Synthesis, antimicrobial and cytotoxic activities, and structure-activity relationships of gypsogenin derivatives against human cancer cells

Article abstract of DOI:10.1016/j.ejmech.2014.05.084

A series of gypsogenin (1) derivatives (1a-i) was synthesized in good yields, and the derivatives' structures were established using UV, IR, ¹H NMR, ¹³C NMR, and LCMS spectroscopic data. Among the tested compounds, 1a, 1b, 1d, 1e, and gypsogenin (1) showed antimicrobial activities against Bacillus subtilis and Bacillus thrungiensis, with inhibition zones of 10-14 mm. In addition, compounds 1b, 1d, and 1e showed antimicrobial activities against Bacillus cereus, with inhibition zones of 9-14 mm. Using six human cancer cell lines in vitro, the cytotoxic activities of all tested compounds were determined by calculating the IC₅₀ values. Doxorubicin and paclitaxel were used as controls. Among the tested compounds, 1a, 1c, and 1d had inhibitory effects with IC₅₀ values of 3.9 μM (HL-60 cells), 5.15 μM (MCF-7 cells), and 5.978 μM (HL-60), respectively. To determine the type of cell death, Hoechst 33258 (HO) and propidium iodide (PI) double staining was used. Especially, gypsogenin (1) and compound 1a triggered the apoptotic mechanism at a concentration of 20 μM. Thus, gypsogenin (1) and compounds 1a, 1c, and 1d possess varying degrees of biological activities and can be considered as potential antitumor agents.

Full text of DOI:10.1016/j.ejmech.2014.05.084

Accepted Manuscript
Synthesis, antimicrobial and cytotoxic activities, and structure-activity relationships of
gypsogenin derivatives against human cancer cells
Safiye Emirdağ-Öztürk , Tamer Karayıldırım , Aysun Çapcı-Karagöz , Özgen Alankuş-
Çalışkan , Ali Özmen , Esin Poyrazoğlu-Çoban
PII:
S0223-5234(14)00516-9
10.1016/j.ejmech.2014.05.084
EJMECH 7042
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 February 2014
Revised Date: 15 May 2014
Accepted Date: 31 May 2014
Please cite this article as: S. Emirdağ-Öztürk, T. Karayıldırım, A. Çapcı-Karagöz, Ö. Alankuş-Çalışkan,
A. Özmen, E. Poyrazoğlu-Çoban, Synthesis, antimicrobial and cytotoxic activities, and structure-activity
relationships of gypsogenin derivatives against human cancer cells, European Journal of Medicinal
Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.084.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, antimicrobial and cytotoxic activities,
and structure-activity relationships of
gypsogenin derivatives against human cancer
cells
Safiye Emirdağ-Öztürk a,*,Tamer Karayıldırım a, Aysun Çapcı-Karagöz b,Özgen
Alankuş-Çalışkan a, Ali Özmen c and Esin Poyrazoğlu-Çoban c
,
a Chemistry Department, Faculty of Science, Ege University, Bornova, Izmir 35100, Turkey
b Institute of Organic Chemistry I, University of Erlangen-Nuremberg, Henkestrasse 42, 91054 Erlangen, Germany
cBiology Department, Adnan Menderes University, Aydin 09010, Turkey
Abstract
A series of gypsogenin (1) derivatives (1a-i) was synthesized in good yields, and the derivatives’ structures were
established
using
UV,
IR,
1H
NMR,
13C
NMR,
and
LCMS
spectroscopic
data.
Among the tested compounds, 1a, 1b, 1d, 1e, and gypsogenin (1) showed antimicrobial activities against Bacillus
subtilis and Bacillus thrungiensis, with inhibition zones of 10–14 mm. In addition, compounds 1b, 1d, and 1e
showed antimicrobial activities against Bacillus cereus, with inhibition zones of 9–14 mm. Using six human cancer
cell lines in vitro, the cytotoxic activities of all tested compounds were determined by calculating the IC50 values.
Doxorubicin and paclitaxel were used as controls. Among the tested compounds, 1a, 1c, and 1d had inhibitory
effects with IC50 values of 3.9 µM (HL-60 cells), 5.15 µM (MCF-7 cells), and 5.978 µM (HL-60), respectively. To
determine the type of cell death, Hoechst 33258 (HO) and propidium iodide (PI) double staining was used.
Especially, gypsogenin (1) and compound 1a triggered the apoptotic mechanism at a concentration of 20 µM.
Thus, gypsogenin (1) and compounds 1a, 1c, and 1d possess varying degrees of biological activities and can be
considered as potential antitumor agents.
Keywords: Gypsogenin; saponin; Gypsophila; cytotoxic; apoptosis
1. Introduction
species [20-22].
Some saponins from Gypsophila have shown a
variety of biological activities including anticarcinogenic
[23], immunostimulatory [24], and cytotoxic activities
[25].
Gypsophila accumulate gypsogenin aglycone with
sugar chains, which has been attributed to various
biological properties. For example, these compounds
have exhibited inhibitory activity [26] and have shown
significant growth inhibition in in vitro cultures [27]. In
addition, some of them have shown very high activity
against different human cancer cell lines [28, 29].
Thus, there is strong evidence that gypsogenin has
anticancer activity.
Saponins, which are detected in a number of plant
families, are glycosides with a polycyclic aglycone and
a sugar moiety. Sugars can attach to aglycone, which is
also called sapogenin, at one or two different positions;
thus, they are named monodesmosidic and
bidesmosidic saponins, respectively [1-3]. These
secondary
metabolites
have
hydrophilic
and
hydrophobic sides; hence, they have surface activity,
and saponin-containing plants are used as soap.
Saponins
have
various
structure-dependent
biological activities such as glucosidase inhibiting [4],
antiviral [5, 6], anti-inflammatory [7], spermicidal [8],
hypocholesterolemic
[9],
antitumor
[10,
11],
Gypsogenin
aglycone
is
found
at
high
anticarcinogenic [12], and antioxidant activities [13].
Moreover, saponins have been evaluated against
cancer cells for anticancer activity [14, 15].
Triterpene saponins can be found in many plant
species [16-19], and several recent studies have
reported on saponins produced from Gypsophila
concentrations in Gypsophila [30]; therefore, it can be
obtained with ease [31]. In this study, nine new
gypsogenin derivatives (1a-i) were synthesized from
gypsogenin aglycone (1). In addition, they were
evaluated for their antibacterial and antifungal activities
as well as cytotoxic activities against six different
human cancer cell cultures.
* Corresponding author:
S.Emirdağ-Öztürk.,Tel.: (+) 90 232 311 2371 Fax: (+) 90 232 388 8264;
E-mail: safiyemirdag_ozturk@hotmail.com, safiye.ozturk@ege.edu.tr
1

