Synthesis and evaluation of anticancer activities of novel C-28 guanidine-functionalized triterpene acid derivatives

Article abstract of DOI:10.3390/molecules23113000

Triterpene acids, namely, 20,29-dihydrobetulinic acid (BA), ursolic acid (UA) and oleanolic acid (OA) were converted into C-28-amino-functionalized triterpenoids 4–7, 8a, 15, 18 and 20. These compounds served as precursors for the synthesis of novel guani

Full text of DOI:10.3390/molecules23113000

molecules
Article
Synthesis and Evaluation of Anticancer Activities of
Novel C-28 Guanidine-Functionalized Triterpene
Acid Derivatives
Anna Spivak * , Rezeda Khalitova, Darya Nedopekina, Lilya Dzhemileva *,
Milyausha Yunusbaeva, Victor Odinokov, Vladimir D’yakonov and Usein Dzhemilev
Institute of Petrochemistry and Catalysis, Russian Academy of Sciences, 141 Prospekt Oktyabrya, Ufa 450075,
Russia; rezedamuf@yandex.ru (R.K.); dashana25@gmail.com (D.N.); milyausha_ufa@mail.ru (M.Y.);
odinokov@anrb.ru (V.O.); DyakonovVA@gmail.com (V.D.); dzhemilev@anrb.ru (U.D.)
* Correspondence: spivak.ink@gmail.com (A.S.); dzhemilev@mail.ru (L.D.);
Tel.: +7-917-421-71-06 (A.S.); +7-917-458-47-29 (L.D.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Carla Boga and Gabriele Micheletti
Received: 8 October 2018; Accepted: 13 November 2018; Published: 16 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Triterpene acids, namely, 20,29-dihydrobetulinic acid (BA), ursolic acid (UA) and oleanolic
acid (OA) were converted into C-28-amino-functionalized triterpenoids
These compounds served as precursors for the synthesis of novel guanidine-functionalized triterpene
acid derivatives 9b 12b 15c 18c and 20c. The influence of the guanidine group on the antitumor
4–7, 8a, 15, 18 and 20.
–
,
,
properties of triterpenoids was investigated. The cytotoxicity was tested on five human tumor
cell lines (Jurkat, K562, U937, HEK, and Hela), and compared with the tests on normal human
fibroblasts. The antitumor activities of the most tested guanidine derivatives was lower, than that of
corresponding amines, but triterpenoids with the guanidine group were less toxic towards human
fibroblasts. The introduction of the tris(hydroxymethyl)aminomethane moiety into the molecules of
triterpene acids markedly enhanced the cytotoxic activity of the resulting conjugates 15
, 15c, 18b,c and
20b irrespective of the triterpene skeleton type. The dihydrobetulinic acid amine 15, its guanidinium
,
c
derivative 15c and guanidinium derivatives of ursolic and oleanolic acids 18c and 20c were selected
for extended biological investigations in Jurkat cells, which demonstrated that the antitumor activity
of these compounds is mediated by induction of cell cycle arrest at the S-phase and apoptosis.
Keywords: triterpenoids; betulinic acid; ursolic acid; oleanolic acid; amino group; guanidine group;
cytotoxicity; apoptosis; cell cycle
1. Introduction
Among natural products of plant origin that are considered as abundant sources of lead structures
for the discovery of new drugs, the pentacyclic lupane, ursane, and oleanane triterpenoids occupy a
prominent place [
for pharmacological research, as they exhibit a variety of biological activities including antimicrobial,
antiparasitic, antitumor, and antiviral, in particular, anti-HIV, types of activity [ ]. Among these
properties of triterpenoids, of special interest is their anticancer activity and the ability to trigger the
mitochondrial apoptosis pathway in various types of human cancer cells [ ]. Thus, betulinic acid
is capable of inducing apoptosis in tumor cells, such as melanoma, adenocarcinoma, neuroblastoma,
medulloblastoma, glioblastoma and neuroectodermal tumors [ ]. The in vivo anticancer activity
of betulinic acid was identified using xenograft models [10 11]. The ursolic acid can also induce
1,2]. Triterpene acids (betulinic, ursolic, and oleanolic acids, Figure 1) are of interest
3–6
5–9
7–9
,
apoptosis, autophagy, and cell cycle arrest through various pathways, such as inhibition of DNA
replication, stimulation of reactive oxygen species (ROS) production, and affecting the balance between
proapoptotic and antiapoptotic proteins [6,12,13].
Molecules 2018, 23, 3000; doi:10.3390/molecules23113000
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Molecules 2018, 23, 3000
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The useful pharmacological properties of triterpene acids are successfully combined with their
acceptable systemic toxicity towards animals. However, the relatively low anticancer potential and
high hydrophobicity of these secondary metabolites markedly hamper their advancement as anticancer
drug candidates. For this reason, active search is in progress for analogues of natural triterpenoids
with a higher biological potential and enhanced pharmacological characteristics (hydrophilicity,
bioavailability) [
derivatives such as quaternary ammonium [16
salts [20 25] may serve as an efficient approach to improving bioavailability and selectivity of their
8,
14
,
15]. It has been shown [16
–25] that conversion of triterpene compounds to cationic
,
17], pyridinium [18 19] or triphenylphosphonium
,
–
biological action. Our recent study has shown that triphenylphosphonium derivatives of betulinic
and ursolic acids are substantially superior over their prototypes in the antitumor activity and in the
triggering mitochondria-dependent apoptosis of cancer cells [24,25].
Figure 1. Betulinic, ursolic and oleanolic acids.
However, the cytotoxic activity of the phosphonium salts was comparable with their cytotoxic
activity against normal peripheral blood cells. In continuation of the search for efficient and selective
antitumor agents, we have investigated novel cationic derivatives of pentacyclic triterpenoids containing
guanidine groups, which are readily protonated at a physiological pH level. The introduction of hydrophilic
guanidine groups into hydrophobic triterpene acid molecules may enhance their transmembrane transport
and physicochemical characteristics. Meanwhile, the new hybrid molecules may preserve the selectivity
of cytotoxic action against normal cells inherent in the natural triterpene acids. The guanidine group is
a common key unit in various natural and synthetic compounds demonstrating antimicrobial, antiviral,
and antitumor activities [26]. High symmetry of the Y-shaped guanidinium group promotes the formation
of two parallel hydrogen bonds with the biologically relevant counterparts. Unlike ammonium groups,
in which the charge is localized on one nitrogen atom (hard cations), guanidinium groups with a delocalized
charge actively interact through hydrogen bonds with soft ions such as phosphates and sulfates. This feature
of the guanidinium cation induce the efficient transport of biologically active substances through liposomal
and cell membranes [27–29]. Furthermore, because of high basicity (pKa 13.5), the guanidinium group
is important for selective delivery of cytotoxic molecules to tumor cells. Guanidine derivatives can be
accumulated in the mitochondria of tumor cells, thus destroying the mitochondrial potential and inhibiting
the mitochondrial respiratory chain [29,30].
Polyamines, which are precursors of aminoalkylguanidines, are also used to develop
chemotherapeutic agents, including antibacterial and antitumor compounds [31,32]. Structurally,
polyamine molecules contain positively charged nitrogen atoms at physiological pH value and
can serve as electrostatic bridges between negatively charged phosphates. They are able to bind
to negatively charged DNA macromolecules. However, some of physiological diamines, polyamines,
and their synthetic analogues have exhibited high toxicity toward normal cells. A large body of data
has now been accumulated on the biological activity of polyaminosterols, among which squalamine,
trodusquemine, and their synthetic analogues are best known [33
–36]. The synthesis and biological
properties of polyamino triterpene acids are described in several publications [
6,37–40]; the effect
of introduction of the guanidine group into triterpenoid molecules has not been studied so far.
Here we describe the synthesis and comparative evaluation of the cytotoxic and apoptosis-inducing
activities of new guanidine derivatives of pentacyclic lupane, ursane, and oleanane triterpenoids

Molecules 2018, 23, 3000
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and their precursors—C-28 conjugates of triterpene acids with some linear and branched mono-, di-,
and triaminoalkanes.
2. Results and Discussion
2.1. Chemistry
While synthesizing the target compounds, we found that the Boc-deprotection of guanidine
derivatives of betulinic and betulonic acids in acid medium (50% TFA in CH2Cl2) is complicated
by skeletal rearrangements of the lupane skeleton. It is known from the literature [41,42] that
hydrogenation of the C-20 double bond of lupane triterpenoids does not considerably affect their
cytotoxic activity and selectivity between normal and tumor cell lines; therefore, in the subsequent
experiments, we used 20,29-dihydrobetulinic and 20,29-dihydrobetulonic acids
1
and
2
to prepare
target compounds 9b–12b and 14 (Scheme 1).
H
H
N
a
COR2
N
NHBoc
COOH
d
X
R1
R
O
R1
R
NBoc
R1
R
4-7, 8a
1, 3
9-13
1: R + R1= O
2: R= OH, R1= H
3: R= AcO, R1= H
b
4: R+R1= O; R2= NH (CH2)4 NH2
e
5: R= AcO, R1= H; R2= NH (CH2)2 NH2
6: R= AcO, R1= H; R2= NH (CH2)4 NH2
7: R= AcO, R1= H; R2= NH (CH2)2 N (CH2)2 NH2
(CH2)2 NH2
H
H
N
N
NH2
X
8: R= AcO, R1= H; R2=
8a: R= AcO, R1= H; R2=
TFA
HN (CH2)3
(CH2)3 NHBoc
N
N
N
N
O
NH
R1
c
HN (CH2)3
(CH2)3 NH2
R
9a-13a
9, 9a, 9b: R+R1= O; X= (CH2)4
10, 10a, 10b: R= AcO, R1= H; X= (CH2)2
11, 11a, 11b: R= AcO, R1= H; X= (CH2)4
f
12: R= AcO, R1= H; X = (CH2)2 N (CH2)2
H
(CH2)2
N
NHBoc
NBoc
H
H
N
N
NH2
X
12a: R= AcO, R1= H; X= (CH2)2 N (CH2)2
HCl
O
NH
9b-12b
R1
R
H
(CH2)2
N
NH2
TFA
NH
12b: R= AcO, R1= H; X= (CH2)2 N (CH2)2
H
(CH2)2
N
NH2
HCl
11b
14
NH
N
g
13, 13a: R= AcO, R1= H; X=
14: R= OH, R1= H; X= (CH2)4
N
(CH2)3
(CH2)3
Scheme 1. Synthesis of target compounds 9b
2. 1,2-diaminoethane or 1,4-diaminobutane or tris(2-aminoethyl)amine or N-tert-butoxy-carbonyl-1,4-bis(3-
aminopropyl)piperazine, Et3N, CH2Cl2, r.t.; ( ) AcCl, THF, Py, DMAP, r.t.; ( ) 10% TFA, CH2Cl2, 3 h, r.t.;
) 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine, Et3N, CH2Cl2; ( ) 50% TFA, CH2Cl2, 2–4 h, r.t.; ( ) 5 M
HCl, MeOH; (g) 4 N NaOH, MeOH, THF.
–12b and 14. Reagents and conditions: (a) 1. (COCl)2, CH2Cl2;
b
c
(d
e
f
Acetate
were converted to C-28 amide derivatives
The reactions were carried out with 1,2-diaminoethane, 1,4-diaminobutane (putrescine),
3
obtained upon hydroxyl group protection in dihydrobetulinic acid
2, and dihydrobetulonic
acid
1
4–7
and 8a via relatively unstable acid chlorides (Scheme 1).
tris(2-aminoethyl)amine, and N-tert-butyloxycarbonyl-1,4-bis(3-aminopropyl)piperazine. The use of
branched triamine resulted in two guanidine functions being introduced into the triterpenoid molecule.

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Amides 4–7 were synthesized using a 3-fold molar excess of amines over triterpenes in order to avoid
the formation of dimeric products. Compound 8a was formed in a good yield only with the use of
N-Boc bis-aminopropylpiperazine, which was synthesized as described in reference [43]. The N-Boc
protection was then removed by treatment with 10% trifluoroacetic acid in CH2Cl2. Guanylation of
amines
available N,N0-di-Boc-N”-triflylguanidine. The expected compounds
yields after column chromatography on SiO2. The subsequent treatment of Boc derivatives
with CF3COOH and then with HCl/MeOH gave hydrochlorides 9b 13b. Dihydrobetulinic acid
guanidinium hydrochloride 14 was prepared by saponification of the 3-OAc function in conjugate 11b
A well studied and promising betulinic acid derivative is 2-amino-3-hydroxy-2-(hydroxymethyl)propyl
3-O-acetyl betulinate, known as the anticancer agent NVX-207 [45 48]. We have synthesized its C-20,29
4–
7
and 8a was carried out according to the typical procedure [44] by using commercially
13 were obtained in 60–88%
13
9
–
9
–
–
.
–
hydrogenated derivative 15. Ursolic and oleanolic acetates 16 and 17 were converted into NVX-207
analogues 18 and 20 with ursane and oleanane skeletons (Scheme 2).
Scheme 2. Synthesis of compounds 15a
2. tris(hydroxymethyl)aminomethane, DMAP, Py, CH2Cl2; (
guanidine, Et3N, CHCl3, reflux; (c) 50% TFA, CH2Cl2, 2–4 h, r.t.; (d) 5M HCl, MeOH.
–c,
18a–c,
20a–c. Reagents and conditions: (a) 1. (COCl)2, CH2Cl2;
b) 1,3-di-Boc-2-(trifluoromethyl-sulfonyl)
The reactions under reported conditions [45] afforded the desired esters 15
, 18, and 20 containing
the terminal NH2 group in the branched C-28 chain and amides 19 and 21 in 15–23% and 24–32%
yields, respectively. Guanylation of the free amino groups in the resulting amines 15
,
18, and 20 on
0
treatment with N,N -di-Boc-N”-triflylguanidine gave rise to guanidine derivatives 15a
, 18a, and 20a in
63–79% yields. The Boc-deprotection with 50% TFA/CH2Cl2 and the subsequent treatment of salts
15b, 18b, and 20b with 5M HCl in MeOH yielded hydrochlorides 15c, 18c, and 20c.

