Synthesis, characterization and cytotoxic activity evaluation of ginsengdiol oxidation and nitrogen hybrid derivatives

Article abstract of DOI:10.1039/C8MD00387D

Panaxadiol (PD), a diol-type ginseng saponin, with a dammarane skeleton plays a potential role in the apoptosis of tumor cells. In this study, 28 oxidation and nitrogen hybrid derivatives of PD were synthesized, of which 20 were novel compounds. All the obtained compounds were screened for their cytotoxic activity in six cell lines. As compared with the positive control, some compounds showed better anti-proliferative activities while having much weaker effect on the growth of normal cells. Among them, ring-A fused pyrazoline of PD (1j) displayed impressive cytotoxic activity with IC₅₀ 9.62 ± 1.34, 11.65 ± 1.71, and 13.45 ± 1.60 μM against A549, HeLa and 8901 cell lines, respectively. Additionally, compound 2f exhibited the most potent activity with an IC₅₀ value of 8.93 ± 1.11 μM against cell line A549. Therefore, our results indicated that 1j and 2f can be promising lead candidates for further studies.

Full text of DOI:10.1039/C8MD00387D

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RESEARCH ARTICLE
Synthesis, characterization and cytotoxic activity
evaluation of ginsengdiol oxidation and nitrogen
hybrid derivatives†
Cite this: Med. Chem. Commun.,
2018, 9, 1910
Yumeng Zhang,a Weihui Yuan,a Xude Wang,a Hongyu Zhang,a Yuanyuan Sun,a
ab
ab
*
*
Xiaoshu Zhang
and Yuqing Zhao
Panaxadiol (PD), a diol-type ginseng saponin, with a dammarane skeleton plays a potential role in the apo-
ptosis of tumor cells. In this study, 28 oxidation and nitrogen hybrid derivatives of PD were synthesized, of
which 20 were novel compounds. All the obtained compounds were screened for their cytotoxic activity in
six cell lines. As compared with the positive control, some compounds showed better anti-proliferative ac-
tivities while having much weaker effect on the growth of normal cells. Among them, ring-A fused
pyrazoline of PD (1j) displayed impressive cytotoxic activity with IC50 9.62 1.34, 11.65 1.71, and 13.45
1.60 μM against A549, HeLa and 8901 cell lines, respectively. Additionally, compound 2f exhibited the most
potent activity with an IC50 value of 8.93 1.11 μM against cell line A549. Therefore, our results indicated
that 1j and 2f can be promising lead candidates for further studies.
Received 6th August 2018,
Accepted 14th September 2018
DOI: 10.1039/c8md00387d
rsc.li/medchemcomm
5-fluorouracil16 could significantly increase its anti-tumor ac-
tivity in human cancer cell lines. Moreover, previous studies
Introduction
Ginseng has been traditionally used as a medicine in Eastern
Asia for thousands of years. Ginsenosides are the main bioac-
tive components of Panax ginseng, which own a range of bio-
logical activities including anticancer,1–3 antioxidative,4 im-
munomodulatory,5 analgesic and anti-inflammatory.6,7
Especially regarding cancer chemoprevention, ginsenosides
are considered to be the main bioactive constituents.8 It is
classified into three subclasses: panaxadiol, panaxatriol, and
oleanic acid-type ginsenosides.9 Moreover, recent studies have
found that the protopanaxadiol saponin possessed excellent
anti-tumor activity with low toxicity and side effects.10 In our
previous studies, we found that many panaxadiol derivatives
possessed more potent anti-tumor activities, such as 25-
methoxylprotopanaxadiol and 25-hydroxyprotopanaxadiol.11,12
Panaxadiol (PD), a diol-type ginseng saponin, with a
dammarane skeleton plays a potential role in the apoptosis
of tumor cells.13,14 It has been reported that PD in combina-
tion with anti-cancer drugs such as cyclophosphamide15 and
have shown that PD derivatives (3β-acetoxy-PD, 3β-palmitic
acid aceloxy-PD and 3β-octadecanoic-PD) possessed more po-
tent anti-tumor activity than PD.17 Additionally, synthetic
panaxadiol monoside possesses higher physiological activity
than the original panaxadiol.18 Therefore, PD derivatives as
potential effective anti-tumor agents need to be developed.
In our previous study, some modified ginsenosides with a
series of small molecular compounds were obtained, and
some of them displayed potent or similar cytotoxic activities.
Based on the structure–activity relationship of these deriva-
tives, we concluded that C-3 was an active site of dammarane-
type sapogenins and the hydroxyl substitutions at C-3 were
crucial for biological activity.19,20 Among the natural products,
nitrogen heterocyclic compounds mainly distributed in sea
worms possess outstanding cytotoxic effects.21 It was reported
that pyrazine and benzopyrazine compounds have significant
antitumor effects.22 Interestingly, the azole compounds of
betulinic acid were extremely sensitive to HCT116 cells and
had a low effect on human normal cells.23 Mallavadhani et al.
found that pyrimidine compounds possessed general inhibi-
tory activity in breast cancer cells during the structural modi-
fication of oleanolic acid, and its activity was superior to the
positive drug etoposide.24 Meanwhile, it was reported that the
oxidation and A ring-opening products of betulinic acid
exhibited good activity in inhibiting tumor proliferation,
while the oxidation product of PD and the ring-opening prod-
uct of A ring can inhibit HIV and HCV protease.25,26 There-
fore, we have carried out a series of beneficial synthetic
a Department of Traditional Chinese Medicine Chemical, Shenyang
Pharmaceutical University, Shenyang 110016, China.
E-mail: xiaoshu2397@163.com, zyq4885@126.com; Fax: +86 24 43520300;
Tel: +86 24 43520309
b Key Laboratory of Structure-based Drug Design and Discovery, Ministry of Edu-
cation, Shenyang Pharmaceutical University, Shenyang 110016, China
† Electronic supplementary information (ESI) available: Experimental procedures
and characterization data for 27 compounds, copies of NMR spectra. See DOI:
10.1039/c8md00387d
1910 | Med. Chem. Commun., 2018, 9, 1910–1919
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reactions on the available PD in order to improve its anti-
tumor activity.