ACCEPTED MANUSCRIPT
2. Results and discussion
Usually, this rection is preferred to protect the carboxyl
group of aglycone [33-35]. These intermediates were
then reacted with thiosemicarbazide in 1:1 methanol:
water to yield compounds 1e and 1g, respectively.
Compounds 1b and 1c were converted into compounds
1d and 1f, respectively, by treatment with
hydroxylamine hydrochloride at room temperature.
Acetylation of compound 1c at C-3 gave compound 1h,
which was further reacted with hydroxyl hydrochloride
to afford compound 1i.
2.1. Chemistry
Nine gypsogenin derivatives (1a-i) were synthesized
by a series of reactions as outlined in Scheme 1.
The starting material, gypsogenin (1), was obtained
from the commercially available Gypsophila arrostii root
extract, and its isolation has been explained in our
previous work [32].
All compounds were obtained in good yields, as
shown in Table 1.
The nine synthesized compounds were established
1
1
by IR, UV, H NMR, 13C NMR, and LCMS analyses. H
NMR spectroscopy was especially useful to confirm the
structures. For example, for compound 1a, the signal
for the aldehyde proton at δH 10.75 was converted to an
1
oxime group at δH 7.88. The H NMR spectrum also
showed a doublet of doublets at δH 4.17 (J=16.0 and
5.2 Hz) attributed to H-3.
In the 1H NMR for compound 1b, the proton signal of
H-3 at δH 5.51 was observed instead of at δH 5.20 ppm.
This conversion was also seen for compound 1h. The
1H NMR spectrum of compound 1c showed the extra
proton signal of OCH2-C6H5 at δH 5.16 ppm. In the 13C
NMR, the signal of OCH2-C6H5 appeared at δC 66.46
ppm. For compound 1e, the signals for –N=CH and
NH2C=S could be seen at δH 7.51 and δH 9.73,
respectively. These signals were also evident by the 13
C
NMR spectrum showing –N=CH and NH2C=S at δC
153.76 and δC 181.00, respectively.
These spectral data are summarized in Tables 2 and
3 and the Supplementary data (Figure 1).
Table 1
Structures and yields of gypsogenin (1) and its derivatives
(compounds 1a-i).
2.2. Biological activity
Compound
R1
R2
R3
Yield
(%)
2.2.1. Antimicrobial activity
Gypsogenin
-OH
-COH
-COOH
-
(1)
The antimicrobial activities of gypsogenin (1) and its
nine new derivatives were analyzed by the disc
diffusion method [36] and minimum inhibitory
1a
1b
1c
1d
1e
1f
-OH
-CH=NOH
-CHO
-COOH
70.2
97.8
54.6
97.2
45.8
88.3
56.9
97.7
91.2
concentration
(MIC)
determination
[37].
The
-OCOCH3
-OH
-COOH
antimicrobial activities of these compounds were
evaluated against different strains of Gram-negative
bacteria (Escherichia coli ATCC 25922, Salmonella
typhimurium ATCC 14028, Klebsiella pneumonia ATCC
13882, Pseudomonas aeruginosa ATCC 35032, and
Proteus vulgaris ATCC 33420), Gram-positive bacteria
(Micrococcus luteus ATCC 9341, Stapylococcus aureus
ATCC 25923, Stapylococcus epidermidis ATCC 12228,
Bacilllus cereus ATCC 11778, Bacillus subtilis ATCC
6633, Bacillus thrungiensis, and Entereococcus faecalis
ATCC 29212), and yeast (Candida utilis ATCC 9950,
Candida albicans ATCC 10231, Candida glabrata,
Candida trophicalis, and Saccharomyces cerevisiae
ATCC 9763).
-CHO
-COOCH2C6H5
-COOH
-OCOCH3
-OCOCH3
-OH
-CH=NOH
-CH=NNHCSNH2
-CH=NOH
-CH=NNHCSNH2
-CHO
-COOH
-COOCH2C6H5
-COOCH2C6H5
-COOCH2C6H5
-COOCH2C6H5
1g
1h
1i
-OH
-OCOCH3
-OCOCH3
-CH=NOH
The results of the in vitro antimicrobial activities of
the tested compounds, along with known antibiotic and
antifungal reagents for comparison, are reported as
inhibition zone (mm) and MIC values and are listed in
Tables 4 and 5, respectively.
Gypsogenin (1) was treated with hydroxylamine
hydrochloride and sodium acetate in 3:1 acetonitrile:
water at room temperature to provide compound 1a.
The intermediate compounds 1b and 1c were
synthesized by substitution reactions involving
acetylation at C-3 and benzylation at C-28, respectively.
2

ACCEPTED MANUSCRIPT
1d
1b
1e
1a
(1)
1f
1c
1g
1h
1i
Scheme 1. Reagents and conditions: (a) hydroxylamine hydrochloride (NH2OH·HCl), sodium acetate, 3:1 acetonitrile: water, rt; (b) acetic
anhydride, pyridine, rt; (c) benzyl bromide, triethylamine, reflux; (d) thiosemicarbazide, 1:1 MeOH: water, reflux.
3