Molecules 2018, 23, 3000
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The structures of all products were confirmed by 1D (1H, 13C, 19F) and 2D (HSQC, HMBC, COSY)
NMR experiments and MALDI TOF MS. The 13C-NMR spectra of compounds
8a 15 18, and 20
show signals for amide carbon atoms at 176.3–179.3 ppm, while the amide group protons resonate at
5.86–6.79 ppm in the 1H-NMR spectra. In the spectra of guanidine derivatives
13 15a 18a, and 20a,
4
–7,
,
,
9–
,
,
the NH-C=N and NH-Boc proton signals occurred at 8.30–9.06 ppm and 11.47–11.53 ppm, respectively.
The signals for the guanidine group carbon atoms were observed at 161.8–163.6 ppm.
2.2. Biological Evaluation
The in vitro cytotoxic activity of triterpene acids (dihydrobetulinic, ursolic and oleanolic acids),
twelve guanidinium salts, and some of their precursors, primary amines 4–6, 8a, and 15, was evaluated
on five human tumor cell lines: Jurkat (T-lymphoblastic leukemia), K562 (chronic myeloid leukemia),
U937 (histiocytic lymphoma), HEK 293 (embryonic kidney), and HeLa (cervical cancer). The possible
cell toxicity was assessed against normal human fibroblasts. Most of the tested compounds showed
moderate or significant activity against Jurkat, K562, and U937 cells as compared to triterpenoic acids
(Table 1).
Table 1. Cytotoxicity of dihydrobetulinic-, ursolic- and oleanolic acids and compounds
4–6,
8a, 9b–12b,
12a
,
13a, 14, 15, 15c, 18b,c, and 20b,c against Jurkat, K562, Hek293, HeLa, U937, and normal fibroblast cells.
IC50 (µmol/L) a
Compound
Jurkat
K562
U937
HEK293
HeLa
Fibroblasts
Dihydro BA
59 ± 0.31
23 ± 0.34
44 ± 0.24
68 ± 0.11
235 ± 0.24
1.3 ± 0.49
2.8 ± 0.23
1.8 ± 0.12
3.0 ± 0.19
57 ± 0.36
98 ± 0.35
55 ± 0.38
89 ± 0.18
52 ± 0.26
11 ± 0.33
36 ± 0.42
2.1 ± 0.13
2.3 ± 0.34
40 ± 0.29
11 ± 0.18
20 ± 0.14
13 ± 0.45
39 ± 0.38
17 ± 0.12
186 ± 0.18
1.7 ± 0.36
1.9 ± 0.34
4.5 ± 0.43
8.2 ± 0.13
14 ± 0.16
82 ± 0.14
63 ± 0.29
28 ± 0.25
13 ± 0.06
18 ± 0.21
20 ± 0.12
6.6 ± 0.23
15 ± 0.23
2.1 ± 0.41
5.3 ± 0.29
6.1 ± 0.43
6.8 ± 0.11
128 ± 0.32
96 ± 0.22
247 ± 0.27
2.4 ± 0.35
4.8 ± 0.25
6.7 ± 0.21
5.9 ± 0.14
92 ± 0.34
128 ± 0.35
98 ± 0.27
112 ± 0.19
68 ± 0.29
74 ± 0.15
59 ± 0.21
17 ± 0.25
16 ± 0.27
13 ± 0.19
14 ± 0.34
12 ± 0.23
19 ± 0.17
132 ± 0.47
88 ± 0.35
258 ± 0.31
2.8 ± 0.28
3.9 ± 0.41
7.1 ± 0.18
6.1 ± 0.38
81 ± 0.19
152 ± 0.49
106 ± 0.51
101 ± 0.22
72 ± 0.34
79 ± 0.29
62 ± 0.24
19 ± 0.49
14 ± 0.31
11 ± 0.27
12 ± 0.29
13 ± 0.41
17 ± 0.32
517 ± 0.26
324 ± 0.16
694 ± 0.17
3.4 ± 0.19
5.3 ± 0.27
8.3 ± 0.29
7.3 ± 0.43
179 ± 0.43
354 ± 0.46
117 ± 0.38
149 ± 0.43
106 ± 0.23
104 ± 0.26
96 ± 0.17
31 ± 0.29
27 ± 0.14
47 ± 0.15
51 ± 0.26
49 ± 0.25
54 ± 0.12
UA
OA
4
5
6
8a
9b
10b
11b
12a
12b
13a
14
271 ± 0.19
1.4 ± 0.41
2.3 ± 0.11
7.7 ± 0.31
4.5 ± 0.29
21 ± 0.24
67 ± 0.21
16 ± 0.18
22 ± 0.21
14 ± 0.37
24 ± 0.27
17 ± 0.34
3.3 ± 0.11
3.1 ± 0.41
6.8 ± 0.32
3.8 ± 0.23
6.7 ± 0.15
7.6 ± 0.33
15
15c
18b
18c
20b
20c
a
IC50
(µM) is the half maximal inhibitory concentration for viable cells. Each IC50 (mean
±
SE) has been derived
from at least three experiments in duplicate.
Amino derivatives
values of 1.3–8 M. However, these compounds were also cytotoxic against fibroblasts. Contrary
to our expectations, guanylation of the terminal amino groups of lupane triterpenoids with C-28
linear aminoalkane chains (compounds 9b 11b 13a, and 14) or the branched tris-aminoethyl moiety
(compounds 12a ) did not considerably enhance the cytotoxic action. Of the listed guanidinium
salts, only compounds 9b 11b, and 14 showed an approximately 3-fold increase in the cytotoxic
activity against Jurkat cells in comparison with dihydrobetulinic acid and also showed selectivity
4–6 and 8a showed cytotoxic activity against all tumor cell cultures with IC50
µ
–
,
,b
,
2
towards Jurkat cells with an SI of 8.5, 7.3, and 5.6 respectively (SI = IC50 fibroblasts/IC50 Jurkat cells).
The introduction of the tris(hydroxymethyl)aminomethane moiety into the molecules of triterpene
acids
and 20b
and oleanolic acids with amino and guanidine groups in the ester side chain had from moderate to
3
,
16 and 17 markedly enhanced the cytotoxic activities for the resulting conjugates 15
, 15c, 18b,c,
,
c, irrespective of the triterpene skeleton type. The C-28 esters of dihydrobetulinic, ursolic,

Molecules 2018, 23, 3000
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good activity against Jurkat and K562 cell lines. For example, the IC50 values of compounds 15c and
18c were 3.1 and 3.8 M for T-lymphoblastic leukemia cells and 2.3 and 11.0 M for chronic myeloid
leukemia cells. The most pronounced differences in the antitumor activity were found for oleanolic
acid and its conjugates 20b . Indeed, the IC50 values of oleanolic acid, 20b, and 20c for Jurkat cells
were 271, 6.7, and 7.6 M, respectively. It is worth noting that amine 15, its guanidine derivative 15c
µ
µ
,
c
µ
,
and guanidinium salts of ursolic and oleanolic acids 18b
,
c
and 20b
,c
showed acceptable selectivity
towards Jurkat tumor cell with an SI from 6.9 to 13.4.
The identified lead compounds with the highest cytotoxicity characteristics, 15
,
15c 18c, and 20c,
,
were further evaluated for the possible apoptosis induction in tumor cell cultures. The measurements
were done by flow cytometry (Figure 2).
Figure 2. AnnexinV/7-AAD staining upon induction of apoptosis in Jurkat cells. Cells were treated
with compounds at their IC50 concentration for 24 and 48 h. Then, the cells were harvested, stained
with Annexin V/7-AAD and analyzed by flow cytometry. The experiments were performed three times,
and the results of the representative experiments are shown. The first cytogram represents an untreated
cell sample (
with compound 15 for 24 h (
for 48 h ( ); after incubation with compound 18c for 24 h (
compound 20c for 24 h ( ) and for 48 h ( ). Q7-1, necrotic cells; Q7-2, late apoptotic cells; Q7-3, living
cells; Q7-4, early apoptotic cells.
A
); after incubation with dihydrobetulinic acid for 24 h (
) and for 48 h ( ); after incubation with compound 15c for 24 h (
) and for 48 h ( ); after incubation with
B
) and for 48 h (
C
); after incubation
D
E
F) and
G
H
I
J
K
The highest percentage of late apoptosis (91.7%) was detected upon the treatment of Jurkat
cells with the test compound 15 at IC50 concentration exposure for 48 h as depicted in Figure 2E.
Compounds 15c
case, the apoptotic effect of these guanidine derivatives was notably weaker. Thus, after treatment of
Jurkat cells with compound 15c at IC50 concentration (4 M) the number of vital cells is decreased from
, 18c and 20c also showed apoptotic mode of cell death on Jurkat cells line, but, in this
µ

Molecules 2018, 23, 3000
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96.2% (control) to 54.5%. Total number of apoptotic cells population constituted 23.7% (7.2% and 16.5%
of early and late apoptotic cells, respectively) and number of necrotic cells was 21.7%. Comparable
results were obtained with the guanidine derivative of ursolic acid 18c (6.8, 14.3, and 20.7% of early,
late apoptotic cells and necrotic cells, respectively). Treatment of the Jurkat cells with 20c resulted in
about 15.5% apoptotic cells and 6.1% necrotic cells, with 78.4% of the cells still being considered vital
(Figure 2K). Next, we analyzed the ability of dihydrobetulinic acid to stimulate apoptosis. Our results
showed that dihydrobetulinic acid triggers apoptosis in Jurkat cells at higher doses as compared to
derivatives 15, 15c, and 18c. The number of apoptotic cells on treatment with dihydrobetulinic acid
(59 M) for 48 h constituted around 24% (4.0% of early-stage and 20.5% of secondary necrotic/late-stage
µ
apoptotic), while the number of vital cells was 73.1% (Figure 2C).
In summary, our results indicate that the apoptosis is induced in Jurkat cells by all test compounds.
However, we observed a higher rate of necrosis after 15c, 18c and 20c incubation compared to derivative 15.
DNA flow cytometry was also used to analyze the cell cycle kinetics in Jurkat cells pre-incubated
with dihydrobetulinic acid and derivatives 15, 15c, 18c, or 20c at their IC50 concentration for 24 and
48 h (Figure 3).
Figure 3. Cell cycle analysis in Jurkat cells. Jurkat cells treated with tested compounds at the IC50
concentration for 24 and 48 h. The cells were trypsinized, harvested and washed three times with
ice-PBS for PI-stained DNA content detected by flow cytometry. The experiments were performed
three times, and the results of the representative experiments are shown. The first cytogram represents
an untreated cell sample (
with compound 15 for 24 h (
for 48 h ( ); after incubation with compound 18c for 24 h (
compound 20c for 24 h (I) and for 48 h (J).
A
); after incubation with dihydrobetulinic acid for 48 h (
) and for 48 h ( ); after incubation with compound 15c for 24 h (
) and for 48 h ( ); after incubation with
B
); after incubation
C
D
E) and
F
G
H
The distribution of DNA content in Jurkat cells reveals whether cell proliferation is arrested at one
checkpoint. The results of all experiments showed in significant S-phase arrest in cells after treating
with these compounds. Thus, the ratio of cells in the S phase increased from 38.3% (control) to 72.7% in

Molecules 2018, 23, 3000
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cells treated with 15 (4
µM for 48 h) and increased to 47.2–56.8% in cells treated with dihidrobetulinic
acid, 15c, 18c, or 20c. The appropriate decrease of the number of cells in the G2/M phase was observed.
For example, the treatment of Jurkat cells with 15 and 15c resulted in a decrease of cells in the G2/M
phase from 14.5% (control) to 7.0 and 1.5% (Figure 3D,F). Considering these results, we assume that
dihydrobetulinic acid and compounds 15, 15c, 18c, or 20c are able to trigger programmed cell death,
including apoptotic mechanisms and cell cycle arrest in the S-phase.
3. Materials and Methods
3.1. Chemistry
IR spectra (thin films or solutions in CHCl3) were obtained with use of a Vertex 70v spectrometer
1
(Bruker, Karlsruhe, Germany). H- and 13C-NMR spectra were recorded in CDCl3, in MeOD or in
d6-DMSO with Me4Si as the internal standard on an AVANCE–500 instrument (500.13 (1H), 125.78 MHz
(13C), 470.59 MHz (19F)) or on an AVANCE-400 (400.13 (1H), 100.62 MHz (13C), 376.50 MHz (19F))
(Bruker). Mass spectra of new compounds were recorded on a Bruker–Autoflex III spectrometer
(MALDI TOF, positive ion mode, sinapic acid as the matrices). Optical rotation was determined on a
−1
141 polarimeter (Perkin–Elmer, Beaconsfield, UK). Specific rotation [α]D is expressed in (deg
·mL)/(g·dm)
;
the concentration of the solution c is expressed in g/100 mL. Elemental analysis was carried out on a
1106 analyzer (Carlo Erba, Milan, Italy). TLC was carried out on Sorbfil plates (Sorbpolimer, Krasnodar,
Russia) in hexane–EtOAc and chloroform–methanol, spots were visualized with anisaldehyde. Silica gel L
(KSKG grade, 50–160 µm) was employed for column chromatography. All reagents and solvents were of the
purest grade available, and generally were used without further treatment. The starting compounds ursolic,
oleanolic acids and reagents: sodium borohydride (NaBH4), acetyl chloride, 10% Pd/C, oxalyl chloride,
1,2-diaminoethane, 1,4-diaminobutane, tris(2-aminoethyl)amine, 1,4-bis(3-aminopropyl)piperazine,
tris(hydroxymethyl)aminomethane, 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine, triethylamine (Et3N),
dimethylaminopyridine (DMAP), trifluoroacetic acid (TFA) were purchased from Acros Organics (Geel,
Belgium). Dihydrobetulonic and dihydrobetulinic acids were obtained from betulin according to the
known procedures [41]. Acetates of oleanolic, ursolic, dihydrobetulonic and dihydrobetulinic acids were
synthesized according to the typical procedures. Mono-Boc-protected bis-aminopropylpiperazine and
compounds 19–23 were prepared by a reported methods [43,
45]. NMR 1H and 13C spectra of all new
compounds are in Supplementary Materials.
3.1.1. General Procedure for the Synthesis of Amines 4–7
Oxalyl chloride (0.13 mL, 1.5 mmol) was a◦dded with stirring to a solution of compounds
1 or 3
(0.5 mmol) in dry CH2Cl2 (5 mL) precooled to 0 C, and stirring of the reaction mixture was continued
at room temperature for 2 h. Then the solvent and excess oxalyl chloride were removed under vacuum.
The amine (1.5 mmol) was dissolved in dry CH2Cl2 (2 mL) and under vigorous stirring were added
Et3N (0.2 mL, 1.5 mmol) and a solution of the acid chloride of
The mixture was stirred for 24 h (monitoring by TLC). The mixture was then poured into cold H2O
and extracted with CH2Cl2 (2 15 mL each). The organic phases washed brine and were dried over
1 or 3 (0.5 mmol) in dry CH2Cl2 (4 mL).
×
Na2SO4 and evaporated under reduced pressure. The residue was chromatographed on silica gel,
using CH2Cl2/MeOH 30:1→1:1 (v/v), to obtain pure compounds 4–7.
N-(4-Aminobuthyl)-3-oxolupane-28-amide (
+4◦ (c 0.23, CH2Cl2); IR (CHCl3) 1641, 1702 (C=O), 3357 (NH) cm−1; 1H-NMR (500 MHz, CDCl3)
4 :
), White powder, 78% yield; mp 170–172 ◦C (EtOH); [α]D19
ν
max
δ
: 5.86 (t, J = 5.5 Hz, 1H, CONH), 3.32–3.20 (m, 2H, H-10), 2.76 (t, J = 6.5 Hz, 2H, H-40), 2.55–1.14 (m,
26H, CH, CH2 in pentacyclic skeleton, 4H, H-20, H-30), 1.08, 1.03, 0.98, 0.97, 0.94 (all s, 3H each,
H-23–H-27), 0.87 (d, J = 6.5 Hz, 3H, H-29), 0.75 (d, J = 7.0 Hz, 3H, H-30); 13C-NMR (125 MHz, CDCl3)
δ
:
218.2 (C-3), 176.3 (C-28), 56.0 (C-17), 55.0 (C-5), 49.8 (C-9), 49.5 (C-19), 47.4 (C-4), 44.2 (C-18), 42.7 (C-14),
41.6 (C-40), 40.7 (C-8), 39.6 (C-1), 39 (C-22), 38.8 (C-10), 37.7 (C-13), 36.9 (C-10), 34.2 (C-2), 33.8 (C-16),
0
33.6 (C-7), 30.6 (C-3 ), 29.9 (C-20), 29.4 (C-15), 27.2 (C-23), 27.0 (C-2’), 26.6 (C-12), 23.1 (C-29), 23.0 (C-21),