For HeLa, compounds 1j, 1k, 2, and 3b exhibited better ac-
tivity with IC50 values of 11.65–19.60 μM, wherein compound
1j showed an anti-proliferative activity which is 2.5-fold that
of 5-fluorouracil. In addition, most of the compounds showed
low toxicity in IOSE144 cell line.
Results and discussion
In this study, 28 oxidation and ring-A fused nitrogen
heterocycle derivatives of PD were designed, synthesized, and
evaluated for their anti-tumor activity. Some of these com-
pounds displayed potent or similar cytotoxic activity in vitro
compared with PD and 5-fluorouracil. Interestingly, among
the synthesized compounds, the pyrazoline compound 1j
showed much higher activity than the other derivatives with
IC50 values of 9.62 1.34, 11.65 1.71, and 13.45 1.60 μM
against A549, HeLa, and 8901 cell lines, respectively. We
speculated that it was related with its special ring-A fused
pyrazoline structure. Furthermore, according to the SAR anal-
ysis, the pyrazine compound (2f) with C (17)-fused chain frag-
ment owned excellent antiproliferative activity with IC50 8.93–
17.73 μM than C (17)-fused cycle fragment (1d). Moreover,
the effect of compound 2f was greatly improved compared to
2g, which may be due to its C-12 position being attached to
the hydroxyl group instead of the carbonyl group. In addi-
tion, compared with the parent compound PD, the activity of
1k underwent beneficial changes due to its C-2 position
linked with the cyano group.
Screening for cytotoxicity of the tested compounds (Table 1)
in U87 cell line revealed that compounds 1a, 3, and 3d were
more potent than the positive control. Compound 3 showed
stronger anti-proliferative activity with IC50 = 19.51
1.00
μM. In addition, it exhibited more potent anti-cancer activi-
ties than panaxadiol, similar to the results of previous stud-
ies.12 The values obtained for other compounds showed weak
activity.
Data on the MCF-7 cell line showed similar results. Com-
pounds 1a, 1j, and 2 showed superior anti-tumor activity
than 5-fluorouracil (29.4
0.70 μM) and the parent com-
pound PD (52.62 2.39 μM) with IC50 values of 17.73–23.58
μM. IC50 values of other compounds were greater than 50
μM.
For 8901 and A549, compounds 1j, 1k, and 2f displayed
excellent anti-proliferative activity than 5-fluorouracil (23.5–
33.27 μM), with IC50 values of 8.93–13.45 μM. In particular,
compound 2f (8.93 1.11 μM) possessed excellent activity,
which was 2.6-fold that of the positive control 5-fluorouracil.
Table 1 Anti-proliferative activity (IC50 SD values, μM, 48 h) of compounds in different human tumor cell lines
IC50 (μM)
No.
Compounds
U87
MCF-7
8901
A549
HeLa
IOSE144
1
2
3
4
5
6
7
8
1
1a
89.99 4.01
20.17 1.01
>100
76.43 3.23
>100
>100
>100
>100
30.55 1.23
>100
26.23 2.69
41.60 1.19
82.64 3.14
86.34 2.77
25.06 1.40
>100
>100
>100
45.39 2.48
>100
38.09 2.09
>100
78.06 2.87
23.58 2.34
>100
91.90 7.72
>100
>100
>100
>100
28.31 2.03
>100
18.90 1.42
29.65 3.34
27.95 1.60
82.95 4.74
49.58 5.42
65.10 2.87
>100
66.62 3.64
46.22 4.05
>100
17.73 1.82
39.10 2.11
44.59 2.14
98.51 6.63
44.90 2.89
50.34 2.94
35.69 1.25
>100
>100
50.11 3.11
>100
>100
>100
>100
>100
>100
30.12 2.52
>100
13.45 1.60
25.68 1.28
45.69 3.09
>100
46.47 2.72
>100
66.48 3.57
>100
87.83 5.61
>100
13.15 2.11
>100
38.53 1.90
>100
81.32 3.12
78.60 2.87
25.91 3.33
>100
>100
39.99 2.88
>100
86.36 3.97
>100
>100
66.43 3.56
25.03 1.98
>100
>100
>100
>100
>100
>100
31.92 3.00
>100
11.65 1.71
12.69 2.56
69.39 3.35
>100
19.60 2.23
>100
>100
>100
50.92 2.68
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
34.80 2.23
1b
1cb
1db
1e
1fb
1gb
1hb
1ib
1jb
1kb
1mb
1nb
2
>100
>100
9
18.47 2.67
67.35 4.55
9.62 1.34
12.93 1.67
26.89 2.99
>100
27.91 2.14
89.98 4.29
50.57 2.72
25.74 3.54
24.31 3.88
>100
8.93 1.11
19.70 2.51
29.28 2.28
>100
37.12 6.59
39.94 1.11
21.57 1.20
85.00 2.83
44.57 3.01
23.57 1.80
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2a
2bb
2c
2db
2eb
2fb
2gb
3
>100
29.36 3.06
>100
18.03 1.99
>100
39.00 2.79
>100
19.51 1.00
>100
32.74 1.38
>100
20.71 1.58
>100
>100
3ab
3bb
3cb
3db
3eb
PD
5-Fua
52.62 2.39
29.41 0.70
>100
33.27 1.37
>100
28.22 1.91
22.00 0.98
a
Positive control. b Novel compound.
This journal is © The Royal Society of Chemistry 2018
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Since few studies on oxidation and nitrogen hybrid or SAR
in tetracyclic triterpene dammarane and as PD in previous
study showed very low cytotoxicity against human normal
cells, this study may provide useful data for researching and
developing the study on structure–activity relationship of
anticancer agents.
(3H, s, CH3), 1.05 (3H, s, CH3), 1.04 (3H, s, CH3), 0.99 (3H, s,
CH3), 0.91 (3H, s, CH3); 13C-NMR (150 MHz, CDCl3): δ 39.86
(C-1), 34.18 (C-2), 217.91 (C-3), 47.51 (C-4), 55.44 (C-5), 18.90
(C-6), 34.82 (C-7), 39.77 (C-8), 49.56 (C-9), 36.92 (C-10), 31.11
(C-11), 71.34 (C-12), 48.70 (C-13), 51.80 (C-14), 31.74 (C-15),
26.85 (C-16), 49.97 (C-17), 15.50 (C-18), 16.09 (C-19), 74.55 (C-
20), 22.11 (C-21), 42.81 (C-22), 19.78 (C-23), 46.67 (C-24),
70.98 (C-25), 32.52 (C-26), 32.72 (C-27), 26.43 (C-28), 21.16 (C-
29), 17.19 (C-30).