ACCEPTED MANUSCRIPT
Table 2
1H NMR data of gypsogenin (1) and compounds 1a-i (400 MHz, δ ppm, in pyridine).
Position
Gypsogenin (1)a,b [32]
Compound
1aa
1ba
1ca
1da
1ea
1fa
1ga
1ha
1ia
2.66 (m), 2.17 (m)
3.23 (m), 2.26 (m)
1.92 (m), 1.36 (m)
1.84 (m), 1.36 (m)
1.91 (m), 1.25 (m)
2.33(m), 1.35 (m)
1.82 (m), 1.34 (m)
2.40 (m), 1.47 (m)
1.76 (m), 1.20 (m)
2.40 (m), 1.22 (m)
1.66 (m), 1.05 (m)
1.89 (m), 0.08 (m)
1.69 (m), 1.04 (m)
1.67 (m), 1.00 (m)
2.02 (m), 1.05 (m)
1.56 (m), 1.01 (m)
2.02 (m), 1.06 (m)
1
2
2.40 (m), 1.46 (m)
2.37 (m), 1.37 (m)
2.02 (m), 1.07 (m)
4.17 (dd),
(J=16.0, 5.2 Hz)
5.51 (dd),
(J=16.8, 5.6 Hz)
3.92 (dd),
(J=16.0, 5.2 Hz)
3
5.20 (m)
4.36 (m)
5.44 (m)
5.53 (m)
4.02 (m)
5.22 (m)
5.16 (m)
4
-
-
-
-
-
-
-
-
-
-
5
2.58 (m)
1.80 (m)
1.63 (m)
1.73 (m)
1.71 (m)
1.60 (m)
1.56 (m)
1.49 (m)
1.45 (m)
1.43 (m)
6
2.61 (m), 2.29 (m)
1.87 (m), 1.54 (m)
1.75 (m), 1.43 (m)
1.79 (m), 1.37 (m)
1.76 (m), 1.49 (m)
1.70 (m), 1.24 (m)
1.61 (m),1.14 (m)
1.63 (m), 1.12 (m)
1.55 (m), 1.14 (m)
1.52 (m), 1.12 (m)
7
2.59 (m), 2.31 (m)
1.83 (m), 1.56 (m)
1.70 (m), 1.43 (m)
1.76 (m), 1.43 (m)
1.72 (m), 1.51 (m)
1.68 (m), 1.24 (m)
1.58 (m), 1.16 (m)
1.60 (m), 1.16 (m)
1.53 (m), 1.16 (m)
1.46 (m), 1.15 (m)
8
-
-
-
-
-
-
-
-
-
-
9
2.89 (m)
2.04 (m)
2.04 (m)
2.02 (m)
1.99 (m)
2.02 (m)
1.72 (m)
1.77 (m)
1.81 (m)
1.66 (s)
10
11
12
13
14
15
16
17
-
-
-
-
-
-
-
-
-
-
2.98 (m), 2.46 (m)
6.62 (br s)
-
-
2.61 (m)
3.25 (m), 3.06 (m)
-
2.26 (m), 1.73 (m)
5.80 (br s)
-
-
1.91 (m)
2.43 (m), 2.31 (m)
-
2.22 (m), 1.55 (m)
5.76 (br s)
-
-
1.80 (m)
2.39 (m), 2.30 (m)
-
2.10 (m), 1.61 (m)
5.72 (br s)
-
-
1.82 (m)
2.34 (m), 2.19 (m)
-
2.21 (m), 1.61 (m)
5.76 (br s)
-
-
1.79 (m)
2.42 (m), 1.77 (m)
-
2.26 (m), 1.50 (m)
5.76 (br s)
-
-
1.73 (m)
2.42 (m), 2.30 (m)
-
1.80 (m),1.41 (m)
5.44 (br s)
-
-
1.64 (m)
1.96 (m), 1.83 (m)
-
1.93 (m), 1.36 (m)
5.43 (br s)
-
-
1.67 (m)
2.05 (m), 1.95 (m)
-
1.88 (m), 1.23 (m)
5.42 (br s)
-
-
1.64 (m)
2.06 (m), 1.89 (m)
-
1.82 (m), 1.34 (m)
5.42 (br s)
-
-
1.53 (m)
2.06 (m), 1.88 (m)
-
4.43 (dd),
(J=13.6, 4.0 Hz)
2.92 (m), 2.45 (m)
-
3.60 (dd),
(J=18.0, 4.0 Hz)
2.12 (m), 1.63 (m)
-
3.59 (dd),
(J=18.0, 3.6 Hz)
2.11 (m), 1.51 (m)
-
3.45 (dd),
(J=18.4, 4.4 Hz)
2.05 (m), 1.57 (m)
-
3.58 (dd),
(J=17.6, 4.4 Hz)
2.11 (m), 1.59 (m)
-
3.59(dd),
(J=17.6, 3.6 Hz)
2.10 (m), 1.45 (m)
-
3.17 (dd),
(J=17.6, 3.6 Hz)
1.74 (m), 1.28 (m)
-
3.17 (dd),
(J=17.6, 3.6 Hz)
1.81 (m), 1.27 (m)
-
3.17 (dd),
(J=17.6, 3.6 Hz)
1.77 (m), 1.22 (m)
-
3.16 (dd)
(J=17.6, 4.0 Hz)
1.77 (m), 1.25 (m)
-
18
19
20
21
22
23
24
25
26
27
28
2.56 (m), 2.35 (m)
3.15 (m), 2.93 (m)
10.75 (s)
2.48 (s)
2.01 (s)
2.11 (s)
1.78 (m), 1.56 (m)
2.35 (m), 2.19 (m)
-
1.75 (s)
1.25 (s)
1.30 (s)
1.58 (s)
-
1.61 (m), 1.45 (m)
2.33 (m), 2.19 (m)
9.79 (s)
1.58 (s)
1.15 (s)
1.27 (s)
1.47 (s)
-
1.70 (m), 1.45 (m)
2.23 (m), 2.05 (m)
9.91 (s)
1.66 (s)
1.06 (s)
1.23 (s)
1.52 (s)
-
1.69 (m), 1.54 (m)
2.33 (m), 2.18 (m)
-
1.64 (s)
1.20 (s)
1.26 (s)
1.57 (s)
-
1.56 (m), 1.25 (m)
2.34 (m), 2.18 (m)
-
1.54 (s)
1.18 (s)
1.41 (s)
1.31 (s)
-
1.53 (m), 1.19 (m)
1.87 (m), 1.78 (m)
-
1.49 (s)
0.95 (s)
0.98 (s)
1.23 (s)
-
1.46 (m), 1.19 (m)
1.97 (m), 1.84 (m)
-
1.41 (s)
0.80 (s)
0.93 (s)
1.21 (s)
-
1.28 (m), 1.20 (m)
1.90 (m), 1.83 (m)
9.52 (s)
1.24 (s)
0.76 (s)
0.94 (s)
1.21 (s)
-
140 (m), 1.19 (m)
1.90 (m),1.79 (m)
-
1.38 (s)
0.77 (s)
0.96 (s)
1.23 (s)
-
2.40 (s)
-
29
30
2.08 (s)
2.13 (s)
1.27 (s)
1.32 (s)
1.25 (s)
1.32 (s)
1.22 (s)
1.24 (s)
1.25 (s)
1.32 (s)
1.25 (s)
1.27 (s)
0.96 (s)
1.00 (s)
0.94 (s)
0.98 (s)
0.90 (s)
0.96 (s)
0.94 (s)
0.98 (s)
-CHNOH
-CH3COO
-CH2-C6H5
7.88 (s)
7.60 (s)
2.24 (s)
7.75 (s)
7.60 (s)
1.97 (s)
4.90
2.23 (s)
2.20 (s)
1.95 (s)
4.85
5.16
5.12
5.29
7.55 (d),
(J=8.0 Hz)
7.45 (t),
(J=8.0, 8.0 Hz)
7.37 (t),
(J=8.0, 8.0 Hz)
7.55 (d),
(J=8.0 Hz)
7.44 (t),
(J=8.0, 7.2 Hz)
7.37 (t),
(J=7.2, 7.2 Hz)
7.55 (d),
(J=7.6 Hz)
7.45 (t),
(J=8.0, 7.2 Hz)
7.37 (t),
(J=7.2, 7.2 Hz)
7.23 (s)
7.55 (d),
(J=8.0 Hz)
7.45 (t),
(J=8.0, 8.0 Hz)
7.37 (t),
(J=8.0, 8.0 Hz)
7.54 (d),
(J=7.2 Hz)
7.45 (t),
(J=8.0, 7.2 Hz)
7.37 (t),
B (C6H5)
C (C6H5)
D (C6H5)
(J=8.0, 8.0 Hz)
-CHNNH
-CSNH
-CSNH2
7.51 (s)
8.60 (br s)
9.73 (br s)
8.74 (br s)
9.38 (br s)
aMultiplicity of signals is given in parentheses: s, singlet; d, doublet; t, triplet; m, multiplet; and br, broad.
bIdentified from the HMBC, HMQC, and NOESY spectra.
4

