Synthesis and pharmacological effects of diosgenin—Betulinic acid conjugates

Article abstract of DOI:10.3390/molecules25153546

The target diosgenin–betulinic acid conjugates are reported to investigate their ability to enhance and modify the pharmacological effects of their components. The detailed synthetic procedure that includes copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (click reaction), and palladium-catalyzed debenzylation by hydrogenolysis is described together with the results of cytotoxicity screening tests. Palladium-catalyzed debenzylation reaction of benzyl ester intermediates was the key step in this synthetic procedure due to the simultaneous presence of a 1,4-disubstituted 1,2,3-triazole ring in the molecule that was a competing coordination site for the palladium catalyst. High pressure (130 kPa) palladium-catalyzed procedure represented a successful synthetic step yielding the required products. The conjugate 7 showed selective cytotoxicity in human T-lymphoblastic leukemia (CEM) cancer cells (IC₅₀ = 6.5 ± 1.1 μM), in contrast to the conjugate 8 showing no cytotoxicity, and diosgenin (1), an adaptogen, for which a potential to be active on central nervous system was calculated in silico. In addition, 5 showed medium multifarious cytotoxicity in human T-lymphoblastic leukemia (CEM), human cervical cancer (HeLa), and human colon cancer (HCT 116). Betulinic acid (2) and the intermediates 3 and 4 showed no cytotoxicity in the tested cancer cell lines. The experimental data obtained are supplemented by and compared with the in silico calculated physico-chemical and absorption, distribution, metabolism, and excretion (ADME) parameters of these compounds.

Full text of DOI:10.3390/molecules25153546

molecules
Article
Synthesis and Pharmacological Effects of
Diosgenin—Betulinic Acid Conjugates
Zülal Özdemir 1,2 , Michaela Rybková 2, Martin Vlk 1,3 , David Šaman 4, Lucie Rárová 5 and
Zdeneˇk Wimmer 1,2,
*
1
Isotope Laboratory, Institute of Experimental Botany of the Czech Academy of Sciences, Vídenˇská 1083,
14220 Prague 4, Czech Republic; zulalozdemr@gmail.com (Z.Ö.); martin.vlk@fjfi.cvut.cz (M.V.)
Department of Chemistry of Natural Compounds, University of Chemistry and Technology in Prague,
Technická 5, 16628 Prague 6, Czech Republic; rybkova.misa@seznam.cz
Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Brˇehová 7,
11519 Prague 1, Czech Republic
Department of NMR Spectroscopy, Institute of Organic Chemistry and Biochemistry of the Czech Academy
of Sciences, Flemingovo námeˇstí 2, 16610 Prague 6, Czech Republic; nmrsaman@gmail.com
Laboratory of Growth Regulators, Faculty of Science, Palacký University, and Institute of Experimental
Botany of the Czech Academy of Sciences, Šlechtitelu˚ 27, 78371 Olomouc, Czech Republic;
lucie.rarova@upol.cz
2
3
4
5
*
Correspondence: wimmer@biomed.cas.cz or wimmerz@vscht.cz; Tel.: +420-241-062-457
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 July 2020; Accepted: 30 July 2020; Published: 3 August 2020
Abstract: The target diosgenin–betulinic acid conjugates are reported to investigate their ability
to enhance and modify the pharmacological effects of their components. The detailed synthetic
procedure that includes copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (click reaction),
and palladium-catalyzed debenzylation by hydrogenolysis is described together with the results of
cytotoxicity screening tests. Palladium-catalyzed debenzylation reaction of benzyl ester intermediates
was the key step in this synthetic procedure due to the simultaneous presence of a 1,4-disubstituted
1,2,3-triazole ring in the molecule that was a competing coordination site for the palladium
catalyst. High pressure (130 kPa) palladium-catalyzed procedure represented a successful synthetic
step yielding the required products. The conjugate
T-lymphoblastic leukemia (CEM) cancer cells (IC50 = 6.5
showing no cytotoxicity, and diosgenin ( ), an adaptogen, for which a potential to be active on central
nervous system was calculated in silico. In addition, showed medium multifarious cytotoxicity in
human T-lymphoblastic leukemia (CEM), human cervical cancer (HeLa), and human colon cancer
(HCT 116). Betulinic acid ( ) and the intermediates and showed no cytotoxicity in the tested
7
showed selective cytotoxicity in human
±
1.1 M), in contrast to the conjugate
µ
8
1
5
2
3
4
cancer cell lines. The experimental data obtained are supplemented by and compared with the in
silico calculated physico-chemical and absorption, distribution, metabolism, and excretion (ADME)
parameters of these compounds.
Keywords: betulinic acid; diosgenin; Huisgen copper(I)-catalyzed 1,3-dipolar cycloaddition;
catalytic hydrogenation; conjugate; adaptogen; cytotoxicity; ADME parameters
1. Introduction
Saponins belong among plant products displaying multiple biological and pharmacological
activity, and many of them are considered to be adaptogens. This term is used to describe a plant
product capable of (a) producing a nonspecific response resulting in increasing the power of resistance
against multiple (physical, chemical, or biological) stressors; (b) having a normalizing effect, irrespective
Molecules 2020, 25, 3546; doi:10.3390/molecules25153546
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3546
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of the nature of the pathology, so thus being non-toxic; and (c) being harmless and not influencing
normal body functions more than required [1].
Diosgenin, (3β,25R)-spirost-5-en-3-ol (1, Figure 1), is a steroid sapogenin part of the saponin
dioscin found in the tubers of Dioscorea zingiberensis C. H. Wright or Trigonella foenum-graecum L. and
in numbers of legumes. Diosgenin is a widely used precursor in the synthesis of sexual hormones,
peroral contraceptives and other steroids in the pharmaceutical industry [2]. It is an adaptogen,
displaying non-steroidogenic activity along with other beneficial effects [ ]. Diosgenin (
3
1
) is unable to
bind metal ions, and therefore, the change made from more traditional cholesterol/cholesterylamine
system to diosgenin could influence the overall conformation of the bivalent structures, modifying
the metal ions chelating properties. Saponins are always species formed from an aglycone and
several monosaccharide units, the presence of which increases the solubility of saponins in natural
aqueous media [4].
Figure 1. Structure of diosgenin (1). Note: carbon atoms are double-primed as in evaluation of the
NMR spectra of diosgenin derivatives.
During the past decade, a number of preclinical investigations have been made to elucidate
chemopreventive and therapeutic effects of diosgenin [4]. In general, chemoprevention of cancer has
been focused on a regression of multistage process of carcinogenesis. For that purpose, phytochemicals
have been intensively applied due to their high safety, low toxicity, and almost no side effects
(cf. definition for adaptogens above). So far, the main antitumor effect of diosgenin has been observed
in treatment of colon cancer [
in HT-29 human colon cancer cell line [
gene Bcl-2 [ ]. Diosgenin is not metabolized in the human body, and it is considered to represent a
5]. It affects cell growth and differentiation, and it also induces apoptosis
5]. It induces caspase-3 expression and suppresses anti-apoptotic
5
safe natural drug. It has also been investigated for treating hyperglycemia, hypercholesterolemia,
hypertriacylglycerolemia, and Alzheimer’s disease [4,6].
Betulinic acid, 3
perspective triterpenoid plant product with a broad spectrum of effects, e.g., antitumor, anti-HIV,
cytostatic, and anti-inflammatory [ ]. It can be obtained from the bark of Betula pendula Roth,
β-hydroxylup-20(29)-en-28-oic acid (2; Scheme 1), is a pharmacologically
7,8
widely distributed in Europe, and from a number of subtropical and tropical plants [7,8]. In 1995,
a role of betulinic acid (2) in cell apoptosis was described [9].
The objectives of this investigation were: (a) to design simple conjugates on the basis of diosgenin (
1)
and betulinic acid ( ) connected by a spacer bearing 1,4-disubstitued 1,2,3-triazole ring, (b) to investigate
2
the key reaction steps of the synthetic procedure and to optimize them, as well as (c) to investigate
the ability of such a kind of conjugate to enhance and/or to change the nature of the pharmacological
effects of its components.