Experimental section
General
20IJR)-20,25-Epoxy-[2,3-b]-pyrazine-damran-12-ol
(1d).
20IJR)-20,25-Epoxy-12β-hydroxydammaran-3-one (1). Yield:
85.4%, Mp: 228–230 °C, [α]D20 + 22.5 (0.10, MeOH); ESI-MS: m/
Yield: 68.0%, Mp: 257–259 °C, HR-ESI-TOF-MS: m/z 495.3948
[M + H]+ (calcd for C32H51N2O2, 495.3945); 1H-NMR (400
MHz, CDCl3): δ 8.43 (1H, s, N–CH), 8.32 (1H, s, N–CH),
3.60 (1H, td, J = 10.3, 5.0 Hz, H-12), 3.14 (1H, d, J = 16.7 Hz,
H-1), 2.54 (1H, d, J = 16.8 Hz, H-1), 1.32 (6H, s, 2 × CH3), 1.28
(3H, s, CH3), 1.24 (3H, s, CH3), 1.20 (3H, s, CH3), 1.06 (3H, s,
CH3), 0.93 (3H, s, CH3), 0.87 (3H, s, CH3); 13C-NMR (100
MHz, CDCl3): δ 48.80 (C-1), 150.81 (C-2), 159.89 (C-3), 39.80
(C-4), 54.86 (C-5), 20.26 (C-6), 34.18 (C-7), 39.74 (C-8), 49.46
(C-9), 36.86 (C-10), 31.10 (C-11), 69.90 (C-12), 48.36 (C-13),
51.43 (C-14), 31.28 (C-15), 25.32 (C-16), 53.60 (C-17), 15.30 (C-
18), 16.29 (C-19), 76.80 (C-20), 19.59 (C-21), 35.92 (C-22),
16.44 (C-23), 36.63 (C-24), 73.31 (C-25), 33.21 (C-26), 27.30 (C-
27), 31.71 (C-28), 24.15 (C-29), 17.15 (C-30), 141.38 (N–CH),
142.47 (N–CH).
1
z 459.4 [M + H]+; H-NMR (400 MHz, CDCl3): δ 3.55 (1H, td, J
= 10.3, 5.0 Hz, H-12), 1.27 (3H, s, CH3), 1.22 (3H, s, CH3),
1.19 (3H, s, CH3), 1.08 (3H, s, CH3), 1.04 (3H, s, CH3), 1.02
(3H, s, CH3), 0.98 (3H, s, CH3), 0.89 (3H, s, CH3); 13C-NMR
(100 MHz, CDCl3): δ 39.80 (C-1), 34.28 (C-2), 218.24 (C-3),
47.51 (C-4), 55.44 (C-5), 19.53 (C-6), 34.32 (C-7), 39.83 (C-8),
49.36 (C-9), 36.93 (C-10), 31.02 (C-11), 69.97 (C-12), 49.36 (C-
13), 51.34 (C-14), 31.26 (C-15), 25.26 (C-16), 54.78 (C-17),
15.47 (C-18), 16.07 (C-19), 77.16 (C-20), 19.80 (C-21), 35.84 (C-
22), 16.38 (C-23), 36.55 (C-24), 73.33 (C-25), 33.14 (C-26),
27.24 (C-27), 26.80 (C-28), 21.15 (C-29), 17.09 (C-30).
20IJR)-20,25-Epoxy-2,12β-dihydroxydammaran-1-en-3-one
(1a). Yield: 93.6%, Mp: 195–198 °C, [α]2D0 + 23.0 (0.10, MeOH);
ESI-MS: m/z 473.4 [M + H]+; 1H-NMR (400 MHz, CDCl3): δ
6.52 (1H, s, H-1), 3.56 (1H, td, J = 10.4, 5.0 Hz, H-12), 1.27
(3H, s, CH3), 1.23 (3H, s, CH3), 1.20 (3H, s, CH3), 1.19 (3H, s,
CH3), 1.18 (3H, s, CH3), 1.12 (3H, s, CH3), 1.04 (3H, s, CH3),
0.88 (3H, s, CH3); 13C-NMR (100 MHz, CDCl3): δ 129.02 (C-1),
144.03 (C-2), 201.29 (C-3), 44.18 (C-4), 54.72 (C-5), 19.51 (C-6),
34.52 (C-7), 40.67 (C-8), 45.16 (C-9), 38.73 (C-10), 30.62 (C-11),
69.72 (C-12), 49.40 (C-13), 51.38 (C-14), 31.11 (C-15), 25.23 (C-
16), 54.61 (C-17), 16.18 (C-18), 18.89 (C-19), 77.16 (C-20),
20.34 (C-21), 35.83 (C-22), 16.37 (C-23), 36.55 (C-24), 73.35 (C-
25), 33.16 (C-26), 27.30 (C-27), 27.28 (C-28), 21.71 (C-29),
17.05 (C-30).
20IJR)-20,25-Epoxy-[2,3-b]-indol-12β-dammaranol
(1e).
Yield: 39.3%, Mp: 206–208 °C. ESI-MS: m/z 530.1 [M − H]−;
1H-NMR (400 MHz, pyridine-d5): δ 11.67 (1H, s, -NH), 7.75
(1H, m, Ar–H), 7.49 (1H, m, Ar–H), 7.30 (1H, m, Ar–H), 7.28
(1H, m, Ar–H), 3.89 (1H, td, J = 10.1, 5.0 Hz, H-12), 3.12 (1H,
d, J = 14.9 Hz, H-1), 2.38 (1H, d, J = 14.8 Hz, H-1), 1.51 (3H, s,
CH3), 1.38 (3H, s, CH3), 1.33 (3H, s, CH3), 1.27 (3H, s, CH3),
1.25 (3H, s, CH3), 1.11 (3H, s, CH3), 1.01 (3H, s, CH3), 0.95
(3H, s, CH3); 13C-NMR (100 MHz, pyridine-d5): δ 38.54 (C-1),
121.34 (C-2), 142.62 (C-3), 38.88 (C-4), 55.30 (C-5), 20.08 (C-6),
34.95 (C-7), 40.47 (C-8), 49.50 (C-9), 36.97 (C-10), 31.50 (C-11),
70.66 (C-12), 50.42 (C-13), 51.85 (C-14), 31.76 (C-15), 25.80 (C-
16), 54.75 (C-17), 15.95 (C-18), 16.93 (C-19), 77.30 (C-20),
20.05 (C-21), 35.20 (C-22), 17.01 (C-23), 36.24 (C-24), 73.43 (C-
25), 33.54 (C-26), 27.79 (C-27), 32.18 (C-28), 23.78 (C-29),
17.64 (C-30), 138.18 (Ar–C), 129.47 (Ar–C), 119.20 (Ar–C),
118.79 (Ar–C), 111.61 (Ar–C), 106.83 (Ar–C).
