Picolyl amides of betulinic acid as antitumor agents causing tumor cell apoptosis

Article abstract of DOI:10.1016/j.ejmech.2017.12.096

A series of picolyl amides of betulinic acid (3a–3c and 6a–6c) was prepared and subjected to the cytotoxicity screening tests. Structure-activity relationships studies resulted in finding differences in biological activity in dependence on o-, m- and p-substitution of the pyridine ring in the target amides, when cytotoxicity data of 3a–3c and 6a–6c were obtained and compared. The amides 3b and 3a displayed cytotoxicity (given in the IC₅₀ values) in G-361 (0.5 ± 0.1 μM and 2.4 ± 0.0 μM, respectively), MCF7 (1.4 ± 0.1 μM and 2.2 ± 0.2 μM, respectively), HeLa (2.4 ± 0.4 μM and 2.3 ± 0.5 μM, respectively) and CEM (6.5 ± 1.5 μM and 6.9 ± 0.4 μM, respectively) tumor cell lines, and showed weak effect in the normal human fibroblasts (BJ). Selectivity against all tested cancer cells was determined and compared to normal cells with therapeutic index (TI) between 7 and 100 for compounds 3a and 3b. The therapeutic index (TI = 100) was calculated for human malignant melanoma cell line (G-361) versus normal human fibroblasts (BJ). The cytotoxicity of other target amides (3c and 6a–6c) revealed lower effects than 3a and 3b in the tested cancer cell lines.

Full text of DOI:10.1016/j.ejmech.2017.12.096

Accepted Manuscript
Picolyl amides of betulinic acid as antitumor agents causing tumor cell apoptosis
Uladzimir Bildziukevich, Lucie Rárová, David Šaman, Zdeněk Wimmer
PII:
S0223-5234(17)31126-1
10.1016/j.ejmech.2017.12.096
EJMECH 10074
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 November 2017
Revised Date: 13 December 2017
Accepted Date: 30 December 2017
Please cite this article as: U. Bildziukevich, L. Rárová, D. Šaman, Zdeně. Wimmer, Picolyl amides
of betulinic acid as antitumor agents causing tumor cell apoptosis, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.096.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

G-361, I5 ꢀ ꢁ2. ꢂ 020 mM, ꢃ ꢀ 1ꢄ23ACCEPTED MAG-N36U1,SICRꢀIP0T25 ꢂ 021 mM, ꢃ ꢀ 100
0
50
MIF7, I5 ꢀ ꢁ2ꢁ ꢂ 02ꢁ mM
MIF7, I50 ꢀ 12. ꢂ 021 mM
0
H
H
H
COOH
H
COOH
O
O
H
H
NH
N
NH
N
O
O
H
H
3a
3b
O
O

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Picolyl amides of betulinic acid as antitumor agents causing tumor cell apoptosis
a,b
c
d
a,b,
Uladzimir Bildziukevich, Lucie Rárová, David Šaman, and Zdeněk Wimmer
*
a
University of Chemistry and Technology, Department of Chemistry of Natural Compounds,
Technická 5, 166 28 Prague 6, Czech Republic;
b
Institute of Experimental Botany AS CR, v.v.i., Isotope Laboratory, Vídeňská 1083, 142 20
Prague 4, Czech Republic;
c
Palacký University, Centre of the Region Haná for Biotechnological and Agricultural
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
Research, Department of Chemical Biology and Genetics, Šlechtitelů 27, 783 71 Olomouc,
Czech Republic;
d
Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Flemingovo náměstí 2,
166 10 Prague 6, Czech Republic
Corresponding author at: University of Chemistry and Technology, Department of Chemistry
of Natural Compounds, Technická 5, 166 28 Prague 6 & Institute of Experimental Botany AS
CR, v.v.i., Isotope Laboratory, Vídeňská 1083, 14220 Prague 4, Czech Republic.
Tel.: +420 220 444 398, +420 241 062 457; fax: +420 220 444 260.
E-mail addresses: Zdenek.Wimmer@vscht.cz, wimmer@biomed.cas.cz (Z. Wimmer).
1

ACCEPTED MANUSCRIPT
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Abstract
A series of picolyl amides of betulinic acid (3a–3c and 6a–6c) was prepared and subjected to
the cytotoxicity screening tests. Structure-activity relationships studies resulted in finding
differences in biological activity in dependence on o-, m- and p-substitution of the pyridine
ring in the target amides, when cytotoxicity data of 3a–3c and 6a–6c were obtained and
compared. The amides 3b and 3a displayed cytotoxicity (given in the IC values) in G-361
5
0
(0.5 ± 0.1 µM and 2.4 ± 0.0 µM, respectively), MCF7 (1.4 ± 0.1 µM and 2.2 ± 0.2 µM,
respectively), HeLa (2.4 ± 0.4 µM and 2.3 ± 0.5 µM, respectively) and CEM (6.5 ± 1.5 µM
and 6.9 ± 0.4 µM, respectively) tumor cell lines, and showed weak effect in the normal human
fibroblasts (BJ). Selectivity against all tested cancer cells was determined and compared to
normal cells with therapeutic index (TI) between 7 and 100 for compounds 3a and 3b. The
therapeutic index (TI = 100) was calculated for human malignant melanoma cell line (G-361)
versus normal human fibroblasts (BJ). The cytotoxicity of other target amides (3c and 6a–6c)
revealed lower effects than 3a and 3b in the tested cancer cell lines.
Key words: betulinic acid; picolyl amine; amide; cytotoxicity; therapeutic index
1. Introduction
In the past years, we have published a paper dealing with the investigation of heteroaromatic
amides of selected steryl hemiesters [1]. Several of those compounds were based on o-, m-
and p-picolyl amines, displaying only moderate cytotoxicity. Those compounds suffered from
low polarity of the steryl skeleton. Nevertheless, even in that range of biological activity,
difference in cytotoxicity values could be seen, based on o-, m- or p-substitution in picolyl
amides. More recently, our attention has been focused at the investigation of triterpenoid acid
and their derivatives, which mostly showed considerable cytotoxicity and high and sometimes
2

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
selective antimicrobial activity. The compounds completed a majority of ADME parameters
required for prediction of potential drugs, and – in several cases – displayed important
supramolecular characteristics, among which formation of supramolecular hydrogels was one
of the most important features [2,3].
Betulinic acid, (3β)-3-hydroxylup-20(29)-en-28-oic acid, is practically insoluble in water,
mainly due to the presence of large lipophilic backbone and inadequate number of hydrophilic
groups [4]. Even with this feature, which means, in fact, that this natural product does not
correspond to the Lipinski [5] and Ghose [6] rules describing potentially biologically active
compounds, it displays anti-HIV activity [7], antitumor [8,9] and antidiabetic activity [10,11].
Nature itself possesses natural mechanisms for increasing bioavailability of natural products
by forming their water-soluble conjugates (e.g. glycosides) [12]. To improve its
pharmacological characteristics in forms of the novel compounds has always been a challenge
for researchers, to find its new derivatives and related compounds [12]. Betulinic acid is a
plant product, to be found in genus Betulla, Diospiros, Paeonia, Syzigium or Ziziphus [13].
Concerning the antitumor effect, derivatives of betulinic acid have been used against a variety
of tumor cell lines: malignant brain tumor, primitive neuroectodermal tumor [14], human
chronic myelogenous leukemia, and against most of prevalent human cancer types, such as
cervical, prostate, breast, lung or colorectal cancer [12].
Many bioactive secondary metabolites of plants, including betulinic acid, induce apoptosis
pathway in cancer cells to exert their selective cytotoxic effects [15]. The cysteine-aspartic
acid protease (caspase) family proteins play a central role in the execution phase of apoptosis,
and the mechanism of initiation of apoptosis is mediated by the caspase cascade activation
[16]. Caspase-3 and caspase-7 are downstream factors activated by caspase-8 and caspase-9,
which – in turn – are activated predominantly by the extrinsic (death receptor) and intrinsic
(mitochondrial) pathways, respectively [17]. Caspase-3 is an executioner protease that results
3

ACCEPTED MANUSCRIPT
7
7
7
7
8
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
9
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
in cleavage of poly-(ADP-ribose) polymerase (PARP), subsequent DNA degradation and
apoptotic death [18]. Previous studies showed that betulinic acid also displays anti-
inflammatory, anti-HIV, anti-helminthic, anti-nociceptive activities [19]. Several studies were
done to explain the molecular mechanisms of betulinic acid mediated antitumor activity. The
process seems to be largely dependent on the ability of betulinic acid to trigger the
mitochondrial pathway of apoptosis in cancer cells. Betulinic acid shows direct effect on
mitochondria. Successive treatment of betulinic acid on cancer cells disrupts the
mitochondrial transmembrane potential, which facilitates the cells to undergo apoptosis.
Involvement of excessive amount of reactive oxygen species was found to be the most
important factor for loss of mitochondrial membrane integrity of melanoma cells treated with
betulinic acid. It directly targets to mitochondria, which – in turn – regulates the downstream
caspase activation and side by side overcomes resistance property. Thus, betulinic acid has
not developed resistance in cancer cells, and, thereby, it became permissible agents for future
cancer therapy [15].
Aminomethylpyridines (picolyl amines), and related heteroaromatic amines, including
numbers of their derivatives, and also the derived N-oxides, had already been investigated for
their pharmacological activity [1,20-22], as markers of solid tumors [23] or agents in
supramolecular self-assembly [24]. Lipophilic derivation of picolyl amines have resulted in
obtaining conjugates, bearing ester and amide bond in each molecule; that type of study was
made in our previous work [1]. The importance of such compounds consists in enabling
transportation of potentially biologically active compounds through biomembrane and they
often form cationic immune-stimulating complexes [25,26]. The derivatives of picolyl amines
have also been found to be involved in activation of the caspase cascade and promote cell
death [27].
4