ACCEPTED MANUSCRIPT
Table 3
13C NMR data of gypsogenin (1) and compounds 1a-i (100 MHz, δ ppm, in pyridine).
Position
Gypsogenin (1)a [32]
Compound
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
38.24, CH2
28.02, CH2
71.38, CH
56.04, qC
47.46, CH
20.97, CH2
32.78, CH2
39.81, qC
47.84, CH
35.81, qC
26.82, CH2
122.02, CH
144.64, qC
42.21, qC
30.85, CH2
23.82, CH2
46.62, qC
41.76, CH
46.22, CH2
32.32, qC
35.50, CH2
33.05, CH2
206.87, qC
9.51, CH3
15.34, CH3
17.35, CH3
25.93, CH3
179.85, qC
33.97, CH3
23.54, CH3
39.04
28.64
75.57
52.32
47.40
20.57
33.51
40.38
48.58
37.24
27.44
122.73
145.22
42.56
31.29
24.14
46.99
42.37
46.81
33.22
37.20
33.61
-
38.09
28.57
73.84
54.94
48.11
21.07
33.48
40.33
48.12
36.34
26.49
122.42
145.22
42.54
31.29
23.99
46.94
42.30
46.78
32.71
36.30
33.60
204.93
10.07
15.78
17.60
24.10
180.40
34.56
23.14
38.85
28.31
72.01
56.57
48.03
21.33
33.09
40.35
48.23
36.46
27.40
123.06
144.42
42.41
31.14
23.99
47.30
42.27
46.43
32.82
36.16
33.45
207.53
10.02
16.05
17.55
26.38
177.46
34.30
23.33
38.33
28.59
77.38
52.73
46.94
20.28
33.50
40.30
48.32
37.09
26.52
122.53
145.23
42.52
31.29
24.04
46.76
42.30
45.72
32.99
36.76
33.60
-
13.02
16.13
17.64
24.10
180.42
34.56
23.62
157.46
21.39
170.47
38.14
28.60
76.66
52.66
47.00
20.36
33.50
40.28
48.28
37.11
26.50
122.50
145.20
42.50
31.29
24.01
46.94
42.29
46.75
32.98
36.06
33.59
-
39.06
28.34
75.57
52.26
47.39
20.52
33.12
40.33
48.48
37.20
27.44
123.20
144.43
42.40
31.13
23.99
47.32
42.28
46.42
32.55
36.07
33.45
-
38.93
28.34
75.24
52.20
48.48
20.65
33.10
40.32
48.70
37.07
27.55
123.16
144.41
42.36
31.13
23.97
47.32
42.27
46.42
33.12
36.05
33.45
-
38.10
28.28
73.85
54.95
48.00
21.04
33.09
40.29
48.09
36.30
26.38
122.90
144.43
42.40
31.14
23.68
47.29
42.23
46.39
32.64
36.00
33.44
204.95
10.10
15.81
17.48
23.97
177.47
34.28
23.14
38.36
28.29
77.38
52.68
47.29
20.24
33.11
40.26
48.22
37.06
28.30
123.00
144.45
42.37
31.14
23.71
46.38
42.21
45.73
32.90
36.00
33.45
-
13.05
16.17
17.51
26.41
177.49
34.29
23.61
157.43
21.40
170.49
66.43
137.61
129.22
128.68
128.76
12.65
16.29
17.74
26.51
180.45
34.57
24.02
159.77
12.70
16.08
17.64
24.10
180.42
34.55
23.63
12.67
16.33
17.61
26.41
177.50
34.30
23.74
159.72
12.20
16.27
17.59
26.37
177.49
34.30
23.72
-CHNOH
-CH3COO
-CH3COO
-CH2-C6H5
A (C6H5)
B (C6H5)
C (C6H5)
D (C6H5)
-CSNH2
-CHNNH
21.11
170.41
21.30
170.76
21.11
170.43
66.47
137.61
129.23
128.71
128.78
66.46
66.44
66.46
137.59
129.22
128.69
128.76
137.62
129.22
128.68
128.76
137.61
129.23
128.71
128.78
180.65
156.98
181.00
153.76
aIdentified from the HMBC, HMQC, and NOESY spectra.
5

ACCEPTED MANUSCRIPT
Table 4
Antimicrobial activities of gypsogenin (1) and compounds 1a-i as determined by inhibition zone measurements.
Inhibition zone (mm)
Compounds
Reference antibiotics
Test Microorganisms
1a
1b
1c
1d
1e
1f
1g
1h
1i
Gypsogenin (1)
C30
CN10
TE30
E15
AMP10
NS100
Escherichia coli
ATCC 25922
-
-
-
-
-
-
-
-
-
-
24
21
15
11
-
NT
Salmonella typhimirium
ATCC 14028
Micrococcus luteus
ATCC 9341
Stapylococcus aureus
ATCC 25923
Stapylococcus epidermidis
ATCC 12228
Klebsiella pneumoniae
ATCC 13882
Pseudomonas aeruginosa
ATCC 35032
Proteus vulgaris
ATCC 33420
Entereococcus faecalis
ATCC 29212
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
17
25
23
22
21
22
17
16
23
16
15
20
17
19
20
24
11
24
15
26
22
19
20
20
16
19
25
8
8
28
20
17
-
NT
NT
NT
NT
NT
NT
NT
NT
NT
30
23
11
14
21
20
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
14
-
Bacilllus cereus
ATCC 11778
9
9
12
14
13
26
Bacillus subtilis
ATCC 6633
Bacillus thrungiensis*
Candida albicans
ATCC 10231
11
10
-
11
10
-
-
-
-
13
12
-
14
14
-
-
-
-
-
-
-
-
-
-
-
-
-
13
13
-
22
26
20
21
12
15
25
28
-
-
NT
NT
22
NT
NT
NT
NT
NT
Candida utilis
ATCC 9950
-
-
-
-
-
-
-
-
-
-
NT
NT
NT
NT
NT
21
Candida trophicalis*
Candida glabrata*
Saccharomyces cerevisiae
ATCC 9763
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
15
16
-
-
-
-
-
-
-
-
-
-
NT
NT
NT
NT
NT
15
(-): Zone did not occur. NT: Not tested;
C30: Chloramphenicol (30 mg, Oxoid); CN10: Gentamycin (10 mg, Oxoid); TE30: Tetracycline (30 mg, Oxoid); E15: Erytromycin (15 mg,
Oxoid); AMP10: Ampicillin (10 mg, Oxoid); NS: Nystatin (100 mg, Oxoid);
*Special gift from the Faculty of Medicine, Adnan Menderes University.
Compounds 1a, 1b, 1d, 1e, and gypsogenin (1) 2.2.2. Cytotoxic activity
showed moderate effects against B. cereus ATCC
11778, B. subtilis ATCC 6633, and B. thrungiensis, with
The antiproliferative activities of gypsogenin (1) and
compound 1e exhibiting the strongest activity against its derivatives were analyzed by an MTT assay using
these strains (inhibition zone = 14 mm) (Table 4). The the following human cancer cell lines in cell culture: HL-
MIC determination results also showed that these same 60 (an acute promyelocytic leukemia cell line), HT-29
compounds exhibited moderate antimicrobial activity and Caco-2 (colorectal adenocarcinoma cell lines),
against the tested microorganisms (Table 5). Again, Saos-2 (an osteosarcoma cell line), MCF-7 (a breast
compound 1e had the highest activity (32 ꢀg mL-1), cancer cell line), and HeLa (a cervical cancer cell line).
which was similar to that of gypsogenin (1) and more The results are shown in Table 6. The IC50 values were
potent than that of streptomycin.
determined for each compound and cell line. In
addition, the activities of doxorubicin and paclitaxel
were determined to serve as comparison compounds.
Only the significant (IC50<10 µM) antiproliferative
activities were considered for interpreting the
compounds as anticancer agents.
Table 5
Antimicrobial activities of compounds as determined by MIC values
(µg mL-1).
Gypsogenin
Test microorganisms
1a
1b
1d
1e
Str
(1)
Gypsogenin (1) showed antiproliferative activity
against two cancer cell lines (Saos-2 and MCF-7), with
IC50 values less than 10 µM. Interestingly, compounds
1a, 1c, 1d, and 1g were effective against more cell
types than gypsogenin (1). The highest activities were
found for compounds 1a, 1c, and 1d against HL-60,
MCF-7, and HL-60 cell lines, respectively, with IC50
values of 3.90 µM, 5.15 µM, and 5.98 µM, respectively.
Thus, these compounds could be possible anticancer
agents, with IC50 values comparable to doxorubicin and
paclitaxel. Compounds 1b, 1e, 1f, 1h, and 1i were not
active against the cell lines tested.
Bacilllus cereus
ATCC 11778
Bacillus subtilis
ATCC 6633
128
128
64
32
32
64
64
64
32
64
32
32
32
32
64
64
Bacillus thrungiensis*
128
128
Compounds 1c, 1f, 1g, 1h, and 1i did not show antibacterial activity.
Str = Streptomycin
(-) = No effect.
* From the Faculty of Medicine, Adnan Menderes University.
6