20IJR)-20,25-Epoxy-2,3-seco-12β-hydroxydammaran-2,3-dioic
acid (1b). Yield: 40.3%, Mp: 219–221 °C, [α]2D0 + 17.2 (0.10,
1
MeOH); ESI-MS: m/z 505.2 [M − H]−; H-NMR (400 MHz, pyri-
dine-d5): δ 3.90 (1H, td, J = 10.1, 4.8 Hz, H-12), 1.61 (3H, s,
CH3), 1.55 (3H, s, CH3), 1.25 (3H, s, CH3), 1.23 (3H, s, CH3),
1.21 (3H, s, CH3), 1.17 (3H, s, CH3), 1.10 (3H, s, CH3), 1.04
(3H, s, CH3); 13C-NMR (100 MHz, pyridine-d5): δ 43.44 (C-1),
174.83 (C-2), 182.72 (C-3), 47.47 (C-4), 49.17 (C-5), 20.05 (C-6),
34.94 (C-7), 40.31 (C-8), 41.99 (C-9), 42.55 (C-10), 31.10 (C-11),
70.60 (C-12), 50.46 (C-13), 52.23 (C-14), 32.60 (C-15), 25.80 (C-
16), 55.15 (C-17), 16.07 (C-18), 22.17 (C-19), 77.22 (C-20),
20.83 (C-21), 36.10 (C-22), 16.83 (C-23), 36.86 (C-24), 73.32 (C-
25), 33.51 (C-26), 27.74 (C-27), 28.05 (C-28), 25.32 (C-29),
17.78 (C-30).
20IJR)-20,25-Epoxy-[2,3-b]-quinoxaline-dammaran-12β-ol (1f).
Yield: 15.0%, Mp: 228–230 °C, HR-ESI-TOF-MS: m/z 459.3829
[M + H]+ (calcd for C30H51O3, 459.3933); 1H-NMR (600 MHz,
CDCl3): δ 3.54 (1H, td, J = 10.4, 5.1 Hz, H-12), 2.43 (1H, dd, J
= 12.6, 1.9 Hz, H-1), 2.26 (1H, d, J = 13.3 Hz, H-3), 2.15 (1H,
dd, J = 13.3, 1.9 Hz, H-3), 1.98 (1H, d, J = 12.8 Hz, H-1), 1.26
(3H, s, CH3), 1.21 (3H, s, CH3), 1.18 (3H, s, CH3), 1.04 (3H, s,
CH3), 0.99 (3H, s, CH3), 0.91 (3H, s, CH3), 0.89 (3H, s, CH3),
0.88 (3H, s, CH3); 13C-NMR (150 MHz, CDCl3): δ 56.41 (C-1),
212.17 (C-2), 56.68 (C-3), 39.24 (C-4), 56.34 (C-5), 19.20 (C-6),
34.55 (C-7), 40.35 (C-8), 49.78 (C-9), 42.95 (C-10), 30.75 (C-11),
69.82 (C-12), 49.22 (C-13), 51.39 (C-14), 31.27 (C-15), 25.27 (C-
16), 54.82 (C-17), 15.43 (C-18), 17.40 (C-19), 76.80 (C-20),
19.54 (C-21), 35.88 (C-22), 16.40 (C-23), 36.59 (C-24), 73.33 (C-
20IJR)-12,25-Epoxy-20-hydroxydammaran-3-one (1c). Yield:
10.2%, Mp: 196–199 °C, [α]2D0 + 34.0 (0.07, MeOH); HR-ESI-
TOF-MS: m/z 481.3656 [M + Na]+ (calcd for C30H50NaO3,
1
481.3760); H-NMR (600 MHz, CDCl3): δ 3.65 (1H, td, J = 10.3,
5.0 Hz, H-12), 1.59 (6H, s, 2 × CH3), 1.18 (3H, s, CH3), 1.09
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25), 33.51 (C-26), 27.25 (C-27), 33.17 (C-28), 23.18 (C-29),
17.18 (C-30).
16), 54.81 (C-17), 15.36 (C-18), 16.03 (C-19), 76.84 (C-20),
19.58 (C-21), 36.62 (C-22), 16.43 (C-23), 36.94 (C-24), 73.28 (C-
25), 33.18 (C-26), 27.28 (C-27), 31.13 (C-28), 24.07 (C-29),
17.16 (C-30), 132.74 (NC).
20IJR)-20,25-Epoxy-12β-hydroxy-dammar-3-one oxime (1g).
Yield: 83.3%, Mp: 278–280 °C, HR-ESI-TOF-MS: m/z 474.3913
1
[M + H]+ (calcd for C30H51NO3, 474.3942); H-NMR (400 MHz,
20IJR)-20,25-Epoxy-2-cyano-2-ene-dammarane-3,12β-diol
(1k). Yield: 85.0%, Mp: 236–238 °C, HR-ESI-TOF-MS: m/z
484.3824 [M + H]+ (calcd for C31H50NO3, 484.3785); 1H-NMR
(600 MHz, pyridine-d5): δ 3.76 (1H, m, H-12), 2.32 (1H, d, J =
15.1 Hz, H-1), 1.93 (1H, d, J = 15.1 Hz, H-1), 1.34 (3H, s,
CH3), 1.29 (3H, s, CH3), 1.24 (3H, s, CH3), 1.23 (3H, s, CH3),
1.21 (3H, s, CH3), 0.99 (3H, s, CH3), 0.90 (3H, s, CH3), 0.89
(3H, s, CH3); 13C-NMR (150 MHz, pyridine-d5): δ 42.84 (C-1),
80.12 (C-2), 172.90 (C-3), 39.51 (C-4), 53.35 (C-5), 20.01 (C-6),
34.57 (C-7), 40.12 (C-8), 48.80 (C-9), 36.55 (C-10), 31.93 (C-11),
70.38 (C-12), 50.06 (C-13), 51.40 (C-14), 31.65 (C-15), 25.72 (C-
16), 55.25 (C-17), 15.74 (C-18), 16.12 (C-19), 77.25 (C-20),
20.18 (C-21), 36.21 (C-22), 16.90 (C-23), 36.94 (C-24), 73.43 (C-
25), 33.51 (C-26), 27.73 (C-27), 28.41 (C-28), 20.40 (C-29),
17.51 (C-30), 121.57 (–CN).