Molecules 2020, 25, 3546
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Scheme 1. Synthetic procedure. Reagents used: i: benzyl bromide, K2CO3, DMF; ii: propargyl bromide
,
NaH, THF; iii: azidovaleric acid, CuSO4 5H2O, TBTA, sodium ascorbate, water/CH2Cl2 mixture;
·
iv: H2 (Pd/C), 130 kPa, ethanol/THF mixture; v:
ethanol/THF mixture.
1, DCC, CH2Cl2; vi: H2 (Pd/C), 130 kPa,
2. Results and Discussion
In this synthetic procedure (Scheme 1), several reaction steps were found requiring use of other
approaches than originally planned ones. Either different reagents for successful completion of the
target molecules had to be used or different approaches had to be selected. A selection of the reagents
was often based on analogous synthetic procedures done earlier during our projects focused on
steroids and triterpenoid acids [10]. Therefore, a more detailed investigation and/or optimization in
the synthesis of
8 and 6 was made. We mostly focused on a behavior of the source molecules (7 and 5)
during the synthesis, in which several procedures, earlier developed in our team, were used in the

Molecules 2020, 25, 3546
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synthesis of triterpenoid acid derivatives, and in analogous reactions [10
–
16]. A hypothesis on the
course of the palladium-catalyzed hydrogenation in its employed modifications is postulated.
The first step of the synthetic procedure (Scheme 1) consisted in a conversion of the carboxyl
group in
presence of potassium carbonate [11
into the corresponding propargyl ether
hydride in THF [18]. Note that it is important to mix
before adding propargyl bromide, to enable a formation of the sodium salt of
2
into its benzyl ester
3
by reacting
17]. Subsequently, the C(3)-OH group of
using propargyl bromide, in the presence of sodium
with sodium hydride at least 30 min
. The originally
2
with benzyl bromide in DMF, under the
,
3
was converted
4
3
3
used methods of etherification, employing either propargyl chloride [18] or propargylic alcohol
in chloroform under the presence of Montmorillonit K [19], were not successful in this case.
The propargyl ether
cycloaddition, CuAAC) with azidovaleric acid under a catalysis with copper(II) sulfate in
a water-dichloromethane mixture, yielding . TBTA was used as stabilizer of the Cu(I)
oxidation state in aqueous solutions as well as acted as a rate-accelerating ligand [20 21].
4 was subjected to a click reaction (Huisgen copper(I)-catalyzed 1,3-dipolar
5
,
To stabilize Cu(I) oxidation state is important because Cu(II) ions are harmful to this reaction due
to the fact that Cu(II) ions catalyze the oxidative coupling of the alkyne substrates to give diynes as
undesired by-products [20]. Sodium ascorbate acts as a reducing agent.
The main quantity of
esterification with under the presence of N,N’-dicyclohexylcarbodiimide (DCC) as coupling agent,
and 4-(dimethylamino)pyridine (DMAP) as reaction enhancer, to get . This reaction is an example
5 was used for modification of the diosgenin molecule (1) by Steglich
5
7
of esterification that was not mediated successfully with 1-propanephosphonic anhydride (T3P),
another coupling agent. T3P had often been used for formation of ester or amide bonds in our team [11].
Generally, we prefer using T3P over DCC since phosphonic acid products resulting from the T3P
hydrolysis are soluble in water and are not extracted with organic solvents in contrast to dicyclohexyl
urea resulting from DCC. We also tried to convert
this method did not yield the required product. Finally, the resulting product
5
to its acyl chloride with oxalyl chloride; however,
was subjected to a
7
high-pressure (130 kPa) hydrogenation on 10% Pd/C in an ethanol-THF mixture, yielding the target
diosgenin conjugate 8.
A deprotection of
in our laboratory (entry 1, Table 1) due to its failure. This result inspired us to search for other
methods of deprotection of and . First, we have employed hydrogenation under atmospheric
pressure (entries 2, 3 and 4 [22]) to obtain . However, the hydrogenation has not been catalyzed
5 and 7 was carried out in a different way than customary used procedure [10]
5
7
8
quantitatively. Using BCl3 in dichloromethane (entry 5 [23]) showed no reaction, and using HBr in
acetic acid (entry 6 [24]) resulted in a decomposition of the starting material. Originally, the main
reason for employing hydrogenation reaction under atmospheric pressure was to prevent possible
saturation of the C(20)=C(29) double bond in the betulinic acid unit of
did not furnish and , we have investigated hydrogenation under slightly higher pressure than
atmospheric pressure, using a different palladium catalyst (entry 7). Nevertheless, it also did not
catalyze the reaction in the required way. Finally, the high pressure hydrogenation procedure [17
was the successful method for deprotection of and and it occurred without saturating the C(20)=C(29)
6 and 8. When this reaction
6
8
]
5
7
double bond (entries 8 and 10). However, when the pressure increased the value of 130 kPa (entry 9),
saturation of the double bond appeared, indicating that 130 kPa is the critical pressure to keep the
C(20)=C(29) double bond in
8 unsaturated. Saturation of the C(20)=C(29) double bond was proven by
the presence of a peak belonging to the saturated product in the HR-MS spectra. Saturated product was
isolated from the TLC plate and dissolved in methanol prior to measurement. It was an unexpected
result, because transferring hydrogenation under atmospheric pressure was used successfully for
removal of protecting benzyl group from carboxylic benzyl esters several times [10
for the failing of transferring hydrogenation in the removal of the benzyl group from
,
5
11
and
,
13]. The reason
to get and
7
6
8
can be explained by a simultaneous presence of the 1,4-disubstituted 1,2,3-triazole ring in the molecule,
a phenomenon that had already been observed earlier in the literature [25]. The 1,4-disubstituted