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
The objectives of the current research consist in (a) a synthesis of a series of picolyl amides of
betulinic acid both, at C(17)-COOH and at C(3)-OH (in the latter subseries using succinic
acid as a junction unit), and (b) investigation of cytotoxicity of the target compounds. A
comparison of the current series of compounds with those published earlier [1] will also be
discussed.
2. Experimental part
2.1. General
1
13
The H NMR and the C NMR spectra were recorded on a Bruker AVANCE 600 MHz
spectrometer at 600.13 MHz and 150.90 MHz in deuterochloroform, using tetramethylsilane
1
(δ = 0.0) as internal reference. H NMR data are presented in the following order: chemical
shift (δ) expressed in ppm, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet), coupling constants in Hertz, number of protons. For unambiguous assignment of
1
13
1
13
both H and C signals 2D NMR H, C gHSQC and gHMBC spectra were measured using
standard parameters sets and pulse programs delivered by producer of the spectrometer.
Copies of the NMR spectra are presented in the Supplementary material. Infrared spectra (IR)
were measured with a Nicolet 205 FT-IR spectrometer. Mass spectra (MS) were measured
with a Waters ZMD mass spectrometer in a positive ESI mode. Optical rotation was measured
on an Autopol IV instrument (Rudolph Research Analytical, USA) at 589 nm wavelength, and
the value was corrected to 20°C. The PE 2400 Series II CHNS/O Analyzer (Perkin Elmer,
USA) was used for simultaneous determination of C, H, and N (accuracy of CHN
determination better than 0.30 % abs.). TLC was carried out on silica gel plates (Merck
60F ) and the visualization was performed by the UV detection and by spraying with the
2
54
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
5

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
solvents were purchased from regular commercial sources in analytical grade and the solvents
were purified by general methods before use.
2.2. (3β)-3-[(3-Carboxypropanoyl)oxy]lup-20(29)-en-28-oic acid (2)
Succinic anhydride (1.62 g; 16.2 mmol; 3.5 eq) and DMAP (170 mg; 1.39 mmol, 0.30 eq)
were added to a solution of betulinic acid (1; 2.1 g; 4.6 mmol) in dry pyridine (20 mL). The
reaction mixture was stirred at r.t. for 5 days. After stopping the reaction, the resulting
mixture was poured onto ice, hydrochloric acid was added to adjust pH = 7, the organic layer
was extracted with chloroform, and the extract was dried over sodium sulfate. Evaporation of
the solvent afforded a solid that was purified by column chromatography, yielding 1.93 g
(75%) of 2.
1
H NMR: δ 0.79 (3H, s, H23), 0.79 (3H, s, H24), 0.80 (3H, s, H25), 0.87 (3H, s, H26), 0.94
(3H, s, H27), 1.64 (3H, dd, J = 0.7 Hz, J = 1.4 Hz, H29), 2.09-2.13 (2H, m, H16), 2.22 (1H,
1
2
ddd, J = 3.6 Hz, J = 11.6 Hz, J = 12.9 Hz, H13), 2.44-2.52 (4H, m, H2'-H3'), 2.95 (1H, dt, J
1
1
2
3
= 4.8 Hz, J = 11.0 Hz, J = 11.0 Hz, H19), 4.38 (1H, dd, J = 4.7 Hz, J = 11.6 Hz, H3), 4.56
2
3
1
2
(1H, dq, J = 1.4 Hz, J = 1.4 Hz, J = 1.4 Hz, J = 2.4 Hz, H30), 4.69 (1H, dq, J = 0.7 Hz, J =
1
2
3
4
1
2
1
3
0.7 Hz, J = 0.7 Hz, J = 2.4 Hz, H30). C NMR: δ 14.31 (q, C27), 15.65 (q, C24), 15.81 (q,
3
4
C25), 16.35 (q, C26), 17.67 (t, C6), 18.90 (q, C29), 20.42 (t, C11), 23.27 (t, C2), 25.00 (t,
C12), 27.56 (q, C23), 28.77 (t, C2'), 29.15 (t, C21), 29.15 (t, C3'), 30.06 (t, C15), 31.64 (t,
C16), 33.68 (t, C22), 36.28 (t, C1), 36.60 (s, C10), 37.38 (s, C4), 37.54 (d, C13), 37.67 (t,
C7), 40.21 (s, C8), 41.99 (s, C14), 46.57 (d, C19), 48.50 (d, C18), 49.61 (d, C9), 54.62 (d,
C5), 55.36 (s, C17), 79.98 (d, C3), 109.58 (t, C30), 150.26 (s, C20), 171.56 (s, C1'), 173.32 (s,
-
-1
C4'), 177.17 (s, C28). MS: m/z 555.2 [M-H] . IR (KBr; cm ): 2946 (C–H val.), 2872 (–CH
3
val.), 1734 (–C=O val.). For C H O (556.77) calculated C (73.34), H (9.41), found C
3
4
52
6
o
(73.37), H (9.40). M.p. 244-246 C.
6

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
2.3. (3β)-3-({4-Oxo-4-[(pyridin-n-ylmethyl)amino]butanoyl}oxy)lup-20(29)-en-28-oic acid
[3a (n=2), 3b (n=3) and 3c (n=4)]
o-, m- or p-Picolyl amine (20 µl; 0.198 mmol; 1.1 eq) and T3P (0.32 ml; 0.54 mmol; 3 eq)
were added to a solution of 2 (100 mg; 0.18 mmol) in dry pyridine (2 mL), and the reaction
mixture was stirred at r.t. for 1 day. After stopping the reaction, saturated solution of sodium
bicarbonate (4 mL) was added to reaction mixture, and stirring continued for several more
hours. The resulting mixture was extracted with chloroform, and dried over sodium sulfate.
Evaporation of the solvent afforded a solid, which was purified by column chromatography.
Yields: 3a (85 %), 3b (91 %), 3c (89 %).
1
3a: H NMR: δ 0.77 (3H, s, H23), 0.78 (3H, s, H24), 0.80 (3H, s, H25), 0.87 (3H, s, H26),
0.95 (3H, s, H27), 1.65 (3H, dd, J = 0.7 Hz, J = 1.3 Hz, H29), 2.09-2.14 (2H, m, H16), 2.23
1
2
(1H, ddd, J = 4.7 Hz, J = 11.7 Hz, J = 12.9 Hz, H13), 2.44-2.55 (4H, m, H2'-H3'), 2.95 (1H,
1
2
3
dt, J = 5.3 Hz, J = 11.0 Hz, J = 11.0 Hz, H19), 4.31 (1H, dd, J = 6.0 Hz, J = 15.9 Hz, H5'),
1
2
3
1
2
4.35 (1H, dd, J = 6.0 Hz, J = 15.9 Hz, H5'), 4.38 (1H, dd, J = 4.8 Hz, J = 11.6 Hz, H3),
1
2
1
2
4.56 (1H, dq, J = 1.3 Hz, J = 1.3 Hz, J = 1.3 Hz, J = 2.4 Hz, H30), 4.69 (1H, bd, J = 2.4 Hz,
1
2
3
4
H30), 7.24 (1H, dddt, J = 0.6 Hz, J = 0.6 Hz, J = 1.2 Hz, J = 4.8 Hz, J = 7.5 Hz, H8'), 7.26
1
2
3
4
5
(1H, dtt, J = 0.6 Hz, J = 0.6 Hz, J = 1.1 Hz, J = 1.1 Hz, J = 7.8 Hz, H10'), 7.72 (1H, dt, J
1
1
2
3
4
5
= 1.8 Hz, J = 7.7 Hz, J = 7.7 Hz, H9'), 8.45 (1H, t, J = 6.0 Hz, NH), 8.48 (1H, ddd, J = 0.9
2
3
1
1
3
Hz, J = 1.8 Hz, J = 4.8 Hz, H7'). C NMR: δ 14.32 (q, C27), 15.66 (q, C24), 15.82 (q, C25),
2
3
16.36 (q, C26), 17.67 (t, C6), 18.90 (q, C29), 20.43 (t, C11), 23.29 (t, C2), 25.01 (t, C12),
27.58 (q, C23), 29.16 (t, C21), 29.35 (t, C2'), 29.87 (t, C3'), 30.06 (t, C15), 31.66 (t, C16),
33.70 (t, C22), 36.29 (t, C1), 36.59 (s, C10), 37.38 (s, C4), 37.54 (d, C13), 37.69 (t, C7),
40.22 (s, C8), 41.99 (s, C14), 44.17 (t, C5'), 46.58 (d, C19), 48.50 (d, C18), 49.62 (d, C9),
54.63 (d, C5), 55.37 (s, C17), 79.87 (d, C3), 109.58 (t, C30), 120.88 (d, C10'), 121.97 (d, C8'),
7