CDCl3): δ 3.55 (1H, td, J = 10.5, 5.1 Hz, H-12), 3.00 (1H, m, H-
2), 2.40 (1H, m, H-2), 1.26 (3H, s, CH3), 1.21 (3H, s, CH3),
1.20 (3H, s, CH3), 1.18 (3H, s, CH3), 1.11 (3H, s, CH3), 1.00
(3H, s, CH3), 0.97 (3H, s, CH3), 0.86 (3H, s, CH3); 13C-NMR
(100 MHz, CDCl3): δ 38.90 (C-1), 27.53 (C-2), 169.38 (C-3),
39.95 (C-4), 56.06 (C-5), 18.02 (C-6), 34.55 (C-7), 40.65 (C-8),
49.59 (C-9), 37.31 (C-10), 30.95 (C-11), 69.96 (C-12), 49.38 (C-
13), 51.35 (C-14), 31.25 (C-15), 25.29 (C-16), 54.83 (C-17),
15.65 (C-18), 15.95 (C-19), 77.16 (C-20), 19.56 (C-21), 35.89 (C-
22), 16.42 (C-23), 36.60 (C-24), 73.30 (C-25), 33.17 (C-26),
27.29 (C-27), 22.95 (C-28), 19.25 (C-29), 17.11 (C-30).
20IJR)-2-Formyl-20,25-epoxy-12β-hydroxy-dammar-3-one
(1h). Yield: 67.9%, Mp: 237–240 °C, ESI-MS: m/z 485.1 [M −
H]−; 1H-NMR (400 MHz, CDCl3): δ 14.87 (1H, d, J = 2.3 Hz,
CHO), 8.59 (1H, d, J = 1.6 Hz, H-2), 3.57 (1H, td, J = 10.3, 5.0
Hz, H-12), 1.27 (3H, s, CH3), 1.23 (3H, s, CH3), 1.20 (3H, s,
CH3), 1.18 (3H, s, CH3), 1.11 (3H, s, CH3), 1.02 (3H, s, CH3),
0.90 (3H, s, CH3), 0.88 (3H, s, CH3); 13C-NMR (100 MHz,
CDCl3): δ 40.31 (C-1), 105.85 (C-2), 190.14 (C-3), 35.86 (C-4),
52.60 (C-5), 19.54 (C-6), 34.08 (C-7), 39.94 (C-8), 48.24 (C-9),
39.68 (C-10), 31.02 (C-11), 69.98 (C-12), 49.34 (C-13), 51.42 (C-
14), 31.26 (C-15), 25.26 (C-16), 54.74 (C-17), 15.23 (C-18),
15.36 (C-19), 76.80 (C-20), 19.62 (C-21), 36.53 (C-22), 16.38 (C-
23), 36.57 (C-24), 73.36 (C-25), 33.11 (C-26), 27.26 (C-27),
28.41 (C-28), 20.76 (C-29), 17.10 (C-30), 189.24 (CHO).
20IJR)-20,25-Epoxy-2′-amino-[6,5-b]-pyrimidine-12β-
dammaranol (1m). Yield: 61.2%, Mp: 270–272 °C, HR-ESI-
TOF-MS: m/z 510.4052 [M + H]+ (calcd for C32H52N3O2,
1
510.4054); H-NMR (400 MHz, pyridine-d5): δ 8.16 (1H, s, –N
CH), 6.97 (2H, s, NH2), 3.82 (1H, m, H-12), 2.63 (1H, d, J =
15.1 Hz, H-1), 2.07 (1H, d, J = 14.5 Hz, H-1), 1.42 (3H, s,
CH3), 1.33 (3H, s, CH3), 1.31 (3H, s, CH3), 1.26 (3H, s, CH3),
1.25 (3H, s, CH3), 1.05 (3H, s, CH3), 0.92 (3H, s, CH3), 0.80
(3H, s, CH3); 13C-NMR (100 MHz, pyridine-d5): δ 42.43 (C-1),
116.77 (C-2), 173.17 (C-3), 39.96 (C-4), 54.12 (C-5), 20.82 (C-6),
34.77 (C-7), 40.21 (C-8), 48.84 (C-9), 36.95 (C-10), 31.48 (C-11),
70.53 (C-12), 50.36 (C-13), 51.79 (C-14), 31.68 (C-15), 25.76 (C-
16), 55.28 (C-17), 15.78 (C-18), 15.97 (C-19), 77.28 (C-20),
20.04 (C-21), 36.22 (C-22), 16.91 (C-23), 36.80 (C-24), 73.43 (C-
25), 33.56 (C-26), 27.74 (C-27), 32.05 (C-28), 24.06 (C-29),
17.57 (C-30), 164.42 (–CN), 160.12 (–NCH).