ACCEPTED MANUSCRIPT
Table 6
Antiproliferative activities of investigated compounds against human cancer cell lines.
IC50
(µM)
Cell line
1a
1b
1c
1d
1e
1f
1g
1h
1i
Gypsogenin (1)
Doxorubicin
Paclitaxel
HL-60
3.90
10.77
±2.17
11.14
±2.00
17.38
±2.21
8.24
8.14
5.98
12.82
±1.87
13.34
±1.35
30.42
±2.97
10.10
±1.48
7.50
10.62
±1.86
8.01
6.21
6.74
8.68
10.40
±2.77
11.90
±1.78
16.67
±3.77
7.85
0.33
0.32
±1.13
10.89
±2.35
28.08
±2.68
7.93
±1.44
8.51
±1.69
6.71
±1.00
14.98
±2.13
36.50
±3.65
6.97
±0.99
10.31
±2.10
51.14
±6.89
10.74
±3.22
65.18
±1.32
286.51
±12.18
±0.67
21.43
±2.56
20.28
±3.08
11.70
±2.46
9.90
±1.62
0.31
±0.79
0.29
HT 29
Caco-2
Saos-2
MCF-7
HeLa
±1.11
11.06
±3.26
12.70
±1.72
5.15
±0.54
28.24
±2.04
8.95
±1.63
16.13
±2.73
10.28
±1.97
6.67
±1.96
0.35
±0.66
0.31
±1.77
0.33
±1.12
0.35
±1.87
7.50
±2.15
20.51
±3.16
35.00
±8.70
±1.64
10.77
±2.19
116.23
±7.26
±0.46
6.05
±1.45
9.02
±2.00
0.35
±0.93
0.33
±2.14
8.71
±1.18
74.22
±15.00
±2.03
110.00
±6.45
±2.64
28.85
±2.67
±1.28
23.62
±3.46
±1.33
40.33
±4.70
±2.49
22.48
±2.60
±1.51
0.34
±1.53
0.29
±2.06
±1.56
±1.44
Results are expressed as means ± standard deviation.
The structure-activity relationship was also analyzed. to mid-apoptotic stage, or pink (interference of blue and
When a different substituent was attached to the red) when the cells are in a late apoptotic stage or are
necrotic. Thus, HO/PI staining facilitates distinguishing
between apoptosis and necrosis.
The highest apoptotic rates were determined with
gypsogenin (1) and compound 1a. At 48 h after the
gypsogenin aglycone (1), the activity of the compound
changed. For example, compound 1a, which has an
oxime group on gypsogenin (1), showed higher activity
than compound 1b, which contains an acetyl group. In
addition, compound 1d (with both an acetyl group and application of these compounds, between 20% and
an oxime group) was more active than compound 1f 36% of the cells died through an apoptotic pathway in
(with both a benzyl group and an oxime group). the different cell lines tested. Paclitaxel, a cytoskeletal
Compound 1i, which contains oxime, acetyl, and benzyl drug that targets tubulin and stabilizes the microtubule
polymer, was used as a positive control. This drug
blocks the progression of mitosis, thus triggering the
apoptotic pathway at the mitotic checkpoint. Paclitaxel
showed approximately 50% apoptosis at a dose of 0.5
µM. In this study, only gypsogenin (1) and compound 1a
approached these levels, with 20–36% apoptosis
achieved at a concentration of 20 µM and very low
necrosis rates (Table 7).
substituents on the gypsogenin core (1), was not
suitable for biological activity, even though compound
1c, which contains
a benzyl group, did exhibit
antiproliferative activity.
2.2.3. Cell death assay
Thus, gypsogenin (1) and compound 1a triggered
The apoptotic effects of gypsogenin (1) and
the apoptotic pathway in cancer cell lines. Normally,
compounds 1a-i were determined by the Hoechst approximately 50% of cancers have their apoptotic cell
33258 (HO)/propidium iodide (PI) double-staining death mechanism disabled. Therefore, this endpoint is
method. As only the very late phenotypes of apoptosis an important approach to evaluate the effects of such
can be visualized by phase contrast microscopy, the compounds.
quantification of cell death impossible using microscopy
In contrast, compound 1b was found to have
alone. However, the combination of microscopic necrotic effects with a high cell death value (20–55%) in
examination together with a double-staining method all tested cell lines. Compounds 1c, 1f, and 1i could be
with fluorescent dyes is both a reliable and sensitive described as apoptotic/necrotic agents, but the necrotic
method to determine the type of cell death and to ratios were higher, causing cell death at high levels.
quantify it. HO and PI both stain chromatin. When Compounds 1f and 1i were obtained from compound
HO/PI-stained cells are examined under a microscope, 1c, implying that the activities of compounds 1f and 1i
the early stages of death are also detected and can be are related to those of compound 1c. Meanwhile,
quantified. In combination with the examination of compounds 1d, 1e, 1g, and 1h showed mostly necrotic
condensed chromatin, this method allows discrimination effects in all cell lines. Only a few apoptotic cells were
between early or late apoptosis, necrosis, and intact observed by microscopic observations.
cells because administration of both dyes either stains
the nuclei blue when the cells are viable or in an early-
7