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
136.56 (d, C9'), 148.71 (d, C7'), 150.28 (s, C20), 158.64 (s, C6'), 170.88 (s, C1'), 171.88 (s,
+
-
-1
C4'), 177.18 (s, C28). MS: m/z 647.3 [M+H] , 645.0 [M-H] . IR (KBr; cm ): 3377 (N–H val.
amide), 2945 (C–H val.), 2871 (–CH val.), 1728 (–C=O val. ester), 1659 (–C=O val. amide).
3
For C H N O (646.90) calculated C (74.27), H (9.04), N (4.33), found C (74.29), H (9.03),
4
0
58
2
5
o
20
N (4.35). M.p. 121-124 C. [α]D = +15.5 (c 0.323).
1
3b: H NMR: δ 0.70-0.73 (1H, m, H5), 0.76 (3H, s, H24), 0.76 (3H, s, H23), 0.78 (3H, d, J =
0.6 Hz, H25), 0.88 (3H, s, H26), 0.92 (3H, d, J = 0.7 Hz, H27), 1.63 (3H, dd, J = 0.7 Hz, J =
1
2
1.3 Hz, H29), 2.17 (1H, ddd, J = 3.7 Hz, J = 11.6 Hz, J = 12.9 Hz, H13), 2.22 (2H, ddd, J =
1
2
3
1
2.9 Hz, J = 4.0 Hz, J = 13.1 Hz, H16), 2.46 (2H, t, J = 6.6 Hz, H3'), 2.58-2.66 (2H, m, H2'),
2
3
2.94 (1H, dt, J = 5.0 Hz, J = 11.0 Hz, J = 11.0 Hz, H19), 4.39 (1H, dd, J = 5.9 Hz, J = 14.9
1
2
3
1
2
Hz, H5'), 4.41 (1H, dd, J = 5.7 Hz, J = 14.9 Hz, H5'), 4.42 (1H, dd, J = 6.2 Hz, J = 10.1 Hz,
1
2
1
2
H3), 4.55 (1H, dq, J = 1.3 Hz, J = 1.3 Hz, J = 1.3 Hz, J = 2.4 Hz, H30), 4.68 (1H, dq, J =
1
2
3
4
1
0.7 Hz, J = 0.7 Hz, J = 0.7 Hz, J = 2.3 Hz, H30), 6.20 (1H, bt, J = 5.8 Hz, NH), 7.23 (1H,
2
3
4
ddd, J = 0.8 Hz, J = 4.8 Hz, J = 7.8 Hz, H10'), 7.63 (1H, ddt, J = 0.7 Hz, J = 0.7 Hz, J =
1
2
3
1
2
3
1.8 Hz, J = 2.1 Hz, J = 7.8 Hz, H11'), 8.48 (1H, bdd, J = 1.8 Hz, J = 4.8 Hz, H9'), 8.50 (1H,
4
5
1
2
1
3
bs, H7'). C NMR: δ 14.71 (q, C27), 16.13 (q, C24), 16.17 (q, C25), 16.51 (q, C26), 18.24 (t,
C6), 19.41 (q, C29), 20.99 (t, C11), 23.77 (t, C2), 25.60 (t, C12), 28.02 (q, C23), 29.78 (t,
C21), 29.78 (t, C2'), 30.72 (t, C3'), 31.25 (t, C15), 32.29 (t, C16), 34.38 (t, C22), 37.09 (t, C1),
37.23 (s, C10), 37.95 (s, C4), 38.49 (d, C13), 38.49 (t, C7), 40.85 (t, C5'), 41.09 (s, C8), 42.56
(s, C14), 47.01 (d, C19), 49.46 (d, C18), 50.57 (d, C9), 55.56 (d, C5), 56.38 (s, C17), 81.68
(d, C3), 109.64 (t, C30), 123.82 (d, C10'), 134.65 (s, C6'), 136.46 (d, C11'), 147.92 (d, C9'),
148.35 (d, C7'), 150.45 (s, C20), 171.74 (s, C1'), 172.73 (s, C4'), 179.99 (s, C28). MS: m/z
+
-
-1
647.3 [M+H] , 645.3 [M-H] . IR (KBr; cm ): 3350 (N–H val. amide), 2946 (C–H val.), 2871
(–CH val.), 1729 (–C=O val. ester), 1657 (–C=O val. amide). For C H N O (646.90)
3
40 58
2
5
8

ACCEPTED MANUSCRIPT
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
calculated C (74.27), H (9.04), N (4.33), found C (74.30), H (9.02), N (4.35). M.p. 131-134
o
20
C. [α]D = +12.8 (c 0.383).
1
3c: H NMR: δ 0.70-0.74 (1H, m, H5), 0.76 (3H, s, H23), 0.76 (3H, s, H24), 0.78 (3H, s,
H25), 0.87 (3H, s, H26), 0.91 (3H, bd, H27), 0.98 (2H, dq, J = 4.0 Hz, J = 13.0 Hz, J = 13.0
1
2
3
Hz, J = 13.0 Hz, H12), 1.62 (3H, dd, J = 0.7 Hz, J = 1.3 Hz, H29), 2.16 (1H, ddd, J = 3.6
4
1
2
1
Hz, J = 11.5 Hz, J = 12.8 Hz, H13), 2.21 (2H, ddd, J = 3.2 Hz, J = 3.8 Hz, J = 13.0 Hz,
2
3
1
2
3
H16), 2.48 (1H, dt, J = 6.5 Hz, J = 6.5 Hz, J = 15.0 Hz, H3'), 2.50 (1H, dt, J = 5.7 Hz, J =
1
2
3
1
2
5.7 Hz, J = 15.0 Hz, H3'), 2.62 (1H, dt, J = 5.7 Hz, J = 5.7 Hz, J = 17.3 Hz, H2'), 2.66 (1H,
3
1
2
3
dt, J = 6.6 Hz, J = 6.6 Hz, J = 17.3 Hz, H2'), 2.95 (1H, dt, J = 5.0 Hz, J = 10.9 Hz, J =
1
2
3
1
2
3
10.9 Hz, H19), 4.38 (1H, bdd, J = 6.1 Hz, J = 16.0 Hz, H5'), 4.42 (1H, bdd, J = 6.1 Hz, J =
1
2
1
2
16.0 Hz, H5'), 4.43 (1H, dd, J = 5.5 Hz, J = 11.0 Hz, H3), 4.54 (1H, dq, J = 1.3 Hz, J = 1.3
1
2
1
2
Hz, J = 1.3 Hz, J = 2.3 Hz, H30), 4.67 (1H, dq, J = 0.7 Hz, J = 0.7 Hz, J = 0.7 Hz, J = 2.3
3
4
1
2
3
4
1
3
Hz, H30), 6.30 (1H, t, J = 6.1 Hz, NH), 7.16-7.18 (2H, m, H7'), 8.47-8.49 (2H, m, H8'). C
NMR: δ 14.68 (q, C27), 16.06 (q, C24), 16.15 (q, C25), 16.49 (q, C26), 18.18 (t, C6), 19.37
(q, C29), 20.92 (t, C11), 23.75 (t, C2), 25.50 (t, C12), 28.01 (q, C23), 29.73 (t, C21), 29.94 (t,
C2'), 30.64 (t, C3'), 31.16 (t, C15), 32.26 (t, C16), 34.28 (t, C22), 37.08 (t, C1), 37.16 (s,
C10), 37.90 (s, C4), 38.38 (t, C7), 38.40 (d, C13), 40.75 (s, C8), 42.42 (s, C14), 42.48 (t, C5'),
46.97 (d, C19), 49.33 (d, C18), 50.47 (d, C9), 55.48 (d, C5), 56.34 (s, C17), 81.73 (d, C3),
109.63 (t, C30), 122.47 (d, C7'), 148.49 (s, C6'), 149.08 (d, C8'), 150.49 (s, C20), 171.98 (s,
+
-
-1
C1'), 172.91 (s, C4'), 180.46 (s, C28). MS: m/z 647.2 [M+H] , 645.3 [M-H] . IR (KBr; cm ):
3358 (N–H val. amide), 2946 (C–H val.), 2870 (–CH val.), 1729 (–C=O val. ester), 1663 (–
3
C=O val. amide). For C H N O (646.90) calculated C (74.27), H (9.04), N (4.33), found C
4
0
58
2
5
o
20
D
(74.26), H (9.05), N (4.31). M.p. 145-147 C. [α] = +12.8 (c 0.188).
2.4. (3β)-3-(Acetyloxy)lup-20(29)-en-28-oic acid (4)
9

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Acetic anhydride (0.45 mL; 4.7 mmol; 1.44 eq), DMAP (52 mg; 0.43 mmol, 0.13 eq) and
EDIPA (1 mL) were added to a solution of betulinic acid (1; 1.5 g; 3.3 mmol) in dry THF (10
mL). The reaction mixture was refluxed for 3 h. After stopping the reaction, water (10 mL)
was added and the resulting mixture was stirred for 1 hour. The resulting mixture was
extracted with chloroform, and the extract was dried over sodium sulfate. Evaporation of the
solvent afforded a solid, which was purified by column chromatography, resulting finally in
1.55 g (95 %) of 4.
1
H NMR: δ 0.83 (3H, s, H23), 0.84 (3H, s, H25), 0.85 (3H, bs, H24), 0.93 (3H, s, H26), 0.97
(3H, bs, H27), 1.05 (2H, dq, J = 4.9 Hz, J = 13.0 Hz, J = 13.0 Hz, J = 13.0 Hz, H12), 1.19
1
2
3
4
(2H, dt, J = 3.2 Hz, J = 3.2 Hz, J = 13.6 Hz, H21), 1.70 (3H, dd, J = 0.7 Hz, J = 1.4 Hz,
1
2
3
1
2
H29), 2.04 (3H, s, OAc), 2.13 (2H, ddd, J = 3.7 Hz, J = 11.6 Hz, J = 12.8 Hz, H1), 2.27
1
2
3
(2H, dt, J = 3.8 Hz, J = 3.8 Hz, J = 13.1 Hz, H16), 3.01 (1H, dt, J = 5.0 Hz, J = 10.9 Hz, J
3
1
2
3
1
2
= 10.9 Hz, H19), 4.47 (1H, dd, J = 5.5 Hz, J = 10.9 Hz, H3), 4.61 (1H, dq, J = 1.4 Hz, J =
1
2
1
2
1.4 Hz, J = 1.4 Hz, J = 2.3 Hz, H30), 4.74 (1H, dq, J = 0.7 Hz, J = 0.7 Hz, J = 0.7 Hz, J =
3
4
1
2
3
4
1
3
2.3 Hz, H30). C NMR: δ 14.64 (q, C27), 16.02 (q, C24), 16.17 (q, C25), 16.46 (q, C26),
18.15 (t, C6), 19.32 (q, OAc), 19.33 (q, C29), 20.84 (t, C11), 23.68 (t, C2), 25.43 (t, C12),
27.94 (q, C23), 29.68 (t, C21), 30.54 (t, C15), 32.13 (t, C16), 34.22 (t, C22), 37.03 (d, C13),
37.11 (s, C10), 37.79 (t, C7), 38.37 (s, C4), 38.39 (t, C1), 40.68 (s, C8), 42.41 (s, C14), 46.92
(d, C19), 49.25 (d, C18), 50.38 (d, C9), 55.40 (d, C5), 56.35 (s, C17), 80.94 (d, C3), 109.74 (t,
-
-
C30), 150.36 (s, C20), 171.06 (s, OAc), 181.47 (s, C28). MS: m/z 497.1 [M-H] . IR (KBr; cm
1
): 2945 (C–H val.), 2871 (–CH val.), 1735 (–C=O val. ester), 1694 (–C=O val. conjugated
3
acid). For C H O (498.74) calculated C (77.06), H (10.10), found C (77.09), H (10.08).
3
2
50
4
o
M.p. 263-266 C.
1
0

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
2.5. (3β)-28-Oxo-28-[(pyridin-n-ylmethyl)amino]lup-20(29)-en-3-yl acetate [5a (n=2), 5b
(n=3) and 5c (n=4)]
A solution of oxalyl chloride in dry dichloromethane (2 M; 1.4 mL; 2.8 mmol; 7 eq) was
added to a solution of 4 (200 mg; 0.4 mmol) in dry dichloromethane (4 mL), the reaction
mixture was stirred for 3 h, then evaporated and dissolved again in dry dichloromethane (4
mL). EDIPA (0.182 mL; 1.04 mmol; 2.6 eq) and o-, m- or p-picolyl amine (0.049 mL; 0.48
mmol; 1.2 eq) were added, and the reaction mixture was stirred at r.t. through night (for 12 h).
After stopping the reaction, evaporation of the solvent afforded a solid, which was purified by
column chromatography. Yields: 5a (86 %), 5b (99 %), 5c (87 %).
1
5a: H NMR: δ 0.72 (1H, dd, J = 2.1 Hz, J = 11.7 Hz, H5), 0.75 (3H, s, H24), 0.77 (3H, s,
1
2
H25), 0.77 (3H, s, H23), 0.78 (3H, s, H26), 0.90 (3H, d, J = 0.6 Hz, H27), 1.08 (2H, dt, J =
1
3.2 Hz, J = 3.2 Hz, J = 13.4 Hz, H21), 1.64 (3H, dd, J = 0.7 Hz, J = 1.4 Hz, H29), 1.99
2
3
1
2
(3H, s, H2'), 2.04 (2H, dt, J = 3.3 Hz, J = 3.3 Hz, J = 13.6 Hz, H16), 2.38 (1H, ddd, J = 3.7
1
2
3
1
Hz, J = 11.5 Hz, J = 12.9 Hz, H13), 3.10 (1H, dt, J = 4.6 Hz, J = 11.1 Hz, J = 11.1 Hz,
2
3
1
2
3
H19), 4.41 (1H, dd, J = 5.5 Hz, J = 10.0 Hz, H3), 4.46 (1H, dd, J = 5.2 Hz, J = 15.9 Hz,
1
2
1
2
H3'), 4.52 (1H, dd, J = 5.2 Hz, J = 15.9 Hz, H3'), 4.54 (1H, dq, J = 1.4 Hz, J = 1.4 Hz, J =
1
2
1
2
3
1.4 Hz, J = 2.4 Hz, H30), 4.69 (1H, dq, J = 0.7 Hz, J = 0.7 Hz, J = 0.7 Hz, J = 2.4 Hz,
4
1
2
3
4
H30), 6.85 (1H, t, J = 5.2 Hz, NH), 7.17 (1H, m, J = 0.6 Hz, J = 0.6 Hz, J = 1.2 Hz, J = 4.9
1
2
3
4
Hz, J = 7.5 Hz, H7'), 7.29 (1H, m, J = 0.6 Hz, J = 0.6 Hz, J = 1.2 Hz, J = 7.8 Hz, H9'),
5
1
2
3
4
7.64 (1H, dt, J = 1.8 Hz, J = 7.6 Hz, J = 7.6 Hz, H8'), 8.49 (1H, ddd, J = 0.8 Hz, J = 1.8
1
2
3
1
2
1
3
Hz, J = 4.9 Hz, H6'). C NMR: δ 14.58 (q, C27), 15.98 (q, C24), 16.17 (q, C25), 16.45 (q,
3
C26), 18.15 (t, C6), 19.42 (q, C29), 20.91 (t, C11), 21.31 (q, C2'), 23.67 (t, C2), 25.56 (t,
C12), 27.91 (q, C23), 29.41 (t, C21), 30.86 (t, C15), 33.65 (t, C16), 34.26 (t, C22), 37.08 (s,
C10), 37.71 (d, C13), 37.76 (s, C4), 38.33 (t, C1), 38.35 (t, C7), 40.70 (s, C8), 42.45 (s, C14),
44.17 (t, C3'), 46.78 (d, C19), 50.06 (d, C18), 50.49 (d, C9), 55.40 (d, C5), 55.76 (s, C17),
1
1

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
80.93 (d, C3), 109.35 (t, C30), 122.34 (d, C7'), 122.64 (d, C9'), 137.12 (d, C8'), 148.50 (d,
C6'), 150.98 (s, C20), 157.08 (s, C4'), 171.01 (s, C1'), 176.39 (s, C28). MS: m/z 589.2
+
-
-1
[M+H] , 587.2 [M-H] . IR (KBr; cm ): 2946 (C–H val.), 2870 (–CH val.), 1734 (–C=O val.
3
ester), 1651 (–C=O val. amide). For C H N O (588.86) calculated C (77.51), H (9.59), N
3
8
56
2
3
o
(4.76), found C (77.53), H (9.58), N (4.75). M.p. 110-115 C.
1
5b: H NMR: δ 0.72 (1H, dd, J = 2.1 Hz, J = 11.0 Hz, H5), 0.77 (3H, s, H24), 0.78 (3H, d, J
1
2
= 0.7 Hz, H25), 0.78 (3H, s, H23), 0.79 (3H, s, H26), 0.90 (3H, s, H27), 1.07 (2H, dt, J = 3.3
1
Hz, J = 3.3 Hz, J = 13.5 Hz, H21), 1.64 (3H, dd, J = 0.7 Hz, J = 1.4 Hz, H29), 1.89 (2H, dt,
2
3
1
2
J = 3.4 Hz, J = 3.4 Hz, J = 13.7 Hz, H16), 1.99 (3H, s, H2'), 2.39 (1H, ddd, J = 3.7 Hz, J =
1
2
3
1
2
11.5 Hz, J = 12.9 Hz, H13), 3.09 (1H, dt, J = 4.5 Hz, J = 11.2 Hz, J = 11.2 Hz, H19), 4.31
3
1
2
3
(1H, dd, J = 5.9 Hz, J = 14.9 Hz, H3'), 4.41 (1H, dd, J = 5.3 Hz, J = 9.6 Hz, H3), 4.48 (1H,
1
2
1
2
dd, J = 6.1 Hz, J = 14.9 Hz, H3'), 4.55 (1H, dq, J = 1.4 Hz, J = 1.4 Hz, J = 1.4 Hz, J = 2.4
1
2
1
2
3
4
Hz, H30), 4.69 (1H, dq, J = 0.7 Hz, J = 0.7 Hz, J = 0.7 Hz, J = 2.4 Hz, H30), 6.08 (1H, t, J
1
2
3
4
= 6.0 Hz, NH), 7.24 (1H, ddd, J = 0.9 Hz, J = 4.9 Hz, J = 7.8 Hz, H8'), 7.66 (1H, bddd, J =
1
2
3
1
1.8 Hz, J = 2.2 Hz, J = 7.8 Hz, H9'), 8.47 (1H, dd, J = 1.8 Hz, J = 4.9 Hz, H7'), 8.52 (1H,
2
3
1
2
1
3
bd, J = 2.2 Hz, H5'). C NMR: δ 14.57 (q, C27), 16.01 (q, C24), 16.21 (q, C25), 16.45 (q,
C26), 18.14 (t, C6), 19.45 (q, C29), 20.91 (t, C11), 21.31 (q, C2'), 23.67 (t, C2), 25.56 (t,
C12), 27.91 (q, C23), 29.39 (t, C21), 30.81 (t, C15), 33.68 (t, C16), 34.29 (t, C22), 37.09 (s,
C10), 37.68 (d, C13), 37.77 (s, C4), 38.32 (t, C1), 38.37 (t, C7), 40.71 (t, C3'), 40.73 (s, C8),
42.44 (s, C14), 46.65 (d, C19), 50.10 (d, C18), 50.51 (d, C9), 55.43 (d, C5), 55.66 (s, C17),
80.91 (d, C3), 109.48 (t, C30), 123.66 (d, C8'), 135.15 (s, C4'), 136.22 (d, C9'), 148.24 (d,
C7'), 148.73 (d, C5'), 150.74 (s, C20), 171.03 (s, C1'), 176.26 (s, C28). MS: m/z 589.2
+
-
-1
[M+H] , 587.2 [M-H] . IR (KBr; cm ): 3270 (N–H val. amide), 2941 (C–H val.), 2871 (–CH
3
val.), 1725 (–C=O val. ester), 1659 (–C=O val. amide). For C H N O (588.86) calculated C
3
8
56
2
3
o
(77.51), H (9.59), N (4.76), found C (77.50), H (9.60), N (4.78). M.p. 241-244 C.
1
2