Molecules 2018, 23, 3000
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21.5 (C-24), 21.1 (C-11), 19.7 (C-6), 16.0 (C-25), 15.9 (C-26), 14.6 (C-30), 14.5 (C-27); Anal. Calcd. for
C34H58N2O2: C, 77.51, H, 11.10. Found: C, 78.03, H, 11.02%. MS: m/z 527.45 [M + H]+ (calcd. for
C34H58N2O2, 526.45).
3
β
-N-(2-Aminoethyl)-3-O-acetyl-lupane-28-amide (
[α]D19 8◦ (c 0.24, CH2Cl2); IR (CHCl3) νmax 1647, 1735 (C=O), 3367 (NH) cm−1; 1H-NMR (500 MHz,
CDCl3)
5
), White powder, 74% yield; mp 120–122 ◦C (EtOH);
−
δ
: 6.14 (t, J = 5.5 Hz, 1H, CONH), 4.48–4.44 (m, 1H, H-3), 3.35–3.21 (m, 2H, H-10), 2.81 (t,
J = 6.0 Hz, 2H, H-20), 2.43–0.97 (m, 25H, CH, CH2 in pentacyclic skeleton), 2.01 (s, 3H, CH3CO–),
0.94, 0.92, 0.85, 0.84, 0.83, 0.82 (all s, 3H each, H-23–H-27 and H-29), 0.77 (d, J = 10.0 Hz, 1H, H-5),
0.73 (d, J = 7.0 Hz, 3H, H-30); 13C-NMR (125 MHz, CDCl3)
δ: 176.7 (C-28), 171.0 (COCH3), 80.9 (C-3),
56.2 (C-17), 55.4 (C-5), 50.3 (C-9), 49.5 (C-19), 44.3 (C-18), 42.6 (C-14), 41.8 (C-20), 41.7 (C-10), 40.8 (C-8),
38.7 (C-1), 38.4 (C-22), 37.8 (C-4), 37.6 (C-13), 37.1 (C-10), 34.4 (C-7), 33.6 (C-16), 29.9 (C-20), 29.5 (C-15),
27.9 (C-23), 27.0 (C-2), 23.7 (C-12), 23.1 (C-21, C-29), 21.3 (COCH3), 21.0 (C-11), 18.2 (C-6), 16.5 (C-25),
16.2 (C-24, C-26), 14.6 (C-30), 14.5 (C-27); Anal. Calcd. for C34H58N2O3: C, 75.23, H, 8.84. Found: C,
75.74, H, 8.79%. MS: m/z 543.40 [M + H]+ (calcd. for C34H58N2O3, 542.44).
◦
3
β
-N-(4-Aminobuthyl)-3-O-acetyl-lupane-28-amide (
[α]D19 11◦ (c 0.69, CHCl3); IR (CHCl3) νmax 1646, 1734 (C=O), 3367 (NH) cm−1
MHz, CDCl3)
6
), White powder, 58% yield; mp 104–106 C (EtOH);
−
;
1H-NMR (500
δ
: 5.86 (t, J = 5.5 Hz, 1H, CONH), 4.48–4.45 (m, 1H, H-3), 3.30–3.17 (m, 2H, H-10),
2.73 (t, J = 6.5 Hz, 2H, H-40), 2.47–0.98 (m, 25H, CH, CH2 in pentacyclic skeleton, 4H, H-20, H-30),
2.03 (s, 3H, CH3CO–), 0.95, 0.93, 0.92, 0.84, 0.83, 0.82, (all s, 3H each, H-23–H-27 and H-29), 0.78 (d,
J = 10.0 Hz, 1H, H-5), 0.73 (d, J = 7.0 Hz, 3H, H-30); 13C-NMR (125 MHz, CDCl3)
δ: 176.3 (C-28),
171.0 (COCH3), 80.9 (C-3), 56.0 (C-17), 55.4 (C-5), 50.3 (C-9), 49.5 (C-19), 44.3 (C-18), 42.6 (C-14),
41.6 (C-40), 40.8 (C-80), 39.0 (C-10), 38.7 (C-22), 38.4 (C-1), 37.8 (C-4), 37.6 (C-13), 37.1 (C-10), 34.4 (C-7),
0
33.6 (C-16), 30.7 (C-3 ), 29.9 (C-20), 29.4 (C-15), 27.9 (C-23), 27.2 (C-2 ), 27.0 (C-2), 23.7 (C-12), 23.0 (C-21,
C-29), 21.3 (COCH3), 21.0 (C-11), 18.2 (C-6), 16.5 (C-25), 16.2 (C-26, C-24), 14.6 (C-30), 14.5 (C-27); Anal.
Calcd. for C36H62N2O3: C, 75.74, H, 10.95. Found: C, 76.01, H, 10.89%. MS: m/z 609.43 [M + K]+ (calcd.
for C36H62N2O3, 570.48).
3
β
-N-[2-(N,N0-bis-Aminoethyl)-aminoethyl]-3-O-acetyl-lupane-28-amide (
mp 108–110 ◦C (EtOH); [α]D19 5◦ (c 0.16, C2H5OH); IR (CHCl3) νmax 1648, 1735 (C=O), 3441 (NH)
cm−1; 1H-NMR (400 MHz, MeOD)
7), White powder, 69% yield;
−
δ
: 4.49–4.45 (m, 1H, H-3), 3.33–3.27 (m, 2H, H-10), 2.74–2.72 (m,
4H, H-40, H-42”), 2.60–2.58 (m, 6H, H-20, H-30, H-3”), 2.32–0.84 (m, 26H, CH, CH2 in pentacyclic
skeleton), 2.05 (s, 3H, CH3CO–), 1.02, 1.00, 0.93, 0.90, 0.89, 0.88 (all s, 3H each, H-23–H-27 and H-29),
0.79 (d, J = 6.4 Hz, 3H, H-30); 13C-NMR (100 MHz, MeOD)
δ: 179.3 (C-28), 172.8 (COCH3), 82.5 (C-3),
57.7 (C-17), 57.4 (C-5), 57.0 (C-30, C-3”), 55.2 (C-2’), 51.9 (C-9), 51.1 (C-19), 45.5 (C-18), 43.8 (C-14),
42.2 (C-8), 40.2 (C-22), 39.9 (C-1), 39.8 (C-40, C-4”), 39.0 (C-4), 38.8 (C-10), 38.4 (C-13), 38.3 (C-10),
35.8 (C-7), 34.1 (C-16), 31.4 (C-20), 30.9 (C-15), 28.7 (C-23), 28.5 (C-2), 24.8 (C-12), 24.2 (C-29), 23.8 (C-21),
22.4 (COCH3), 21.4 (C-11), 19.4 (C-6), 17.2 (C-25), 17.0 (C-24, C-26), 15.3 (C-30, C-27); Anal. Calcd. for
C38H68N4O3: C, 72.56, H, 10.90. Found: C, 73.04, H, 10.87%. MS: m/z 629.56 [M + H]+ (calcd. for
C38H68N4O3, 628.53).
3
β-N-{[3-(3-Aminopropyl)piperazinyl]propyl}-3-O-acetyl-lupane-28-amide (8a), The acid chloride of
3 was
synthesized according to the general procedure for synthesis of amines . Then the N-tert-butoxycarbonyl-
4–7
1,4-bis(3-aminopropyl)piperazine (0.45 g, 1.5 mmol) and Et3N (0.25 mL, 1.8 mmol) were added to acid
chloride of 3 (0.50 g, 1 mmol) in dry CH2Cl2 (10 mL). The mixture was stirred at room temperature
for 16 h (monitoring by TLC), then was poured into cold H2O and extracted with CH2Cl2 (2
×
15 mL).
The organic phases washed brine, dried over Na2SO4 and evaporated under reduced pressure to obtain
compound 8. Then the compound 8 was dissolved in CH2Cl2 (7 mL), acidified with TFA 10% (v/v)
in CH2Cl2 (17 mL) for deprotection reaction and stirred for around 5 h. The reaction was quenched
using saturated aqueous potassium carbonate solution (20 mL). The aqueous phase was extracted with
CH2Cl2 (2 × 15 mL). The organic phases washed brine, dried over Na2SO4 and evaporated under reduced

Molecules 2018, 23, 3000
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pressure. The residue was chromatographed on silica gel, using CH2Cl2/MeOH 50:1
→
5:1, to obtain pure
compound 8a. White powder, 78% yield; mp 119–121 ◦C (EtOH); [α]D21
−
7.4◦ (c 0.5, CHCl3); IR (CHCl3)
1
νmax 1735 (C=O), 3361 (NH) cm−1; H-NMR (500 MHz, CDCl3)
δ
: 6.79 (t, J = 5.5 Hz, 1H, CONH),
4.47–4.44 (m, 1H, H-3), 3.31–3.25 (m, 2H, H-10), 2.78–2.77 (m, 2H, H-1”), 2.50–2.42 (m, 12H, H-30–H-50,
H-3”–H-5”), 2.41–0.98 (m, 25H, CH, CH2 in pentacyclic skeleton, 4H, H-20, H-2”), 2.02 (s, 3H, CH3CO–),
0.93, 0.92, 0.91, 0.83, 0.82, 0.81 (3H each, all s, H-23–H-27 and H-29), 0.77 (d, J = 10.5 Hz, 1H, H-5), 0.72 (d,
J = 6.5 Hz, 3H, H-30); 13C-NMR (125 MHz, CDCl3)
δ
: 176.3 (C-28), 171.0 (
C
OCH3), 80.9 (C-3), 57.8 (C-30),
0
0
56.7 (C-3”), 55.9 (C-17), 55.4 (C-5), 53.5 (C-5 , C-5”), 53.3 (C-4 ), 53.3 (C-4”), 50.3 (C-9), 49.6 (C-19), 44.1 (C-18),
42.6 (C-14), 40.9 (C-1”), 40.8 (C-8), 39.3 (C-10), 38.8 (C-22), 38.4 (C-1), 37.8 (C-4), 37.4 (C-13), 37.1 (C-10),
34.4 (C-7), 33.7 (C-16), 29.9 (C-20), 29.8 (C-20), 29.7 (C-15), 27.9 (C-23), 26.9 (C-2), 25.3 (C-2”), 23.7 (C-12),
23.0 (C-21, C-29), 21.3 (COCH3), 21.0 (C-11), 18.2 (C-6), 16.5 (C-25), 16.3 (C-24), 16.2 (C-26), 14.6 (C-30),
14.5 (C-27); Anal. Calcd. for C42H74N4O3: C, 73.85, H, 10.92. Found: C, 74.13, H, 10.84%. MS: m/z 683.52
[M + H]+ (calcd. for C42H74N4O3, 682.58).
3.1.2. General Procedure for the Synthesis of Compounds 15, 18–21
Acid chlorides of
3
,
16
,
17 was synthesized according to the general procedure for synthesis
16 17 (1 mmol) was dissolved in a mixture of pyridine
of amines . Then the acid chlorides of
4–
7
3
,
,
(4 mL), CH2Cl2 (1 mL) and DMAP (0.09 g, 0.7 mmol) was added. After complete dissolution of DMAP,
a solution containing of TRIS (tris(hydroxymethyl)aminomethane) (0.24 g, 2 mmol) in pyridine (0,5 mL)
was added. The mixture was stirred for 10 h at room temperature and the solvent was removed rapidly
under vacuum. The residue was chromatographed on silica gel, using CH2Cl2/MeOH 30:1
to obtain pure compounds 15, 18–21.
→
1:1,
3β-[2-Amino-3-hydroxy-2-(hydroxymethyl)propyl]-3-O-acetyl-lupane-28-oate (15), White powder, 16% yield;
mp 134–136 C (EtOH); [α]D21
cm−1; 1H-NMR (400 MHz, CDCl3)
−
13.1 (c 0.51, CHCl3); IR (CHCl3) νmax 1731 (C=O), 3366 (OH), 3446 (NH)
◦
◦
δ
: 4.49–4.45 (m, 1H, H-3), 4.09, 4.03 (both d, J = 11.6 Hz, 1H each,
H-10), 3.52 (br s, 4H, H-30, H-40), 2.70 (br s, 4H, NH2, OH), 2.05 (s, 3H, CH3CO–), 2.24–0.81 (m, 26H,
CH, CH2 in pentacyclic skeleton), 0.95, 0.91, 0.85, 0.84, 0.83, 0.82 (all s, 3H each, H-23–H-27 and H-29),
0.76 (d, J = 6.8 Hz, 3H, H-30); 13C-NMR (100 MHz, CDCl3)
δ: 177.0 (C-28), 171.1 (COCH3), 80.9 (C-3),
64.9 (C-10), 64.2 (C-30, C-40), 57.3 (C-17, C-20), 55.4 (C-5), 50.2 (C-9), 48.8 (C-19), 44.2 (C-18), 42.6 (C-14),
40.7 (C-8), 38.4 (C-22), 38.1 (C-1), 37.8 (C-4), 37.5 (C-13), 37.1 (C-10), 34.3 (C-7), 32.0 (C-16), 29.7 (C-15,
C-20), 27.9 (C-23), 26.9 (C-2), 23.7 (C-12), 23.0 (C-29), 22.8 (C-21), 21.3 (COCH3), 20.9 (C-11), 18.2 (C-6),
16.5 (C-25), 16.2 (C-24), 16.1 (C-26), 14.7 (C-30), 14.6 (C-27); Anal. Calcd. for C36H61NO6: C, 71.60, H,
10.18. Found: C, 72.09, H, 10.11%. MS: m/z 604.42 [M + H]+ (calcd. for C36H61NO6, 603.45).
3β-[2-Amino-3-hydroxy-2-(hydroxymethyl)propyl]-3-O-acetylurs-12-en-28-oate (18), White powder, 15% yield;
mp 129–131 ◦C (EtOH); [α]D18 +40◦ (c 0.74, CHCl3); IR (CHCl3) νmax 1721 (C=O), 3444 (NH), 3468 (OH)
1
cm−1; H-NMR (400 MHz, CDCl3)
δ
: 5.25 (br s, 1H, H-12), 4.51–4.47 (m, 1H, H-3), 4.02, 3.93 (both d,
J = 12 Hz, 1H each, H-10), 3.48 (br s, 4H, H-30, H-40), 2.68 (br s, 4H, NH2, OH), 2.19 (d, J = 11.2 Hz, 1H,
H-18), 2.05 (s, 3H, CH3CO–), 1.99–0.81 (m, 22H, CH, CH2 in pentacyclic skeleton), 1.08, 0.96, 0.94, 0.88,
0.87, 0.86, 0.74 (all s, 3H each, H-23–H-27, H-29 and H-30); 13C-NMR (100 MHz, CDCl3)
δ
: 178.1 (C-28),
171.1 (C
OCH3), 138.3 (C-13), 125.6 (C-12), 80.9 (C-3), 65.7 (C-10), 64.4 (C-30, C-40), 56.6 (C-20), 55.3 (C-5),
53.0 (C-18), 48.6 (C-17), 47.4 (C-9), 42.1 (C-14), 39.5 (C-4, C-8), 38.9 (C-19), 38.3 (C-20), 37.7 (C-1), 37.1 (C-22),
36.8 (C-10), 32.9 (C-7), 30.6 (C-21), 28.1 (C-23), 27.9 (C-15), 24.3 (C-16, C-27), 23.5 (C-2, C-11), 21.3 (COCH3),
21.1 (C-30), 18.2 (C-6), 17.2 (C-29), 17.0 (C-26), 16.7 (C-24), 15.5 (C-25); Anal. Calcd. for C36H59NO6: C,
71.84, H, 9.88. Found: C, 72.13, H, 9.83%. MS: m/z 602.40 [M + H]+ (calcd. for C36H59NO6, 601.43).
3
β
-N-[(1◦0,10,10-tris-Hidroxymethyl)methyl]-3-O-acetyl-ursolamide (19), White powder, 24% yield; mp
203–205 C (EtOH); [α]1D8 +26.9 (c 0.74, CH3OH); IR (CH3OH) νmax 1734 (C=O), 2923, 2871, 2852 (OH),
◦
3352 (NH) cm−1; 1H-NMR (500 MHz, MeOD)
δ: 5.38 (br s, 1H, H-12), 4.50–4.47 (m, 1H, H-3), 3.66,
3.58 (both d, J = 10 Hz, 3H each, H-20–H-40), 2.12–0.88 (m, 23H, CH, CH2 in pentacyclic skeleton),
2.05 (s, 3H, CH3CO–), 1.18, 1.04, 0.99, 0.95, 0.93, 0.92, 0.91 (all s, 3H each, H-23–H-27, H-29 and H-30);