ACCEPTED MANUSCRIPT
Table 7
Apoptosis and necrosis ratios in different cell lines after the application of 20 µM test compound or 0.5 µM control.
Apoptosis/Necrosis
(%)
1a
1b
1c
1d
1e
1f
(20 µM)
1g
1h
1i
Gypsogenin (1)
Doxorubicin
Paclitaxel
Cell line
(0.5 µM)
HL-60
HT-29
Caco
30/3
27/0.7
23/3
8/25
0.8/20
3/55
6/48
1/22
1/15
9/30
4/57
4/45
6/52
0.6/28
0.0/17
1/46
2/67
3/26
3/29
2/61
6/63
2/65
8/41
12/29
11/32
9/23
2/64
2/32
4/48
5/41
4/63
5/55
3/51
0.7/29
1/37
5/50
24/14
19/33
24/44
10/48
15/50
33/4
21/2
2/45
5/49
4/47
2/34
1/44
2/28
53/0.0
52/2
52/2
42/1
50/1
44/2
20/0.0
36/2
Saos
27/3
3/44
0.0/45
5/36
MCF-7
HeLa
26/4
5/29
5/55
12/54
11/32
30/4
26/3
4/33
0.0/64
2/68
29/0.0
3. Conclusion
LC-MS was recorded on an AGILENT 1200 Capillary
spectrometer. Column chromatography was carried out
using 60 Å silica gel (Merck 7734). TLC was carried out
using 60 Å silica gel on F254 aluminum plates (Merck
5554).
In summary, we synthesized nine gypsogenin
derivatives in good yield. Gypsogenin (1) and its
derivatives were evaluated for their antimicrobial
(against twelve bacteria and five yeasts) and cytotoxic
activities. Furthermore, gypsogenin (1) and compounds
1a, 1b, 1d, and 1e were assessed against three
Bacillus species using the MIC method.
4.2. General method for the preparation of gypsogenin
(1)
Among all of these compounds, 1a, 1b, 1d, and 1e
as well as gypsogenin (1) showed activities only against
Gram-positive bacteria (B. cereus ATCC 11778, B.
subtilis ATCC 6633, and B. thrungiensis). According to
our results, compounds 1d and 1e had stronger effects
against B. subtilis ATCC 6633 compared to the other
compounds. In addition, compound 1e and gypsogenin
(1) were more potent against B. cereus ATCC 11778,
B. subtilis ATCC 6633, and B. thrungiensis, compared
to the other compounds. However, none of the
compounds showed antifungal effects.
The synthesized compounds were tested against six
human cancer cell lines, and compounds 1a, 1c, and
1d could be considered as possible anticancer agents
as they were shown to affect the cell cycle or check
points, causing cell cycle arrest and cell death. In
particular, gypsogenin (1) and compound 1a triggered
the apoptotic pathway in cancer cells, showing high
apoptosis ratios. As cell cycle arrest and apoptosis are
significant endpoints/targets in cancer therapy, these
novel gypsogenin derivatives have potential as
anticancer therapeutics.
The commercially available water extract of G.
arrostii roots (400 mL) was mixed with ethanol (100 mL)
and hydrolyzed with 10% KOH at 100 °C for two days.
The reaction mixture was collected and neutralized with
HCl. Then, this mixture was hydrolyzed with 10% HCl
for three days before being neutralized with KOH and
extracted with CH2Cl2. The CH2Cl2 phase was
concentrated to give 1,4504 g of crude gypsogenin (1).
The crude product was purified by column
chromatography with hexane/ethyl acetate (6/4) as the
eluent to afford gypsogenin (1) (325 mg) [32].
4.2.1. 3-Hydroxy-23-oxoolean-12-en-28-oic acid
(gypsogenin (1))
mp: 273–274 °C; LC/MS (ESI-MS) m/z = 469.20
(M-1) (negative ion mode).
4.3. General procedure for the synthesis of compounds
1a, 1d, 1f, and 1i
Gypsogenin (1) (100 mg, 2.13×10-4 mol) and
compounds 1b (60 mg, 1.17×10-4 mol), 1c (75 mg,
1.34×10-4 mol), and 1h (60 mg, 9.97×10-5 mol) were
mixed with hydroxylamine hydrochloride (NH2OH·HCl)
(6 mg, 20 mg, 30 mg, 13 mg, respectively) and sodium
acetate and dissolved in acetonitrile (6 mL) and water
(2 mL). The reaction mixture was stirred at room
temperature for 24 h. After acetonitrile evaporation,
water (10 mL) was added to this mixture, and the
mixture was extracted with CH2Cl2 (3×15 mL). The
4. Experimental
4.1. General
Melting points of all compounds were recorded on a
Gallenkamp electrothermal melting point apparatus and
are uncorrected. IR spectra were recorded on a Perkin-
Elmer Spectrum 100 FT-IR spectrometer. NMR spectra
1
were measured in pyridine-d5 at 400 MHz for H NMR, organic layer was dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo. The residue was
purified by column chromatography using hexane/ethyl
HMBC, HMQC, and NOESY experiments and at 100
MHz for 13C NMR on a Varian AS-400 spectrometer.
8