ACCEPTED MANUSCRIPT
1
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
5c: H NMR: δ 0.72 (1H, dd, J = 2.1 Hz, J = 11.1 Hz, H5), 0.78 (3H, s, H24), 0.79 (3H, s,
1 2
H25), 0.79 (3H, s, H23), 0.82 (3H, s, H26), 0.91 (3H, s, H27), 1.11 (2H, dt, J = 3.4 Hz, J =
1
2
3.4 Hz, J = 13.5 Hz, H21), 1.64 (3H, dd, J = 0.7 Hz, J = 1.4 Hz, H29), 1.91 (2H, dt, J = 3.3
3
1
2
1
Hz, J = 3.3 Hz, J = 13.9 Hz, H16), 1.99 (3H, s, H2'), 2.39 (1H, ddd, J = 3.7 Hz, J = 11.6
2
3
1
2
Hz, J = 13.0 Hz, H13), 3.08 (1H, dt, J = 4.5 Hz, J = 11.1 Hz, J = 11.1 Hz, H19), 4.31 (1H,
3
1
2
3
dd, J = 6.0 Hz, J = 15.6 Hz, H3'), 4.42 (1H, dd, J = 5.9 Hz, J = 10.7 Hz, H3), 4.45 (1H, dd,
1
2
1
2
J = 6.1 Hz, J = 15.6 Hz, H3'), 4.55 (1H, dq, J = 1.4 Hz, J = 1.4 Hz, J = 1.4 Hz, J = 2.4 Hz,
1
2
1
2
3
4
H30), 4.69 (1H, dq, J = 0.7 Hz, J = 0.7 Hz, J = 0.7 Hz, J = 2.4 Hz, H30), 6.10 (1H, t, J =
1
2
3
4
1
3
6.1 Hz, NH), 7.18-7.21 (2H, m, H5'), 8.48-8.51 (2H, m, H6'). C NMR: δ 14.58 (q, C27),
16.09 (q, C24), 16.22 (q, C25), 16.45 (q, C26), 18.15 (t, C6), 19.45 (q, C29), 20.91 (t, C11),
21.31 (q, C2'), 23.66 (t, C2), 25.55 (t, C12), 27.91 (q, C23), 29.46 (t, C21), 30.80 (t, C15),
33.72 (t, C16), 34.34 (t, C22), 37.09 (s, C10), 37.68 (d, C13), 37.77 (s, C4), 38.38 (t, C7),
38.38 (t, C1), 40.77 (s, C8), 42.24 (s, C14), 42.46 (t, C3'), 46.60 (d, C19), 50.10 (d, C18),
50.52 (d, C9), 55.44 (d, C5), 55.72 (s, C17), 80.91 (d, C3), 109.55 (t, C30), 122.57 (d, C5'),
148.98 (s, C4'), 149.41 (d, C6'), 150.65 (s, C20), 171.04 (s, C1'), 176.38 (s, C28). MS: m/z
+
-
-1
589.2 [M+H] , 587.2 [M-H] . IR (KBr; cm ): 3378 (N–H val. amide), 2946 (C–H val.), 2869
(–CH val.), 1734 (–C=O val. ester), 1642 (–C=O val. amide). For C H N O (588.86)
3
38 56
2
3
calculated C (77.51), H (9.59), N (4.76), found C (77.49), H (9.57), N (4.78). M.p. 105-108
o
C.
2.6. (3β)-3-Hydroxy-N-(pyridin-n-ylmethyl)lup-20(29)-en-28-amide [6a (n=2), 6b (n=3) and
6c (n=4)]
LiOH·H O (54 mg; 1.3 mmol; 5 eq) was added to a solution of 5a, 5b or 5c (151 mg; 0.26
2
mmol) in methanol (15 mL), and the reaction mixture was refluxed for 5 h. After stopping the
reaction, the solvent was evaporated, and the residue was dissolved in chloroform and filtered.
1
3

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Evaporation of the solvent afforded a solid, which was purified by column chromatography.
Yields: 6a (82 %), 6b (84 %), 6c (83 %).
1
6a: H NMR: δ 0.62 (1H, dd, J = 2.3 Hz, J = 11.5 Hz, H5), 0.70 (3H, s, H24), 0.74 (3H, d, J
1
2
= 0.8 Hz, H25), 0.75 (3H, s, H23), 0.90 (3H, s, H26), 0.91 (3H, d, J = 0.6 Hz, H27), 1.08 (2H,
dt, J = 3.1 Hz, J = 3.1 Hz, J = 13.5 Hz, H21), 1.64 (3H, dd, J = 0.7 Hz, J = 1.4 Hz, H29),
1
2
3
1
2
2.04 (2H, dt, J = 3.3 Hz, J = 3.3 Hz, J = 13.5 Hz, H16), 2.38 (1H, ddd, J = 3.6 Hz, J = 11.6
1
2
3
1
2
Hz, J = 13.1 Hz, H13), 3.10 (1H, dt, J = 4.6 Hz, J = 11.1 Hz, J = 11.1 Hz, H19), 3.13 (1H,
3
1
2
3
dd, J = 4.9 Hz, J = 11.5 Hz, H3), 4.46 (1H, dd, J = 5.1 Hz, J = 12.0 Hz, H1'), 4.53 (1H, dd,
1
2
1
2
J = 5.1 Hz, J = 12.0 Hz, H1'), 4.54 (1H, dq, J = 1.4 Hz, J = 1.4 Hz, J = 1.4 Hz, J = 2.4 Hz,
1
2
1
2
3
4
H30), 4.69 (1H, dq, J = 0.7 Hz, J = 0.7 Hz, J = 0.7 Hz, J = 2.4 Hz, H30), 6.85 (1H, bt, J =
1
2
3
4
5.1 Hz, NH), 7.17 (1H, ddd, J = 1.3 Hz, J = 5.0 Hz, J = 7.6 Hz, H5'), 7.29 (1H, bdt, J = 1.0
1
2
3
1
Hz, J = 1.0 Hz, J = 7.7 Hz, H7'), 7.64 (1H, dt, J = 1.8 Hz, J = 7.6 Hz, J = 7.6 Hz, H6'),
2
3
1
2
3
1
3
8.49 (1H, ddd, J = 1.0 Hz, J = 1.8 Hz, J = 5.0 Hz, H4'). C NMR: δ 14.63 (q, C27), 15.34
1
2
3
(q, C24), 15.98 (q, C25), 16.12 (q, C26), 18.28 (t, C6), 19.46 (q, C29), 20.90 (t, C11), 25.61
(t, C12), 27.40 (t, C2), 27.99 (q, C23), 29.43 (t, C15), 33.67 (t, C16), 33.88 (t, C21), 34.34 (t,
C22), 37.17 (s, C10), 37.74 (d, C13), 38.34 (t, C7), 38.69 (t, C1), 38.83 (s, C4), 40.69 (s, C8),
42.46 (s, C14), 44.21 (t, C1'), 46.77 (d, C19), 50.07 (d, C18), 50.59 (d, C9), 55.34 (d, C5),
55.77 (s, C17), 78.95 (d, C3), 109.30 (t, C30), 122.33 (d, C4'), 122.61 (d, C7'), 137.05 (d,
C6'), 148.58 (d, C3'), 151.02 (s, C20), 157.12 (s, C2'), 176.36 (s, C28). MS: m/z
+
-
-1
547.3[M+H] , 545.3 [M-H] . IR (KBr; cm ): 3368 (N–H val. amide), 2942 (C–H val.), 2868
(–CH val.), 1649 (–C=O val. amide). For C H N O (546.83) calculated C (79.07), H
3
36 54
2
2
o
20
(9.95), N (5.12), found C (79.09), H (9.93), N (5.14). M.p. 210-211 C. [α]D = +10.3 (c
0.350).
1
6b: H NMR: δ 0.61 (1H, dd, J = 2.3 Hz, J = 11.0 Hz, H5), 0.70 (3H, s, H24), 0.76 (3H, d, J
1
2
= 0.7 Hz, H25), 0.79 (3H, s, H23), 0.91 (3H, s, H26), 0.91 (3H, d, J = 0.8 Hz, H27), 1.07 (2H,
1
4

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
dt, J = 3.2 Hz, J = 3.2 Hz, J = 13.4 Hz, H21), 1.63 (3H, dd, J = 0.7 Hz, J = 1.4 Hz, H29),
1
2
3
1
2
1.88 (2H, dt, J = 3.3 Hz, J = 3.3 Hz, J = 13.5 Hz, H16), 2.39 (1H, ddd, J = 3.7 Hz, J = 11.5
1
2
3
1
2
Hz, J = 13.0 Hz, H13), 3.09 (1H, dt, J = 4.4 Hz, J = 11.0 Hz, J = 11.0 Hz, H19), 3.12 (1H,
3
1
2
3
dd, J = 4.9 Hz, J = 11.4 Hz, H3), 4.31 (1H, dd, J = 5.8 Hz, J = 15.0 Hz, H1'), 4.47 (1H, dd,
1
2
1
2
J = 6.1 Hz, J = 15.0 Hz, H1'), 4.55 (1H, dq, J = 1.4 Hz, J = 1.4 Hz, J = 1.4 Hz, J = 2.4 Hz,
1
2
1
2
3
4
H30), 4.69 (1H, dq, J = 0.7 Hz, J = 0.7 Hz, J = 0.7 Hz, J = 2.4 Hz, H30), 6.06 (1H, bt, J =
1
2
3
4
6.0 Hz, NH), 7.22 (1H, ddd, J = 0.9 Hz, J = 4.9 Hz, J = 7.9 Hz, H6'), 7.64 (1H, bddd, J =
1
2
3
1
1.7 Hz, J = 2.3 Hz, J = 7.6 Hz, H7'), 8.49 (1H, bdd, J = 1.7 Hz, J = 4.9 Hz, H5'), 8.50 (1H,
2
3
1
2
1
3
bdq, J = 0.7 Hz, J = 0.7 Hz, J = 0.7 Hz, J = 2.3 Hz, H3'). C NMR: δ 14.62 (q, C27), 15.34
1
2
3
4
(q, C24), 16.02 (q, C25), 16.15 (q, C26), 18.26 (t, C6), 19.49 (q, C29), 20.90 (t, C11), 25.60
(t, C12), 27.39 (t, C2), 27.96 (q, C23), 29.41 (t, C15), 30.83 (t, C21), 33.70 (t, C16), 34.37 (t,
C22), 37.17 (s, C10), 37.71 (d, C13), 38.33 (t, C7), 38.70 (t, C1), 38.83 (s, C4), 40.42 (s, C8),
40.72 (t, C1'), 42.45 (s, C14), 46.63 (d, C19), 50.12 (d, C18), 50.61 (d, C9), 55.35 (d, C5),
55.67 (s, C17), 78.94 (d, C3), 109.42 (t, C30), 123.62 (d, C6'), 135.04 (s, C2'), 136.06 (d,
C7'), 148.41 (d, C5'), 148.83 (d, C3'), 150.77 (s, C20), 176.23 (s, C28). MS: m/z
+
-
-1
547.3[M+H] , 545.3 [M-H] . IR (KBr; cm ): 3284 (N–H val. amide), 2933 (C–H val.), 2867
(–CH val.), 1640 (–C=O val. amide). For C H N O (546.83) calculated C (79.07), H
3
36 54
2
2
o
20
(9.95), N (5.12), found C (79.05), H (9.96), N (5.11). M.p. 254-256 C. [α]D = +11.1 (c
0.307).
1
6c: H NMR: δ 0.62 (1H, dd, J = 2.2 Hz, J = 11.1 Hz, H5), 0.71 (3H, s, H24), 0.77 (3H, bs,
1
2
H25), 0.82 (3H, s, H23), 0.92 (3H, bs, H27), 0.92 (3H, s, H26), 1.12 (2H, dt, J = 3.5 Hz, J =
1
2
3.5 Hz, J = 13.5 Hz, H21), 1.64 (3H, dd, J = 0.7 Hz, J = 1.4 Hz, H29), 1.74 (2H, ddd, J =
3
1
2
1
0.9 Hz, J = 8.1 Hz, J = 12.3 Hz, H7), 2.39 (1H, ddd, J = 3.6 Hz, J = 11.6 Hz, J = 12.8 Hz,
2
3
1
2
3
H13), 3.09 (1H, dt, J = 4.6 Hz, J = 11.1 Hz, J = 11.1 Hz, H19), 3.13 (1H, dd, J = 4.9 Hz, J
2
1
2
3
1
= 11.4 Hz, H3), 4.32 (1H, dd, J = 6.0 Hz, J = 15.6 Hz, H1'), 4.45 (1H, dd, J = 6.1 Hz, J =
1
2
1
2
1
5

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
15.6 Hz, H1'), 4.55 (1H, dq, J = 1.4 Hz, J = 1.4 Hz, J = 1.4 Hz, J = 2.4 Hz, H30), 4.69 (1H,
1
2
3
4
dq, J = 0.7 Hz, J = 0.7 Hz, J = 0.7 Hz, J = 2.4 Hz, H30), 6.05 (1H, bt, J = 6.0 Hz, NH),
1
2
3
4
1
3
7.16-7.18 (2H, m, H4'), 8.49-8.51 (2H, m, H3'). C NMR: δ 14.63 (q, C27), 15.33 (q, C24),
16.10 (q, C25), 16.16 (q, C26), 18.27 (t, C6), 19.49 (q, C29), 20.90 (t, C11), 25.60 (t, C12),
27.39 (t, C2), 27.96 (q, C23), 29.47 (t, C15), 30.82 (t, C21), 33.75 (t, C16), 34.42 (t, C22),
37.18 (s, C10), 37.71 (d, C13), 38.39 (t, C7), 38.71 (t, C1), 38.84 (s, C4), 40.76 (s, C8), 42.21
(t, C1'), 42.48 (s, C14), 46.59 (d, C19), 50.12 (d, C18), 50.61 (d, C9), 55.37 (d, C5), 55.73 (s,
C17), 78.96 (d, C3), 109.49 (t, C30), 122.47 (d, C3'), 148.49 (s, C2'), 149.78 (d, C4'), 150.70
+
-
-1
(s, C20), 176.23 (s, C28). MS: m/z 547.3[M+H] , 545.3 [M-H] . IR (KBr; cm ): 3352 (N–H
val. amide), 2942 (C–H val.), 2871 (–CH val.), 1670 (–C=O val. amide). For C H N O
2
3
36 54
2
(546.83) calculated C (79.07), H (9.95), N (5.12), found C (79.06), H (9.94), N (5.15). M.p.
o
20
246-248 C. [α] = +3.7 (c 0.454).
D
2.7. Cell Cultures
The screening cell lines: T-lymphoblastic leukemia CEM; breast carcinoma MCF7; cervical
carcinoma HeLa; malignant melanoma G-361; 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, MO, USA). Media used
were supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1% penicillin-
streptomycin. The cell lines were maintained under standard cell culture conditions at 37 °C
and 5% CO in humid environment. Cells were subcultured twice or three times a week using
2
the standard trypsinization procedure.
2.8. Cytotoxicity screening tests
Description of the experimental procedure used in cytotoxicity test was already published
1
6

ACCEPTED MANUSCRIPT
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
[1,2]. IC values obtained with the compounds 3a–3c and 6a–6c are shown in Table 1.
5
0
2.9. Flow cytometry analysis
G-361 cancer cells were trypsinized, seeded in 100 mm culture dishes, and after 24 h of
stabilization incubated with the tested compounds. After additional 24 h, the cells were again
-1
detached with trypsin, washed, fixed and stained in 0.1% [v/v] Triton X-100, 0.2 mg.mL
-1
RNase A and 10 µg.mL propidium iodide in PBS. Their DNA contents were then assessed
TM
by a flow cytometer (FACS Verse , Becton Dickinson, NJ, USA), and the distribution of
cells in subG (“apoptotic cells”), G /G , S and G /M peaks were quantified by histogram
1
0
1
2
analysis using BD FACSuite software (Becton Dickinson, NJ, USA). The experiments were
repeated three times. The differences between control and treated cells were analyzed by one-
way analysis of variance ANOVA using Microsoft Excel 2010. Signification was marked
with asterisk (p ˂ 0.05).
2.10. SDS–polyacrylamide gel electrophoresis and immunoblotting
Cells (G-361) were seeded into culture medium in 100-mm culture dishes at a density of 1.5 ×
6
-1
10 cells.mL and after 24 h of stabilization treated with tested compounds. DMSO was used
as a vehicle for controls. After another 24 h of treatment, the cells were washed three times
with cold PBS (10 mM, pH 7.4) and lysed in ice-cold RIPA protein extraction buffer (20 mM
Tris-HCl, pH 7.4, 5 mM EDTA, 2 mM EGTA, 100 mM NaCl, 2 mM NaF, 0.2% Nonidet P-
-1
40, 30 mM PMSF, 1 mM DTT, 10 mg.mL of aprotinin and leupeptin). The lysate was
collected into a microfuge tube and incubated on ice for 1 h. It was then cleared by
centrifugation at 18,000 × g for 30 min at 4 ºC and the supernatant was collected. Proteins in
lysates were quantified by the Bradford method and diluted with Laemmli electrophoresis
buffer. The proteins were then separated on 10% or 12% SDS-polyacrylamide gels,
1
7

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
transferred to nitrocellulose membranes (Bio-Rad Laboratories, CA, USA) and stained with
Ponceau S (Sigma Aldrich, St. Louis, MO, USA) to check equal protein loading. The
membranes were blocked with 5% (w/v) non-fat dry milk and 0.1% Tween-20 in PBS for 2 h
and probed with specific primary antibodies (Cell Signaling Technology, Danvers, MA, USA;
Santa Cruz Biotechnology, CA, USA) overnight. Anti-caspase-3 and anti-caspase-7
recognized zymogen and also its fragment. After washing in PBS and PBS with 0.1% Tween-
20, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies
and visualized with West Pico Supersignal chemiluminescent detection reagent (Thermo
Fisher Scientific, Rockford, USA). Signal was detected by a CCD camera (Fujifilm, Tokio,
Japan). To confirm equal protein loading, immunodetection was performed with anti-β-actin
monoclonal antibody (Santa Cruz Biotechnology, CA, USA). The experiments were repeated
three times. The protein expression in treated cells was compared to untreated controls.
2.11. Activities of caspase 3/7
Treated G-361 cells were harvested by centrifugation and homogenized in an extraction buffer
(10 mM KCl, 5 mM HEPES, 1 mM EDTA, 1 mM EGTA, 0.2% CHAPS, plus protease
inhibitors: aprotinin, leupeptin, PMSF; pH 7.4) on ice for 20 min. The resulting homogenates
were clarified by centrifugation at 18,000 × g for 20 min at 4°C. The protein contents of the
samples were quantified by the Bradford method and they were diluted to equivalent protein
concentrations. Lysates were then incubated for 1 h with 100 mM Ac-DEVD-AMC as a
substrate (Sigma, MO, USA) in an assay buffer (25 mM PIPES, 2 mM EGTA, 2 mM MgCl2,
5 mM DTT, pH 7.3). For negative controls, the lysates were supplemented with 100 mM Ac-
DEVD-CHO (Sigma, MO, USA) as a caspase-3/7 inhibitor. The fluorescence of the product
was measured using a Fluoroskan Ascent microplate reader (Labsystems, Vantaa, Finland) at
346/442 nm (ex/em). Experiments were repeated three times in triplicates. The differences
1
8