Molecules 2018, 23, 3000
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13C-NMR (125 MHz, CDCl3)
δ
: 178.5 (C-28), 170.7 (
C
OCH3), 138.1 (C-13), 125.8 (C-12), 80.4 (C-3),
61.8 (C-10), 60.8 (C-20–C-40), 55.1 (C-5), 53.6 (C-18), 48.3 (C-17), 47.4 (C-9), 42.3 (C-14), 39.5 (C-4, C-8),
38.9 (C-19), 38.4 (C-20), 37.7 (C-22, C-1), 36.8 (C-10), 33 (C-7), 30.9 (C-21), 28.3 (C-23), 27.8 (C-15),
24.5 (C-16), 23.7 (C-2), 23.4 (C-11, C-27), 21.5 (COCH3), 21.4 (C-30), 18.2 (C-6), 17.5 (C-29, C-26),
17.1 (C-24), 15.7 (C-25); Anal. Calcd. for C36H59NO6: C, 71.84, H, 9.88. Found: C, 72.09, H, 9.79%. MS:
m/z 602.41 [M + H]+ (calcd. for C36H59NO6, 601.43).
3
β
-[2-Amino-3-hyd◦roxy-2(hydroxymethyl)propyl]-3-O-acetylolean-12-en-28-oate (20), White powder, 23%
yield; mp 141–143 C (EtOH); [α]1D8 +49 (c 0.75, CHCl3); IR (CHCl3) νmax 1618, 1727 (C=O), 3447 (NH)
◦
cm−1; 1H-NMR (500 MHz, CDCl3)
δ: 5.30 (br s, 1H, H-12), 4.51–4.48 (m, 1H, H-3), 4.04, 3.99 (both d,
J = 11.5 Hz, 1H each, H-10), 3.50 (br s, 4H, H-30, H-40), 2.84 (d, J = 10.0 Hz, 1H, H-18), 2.68–2.60 (m,
4H, NH2, OH), 2.05 (s, 3H, CH3CO–), 2.01–0.83 (m, 22H, CH, CH2 in pentacyclic skeleton), 1.14, 0.94,
0.93, 0.92, 0.87, 0.86, 0.73 (all s, 3H each, H-23–H-27, H-29 and H-30); 13C-NMR (125 MHz, CDCl3)
δ
: 178.2 (C-28), 171.1 (C
OCH3), 143.7 (C-13), 122.6 (C-12), 80.9 (C-3), 65.7 (C-10), 64.6 (C-30, C-40),
56.6 (C-20), 55.3 (C-5), 47.5 (C-9), 47.2 (C-17), 45.7 (C-19), 41.8 (C-14), 41.5 (C-18), 39.3 (C-8), 38.1 (C-1),
37.7 (C-4), 36.9 (C-10), 33.8 (C-22), 33.0 (C-30), 32.8 (C-7), 32.7 (C-21), 30.7 (C-20), 28.0 (C-23), 27.6 (C-15),
25.8 (C-27), 23.6 (C-29), 23.5 (C-11), 23.4 (C-2), 23.1 (C-16), 21.3 (COCH3), 18.2 (C-6), 17.1 (C-26),
16.7 (C-24), 15.4 (C-25); Anal. Calcd. for C36H59NO6: C, 71.84, H, 9.88. Found: C, 72.04, H, 9.82%. MS:
m/z 602.40 [M + H]+ (calcd. for C36H59NO6, 601.43).
3
β
-N-[(10◦,10,10-tris-Hydroxymethyl)methyl]-3-O-acetyl-oleanolamide (21), White powder, 32% yield; mp
249–251 C (EtOH); [α]1D8 +33.9◦ (c 0.61, CH3OH); IR (CH3OH) νmax 1732(C=O), 2945, 2927 (OH),
3353 (NH) cm−1; H-NMR (500 MHz, d6-DMSO)
J = 5 Hz, 3H, –OH), 4.42–4.38 (m, 1H, H-3), 3.49–3.42 (m, 6H, H-2 –H-4 ), 2.61 (d, J = 10.0 Hz, 1H, H-18),
δ
: 6.62 (s, 1H, NH), 5.24 (br s, 1H, H-12), 4.97 (t,
1
0
0
2.00 (s, 3H, CH3CO–), 1.97–0.84 (m, 22H, CH, CH2 in pentacyclic skeleton), 1.91, 0.90, 0.88, 0.87, 0.82,
0.81, 0.78 (all s, 3H each, H-23–H-27, H-29 and H-30); 13C-NMR (125 MHz, d6-DMSO)
δ
: 178.3 (C-28),
170.6 (
C
OCH3), 143.7 (C-13), 122.6 (C-12), 80.4 (C-3), 61.8 (C-10), 60.8 (C-20–C-40), 55 (C-5), 47.4 (C-9),
46.8 (C-17), 46.6 (C-19), 42.0 (C-14), 41.9 (C-18), 39.5 (C-8), 38.2 (C-1), 37.7 (C-4), 36.9 (C-10), 34.1 (C-22),
33.3 (C-7), 33.2 (C-30), 32.7 (C-21), 30.8 (C-20), 28.2 (C-23), 27.4 (C-15), 25.8 (C-27), 23.8 (C-29), 23.7 (C-2),
23.4 (C-11, C-16), 21.4 (COCH3), 18.2 (C-6), 17.4 (C-26), 17.1 (C-24), 15.6 (C-25); Anal. Calcd. for
C36H59NO6: C, 71.84, H, 9.88. Found: C, 72.11, H, 9.85%. MS: m/z 602.39 [M + H]+ (calcd. for
C36H59NO6, 601.43).
3.1.3. General Procedure for the Guanilation of Amines 4–7, 8a, 15, 18 and 20
The amine (0.5 mmol) is added neat to a solution of 1,3-di-Boc-2-(trifluoromethyl-sulfonyl)guanidine
(0.18 g, 0.45 mmol) or (0.60 g, 0.9 mmol) for amine
7
and Et3N (0.07 mL, 0.5 mmol) in CH2Cl2 (at r.t.) of
18 20 (10 mL). The mixture was stirred for
compounds 8a or in CHCl3 (at reflux) of compounds 15
4–7,
,
,
2–12 h (TLC monitoring CH2Cl2/MeOH 20:1). After completion of the reaction, the mixture is diluted with
CH2Cl2 and washed witH-NH4Cl, NaHCO3 and brine. After drying with sodium sulfate and filtering
the solvent is removed under reduced pressure. The residue was chromatographed on silica gel, using
hexane/EtOAc 10:1→1:1, to obtain pure compounds 9–13, 15a, 18a and 20a.
N-[4-tert-Butyloxycarbonyl buthylguanidine]-3-oxo-lupane-28-amide (
9), White powder, 88% yield; mp
158–160 ◦C (EtOH); [α]2D3 +3.4◦ (c 0.59, CH2Cl2); IR (CHCl3) νmax 1637, 1718 (C=O), 3337 (NH) cm−1
;
1H-NMR (500 MHz, CDCl3)
δ: 11.52 (s, 1H, NH in Boc), 8.30 (br s, 1H, NH–C=N), 5.70 (br s, 1H,
CONH), 3.48–3.41 (m, 2H, H-20), 3.37–3.20 (m, 2H, H-10), 1.51, 1.48 (both s, 9H each, CH3 in Boc),
2.54–1.15 (m, 26H, CH, CH2 in pentacyclic skeleton, 4H, H-20, H-30), 1.08, 1.03, 0.98, 0.97, 0.95 (all
s, 3H each, H-23–H-27), 0.87 (d, J = 6.5 Hz, 3H, H-29), 0.75 (d, J = 6.5 Hz, 3H, H-30); 13C-NMR
(125 MHz, CDCl3) δ: 218.1 (C-3), 176.3 (C-28), 163.6 (C=N), 156.1, 153.3 (CONH-Boc), 83.0, 79.2 (C
in Boc), 56.0 (C-17), 55.0 (C-5), 49.8 (C-9), 49.5 (C-19), 47.3 (C-4), 44.1 (C-18), 42.6 (C-14), 40.7 (C-8),
40.5 (C-40), 39.6 (C-1), 38.7 (C-22, C-10), 37.6 (C-13), 36.9 (C-10), 34.1 (C-2), 33.8 (C-16), 33.6 (C-7),
29.9 (C-20), 29.4 (C-15), 28.4, 28.0 (CH3 in Boc), 27.7 (C-23), 27.0 (C-20, C-30), 26.6 (C-12), 23.0 (C-21,