acetate (6/4) to give compounds 1a, A1dC, C1Ef, PaTndED1i,MANUSCRIPT
4.4. General procedure for the synthesis of compounds
1b and 1h
respectively.
4.3.1. 3-Hydroxy-23-(hydroxyimino)olean-12-en-28-oic
acid (1a)
Acetic anhydride (5 mL) was added to gypsogenin
(1) (125 mg, 2.66×10-4 mol) in pyridine (1 mL) and to
compound 1c (100 mg, 1.79×10-4 mol) in pyridine (2
mL). The mixture was allowed to stir for 48 h at room
temperature. After stirring, the mixture was extracted
with CH2Cl2 (3×10 mL). The organic layer was dried
over anhydrous sodium sulfate and evaporated to
dryness. The residue was purified by silica gel
chromatography using hexane/ethyl acetate (6/4) to
give compounds 1b and 1h, respectively.
Prepared gypsogenin (1) (100 mg, 2.13×10-4 mol),
hydroxylamine hydrochloride (NH2OH·HCl) (6 mg,
8.63×10-5 mol), and sodium acetate (13.7 mg, 1.67×10-4
mol) were mixed according to the above general
procedure to provide 72.3 mg of compound 1a as a
white solid. Yield: 70.2%; m.p. 268.1–270.0 °C; UV λmax
(CH2Cl2): 205.0 nm; IR νmax (CH2Cl2) cm-1: 3912, 3375,
2854, 1686, 1463, 1363, 1273, 957, 817, 647, 520, 475;
LC/MS (ESI-MS) m/z = 484.20 (M-1) (negative ion
mode).
4.4.1. 3-(Acetyloxy)-23-oxoolean-12-en-28-oic acid (1b)
Prepared gypsogenin (1) (125 mg, 2.66×10-4 mol),
pyridine (1 mL), and acetic anhydride were mixed
4.3.2. 3-(Acetyloxy)-23-(hydroxyimino)olean-12-en-28-
oic acid (1d)
Prepared compound 1b (60 mg, 1.17×10-4 mol), according to the above general procedure to provide
hydroxylamine hydrochloride (NH2OH·HCl) (20 mg, 133 mg of compound 1b as a white solid. Yield: 97.8%;
2.88×10-4 mol), and sodium acetate (40 mg, 4.87×10-4 m.p. 164.8–166.1 °C; UV λmax (CH2Cl2): 208.0 nm; IR
mol) were mixed according to the above general νmax (CH2Cl2) cm-1: 3848, 3573, 3353, 2946, 1736,
procedure to provide 60 mg of compound 1d as a light 1694, 1463, 1370, 1238, 1030, 1009, 736, 688, 645,
yellow solid. Yield: 97.2%; m.p. 251.4–253.0 °C; UV
603, 509, 485; LC/MS (ESI-MS) m/z = 511.20 (M-1)
λmax (CH2Cl2): 209.0 nm; IR νmax (CH2Cl2) cm-1: 3817, (negative ion mode).
3649, 3254, 2950, 2639, 1730, 1689, 1459, 1369, 1265,
1242, 1032, 958, 818, 736, 690, 645, 568; LC/MS (ESI-
MS) m/z = 526.20 (M-1) (negative ion mode).
4.4.2. Benzyl 3-(acetyloxy)-23-oxoolean-12-en-28-oate
(1h)
Prepared compound 1c (100 mg, 1.79×10-4 mol),
4.3.3. Benzyl 3-hydroxy-23-(hydroxyimino)olean-12-en- pyridine (2 mL), and acetic anhydride were mixed
28-oate (1f)
according to the above general procedure to provide
Prepared compound 1c (75 mg, 1.34×10-4 mol), 105 mg of compound 1h as a white solid. Yield: 97.7%;
hydroxylamine hydrochloride (NH2OH·HCl) (30 mg, m.p. 159.6–161.1 °C; UV λmax (CH2Cl2): 208.0, 290.0
4.32×10-4 mol), and sodium acetate (60 mg, 7.31×10-4 nm; IR νmax (CH2Cl2) cm-1: 3839, 3736, 3100, 2947,
mol) were mixed according to the above general 2876, 1733, 1686, 1458, 1369, 1238, 1159, 1121, 1030,
procedure to provide 68 mg of compound 1f as a light 1010, 976, 897, 736, 647, 604, 558; LC/MS (ESI-MS)
yellow solid. Yield: 88.3%; m.p. 139.4–141.0 °C; UV
λmax (CH2Cl2): 209.0 nm; IR νmax (CH2Cl2) cm-1: 3806,
3300, 2945, 2590, 2297, 1738, 1723, 1456, 1385, 1365,
m/z = 603.40 (M+1) (positive ion mode).
1261, 1230, 1216, 1159, 1121, 1084, 1030, 934, 819, 4.5. General procedure for the synthesis of compound
736, 695, 529, 463; LC/MS (ESI-MS) m/z = 576.40 1c
(M+1) (positive ion mode).
A mixture of gypsogenin (1) (400 mg, 8.51×10-4) and
triethylamine (3 mL) was stirred at room temperature for
4.3.4. Benzyl 3-(acetyloxy)-23-(hydroxyimino)olean-12-
en-28-oate (1i)
1 h before benzyl bromide (2 mL) was added. The
mixture was refluxed for 3 h and then stirred at room
temperature for 48 h. Water (10 mL) was added to the
reaction mixture, and the mixture was extracted with
CH2Cl2 (3×10 mL). The organic layer was dried over
anhydrous sodium sulfate and evaporated to dryness.
The residue was purified by column chromatography on
silica gel using hexane/ethyl acetate (4/6) to give
compound 1c as an amorphous powder (260 mg).
Prepared compound 1h (60 mg, 9.97×10-5 mol),
hydroxylamine hydrochloride (NH2OH·HCl) (13 mg,
1.87×10-4 mol), and sodium acetate (45 mg, 5.49×10-4
mol) were mixed according to the above general
procedure to provide 56 mg of compound 1i as a white
solid. Yield: 91.2%; m.p. 224.5–226.0 °C; UV λmax
(CH2Cl2): 205.0, 290.0 nm; IR νmax (CH2Cl2) cm-1: 3696,
3350, 2920, 2850, 2130, 1696, 1635, 1534, 1463, 1386,
1364, 1278, 1182, 1016, 956, 720, 601, 529, 464, 457;
LC/MS (ESI-MS) m/z = 618.40 (M+1) (positive ion
mode).
4.5.1. Benzyl 3-hydroxy-23-oxoolean-12-en-28-oate
(1c)
Yield: 54.6%; UV λmax (CH2Cl2): 207.0 nm; IR νmax
(CH2Cl2) cm-1: 3290, 2964, 2855, 2700, 1750, 1466,
1458, 1383, 1358, 1317, 1258, 1217, 1158, 1117, 1075,
1042, 1025, 958, 833, 733, 683, 608, 542; LC/MS (ESI-
MS) m/z = 561.40 (M+1) (positive ion mode).
9