Molecules 2020, 25, 3546
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1,2,3-triazole ring probably shows its competing effect in coordination of palladium catalyst over
the target site, requiring a different approach than usually used for the successful course of the
debenzylation reaction. See also Scheme S1 in the Supplementary Material.
Table 1. Applied methods of conversion of 7 to 8.
Entry
Catalyst
Equiv.
Solvent
H-donor
Press.
Temp.
Result a
1
2
3
4
5
6
7
8
9
10% Pd/C
10% Pd/C
PdO
0.67 [10]
0.67
EtOH/THF 1,4-cyclohexadiene
atm
atm
atm
atm
atm
25 ◦C
25 ◦C
25 ◦C
25 ◦C
25 ◦C
60 ◦C
25 ◦C
25 ◦C
25 ◦C
25 ◦C
no rxn b
c
EtOH/THF
EtOH/EtOAc
CH2Cl2
H2
H2
H2
-
n. c.
n. c.
c
2.0
c
Pd(OAc)2
0.05 [22]
1.05 [23]
1.0 [24]
0.05
0.67
0.67
n. c.
no rxn b
BCl3
HBr
Pd/BaSO4
10% Pd/C
10% Pd/C
10% Pd/C
CH2Cl2
d
AcOH
-
atm
dec.
n. c.
c
EtOH/dioxane
EtOH/THF
EtOH/THF
EtOH/THF
H2
H2
H2
H2
101.3 kPa
<130 kPa
>130 kPa
130 kPa
success
e
sat.
10
0.67
success
a
result of the course of the reaction, b no reaction observed, c reaction not completed, d decomposition (cleavage of
the molecule), e saturation of the C(20)=C(29) double bond.
Removal of the benzyl protecting group from
hydrogenation was also made to prepare , the other target product for pharmacological screening.
HR-MS ESI supported successful removal of the benzyl protecting group of
C(20)=C(29) double bond unsaturated. In HR-MS ESI of and , typical adducts [M
C38H59O5N3 (for ), or C65H99O7N3 (for ), respectively. In HR-MS of , and
were identified (Table 2).
5 using the above described high pressure (130 kPa)
6
7
, while keeping the
6
8
−
H]− were observed,
6
8
4
,
5
7
, adducts [M − H]+
Table 2. HR-MS ESI of compounds 3–8.
Measured Mass
Theoretical Mass
Mass Error
(ppm)
Calculated
Formula
Compound
(m/z)
(m/z)
3
4
5
6
7
8
547.4172
585.4289
728.4987
636.4413
1124.8025
1033.7533
C37H55O3
C40H57O3
C45H66O5N3
C38H59O5N3
C72H105O7N3
C65H99O7N3
547.4146
585.4302
728.4997
636.4382
1124.8025
1033.7483
+4.87
+2.19
−1.31
−4.85
+0.02
+4.88
The compounds 1–8 were subjected to the cytotoxicity screening tests on cells of human
T-lymphoblastic leukemia (CEM), cells of human breast adenocarcinoma (MCF7), cells of human
cervical cancer (HeLa), and human colon carcinoma (HCT 116 cancer cell lines), using normal human
fibroblasts (BJ) as reference cell lines. Doxorubicin was used as a positive control. While the target
compounds
studied compounds to penetrate into the cell,
all cancer cell lines. Benzyl ester of the main target compound (
6
and
8
were inactive, due to a possible high polarity, causing a decrease in ability of the
showed moderate to weak multifarious cytotoxicity to
) displayed a selective cytotoxicity
M) towards the reference cell line BJ
5
7
on CEM (6.5
(Table 3).
±
1.1
µM), but also weak cytotoxicity (46.2
±
2.8 µ
To support the data obtained experimentally, physico-chemical and ADME parameters of the
synthesized compounds have been calculated using ACD/iLabs software and databases [27] for
(Table 4). The data were compared with the Lipinski rule of five [28] and with the Ghose [29
1–8
]
rule. The Lipinski [28] and Ghose [29] rules were postulated for testing in vivo. Nevertheless,
they are applicable in the screening tests in cancer cell lines, as we have proven several times
in our previous papers [13–15]. However, deviations of experimental and calculated values have
already been observed [16]. The rules describe molecular properties important for a small molecule

Molecules 2020, 25, 3546
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drug pharmacokinetics in the human body, including their absorption, distribution, metabolism,
and excretion (known as ADME parameters). However, the rules do not predict displaying of the
pharmacological activity. The Lipinski rule of five [28] and Ghose rule [29] consider partition coefficient
(log P, range
500), number of atoms in the molecule (20 to 70), and polar surface area (up to 14 nm). However,
the parameters calculated for some of the target compounds correspond only in part to the ranges
given for them by the above Lipinski [28] and Ghose [29] rules (Table 4). Despite those facts, and
−
0.4 to +5.6), molar refractivity (range 40 to 130), molecular weight (range 180 to
1–8
5
7,
the only cytotoxic compounds of this series, displayed cytotoxicity. For more details on calculation,
explanation, and importance of physico-chemical and ADME characteristics in searching for novel
potential drugs cf. Supplementary Materials.
Table 3. Cytotoxicity screening tests (IC50 values (µM), 72 h).
Cytotoxicity (IC50 (µM))
Compound
MW
CEM a
HeLa c
BJ e
MCF7 b
HCT 116 d
1
2
3
4
5
6
7
8
414.62
456.70
546.82
584.87
728.02
637.89
1124.62
1034.50
543.53
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
15.8 ± 0.6
49.5 ± 0.7
20.3 ± 4.4
19.4 ± 2.1
>50
>50
> 50
>50
>50
6.5 ± 1.1
>50
>50
>50
> 50
> 50
>50
>50
46.2 ± 2.8
>50
NT f
Doxorubicin
0.273 ± 0.019 0.868 ± 0.054 0.524 ± 0.177 0.278 ± 0.036
CEM, cells of human T-lymphoblastic leukemia; b MCF7, cells of human breast adenocarcinoma; c HeLa, cells of
human cervical cancer; d HCT 116, human colon carcinoma; e BJ, normal human fibroblasts; f NT = not tested in our
team; however, IC50 value (CEM) 0.02 + 0.00 µM (ref. [26]).
a
Calculated activity on central nervous system (CNS) for
1 and 2 resulted in a finding that 1 is
CNS active, while appears in the area with low or no CNS activity, also made by the ACD/iLabs
2
software [27]. The data obtained show the key parameters for assigning CNS activity, log PS*fu, brain
(the brain/plasma equilibration rate), log BB (a hybrid parameter determined by permeability, plasma
and brain tissue binding and active transport mechanism; range +1.2 to
brain penetration parameter, indicating if the drug might be CNS active or CNS inactive; range +2
(CNS active) to 2 (CNS inactive); cf. Table 4 and Figure S1 in the Supplementary Material). Based on
the values of log PS*fu,brain, log PB and log BB (Table 4), when liberated from or by their metabolic
−
3.0), and log PB (the extent of
−
1
7
8
decomposition, may display certain activity on central nervous system, which may result in additional
positive adaptogenic effect on organism if applied in vivo.
Aside from
1, no one of the studied compounds (2–8) appeared to have any potential for CNS
activity at all, as shown in Figure S2 in the Supplementary Material.

Molecules 2020, 25, 3546
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Table 4. Physico-chemical and absorption, distribution, metabolism, and excretion (ADME) parameters of 1–8 calculated using the ACD/iLabs software [27].
ADME Parameters c
Physico-Chemical Parameters a,b
Compd. or
Recom. Range
MW
Bioav.
(%)
log PS *
fu,brain
PPB
(%)
log P
log D (pH 7.4)
log S (pH 7.4)
Hacc/Hdon/n.m.b.
log PS
log PB
log BB
1
2
3
4
5
6
7
8
414.62
456.70
546.82
584.87
728.01
637.89
1124.62
1034.50
+6.01
+6.26
+8.92
+9.87
+9.12
+6.65
+15.11
+13.15
+6.01
+3.77
+8.92
+9.87
+6.62
+1.96
+15.11
+10.66
-
−5.65
−3.34
3/1/0
3/2/2
3/1/5
30–70
30–70
<30
<30
<30
30–70
<30
<30
-
−3.5
−4.4
−5.2
−5.7
−6.9
−5.2
−10.5
−10.1
-
−1.2
−2.6
−2.9
−3.4
−5.1
−3.4
−8.1
−8.3
-
+0.29
−0.35
−0.58
−0.92
−2.00
−0.88
−2.00
−2.00
+0.28
−0.20
−0.58
−0.92
−2.00
−0.88
−2.00
−2.00
98.97
99.29
99.87
99.94
99.93
99.79
100.00
100.00
-
−8.10
−9.22
3/0/8
−6.07
8/1/13
8/2/10
10/0/15
10/1/12
10/5/-
−2.21
−10.72
−7.51
recom. range
180/500 −0.4/+5.6
−6.5/+0.5
−1.5/+1.5 −3.0/+1.2
a
log P—partition coefficient; log D—distribution coefficient; log S—predicted aqueous solubility; Hacc/Hdon/n.m.b. = number of H-bond acceptors/number of H-bond donors/number
of movable bonds; Lipinski rule [28]: MW = max. 500, log P = max. 5.0, Hacc/Hdon/n.m.b. = max. 10/max. 5/ , and Ghose rule [29]: MW = max. 500, log P = max. 5.6;
bioav. = bioavailability—the degree of availability of a chemical by the target tissue; log PS * fu,brain—the brain/plasma equilibration rate, the parameter that is a mathematical modeling
parameter based on time required for reaching brain equilibrium; log PS—logarithm of the permeability-surface area coefficient; log PB—the extent of brain penetration parameter;
log BB—a hybrid parameter determined by permeability, plasma and brain tissue binding, and active transport mechanism; PPB—plasma protein binding.
b
−
c

Molecules 2020, 25, 3546
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3. Material and Methods
3.1. General
The NMR measurements were performed either on a Bruker AVANCE 500 MHz or on a Bruker
AVANCE II 600 MHz spectrometer equipped with a 5 mm TCI cryoprobe in a 5 mm tube in different
1
solvents. The H NMR and the 13C NMR spectra were recorded at 499.98 MHz and 125.73 MHz
(AVANCE 500 MHz) or at 600.13 MHz and 150.90 MHz (AVANCE II 600 MHz) in CDCl3 or CD3OD
using tetramethylsilane (
as internal references. H NMR data are presented in the following order: chemical shift (
δ
= 0.0
−
CDCl3) or signal of solvent (
δ
= 3.31 or 49.50 for 1H/13C
−
CD3OD)
1
δ) expressed
in ppm, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), number of protons,
1
coupling constants in Hertz. For unambiguous assignment of both H and 13C signals, 2D NMR
1H,13C gHSQC and gHMBC spectra were measured using standard parameters sets and pulse
programs delivered by producer of the spectrometer. Infrared spectra were measured with a Nicolet
205 FT-IR spectrometer (Thermofisher Scientific, Waltham, MA, USA). HR-MS was acquired on
Exactive Plus Orbitrap Mass Spectrometer with HESI (coin voltage 4.5 kV, resolution 100,000 @ 1 Hz,
capillary temperature 300 ◦C, probe temperature 30−40 ◦C) and were processed in Xcalibur™ 2.1 SW
(ThermoFisher Scientific, Waltham, MA, USA). Samples were dissolved in methanol. See Supplementary
Material for more details on HR-MS (ESI) analyses (Table 1). TLC was carried out on silica gel plates
(Merck 60F254, Merck, Prague, Czech Republic) and the visualization was performed by both the
UV detection and spraying with the methanolic solution of phosphomolybdic acid (5%) followed
by heating. For column chromatography, silica gel 60 (0.063–0.200 mm) from Merck was used.
All chemicals and solvents were purchased from regular commercial sources in analytical grade
and the solvents were purified by general methods before use. Betulinic acid was purchased from
Dr. Jan Šarek—Betulinines (www.betulinines.com). Diosgenin was purchased from Wako Chemicals
(Osaka, Japan). Analytical HPLC was carried out on a TSP (Thermoseparation Products, Boston,
MA, USA) instrument equipped with a ConstaMetric 4100 Bio pump and a SpectroMonitor 5000 UV
DAD. The analyses of the products were performed on a reverse phase Nucleosil 120-5 C18 column
(
250 × 4 mm; Watrex, Prague, Czech Republic) using a methanol/water mixture (9:1, v/v) as mobile
phase at 0.5 to 1.0 mL.min−1. The eluate was monitored at 220, 254, and 275 nm, and the UV spectra
were run from 200 to 300 nm.
3.2. Benzyl (3β)-3-hydroxylup-20(29)-en-28-oate (3)
Benzyl bromide (0.65 mL; 5.48 mmol) was added to a solution of betulinic acid (2; 1.0 g; 2.19 mmol)
in acetone (40 mL), in a presence of potassium carbonate (0.45 g; 3.28 mmol), and the reaction mixture
was stirred at r.t. for 24 h. The mixture was worked-up by diluting it with chloroform and extracted
with water. Organic layer was dried with sodium sulfate, then the solvent was evaporated under
reduced pressure, and the residue was purified by chromatography using chloroform/EtOH mixture
1
as mobile phase yielding
3
as white powder in a 99% yield (1.19 g). H-NMR (499.98 MHz, CDCl3):
δ
[ppm] 0.61 (dd, 1H, J = 2.5; 11.3 Hz, H-5), 0.70 (s, 3H, H-23), 0.71 (s, 3H, H-24), 0.75 (d, 3H, J = 0.7
Hz, H-25), 0.90 (d, 3H, J = 0.6 Hz, H-26), 0.91 (s, 3H, H-27), 1.05 (dt, 2H, J = 3.0; 3.0; 13.2 Hz, H-21),
1.63 (dd, 3H, J = 0.7; 1.4 Hz, H-30), 2.14 (ddd, 1H, J = 3.7; 11.6; 12.9 Hz, H-13), 2.23 (dt, 2H, J = 3.4;
3.4; 12.5 Hz, H-16), 2.97 (dt, 1H, J = 4.6; 11.0; 11.0 Hz, H-19), 3.13 (dd, 1H, J = 4.9; 11.4 Hz, H-3),
4.55 (dq, 2H, J = 1.4; 1.4; 1.4; 2.4 Hz, H-29), 4.68 (dq, 2H, J = 0.7; 0.7; 0.7; 2.4 Hz, H-29), 5.05 (d, 2H,
J = 12.3 Hz, H-10), 5.10 (d, 2H, J = 12.3 Hz, H-10), 7.24–7.35 (m, 2H, H-30,H-40,H-50). 13C-NMR (125.73
MHz, CDCl3):
δ [ppm] 14.66 (q, C-27), 15.34 (q, C-24), 15.81 (q, C-25), 16.11 (q, C-26), 18.27 (t, C-6),
19.35 (q, C-29), 20.85 (t, C-11), 25.51 (t, C-12), 27.39 (t, C-2), 27.96 (q, C-23), 29.55 (t, C-21), 30.56 (t, C-15),
32.10 (t, C-16), 34.28 (t, C-22), 36.92 (t, C-7), 37.15 (s, C-10), 38.18 (d, C-13), 38.69 (t, C-1), 38.83 (s, C-4),
40.62 (s, C-8), 42.37 (s, C-14), 46.91 (d, C-19), 49.42 (d, C-18), 50.53 (d, C-9), 55.32 (d, C-5), 56.53 (s, C-17)
,
65.71 (t, C-10), 78.96 (d, C-3), 109.56 (t, C-30), 128.04 (d, C-50), 128.22 (d, C-40), 128.47 (d, C-30),
136.47 (s, C-20), 150.57 (s, C-20), 175.80 (s, C-28). IR (KBr): [cm−1] 3533 s (O-H) stretching, 2940 s

Molecules 2020, 25, 3546
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(ring system, sp3, C-H) stretching, 1692 s (C=O) stretching, 1647 w (benzene, C=C) stretching, 1453 m
(benzene, C=C) stretching, 1184, 757, 699. HR-MS (ESI) m/z 547.4172, calcd. 547.4146 for C37H55O3.
3.3. Benzyl (3β)-3-(prop-2-yn-1-yloxy)lup-20(29)-en-28-oate (4)
A portion of sodium hydride (0.48 g; 20.44 mmol) was added to a solution of 3 (0.325 g; 0.60 mmol)
in THF (15 mL), and the mixture was stirred at r.t. for 30 min. Then, an additional portion of
sodium hydride (0.48 g; 20.44 mmol) was added, and the mixture was stirred for an additional 30 min.
Propargyl bromide (0.176 mL; 2.32 mmol) was added, and the mixture was stirred at r.t. for 24 h. Then,
additional propargyl bromide (0.137 mL; 1.80 mmol) was added, and the mixture was stirred at r.t.
for an additional 24 h. The reaction was then worked-up by extraction with chloroform (4 × 15 mL).
The extract was washed with water, dried over sodium sulfate, filtered, and the solvent was evaporated
under reduced pressure. The residue was purified by column chromatography on silica gel, using
petroleum ether/chloroform as mobile phase, yielding
MHz, CDCl3): [ppm] 0.61 (dd, 1H, J = 2.2; 11.3 Hz, H-5), 0.68 (s, 3H, H-23), 0.69 (s, 3H, H-24),
4
in a 96% (0.336 g) yield. 1H-NMR (499.98
δ
0.73 (s, 3H, H-25), 0.87 (d, 3H, J = 0.6 Hz, H-26), 0.89 (s, 3H, H-27), 1.02 (dt, 2H, J = 3.2; 3.2; 13.1 Hz,
H-21), 1.61 (dd, 3H, J = 0.7; 1.4 Hz, H-30), 2.11 (ddd, 1H, J = 3.7; 11.5; 12.9 Hz, H-13), 2.21 (dt, 2H,
J = 2.9; 2.9; 12.3 Hz, H-16), 2.29 (t, 1H, J = 2.4 Hz, H-30), 2.92 (dd, 1H, J = 4.4; 11.8 Hz, H-3), 2.95 (dt, 1H,
J = 5.8; 11.2; 11.2 Hz, H-19), 4.07 (dd, 2H, J = 2.4; 16.0 Hz, H-10), 4.15 (dd, 2H, J = 2.4; 16.0 Hz, H-10),
4.52 (dq, 2H, J = 1.4; 1.4; 1.4; 2.4 Hz, H-29), 4.66 (dq, 2H, J = 0.7; 0.7; 0.7; 2.4 Hz, H-29), 5.02 (d, 2H,
J = 12.3 Hz, H-40), 5.07 (d, 2H, J = 12.3 Hz, 40), 7.23–7.32 (m, 2H, H-60, H-70, H-80). 13C-NMR
(125.73 MHz, CDCl3): δ [ppm] 14.62 (q, C-27), 15.82 (q, C-24), 16.10 (q, C-25), 16.17 (q, C-26), 18.21
(t, C-6), 19.37 (q, C-29), 20.89 (t, C-11), 25.54 (t, C-12), 27.96 (t, C-2), 29.54 (q, C-23), 30.17 (t, C-21),
30.57 (t, C-15), 32.10 (t, C-16), 34.29 (t, C-22), 36.93 (t, C-7), 37.11 (t, C-10), 38.18 (d, C-13), 38.53 (t, C-1),
38.56 (s, C-4), 40.67 (s, C-8), 42.37 (s, C-14), 46.92 (d, C-19), 49.44 (d, C-18), 50.52 (d, C-9), 55.89 (d, C-5),
56.38 (t, C-10),0 56.53 (s, C-17), 65.71 (t, C-40), 73.39 (d, C-3), 80.83 (d, C-30), 80.95 (s, C-20), 109.55 (t, C-30)
,
128.04 (d, C-8 ), 128.23 (d, C-70), 128.47 (d, C-60), 136.47 (s, C-50), 150.60 (s, C-20), 175.79 (s, C-28).
IR (KBr): [cm−1] 3305 m (sp, C-H) stretching, 2957 s (ring system, sp3, C-H) stretching, 1715 s (C=O),
1644 w (benzene, C=C) stretching, 1453 m (benzene, C=C), 1128, 1080, 755, 628. HR-MS (ESI) m/z
585.4289, calcd. 585.4302 for C40H57O3.
3.4. 5-[4-({[(3β)-28-(Benzyloxy)-28-oxolup-20(29)-en-3-yl]oxy}-methyl)-1H-1,2,3-triazol-1-yl]pentanoic
acid (5)
5-Azidovaleric acid (61.27 mg; 0.43 mmol), sodium ascorbate (16.94 mg; 0.09 mmol) and a solution
of CuSO4 5H2O with TBTA in water (7.61 mL; 0.89 mmol) was added to a solution of 4 (200 mg;
·
0.34 mmol) in CH2Cl2 (5 mL), and the mixture was stirred at r.t. for 30 h. The mixture was worked-up by
extraction with methylene chloride, the extract was washed with water, and dried over sodium sulfate.
After filtration, the solvent was evaporated under reduced pressure, and the residue was purified by
column chromatography on silica gel, using petroleum ether/chloroform mixture as mobile phase,
1
yielding
5
in a 95% yield (0.26 g). H-NMR (499.98 MHz, CDCl3):
δ
[ppm] 0.58 (dd, 1H, J = 2.3; 11.2 Hz,
H-5), 0.68 (s, 3H, H-23), 0.68 (s, 3H, H-24), 0.73 (s, 3H, H-25), 0.81 (s, 3H, H-26), 0.86 (s, 3H, H-27)
,
1.02 (dt, 2H, J = 3.2; 3.2; 13.5 Hz, H-21), 1.52 (t, 1H, J = 11.4 Hz, H-18), 1.53–1.65 (m, 2H, H-50, H-60),
1.61 (dd, 3H, J = 0.7; 1.4 Hz, H-30), 2.10 (ddd, 1H, J = 3.6; 11.5; 12.8 Hz, H-13), 2.20 (dt, 2H, J = 3.1; 3.1;
12.5 Hz, H-16), 2.33 (t, 2H, J = 7.3 Hz, H-70), 2.87 (dd, 1H, J = 4.4; 11.8 Hz, H-3), 2.95 (dt, 1H, J = 4.8;
11.0; 11.0 Hz, H-19), 4.30 (t, 2H, J = 7.1 Hz, H-40), 4.49 (dd, 2H, J = 0.6; 12.5 Hz, H-10), 4.53 (dq, 2H,
J = 1.4; 1.4; 1.4; 2.4 Hz, H-29), 4.65 (dq, 2H, J = 0.7; 0.7; 0.7; 2.4 Hz, H-29), 4.70 (dd, 2H, J = 0.6; 1.2
Hz, H-10), 5.02 (d, 2H, J = 12.3 Hz, H-90), 5.07 (d, 2H, J = 12.3 Hz, H-90), 7.22–7.32 (m, 2H, H-110,
H-130), 7.44 (s, 1H, H-30). 13C-NMR (125.73 MHz, CDCl3):
δ [ppm] 14.61 (q, C-27), 15.81 (q, C-24),
16.12 (q, C-25), 16.25 (q, C-26), 18.20 (t, C-6), 19.34 (q, C-29), 20.87 (t, C-11), 21.52 (t, C-60), 22.89 (t, C-50),
25.51 (t, C-12), 27.99 (q, C-23), 28.19 (t, C-2), 29.51 (t, C-15), 30.55 (t, C-21), 32.09 (t, C-16), 32.97 (t, C-70),
34.26 (t, C-22), 36.92 (t, C-7), 37.11 (s, C-10), 38.17 (d, C-13), 38.50 (t, C-1), 38.78 (s, C-4), 40.65 (s, C-8),

Molecules 2020, 25, 3546
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42.35 (s, C-14), 46.92 (d, C-19), 49.42 (d, C-18), 49.85 (t, C-40), 50.47 (d, C-9), 55.70 (d, C-5), 56.53 (s, C-17),
63.18 (t, C-10), 65.70 (t, C-90), 86.61 (d, C-3), 109.57 (t, C-30), 122.09 (d, C-30), 128.02 (d, C-130), 128.21
(d, C-120), 128.46 (d, C-110), 136.46 (s, C-100), 146.55 (s, C-20), 150.56 (s, C-20), 175.81 (s, C-28), 177.46
(s, C-80). IR (KBr): [cm−1] 2945 s br (ring system, sp3 C-H and O-H overlap) stretching, 1732 s (C=O)
stretching, 1642 w (benzene, C=C) stretching, 1453 m (benzene, C=C) stretching, 1130. HR-MS (ESI)
m/z 728.4987, calcd. 728.4997 for C45H66O5N3.
3.5. (3β)-3-{[1-(4-Carboxybutyl)-1H-1,2,3-triazol-4-yl]methoxy}lup-20(29)-en-28-oic Acid (6)
The compound 5 (75 mg, 0.105 mmol) was subjected to a removal of the benzyl protecting group by
hydrogenation on a 10% Pd/C (75 mg, 0.705 mmol) in a THF/ethanol (1:1) mixture (8 mL) at 130 kPa at
r.t. for 30 min. After filtering the mixture off to remove the catalyst, and evaporation of the solvent,
the residue was purified by column chromatography, using petroleum ether/ethyl acetate as mobile
phase, yielding
6 δ [ppm] 0.61 (dd, 1H,
in a 92% yield (61.3 mg). 1H-NMR (600.13 MHz, CDCl3):
J = 1.9; 11.4 Hz, H-5), 0.68 (s, 3H, H-25), 0.75 (s, 3H, H-24), 0.81 (d, 3H, J = 0.6 Hz, H-23), 0.84 (d, 3H,
J = 0.8 Hz, H-26), 0.89 (d, 3H, J = 0.5 Hz, H-27), 1.54 (t, 1H, J = 11.4 Hz, H-18), 1.58 (dd, 3H,
J = 0.7; 1.4 Hz, H-30), 2.09 (ddd, 1H, J = 3.8; 11.8; 12.8 Hz, H-13), 2.19 (ddd, 2H, J = 3.2; 3.7; 13.1 Hz,
H-16), 2.34 (t, 2H, J = 7.2 Hz, H-70), 2.89 (dd, 1H, J = 4.4; 11.7 Hz, H-3), 2.92 (dt, 1H, J = 4.9; 11.0;
11.5 Hz, H-19), 4.31 (dt, 2H, J = 1.0; 7.0; 7.0 Hz, H-40), 4.48 (dd, 2H, J = 0.7; 12.5 Hz, H-10), 4.54 (dq, 2H,
J = 1.4; 1.4; 1.4; 2.4 Hz, H-29), 4.67 (dq, 2H, J = 0.7; 0.7; 0.7; 2.4 Hz, H-29), 4.72 (dd, 2H, J = 0.6; 12.5 Hz,
H-10), 7.46 (s, 1H, H-30). 13C-NMR (150.91 MHz, CDCl3):
δ [ppm] 14.63 (q, C-27), 16.10 (q, C-24),
16.13 (q, C-26), 16.24 (q, C-25), 18.21 (t, C-6), 19.36 (q, C-29), 20.84 (t, C-60), 21.51 (t, C-11), 22.93 (t, C-50),
25.45 (t, C-12), 28.01 (q, C-23), 29.52 (t, C-2), 29.68 (t, C-15), 30.56 (t, C-21), 32.13 (t, C-16), 33.14 (t, C-70),
34.25 (t, C-22), 37.05 (t, C-7), 37.14 (s, C-10), 38.40 (d, C-13), 38.50 (t, C-1), 38.80 (s, C-4), 40.67 (s, C-8),
42.38 (s, C-14), 46.90 (t, C-19), 49.28 (d, C-18), 49.91 (t, C-40), 50.38 (d, C-9), 55.70 (d, C-5), 56.42 (s, C-17),
63.31 (t, C-10), 86.73 (d, C-3), 109.69 (t, C-30), 122.10 (d, C-30), 146.60 (s, C-20), 150.39 (s, C-20),
178.37 (s, C-28), 182.46 (s, C-80). IR (KBr): [cm−1] 2944 s, (ring system, sp3 C-H and O-H overlap)
stretching, 1705 s (C=O) stretching. HR-MS (ESI) m/z 636.4413, calcd. 636.4382 for C38H59O5N3.
3.6. Benzyl (3
β)-3-[(1-{5-oxo-5-[(3β,25R)-spirost-5-en-3-yloxy]pentyl}-1H-1,2,3-triazol-4-yl)methoxy]lup-
20(29)-en-28-oate (7)
DCC (167.0 mg, 0.81 mmol) and DMAP (152.5 mg, 1.25 mmol) were added to a solution of
5
(
168 mg, 0.23 mmol) in methylene chloride (10 mL) under stirring. Then, diosgenin ( ; 143 mg,
1
0.35 mmol) was added, and stirring continued at r.t. for 24 h. The crude product was purified by
column chromatography on silica gel using petroleum ether/ethyl acetate as mobile phase, yielding
1
white, crystalline
7
in a 72% yield (186 mg). H-NMR (600.13 MHz, CDCl3):
δ
[ppm] 0.64 (dd, 1H,
J = 2.1; 11.2 Hz, H-5), 0.74 (s, 3H, H-23), 0.74 (s, 3H, H-19”), 0.76 (s, 3H, H-25), 0.78 (d, 3H, H-27”),
0.86 (s, 3H, H-24), 0.92 (s, 3H, H-26), 0.96 (d, 3H, J = 7.0 Hz, H-21”), 1.02 (s, 3H, H-27), 1.02 (s, 3H,
H-18”), 1.67 (dd, 3H, J = 0.7; 1.4 Hz, H-30), 2.16 (ddd, 1H, J = 3.5; 11.6; 12.8 Hz, H-13), 2.26 (dt, 2H,
J = 3.1; 3.1; 12.7 Hz, H-16), 2.31 (t, 2H, J = 7.3 Hz, H-120), 2.93 (dd, 1H, J = 4.3; 11.7 Hz, H-3),
3.01 (dt, 1H, J = 4.9; 10.9; 10.9 Hz, H-19), 3.37 (t, 2H, J = 11.0 Hz, H-26”), 3.44–3.50 (m, 2H, H-26”),
3.44–3.50 (m, 1H, H-20”), 4.35 (t, 2H, J = 7.0 Hz, H-90), 4.40 (ddd, 1H, J = 6.5; 7.6; 8.5 Hz, H-16”),
4.54 (dd, 2H, J = 0.5; 12.5 Hz, H-60), 4.58 (dq, 2H, J = 1.4; 1.4; 1.4; 2.3 Hz, H-29), 4.59 (dddd, 2H, J = 4.3
;
6.5; 10.6; 11.4 Hz, H-3”), 4.71 (dq, 2H, J = 0.7; 0.7; 0.7; 2.3 Hz, H-29), 4.76 (dd, 2H, J = 0.5; 12.5 Hz,
H-60), 5.08 (dd, 2H, J = 0.5; 12.5 Hz, H-10), 5.13 (dd, 2H, J = 0.5; 12.5 Hz, H-10), 5.36 (dt, 2H, J = 0.9; 0.9;
5.3 Hz, H-6”), 7.28–7.37 (m, 2H, H-30-H-50). 13C-NMR (150.91 MHz, CDCl3):
δ [ppm] 14.51 (q, C-21”),
14.62 (q, C-27), 16.12 (q, C-25), 16.25 (q, C-18”), 16.26 (q, C-24), 17.12 (q, C-27”), 18.22 (t, C-6),
19.32 (q, C-29), 19.34 (q, C-19”), 20.77 (t, C-11”), 20.87 (t, C-11), 21.84 (t, C-100), 22.89 (t, C-110),
23.82 (t, C-2), 25.51 (t, C-12), 25.82 (q, C-26), 27.74 (q, C-23), 28.78 (t, C-24”), 29.53 (t, C-21), 29.62 (t, C-23”)
,
30.28 (d, C-8”), 30.56 (t, C-15), 31.37 (t, C-2”), 31.37 (d, C-25”), 31.81 (t, C-7”), 32.02 (t, C-15”),
32.10 (t, C-16), 33.89 (t, C-120), 33.89 (t, C-12”), 34.28 (t, C-22), 36.70 (s, C-10”), 36.91 (s, C-10),

Molecules 2020, 25, 3546
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36.93 (t, C-7), 37.12 (t, C-1”), 38.08 (s, C-4), 38.18 (d, C-13), 38.51 (t, C-1), 40.66 (s, C-8), 40.83 (s, C-13”)
,
41.59 (t, C-4”), 42.36 (s, C-14), 46.93 (d, C-19), 49.21 (d, C-20”), 49.43 (d, C-18), 49.90 (t, C-90),
49.91 (d, C-9”), 50.48 (d, C-9), 55.71 (d, C-5), 56.41 (d, C-14”), 56.53 (s, C-17), 62.05 (d, C-17”),
63.22 (t, C-60), 65.70 (t, C-10), 66.83 (t, C-26”), 74.01 (d, C-3”), 80.78 (d, C-16”), 86.49 (d, C-3), 109.27
(s, C-22”), 109.58 (t, C-30), 122.01 (d, C-80), 122.44 (d, C-6”), 128.03 (d, C-50), 128.22 (d, C-40), 128.46
(d, C-30), 136.47 (s, C-20), 139.56 (s, C-5”), 146.55 (s, C-70), 150.57 (s, C-20), 175.80 (s, C-28). IR (KBr):
[cm−1] 2945 s, (ring system, sp3, C-H) stretching, 1731 s (C=O), 1626 m (alkene, C=C) stretching, 1456
m (benzene, C=C) stretching, 1128. HR-MS (ESI) m/z 1124.8025, calcd. 1124.8023 for C72H105O7N3.
3.7. (3
β
)-3-[(1-{5-Oxo-5-[(3
β
,25R)-spirost-5-en-3-yloxy]pentyl}-1H-1,2,3-triazol-4-yl]methoxy}lup-20(29)-
en-28-oic Acid (8)
The benzyl ester
7 (186.3 mg, 0.166 mmol) was subjected to a hydrogenation on a 10% Pd/C (118.1 mg,
0.111 mmol) in a THF/ethanol (1:1; 10 mL) mixture at 130 kPa at r.t. for 40 min. The mixture was
then filtered off and the product was purified by column chromatography on silica gel, using a
chloroform/ethanol mixture (200:1) as mobile phase, yielding the product
8 in an 87% yield (149 mg).
1H-NMR (600.13 MHz, CDCl3): [ppm] 0.61 (dd, 1H, J = 1.9; 11.4 Hz, H-5), 0.69 (s, 3H, H-190’),
δ
0.72 (d, 3H, J = 6.4 Hz, H-270’), 0.75 (s, 3H, H-180’), 0.76 (s, 3H, H-25), 0.81 (s, 3H, H-23), 0.86 (s, 3H,
H-24), 0.89 (s, 3H, H-26), 0.89 (s, 3H, H-27), 0.90 (d, 3H, J = 7.0 Hz, H-210’), 0.96 (s, 3H, H-30), 2.12 (ddd,
1H, J = 3.8; 11.8; 12.8 Hz, H-13), 2.28 (ddd, 2H, J = 2.8; 4.6; 12.8 Hz, H-16), 2.88 (dd, 1H, J = 4.4; 11.7
Hz, H-3), 2.94 (dt, 1H, J = 5.0; 10.9; 10.9, H-19), 3.31 (t, 1H, J = 11.0 Hz, H-260’), 3.41 (ddt, 1H, J = 1.8;
1.8; 5.0; 11.0 Hz, H-260’), 4.34 (ddd, 1H, J = 6.4; 7.3; 8.5 Hz, H-160’), 4.50 (d, 1H, J = 12.6 Hz, H-10),
4.53 (dddd, 1H, J = 4.6; 6.3; 10.5; 11.7 Hz, H-30’), 4.54 (dq, 1H, J = 3
J = 2.3 Hz, H-29), 4.70 (d, 1H, J = 12.6 Hz, H-10), 5.30 (dt, 1H, J = 1.9; 1.9; 5.3 Hz, H-60’), 7.45 (s, 1H,
H-30). 13C-NMR (150.91 MHz, CDCl3): [ppm] 14.48 (q, C-210’), 14.65 (q, C-27), 16.08 (q, C-26), 16.17
(q, C-25), 16.27 (q, C-24), 16.27 (q, C-180’), 17.12 (q, C-270’), 18.23 (t, C-6), 19.33 (q, C-29), 19.36 (q, C-190’),
20.81 (t, C-110’), 20.87 (t, C-11), 21.83 (t, C-50), 22.93 (t, C-2), 22.93 (t, C-60), 25.47 (t, C-12), 28.03 (q, C-23)
×
1.4; 2.3 Hz, H-29), 4.67 (bd, 1H,
δ
,
28.77 (t, C-240’), 29.35 (t, C-21), 29.56 (t, C-230’), 30.27 (d, C-80’), 30.56 (t, C-15), 31.36 (t, C-20’),
31.36 (d, C-250’), 31.80 (t, C-70’), 32.01 (t, C-150’), 32.17 (t, C-16), 33.68 (t, C-70), 33.68 (t, C-120’), 34.29
(t, C-22), 36.70 (s, C-100’), 36.93 (s, C-10), 37.13 (t, C-7), 37.15 (t, C-10’), 38.10 (s, C-4), 38.50 (d, C-13),
38.80 (t, C-1), 40.24 (s, C-8), 40.70 (s, C-130’), 41.61 (t, C-40’), 42.41 (s, C-14), 46.91 (d, C-19), 49.26 (d,
C-200’), 49.86 (d, C-18), 49.93 (t, C-40), 49.93 (d, C-90’), 50.42 (d, C-5), 55.70 (d, C-5), 56.25 (d, C-140’),
56.42 (s, C-17), 62.00 (d, C-170’), 63.26 (t, C-10), 66.83 (t, C-260’), 74.02 (d, C-30’), 80.82 (d, C-160’), 86.54
(d, C-3), 109.35 (s, C-220’), 109.68 (t, C-30), 122.01 (d, C-30), 122.45 (d, C-60’), 139.56 (s, C-50’), 146.64
(s, C-20), 150.43 (s, C-20), 172.35 (s, C-80), 179.92 (s, C-28). IR (KBr): [cm−1] 3322 m (O-H) stretching,
2928 s (ring system, sp3, C-H) streching, 1731 s (C=O) stretching, 1620 m (C=C) stretching
1243, 1088. HR-MS (ESI) m/z 1033.7533, calcd. 1033.7483 for C65H99O7N3.
→
alkene,
3.8. Cell Cultures
The screening cell lines, T-lymphoblastic leukemia (CEM), cervical carcinoma (HeLa),
breast carcinoma (MCF7), and human foreskin fibroblasts (BJ) were obtained from the American Type
Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM (Dulbecco’s Modified Eagle
Medium, Sigma Aldrich, Darmstadt, Germany). Media used were supplemented with 10% fetal
bovine serum, 2 mM l-glutamine, and 1% penicillin-streptomycin (all from Sigma Aldrich, Darmstadt,
Germany). The cell lines were maintained under standard cell culture conditions at 37 ◦C and 5%
CO2 in humid environment. Cells were subcultured twice or three times a week using the standard
trypsinization procedure.

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3.9. Cytotoxicity Screening Tests
Description of the experimental procedure used in cytotoxicity assay was already published [11].
The IC50 values in CEM, MCF7, HeLa, HCT 116, and BJ measured with the compounds
of treatment are shown in Table 3.
1–8 after 72 h
4. Conclusions
We have developed an easy and efficient method for constructing the ester bond to connect highly
hindered and/or low reactive components. Several coupling reagents were studied and their application
for a given synthesis has been optimized. Different methods of removal of benzyl protecting group
were tested under different hydrogenation conditions and the main focus was given on prevention
of any double bond reduction. Removal of benzyl protecting group in
5 and 7 in the presence of
a 1,4-disubstituted 1,2,3-triazole ring in the molecule has seemed to be a competing element for
coordination of palladium, disabling the required palladium-catalyzed reaction. Palladium-catalyzed
removal of the benzyl group is enabled only under high pressure (130 kPa), and this pressure value
sets as a critical one. In the cytotoxicity screening tests, we found that less polar conjugates display
higher cytotoxicity than more polar ones (cf. compound 5 versus 6, and compound 7 versus 8).
Diosgenin (
1), betulinic acid (2) and the synthetic intermediates
3 and 4 were not cytotoxic,
i.e., their IC50 values are higher than 50
µM against all tested cell lines. This finding resulted in
a conclusion that the synthesized type of a conjugate is capable of enhancing the pharmacological
activity of its components, and they act in a synergy. Moreover, selectivity towards cancer cells was
observed, which augments the importance of the target conjugates. In turn, conjugation of diosgenin
(
1
) with betulinic acid (
activity of the conjugates 7 and 8 in comparison with that of 1 (cf. Supplementary Material).
Even if most of the physico-chemical and ADME parameters calculated for do not correspond
to some of their limits given by the Lipinski [28] and Ghose [29] rules, still showed selective cytotoxicity
and showed medium to weak multifarious cytotoxicity. This finding clearly demonstrates that in silico
2) in 7 or 8 resulted in the theoretical in silico calculation showing no CNS
1–8
5
7
calculations should always be accompanied by the experimental work for comparing both approaches
and for confronting theoretical calculations with practical results. They are applicable in the screening
tests in cancer cell lines, as we have proven several times in our previous papers [13
–15]. In turn,
we have already observed deviation of the experimental and calculated values [16]. Diosgenin (
1
)
displays certain potential for a weak CNS active drug (cf. Supplementary Material), which finding is
in a good agreement with identifying 1 as an adaptogen [3].
The importance of phytochemicals, namely terpenoids, saponins, polyphenols, and alkaloids,
has recently been reviewed and evaluated [30]. Plant products represent an important source of
compounds capable of displaying a broad variety of effects on human health, among which cancer
chemotherapy is the most important. This review paper [30] also supports findings and conclusions
presented herewith.
Supplementary Materials: Electronic Supplementary Material is available. Details on a graphical image of
conversion of
7 to 8, and physico-chemical and ADME parameters of the target compounds are presented therein,
and it can be found online. Scheme S1: Applied methods of conversion of
A comparison of the calculated position of diosgenin ( ; green dot) and betulinic acid (
of the currently used CNS active drugs (blue dots) and CNS inactive drugs (orange dots). Figure S2: Calculated
7
to
8
, graphical image. Figure S1:
1
2; green dot) in the diagram
CNS activity of
C and D).
5 (A), 6 (B), 7 (C) and 8 (D). Green dots are located in the left bottom corners of the pictures (for A,
Author Contributions: Conceptualization, Z.W.; methodology, Z.W., Z.Ö.; validation, M.V., D.Š.; investigation,
M.R., Z.Ö. L.R.; resources, Z.W.; data curation, Z.W., M.R., D.Š., L.R.; writing—original draft preparation, Z.W.,
Z.Ö.; writing—review and editing, Z.W., Z.Ö.; supervision, Z.W.; project administration, Z.W.; funding acquisition,
Z.W. All authors have read and agreed to the published version of the manuscript.
Funding: The authors thank for funding of this research through the grants from MPO: FV10599 (Z.Ö.) and FV30300
(Z.W.); from the European Regional Development Fund: Project ENOCH No. CZ.02.1.01/0.0/0.0/16_019/0000868

Molecules 2020, 25, 3546
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(L.R.); from CTU in Prague: SGS16/251/OHK4/3T/14 (M.V.). Special thanks to The Czech Institute of Informatics,
Robotics and Cybernetics, CTU in Prague for the HR-MS instrumentation.
Conflicts of Interest: There are no conflicts to declare.
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Sample Availability: Samples of the compounds are available from the authors.
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