Molecules 2018, 23, 3000
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C-29), 21.5 (C-24), 21.0 (C-11), 19.6 (C-6), 16.0 (C-25), 15.9 (C-26), 14.6 (C-30), 14.5 (C-27); Anal. Calcd.
for C45H76N4O6: C, 70.27, H, 9.96. Found: C, 70.62, H, 9.91%. MS: m/z 791.56 [M + Na]+ (calcd. for
C45H76N4O6, 768.58).
3β-N-(2-tert-Butyloxycarbonylethylguanidine)-3-O-acetyl-lupane-28-amide (10), White powder, 86% yield;
mp 176–177 C (EtOH); [α]D23 +3.8◦ (c 0.56, CHCl3); IR (CHCl3) νmax 1724 (C=O), 3329 (NH) cm−1
;
◦
1H-NMR (500 MHz, CDCl3)
δ
: 11.53 (s, 1H, NH in 0Boc), 8.65 (br s, 1H, NH–C=N), 6.88 (br s, 1H,
0
CONH), 4.50–4.47 (m, 1H, H-3), 3.69–3.54 (m, 2H, H-2 ), 3.47–3.35 (m, 2H, H-1 ), 2.05 (s, 3H, CH3CO–),
1.51, 1.50 (both s, 9H each, CH3 in Boc), 2.46–0.96 (m, 25H, CH, CH2 in pentacyclic skeleton), 0.94,
0.90, 0.87, 0.86, 0.85, 0.84 (all s, 3H each, H-23–H-27 and H-29), 0.79 (d, J = 9.5 Hz, 1H, H-5), 0.75 (d,
J = 7.0 Hz, 3H, H-30); 13C-NMR (125 MHz, CDCl3)
δ: 176.9 (C-28), 171.0 (COCH3), 163.1 (C=N),
153.5, 153.0 (CONH-Boc), 83.5 (C in Boc), 80.9 (C-3), 77.3 (C in Boc), 56.0 (C-17), 55.4 (C-5), 50.3 (C-9),
49.7 (C-19), 44.2 (C-18), 42.5 (C-14), 41.2 (C-20), 40.7 (C-8), 39.7 (C-10), 38.6 (C-22), 38.4 (C-1), 37.8 (C-4),
37.5 (C-13), 37.1 (C-10), 34.3 (C-7), 33.4 (C-16), 29.8 (C-20), 29.4 (C-15), 28.3, 28.0 (CH3 in Boc), 27.9 (C-23),
26.9 (C-2), 23.7 (C-12), 23.1 (C-29), 23.0 (C-21), 21.3 (COCH3), 21.0 (C-11), 18.3 (C-6), 16.5 (C-25),
16.2 (C-24), 16.1 (C-26), 14.7 (C-30), 14.6 (C-27); Anal. Calcd. for C45H76N4O7: C, 68.84, H, 9.76. Found:
C, 69.23, H, 9.69%. MS: m/z 807.54 [M + Na]+ (calcd. for C45H76N4O7, 784.57).
3
β
-N-(4-tert-Butyloxycarbonylbutylguanidine)-3-O-acetyl-lupane-28-amide (11), White powder, 82% yield;
mp 148–150 C (EtOH); [α]D17
−
4◦ (c 0.52, CHCl3); IR (CHCl3) νmax 1640, 1720 (C=O), 3288, 3337,
◦
3410 (NH) cm−1; H-NMR (500 MHz, CDCl3)
δ
: 11.48 (s, 1H, NH in Boc), 8.30 (t, J = 5.0 Hz, 1H,
1
0
NH–C=N), 5.73 (t, J = 5.5 Hz, 1H, CONH), 4.46–4.43 (m, 1H, H-3), 3.42–3.37 (m, 2H, H-2 ), 3.31–3.16 (m,
2H, H-10), 2.01 (s, 3H, CH3CO–), 1.47, 1.46 (both s, 9H each, CH3 in Boc), 2.45–0.94 (m, 25H, CH, CH2
in pentacyclic skeleton, 4H, H-20, H-30), 0.91, 0.90, 0.83, 0.82, 0.81, 0.80 (all s, 3H each, H-23–H-27 and
H-29), 0.75 (d, J = 9.5 Hz, 1H, H-5), 0.71 (d, J = 7.0 Hz, 3H, H-30); 13C-NMR (125 MHz, CDCl3)
δ:
176.2 (C-28), 170.9 (COCH3), 163.6 (C=N), 156.1, 153.3 (CONH-Boc), 83.0 (C in Boc), 81.0 (C-3), 79.2 (C
in Boc), 56.0 (C-17), 55.4 (C-5), 50.3 (C-9), 49.5 (C-19), 44.2 (C-18), 42.6 (C-14), 40.8 (C-8), 40.5 (C-40),
38.7 (C-10, C-22), 38.4 (C-1), 37.8 (C-4), 37.5 (C-13), 37.1 (C-10), 34.4 (C-7), 33.6 (C-16), 29.9 (C-30),
0
29.4 (C-20), 28.3, 28.0 (CH3 in Boc), 27.8 (C-15), 27.3 (C-23), 26.9 (C-2 ), 26.5 (C-2), 23.7 (C-12), 23.0 (C-21,
C-29), 21.3 (COCH3), 21.0 (C-11), 18.2 (C-6), 16.5 (C-25), 16.2 (C-24, C-26), 14.6 (C-30), 14.5 (C-27); Anal.
Calcd. for C47H80N4O7: C, 69.42, H, 9.92. Found: C, 69.74, H, 9.85%. MS: m/z 835.51 [M + Na]+ (calcd.
for C47H80N4O7, 812.60).
0
3
β
-N-[2-(N,N -bis-tert-Butyloxycarbonylethylgyanidine)-aminoethyl]-3-O-acetyl-lupane-28-amide (12), White
powder, 87% yield; mp 188–190 C (EtOH); [α]1D9 +1.02◦ (c 0.96, CHCl3); IR (CHCl3) νmax 1641,
◦
1722 (C=O), 3335, 3442 (NH) cm−1; 1H-NMR (500 MHz, CDCl3)
δ: 11.52 (s, 2H, NH in Boc), 8.52 (br s,
2H, NH–C=N), 6.35 (t, 1H, J = 5.5 Hz, CONH), 4.49–4.46 (m, 1H, H-3), 3.53–3.43 (m, 4H, H-40, H-4”),
3.41–3.22 (m, 2H, H-10), 2.65–2.54 (m, 6H, H-20, H-30, H-3”), 2.04 (s, 3H, CH3CO–), 1.50, 1.48 (both br s,
18H each, CH3 in Boc), 2.32–0.98 (m, 25H, CH, CH2 in pentacyclic skeleton), 0.93, 0.92, 0.85, 0.84, 0.83,
0.82 (all s, 3H each, H-23–H-27 and H-29), 0.78 (d, J = 9.0 Hz, 1H, H-5), 0.73 (d, J = 6.5 Hz, 3H, H-30);
13C-NMR (125 MHz, CDCl3)
δ: 176.7 (C-28), 171.0 (COCH3), 163.5 (C=N), 155.9, 153.2 (CONH-Boc),
0
0
82.9 (C in Boc), 81.0 (C-3), 79.2 (C in Boc), 55.9 (C-17), 55.5 (C-5), 54.8 (C-2 ), 53.8 (C-3 , C-3”), 50.4 (C-9),
49.6 (C-19), 43.8 (C-18), 42.5 (C-14), 40.8 (C-8), 39.0 (C-40, C-4”), 38.9 (C-22), 38.4 (C-1), 38.0 (C-13),
37.8 (C-10), 37.2 (C-4), 37.1 (C-10), 34.4 (C-7), 33.4 (C-16), 29.8 (C-20), 29.5 (C-15), 28.3, 28.1 (CH3 in
Boc), 28.0 (C-23), 27.0 (C-2), 23.7 (C-12), 23.1 (C-29), 23.0 (C-21), 21.3 (COCH3), 21.0 (C-11), 18.3 (C-6),
16.5 (C-25), 16.4 (C-24), 16.2 (C-26), 14.7 (C-30), 14.4 (C-27); Anal. Calcd. for C60H104N8O11: C, 64.72; H,
9.41. Found: C, 65.02; H, 9.34%. MS: m/z 1135.71 [M + Na]+ (calcd. for C60H104N8O11, 1112.78).
3β-N-{[3-(3-tert-Butyloxycarbonylpropylgyanidine)piperazinyl]propyl}-3-O-acetyl-lupane-28-amide (13), White
powder, 60% yield; mp 131–134 C (EtOH); [α]D21
3289, 3333 (NH) cm−1; 1H-NMR (500 MHz, CDCl3)
6.86 (br s, 1H, CONH), 4.47–4.45 (m, 1H, H-3), 3.51–3.42 (m, 2H, H-1 ), 3.32–3.27 (m, 2H, H-1 ), 2.48–2.41 (m,
−
7.9 (c 0.57, CHCl3); IR (CHCl3) νmax 1640, 1722 (C=O),
◦
◦
δ
: 11.50 (br s, 1H, NH in Boc), 8.53 (br s, 1H, NH–C=N),
00
0

Molecules 2018, 23, 3000
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14H, H-30–H-50, H-2”–H-5”), 2.06 (s, 3H, CH3CO–), 1.49 (br s, 18H, CH3 in Boc), 2.33–0.99 (m, 25H, CH,
0
CH2 in pentacyclic skeleton, 2H, H-2 ), 0.93, 0.92, 0.85, 0.84, 0.83, 0.81 (all s, 3H each, H-23–H-27 and H-29),
0.78 (d, J = 10.5 Hz, 1H, H-5), 0.73 (d, J = 6.5 Hz, 3H, H-30); 13C-NMR (125 MHz, CDCl3)
δ
: 176.4 (C-28),
0
171.0 (
C
OCH3), 163.7 (C=N), 156.1, 153.0 (CONH-Boc), 82.8 (C in Boc), 80.9 (C-3), 79.2 (C in Boc), 56.4 (C-3 ),
0
0
55.9 (C-17), 55.9 (C-3”), 55.4 (C-5), 53.1 (C-4 , C-4”, C-5 , C-5”), 50.3 (C-9), 49.6 (C-19), 44.1 (C-18), 42.6 (C-14),
40.8 (C-8), 39.8 (C-22, C-1”), 39.2 (C-10), 38.8 (C-1), 38.4 (C-4), 37.8 (C-10), 37.4 (C-13), 34.5 (C-7), 33.7 (C-16),
29.8 (C-20), 29.4 (C-15), 28.3, 28.1 (CH3 in Boc), 27.9 (C-23), 26.9 (C-2), 26.9 (C-20), 25.9 (C-2”), 23.7 (C-12),
23.1 (C-21), 23.0 (C-29), 21.3 (COCH3), 21.0 (C-11), 18.2 (C-6), 16.5 (C-25), 16.3 (C-24), 16.2 (C-26), 14.6 (C-30),
14.5 (C-27); Anal. Calcd. for C53H92N6O7: C, 68.79, H, 10.02. Found: C, 69.04, H, 9.96%. MS: m/z 947.58
[M + Na]+ (calcd. for C53H92N6O7, 924.70).
3
β
-[2-tert-Butyloxycarbonylguanidine-3-hydroxy-2-(hydroxymethyl)propyl]-3-O-acetyl-lupane-28-oate (15a),
White powder, 63% yield; mp 106–108 ◦C (EtOH); [α]2D1 5.6◦ (c 0.48, CHCl3); IR (CHCl3) νmax 1655,
1714 (C=O), 3271, 3437 (NH) cm−1; 1H-NMR (400 MHz, CDCl3)
: 11.47 (s, 1H, NH in Boc), 9.05 (s,
−
δ
1H, NH–C=N), 4.49–4.45 (m, 1H, H-3), 4.25 (br s, 2H, H-10), 3.82-3.77, 3.57–3.53 (both m, 2H each,
H-30, H-40), 2.05 (s, 3H, CH3CO–), 1.49, 1.47 (both br s, 9H each, CH3 in Boc), 2.30–0.98 (m, 25H, CH,
CH2 in pentacyclic skeleton), 0.94, 0.90, 0.88, 0.86, 0.85, 0.83 (all s, 3H each, H-23–H-27 and H-29),
0.79 (d, J = 9.6 Hz, 1H, H-5), 0.75 (d, J = 6.4 Hz, 3H, H-30); 13C-NMR (100 MHz, CDCl3)
171.1 (
δ
: 176.3 (C-28),
C
OCH3), 161.8 (C=N), 155.7, 152.8 (CONH-Boc), 83.8 (C in Boc), 80.9 (C-3), 80.1 (C in Boc),
62.9 (C-10), 62.8 (C-40), 62.8 (C-30), 61.9 (C-20), 57.3 (C-17), 55.4 (C-5), 50.2 (C-9), 49.0 (C-19), 44.0 (C-18),
42.5 (C-14), 40.7 (C-8), 38.4 (C-22), 38.0 (C-1), 37.8 (C-4), 37.1 (C-13, C-10), 34.3 (C-7), 31.8 (C-16),
29.8 (C-20), 29.7 (C-15), 28.1 (CH3 in Boc), 28.0 (CH3 in Boc, C-23), 26.9 (C-2), 23.7 (C-12), 23.0 (C-29),
22.7 (C-21), 21.3 (COCH3), 20.9 (C-11), 18.2 (C-6), 16.5 (C-25), 16.2 (C-24), 15.9 (C-26), 14.7 (C-30),
14.6 (C-27); Anal. Calcd. for C47H79N3O10: C, 66.72, H, 9.41. Found: C, 67.13, H, 9.36%. MS: m/z 868.45
[M + Na]+ (calcd. for C47H79N3O10, 845.58).
3β-[2-tert-Butyloxycarbonylguanidine-3-hydroxy-2-(hydroxymethyl)propyl]-3-O-acetyl-urs-12-en-28-oate
◦
(
18a), White powder, 75% yield; mp 118–120 C (EtOH); [α]2D1 +31.3◦ (c 0.53, CHCl3); IR (CHCl3)
νmax 1653, 1729 (C=O), 3271, 3443 (NH) cm−1; H-NMR (500 MHz, CDCl3)
δ
: 11.50 (s, 1H, NH in
1
Boc), 9.06 (s, 1H, NH–C=N), 5.26 (br s, 1H, H-12), 4.51–4.48 (m, 1H, H-3), 4.13 (br s, 2H each, H-10),
3.82–3.76 (m, 2H, H-30), 3.54–3.52 (m, 2H, H-40), 2.26 (d, J = 11.0 Hz, 1H, H-18), 2.04 (s, 3H, CH3CO–),
1.99–0.81 (m, 22H, CH, CH2 in pentacyclic skeleton), 1.50, 1.47 (both br s, 9H each, CH3 in Boc), 1.07,
0.94, 0.93, 0.86, 0.85, 0.83, 0.75 (all s, 3H each, H-23–H-27, H-29 and H-30); 13C-NMR (125 MHz, CDCl3)
δ
: 177.4 (C-28), 171.0 (COCH3), 161.8 (C=N), 155.6, 152.8 (CONH-Boc), 138.1 (C-13), 125.8 (C-12),
83.7 (C in Boc), 80.9 (C-3), 79.9 (C in Boc), 63.4 (C-10), 62.9 (C-30), 62.6 (C-40), 61.6 (C-20), 55.3 (C-5),
52.8 (C-18), 48.5 (C-17), 47.5 (C-9), 42.0 (C-14), 39.5 (C-8), 39.0 (C-4), 38.7 (C-19), 38.3 (C-20), 37.7 (C-1),
36.8 (C-22), 36.5 (C-10), 32.9 (C-7), 30.6 (C-21), 28.1 (CH3 in Boc), 28.0 (C-15), 28 (C-23), 24.1 (C-16),
23.6 (C-2), 23.5 (C-27), 23.3 (C-11), 21.3 (C-30), 21.0 (COCH3), 18.2 (C-6), 17.0 (C-29), 16.9 (C-26),
16.7 (C-24), 15.5 (C-25); Anal. Calcd. for C47H77N3O10: C, 66.87, H, 9.19. Found: C, 67.27, H, 9.14%.
MS: m/z 866.43 [M + Na]+ (calcd. for C47H77N3O10, 843.56).
3
β
-[2-tert-Butyloxycarbonylguanidine-3-hydroxy-2-(hydroxymethyl)propyl]-3-O-acetyl-olean-12-en-28-oate
20a), White powder, 79% yield; mp 140–142 ◦C (EtOH); [α]1D9 +30.4◦ (c 0.56, CHCl3); IR (CHCl3) νmax
1618, 1727 (C=O), 3434 (NH) cm−1; 1H-NMR (400 MHz, CDCl3)
: 11.49 (s, 1H, NH in Boc), 9.03 (s,
(
δ
1H, NH–C=N), 5.30 (br s, 1H, H-12), 5.10 (br s, 1H, OH), 4.49–4.45 (m, 1H, H-3), 4.26, 4.15 (both d,
J = 11.6 Hz, 1H each, H-10), 3.77, 3.54 (both d, J = 12.0 Hz, J = 11.6 Hz, 2H each, H-30, H-40), 2.85 (d,
J = 10.0 Hz, 1H, H-18), 2.03 (s, 3H, CH3CO–), 1.99–0.80 (m, 22H, CH, CH2 in pentacyclic skeleton), 1.51,
1.48 (both br s, 9H each, CH3 in Boc), 1.12, 0.91, 0.90, 0.89, 0.85, 0.84, 0.71 (3H each, all s, H-23–H-27,
H-29 and H-30); 13C-NMR (100 MHz, CDCl3)
δ: 177.4 (C-28), 171.0 (COCH3), 161.8 (C=N), 155.6,
152.8 (CONH-Boc), 138.1 (C-13), 125.8 (C-12), 83.7 (C in Boc), 80.9 (C-3), 79.9 (C in Boc), 63.4 (C-10),
62.9 (C-30), 62.6 (C-40), 61.6 (C-20), 55.3 (C-5), 52.8 (C-18), 48.5 (C-17), 47.5 (C-9), 42.0 (C-14), 39.5 (C-8),
39.0 (C-4), 38.7 (C-19), 38.3 (C-20), 37.7 (C-1), 36.8 (C-22), 36.5 (C-10), 32.9 (C-7), 30.6 (C-21), 28.1 (CH3

Molecules 2018, 23, 3000
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in Boc), 28.0 (C-15, C-23), 24.1 (C-16), 23.6 (C-2), 23.5 (C-27), 23.3 (C-11), 21.3 (C-30), 21.0 (COCH3),
18.2 (C-6), 17.0 (C-29), 16.9 (C-26), 16.7 (C-24), 15.5 (C-25); Anal. Calcd. for C47H77N3O10: C, 66.87, H,
9.19. Found: C, 67.24, H, 9.14%. MS: m/z 866.47 [M + Na]+ (calcd. for C47H77N3O10, 843.56).
3.1.4. General Procedure for the Synthesis of Compounds 9a–13a, 15b, 18b and 20b
Compounds 9–13 and 15a, 18a, 20a (0.2 mmol) in 1 mL of dry CH2Cl2 were treated with TFA
(1 mL) and the mixture was stirred for 4–6 h at room temperature (TLC control, hexane:EtOAc, 1:1,
v/v). The solution was evaporated to dryness to obtain pure compounds 9a–13a, 15b, 18b and 20b.
N-(4-Butylgyanidine)-3-oxolupane-28-amide trifluoroacetate (9a), White powder, 96% yield; mp 142–144 ◦C
(EtOH); [α]1D9
(376.50 MHz, CDCl3)
1H, NH–C=N), 6.82 (br s, 2H, NH2), 6.39 (br s, 1H, CONH), 3.26 (m, 4H, H-1 , H-4 ), 2.45–1.19 (m, 26H,
−
0.6◦ (c 0.34, CHCl3); IR (CHCl3) νmax 1669 (C=O), 3206, 3437 (NH) cm−1 19F-NMR
;
δ
:
−
75.91; 1H-NMR (400 MHz, CDCl3)
δ: 11.13 (br s, 1H, NH in Boc), 7.50 (br s,
0
0
0
0
CH, CH2 in pentacyclic skeleton, 4H, H-2 , H-3 ), 1.07, 1.01, 0.96, 0.92, 0.90 (all s, 3H each, H-23–H-27),
0.85 (d, J = 6.0 Hz, 3H, H-29), 0.75 (d, J = 6.0 Hz, 3H, H-30); 13C-NMR (100 MHz, CDCl3)
δ
: 220.4 (C-3),
178.8 (C-28), 161.4 (q, JC,F = 37 Hz), 157.2 (C=N), 117.5 (q, JC,F = 288 Hz), 56.4 (C-17), 54.8 (C-5),
49.6 (C-9), 49.2 (C-19), 47.4 (C-4), 44.4 (C-18), 42.7 (C-14), 40.9 (C-1, C-40), 40.7 (C-8), 38.8 (C-22),
0
0
38.1 (C-1 ), 38.0 (C-13), 36.8 (C-10), 34.2 (C-2), 33.6 (C-16), 33.3 (C-7), 29.9 (C-20), 29.7 (C-3 ), 29.4 (C-15),
27.0 (C-20), 26.7 (C-23), 25.5 (C-12), 23.0 (C-21), 22.9 (C-29), 20.9 (C-11, C-24), 19.6 (C-6), 15.8 (C-25),
15.6 (C-26), 14.5 (C-30), 14.4 (C-27); Anal. Calcd. for C37H61F3N4O4: C, 65.08, H, 9.00. Found: C, 65.52,
H, 8.94%. MS: m/z 569.43 [M + H]+ (calcd. for C35H60N4O2, 568.47).
3
β
-N-(2-◦Ethylgyanidine)-3-O-a◦cetyl-lupane-28-amide trifluoroacetate (10a), White powder, 93% yield; mp
132–134 C (EtOH); [α]D19
−
5.6 (c 0.31, CHCl3); IR (CHCl3) νmax 1671 (C=O), 3351 (NH) cm
;
19F-NMR
−1
(470.59 MHz, CDCl3)
δ
:
−
75.80; 1H-NMR (500 MHz, CDCl3)
δ: 10.63 (br s, 1H, NH in Boc), 8.05 (br
s, 1H, NH–C=N), 7.00 (br s, 2H, NH2), 6.79 (br s, 1H, CONH), 4.48–4.45 (m, 1H, H-3), 3.39–3.28 (m,
4H, H-10, H-20), 2.09 (s, 3H, CH3CO–), 2.31–1.01 (m, 25H, CH, CH2 in pentacyclic skeleton), 0.95,
0.92, 0.89, 0.86, 0.85, 0.83 (all s, 3H each, H-23–H-27 and H-29), 0.79 (d, J = 11.0 Hz, 1H, H-5), 0.75 (d,
J = 6.0 Hz, 3H, H-30); 13C-NMR (125 MHz, CDCl3)
δ: 179.6 (C-28), 171.6 (COCH3), 161.2 (q, JC,F = 37
Hz), 157.9 (C=N), 117.2 (q, JC,F = 287 Hz), 81.2 (C-3), 56.4 (C-17), 55.4 (C-5), 50.2 (C-9), 49.3 (C-19), 44.3
(C-18), 42.6 (C-14), 40.7 (C-1, C-20), 40.6 (C-8), 38.6 (C-10), 38.4 (C-22), 37.8 (C-4, C-13), 37.0 (C-10), 34.2
(C-7), 33.1 (C-16), 29.9 (C-20), 29.3 (C-15), 27.9 (C-23), 26.9 (C-2), 23.6 (C-12), 23.0 (C-21, C-29), 21.3
(COCH3), 21.0 (C-11), 18.1 (C-6), 16.4 (C-25), 16.0 (C-24), 15.9 (C-26), 14.5 (C-27, C-30); Anal. Calcd.
for C37H61F3N4O5: C, 63.59, H, 8.80. Found: C, 63.88, H, 8.73%. MS: m/z 585.62 [M + H]+ (calcd. for
C35H60N4O3, 584.47).
3β-N-(4-Butylgyanidine)-3-O-acetyl-lupane-28-amide trifluoroacetate (11a), White powder, 95% yield; mp
148–150 ◦C (EtOH); [α]1D7
−
12◦ (c 0.49, CHCl3); IR (CHCl3) νmax 1672 (C=O), 3207, 3354 (NH) cm−1
;
1
19F-NMR (470.59 MHz, C0DCl3)
δ
:
−
77.19; H-NMR (500 MHz, MeOD)
δ
: 4.48–4.45 (m, 1H, H-3),
0
3.28–3.15 (m, 4H, H-1 , H-4 ), 2.04 (s, 3H, CH3CO–), 2.61–1.04 (m, 25H, CH, CH2 in pentacyclic skeleton,
0
0
4H, H-2 , H-3 ), 1.02, 0.98, 0.92, 0.90, 0.89, 0.88 (3H each, all s, H-23–H-27 and H-29), 0.84 (d, J = 11.0 Hz
,
1H, H-5), 0.79 (d, J = 7.0 Hz, 3H, H-30); 13C-NMR (125 MHz, MeOD)
δ
: 179.6 (C-28), 172.9 ( OCH3),
C
161.2 (q, JC,F = 37 Hz), 158.8 (C=N), 117.2 (q, JC,F = 287 Hz), 82.6 (C-3), 57.5 (C-17), 57.0 (C-5), 51.9 (C-9),
51.1 (C-19), 45.5 (C-18), 43.8 (C-14), 42.2 (C-8, C-40), 40.0 (C-22), 39.8 (C-1), 39.4 (C-10), 39.0 (C-4),
0
0
38.9 (C-13), 38.4 (C-10), 35.8 (C-7), 34.1 (C-16), 31.3 (C-20), 30.7 (C-3 ), 28.7 (C-15), 28.5 (C-23), 28.1 (C-2 ),
27.4 (C-2), 24.8 (C-12), 24.2 (C-29), 23.7 (C-21), 22.5 (C-11), 21.3 (COCH3), 19.4 (C-6), 17.2 (C-25),
17.0 (C-24, C-26), 15.3 (C-30), 15.2 (C-27); Anal. Calcd. for C39H65F3N4O5: C, 64.44, H, 9.01. Found: C,
64.87, H, 8.94%. MS: m/z 613.48 [M + H]+ (calcd. for C37H64N4O3, 612.50).
3
β
-N-[2-(N,N0-bis-Ethylgyanidine)-aminoethyl]-3-O-acet◦yl-lupane-28-amide trifluoroacetate (12a), White
powder, 96% yield; mp 116–118 C (EtOH); [α]D17
−
10.5 (c 0.2, C2H5OH); IR (CHCl3) νmax 1681 (C=O),
◦
3199, 3362 (NH) cm−1
;
19F-NMR (470.59 MHz, MeOD)
δ
:
−
76.98; H-NMR (500 MHz, MeOD)
δ
:
1

Molecules 2018, 23, 3000
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4.48–4.44 (m, 1H, H-3), 3.73–3.70 (m, 4H, H-40, H-4”), 3.56–3.53 (m, 2H, H-10), 3.46 (t, J = 6.5 Hz, 4H,
H-30, H-3”), 3.29–3.26 (m, 2H, H-20), 2.04 (s, 3H, CH3CO–), 2.52–1.06 (m, 25H, CH, CH2 in pentacyclic
skeleton), 1.02, 0.97, 0.92, 0.90, 0.88, 0.87 (all s, 3H each, H-23–H-27 and H-29), 0.85 (d, J = 11.5 Hz, 1H,
H-5), 0.79 (d, J = 6.5 Hz, 3H, H-30); 13C-NMR (125 MHz, MeOD)
δ: 181.2 (C-28), 173.0 (COCH3), 161.7 (q,
JC,F = 37 Hz), 159.1 (C=N), 117.5 (q, JC,F = 288 Hz), 35.6 (C-4”), 53.4 (C-3”), 82.6 (C-3), 57.6 (C-17),
56.9 (C-5), 53.9 (C-20), 53.4 (C-30), 51.8 (C-9), 51.0 (C-19), 45.5 (C-18), 43.8 (C-14), 42.2 (C-8), 39.7 (C-1),
0
0
39.7 (C-22), 39.0 (C-13, C-4), 38.4 (C-1 ), 37.8 (C-10), 35.6 (C-4 , C-7), 33.8 (C-16), 31.3 (C-20), 30.7 (C-15),
28.6 (C-23), 28.4 (C-2), 24.8 (C-12), 24.1 (C-21), 23.5 (C-29), 22.3 (C-11), 21.3 (COCH3), 19.4 (C-6),
17.1 (C-25), 17.0 (C-24), 16.9 (C-26), 15.2 (C-30), 15.1 (C-27); Anal. Calcd. for C44H74F6N8O7: C, 56.16,
H, 7.93. Found: C, 56.63, H, 7.86%. MS: m/z 713.59 [M + H]+ (calcd. for C40H72N8O3, 712.57).
3β-N-{[3-(3-Propylguanidine)piperazinyl]propyl}-3-O-acetyl-lupane-28-amide trifluoroacetate (13a), White
powder, 96% yield; mp 92–94 ◦C (EtOH); [α]D19 +8◦ (c 0.54, CHCl3); IR (CHCl3) νmax 1673, 1773 (C=O),
1
3190, 3367 (NH) cm−1
;
19F-NMR (470.59 MHz, MeOD)
δ
:
−
77.25; H-NMR (500 MHz, MeOD)
δ
:
4.48–4.45 (m, 1H, H-3), 3.49–3.40 (m, 4H, H-2”, H-3”), 3.31–3.22 (m, 8H, H-40, H-4”, H-50, H-5”),
3.13–3.10 (m, 2H, H-1”), 3.00–2.98 (m, 2H, H-10), 2.58–0.84 (m, 26H, CH, CH2 in pentacyclic skeleton,
4H, H-20, H-30), 2.08 (s, 3H, CH3CO–), 1.02, 0.99, 0.92, 0.89, 0.86, 0.84 (all s, 3H each, H-23–H-27 and
H-29), 0.80 (d, J = 7.0 Hz, 3H, H-30); 13C-NMR (125 MHz, MeOD)
δ: 180.2 (C-28), 173.0 (COCH3),
161.7 (q, JC,F = 37 Hz), 158.9 (C=N), 117.5 (q, JC,F = 288 Hz), 82.6 (C-3), 57.6 (C-17), 57.0 (C-5), 56.0 (C-3”),
55.2 (C-30), 51.9 (C-9), 51.0 (C-19), 50.1 (C-40, C-4”, C-50, C-5”), 45.5 (C-18), 43.8 (C-14), 42.2 (C-8),
39.9 (C-10), 39.8 (C-1”), 39.6 (C-1, C-22), 39.0 (C-4), 38.9 (C-13), 38.4 (C-10), 35.7 (C-7), 34.0 (C-16),
31.3 (C-20), 30.7 (C-15), 28.6 (C-23), 28.5 (C-2”), 25.6 (C-20), 24.9 (C-2), 24.2 (C-12), 23.6 (C-21, C-29),
22.4 (C-11), 21.3 (COCH3), 19.4 (C-6), 17.1 (C-25), 17.0 (C-24), 16.9 (C-26), 15.2 (C-30), 15.1 (C-27); Anal.
Calcd. for C45H77F3N6O5: C, 64.41, H, 9.25. Found: C, 64.88, H, 9.19. MS: m/z 747.58 [M + Na]+ (calcd.
for C43H76N6O3, 724.60).
3
β
-[2-Guanidine-3-hydroxy-2-(hydroxymethyl)propyl]-3-O-acetyl-lupane-28-oate trifluoroacetate (15b), White
powder, 95% yield; mp 126–129 C (EtOH); [α]D19
−
9.5◦ (c 0.34, CHCl3); IR (CHCl3) νmax 1620,
◦
1698 (C=O), 3437 (NH) cm−1
: 4.50–4.47 (m, 1H, H-3), 4.31 (2H, m, H-10), 3.75 (m, 4H, H-30, H-40), 2.18–0.99 (m, 26H, CH, CH2
in pentacyclic skeleton), 2.08 (s, 3H, CH3CO–), 0.98, 0.97, 0.96, 0.87, 0.86 (all s, 3H each, H-23–H-27),
0.89 (d, J = 6.0 Hz, 3H, H-29), 0.76 (d, J = 6.0 Hz, 3H, H-30); 13C-NMR (125 MHz, MeOD)
: 177.4 (C-28),
172.3 ( OCH3), 161.4 (q, JC,F = 37 Hz), 157.5 (C=N), 117.5 (q, JC,F = 288 Hz), 81.7 (C-3), 63.1 (C-3 , C-4 ),
;
19F-NMR (470.59 MHz, CDCl3)
δ
:
−
75.97; 1H-NMR (500 MHz, MeOD)
δ
δ
0
0
C
62.0 (C-20), 60.2 (C-10), 57.5 (C-17), 55.4 (C-5), 50.2 (C-9), 48.8 (C-19), 44.1 (C-18), 42.6 (C-14), 40.7 (C-8),
38.4 (C-22), 38.2 (C-1), 37.8 (C-4, C-13), 37.0 (C-10), 34.2 (C-7), 31.9 (C-16), 29.7 (C-20), 29.0 (C-15),
27.9 (C-23), 26.8 (C-2), 23.6 (C-12), 22.9 (C-29, C-21), 21.3 (COCH3), 20.9 (C-11), 18.1 (C-6), 16.4 (C-25),
16.1 (C-24), 15.8 (C-26), 14.6 (C-27, C-30); Anal. Calcd. for C39H64F3N3O8: C, 61.64; H, 8.49. Found: C,
62.34; H, 8.43%. MS: m/z 646.39 [M + H]+ (calcd. for C37H63N3O6, 645.47).
3
β
-[2-Guanidine-3-hydroxy-2-(hydroxymeth◦yl)propyl]-3-O-acetyl-urs-12-en-28-oate trifluoroacetate (18b),
White powder, 98% yield; mp 124–126 C (EtOH); [α]1D9 +30.0◦ (c 0.72, CH2Cl2); IR (CHCl3) νmax
1619, 1682 (C=O), 3438 (NH) cm−1
;
19F-NMR (470.59 MHz, MeOD)
δ
:
−
77.17; 1H-NMR (500 MHz,
MeOD) δ
: 5.29 (br s, 1H, H-12), 4.49–4.46 (m, 1H, H-3), 4.31, 4.17 (both d, J = 11.5 Hz, 1H each, H-10),
3.75 (m, 4H, H-30, H-40), 2.24 (d, J = 11.0 Hz, 1H, H-18), 2.04 (s, 3H, CH3CO–), 2.13–0.87 (m, 22H,
CH, CH2 in pentacyclic skeleton), 1.15, 1.01, 0.97, 0.98, 0.92, 0.90, 0.81 (3H each, all s, H-23–H-27,
H-29 and H-30); 13C-NMR (125 MHz, MeOD)
δ: 179.2 (C-28), 173.0 (COCH3), 161.4 (q, JC,F = 37 Hz),
159.2 (C=N), 140.1 (C-13), 127.2 (C-12), 117.5 (q, JC,F = 288 Hz), 82.5 (C-3), 64.3 (C-30), 64.2 (C-40),
62.7 (C-20), 62.2 (C-10), 56.8 (C-5), 54.4 (C-18), 49.5 (C-17), 48.6 (C-9), 43.3 (C-14), 40.9 (C-8), 40.4 (C-4,
C-19), 39.6 (C-20), 38.8 (C-1), 38.1 (C-22), 38.0 (C-10), 34.2 (C-7), 31.7 (C-21), 29.2 (C-15), 28.7 (C-23),
25.5 (C-16), 24.6 (C-2), 24.5 (C-27), 24.4 (C-11), 21.6 (C-30), 21.3 (COCH3), 19.4 (C-6), 17.9 (C-29),
17.8 (C-26), 17.3 (C-24), 16.2 (C-25); Anal. Calcd. for C39H62F3N3O8: C, 61.80, H, 8.25. Found: C, 62.33,
H, 8.19%. MS: m/z 644.47 [M + H]+ (calcd. for C37H61N3O6, 643.46).

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3β-[2-Guanidine-3-hydroxy-2-(hydroxymethyl)propyl]-3-O-acetyl-olean-12-en-28-oate trifluoroacetate (20b),
White powder, 97% yield; mp 130–132 C (EtOH); [α]1D7 +26.6 (c 0.53, C2H5OH); IR (CHCl3) νmax 1619,
◦
◦
−1
1682 (C=O), 3438 (NH) cm
;
19F-NMR (470.59 MHz, CDCl3)
5.31 (br s, 1H, H-12), 4.49–4.46 (m, 1H, H-3), 4.33, 4.20 (d, J = 11.5 Hz, 1H each, H-1 ), 3.75 (m, 4H, H-3 ,
H-40), 2.89 (d, 1H, J = 9.5 Hz, H-18), 2.09–0.88 (m, 22H, CH, CH2 in pentacyclic skeleton), 2.04 (s, 3H,
CH3CO–), 1.19, 1.00, 0.97, 0.95, 0.90, 0.89, 0.79 (all s, 3H each, H-23–H-27, H-29 and H-30); 13C-NMR
δ
:
−
77.24; 1H-NMR (500 MHz, MeOD)
δ
0
:
0
(125 MHz, MeOD)
δ
: 178.9 (C-28), 173.0 (
C
OCH3), 1610.4 (q, JC,F =037 Hz), 159.2 (C=N), 145.0 (C-13),
0
0
124.1 (C-12), 117.5 (q, JC,F = 288 Hz), 82.6 (C-3), 64.3 (C-4 ), 64.2 (C-3 ), 62.7 (C-2 ), 62.3 (C-1 ), 56.9 (C-5),
48.5 (C-9, C-17), 47.1 (C-19), 43.0 (C-14), 42.9 (C-18), 40.8 (C-8), 39.5 (C-1), 38.9 (C-4), 38.3 (C-10),
34.9 (C-22), 33.9 (C-30), 33.7 (C-7), 33.6 (C-21), 31.7 (C-20), 28.9 (C-15), 28.7 (C-23), 26.6 (C-27), 24.7 (C-11,
C-29), 24.3 (C-2), 24.2 (C-16), 21.3 (COCH3), 19.5 (C-6), 17.9 (C-26), 17.3 (C-24), 16.1 (C-25); Anal. Calcd.
for C39H62F3N3O8: C, 61.80, F, 7.52, H, 8.25. Found: C, 62.12, H, 8.21%. MS: m/z 644.57 [M + H]+
(calcd. for C37H61N3O6, 643.46).
3.1.5. General Procedure for the Synthesis of Compounds 9b–12b, 15c, 18c and 20c
The compounds 9a–12a, 15b, 18b, 20b (0.2 g) were dissolved in 2 mL MeOH and 5M HCl was added
dropwise until the precipiate formed. The solution was evaporated to dryness and this procedure was
repeated three times. The precipitate which formed was filtered off and washed with water to pH = 7.
The salts 9b–12b and 15c, 18c, 20c were obtained as white solids with a quantitative yield.
◦
N-(4-Butylgyanidine)-3-oxolupane-28-amide dihydrochloride (9b), White powder, 87% yield; mp 176–178 C
(EtOH); [α]2D1
−
11◦ (c 0.29, C2H5OH); IR (CHCl3) νmax 1721 (C=O), 3342 (NH) cm−1 1H-NMR
;
(500 MHz, d6-DMSO) δ
: 7.71 (br s, 1H, NH), 7.59 (br s, 1H, CONH), 3.10–3.01 (m, 4H, H-10, H-40),
2.61–1.04 (m, 26H, CH, CH2 in pentacyclic skeleton, 4H, H-2 , H-3 ), 0.99, 0.93, 0.91, 0.87, 0.86 (3H each,
all s, H-23–H-27), 0.81 (d, J = 6.5 Hz, 3H, H-29), 0.71 (d, J = 7.0 Hz, 3H, H-30); 13C-NMR (125 MHz,
0
0
d6-DMSO)
δ: 217.1 (C-3), 176.1 (C-28), 157.4 (C=N), 55.7 (C-17), 54.3 (C-5), 49.6 (C-9), 49.5 (C-19),
47.0 (C-4), 43.8 (C-18), 42.6 (C-14), 40.9 (C-40), 40.3 (C-8), 39.7 (C-1), 38.1 (C-22, C-10), 37.0 (C-13),
0
0
36.8 (C-10), 34.1 (C-2), 33.8 (C-16), 32.7 (C-7), 30.0 (C-20), 29.4 (C-15), 27.2 (C-3 ), 27.0 (C-2 ), 26.9 (C-23),
26.4 (C-12), 23.6 (C-29), 23.1 (C-21), 21.7 (C-11), 21.2 (C-24), 19.7 (C-6), 16.2 (C-25), 16.1 (C-26), 15.0 (C-30),
14.6 (C-27); Anal. Calcd. for C35H62Cl2N4O2: C, 65.50, Cl, 11.05, H, 9.74. Found: C, 65.97, Cl, 11.78, H,
9.68%. MS: m/z 569.49 [M + H]+ (calcd. for C35H60N4O2, 568.47).
3β-N-(2-Ethylgyanidine)-3-O-acetyl-lupane-28-amide hydrochloride (10b), White powder, 82% yield; mp
192–194 ◦C (EtOH); [α]1D7
−
16◦ (c 0.23, C2H5OH); IR (CHCl3) νmax 1652, 1716 (C=O), 3155, 3327 (NH)
: 4.48–4.45 (m, 1H, H-3), 3.43–3.16 (m, 4H, H-10, H-20), 2.04 (s,
1
cm−1; H-NMR (500 MHz, MeOD)
δ
3H, CH3CO–), 2.60–0.81 (m, 25H, CH, CH2 in pentacyclic skeleton), 1.02, 0.98, 0.92, 0.90, 0.88, 0.87 (all
s, 3H each, H-23–H-27 and H-29), 0.80 (d, J = 6.5 Hz, 3H, H-30); 13C-NMR (125 MHz, MeOD)
180.8 (C-28), 173.0 ( OCH3), 159.0 (C=N), 82.6 (C-3), 57.6 (C-17), 57.0 (C-5), 51.1 (C-9, C-19), 45.5 (C-18),
δ
:
C
42.7 (C-14, C-10), 42.2 (C-8), 39.8 (C-1), 39.7 (C-20), 39.4 (C-22), 39.0 (C-4), 38.9 (C-13), 38.4 (C-10),
35.7 (C-7), 34.0 (C-16), 31.4 (C-20), 30.7 (C-15), 28.6 (C-23), 28.5 (C-2), 24.8 (C-12), 24.1 (C-21), 23.6 (C-29),
22.4 (C-11), 21.3 (COCH3), 19.4 (C-6), 17.1 (C-25), 16.9 (C-24, C-26), 15.2 (C-30), 15.1 (C-27); Anal. Calcd.
for C35H61ClN4O3: C, 67.66, Cl, 5.71, H, 9.90. Found: C, 67.99, Cl, 5.40, H, 9.84%. MS: m/z 585.54
[M + H]+ (calcd. for C35H60N4O3, 584.47).
3
β
-N-(4-◦Butylgyanidine)-3-O-acetyl-lupane-28-amide hydrochloride (11b), White powder, 79% yield; mp
156–158 C (EtOH); [α]D22
−
14.5 (c 0.53, C2H5OH); IR (CHCl3) νmax 1645, 1716 (C=O), 3168, 3338 (NH)
◦
0
cm−1; 1H-NMR (500 MHz, MeOD)
δ: 7.45 (br s, 1H, NH), 4.48–4.45 (m, 1H, H-3), 3.23–3.18 (m, 4H, H-1 ,
H-40), 2.04 (s, 3H, CH3CO–), 2.61–0.84 (m, 26H, CH, CH2 in pentacyclic skeleton, 4H, H-20, H-30), 1.01,
0.98, 0.92, 0.89, 0.88, 0.86 (all s, 3H each, H-23–H-27 and H-29), 0.79 (d, J = 7.0 Hz, 3H, H-30); 13C-NMR
(125 MHz, MeOD) δ: 179.6 (C-28), 172.9 (COCH3), 158.8 (C=N), 82.6 (C-3), 57.5 (C-17), 57.0 (C-5),
51.9 (C-9), 51.1 (C-19), 45.5 (C-18), 43.8 (C-14), 42.4 (C-8), 42.2 (C-40), 40.00(C-22, C-10), 39.8 (C-1),
39.0 (C-4), 38.9 (C-13), 38.4 (C-10), 35.8 (C-7), 34.1 (C-16), 31.4 (C-20), 30.8 (C-3 ), 28.6 (C-15), 28.2 (C-23,