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4.6. General procedure for the synthesis of compounds
1e and 1g
The antibacterial and antifungal activities of all nine
synthesized compounds were analyzed by the disc
diffusion method and the broth dilution method to
A solution of thiosemicarbazide in water (7 mL) was determine the MIC.
added to a solution of compounds 1b (125 mg,
2.44×10-4) and 1c (87 mg, 1.55×10-4) in MeOH (7 mL).
The mixture was refluxed for 13 h. After cooling, the 4.7.1.1. Disc diffusion method
mixture was extracted with CH2Cl2 (3×10 mL). The
organic layer was dried over anhydrous sodium sulfate
The antimicrobial activities were determined
and evaporated to dryness. The residue was purified by according to the standard Antimicrobial Disc
column chromatography on silica gel using Susceptibility Tests summarized by the National
hexane/ethyl acetate (6/4) to give compounds 1e and Committee for Clinical Laboratory Standards [37]. Fresh
1g, respectively.
stock solutions (1000 µg mL-1) of all the synthesized
compounds were prepared in DMSO according to the
desired concentrations for the experiments. The
inoculum suspensions of the tested bacteria and yeasts
were prepared from the broth cultures (18–24 h), and
4.6.1. 3-(Acetyloxy)-23-[(aminocarbonothioyl)
hydrazono]olean-12-en-28-oic acid (1e)
Prepared compound 1b (125 mg, 2.44×10-4) and the turbidity was adjusted to be equivalent to a 0.5
thiosemicarbazide (120 mg, 1.32×10-3 mol) were mixed McFarland standard tube to give a concentration of
according to the above general procedure to provide 65 1×108 bacterial cells and 1×106 yeast cells mL-1,
mg of compound 1e as a light yellow solid. Yield: respectively. To test the antimicrobial activity of all the
45.8%; m.p. 205.3–207.4 °C; UV λmax (CH2Cl2): 211.0, synthesized compounds, a Mueller Hinton Agar (MHA)
261.0 nm; IR νmax (CH2Cl2) cm-1: 3854, 3629, 3434, plate was inoculated with 0.1 mL of broth culture of
3267, 3164, 2950, 1734, 1691, 1597, 1534, 1459, 1366, bacteria or yeast. Then, a hole of 6 mm in diameter and
1274, 1260, 1245, 1088, 1028, 1008, 1055, 839, 820, depth was made on top of the agar with a sterile stick
764, 749, 642, 530, 462; LC/MS (ESI-MS) m/z = 584.20 and filled with 30 ꢀL of each synthesized compound,
(M-1) (negative ion mode).
respectively.
Plates inoculated with E. coli, S. typhimurium, S.
aureus, S. epidermidis, E. faecalis, K. pneumonia, P.
4.6.2. Benzyl 23-[(aminocarbonothioyl)hydrazono]-3- aeruginosa, and P. vulgaris were incubated at 37 °C for
hydroxyolean-12-en-28-oate (1g) 24 h, while those with M. luteus, B. subtilis, B. cereus,
Prepared compound 1c (87 mg, 1.55×10-4) and B. thrungiensis, S. cerevisiae, C. albicans, C. utilis, C.
thiosemicarbazide (50 mg, 5.48×10-4 mol) were mixed glabrata, and C. trophicalis were incubated at 30 °C for
according to the above general procedure to provide 56 24 h. At the end of the incubation time, the diameters of
mg of compound 1g as a white solid. Yield: 56.9%; m.p. the inhibition zones formed on the MHA were evaluated
138.1–139.0 °C; UV λmax (CH2Cl2): 207.0; 270.0 nm; IR in millimeters. Discs of chloramphenicol (C30),
νmax (CH2Cl2) cm-1: 3901, 3819, 3567, 3244, 2970, gentamycine (CN10), tetracycline (TE30), erytromycine
2300, 1743, 1593, 1531, 1365, 1276, 1229, 1216, 1158, (E15), ampicillin (AMP10), and nystatine (NS100) were
1082, 958, 832, 749, 662, 529, 474, 464, 457; LC/MS used as positive controls. The measured inhibition
(ESI-MS) m/z = 634.40 (M+1) (positive ion mode).
zones of the study compounds were compared with
those of the reference discs.
4.7. Pharmacology
4.7.1.2. Dilution method
4.7.1. Antibacterial and antifungal activity
Screening for antibacterial activities was carried out
by preparing
a microdilution broth, following the
All of the compounds used in this study were tested procedure outlined in the Manual of Clinical
for their in vitro antibacterial and antifungal effects Microbiology [38]. All the bacteria were inoculated in the
(antimicrobial activity) following standard methods nutrient broth and incubated at 30 °C for 24 h. The
(Supplementary data, Figure 2).
compounds were dissolved in DMSO (2 mg mL-1) and
In this study, twelve bacterial cultures and five then diluted in Mueller Hinton broth. Two-fold serial
different yeasts were used. Eleven bacterial strains and dilutions of the compounds were employed to determine
three yeast strains were obtained from the American the MIC, ranging from 256 to 0.125 µg mL-1. Cultures
Type Culture Collection (ATCC; Rockville, MD, USA). were grown at 30 °C for 18–20 h, and the final inoc ulum
The other bacterial strain, B. thrungiensis, was isolated was approximately 106 cfu mL-1. Test cultures were
from human fecal samples obtained from Adnan incubated at 37 °C for 24 h. The lowest concentrati on of
Menderes University (Clinical Microbiology Laboratory, antimicrobial agent that resulted in complete inhibition
Faculty of Medicine) and was cultured in Nutrient Broth of the microorganisms was represented as the MIC (µg
(Merck) at 30 °C for 24 h. The other two yeast strains,
mL-1). Streptomycin (I.E. Ulagay) was used as a
C. glabrata and C. trophicalis, were cultured in Malt positive control in the dilution method.
Extract Broth (Merck, USA) at 30 °C for 24 h.
10

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Appendix A. Supplementary data
4.7.2. Cell culture
Supplementary data related to this article can be
found at http://
HL-60, HT-29, Caco-2, Saos-2, MCF-7, and HeLa
cell lines were purchased from ATCC. Growth media
and its supplements were purchased from Life Science
Tech. (Sacem, Turkey). Cells were grown in RPMI-
1540, Dulbecco’s modified Eagle’s medium, or McCoys
5A medium supplemented with 10–15% heat
inactivated fetal calf serum, 1% L-glutamine, 1%
penicillin/streptomycin, and nonessential amino acids
and cultivated at 37 °C in a humidified atmosphere
containing 5% CO2. Control chemicals doxorubicin and
paclitaxel were purchased from Cell Signalling Tech.,
Inc. (Beverly, MA, USA).
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12

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Highlights
•
•
•
•
Nine novel gypsogenin derivatives (1a-i) were semisynthesized.
1b, 1d, 1e showed the best antimicrobial activities against Bacillus cereus.
1a 3.9 µM, 1c 5.15 µM, 1d 5.98 µM showed remarkable cytotoxic activity.
Gypsogenin and 1a triggered the apoptotic mechanism at a concentration of 20 µM.

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Figure 1: 1H NMR and 13C NMR, ESI mass spectrum and IR spectra for compound 1a-i.
1H NMR spectrum of compound 1a recorded on a Varian AS-400 spectrometer
(400 MHz, pyridine-d5):

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13C NMR spectrum of compound 1a recorded on a Varian AS-400 spectrometer
(100 MHz, pyridine-d5):

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ESI mass spectrum of compound 1a recorded on an AGILENT 1200 Capillary
spectrometer:
For compound 1a, IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR
spectrometer:

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1H NMR spectrum of compound 1b recorded on a Varian AS-400 spectrometer
(400 MHz, pyridine-d5):

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13C NMR spectrum of compound 1b recorded on a Varian AS-400 spectrometer
(100 MHz, pyridine-d5):

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ESI mass spectrum of compound 1b recorded on an AGILENT 1200 Capillary
spectrometer:
Intens.
x10
1.
-MS, 9.7-12.6min
8
511.2
2.5
2.0
1.5
1.0
0.5
0.0
200
300
400
500
600
m/z
For compound 1b, IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR
spectrometer:

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1H NMR spectrum of compound 1c recorded on a Varian AS-400 spectrometer
(400 MHz, pyridine-d5):

ACCEPTED MANUSCRIPT
13C NMR spectrum of compound 1c recorded on a Varian AS-400 spectrometer
(100 MHz, pyridine-d5):

ACCEPTED MANUSCRIPT
ESI mass spectrum of compound 1c recorded on an AGILENT 1200 Capillary
spectrometer:
For compound 1c, IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR
spectrometer:

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1H NMR spectrum of compound 1d recorded on a Varian AS-400 spectrometer
(400 MHz, pyridine-d5):

ACCEPTED MANUSCRIPT
13C NMR spectrum of compound 1d recorded on a Varian AS-400 spectrometer
(100 MHz, pyridine-d5):

ACCEPTED MANUSCRIPT
ESI mass spectrum of compound 1d recorded on an AGILENT 1200 Capillary
spectrometer:
For compound 1d, IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR
spectrometer:

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1H NMR spectrum of compound 1e recorded on a Varian AS-400 spectrometer
(400 MHz, pyridine-d5):

ACCEPTED MANUSCRIPT
13C NMR spectrum of compound 1e recorded on a Varian AS-400 spectrometer
(100 MHz, pyridine-d5):

ACCEPTED MANUSCRIPT
ESI mass spectrum of compound 1e recorded on an AGILENT 1200 Capillary
spectrometer:
For compound 1e, IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR
spectrometer: