1685,
(d, J ¼ 16.0 Hz, 1H, H-31), 6.64 (d, J ¼ 16.0 Hz, 1H, H-30), 5.11, 5.06
(2H, all s, 2 ꢀ H-29), 3.30 (td, J1 ¼ 11.1, J2 ¼ 4.7 Hz, 1H, H-19 ), 3.19
), 2.38 (s, 3H, eCH3), 2.03 (dd,
b
(dd, J1 ¼ 11.5, J2 ¼ 4.5 Hz, 1H, H-3
a
J1 ¼12.6, J2 ¼ 8.0 Hz, 1H), 1.92 (t, J ¼ 11.3 Hz, 1H), 1.02 (s, 3H), 0.97 (s,
3H), 0.96 (s, 3H), 0.82 (s, 3H), 0.75 (s, 3H) ppm; 13C NMR (126 MHz,
1602, 1463 cmꢂ1
.
CDCl3)
d 182.32, 152.75, 136.88, 135.78, 132.79, 130.42, 127.43,
126.46, 126.26, 125.54, 112.69, 79.18, 56.63, 55.49, 50.65, 50.51,
42.57, 41.17, 40.87, 39.00, 38.86, 38.70, 37.34, 37.09, 34.52, 32.91,
32.35, 30.01, 28.14, 27.44, 27.06, 21.19, 20.04, 18.42, 16.26, 16.23,
15.47, 14.93 ppm; HRMS (ESIþ) m/z calcd for C38H55O3 [MþH]þ
559.4146, found 559.4147; IR (DRIFT) nmax 3660, 3447, 2938, 2869,
4.1.5.9. 30-(2-Iodobenzylidene)betulinic acid 4.15. The product 4.15
was obtained as white solid, 177 mg (83%) by the general procedure
(orange ylide color), while the reaction time was 18 h: m. p.
156e159 ꢁC; ½a 2D2
ꢃ
þ6ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
2615, 1911, 1688, 1602, 1449 cmꢂ1
.
d
7.85e7.81 (m, 1H, arom. ring), 7.54e7.48 (m, 1H, arom. ring),
7.33e7.28 (m, 1H, arom. ring), 6.95e6.87 (m, 2H, 1 ꢀ arom. ring and
4.1.5.6. 30-(2-Fluorobenzylidene)betulinic acid 4.12. The product
4.12 was obtained as white solid, 170 mg (95%) by the general
procedure (red ylide color) while the reaction time was 4 h: m. p.
H-31), 6.60 (d, J ¼ 16.0 Hz, 1H, H-30), 5.14, 5.12 (2H, all s, 2 ꢀ H-29),
3.34 (td, J1 ¼ 11.2, J2 ¼ 4.9 Hz, 1H, H-19 ), 3.18 (dd, J1 ¼ 11.4,
b
J2 ¼ 4.7 Hz, 1H, H-3 ), 2.22 (td, J1 ¼ 12.4, J2 ¼ 3.3 Hz, 1H), 2.04 (dd,
a
161e164 ꢁC (methanol); ½a 2D2
ꢃ
ꢂ8ꢁ (c 0.29, THF); 1H NMR (500 MHz,
J1 ¼12.7, J2 ¼ 8.0 Hz, 1H), 1.94 (t, J ¼ 11.4 Hz, 1H), 1.01 (s, 3H), 0.96 (s,
3H), 0.95 (s, 3H), 0.80 (s, 3H), 0.75 (s, 3H) ppm; 13C NMR (126 MHz,
CDCl3)
d 7.54e7.48 (m, 1H, arom. ring), 7.21e7.15 (m, 1H, arom.
CDCl3)
d 181.24, 152.29, 140.79, 139.66, 134.19, 132.68, 128.87,
ring), 7.11e7.06 (m, 1H, arom. ring), 7.05e6.99 (m, 1H, arom. ring),
6.84 (s, 2H, H-31 and H-30), 5.14, 5.10 (2H, all s, 2 ꢀ H-29), 3.28 (td,
128.46, 126.32, 114.38, 100.80, 79.18, 56.57, 55.50, 50.66, 50.44,
42.62, 41.45, 40.89, 39.01, 38.87, 38.75, 37.36, 37.07, 34.52, 32.75,
32.31, 29.95, 28.15, 27.54, 27.07, 21.19, 18.44, 16.29, 16.28, 15.50,
15.02 ppm; HRMS (ESIþ) m/z calcd for C37H52O3I [MþH]þ 671.2956,
found 671.2954; IR (DRIFT) nmax 3439, 2939, 2867, 2363, 1917, 1687,
J1 ¼11.2, J2 ¼ 4.7 Hz,1H, H-19 ), 3.18 (dd, J1 ¼11.4, J2 ¼ 4.8 Hz,1H, H-
b
3
a
), 2.34 (dt, J1 ¼12.5, J2 ¼ 2.8 Hz, 1H), 2.02 (dd, J1 ¼12.7, J2 ¼ 7.9 Hz,
1H), 1.91 (t, J ¼ 11.4 Hz, 1H), 1.00 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H),
0.81 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
181.18,
d
1624, 1458 cmꢂ1
.
160.53 (d, JCF ¼ 249.5 Hz), 152.75, 133.85, 128.62 (d, JCF ¼ 8.3 Hz),
127.28 (d, JCF ¼ 3.4 Hz), 125.62 (d, JCF ¼ 12.0 Hz), 124.21 (d,
JCF ¼ 3.1 Hz), 120.68, 115.87 (d, JCF ¼ 22.2 Hz), 113.51, 79.20, 56.52,
55.51, 50.74, 50.67, 42.57, 40.88, 40.52, 39.01, 38.86, 38.64, 37.36,
37.07, 34.53, 33.06, 32.37, 29.96, 28.14, 27.52, 27.15, 21.17, 18.43,
16.25, 15.48, 14.90 ppm; HRMS (ESIꢂ) m/z calcd for C37H50O3F
[MꢂH]ꢂ 561.3739, found 561.3748; IR (DRIFT) nmax 3639, 3430,
4.1.5.10. 30-(2-Cyanobenzylidene)betulinic acid 4.16. The product
4.16 was obtained as white solid, 74 mg (41%) by the general pro-
cedure (yellow ylide color) while the reaction time was 18 h: m. p.
146e149 ꢁC; ½a 2D2
ꢃ
ꢂ8ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
d
7.68e7.64 (m, 1H, arom. ring), 7.63e7.59 (m, 1H, arom. ring),
3176, 2931, 2867, 2607, 1946, 1684, 1485, 1452 cmꢂ1
.
7.55e7.50 (m, 1H, arom. ring), 7.31e7.27 (m, 1H, arom. ring), 7.03 (d,
J ¼ 16.2 Hz, 1H, H-31), 6.91 (d, J ¼ 16.1 Hz, 1H, H-30), 5.22, 5.19 (2H,
4.1.5.7. 30-(2-Chlorobenzylidene)betulinic acid 4.13. The product
4.13 was obtained as white solid, 181 mg (98%) by the general
procedure (orange ylide color), while the reaction time was 6 h: m.
all s, 2 ꢀ H-29), 3.31 (td, J1 ¼ 11.2, J2 ¼ 4.8 Hz, 1H, H-19
b), 3.19 (dd,
J1 ¼ 11.4, J2 ¼ 4.7 Hz, 1H, H-3 ), 2.22 (td, J1 ¼ 12.6, J2 ¼ 3.2 Hz, 1H),
a
2.04 (dd, J1 ¼ 12.7, J2 ¼ 8.1 Hz, 1H), 1.92 (t, J ¼ 11.4 Hz, 1H), 1.00 (s,
3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.81 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR
p. 153e156 ꢁC (methanol); ½a 2D2
ꢃ
ꢂ5ꢁ (c 0.29, THF); 1H NMR
(126 MHz, CDCl3)
d 181.31, 151.98, 141.12, 136.09, 133.22, 132.76,
(500 MHz, CDCl3) d 7.59e7.55 (m, 1H, arom. ring), 7.36e7.32 (m, 1H,
127.41, 125.71, 124.39, 118.10, 115.66, 111.28, 79.20, 56.51, 55.48,
50.60, 50.53, 42.58, 41.49, 40.86, 38.99, 38.85, 38.67, 37.34, 37.00,
34.49, 32.63, 32.28, 29.89, 28.13, 27.47, 27.15, 21.11, 18.41, 16.26,
16.24, 15.48, 14.87 ppm; HRMS (ESIꢂ) m/z calcd for C38H50O3N
[MꢂH]ꢂ 568.3785, found 568.3800; IR (DRIFT) nmax 3447, 2939,
arom. ring), 7.24e7.20 (m, 1H, arom. ring), 7.17e7.13 (m, 1H, arom.
ring), 7.08 (d, J ¼ 16.2 Hz, 1H, H-31), 6.72 (d, J ¼ 16.2 Hz, 1H, H-30),
5.15, 5.12 (2H, all s, 2 ꢀ H-29), 3.31 (td, J1 ¼ 11.2, J2 ¼ 4.8 Hz, 1H, H-
19
b
), 3.19 (dd, J1 ¼ 11.4, J2 ¼ 4.8 Hz, 1H, H-3
a), 2.38e2.32 (m, 1H),
2.03 (dd, J1 ¼ 12.7, J2 ¼ 7.9 Hz, 1H), 1.94 (t, J ¼ 11.4 Hz, 1H), 1.01 (s,
2868, 2224 (C≡N), 1685, 1595, 1448 cmꢂ1
.
3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.81 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR
(126 MHz, CDCl3)
d 180.92, 152.49, 135.91, 133.94, 133.58, 129.87,
128.39, 126.94, 126.60, 124.84, 114.18, 79.18, 56.54, 55.51, 50.67,
50.48, 42.60, 41.13, 40.89, 39.02, 38.87, 38.68, 37.37, 37.06, 34.53,
32.77, 32.36, 29.94, 28.15, 27.54, 27.09, 21.16,18.44,16.26 (2C),15.49,
14.92 ppm; HRMS (ESIꢂ) m/z calcd for C37H50O3Cl [MꢂH]ꢂ
577.3443, found 577.3457; IR (DRIFT) nmax 3440, 3255, 2927, 2866,
4.1.5.11. 30-(2-Trifluoromethylbenzylidene)betulinic
The product 4.17 was obtained as white solid, 129 mg (66%) by the
general procedure (orange ylide color), while the reaction time was
acid
4.17.
18 h: m. p. 159e162 ꢁC; ½a D22
ꢃ
ꢂ19ꢁ (c 0.29, THF); 1H NMR (500 MHz,
CDCl3)
d 7.69e7.60 (m, 2H, arom. ring), 7.52e7.46 (m, 1H, arom.