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C-20), 27.4 (C-2), 24.8 (C-12), 24.4 (C-21), 24.2 (C-29), 22.5 (C-11), 21.3 (COCH3), 19.4 (C-6), 17.1 (C-25),
17 (C-24), 16.9 (C-26), 15.2 (C-30), 15.1 (C-27); Anal. Calcd. for C37H65ClN4O3: C, 68.43, Cl, 5.46, H,
10.09. Found: C, 68.88; Cl, 5.12; H, 10.02%. MS: m/z 614.51 [M + 2H]+ (calcd. for C37H64N4O3, 612.50).
3β
-N-[2-(N,N0-bis-Ethylgyanidine)-aminoethyl]-3-O-acetyl-lupane-28-amide dihydrochloride (12b), White
powder, 88% yield; mp 198–200 ◦C (EtOH); [α]D17
−
16◦ (c 0.29, C2H5OH); IR (CHCl3) νmax 1637, 1672,
: 4.48–4.45 (m, 1H, H-3),
1
1734 (C=O), 3162, 3271, 3324 (NH) cm−1; H-NMR (500 MHz, MeOD)
δ
3.90–3.83 (m, 4H, H-40, H-4”), 3.66–3.59 (m, 6H, H-20, H-30, H-3”), 3.38–3.33 (m, 2H, H-10), 2.04 (s, 3H,
CH3CO–), 2.51–0.85 (m, 26H, CH, CH2 in pentacyclic skeleton), 1.03, 0.99, 0.93, 0.90, 0.88, 0.87 (all s, 3H
each, H-23–H-27 and H-29), 0.80 (d, J = 6.5 Hz, 3H, H-30); 13C-NMR (125 MHz, MeOD)
δ: 181.0 (C-28),
172.9 (COCH3), 158.9 (C=N), 82.5 (C-3), 57.7 (C-17), 56.9 (C-5), 53.7 (C-30, C-3”), 53.6 (C-20), 51.8 (C-9),
51.0 (C-19), 45.5 (C-18), 43.8 (C-14), 42.2 (C-8), 39.7 (C-22), 39.0 (C-1, C-13), 38.4 (C-4), 37.7 (C-10, C-10),
35.7 (C-7), 35.0 (C-40, C-4”), 33.9 (C-16), 31.3 (C-20), 30.8 (C-15), 28.6 (C-23), 28.4 (C-2), 24.8 (C-12),
24.2 (C-21), 23.6 (C-29), 22.4 (C-11), 21.3 (COCH3), 19.4 (C-6), 17.1 (C-25, C-24), 16.9 (C-26), 15.2 (C-30),
15.1 (C-27); Anal. Calcd. for C40H74Cl2N8O3: C, 61.13, Cl, 9.02, H, 9.49. Found: C, 61.55, Cl, 11.40, H,
9.42%. MS: m/z 713.51 [M + H]+ (calcd. for C40H72N8O3, 712.57).
3
β
-[2-Guanidine-3-hydroxy-2-(hydroxymethyl)propyl]-3-O-acetyl-lupane-28-oate hydrochloride (15c), White
powder, 68% yield; mp 136–138 C (EtOH); [α]D22
−
14.5◦ (c 0.53, C2H5OH); IR (CHCl3) νmax 1673,
◦
1733 (C=O), 3325 (NH) cm−1; H-NMR (500 MHz, MeOD)
H-10), 3.75 (m, 4H, H-30, H-40), 2.04 (s, 3H, CH3CO–), 2.29–0.85 (m, 26H, CH, CH2 in pentacyclic
δ
: 4.48–4.45 (m, 1H, H-3), 4.31 (2H, m,
1
skeleton), 1.03, 1.00, 0.98, 0.91, 0.88, 0.87 (all s, 3H each, H-23–H-27 and H-29), 0.81 (d, J = 6.5 Hz,
3H, H-30); 13C-NMR (125 MHz, MeOD) OCH3), 159.1 (C=N), 82.6 (C-3), 64.1 (C-30),
δ: 177.1 (C-28, C
0
0
0
64.1 (C-4 ), 62.8 (C-2 ), 61.8 (C-1 ), 58.7 (C-17), 56.9 (C-5), 51.8 (C-9), 50.4 (C-19), 45.8 (C-18), 43.9 (C-14),
42.1 (C-8), 39.7 (C-22), 39.6 (C-1), 39.0 (C-4), 38.4 (C-10, C-13), 35.6 (C-7), 33.2 (C-16), 31.2 (C-20),
30.9 (C-15), 28.6 (C-23), 28.4 (C-2), 24.8 (C-12), 23.9 (C-21), 23.5 (C-29), 22.3 (C-11), 21.3 (COCH3),
19.4 (C-6), 17.1 (C-25), 16.9 (C-24), 16.8 (C-26), 15.2 (C-30), 15.1 (C-27); Anal. Calcd. for C37H64ClN3O6:
C, 65.13, Cl, 5.20, H, 9.45. Found: C, 65.6 4, Cl, 4.50; H, 9.39%. MS: m/z 647.47 [M + 2H]+ (calcd. for
C37H63N3O6, 645.47).
3β-[2-Guanidine-3-hydroxy-2-(hydroxymethyl)propyl]-3-O-acetyl-urs-12-en-28-oate hydrochloride (18c),
White powder, 62% yield; mp 142–144 C (EtOH); [α]1D9 +37◦ (c 0.51, C2H5OH); IR (CHCl3) νmax
◦
1675, 1733 (C=O), 3186, 3335 (NH) cm−1; 1H-NMR (500 MHz, MeOD)
δ: 7.07 (s, 1H, NH), 5.30 (br s,
1H,0H-12), 4.50–4.47 (m, 1H, H-3), 4.29, 4.23 (both d, J = 11.5 Hz, 1H each, H-10), 3.75 (m, 4H, H-30,
H-4 ), 2.25 (d, J = 11.5 Hz, 1H, H-18), 2.05 (s, 3H, CH3CO–), 2.14–0.88 (m, 22H, CH, CH2 in pentacyclic
skeleton), 1.16, 1.02, 1.00, 0.92, 0.91, 0.90, 0.81 (all s, 3H each, H-23–H-27, H-29 and H-30); 13C-NMR
(125 MHz, MeOD) δ: 178.6 (C-28), 173.0 (COCH3), 159.2 (C=N), 139.6 (C-13), 127.3 (C-12), 82.6 (C-3),
64.2 (C-30, C-40), 62.9 (C-20), 62.3 (C-10), 56.8 (C-5), 54.5 (C-18), 49.3 (C-17), 49.0 (C-9), 43.4 (C-14),
41.0 (C-8), 40.5 (C-4, C-19), 39.6 (C-20), 38.9 (C-1), 38.2 (C-22), 38.0 (C-10), 34.2 (C-7), 31.8 (C-21),
29.3 (C-15), 28.8 (C-23), 25.5 (C-16), 24.7 (C-2), 24.5 (C-27), 24.4 (C-11), 21.7 (C-30), 21.3 (COCH3),
19.5 (C-6), 17.9 (C-29), 17.8 (C-26), 17.4 (C-24), 16.2 (C-25); Anal. Calcd. for C37H62ClN3O6: C, 65.32, Cl,
5.21, H, 9.19. Found: C, 65.74, Cl, 4.51, H, 9.14%. MS: m/z 644.34 [M + H]+ (calcd. for C37H61N3O6,
643.46).
3
β
-[2-Guanidine-3-hydroxy-2-(hydroxymethyl)propyl]-3-O-acetyl-olean-12-en-28-oate hydrochloride (20c),
White powder, 53% yield; mp 124–128 ◦C (EtOH); [α]D19 +34◦ (c 0.34, C2H5OH); IR (CHCl3) νmax 1662,
1733 (C=O), 3344 (NH) cm−1; 1H-NMR (500 MHz, MeOD)
δ: 5.32 (br s, 1H, H-12), 4.49–4.46 (m, 1H,
H-3), 4.33, 4.20 (both d, J = 11.5 Hz, 1H each, H-10), 3.76 (m, 4H, H-30, H-40), 2.89 (d, J = 9.5 Hz, 1H,
H-18), 2.13–0.90 (m, 22H, CH, CH2 in pentacyclic skeleton), 2.05 (s, 3H, CH3CO–), 1.20, 1.00, 0.98, 0.96,
0.94, 0.91, 0.88 (all s, 3H each, H-23–H-27, H-29 and H-30); 13C-NMR (125 MHz, MeOD)
δ
: 178.8 (C-28),
OCH3), 159.1 (C=N), 145.0 (C-13), 124.1 (C-12), 82.6 (C-3), 64.2 (C-40), 64.1 (C-30), 62.8 (C-20),
62.3 (C-10), 56.8 (C-5), 49.0 (C-9), 48.5 (C-17), 47.1 (C-19), 43.0 (C-14), 42.9 (C-18), 40.7 (C-8), 39.5 (C-1),
172.9 (
C

Molecules 2018, 23, 3000
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38.9 (C-4), 38.2 (C-10), 34.9 (C-22), 33.9 (C-30), 33.7 (C-7, C-21), 31.7 (C-20), 28.9 (C-15), 28.7 (C-23),
26.6 (C-27), 24.7 (C-11, C-29), 24.3 (C-2), 24.2 (C-16), 21.3 (COCH3), 19.5 (C-6), 17.9 (C-26), 17.3 (C-24),
15.6 (C-25); Anal. Calcd. for C37H62ClN3O6: C, 65.32, Cl, 5.21, H, 9.19. Found: C, 65.74, Cl, 4.80, H,
9.12%. MS: m/z 644.44 [M + H]+ (calcd. for C37H61N3O6, 643.46).
3β-N-(4-Butylgyanidine)-3-hydroxy-lupane-28-amide (14), To a solution of the compound 11b (0.33 g,
0.5 mmol) in MeOH (4 mL) and THF (4 mL) was added 4 N NaOH (4 mL). The reaction mixture
was stirred at room temperature for 24 h (monitoring by TLC) and then neutralized with 20% HCl.
The solution was dried under vacuum and reconstituted with CH2Cl2. The organic layer was washed
with brine and dried over anhydrous MgSO4 and concentrated under reduced pressure to obtain pure
compound 14. White powder, 79% yield; mp 260–262 C (EtOH); [α]D19
−
10 (c 0.24, DMSO); IR (CHCl3)
δ: 7.73 (br s, 1H, NH), 7.58 (br s,
◦
◦
νmax 1731 (C=O), 3366 (NH) cm−1; 1H-NMR (500 MHz, DMSO-d5)
1H, CONH), 4.29 (br s, 1H, OH), 3.09–2.98 (m, 5H, H-3, H-10, H-40), 2.19–1.02 (m, 26H, CH, CH2 in
pentacyclic skeleton, 4H, H-20, H-30), 0.89, 0.87, 0.84, 0.78, 0.66 (all s, 3H each, H-23–H-27), 0.81 (d,
J = 6.5 Hz, 3H, H-29), 0.72 (d, J = 6.5 Hz, 3H, H-30); 13C-NMR (125 MHz, DMSO-d5)
δ: 176.1 (C-28),
157.4 (C=N), 77.2 (C-3), 55.7 (C-17), 55.4 (C-5), 50.3 (C-9), 49.6 (C-19), 43.8 (C-18), 42.5 (C-14), 40.9 (C-8),
40.8 (C-40), 39.0 (C-22), 38.8 (C-1), 38.6 (C-4), 38.1 (C-100), 37.2 (C-13), 36.9 (C-10), 34.6 (C-7), 32.8 (C-16),
0
30.0 (C-20), 29.4 (C-15), 28.6 (C-23), 27.4 (C-2), 27.2 (C-3 ), 27.0 (C-2 ), 26.4 (C-12), 23.6 (C-29), 23.2 (C-21),
21.1 (C-11), 18.5 (C-6), 16.4 (C-25), 16.3 (C-24, C-26), 15.0 (C-30), 14.7 (C-27); Anal. Calcd. for
C35H62N4O2: C, 73.63, H, 10.95. Found: C, 73.74, H, 10.88%. MS: m/z 593.31 [M + Na]+ (calcd.
for C35H62N4O2, 570.49).
3.2. Biology
3.2.1. Cell Culturing
Cells (Jurkat, K562, U937, HeLa, HEK293 and normal Fibroblasts) were purchased from Russian
Cell Culture Collection (Institute of Cytology of the Russian Academy of Sciences, Saint Petersburg,
Russia) and cultured according to standard mammalian tissue culture protocols and sterile technique.
Human cell lines HEK293 and HeLa were obtained from the HPA Culture Collections (Salisbury, UK).
All cell lines used in the study were tested and shown to be free of mycoplasma and viral contamination.
HEK293, HeLa cell lines and fibroblasts were cultured as monolayers and maintained in
Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Waltham, MA, USA) supplemented with
10% foetal bovine serum and 1% penicillin-streptomycin solution at 37 ◦C in a humidified incubator
under a 5% CO2 atmosphere.
Cells were maintained in RPMI 1640 (Jurkat, K562, U937) (Gibco, Thermo Fisher Scientific,
Waltham, MA, USA) supplemented with 4 mM glutamine, 10% FBS (Sigma, Burlington, MA, USA)
and 100 units/mL penicillin-streptomycin (Sigma). All types of cells were grown in an atmosphere
of 5% CO2 at 37 ◦C. The cells were subcultures at 2–3 days intervals. Adherent cells (HEK293, HeLa,
fibroblasts) were suspended using trypsin/EDTA and counted after they have reached 80% confluency.
Cells were then seeded in 24 well plates at 5
K562, U937 cells were subcultured at 2 day intervals with a seeding density of 1
plates in RPMI with 10% FBS.
×
104 cells per well and incubated overnight. Jurkat,
×
105 cells per 24 well
3.2.2. Cytotoxicity Assay
Viability (Live/dead) assessment was performed by staining cells with 7-AAD (7-Aminoactinomycin
D) (Biolegend, San Diego, CA, USA). Cells were treated of test compounds with six different concentrations
(1, 5, 10, 15, 30 and 60
saline (PBS) and centrifuged at 400
staining buffer (PBS without Ca2+ and Mg2+, 2.5% FBS) and stained with 5
15 min at room temperature in the dark. Samples were acquired on NovoCyteTM 2000 FlowCytometry
µ
M). After treatment, cells were harvested, washed 1–2 times with phosphate-buffered
g for 5 min. Cell pellets were resuspended in 200 L of flow cytometry
L of 7-AAD staining solutionfor
×
µ
µ

Molecules 2018, 23, 3000
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System (ACEA, San Diego, CA, USA) equipped with 488 nm argon laser. Detection of 7-AAD emission
was collected through a 675/30 nm filter in FL4 channel.
3.2.3. Viability and Apoptosis
Apoptosis was determined by flow cytometric analysis of Annexin V and 7-aminoactinomycin D
staining. Briefly, 200
cells in 200 L, and the cells were incubated with the reagent for 20 min at room temperature in the
dark. The plates were treated with compounds 15 15c 18c 20c and dihydrobetulinic acid at IC50
concentration (4, 8 and 59 M) for 24 h and 48 h. At the end of incubation, the cells were analyzed
µL of Guava Nexin reagent (Millipore, Bedford, MA, USA) was added to 5 × 105
µ
,
,
,
µ
on NovoCyteTM 2000 FlowCytometry System (ACEA). Different states of cell death were defined as
follows: normal cells are localized in the lower-left quadrant (Annexin V−/PI−); early apoptotic cells
are in the lower-right quadrant (Annexin V+/PI−); late apoptotic cells and necrotic cells are in the
upper-right quadrant (Annexin V+/PI+); and necrotic cells are in the upper-left quadrant (Annexin
V−/PI+).
3.2.4. Cell Cycle Analysis
Cell cycle was analyzed using the method of propidium iodide staining. Briefly, cells were
plated in 24-well round bottom plates at density 10
×
105 cells per well, centrifuged at 450
×
g for
5 min, and fixed with ice-cold 70% ethanol for 24 h at 0 ◦C. Cells were then washed with PBS and
incubated with 250 µL of Guava Cell Cycle Reagent (Millipore, Burlington, MA, USA) for 30 min at
room temperature in the dark. Samples were analyzed on NovoCyteTM 2000 FlowCytometry System
(ACEA, San Diego, CA, USA).
4. Conclusions
Novel betulinic, ursolic, and oleanolic acid derivatives, containing a guanidine moiety have been
designed and synthesized in an attempt to develop potent antitumor agents. These compounds and
their precursors, monoamine, diamine and triamine derivatives, were tested for cytotoxic activity
on various human tumor cell lines. Guanidine-functionalized triterpenoids demonstrated higher
cytotoxicity in Jurkat cells, compared with original triterpenoic acids. Most of the tested guanidine
derivatives showed higher IC50 values than amines, but were less toxic to human fibroblasts. The lead
molecules—dihydrobetulinic acid amine 15, its guanidine derivative 15c, and guanidinium salts of
ursolic and oleanolic acids 18c and 20c were selected for extended biological testing by using flow
cytometry analysis. Our results showed that the antitumor activity of compounds 15, 15c, and 18c is
caused by apoptotic processes and induction of cell cycle arrest in the S-phase. Nevertheless, addition
information concerning the molecular mechanisms and targets of these triterpene acid derivatives
is needed.
Supplementary Materials: The following are available online: 1H-NMR and 13C-NMR spectra of all new compounds.
Author Contributions: Supervision, U.D. and V.O.; validation and writing-review & editing, A.S. and L.D.;
performing the chemistry experiments R.K. and D.N.; performing the biology experiments M.Y., L.D. and V.D.;
The manuscript was prepared through the contributions L.D., V.D. and D.N.
Funding: This work was performed under financial support from the Russian Science Foundation (Grant
16-13-10051).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all compounds are available from the authors.
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