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
between control and treated cells were analyzed by one-way analysis of variance ANOVA
using Microsoft Excel 2010. Signification was marked with asterisk (p ˂ 0.05).
3. Results and Discussion
3.1. Synthetic protocol
The synthesis of both series of the target picolyl amides (3a–3c and 6a–6c) is efficient and
easy. To get 3a–3c (Scheme 1), betulinic acid (1) was first esterified by succinic anhydride
[2,26] to prepare its hemiester 2, which was then subjected to a formation of an amide bond
with o-, m- and p-picolyl amines, using T3P as condensation agent [2,27]. To get 6a–6c
(Scheme 2), protecting of the C(3)-OH group as acetate ester was required, and achieved by
reacting betulinic acid (1) with acetic anhydride in dry THF, using EDIPA as a base and
DMAP as reaction promotor [2]. Free carboxylic group of 1 was then converted to betulinic
acyl chloride by means of oxalyl chloride in dichloromethane [2,28], and used subsequently
without isolation in a formation of amide with o-, m- and p-picolyl amines, using again
EDIPA as a base [27], affording 5a–5c. Protecting acetate group was removed by LiOH.H O
2
in methanol [2], yielding the required amides 6a–6c.
Cytotoxicity: The experimental data obtained (Table 1) revealed high importance of 3b and
3a, while 3c and 6a–6c displayed only moderate cytotoxicity. The cytotoxicity data of 3a–3c
clearly demonstrate the importance of substitution of pyridine ring in structure-activity
relationship studies: while o- and m-substitution of the pyridine ring resulted in highly
cytotoxic compounds 3a and 3b, p-substitution revealed its disadvantage in 3c. Both cytotoxic
compounds, 3a and 3b, showed very low cytotoxicity in normal fibroblasts, and became
potential candidates for cytotoxic agents against G-361 (2.4 ± 0.0 µM and 0.5 ± 0.1 µM,
respectively), MCF7 (2.2 ± 0.2 µM and 1.4 ± 0.1 µM, respectively), HeLa (2.3 ± 0.5 µM and
2.4 ± 0.4 µM, respectively) and CEM (6.9 ± 0.4 µM and 6.5 ± 1.5 µM, respectively) cancer
1
9

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
cell lines. Moreover, 3b was the most active in melanoma cancer cell line G-361 (IC 0.5 ±
5
0
0.1 µM) compared to other tested cancer cell lines with weak activity in normal human cells
(BJ) that is important for wide therapeutic window. We determined selectivity against all
tested cancer cells compared to normal cells with therapeutic index between 7 and 100 for
compounds 3a and 3b. The highest therapeutic index (TI = 100) was calculated for human
malignant melanoma cell line (G-361) versus normal human fibroblasts (BJ) with 3b.
Difluoroderivatives of dihydrobetulinic acid showed selectivity with therapeutic index
between 2 and 8 against cancer lines [29]. In another study, therapeutic index of 9.1 was
measured for conjugate of (3β)-3-O-acetylbetulinic acid with a triazole ring further
substituted by benzaldehyde [30]. Based on the literature search, no derivative of betulinic
acid was as highly active in melanoma cancer cell line G-361 as 3b. In a future, we would like
to design other compounds with higher selectivity, mainly based on the structure of 3b.
Together with obtained results, these derivatives could be promising for in vivo experiments
for development new anticancer drugs.
Concerning 6a–6c, their lower cytotoxicity has been expected due to the substitution of
carboxyl group in these derivatives of betulinic acid. However, the great disadvantage of 6a–
6c is their cytotoxicity in normal fibroblasts, which is sometimes even higher than that in the
cancer cell lines. The cytotoxicity of 6a–6c in CEM, MCF7 and HeLa cancer cell lines is
comparable with the cytotoxicity of the compounds investigated earlier [1]. The equal
moderate cytotoxicity with no selectivity as for 6a–6c was also shown for saponins of
betulinic acid in different cancer cell lines [33].
3.2. Picolyl amides of betulinic acid caused apoptosis
We have observed whether compounds 3a–3c influence the cell cycle and cause apoptosis in
human malignant melanoma cells G-361. Cells were treated with 0.5, 2.5, 12.5 or 25 µM of
2
0

ACCEPTED MANUSCRIPT
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
three substances (3a–3c) for 24 h. Then the cells were harvested, stained and analyzed by
flow cytometry, western blotting or caspase activation assay. Flow cytometric analysis
showed an increase in the subG phase of the cell cycle (apoptotic cells) in G-361 cell line
1
after treatment with 3a and 3b. In turn, 3c did not enhance the number of subG cells and
1
caused no changes in the cell cycle distribution. In these cells, treatment with 3a and 3b
increased the proportion of cells in the G /G phase and decreased their proportion in the
0
1
concomitant phases, mainly G /M (Figure 1). Apoptotic markers were detected by western
2
blotting to examine the antiproliferative activities of tested compounds 3a–3c. The initiation
of apoptosis is mediated by caspase cascade activation [34]. Caspase-3 and caspase-7 are
executioner proteases that result in cleavage of poly-(ADP-ribose) polymerase (PARP) and
subsequent DNA degradation and apoptotic death [35]. The western blots showed degradation
of caspase-7 into cleaved fragments (a part of the apoptotic cascade) after treatments with 3a
and 3b in G-361 cells (Figure 2). PARP cleavage, a final step of caspase activation, was
observed in human malignant melanoma cells treated by 3a and 3b (Figure 2). pRb was
downregulated by 12.5 µM and 25 µM of 3a and 3b, but total Rb also decreased in those
treatments. It is probably due to reaching the cytotoxicity towards human melanoma cells. No
changes in the expressions of anti-apoptotic proteins Bcl-2 and Mcl-1 and also p53 were
observed. Downregulation of phospho-p44/42 MAPK (Erk 1/2) (Thr202/Tyr204) was
detected after the treatment with 12.5 and 25 µM of 3a and 3b for 24 h. Total level of p44/42
was not changed. This kinase is responsible for proliferation, differentiation, motility, and
death of cells. The compounds 3a and 3b inhibited those activities mediated by Erk 1/2. These
data were supplemented by estimation of caspase-3/7 activity in G-361 treated cells using the
fluorogenic substrate Ac-DEVD-AMC and/or caspase-3/7 inhibitor Ac-DEVD-CHO.
Compound 3a increased activity of caspase-3/7 after the treatment with 25 µM up to eighteen
fold. Compound 3b induced the activity of caspase-3/7; after 24 h treatment with
2
1

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
concentration 25 µM, a twelve fold increase in the effector caspases was observed compared
to the untreated control (Figure 3). Unlike 3a and 3b, compound 3c did not affect the activity
of caspases-3/7 after 24 h. These results show that 3a and 3b can induce apoptosis by caspase-
3 activation, and target proliferation and differentiation in G-361 cells.
3.3. Physico-chemical parameters
To support rational design of the target compounds investigated in this work, their physico-
chemical parameters have been calculated using ACD/iLabs software and databases [36]. The
parameters calculated for 3a–3c and 6a–6c were compared with the Lipinski [5] rule of five
and with the Ghose [6] rules. The rules describe molecular properties important for a small
molecule 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. Lipinski [5] rule of five considers partition
coefficient (log P, range -0.4 to +5.6), molar refractivity (range 40 to 130), molecular weight
(range 180 to 500), number of atoms in the molecule (20 to 70) and polar surface area (up to
14 nm). The data for comparison are presented in Table 2. However, the target compounds
3a–3c and 6a–6c do not correspond to the range given for any of the above Lipinski and
Ghose rules [5,6], except of the parameter giving the number of atoms in the molecule.
Despite those facts, 3a–3c and 6a–6c display cytotoxicity. Exceptions are already known,
where pharmacologically active compounds do not correspond to all rules, we have observed
such result as well [1,2].
The most important molecular descriptors are the partition coefficient (log P) and the
distribution coefficient (log D) [37]. In chemical and pharmaceutical sciences, both, log P and
log D are measures of hydrophilicity or hydrophobicity of the studied compound, and are
useful for estimating distribution of a drug within the body, where log D shows the
2
2

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
dependence on the pH of the matrix. Hydrophobic drugs are then preferentially distributed to
hydrophobic compartments (e.g., lipid bilayer of cells), while hydrophilic drugs are
preferentially distributed to hydrophilic compartments (e.g., blood serum). The distribution
coefficient is a pH dependent value, and, therefore, the value at pH = 7.4 (the physiological
pH value of blood serum) is of particular importance (see also Table 2). Thus, log P expresses
a ratio of concentrations of non-ionized compound between two phases, non-polar (octanol)
and polar (water), while log D expresses the ratio of the sum of the concentrations of all forms
of the compound (ionized and non-ionized) in each of the two phases. In pharmacology, log P
and log D indicate how easily the drug can reach its intended target in the body, how strong
its effect will be once it reaches its target and how long it will remain in the body in an active
form. The log P values calculated for either 3a–3c or 6a–6c are identical, and they exceed the
values given by the Lipinski and Ghose rules [5,6] (Table 2). Another supportive parameter is
-3
the predicted aqueous solubility, log S. S (in mol dm ) is the concentration of the solute in a
saturated solution that is in equilibrium with the crystalline solid, and it is a pH dependent
parameter. Standard range for log S at pH 7.4 is –6.5/+0.5. Among the prepared compounds,
only 3a–3c show values for log S in this range (Table 2).
3.4. ADME parameters:
The importance of some of the ADME parameters for evaluation of pharmacokinetic
properties of the prepared compounds is summarized in Table 2. The blood brain barrier
(BBB) and plasma protein binding are two of the important factors affecting distribution of
the compound in the human body. Several parameters assist in evaluation of each potential
drug for its BBB transport [38] The rate of brain penetration, log PS, is a logarithm of the
permeability-surface area coefficient that measures the ability of a drug to cross the BBB and
to move into brain tissue over time. It is one of the relevant parameters for evaluation of the
2
3

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
rate of BBB penetration. The extent of brain penetration parameter, log PB, indicates if the
drug might be active or inactive on the central nervous system (CNS). The typical values for
log PB are –1.5/+1.5. Most of the current drugs show the log PB value up to +2 (active on
CNS) or down to –2 (inactive on CNS). The log PB values calculated for 3a–3c and 6a–6c
indicate that these compounds will be inactive on CNS (Table 2). However, the complex of
the BBB parameters is completed by the brain/plasma equilibration rate, the parameter
expressed as log PS * fu, brain that is a mathematical modeling parameter based on time
required for reaching brain equilibrium. It is dependent on the brain unbound fraction (fu, brain).
This value indicates if the drug may potentially be active on CNS together with the log BB
parameter, the predicted brain/blood partition coefficient (cf. Supplementary material). The
parameter log BB is a hybrid parameter determined by permeability, plasma and brain tissue
binding, and active transport mechanism, standard range –3.0/+1.2. The log BB values
calculated for 3a–3c and 6a–6c appear in the required range of this parameter. Nevertheless,
there are numerous exceptions already known. A supportive parameter, plasma protein
binding (PPB) gives calculated quantity of a drug bound to a protein.
Another important ADME parameter is bioavailability. Among the prepared compounds, the
calculated bioavailability for 3a–3c is lower than that for 6a–6c. However, the experimental
data show that 3a–3c are generally more cytotoxic to the tested cancer cell lines than 6a–6c.
The rules [5,6] describe molecular properties important for a small molecule drug
pharmacokinetics in the human body, including their absorption, distribution, metabolism and
excretion (ADME parameters). However, the rules do not predict whether a compound will
display pharmacological activity. Nevertheless, the importance of several ADME parameters,
namely blood-brain barrier and plasma protein binding, reflects the distribution of the
compound in the human body [39]. A comparison of the calculated physico-chemical
properties with the measured cytotoxicity shows that every time a new class of compound is
2
4

ACCEPTED MANUSCRIPT
5
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
being investigated, no available experimental screening may be intentionally omitted (Tables
1 and 2).
3.5. A comparison of 3a–3c and 6a–6c with the compounds prepared earlier [1, 2]
Earlier prepared compound, bearing picolyl amine moiety in the molecule, derived either
from lanosterol, cholesterol or sitosterol, displayed cytotoxicity in the range of IC = 25–45
5
0
µM, or were inactive (IC > 50 µM). The earlier published data also show that the physico-
5
0
chemical and ADME parameters calculated for those compounds, corresponded to the
Lipinski and Ghose rules [5,6] partially only. The current compounds 3a–3c and 6a–6c show
generally higher cytotoxicity than the earlier series of compounds. Only 3a and 3b display
considerable cytotoxicity, and 3b show the IC value on G361 in the range IC = 0.5 ± 0.1
5
0
50
µM, with the therapeutic index TI = 100. This finding augments the importance of this
particular compound 3b. In turn, even 3a and 3b corresponded to a limited number of
calculated physico-chemical and ADME parameters. Any calculation of those parameters is
useful and often desired in the drug design. However, the conclusions of the calculations
should always be supported by the experimental data for confirmation or correction.
4. Conclusion
Amides 3b and 3a represent the most important members of this series of compounds because
of their high cytotoxicity against human melanoma cell line G-361 (Table 1). The former of
this couple of amides (3b) is active in concentration 0.5 ± 0.1 µM, and, subsequently it does
not damage healthy cells. It also displays high cytotoxicity in other tested cell lines.
Investigating structure-activity relationship in dependence on substitution of pyridine ring, the
conclusions are different for each subseries. In the former subseries (3a–3c), the cytotoxicity
of amides bearing m- and o-substituted pyridine ring (3b and 3a) showed high cytotoxicity in
2
5

ACCEPTED MANUSCRIPT
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
all tested cell lines, while p-substitution of the pyridine ring (3c) resulted in weak cytotoxicity
(Table 1). Our hypothesis is that this result might be connected with different mechanism of
action of 3b and 3a in comparison with 3c due to the modifications in their structures. This
hypothesis will be focused in a future investigation, because its confirmation may be
important for better understanding of structure-activity relationship. However, in the latter
subseries (6a–6c), amides bearing p- and o-substituted pyridine ring showed either
comparable cytotoxicity, or p-substituted amide was more active, and the m-substituted
derivative displayed the lowest cytotoxicity (Table 1). The most important finding with 6a–6c
was their high activity in the normal human cells. Due to that finding, 6a–6c are much less
suitable anti-cancer agents than 3a–3c that are, in turn, potential anti-cancer candidate agents.
Supplementary material is available on the web page of the journal.
Acknowledgment: The authors are grateful to the grants FV10599 (MPO; UB; ZW) and 14-
27669P (GACR; LR) for financial support. Financial support from the specific university
(UCT) research (MSMT No. 20-SVV/2017) is also acknowledged (UB; ZW).
References
[1] U. Bildziukevich, L. Rárová, D. Šaman, L. Havlíček, P. Drašar, Z. Wimmer, Amides
derived from heteroaromatic amines and selected steryl hemiesters, Steroids 78 (2013) 1347–
1352.
[2] U. Bildziukevich, N. Vida, L. Rárová, M. Kolář, D. Šaman, L. Havlíček, P. Drašar, Z.
Wimmer, Polyamine derivatives of betulinic acid and β-sitosterol: a comparative
investigation, Steroids 100 (2015) 27–35.
2
6

ACCEPTED MANUSCRIPT
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
[3] U. Bildziukevich, E. Kaletová, D. Šaman, E. Sievänen, E.T. Kolehmainen, M. Šlouf, Z.
Wimmer, Spectral and microscopic study of self-assembly of novel cationic spermine amides
of betulinic acid, Steroids 117 (2017) 90–96.
[4] B.G. Bag, C. Garai, R. Majumdar, M. Laguerre, Natural triterpenoids as renewable nanos,
Struct. Chem. 23 (2012) 393–398.
[5] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and computational
approaches to estimate solubility and permeability in drug discovery and developmental
settings. Adv. Drug Deliv. Rev. 46 (2001) 3–26.
[6] A.K. Ghose, V.N. Viswanadhan, J.J. Wendoloski, A knowledge-based approach in
designing combinatorial or medicinal chemistry libraries for drug discovery. 1. A qualitative
and quantitative characterization of known drug databases. J. Combin. Chem. 1 (1999) 55–68.
[7] Y. Kashiwada, J. Chiyo, Y. Ikeshiro, T. Nagao, H. Okabe, L.M. Cosentino, K. Fowke,
K.H. Lee, 3,28-Di-O-(dimethylsuccinyl)-betulin isomers as anti-HIV agents, Bioorg. Med.
Chem. Lett. 11 (2001) 183–185.
[8] V. Zuco, R. Supino, S.C. Righetti, L. Cleris, E. Marchesi, C. Gambacorti-Passerini, F.
Formelli, Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells,
Cancer Lett. 175 (2002) 17–25.
[9] Y.J. You, Y. Kim, N.H. Nam, B.Z. Ahn, Synthesis and cytotoxic activity of A-ring
modified betulinic acid derivatives, Bioorg. Med. Chem. Lett. 13 (2003) 3137–3140.
[10] S. Fulda, Betulinic acid for cancer treatment and prevention, Int. J. Mol. Sci. 9 (2008)
1096–1107.
[11] C. Genet, A. Strehle, C. Schmidt, G. Boudjelal, A. Lobstein, K. Schoonjans, M. Souchet,
J. Auwerx, R. Saladin, A. Wagner, Structure-activity relationship study of betulinic acid, a
novel and selective TGR5 agonist, and its synthetic derivatives: potential impact in diabetes,
J. Med. Chem. 53 (2010) 178–190.
2
7

ACCEPTED MANUSCRIPT
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
[12] J. Šarek, M. Kvasnica, M. Vlk, M. Urban, P. Džubák, M. Hajdúch, The potential of
triterpenoids in the treatment of melanoma. In: Research on melanoma – a glimpse into
current directions and future trends. (M. Murph, Ed.). Chapter 7, pp. 125–158. InTech, Rijeka,
Croatia, 2011.
[13] R.H. Cichewicz, S.A. Kouzi, Chemistry, biological activity, and chemotherapeutic
potential of betulinic acid for the prevention and treatment of cancer and HIV infection, Med.
Res. Rev. 24 (2004) 90–114.
[14] Y. Iwamoto, Diagnosis and treatment of Ewing’s sarcoma, Jpn. J. Clin. Oncol. 37 (2007)
79–89.
[15] S.K. Dash, S.S. Dash, S. Chattopadhyay, T. Ghosh, S. Tripathy, S.K. Mahapatra, B.G.
Bag, D. Das, S. Roy, Folate decorated delivery of self-assembled betulinic acid nano fibers: a
biocompatible antileukemic therapy, RSC Adv. 5 (2015) 24144–24157.
[16] I. Budihardjo, H. Oliver, M. Lutter, X. Luo, X. Wang, Biochemical pathways of caspase
activation during apoptosis, Annu. Rev. Cell Dev. Biol. 15 (1999) 269–290.
[17] M. Nihal, C.T. Roelke, G.S. Wood, Anti-melanoma effects of vorinostat in combination
with polyphenolic antioxidant (–)-epigallocatechin-3-gallate (EGCG), Pharm. Res. 27 (2010)
1103–1114.
[18] R.T. Allen, M.W. Cluck, D.K. Agrawal, Mechanisms controlling cellular suicide: role of
Bcl-2 and caspases, Cell Mol. Life Sci. 54 (1998) 427–445.
[19] P. Džubák, M. Hajdúch, D. Vydra, A. Hustová, M. Kvasnica, D. Biedermann, L.
Marková, M. Urban, J. Šarek, Pharmacological activities of natural triterpenoids and their
therapeutic implications, Nat. Prod. Rep. 23 (2006) 394–411.
[20] K. Kaczanowska, K.-H. Wiesmüller, A.-P. Schaffner, Design, synthesis, and in vitro
evaluation of novel aminomethyl-pyridines as DPP-4 inhibitors, ACS Med. Chem. Lett. 1
(2010) 530–535.
2
8