2614, 1912, 1680, 1603, 1465, 1447 cmꢂ1
.
ring), 7.34e7.29 (m,1H, arom. ring), 7.11e7.02 (m,1H, H-31), 6.69 (d,
J ¼ 16.0 Hz, 1H, H-30), 5.15, 5.13 (2H, all s, 2 ꢀ H-29), 3.29 (td,
J1 ¼ 11.2, J2 ¼ 4.8 Hz, 1H), 3.19 (dd, J1 ¼ 11.4, J2 ¼ 4.8 Hz, 1H),
2.38e2.31 (m, 1H), 2.03 (dd, J1 ¼ 12.6, J2 ¼ 8.0 Hz, 1H), 1.91 (t,
J ¼ 11.4 Hz, 1H), 0.99 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.81 (s, 3H),
4.1.5.8. 30-(2-Bromobenzylidene)betulinic acid 4.14. The product
4.14 was obtained as white solid, 191 mg (96%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
p. 155e158 ꢁC; ½a D22
ꢃ
ꢂ2ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
0.75 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
d 182.04, 152.36,
d
7.57e7.52 (m, 2H, arom. ring), 7.30e7.23 (m, 1H, arom. ring),
136.97, 135.25, 131.90, 127.67 (q, JCF ¼ 29.7 Hz), 127.14, 127.06,
125.97 (q, JCF ¼ 5.6 Hz), 124.60, 124.58 (q, JCF ¼ 274.1 Hz, CF3 group),
114.66, 79.23, 56.60, 55.50, 50.65, 50.46, 42.57, 41.51, 40.87, 39.01,
7.10e7.07 (m, 1H, arom. ring), 7.04 (d, J ¼ 15.9 Hz, 1H, H-31), 6.67 (d,
J ¼ 16.2 Hz, 1H, H-30), 5.14, 5.12 (2H, all s, 2 ꢀ H-29), 3.32 (td,
10

ꢀ
J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
38.85, 38.71, 37.35, 37.02, 34.51, 32.49, 32.30, 29.92, 28.14, 27.49,
27.03, 21.11, 18.43, 16.21 (2C), 15.49, 14.84 ppm; HRMS (ESIꢂ) m/z
calcd for C38H50O3F3 [MꢂH]ꢂ 611.3707, found 611.3709; IR (DRIFT)
m/z calcd for C38H55O3 [MþH]þ 559.4146, found 559.4147; IR
(DRIFT) nmax 3660, 3433, 3276, 2935, 2867, 2619, 2362, 1684, 1603,
1449 cmꢂ1
.
nmax 3409, 3188, 2938, 2868, 2556, 1940, 1687, 1603, 1451 cmꢂ1
.
4.1.5.15. 30-(3-Fluorobenzylidene)betulinic acid 4.21. The product
4.21 was obtained as white solid, 102 mg (57%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
4.1.5.12. 30-(2-Nitrobenzylidene)betulinic acid 4.18. The product
4.18 was obtained as a yellow solid, 55 mg (29%) by the modified
general procedure (violet ylide color), while the reaction time was
18 h and the reaction temperature was 70 ꢁC; conversion of the
p. 159e162 ꢁC; ½a D22
ꢃ
ꢂ2ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
d
7.29e7.24 (m, 1H, arom. ring), 7.22e7.18 (m, 1H, arom. ring),
starting material was not complete: m. p. 167e170 ꢁC; ½a D22
ꢃ
þ22ꢁ (c
7.17e7.13 (m, 1H, arom. ring), 6.94e6.88 (m, 1H, arom. ring), 6.79 (d,
J ¼ 16.2 Hz, 1H, H-31), 6.65 (d, J ¼ 16.2 Hz, 1H, H-30), 5.14, 5.11 (2H,
0.29, THF); 1H NMR (500 MHz, CDCl3)
d 7.97e7.91 (m, 1H, arom.
ring), 7.66e7.61 (m, 1H, arom. ring), 7.59e7.53 (m, 1H, arom. ring),
7.40e7.33 (m, 1H, arom. ring), 7.16 (d, J ¼ 16.0 Hz, 1H, H-31), 6.69 (d,
J ¼ 16.0 Hz, 1H, H-30), 5.18 (s, 2H, 2 ꢀ H-29), 3.26 (td, J1 ¼ 11.2,
all s, 2 ꢀ H-29), 3.27 (td, J1 ¼ 11.1, J2 ¼ 4.7 Hz, 1H, H-19
b), 3.20 (dd,
a), 2.30e2.23 (m, 1H), 2.23e2.16 (m,
J1 ¼ 11.3, J2 ¼ 4.7 Hz, 1H, H-3
1H), 2.03 (dd, J1 ¼12.7, J2 ¼ 7.9 Hz, 1H), 1.90 (t, J ¼ 11.3 Hz, 1H), 1.01
J2 ¼ 4.6 Hz,1H, H-19
b
), 3.19 (dd, J1 ¼11.4, J2 ¼ 4.7 Hz,1H, H-3
a), 2.04
(s, 3H), 0.97 (s, 3H), 0.95 (s, 3H), 0.81 (s, 3H), 0.75 (s, 3H) ppm; 13C
(dd, J1 ¼ 12.6, J2 ¼ 8.1 Hz, 1H), 1.91 (t, J ¼ 11.3 Hz, 1H), 1.00 (s, 3H),
NMR (126 MHz, CDCl3)
d
182.19, 163.31 (d, JCF ¼ 245.0 Hz), 152.41,
0.96 (s, 3H), 0.94 (s, 3H), 0.81 (s, 3H), 0.75 (s, 3H) ppm; 13C NMR
140.13 (d, JCF ¼ 7.6 Hz), 133.08, 130.09 (d, JCF ¼ 8.4 Hz), 129.17,
128.36, 127.00, 125.44, 124.81, 122.60 (d, JCF ¼ 1.5 Hz), 114.62, 114.23
(d, JCF ¼ 21.4 Hz), 113.88, 113.66, 112.89 (d, JCF ¼ 21.6 Hz), 79.21,
56.59, 55.48, 50.89, 50.63, 42.54, 40.86, 40.10, 38.99, 38.84, 38.69,
37.34, 37.04, 34.50, 33.26, 32.36, 29.97, 28.14, 27.44, 27.21, 21.18,
18.40, 16.25 (2C), 15.48, 14.91 ppm; HRMS (ESIþ) m/z calcd for
(101 MHz, CDCl3)
d 181.48, 152.33, 148.06, 136.47, 133.76, 133.12,
128.52, 127.83, 124.84, 123.93, 115.39, 79.20, 56.55, 55.48, 50.63,
50.59, 42.58, 41.28, 40.86, 39.00, 38.85, 38.66, 37.34, 37.03, 34.50,
32.67, 32.31, 29.92, 28.13, 27.49, 27.12, 21.12, 18.42, 16.24 (2C), 15.48,
14.86 ppm; HRMS (ESIꢂ) m/z calcd for C37H50O5N [MꢂH]ꢂ
588.3684, found 588.3696; IR (DRIFT) nmax 3620, 3482, 3341, 2939,
C
37H52O3F [MþH]þ 563.3895, found 563.3896; IR (DRIFT) nmax
2868, 1944, 1694, 1605, 1519 (NeO), 1448 cmꢂ1
.
3671, 3430, 3279, 2934, 2867, 2615, 1688, 1609, 1578, 1485,
1446 cmꢂ1
.
4.1.5.13. 30-(Pyridin-2-ylmethylene)betulinic acid 4.19. The product
4.19 was obtained as beige solid, 68 mg (39%) by the modified
general procedure (brown ylide color) without extraction (the re-
action mixture was evaporated after completion followed by pu-
rification) while the reaction time was 18 h. Purification was
4.1.5.16. 30-(3-Chlorobenzylidene)betulinic acid 4.22. The product
4.22 was obtained as white solid, 111 mg (60%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
p. 181e184 ꢁC; ½a D22
ꢃ
þ11ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
performed using mobile phase CHCl3/MeOH 50
: 1: m. p.
d
7.44e7.42 (m, 1H, arom. ring), 7.32e7.28 (m, 1H, arom. ring),
166e170 ꢁC; ½a 2D2
ꢃ
þ13ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
7.26e7.21 (m, 1H, arom. ring), 7.20e7.17 (m, 1H, arom. ring), 6.79 (d,
1H, J ¼ 16.2 Hz, H-31), 6.62 (d, 1H, J ¼ 16.2 Hz, H-30), 5.14, 5.11 (2H,
d
8.60e8.55 (m,1H, H-pyridine), 7.68e7.61 (m,1H, H-pyridine), 7.38
(d, 1H, J ¼ 7.9 Hz, H-pyridine), 7.28 (d, 1H, J ¼ 16.3 Hz, H-31),
7.17e7.10 (m, 1H, H-pyridine), 6.79 (d, 1H, J ¼ 16.1 Hz, H-30), 5.25,
5.20 (2H, all s, 2 ꢀ H-29), 3.31 (td, 1H, J1 ¼ 11.1 Hz, J2 ¼ 4.6 Hz, H-
all s, 2 ꢀ H-29), 3.25 (td, 1H, J1 ¼ 11.1 Hz, J2 ¼ 4.8 Hz, H-19
b
), 3.19
(dd, 1H, J1 ¼ 11.4 Hz, J2 ¼ 4.7 Hz, H-3
a), 2.29e2.22 (m, 1H),
2.22e2.15 (m, 1H), 2.02 (dd, 1H, J1 ¼12.8 Hz, J2 ¼ 7.7 Hz), 1.90 (t, 1H,
J ¼ 11.3 Hz), 1.01, 0.96, 0.95, 0.80, 0.74 (15H, all s, 5 ꢀ eCH3) ppm;
19
b
), 3.18 (dd, 1H, J1 ¼ 11.4 Hz, J2 ¼ 4.6 Hz, H-3
a), 2.21 (td, 1H,
J1 ¼ 12.3 Hz, J2 ¼ 3.1 Hz), 2.01 (dd, 1H J1 ¼ 12.6 Hz, J2 ¼ 7.9 Hz), 1.91
13C NMR (126 MHz, CDCl3)
d 181.80, 152.42, 139.64, 134.70, 133.19,
(t, 1H, J ¼ 11.3 Hz), 1.00, 0.96, 0.94, 0.79, 0.74 (15H, all s, 5 ꢀ eCH3)
129.90, 127.37, 126.75, 126.50, 124.85, 113.80, 79.20, 56.57, 55.49,
50.94, 50.64, 42.54, 40.87, 40.06, 39.01, 38.85, 38.67, 37.36, 37.04,
34.51, 33.25, 32.33, 29.97, 28.14, 27.48, 27.20, 21.17, 18.42, 16.26 (2C),
15.49, 14.92 ppm; HRMS (ESIþ) m/z calcd for C37H52O3Cl [MþH]þ
579.3599, found 579.3605; IR (DRIFT) nmax 2380e3610, 2939, 2869,
ppm; 13C NMR (126 MHz, CDCl3)
d 181.15, 155.95, 152.50, 149.18,
137.00, 136.61, 127.13, 122.06, 115.47, 79.16, 56.53, 55.49, 50.94,
50.66, 42.55, 40.87, 39.96, 39.00, 38.85, 38.64, 37.35, 37.08, 34.52,
33.33, 32.41, 29.99, 28.14, 27.50, 27.31, 21.19, 18.43, 16.26 (2C), 15.49,
14.92 ppm; HRMS (ESIþ) m/z calcd for C36H52O3N [MþH]þ
546.3942, found 546.3947; IR (DRIFT) nmax 2380e3595, 2929, 2867,
1689, 1594, 1574, 1451 cmꢂ1
.
1689, 1587, 1465 cmꢂ1
.
4.1.5.17. 30-(3-Bromobenzylidene)betulinic acid 4.23. The product
4.23 was obtained as white solid, 145 mg (73%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
4.1.5.14. 30-(3-Methylbenzylidene)betulinic acid 4.20. The product
4.20 was obtained as white solid, 135 mg (76%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
p. 165e168 ꢁC; ½a D22
ꢃ
þ3ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
d
7.61e7.56 (m, 1H, arom. ring), 7.36e7.31 (m, 2H, arom. ring),
p. 158e161 ꢁC; ½a D22
ꢃ
ꢂ1ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
7.19e7.15 (m, 1H, arom. ring), 6.78 (d, J ¼ 16.2 Hz, 1H, H-31), 6.60 (d,
d
7.28e7.24 (m, 2H, arom. ring), 7.23e7.18 (m, 1H, arom. ring),
J ¼ 16.2 Hz, 1H, H-30), 5.14, 5.11 (2H, all s, 2 ꢀ H-29), 3.25 (td,
7.06e7.01 (m,1H, arom. ring), 6.79 (d, J ¼ 16.2 Hz,1H, H-31), 6.67 (d,
J1 ¼ 11.2, J2 ¼ 4.7 Hz, 1H, H-19 ), 3.20 (dd, J1 ¼ 11.3, J2 ¼ 4.7 Hz, 1H,
b
J ¼ 16.2 Hz, 1H, H-30), 5.10, 5.06 (2H, all s, 2 ꢀ H-29), 3.28 (td,
H-3
J ¼ 11.3 Hz, 1H), 1.01 (s, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.80 (s, 3H),
0.74 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
182.07, 152.42,
a
), 2.22e2.15 (m, 1H), 2.02 (dd, J1 ¼12.7, J2 ¼ 8.0 Hz, 1H), 1.89 (t,
J1 ¼11.1, J2 ¼ 4.7 Hz, 1H, H19 ), 3.19 (dd, J1 ¼11.4, J2 ¼ 4.6 Hz, 1H, H-
b
3
a
), 2.35 (s, 3H), 2.23e2.15 (m, 1H), 2.02 (dd, J1 ¼ 12.7, J2 ¼ 7.9 Hz,
1H), 1.91 (t, J ¼ 11.4 Hz, 1H), 1.01 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H),
0.80 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
182.14,
d
139.94, 133.28, 130.25, 130.17, 129.45, 126.59, 125.25, 122.96, 113.83,
79.21, 56.57, 55.47, 50.93, 50.62, 42.52, 40.85, 39.99, 38.98, 38.84,
38.68, 37.33, 37.03, 34.49, 33.29, 32.35, 29.96, 28.14, 27.42, 27.21,
21.18, 18.39, 16.25 (2C), 15.48, 14.91 ppm; HRMS (ESIþ) m/z calcd for
d
152.80, 138.23, 137.65, 131.53, 128.62, 128.31, 128.26, 127.41, 123.79,
112.58, 79.21, 56.60, 55.49, 50.87, 50.65, 42.55, 40.87, 40.21, 39.00,
38.85, 38.70, 37.35, 37.08, 34.51, 33.28, 32.39, 30.00, 28.14, 27.47,
27.13, 21.54, 21.19, 18.41, 16.26 (2C), 15.48, 14.93 ppm; HRMS (ESIþ)
C
37H52O3Br [MþH]þ 623.3094, found 623.3093; IR (DRIFT) nmax
3426, 2938, 2867, 1686, 1589, 1559, 1450 cmꢂ1
.
11

ꢀ
J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
4.1.5.18. 30-(3-Iodobenzylidene)betulinic acid 4.24. The product
4.24 was obtained as white solid, 182 mg (85%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
J ¼ 16.2 Hz,1H, H-31), 6.71 (d, J ¼ 16.2 Hz,1H, H-30), 5.21, 5.18 (2H, all
s, 2 ꢀ H-29), 3.27 (td, J1 ¼ 11.1, J2 ¼ 4.7 Hz, 1H, H-19
b), 3.18 (dd,
J1 ¼11.4, J2 ¼ 4.6 Hz,1H, H-3 ), 2.23e2.16 (m,1H), 2.03 (dd, J1 ¼12.8,
a
p. 177e180 ꢁC; ½a D22
ꢃ
þ2ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
J2 ¼ 8.0 Hz, 1H),1.91 (t, J ¼ 11.3 Hz, 1H),1.01 (s, 3H), 0.96 (s, 3H), 0.95
(s, 3H), 0.80 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
d
7.82e7.76 (m, 1H, arom. ring), 7.56e7.50 (m, 1H, arom. ring),
d
182.00, 152.15, 148.83, 139.59, 134.96, 132.35, 129.55, 125.66,
7.41e7.36 (m, 1H, arom. ring), 7.07e7.00 (m, 1H, arom. ring), 6.76 (d,
J ¼ 16.2 Hz, 1H, H-31), 6.56 (d, J ¼ 16.2 Hz, 1H, H-30), 5.13, 5.11 (2H,
121.92, 121.12, 115.00, 79.16, 56.58, 55.46, 51.02, 50.60, 42.52, 40.85,
39.85, 38.98, 38.83, 38.69, 37.34, 36.99, 34.49, 33.33, 32.32, 29.96,
28.13, 27.45, 27.31, 21.17, 18.39, 16.27, 16.24,15.46, 14.90 ppm; HRMS
(ESIꢂ) m/z calcd for C37H52O5N [MꢂH]ꢂ 590.3840, found 590.3837;
IR (DRIFT) nmax 3580, 3433, 3299, 2932, 2867, 2724, 2556,1964,1687,
all s, 2 ꢀ H-29), 3.25 (td, J1 ¼ 11.3, J2 ¼ 4.8 Hz, 1H, H-19
b), 3.20 (dd,
J1 ¼11.3, J2¼4.6 Hz, 1H, H-3 ), 2.21e2.15 (m, 1H), 2.02 (dd, J1 ¼12.6,
a
J2 ¼ 8.0 Hz,1H),1.89 (t, J ¼ 11.3 Hz,1H), 1.01 (s, 3H), 0.97 (s, 3H), 0.94
(s, 3H), 0.80 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
1629, 1604, 1524 (NeO), 1448, 1346 (NeO) cmꢂ1
.
d
182.11, 152.44, 140.01, 136.23, 135.51, 133.19, 130.33,126.47, 125.81,
113.81, 94.88, 79.20, 56.57, 55.47, 50.94, 50.62, 42.53, 40.85, 39.96,
38.98, 38.84, 38.69, 37.34, 37.03, 34.49, 33.31, 32.35, 29.96, 28.15,
27.44, 27.21, 21.18, 18.40, 16.26 (2C), 15.49, 14.92 ppm; HRMS (ESIþ)
m/z calcd for C37H52O3I [MþH]þ 671.2956, found 671.2955; IR
(DRIFT) nmax 3433, 2939, 2868, 1685, 1601, 1584, 1556, 1465,
4.1.5.22. 30-(Pyridin-3-ylbenzylidene)betulinic acid 4.28. The prod-
uct 4.28 was obtained as a white solid, 118 mg (68%) by the
modified general procedure (orange-brown ylide color) without
extraction (the reaction mixture was evaporated after completion
followed by purification) while the reaction time was 18 h. Purifi-
cation was performed using mobile phase CHCl3/MeOH 50 : 1: m. p.
1451 cmꢂ1
.
187e190 ꢁC; ½a 2D2
ꢃ
ꢂ10ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
4.1.5.19. 30-(3-Cyanobenzylidene)betulinic acid 4.25. The product
4.25 was obtained as white solid, 94 mg (52%) by the general
procedure (yellow ylide color) while the reaction time was 18 h: m.
d
8.96e8.89 (m, 1H, arom. ring), 8.41e8.35 (m, 1H, arom. ring),
8.05e7.97 (m, 1H, arom. ring), 7.37e7.30 (m, 1H, arom. ring), 6.93
(d, J ¼ 16.3 Hz, 1H, H-31), 6.89 (d, J ¼ 16.4 Hz, 1H, H-30), 5.21, 5.16
p. 163e166 ꢁC; ½a D22
ꢃ
ꢂ3ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
(2H, all s, 2 ꢀ H-29), 3.59 (td, J1 ¼ 11.1, J2 ¼ 5.0 Hz, 1H, H-19
b), 3.17
d
7.73e7.69 (m, 1H, arom. ring), 7.66e7.62 (m, 1H, arom. ring),
(dd, J1 ¼ 11.3, J2 ¼ 4.8 Hz, 1H, H-3 ), 2.01 (dd, J1 ¼ 12.5, J2 ¼ 7.9 Hz,
1H), 1.88 (t, J ¼ 11.3 Hz, 1H), 1.03 (s, 3H), 0.96 (s, 3H), 0.96 (s, 3H),
0.80 (s, 3H), 0.73 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
179.01,
a
7.50e7.47 (m,1H, arom. ring), 7.43e7.39 (m,1H, arom. ring), 6.83 (d,
J ¼ 16.2 Hz, 1H, H-31), 6.64 (d, J ¼ 16.2 Hz, 1H, H-30), 5.18, 5.16 (2H,
d
all s, 2 ꢀ H-29), 3.26 (td, J1 ¼ 11.1, J2 ¼ 4.7 Hz, 1H, H-19
b), 3.19 (dd,
153.13, 148.97, 145.97, 134.91, 134.30, 132.51, 123.96, 123.45, 114.65,
79.20, 56.40, 55.54, 51.42, 50.78, 42.51, 40.93, 39.94, 39.01, 38.87,
38.57, 37.38, 37.04, 34.58, 34.07, 32.43, 30.19, 28.15, 27.60, 27.56,
21.20,18.49,16.28 (2C),15.49,14.87 ppm; HRMS (ESIþ) m/z calcd for
J1 ¼11.3, J2 ¼ 4.6 Hz,1H, H-3 ), 2.22e2.15 (m,1H), 2.03 (dd, J1 ¼12.7,
a
J2 ¼ 7.9 Hz, 1H), 1.90 (t, J ¼ 11.3 Hz, 1H), 1.01 (s, 3H), 0.96 (s, 3H), 0.94
(s, 3H), 0.80 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
d
181.76, 152.17, 139.01, 134.43, 130.71, 130.60, 130.04, 129.49,
C
36H52O3N [MþH]þ 546.3942, found 546.3946; IR (DRIFT) nmax
125.77, 118.93, 114.73, 112.98, 79.16, 56.57, 55.48, 51.00, 50.62,
42.54, 40.86, 39.95, 39.00, 38.84, 38.69, 37.36, 36.99, 34.50, 33.26,
32.31, 29.96, 28.14, 27.48, 27.28, 21.17, 18.40, 16.26 (2C), 15.47,
14.92 ppm; HRMS (ESIꢂ) m/z calcd for C38H50O3N [MꢂH]ꢂ
568.3785, found 568.3793; IR (DRIFT) nmax 3506, 3436, 2934, 2867,
3431, 2933, 2867, 2539, 2065, 1673, 1586, 1569, 1449 cmꢂ1
.
4.1.5.23. 30-(3-Carboxybenzylidene)betulinic acid 4.29. The product
4.29 was obtained as a mixture of inseparable E and Z isomers in a
ratio approximately 2 : 1, white solid, 120 mg (64%) by the modified
general procedure using double amount of t-BuOK (orange ylide
color) while the reaction time was 18 h. The compound is not very
stable on silica gel and exhibits bright blue fluorescence under UV
light. Purification was performed in CHCl3/MeOH 50 : 1: m. p.
2550, 2236 (C≡N), 1992, 1686, 1601, 1450 cmꢂ1
.
4.1.5.20. 30-(3-Trifluoromethylbenzylidene)betulinic
acid
4.26.
The product 4.26 was obtained as white solid, 127 mg (65%) by the
general procedure (orange ylide color), while the reaction time was
183e186 ꢁC; ½a 2D2
ꢃ
ꢂ3ꢁ (c 0.29, THF); 1H NMR (400 MHz, CD3OD)
18 h: m. p. 168e171 ꢁC; ½a D22
ꢃ
ꢂ1ꢁ (c 0.29, THF); 1H NMR (500 MHz,
d
8.22 (s, 0.5H, H-arom. ring of Z-isomer), 8.09 (s,1H, H-arom. ring of
CDCl3) d 7.69e7.66 (m,1H, arom. ring), 7.62e7.58 (m, 1H, arom. ring),
E-isomer), 7.89e7.82 (m, 1.5H, H-arom. ring of both isomers), 7.65
(m, 1.5H, H-arom. ring of both isomers), 7.41 (t, J ¼ 7.7 Hz, 1H, H-
arom. ring of E-isomer), 7.35 (t, J ¼ 7.7 Hz, 0.5H, H-arom. ring of Z-
isomer), 6.91 (d, J ¼ 16.2 Hz, 1H, H-31 of E-isomer), 6.73 (d,
J ¼ 16.2 Hz,1H, H-30 of E-isomer), 6.52 (d, J ¼ 12.7 Hz, 0.5H, H-31 of Z-
isomer), 6.12 (d, J ¼ 12.5 Hz, 0.5H, H-30 of Z-isomer), 5.14 (d,
J ¼ 17.8 Hz, 2H, 2 ꢀ H-29 of E-isomer), 5.00 (d, J ¼ 22.7 Hz, 2H, 2 ꢀ H-
29 of Z-isomer), 3.15e3.03 (m, 2H), 1.00 (s, 3H, CH3- group of E-
isomer), 0.94 (s, 3H, CH3- group of E-isomer), 0.93 (s, 4.5H, CH3-
groups of both isomers), 0.92 (s, 1.5H, CH3- group of Z-isomer), 0.91
(s, 1.5H, CH3- group of Z-isomer), 0.82 (s, 1.5H, CH3- group of Z-iso-
mer), 0.80 (s, 3H, CH3- groupof E-isomer), 0.73 (s,1.5H, CH3- group of
Z-isomer), 0.72 (s, 3H, CH3- group of E-isomer) ppm; HRMS (ESIꢂ) m/
z calcd for C38H51O5 [MꢂH]ꢂ 587.3731, found 587.3744; IR (DRIFT)
7.48e7.40 (m, 2H, arom. ring), 6.85 (d, J ¼ 16.2 Hz, 1H, H-31), 6.70 (d,
J ¼ 16.2 Hz, 1H, H-30), 5.17, 5.14 (2H, all s, 2 ꢀ H-29), 3.27 (td,
J1 ¼11.1 Hz, J2 ¼ 4.7 Hz, 1H, H-19 ), 3.19 (dd, J1 ¼11.3 Hz, J2 ¼ 4.7 Hz,
b
1H, H-3
a
), 2.39e2.33 (m, 1H), 2.19 (td, J1 ¼ 12.3 Hz, J2 ¼ 3.2 Hz, 1H),
2.03 (dd, J1 ¼12.8 Hz, J2 ¼ 7.9 Hz, 1H), 1.91 (t, J ¼ 11.3 Hz, 1H), 1.01 (s,
3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.80 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR
(126 MHz, CDCl3)
d 182.07, 152.38, 138.52, 133.71, 131.16 (q,
JCF ¼ 32.0 Hz), 129.66, 129.12, 126.61, 124.31 (q, JCF ¼ 272.4 Hz, CF3
group), 123.93 (d, JCF ¼ 3.2 Hz), 123.30 (d, JCF ¼ 3.4 Hz), 114.13, 79.20,
56.59, 55.48, 50.96, 50.63, 42.54, 40.87, 39.90, 38.99, 38.84, 38.70,
37.35, 37.03, 34.50, 33.34, 32.36, 29.97, 28.14, 27.46, 27.27, 21.18,
18.40, 16.27, 16.24 (2C), 15.47, 14.91 ppm; HRMS (ESIþ) m/z calcd for
C
38H52O3F3 [MþH]þ 613.3863, found 613.3862; IR (DRIFT) nmax 3445,
2939, 2869, 1689, 1629, 1606, 1449 cmꢂ1
.
nmax 2939, 2870, 2616, 2362, 2006, 1691, 1602, 1582, 1450 cmꢂ1
.
4.1.5.21. 30-(3-Nitrobenzylidene)betulinic acid 4.27. The product
4.27 was obtained as yellow solid, 147 mg (78%) by the general
procedure (red-brown ylide color) while the reaction time was 18 h:
4.1.5.24. 30-(3-Methoxybenzylidene)betulinic acid 4.30. The prod-
uct 4.30 was obtained as white solid, 70 mg (38%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
m. p.168e171 ꢁC; ½a D22
ꢃ
þ3ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
p. 147e150 ꢁC; ½a D22
ꢃ
þ2ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
d
8.31e8.26 (m, 1H, arom. ring), 8.08e8.03 (m, 1H, arom. ring),
7.75e7.71 (m,1H, arom. ring), 7.50e7.45 (m,1H, arom. ring), 6.91 (d,
d 7.25e7.20 (m, 1H, arom. ring), 7.06e7.02 (m, 1H, arom. ring),
12

ꢀ
J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
7.00e6.97 (m, 1H, arom. ring), 6.81e6.75 (m, 2H, arom. ring and H-
31), 6.66 (d, 1H, J ¼ 16.2 Hz, H-30), 5.12, 5.07 (2H, all s, 2 ꢀ H-29),
3.83 (s, 3H, methoxy eCH3), 3.27 (td, 1H, J1 ¼11.1 Hz, J2 ¼ 4.7 Hz, H-
37.28, 36.96, 34.44, 33.22, 32.28, 29.90, 28.07, 27.42, 27.14, 21.11,
18.34, 16.19 (2C), 15.40, 14.84 ppm; HRMS (ESIþ) m/z calcd for
C
37H52O3Cl [MþH]þ 579.3599, found 579.3602; IR (DRIFT) nmax
19
b), 3.19 (dd, 1H, J1 ¼11.4 Hz, J2 ¼ 4.7 Hz, H-3
a
), 2.38e2.32 (m, 1H),
2380e3650, 2939, 2868, 1691, 1593, 1490, 1451 cmꢂ1
.
2.30e2.23 (m, 1H), 2.23e2.16 (m, 1H), 2.02 (dd, 1H, J1 ¼ 12.8 Hz,
J2 ¼ 7.7 Hz), 1.91 (t, 1H, J ¼ 11.4 Hz), 1.01, 0.96, 0.95, 0.80, 0.74 (15 H,
4.1.5.28. 30-(4-Bromobenzylidene)betulinic acid 4.35. The product
4.35 was obtained as a white solid, 76 mg (38%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
all s, 5 ꢀ eCH3) ppm; 13C NMR (126 MHz, CDCl3)
d 182.01, 159.99,
152.66, 139.19, 132.02, 129.66, 128.09, 119.41, 113.32, 112.91, 111.82,
79.20, 56.59, 55.49, 55.39, 50.87, 50.65, 42.55, 40.87, 40.21, 39.00,
38.84, 38.69, 37.35, 37.07, 34.51, 33.25, 32.37, 29.99, 28.14, 27.48,
27.14, 21.18, 18.41, 16.26 (2C), 15.48, 14.92 ppm; HRMS (ESIþ) m/z
calcd for C38H55O4 [MþH]þ 575.4095, found 575,4090; IR (DRIFT)
p. 188e191 ꢁC; ½a D22
ꢃ
þ7ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
d
7.45e7.41 (m, 2H, arom. ring), 7.32e7.28 (m, 2H, arom. ring), 6.77
(d, 1H, J ¼ 16.2 Hz, H-31), 6.61 (d, 1H, J ¼ 16.2 Hz, H-30), 5.12, 5.09
(2H, all s, 2 ꢀ H-29), 3.25 (td, 1H, J1 ¼ 11.1 Hz, J2 ¼ 4.7 Hz, H-19 ),
), 2.29e2.22 (m, 1H), 2.18
b
nmax 3450, 2939, 2868, 1686, 1599, 1578, 1485, 1452 cmꢂ1
.
3.19 (dd, 1H, J1 ¼11.4 Hz, J2 ¼ 4.7 Hz, H-3
a
(td, 1H, J1 ¼ 12.5 Hz, J2 ¼ 3.6 Hz), 2.01 (dd, 1H, J1 ¼ 12.7 Hz,
J2 ¼ 7.8 Hz), 1.90 (t, 1H, J ¼ 11.3 Hz), 1.01, 0.96, 0.94, 0.80, 0.74 (15H,
4.1.5.25. 30-(4-Methylbenzylidene)betulinic acid 4.32. The product
4.32 was obtained as white solid, 93 mg (52%) by the general
procedure (red ylide color) while the reaction time was 18 h: m. p.
all s, 5 ꢀ eCH3) ppm; 13C NMR (126 MHz, CDCl3)
d 181.82, 152.49,
136.67, 132.53, 131.82 (2C), 128.13 (2C), 126.91, 121.19, 113.42, 79.17,
56.57, 55.49, 50.90, 50.64, 42.55, 40.88, 40.03, 39.01, 38.85, 38.70,
37.36, 37.03, 34.51, 33.30, 32.35, 29.97, 28.15, 27.50, 27.23, 21.18,
18.42, 16.27 (2C), 15.48, 14.92 ppm; HRMS (ESIþ) m/z calcd for
182e185 ꢁC; ½a 2D2
ꢃ
þ5ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
d
7.36e7.32 (m, 2H, arom. ring), 7.14e7.10 (m, 2H, arom. ring), 6.75
(d, 1H, J ¼ 16.2 Hz, H-31), 6.66 (d, 1H, J ¼ 16.2 Hz, H-30), 5.08, 5.03
(2H, all s, 2 ꢀ H-29), 3.27 (td, 1H, J1 ¼ 11.2 Hz, J2 ¼ 4.7 Hz, H-19 ),
), 2.34 (s, 3H, eCH3 on
C
37H52O3Br [MþH]þ 623.3094, found 623.3098; IR (DRIFT) nmax
b
3459, 2938, 2868, 1686, 1589, 1486, 1449 cmꢂ1
.
3.19 (dd, 1H, J1 ¼ 11.4 Hz, J2 ¼ 4.7 Hz, H-3
a
arom. ring), 2.29e2.23 (m, 1H), 2.22e2.16 (m, 1H), 2.01 (dd, 1H,
J1 ¼12.7 Hz, J2 ¼ 7.8 Hz),1.90 (t,1H, J ¼ 11.4 Hz),1.01, 0.96, 0.95, 0.80,
0.74 (15H, all s, 5 ꢀ eCH3) ppm; 13C NMR (126 MHz, CDCl3)
4.1.5.29. 30-(4-Iodobenzylidene)betulinic acid 4.36. The product
4.36 was obtained as a white solid, 90 mg (42%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
d
182.02, 152.82, 137.33, 134.92, 130.72, 129.45 (2C), 128.11, 126.56
p. 175e178 ꢁC; ½a D22
ꢃ
ꢂ1ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
(2C), 112.20, 79.20, 56.60, 55.50, 50.85, 50.66, 42.55, 40.88, 40.27,
39.01, 38.85, 38.70, 37.36, 37.09, 34.52, 33.27, 32.38, 29.99, 28.15,
27.50, 27.11, 21.35, 21.19, 18.43, 16.27 (2C), 15.49, 14.93 ppm; HRMS
(ESIþ) m/z calcd for C38H55O3 [MþH]þ 559.4146, found 559.4146; IR
d
7.66e7.59 (m, 2H, arom. ring), 7.21e7.14 (m, 2H, arom. ring), 6.78
(d, J ¼ 16.2 Hz, 1H, H-31), 6.59 (d, J ¼ 16.2 Hz, 1H, H-30), 5.12, 5.09
(2H, all s, 2 ꢀ H-29), 3.25 (td, J1 ¼ 11.1, J2 ¼ 4.6 Hz, 1H, H-19 ), 3.19
), 2.38e2.31 (m, 1H), 2.22e2.14
b
(DRIFT) nmax 2490e3620, 2939, 2868, 1691, 1605, 1511, 1450 cmꢂ1
.
(dd, J1 ¼ 11.3, J2 ¼ 4.6 Hz, 1H, H-3
a
(m, 1H), 2.01 (dd, J1 ¼ 12.6, J2 ¼ 8.0 Hz, 1H), 1.89 (t, J ¼ 11.3 Hz, 1H),
4.1.5.26. 30-(4-Fluorobenzylidene)betulinic acid 4.33. The product
4.33 was obtained as a white solid, 68 mg (38%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
1.00 (s, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.79 (s, 3H), 0.74 (s, 3H) ppm;
13C NMR (126 MHz, CDCl3)
d 181.83, 152.47, 137.77, 137.24, 132.62,
128.37, 126.98, 113.50, 92.58, 79.17, 56.55, 55.47, 50.89, 50.62, 42.53,
40.86, 39.98, 38.99, 38.83, 38.68, 37.34, 37.03, 34.50, 33.31, 32.35,
29.95, 28.14, 27.47, 27.22, 21.17, 18.40, 16.25 (2C), 15.47, 14.91 ppm;
HRMS (ESIꢂ) m/z calcd for C37H50O3I [MꢂH]ꢂ 669.2799, found
669.2811; IR (DRIFT) nmax 3612, 3430, 3197, 2936, 2867, 2610, 1960,
p. 149e151 ꢁC; ½a D22
ꢃ
þ3ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
d
7.43e7.37 (m, 2H, arom. ring), 7.03e6.97 (m, 2H, arom. ring), 6.71
(d, 1H, J ¼ 16.2 Hz, H-31), 6.64 (d, 1H, J ¼ 16.2 Hz, H-30), 5.10, 5.06
(2H, all s, 2 ꢀ H-29), 3.26 (td, 1H, J1 ¼ 11.1 Hz, J2 ¼ 4.7 Hz, H-19 ),
), 2.39e2.32 (m, 1H),
b
1681, 1602, 1483, 1449 cmꢂ1
.
3.19 (dd, 1H, J1 ¼ 11.4 Hz, J2 ¼ 4.7 Hz, H-3
a
2.29e2.23 (m, 1H), 2.19 (td, 1H, J1 ¼ 12.4 Hz, J2 ¼ 3.3 Hz), 2.02 (dd,
1H, J1 ¼12.8 Hz, J2 ¼ 7.8 Hz), 1.90 (t, 1H, J ¼ 11.4 Hz), 1.01, 0.96, 0.95,
0.81, 0.74 (15 H, all s, 5 ꢀ eCH3) ppm; 13C NMR (126 MHz, CDCl3)
4.1.5.30. 30-(4-Cyanobenzylidene)betulinic acid 4.37. The product
4.37 was obtained as a white solid, 160 mg (88%) by the general
procedure (yellow-orange ylide color), while the reaction time was
d
181.78, 162.35 (d, JCF ¼ 247.0 Hz), 152.56, 133.88 (d, JCF ¼ 2.9 Hz),
18 h: m. p. 171e174 ꢁC; ½a D22
ꢃ
ꢂ1ꢁ (c 0.29, THF); 1H NMR (500 MHz,
131.57, 128.09 (d, JCF ¼ 7.9 Hz), 126.95, 115.64 (d, JCF ¼ 21.5 Hz),
112.78, 79.19, 56.58, 55.50, 50.85, 50.65, 42.55, 40.88, 40.13, 39.01,
38.85, 38.69, 37.37, 37.05, 34.52, 33.27, 32.36, 29.99, 28.15, 27.50,
27.19, 21.19, 18.42, 16.27 (2C), 15.49, 14.93 ppm; HRMS (ESIþ) m/z
calcd for C37H52O3F [MþH]þ 563.3895, found 563.3899; IR (DRIFT)
CDCl3)
d 7.60e7.57 (m, 2H, arom. ring), 7.52e7.48 (m, 2H, arom.
ring), 6.88 (d, J ¼ 16.1 Hz, 1H, H-31), 6.66 (d, J ¼ 16.2 Hz, 1H, H-30),
5.20, 5.19 (2H, all s, 2 ꢀ H-29), 3.26 (td, J1 ¼ 11.1, J2 ¼ 4.7 Hz, 1H, H-
19
b
), 3.19 (dd, J1 ¼ 11.3, J2 ¼ 4.7 Hz, 1H, H-3
a), 2.22e2.15 (m, 1H),
nmax 2450e3650, 2939, 2869, 1693, 1600, 1508, 1452 cmꢂ1
.
2.02 (dd, J1 ¼ 12.7, J2 ¼ 8.0 Hz, 1H), 1.89 (t, J ¼ 11.3 Hz, 1H), 1.01 (s,
3H), 0.96 (s, 3H), 0.93 (s, 3H), 0.80 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR
(126 MHz, CDCl3)
d 181.97, 152.20, 142.26, 135.56, 132.50, 127.01,
4.1.5.27. 30-(4-Chlorobenzylidene)betulinic acid 4.34. The product
4.34 was obtained as a white solid, 81 mg (44%) by the general
procedure (orange ylide color), while the reaction time was 18 h: m.
126.30,119.16,115.30,110.47, 79.14, 56.56, 55.45, 50.95, 50.58, 42.51,
40.84, 39.83, 38.97, 38.82, 38.65, 37.33, 36.97, 34.48, 33.27, 32.30,
29.94, 28.13, 27.42, 27.31, 21.15, 18.38, 16.24 (2C), 15.47, 14.89 ppm;
HRMS (ESIꢂ) m/z calcd for C38H50O3N [MꢂH]ꢂ 568.3785, found
568.3788; IR (DRIFT) nmax 3421, 3261, 2931, 2867, 2613, 2223 (C≡N),
p. 173e176 ꢁC; ½a D22
ꢃ
þ11ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
d
7.39e7.34 (m, 2H, arom. ring), 7.30e7.26 (m, 2H, arom. ring), 6.76
(d, 1H, J ¼ 16.2 Hz, H-31), 6.63 (d, 1H, J ¼ 16.2 Hz, H-30), 5.12, 5.09
(2H, all s, 2 ꢀ H-29), 3.25 (td, 1H, J1 ¼ 11.1 Hz, J2 ¼ 4.7 Hz, H-19 ),
), 2.29e2.22 (m, 1H), 2.18
1724, 1673, 1601, 1503, 1450 cmꢂ1
.
b
3.19 (dd, 1H, J1 ¼11.4 Hz, J2 ¼ 4.7 Hz, H-3
a
4.1.5.31. 30-(4-Trifluoromethylbenzylidene)betulinic
The product 4.38 was obtained as a white solid, 105 mg (54%) by the
acid
4.38.
(td, 1H, J1 ¼ 12.5 Hz, J2 ¼ 3.6 Hz), 2.02 (dd, 1H, J1 ¼ 12.8 Hz,
J2 ¼ 7.8 Hz), 1.90 (t, 1H, J ¼ 11.3 Hz), 1.01, 0.96, 0.94, 0.80, 0.74 (15H,
all s, 5 ꢀ eCH3) ppm; 13C NMR (126 MHz, CDCl3)
d 181.85, 152.42,
general procedure (orange ylide color), while the reaction time was
2 h: m. p. 155e158 ꢁC; ½a D22
ꢃ
ꢂ3ꢁ (c 0.29, THF); 1H NMR (500 MHz,
136.15,132.98,132.34,128.80 (2C),127.73 (2C),126.79,113.24, 79.10,
56.50, 55.41, 50.81, 50.56, 42.47, 40.80, 39.98, 38.93, 38.77, 38.62,
CDCl3)
d
7.57e7.51 (m, 4H, arom. ring), 6.87 (d, J ¼ 16.2 Hz, 1H, H-
13

ꢀ
J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
31), 6.69 (d, J ¼ 16.2 Hz, 1H, H-30), 5.18, 5.15 (2H, all s, 2 ꢀ H-29),
37.95, 35.65, 34.32, 33.27, 30.97, 28.60, 28.51, 28.03, 22.27,19.44,16.71,
16.69, 16.09, 15.14 ppm; HRMS (ESIþ) m/z calcd for C38H53O5 [MþH]þ
589.3888, found 589.3889; IR (DRIFT) nmax 3664, 3599, 3394, 2932,
3.27 (td, J1 ¼ 11.1, J2 ¼ 4.7 Hz, 1H, H-19 ), 3.19 (dd, J1 ¼ 11.4,
b
J2 ¼ 4.7 Hz, 1H, H-3
a), 2.39e2.33 (m, 1H), 2.22e2.16 (m, 1H), 2.03
(dd, J1 ¼ 12.7, J2 ¼ 7.9 Hz, 1H), 1.91 (t, J ¼ 11.3 Hz, 1H), 1.01 (s, 3H),
2868, 2000, 1702, 1674, 1604, 1465, 1449 cmꢂ1
.
0.96 (s, 3H), 0.95 (s, 3H), 0.80 (s, 3H), 0.73 (s, 3H) ppm; 13C NMR
(126 MHz, CDCl3)
d 181.88, 152.37, 141.22, 134.32, 129.19 (q,
4.1.5.35. 30-(4-Methoxybenzylidene)betulinic acid 4.42. The prod-
uct 4.42 was obtained as a white solid, 70 mg (38%) by the general
procedure (red ylide color), while the reaction time was 2 h: m. p.
JCF ¼ 32.4 Hz), 126.71 (2C), 125.66 (d, JCF ¼ 3.6 Hz), 124.39 (q,
JCF ¼ 271.8 Hz, CF3 group), 114.39, 79.19, 56.58, 55.48, 50.94, 50.63,
42.55, 40.87, 39.96, 38.99, 38.84, 38.70, 37.35, 37.03, 34.51, 33.31,
32.35, 29.96, 28.13, 27.46, 27.28, 21.19, 18.41, 16.26, 16.25, 15.46,
14.91 ppm; HRMS (ESIꢂ) m/z calcd for C38H50O3F3 [MꢂH]ꢂ
611.3707, found 611.3718; IR (DRIFT) nmax 3671, 3432, 3216, 2934,
156e159 ꢁC; ½a 2D2
ꢃ
ꢂ1ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
d
7.41e7.34 (m, 2H, arom. ring), 6.87e6.84 (m, 2H, arom. ring), 6.67
(d, J ¼ 16.3 Hz, 1H, H-31), 6.63 (d, J ¼ 16.3 Hz, 1H, H-30), 5.06, 5.01
(2H, all s, 2 ꢀ H-29), 3.81 (s, 3H, methoxy eCH3), 3.26 (td, J1 ¼ 11.1,
2868, 2615, 9112, 1681, 1613, 1450 cmꢂ1
.
J2 ¼ 4.7 Hz, 1H, H-19
b
), 3.19 (dd, J1 ¼ 11.4, J2 ¼ 4.7 Hz, 1H, H-3
a),
2.37e2.32 (m, 1H), 2.01 (dd, J1 ¼ 12.7, J2 ¼ 7.9 Hz, 1H), 1.90 (t,
J ¼ 11.3 Hz, 1H), 1.01 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.80 (s, 3H),
4.1.5.32. 30-(4-Nitrobenzylidene)betulinic acid 4.39. The product
4.39 was obtained as a yellow solid, 90 mg (48%) by the general
procedure (dark violet ylide color), while the reaction time was 18 h
0.74 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
d 181.97, 159.20,
152.84, 130.51, 129.73, 127.78 (2C), 127.59, 114.18 (2C), 111.68, 79.19,
56.55, 55.46, 55.41, 50.75, 50.63, 42.52, 40.84, 40.16, 38.96, 38.82,
38.65, 37.31, 37.07, 34.49, 33.30, 32.37, 29.97, 28.12, 27.39, 27.11,
21.17, 18.40, 16.24 (2C), 15.48, 14.90 ppm; HRMS (ESIꢂ) m/z calcd for
and the reaction temperature was 70 ꢁC: m. p.176e178 ꢁC; ½a 2D2
ꢃ
þ1ꢁ
(c 0.29, THF); 1H NMR (400 MHz, CDCl3)
d
8.21e8.14 (m, 2H, arom.
ring), 7.59e7.53 (m, 2H, arom. ring), 6.94 (d, J ¼ 16.2 Hz, 1H, H-31),
C
38H53O4 [MꢂH]ꢂ 573.3938, found 573.3951; IR (DRIFT) nmax 3447,
6.72 (d, J ¼ 16.2 Hz, 1H, H-30), 5.24, 5.23 (2H, all s, 2 ꢀ H-29), 3.28
2935, 2867, 1680, 1603, 1510, 1450 cmꢂ1
.
(td, J1 ¼ 11.1, J2 ¼ 4.7 Hz, 1H, H-19 ), 3.19 (dd, J1 ¼ 11.1, J2 ¼ 4.7 Hz,
b
1H, H-3
1.02 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.80 (s, 3H), 0.74 (s, 3H) ppm;
13C NMR (101 MHz, CDCl3)
181.78, 152.22, 146.79, 144.29, 136.51,
a
), 2.03 (dd, J1 ¼12.8, J2 ¼ 7.8 Hz,1H),1.91 (t, J ¼ 11.3 Hz,1H),
4.1.5.36. 30-(4-{t-Butyl}benzylidene)betulinic acid 4.43. The prod-
uct 4.43 was obtained as a white solid, 121 mg (63%) by the general
procedure (orange ylide color) while the reaction time was 2 h: m.
d
127.01, 125.92, 124.18, 115.87, 79.14, 56.57, 55.47, 51.00, 50.60, 42.53,
40.86, 39.75, 38.99, 38.84, 38.67, 37.35, 36.98, 34.50, 33.31, 32.31,
29.95, 28.14, 27.46, 27.38, 21.17, 18.40, 16.27 (2C), 15.47, 14.90 ppm;
HRMS (ESIꢂ) m/z calcd for C37H50O5N [MꢂH]ꢂ 588.3684, found
588.3688; IR (DRIFT) nmax 3430, 2934, 2867, 1679, 1592, 1512
p. 165e168 ꢁC; ½a D22
ꢃ
þ4ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
d
7.42e7.37 (m, 2H, arom. ring), 7.36e7.33 (m, 2H, arom. ring), 6.77
(d, J ¼ 16.2 Hz, 1H, H-31), 6.68 (d, J ¼ 16.2 Hz, 1H, H-30), 5.10, 5.05
(2H, all s, 2 ꢀ H-29), 3.29 (td, J1 ¼ 11.1, J2 ¼ 4.7 Hz, 1H, H-19 ), 3.20
), 2.40e2.33 (m, 1H), 2.03 (dd,
b
(NeO), 1450 cmꢂ1
.
(dd, J1 ¼ 11.4, J2 ¼ 4.7 Hz, 1H, H-3
a
J1 ¼12.6, J2 ¼ 8.0 Hz, 1H), 1.91 (t, J ¼ 11.3 Hz, 1H), 1.32 (s, 9H, t-butyl
4.1.5.33. 30-(Pyridin-4-ylbenzylidene)betulinic acid 4.40. The prod-
uct 4.40 was obtained as a yellowish solid, 38 mg (22%) by the
general procedure (brown ylide color) without extraction (the re-
action mixture was evaporated after completion followed by pu-
rification) while the reaction time was 18 h. Purification was
group),1.02 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.81 (s, 3H), 0.75 (s, 3H)
ppm; 13C NMR (126 MHz, CDCl3)
d 182.26, 152.84, 150.62, 134.92,
130.90, 127.97, 126.36 (2C), 125.64 (2C), 112.24, 79.21, 56.60, 55.49,
50.82, 50.65, 42.55, 40.87, 40.30, 38.99, 38.84, 38.68, 37.34, 37.10,
34.71, 34.51, 33.23, 32.39, 31.44 (3C), 29.98, 28.14, 27.45, 27.07, 21.18,
18.42, 16.26 (2C), 15.49, 14.93 ppm; HRMS (ESIꢂ) m/z calcd for
performed using mobile phase CHCl3/MeOH 50
: 1: m. p.
C
41H59O3 [MꢂH]ꢂ 599.4459, found 599.4465; IR (DRIFT) nmax 3672,
166e169 ꢁC; ½a 2D2
ꢃ
ꢂ2ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
3448, 3229, 2936, 2867, 2558, 1898, 1734, 1682, 1601, 1518,
d
8.57e8.53 (m, 2H, arom. ring), 7.38e7.34 (m, 2H, arom. ring), 7.00
1462 cmꢂ1
.
(d, J ¼ 16.2 Hz, 1H, H-31), 6.63 (d, J ¼ 16.2 Hz, 1H, H-30), 5.24, 5.23
(2H, all s, 2 ꢀ H-29), 3.32 (td, J1 ¼ 11.1, J2 ¼ 4.7 Hz, 1H, H-19 ), 3.18
), 2.40e2.34 (m, 1H), 2.03 (dd,
b
(dd, J1 ¼ 11.3, J2 ¼ 4.7 Hz, 1H, H-3
a
4.1.5.37. 30-(3-Chloro-4-fluorobenzylidene)betulinic
acid
4.46.
J1 ¼12.5, J2 ¼ 8.0 Hz, 1H), 1.87 (t, J ¼ 11.3 Hz, 1H), 1.01 (s, 3H), 0.96 (s,
The product 4.46 was obtained as yellowish solid, 150 mg (79%) by
the general procedure (orange ylide color) while the reaction time
3H), 0.95 (s, 3H), 0.80 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR (126 MHz,
was 18 h: m. p. 165e168 ꢁC; ½a D22
ꢃ
þ4ꢁ (c 0.29, THF); 1H NMR
CDCl3)
d 179.98, 152.40, 148.89 (2C), 146.34, 137.35, 125.15, 121.39
(2C), 116.14, 79.12, 56.48, 55.50, 51.03, 50.69, 42.55, 40.97, 39.63,
39.00, 38.86, 38.54, 37.35, 37.04, 34.54, 33.43, 32.45, 30.01, 28.14,
27.50, 21.21, 18.44, 16.27, 16.26, 15.50, 14.89 ppm; HRMS (ESIþ) m/z
calcd for C36H52O3N [MþH]þ 546.3942, found 546.3946; IR (DRIFT)
(500 MHz, CDCl3) d 7.51e7.42 (m, 1H, arom. ring), 7.30e7.26 (m, 1H,
arom. ring), 7.10e7.04 (m, 1H, arom. ring), 6.70 (d, J ¼ 16.2 Hz, 1H,
H-31), 6.57 (d, J ¼ 16.2 Hz, 1H, H-30), 5.12, 5.10 (2H, all s, 2 ꢀ H-29),
3.27e3.16 (m, 2H, H-19b and H-3a), 2.21e2.14 (m, 1H), 2.01 (dd,
nmax 2400e3670, 2938, 2867, 1932, 1700, 1600, 1549, 1450 cmꢂ1
.
J1 ¼12.8, J2 ¼ 7.8 Hz, 1H), 1.89 (t, J ¼ 11.3 Hz, 1H), 1.01 (s, 3H), 0.96 (s,
3H), 0.94 (s, 3H), 0.80 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR (126 MHz,
4.1.5.34. 30-(4-Carboxybenzylidene)betulinic acid 4.41. The product
4.41 was obtained as white solid, 96 mg (51%) by the general pro-
cedure using double amount of t-BuOK (red ylide color) while the
reaction time was 18 h. Purification was performed using mobile
CDCl3)
d
181.90, 157.42 (d, JCF ¼ 249.5 Hz), 152.30, 135.10 (d,
JCF ¼ 3.8 Hz), 132.97, 128.41 (d, JCF ¼ 3.6 Hz), 126.19 (d, JCF ¼ 6.2 Hz),
125.69, 121.32 (d, JCF ¼ 17.9 Hz), 116.76 (d, JCF ¼ 21.5 Hz), 113.74,
79.18, 56.56, 55.47, 50.93, 50.62, 42.53, 40.86, 39.95, 38.99, 38.84,
38.68, 37.34, 37.01, 34.50, 33.29, 32.33, 29.96, 28.14, 27.46, 27.23,
21.17, 18.39, 16.26 (2C), 15.47, 14.91 ppm; HRMS (ESIꢂ) m/z calcd for
phase toluene/MeOH 5 : 1: m. p. 217e220 ꢁC; ½a D22
ꢃ
ꢂ3ꢁ (c 0.29, THF);
1H NMR (500 MHz, CD3OD)
d 7.99e7.94 (m, 2H, arom. ring), 7.57e7.51
C
37H49O3ClF [MꢂH]ꢂ 595.3349, found 595.3354; IR (DRIFT) nmax
(m, 2H, arom. ring), 6.99 (d, J ¼ 16.2 Hz,1H, H-31), 6.76 (d, J ¼ 16.2 Hz,
3438, 2939, 2867, 1688, 1597, 1498, 1451 cmꢂ1
.
1H, H-30), 5.21, 5.18 (2H, all s, 2 ꢀ H-29), 3.11 (dd, J1 ¼11.4, J2 ¼ 4.8 Hz,
1H, H-3a),1.03 (s, 3H), 0.97 (s, 3H), 0.94(s, 3H), 0.84(s, 3H), 0.74(s, 3H)
ppm; 13C NMR (126 MHz, CDCl3)
d
180.08, 170.20, 154 0.35, 143.47,
4.1.5.38. 30-(4-Bromo-2-fluorobenzylidene)betulinic
acid
4.47.
135.32, 131.16 (2C), 129.92, 128.09, 127.32 (2C), 114.80, 79.66, 57.54,
56.86, 52.08, 52.01, 43.53, 41.96, 41.27, 40.06, 39.94, 39.75, 38.33,
The product 4.47 was obtained as a white solid, 196 mg (96%) by the
general procedure (orange ylide color), while the reaction time was
14

ꢀ
J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
6 h: m. p. 177e180 ꢁC; ½a D22
ꢃ
ꢂ8ꢁ (c 0.29, THF); 1H NMR (500 MHz,
prepared and diluted according to the particular cell type and the
expected target cell density (25,000e35,000 cells/mL based on cell
growth characteristics). Cells were added by automatic pipettor
CDCl3)
d 7.40e7.35 (m, 1H, arom. ring), 7.25e7.19 (m, 2H, arom.
ring), 6.83 (d, J ¼ 16.4 Hz, 1H, H-31), 6.74 (d, J ¼ 16.4 Hz, 1H, H-30),
(30
dissolved in 100% DMSO and four-fold dilutions of the intended test
concentration were added in 0.15 L aliquots at time zero to the mi-
mL) into 384 well microtiter plates. All tested compounds were
5.15, 5.13 (2H, all s, 2 ꢀ H-29), 3.25 (td, J1 ¼ 11.1, J2 ¼ 4.7 Hz, 1H, H-
19
b
), 3.18 (dd, J1 ¼ 11.4, J2 ¼ 4.7 Hz, 1H, H-3
a), 2.38e2.31 (m, 1H),
m
2.23e2.17 (m, 1H), 2.02 (dd, J1 ¼ 12.7, J2 ¼ 7.9 Hz, 1H), 1.90 (t,
crotiter plate wells by the echoacustic noncontact liquid handler
Echo550 (Labcyte). The experiments were performed in technical
duplicates and three biological replicates at least. The cells were
incubated with the tested compounds for 72 h at 37 ꢁC, in a 5% CO2
atmosphereat100%humidity. At theendof the incubationperiod, the
J ¼ 11.4 Hz, 1H), 1.00 (s, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.80 (s, 3H),
0.74 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
d 181.52, 160.11 (d,
JCF ¼ 254.0 Hz), 152.55, 134.57, 128.23 (d, JCF ¼ 4.2 Hz), 127.60 (d,
JCF ¼ 3.2 Hz), 124.84 (d, JCF ¼ 12.1 Hz), 120.77 (d, JCF ¼ 9.7 Hz), 119.61
(d, JCF ¼ 4.4 Hz), 119.39, 114.17, 79.18, 56.53, 55.49, 50.78, 50.64,
42.55, 40.87, 40.29, 39.01, 38.85, 38.65, 37.36, 37.03, 34.51, 33.11,
32.35, 29.94, 28.14, 27.51, 27.22, 21.16, 18.42, 16.24 (2C), 15.48,
14.89 ppm; HRMS (ESIꢂ) m/z calcd for C37H49O3BrF [MꢂH]ꢂ
639.2844, found 639.2852; IR (DRIFT) nmax 3655, 3450, 3255, 2930,
cells were assayed by using the MTS test. Aliquots (5 mL) of the MTS
stock solution were pipetted into each well and incubated for an
additional1e4h. Afterthisincubationperiod, theopticaldensity(OD)
was measured at 490 nm with an Envision reader (PerkinElmer).
Tumor cell survival (TCS) was calculated by using the following
equation:
2867, 2615, 1963, 1727, 1687, 1596, 1562, 1481, 1462, 1450 cmꢂ1
.
4.1.5.39. 30-(Pentafluorobenzylidene)betulinic acid 4.48. The prod-
uct 4.48 was obtained as white solid, 160 mg (79%) by the general
procedure (yellow ylide color) while the reaction time was 18 h: m.
ODdrugꢂexposed well
mean ODcontrol wells
TCS ¼
,100 %
(1)
p. 167e170 ꢁC; ½a D22
ꢃ
ꢂ12ꢁ (c 0.29, THF); 1H NMR (500 MHz, CDCl3)
The IC50 value, the drug concentration that is lethal to 50% of the
tumor cells, was calculated from the appropriate dose-response
curves in Dotmatics software.
d
7.05 (d, J ¼ 16.7 Hz, 1H, H-31), 6.55 (d, J ¼ 16.7 Hz, 1H, H-30), 5.21,
5.20 (2H, all s, 2 ꢀ H-29), 3.27e3.16 (m, 2H, H-19
b and H-3a), 2.34
(dd, J1 ¼ 9.7, J2 ¼ 3.0 Hz, 1H), 2.03 (dd, J1 ¼12.8, J2 ¼ 7.8 Hz, 1H), 1.87
(t, J ¼ 11.3 Hz, 1H), 1.00 (s, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.81 (s, 3H),
4.2.2. Cell cycle and apoptosis analysis
0.75 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
d 181.50, 152.32,
This part was performed in the same manner as in our previous
work [21]. Suspension of CCRF-CEM cells, seeded at a density of
106 cells/mL in 6-well panels, were cultivated with 1 ꢀ or 5 ꢀ IC50 of
the tested compound in a humidified CO2 incubator at 37 ꢁC in
RPMI 1640 cell culture medium containing 10% fetal calf serum,
146.00e145.68 (m, 1C), 144.01e143.71 (m, 1C), 140.71,
140.16e139.72 (m, 1C), 139.11e138.45 (m, 1C), 137.06e136.68 (m,
1C), 115.77, 112.99e112.68 (m, 1C), 112.64, 79.18, 56.51, 55.50, 50.79,
50.63, 42.56, 40.87, 40.39, 39.01, 38.86, 38.64, 37.36, 37.01, 34.51,
32.82, 32.30, 29.91, 28.14, 27.50, 27.19, 21.14, 18.41, 16.22 (2C), 15.46,
14.85 ppm; HRMS (ESIꢂ) m/z calcd for C37H46O3F5 [MꢂH]ꢂ
633.3362, found 633.3372; IR (DRIFT) nmax 3682, 3461, 3271, 2934,
2867, 2617, 1731, 1639, 1520, 1494, 1465 cm1.
10 mmol/L glutamine, 100 U/mL penicillin, and 100 mg/mL strep-
tomycin. Together with the treated cells, a control sample con-
taining the vehicle was harvested at the same time point after 24 h.
After another 24 h, cells were then washed with cold PBS and fixed
in 70% ethanol added dropwise, and stored overnight at ꢂ20 ꢁC.
Afterward, cells were washed in hypotonic citrate buffer, treated
4.1.5.40. 30-(3,5-Dimethoxybenzylidene)betulinic
acid
4.49.
The product 4.49 was obtained as white solid, 54 mg (28%) by the
with RNAse (50 mg/mL), and stained with propidium iodide. The
general procedure (red ylide color) while the reaction time was
flow cytometer using a 488 nm single beam laser (FACSCalibur,
Becton Dickinson) was used for measurement. Cell cycle was
analyzed in the program ModFitLT (Verity), and apoptosis was
measured in a logarithmic model expressing a percentage of the
particles with propidium content lower than cells in the G0/G1
phase (<G1) of the cell cycle. Half of the sample was used for
pH3Ser10 antibody (Sigma) labeling and subsequent flow cytometry
analysis of mitotic cells [45].
18 h: m. p. 158e161 ꢁC; ½a D22
ꢃ
ꢂ1ꢁ (c 0.29, THF); 1H NMR (500 MHz,
CDCl3)
d
6.76 (d, J ¼ 16.2 Hz, 1H, H-31), 6.65e6.58 (m, 3H, H-30 and
2 ꢀ H e arom. ring), 6.38e6.35 (m,1H, arom. ring), 5.11, 5.07 (2H, all
s, 2 ꢀ H-29), 3.81 (s, 6H, 2 ꢀ methoxy eCH3), 3.26 (td, J1 ¼ 11.1,
J2 ¼ 4.7 Hz, 1H, H-19
b
), 3.18 (dd, J1 ¼ 11.4, J2 ¼ 4.7 Hz, 1H, H-3
a),
2.38e2.32 (m, 1H), 2.22e2.15 (m, 1H), 2.01 (dd, J1 ¼12.8, J2 ¼ 7.8 Hz,
1H), 1.90 (t, J ¼ 11.3 Hz, 1H), 1.01 (s, 3H), 0.96 (s, 3H), 0.94 (s, 3H),
0.80 (s, 3H), 0.74 (s, 3H) ppm; 13C NMR (126 MHz, CDCl3)
d 181.43,
161.08, 152.60, 139.76, 132.24, 128.16, 113.08, 104.72, 100.06, 79.18,
56.56, 55.52, 50.90, 50.65, 42.55, 40.87, 40.17, 39.01, 38.85, 38.70,
37.36, 37.05, 34.52, 33.27, 32.37, 29.99, 28.14, 27.51, 27.16, 21.19,
18.41, 16.28, 16.25, 15.47, 14.93 ppm; HRMS (ESIþ) m/z calcd for
4.2.3. BrdU incorporation analysis (DNA synthesis)
This part was performed in the same manner as in our previous
work [21]. For this analysis, the same procedure of cultivation as
previously was used. Before harvesting, 10
mmol/L 5-bromo-2-
C
39H57O5 [MþH]þ 605.4201, found 605.4205; IR (DRIFT) nmax 3449,
deoxyuridine (BrdU) was added to the cells for puls-labeling for
30 min. Cells were fixed with ice-cold 70% ethanol and stored
overnight. Before the analysis, cells were washed with PBS and
resuspended in 2 mol/L HCl for 30 min at room temperature to
denature their DNA. Following neutralization with 0.1 mol/L
Na2B4O7 (Borax), cells were washed with PBS containing 0.5%
Tween-20 and 1% BSA. Staining with primary anti-BrdU antibody
(Exbio) for 30 min at room temperature in the dark followed. Cells
were then washed with PBS and stained with secondary anti-
mouse-FITC antibody (Sigma). Cells were then rewashed with PBS
and incubated with propidium iodide (0.1 mg/mL) and RNAse A
(0.5 mg/mL) for 1 h at room temperature in the dark and afterward
analyzed by flow cytometry using a 488 nm single beam laser
(FACSCalibur, Becton Dickinson) [45].
2938, 2867, 1687, 1589, 1455 cmꢂ1
.
4.2. Biology
4.2.1. Cytotoxic MTS assay
MTS assay was performed at the Institute of Molecular and
TranslationalMedicine byaroboticplatform (HighResBiosolutions)in
the same manner as in our previous work [21]. HCT116 and HCT116
p53ꢂ/ꢂ cell lines were obtained from Horizon Discovery Ltd., CCRF-
CEM, K562, A549, U2OS, BJ, MRC-5 cell lines were obtained from
ATCC (American Type Culture Collection), and resistant cancer cell
lines CEM-DNR and K562-TAX were developed in our laboratory ac-
cording to our procedure described in Ref. [44]. Cell suspensions were
15

ꢀ
J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
4.2.4. BrU incorporation analysis (RNA synthesis)
4.2.7. Pharmacological parameters
This part was performed in the same manner as in our previous
work [21]. Cells were cultured and treated as above. Before har-
vesting, pulse-labeling with 1 mmol/L 5-bromouridine (BrU) for
30 min followed. The cells were then fixed in 1% buffered para-
formaldehyde with 0.05% of NP-40 at room temperature for 15 min,
and then stored in 4 ꢁC overnight. Before measurement, they were
washed with PBS with 1% glycine, washed in PBS again, and stained
by primary anti-BrdU antibody cross-reacting to BrU (Exbio) for
30 min at room temperature in the dark. After another washing step
in PBS, cells were stained by a secondary anti-mouse-FITC antibody
(Sigma). Following the staining, cells were washed with PBS and
fixed with 1% PBS buffered paraformaldehyde with 0.05% of NP-40
for 1 h. Cells were washed by PBS, incubated with propidium io-
dide (0.1 mg/mL) and RNAse A (0.5 mg/mL) for 1 h at room tem-
perature in the dark, and finally analyzed by flow cytometry using a
488 nm single beam laser (FACSCalibur, Becton Dickinson) [45].
Same instruments and methods for the measurements of phar-
macological parameters were used as described in our earlier work
[21]. Stability in human plasma, microsomal stability assay, parallel
artificial membrane permeability assay, and protein plasma binding
assay were performed in the same manner as we described earlier
[21].
4.2.7.1. Cell cultures. Caco-2 and MDCK-MDR1 cells were main-
tained in DMEM at pH ¼ 7.4, supplemented with 10% fetal bovine
serum and 0.1% penicillin-streptomycin solution in a humidified
atmosphere (5% CO2, 95% air, 37 ꢁC). The cells were seeded on tissue
culture polyester membrane filters (pore size 0.4
mm for Caco-2 and
1
mm for MDCK-MDR1) in 96-well Transwell® plates (Corning). The
culture medium was added to both the donor and the acceptor
compartment. The medium was changed every second day.
4.2.7.2. Transport studies. Caco-2 and MDCK-MDR1 monolayers
grown in Transwell® (96-well) plates [37,38] and were used for
transport studies when they had differentiated and the monolayer
was intact, as checked by Lucifer Yellow Rejection Assay. Prior to the
experiment, the cells were washed twice with HBSS (Gibco) and pre-
equilibrated for 1 h with HBSS buffered at pH ¼ 7.4. After removing
the medium, the cells were treated with the studied compound
4.2.5. Annexin V/propidium iodide staining
Apoptosis in CCRF-CEM cells was assessed using Annexin V/pro-
pidium iodide double staining as described in detail elsewhere [21].
Briefly, cells were treated with 4.22 or 4.39 derivatives at 1 ꢀ IC50 and
5 ꢀ IC50 concentrations for 24 h. Following washing by ice-cold PBS,
cells were incubated with Annexin V-FITC (5 mL) and PI (5 mL) at room
temperature for 20 min in the dark. After the incubation period, cells
were centrifuged, resuspended in 100 l of annexin binding buffer
(10
mmol/L in HBSS, pH ¼ 7.4) for 1 h for MDCK, and 2 h for Caco-2,
m
respectively. After incubation, followed by lyophilization, the sam-
ples were dissolved in a mobile phase containing internal standard
and analyzed using an Agilent RapidFire 300 High-Throughput Mass
Spectrometry System (Agilent) with subsequent detection on the
mass spectrometer Qtrap 5500 (AB Sciex). All experiments were
done in duplicates. The apparent permeability coefficient Papp (cm/s)
was calculated according to the following equation:
(10 mmol/L HEPES/NaOH of pH ¼ 7.4, 140 mmol/L NaCl, 2.5 mmol/L
CaCl2)and analyzed byFACSAriaII(BectonDickinson)flowcytometer.
At least 10,000 cells per sample were acquired.
4.2.6. Western blot
Western blot analysis was performed in the same manner as we
described earlier [21]. CCRF-CEM cells were treated with the most
active derivatives 4.22 and 4.39 at 1 ꢀ IC50 and 5 ꢀ IC50 concen-
trations for 24 h. Total proteins were extracted from the cells with
RIPA lysis buffer (50 mmol/L Tris-HCl of pH ¼ 8.0, 150 mmol/L NaCl,
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulfate) supplemented with protease inhibitor cocktail (Roche) and
incubated for 30 min on ice with occasional agitation. The lysates
were clarified by centrifugation (14,000g, 20 min, 4 ꢁC) and protein
samples were quantified with a BCA protein assay. Aliquots of ly-
sates were mixed with 4 ꢀ concentrated Laemmli buffer and boiled
dQ
dt C0A
1
Papp
¼
,
(2)
where dQ/dt is the rate of permeation of the drug across the cells
(
(
m
m
mol/s), C0 is the donor compartment concentration at time zero
mol/L), and A is the area of the cell monolayer (cm2). C0 is ob-
tained from the analysis of the dosing solution at the start of the
experiment.
The efflux ratio ER was calculated using the following equation:
at 95 ꢁC for 5 min. An equal amount (15
m
g/well) of protein samples
Papp ðBAÞ
Papp ðABÞ
were separated by 10% SDS-PAGE and transferred onto a 0.2-
mm
ER ¼
(3)
pore size nitrocellulose membrane. The membrane was then
blocked with 5% skimmed milk in Tris buffer saline Tween-20
(TBST) buffer for 1 h at room temperature and probed with spe-
cific primary antibodies (Cell Signaling Technology) at 4 ꢁC over-
night. Primary antibodies were diluted in TBST at the ratio of 1 :
1000. After four cycles (5 min each) of washing with TBST, mem-
branes were incubated with horseradish peroxidase-conjugated
secondary antibodies (mouse or rabbit; Sigma-Aldrich) for 1 h at
room temperature. The chemiluminescence signal was visualized
with the ECL Prime kit (Amersham) and detected by Li-cor Odyssey
(LI-COR Biotechnology) imaging system. As an internal control, an
where Papp (BA) and Papp (AB) represent the apparent permeability of
the test compound from the basal to apical (BeA) and apical to the
basal (AeB) side of the cell monolayer. The compounds having an
efflux ratio ER > 2 are considered as having potential for P-gp
substrate.
The compound recovery can be helpful to interpret permeability
data (Equation (4)). If the recovery is very low, this may indicate
poor solubility, binding of the compound to the hydrophobic plate,
metabolic degradation by cells, or accumulation of the compound
in the cell monolayer.
anti-
b-actin (Sigma-Aldrich) primary antibody at the 1 : 5000
dilution was used.
Total compound in donor and receiver at the end of the experiment ðnmolÞ
Recovery ¼
,100 %
(4)
Initial compound present ðnmolÞ
16

ꢀ
J. Pokorný, D. Olejníkova, I. Frydrych et al.
European Journal of Medicinal Chemistry 224 (2021) 113706
For cell permeability assays, reference drugs with established
Papp and efflux values are used. In Caco-2 cell experiments, three
categories are differentiated according to Papp values: low (Papp < 5
$ 10ꢂ6 cm/s), moderate (5 $ 10ꢂ6 < Papp < 20 $ 10ꢂ6 cm/s) and high
(Papp > 20 $ 10ꢂ6 cm/s). The low Papp standard is rhodamine 123,
while the high Papp reference is verapamil [46]. In MDCK-MDR1 cell
assays atenolol was used as a standard for low Papp (Papp < 10 $
10ꢂ6 cm/s) and propranolol for high Papp reference (Papp > 10 $
10ꢂ6 cm/s) [47].
DRIFT
ESI
FITC
FSC
G0
G1
G2
HBSS
Diffuse reflectance infrared Fourier transform
Electrospray ionization
Fluorescein isothiocyanate
Forward scatter
Gap 0 phase of cell cycle
Gap 1 phase of cell cycle
Gap 2 phase of cell cycle
Hank's balanced salt solution
HCT116 p53ꢂ/ꢂ Colon carcinoma cell line
HCT116
HIV
HRMS
IC50
Colon carcinoma cell line
Author contributions
Human immunodeficiency virus
High-resolution mass spectrometry
Half maximal inhibitory concentration
Infrared
Conceptualization, J.P., M.U., P.D., and M.H.; methodology, J.P.,
ꢁ
S.B., J.S., I.F., J.K., B.L. and S.G.; validation, P.D., and M.U.; formal
IR
analysis, M.U.; investigation, J.P., D.O., S.G., and S.B.; resources, P.D.,
M.H., J.S., and M.U.; writing e original draft preparation, J.P., P.D.
and M.U.; writing e review and editing, P.D., S.B. and M.U.; funding
K562
Chronic myelogenous leukemia cell line
ꢁ
K562-TAX Chronic myelogenous leukemia cell line resistant to
paclitaxel
acquisition, P.D., M.H., and M.U.
LC
Liquid chromatography
M
Mitotic phase of cell cycle
Declaration of competing interest
m. p.
MDCK
MDR
MRC-5
MTS
Melting point
Madin-Darby canine kidney cell line
Multiple drug resistance
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Human noncancer fibroblast cell lines
3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium, tetrazolium salt
Nicotinamide adenine dinucleotide phosphate
Nuclear magnetic resonance
Nonyl phenoxypolyethoxylethanol, commercially
available detergent
Acknowledgment
NADPH
NMR
NP-40
Chemical synthesis and analysis of pharmacological parameters
and detailed protein expression analysis were financially supported
by the Ministry of Health of the Czech Republic (NV19-03-00107),
tests of the cytotoxic activity and cell cycle analysis were supported
by the European Regional Development Fund e Project ENOCH (No.
CZ.02.1.01/0.0/0.0/16_019/0000868), other biological experiments
were funded by the Czech Ministry of Education, Youth and Sports
(CZ-OPENSCREEN e LM2018130 and EATRIS-CZ e LM2018133),
spectral analysis of some final products were supported from the
Czech Science Foundation (21-00902S) stipend for J.P. and D.O. were
paid by the internal grant of Palacký University IGA_PrF_2020_012.
OD
Optical density
PAMPA
PARP
PBS
P-gp
pH
Parallel artificial membrane permeability assay
Poly (ADP-ribose) polymerase
Phosphate buffered saline buffer
P-glycoprotein
Potential of Hydrogen
pH3Ser10 Phospho-histone H3 Serin10
PI
Propidium iodide
r. t.
Room temperature
Appendix A. Supplementary data
RFU
RIPA
RNA
Relative fluorescence unit
Radioimmunoprecipitation assay
Ribonucleic acid
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113706.
RPMI 1640 Growth medium used in cell culture
S
SAR
Synthesis phase of cell cycle
Structure-activity relationship
Abbreviations
SDS-PAGE Sodium dodecylsulphate e polyacrylamide gel
A549
ACN
ADME
APCI
BAX
BCA
Bcl-2
BJ
BrdU
BrU
Lung carcinoma cell line
Acetonitrile
Absorption, distribution, metabolism, and excretion
Atmospheric-pressure chemical ionization
Bcl-2 associated X protein
Bicinchoninic acid assay
B-cell lymphoma family
Human noncancer fibroblast cell line
5-Bromo-2-deoxyuridine
electrophoresis
Side scatter
SSC
TBST
t-BuOK
TCS
THF
TLC
U
U2OS
UV
Tris buffer saline Tween-20
Potassium tert-butoxide
Tumor cell survival
Tetrahydrofuran
Thin-layer chromatography
Unit
Osteosarcoma cell line
Ultraviolet
5-Bromouridine
BSA
Bovine serum albumin
Caco-2
Colorectal adenocarcinoma cell line
References
CCRF-CEM T-lymphoblastic leukemia cell line
CEM-DNR T-lymphoblastic leukemia cell line resistant to
daunorubicin
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