20IJR)-20,25-Epoxy-2′-methyl-[6,5-b]-pyrimidine-12β-
dammarol (1n). Yield: 18.0%, Mp: 291–293 °C, HR-ESI-TOF-
MS: m/z 509.4107 [M + H]+ (calcd for C33H53N2O2, 509.4102);
1H-NMR (600 MHz, pyridine-d5): δ 8.39 (1H, s, –NCH), 3.84
(1H, td, J = 9.7, 5.0 Hz, H-12), 2.72 (1H, d, J = 15.7 Hz, H-1),
2.15 (1H, d, J = 15.4 Hz, H-1), 2.80 (3H, s, CH3), 1.44 (3H, s,
CH3), 1.36 (3H, s, CH3), 1.31 (3H, s, CH3), 1.26 (3H, s, CH3),
1.25 (3H, s, CH3), 1.05 (3H, s, CH3), 0.93 (3H, s, CH3), 0.77
(3H, s, CH3); 13C-NMR (150 MHz, pyridine-d5): δ 42.53 (C-1),
124.72 (C-2), 172.38 (C-3), 40.00 (C-4), 53.95 (C-5), 20.05 (C-6),
34.69 (C-7), 40.23 (C-8), 48.81 (C-9), 36.60 (C-10), 31.50 (C-11),
70.49 (C-12), 50.36 (C-13), 51.80 (C-14), 32.07 (C-15), 25.76 (C-
16), 55.29 (C-17), 15.73 (C-18), 16.09 (C-19), 77.30 (C-20),
20.72 (C-21), 36.22 (C-22), 16.92 (C-23), 36.96 (C-24), 73.46 (C-
25), 33.58 (C-26), 27.75 (C-27), 31.68 (C-28), 24.20 (C-29),
17.56 (C-30), 166.30 (–CN), 158.89 (–NCH), 26.47 (-CH3).
20IJR)-Dammarane-3β,12β,20,25-tetrol (25-OH-PPD) (2).
Mp: 252–254 °C, HR-ESI-TOF: m/z 479.4022 [M + H]+ (calcd
for C30H55O4, 479.4056); 1H-NMR (300 MHz, CDCl3): δ 3.15
(1H, dd, H-3), 3.58 (1H, td, H-12), 1.24 (6H, s, 2 × CH3), 1.15
(3H, s, CH3), 1.00 (3H, s, CH3), 0.98 (3H, s, CH3), 0.90 (6H, s,
20IJR)-20,25-Epoxy-[5,4-b]-isoxazole-12β-dammaranol (1i).
Yield: 70.4%, Mp: 237–239 °C, HR-ESI-TOF-MS: m/z 484.3779
1
[M + H]+ (calcd for C31H50NO3, 484.3785); H-NMR (400 MHz,
CDCl3): δ 7.99 (1H, s, HCN), 3.57 (1H, td, J = 10.3, 5.0 Hz,
H-12), 2.52 (1H, d, J = 15.1 Hz, H-1), 2.01 (1H, d, J = 15.1 Hz,
H-1), 1.30 (3H, s, CH3), 1.27 (3H, s, CH3), 1.23 (3H, s, CH3),
1.21 (3H, s, CH3), 1.20 (3H, s, CH3), 1.03 (3H, s, CH3), 0.90
(3H, s, CH3), 0.86 (3H, s, CH3); 13C-NMR (100 MHz, CDCl3): δ
36.23 (C-1), 109.23 (C-2), 173.04 (C-3), 35.00 (C-4), 54.15 (C-5),
18.96 (C-6), 34.19 (C-7), 39.98 (C-8), 48.73 (C-9), 39.00 (C-10),
31.11 (C-11), 70.00 (C-12), 49.33 (C-13), 51.48 (C-14), 31.47 (C-
15), 25.28 (C-16), 54.88 (C-17), 15.36 (C-18), 16.20 (C-19),
76.80 (C-20), 19.58 (C-21), 35.88 (C-22), 16.40 (C-23), 36.58 (C-
24), 73.33 (C-25), 33.16 (C-26), 27.28 (C-27), 28.87 (C-28),
21.75 (C-29), 17.12 (C-30), 150.54 (HCN).
20IJR)-20,25-Epoxy-[5,4-b]-pyrazole-12β-dammaranol
(1j).
Yield: 80.5%, Mp: 291–293 °C, HR-ESI-TOF-MS: m/z 505.3745
[M + Na]+ (calcd for C31H50N2NaO2, 505.3764); 1H-NMR (400
MHz, CDCl3): δ 3.57 (1H, td, J = 10.2, 4.9 Hz, H-12), 2.70 (1H,
d, J1,2 = 14.9 Hz, H-1), 2.05 (1H, d, J = 14.8 Hz, H-1), 1.30 (3H,
s, CH3), 1.27 (3H, s, CH3), 1.23 (3H, s, CH3), 1.21 (3H, s,
CH3), 1.20 (3H, s, CH3), 1.04 (3H, s, CH3), 0.91 (3H, s, CH3),
0.85 (3H, s, CH3); 13C-NMR (100 MHz, CDCl3): δ 33.73 (C-1),
112.96 (C-2), 150.09 (C-3), 35.91 (C-4), 54.00 (C-5), 18.96 (C-6),
34.28 (C-7), 39.91 (C-8), 48.73 (C-9), 38.62 (C-10), 31.13 (C-11),
70.03 (C-12), 49.43 (C-13), 51.45 (C-14), 31.38 (C-15), 25.32 (C-
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2 × CH3), 0.78 (3H, s, CH3); 13C-NMR (75 MHz, pyridine-d5): δ
40.3 (C-1), 28.0 (C-2), 79.5 (C-3), 40.0 (C-4), 57.3 (C-5), 18.9 (C-
6), 35.9 (C-7), 40.9 (C-8), 50.9 (C-9), 38.2 (C-10), 32.0 (C-11),
71.9 (C-12), 49.5 (C-13), 52.6 (C-14), 32.0 (C-15), 27.1 (C-16),
51.3 (C-17), 16.3 (C-18), 16.8 (C-19), 74.7 (C-20), 22.4 (C-21),
44.0 (C-22), 19.4 (C-23), 45.4 (C-24), 71.5 (C-25), 29.4 (C-26),
29.1 (C-27), 28.6 (C-28), 16.2 (C-29), 17.4 (C-30).
(C-3), 46.15 (C-4), 54.98 (C-5), 19.11 (C-6), 35.01 (C-7), 41.06
(C-8), 45.26 (C-9), 39.08 (C-10), 32.56 (C-11), 70.93 (C-12),
49.88 (C-13), 52.16 (C-14), 31.65 (C-15), 26.99 (C-16), 51.22 (C-
17), 16.54 (C-18), 23.08 (C-19), 73.79 (C-20), 22.29 (C-21),
44.42 (C-22), 19.62 (C-23), 46.00 (C-24), 70.08 (C-25), 30.32 (C-
26), 30.63 (C-27), 28.27 (C-28), 21.14 (C-29), 17.57 (C-30).
20IJR)-2,3-Seco-12β,20,25-trihydroxydammaran-2,3-dioc
acid (2e). Yield: 32.1%, Mp: 154–157 °C, [α]2D0 + 18.0 (0.07,
MeOH); HR-ESI-TOF-MS: m/z 523.3631 [M-H]− (calcd for
C30H51O7, 523.3635); 1H-NMR (600 MHz, pyridine-d5): δ 4.04
(1H, td, J = 8.6, 3.1 Hz, H-12), 1.62 (3H, s, CH3), 1.56 (3H, s,
CH3), 1.43 (3H, s, CH3), 1.42 (3H, s, CH3), 1.16 (3H, s, CH3),
1.14 (3H, s, CH3), 1.05 (3H, s, CH3); 13C-NMR (150 MHz, pyri-
dine-d5): δ 43.55 (C-1), 174.76 (C-2), 182.78 (C-3), 47.53 (C-4),
49.19 (C-5), 19.13 (C-6), 34.95 (C-7), 40.31 (C-8), 42.62 (C-9),
42.44 (C-10), 30.89 (C-11), 71.24 (C-12), 49.76 (C-13), 52.78 (C-
14), 31.87 (C-15), 27.15 (C-16), 51.07 (C-17), 16.08 (C-18),
20.93 (C-19), 73.53 (C-20), 23.31 (C-21), 44.34 (C-22), 19.62 (C-
23), 46.01 (C-24), 70.10 (C-25), 30.57 (C-26), 30.33 (C-27),
28.05 (C-28), 25.37 (C-29), 17.90 (C-30).
20IJR)-20,25-Dihydroxydammaran-3,12-dione (2a). Yield:
26.1%, Mp: 115–118 °C, [α]2D0 + 32.0 (0.09, MeOH); HR-ESI-
TOF-MS: m/z 497.3602 [M + Na]+ (calcd for C30H50NaO4,
1
497.3607); H-NMR (400 MHz, CDCl3): δ 1.22 (6H, s, 2 × CH3),
1.21 (3H, s, CH3), 1.09 (3H, s, CH3), 1.06 (3H, s, CH3), 1.03
(3H, s, CH3), 1.00 (3H, s, CH3), 0.81 (3H, s, CH3); 13C-NMR
(100 MHz, CDCl3): δ 39.10 (C-1), 33.28 (C-2), 217.01 (C-3),
47.52 (C-4), 55.24 (C-5), 18.07 (C-6), 33.90 (C-7), 40.15 (C-8),
52.65 (C-9), 37.20 (C-10), 39.17 (C-11), 214.47 (C-12), 56.71 (C-
13), 54.61 (C-14), 30.89 (C-15), 25.19 (C-16), 43.14 (C-17),
15.61 (C-18), 15.61 (C-19), 73.70 (C-20), 21.92 (C-21), 44.11 (C-
22), 19.76 (C-23), 44.49 (C-24), 71.25 (C-25), 29.48 (C-26),
29.36 (C-27), 26.64 (C-28), 21.19 (C-29), 17.50 (C-30).
20IJR)-3β,20,25-Trihydroxydammaran-12-one (2b). Yield:
22.6%, Mp: 166–169 °C, [α]2D0 + 18.7 (0.07, MeOH); HR-ESI-
TOF-MS: m/z 499.3776 [M + Na]+ (calcd for C30H52NaO4,
20IJR)-[2,3-b]-Pyrazine-dammarane-12β,20,25-triol
(2f).
Yield: 60.0%, Mp: 229–231 °C, HR-ESI-TOF-MS: m/z 513.4026
[M + H]+ (calcd for C32H53N2O3, 513.4051); 1H-NMR (600
MHz, CDCl3): δ 8.42 (1H, d, J = 1.8 Hz, N–CH), 8.26 (1H, d,
J = 2.4 Hz, N–CH), 3.67 (1H, td, J = 10.4, 5.0 Hz, H-12), 3.10
(1H, d, J = 16.5 Hz, H-1), 2.54 (1H, d, J = 15.1 Hz, H-1), 1.31
(3H, s, CH3), 1.30 (3H, s, CH3), 1.24 (3H, s, CH3), 1.23 (3H, s,
CH3), 1.14 (3H, s, CH3), 1.07 (3H, s, CH3), 0.94 (3H, s, CH3),
0.86 (3H, s, CH3); 13C-NMR (150 MHz, CDCl3): δ 48.91 (C-1),
150.60 (C-2), 159.78 (C-3), 39.72 (C-4), 53.58 (C-5), 20.23 (C-6),
34.09 (C-7), 39.70 (C-8), 48.51 (C-9), 36.78 (C-10), 31.97 (C-11),
70.71 (C-12), 48.60 (C-13), 51.85 (C-14), 31.17 (C-15), 26.51 (C-
16), 50.41 (C-17), 15.29 (C-18), 16.3 (C-19), 74.14 (C-20), 21.92
(C-21), 43.11 (C-22), 18.00 (C-23), 44.52 (C-24), 71.12 (C-25),
29.56 (C-26), 29.53 (C-27), 31.70 (C-28), 24.15 (C-29), 17.21 (C-
30), 141.56 (N–CH), 142.62 (N–CH).
20IJR)-20,25-Dihydroxy-[2,3-b]-pyrazine-dammar-12-one
(2g). Yield: 69.4%, Mp: 229–231 °C, HR-ESI-TOF-MS: m/z
533.3701 [M + Na]+ (calcd for C32H50N2NaO3, 533.3714); 1H-
NMR (400 MHz, CDCl3): δ 8.43 (1H, d, J = 1.8 Hz, N–CH),
8.29 (1H, d, J = 2.3 Hz, N–CH), 1.32 (6H, s, 2 × CH3), 1.25
(3H, s, CH3), 1.22 (6H, s, 2 × CH3), 1.01 (3H, s, CH3), 0.90
(3H, s, CH3), 0.85 (3H, s, CH3); 13C-NMR (100 MHz, CDCl3): δ
48.21 (C-1), 149.83 (C-2), 159.31 (C-3), 39.70 (C-4), 54.73 (C-5),
20.24 (C-6), 33.10 (C-7), 40.10 (C-8), 51.72 (C-9), 37.09 (C-10),
39.14 (C-11), 214.10 (C-12), 56.66 (C-13), 53.34 (C-14), 31.00
(C-15), 25.22 (C-16), 43.18 (C-17), 15.32 (C-18), 15.93 (C-19),
73.62 (C-20), 22.03 (C-21), 44.10 (C-22), 18.13 (C-23), 44.57 (C-
24), 71.20 (C-25), 29.54 (C-26), 29.36 (C-27), 31.65 (C-28),
24.15 (C-29), 17.49 (C-30), 141.73 (N–CH), 142.72 (N–CH).
20IJR)-25-Methoxy-dammarane-3β,12β,20-triol (3). Mp: 232–
234 °C, [α]2D0 + 21.0 (0.5, MeOH); HR-ESI-TOF: m/z 493.4168
[M + H]+ (calcd for C31H56O4, 499.3763); 1H-NMR (300 Hz,
CDCl3): δ 3.20 (1H, dd, H-3), 3.58 (1H, td, H-12), 3.12 (3H, s,
–OCH3), 1.42 (3H, s, CH3), 1.24 (3H, s, CH3), 1.15 (3H, s,
CH3), 0.91 (3H, s, CH3), 1.10 (3H, s, CH3), 1.04 (3H, s, CH3),
1
499.3763); H-NMR (400 MHz, pyridine-d5): δ 3.25 (1H, d, J =
9.9 Hz, H-3), 1.43 (6H, s, 2 × CH3), 1.32 (3H, s, CH3), 1.24
(3H, s, CH3), 1.18 (3H, s, CH3), 1.05 (3H, s, CH3), 0.89 (3H, s,
CH3), 0.81 (3H, s, CH3); 13C-NMR (100 MHz, pyridine-d5): δ
39.91 (C-1), 28.44 (C-2), 78.13 (C-3), 39.26 (C-4), 56.47 (C-5),
19.19 (C-6), 34.85 (C-7), 41.00 (C-8), 54.61 (C-9), 38.14 (C-10),
40.09 (C-11), 213.51 (C-12), 57.12 (C-13), 55.80 (C-14), 31.99
(C-15), 25.63 (C-16), 43.92 (C-17), 16.60 (C-18), 16.49 (C-19),
73.93 (C-20), 24.60 (C-21), 45.17 (C-22), 19.61 (C-23), 46.04 (C-
24), 70.12 (C-25), 30.56 (C-26), 30.34 (C-27), 28.98 (C-28),
16.20 (C-29), 17.76 (C-30).
20IJR)-12β,20,25-Trihydroxydammaran-3-one (2c). Yield:
28.6%, Mp: 215–218 °C, [α]2D0 + 19.6 (0.07, MeOH); HR-ESI-
TOF-MS: m/z 499.3778 [M + Na]+ (calcd for C30H52NaO4,
1
499.3763); H-NMR (400 MHz, pyridine-d5): δ 3.93 (1H, td, J =
8.9, 4.9 Hz, H-12), 1.44 (9H, s, 3 × CH3), 1.16 (3H, s, CH3),
1.07 (3H, s, CH3), 1.05 (3H, s, CH3), 0.93 (3H, s, CH3), 0.91
(3H, s, CH3); 13C-NMR (100 MHz, pyridine-d5): δ 40.12 (C-1),
34.66 (C-2), 216.76 (C-3), 47.76 (C-4), 55.61 (C-5), 19.13 (C-6),
34.81 (C-7), 40.28 (C-8), 50.07 (C-9), 37.29 (C-10), 32.92 (C-11),
71.06 (C-12), 49.84 (C-13), 52.13 (C-14), 31.80 (C-15), 27.04 (C-
16), 51.13 (C-17), 15.90 (C-18), 16.44 (C-19), 73.77 (C-20),
23.21 (C-21), 44.47 (C-22), 20.30 (C-23), 46.01 (C-24), 70.09 (C-
25), 30.63 (C-26), 30.34 (C-27), 27.19 (C-28), 21.56 (C-29),
17.64 (C-30).
20IJR)-2,12β,20,25-Tetrahydroxydammaran-1-en-3-one (2d).
Yield: 85.7%, Mp: 222–224 °C, [α]2D0 + 24.4 (0.06, MeOH); HR-
ESI-TOF-MS: m/z 513.3540 [M + Na]+ (calcd for C30H50NaO5,
513.3556); 1H-NMR (400 MHz, pyridine-d5): δ 6.77 (1H, s, H-
1), 3.96 (1H, td, J = 10.0, 5.0 Hz, H-12), 1.45 (3H, s, CH3), 1.44
(6H, s, 2 × CH3), 1.27 (3H, s, CH3), 1.18 (3H, s, CH3), 1.13
(3H, s, CH3), 1.07 (3H, s, CH3), 0.90 (3H, s, CH3); 13C-NMR
(100 MHz, pyridine-d5): δ 130.16 (C-1), 146.15 (C-2), 201.51
1914 | Med. Chem. Commun., 2018, 9, 1910–1919
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0.95 (3H, s, CH3), 0.91 (3H, s, CH3); 13C-NMR (75 MHz,
CDCl3): δ 38.9 (C-1), 27.4 (C-2), 78.9 (C-3), 39.7 (C-4), 55.9 (C-
5), 18.3 (C-6), 34.8 (C-7), 38.9 (C-8), 49.9 (C-9), 37.1 (C-10),
31.2 (C-11), 70.8 (C-12), 49.1 (C-13), 51.6 (C-14), 31.0 (C-15),
26.3 (C-16), 50.1 (C-17), 15.6 (C-18), 16.1 (C-19), 74.4 (C-20),
21.8 (C-21), 43.1 (C-22), 17.3 (C-23), 40.5 (C-24), 74.8 (C-25),
24.9 (C-26), 24.9 (C-27), 28.0 (C-28), 15.3 (C-29), 17.1 (C-30),
48.5 (–OCH3).
2 × CH3), 1.13 (3H, s, CH3), 1.07 (3H, s, CH3), 0.90 (3H, s,
CH3); 13C-NMR (100 MHz, CDCl3): δ 128.68 (C-1), 144.08 (C-
2), 201.19 (C-3), 45.30 (C-4), 54.63 (C-5), 18.87 (C-6), 34.47 (C-
7), 40.65 (C-8), 44.20 (C-9), 38.71 (C-10), 31.22 (C-11), 70.43
(C-12), 49.26 (C-13), 51.77 (C-14), 30.94 (C-15), 26.40 (C-16),
50.01 (C-17), 16.24 (C-18), 20.32 (C-19), 74.77 (C-20), 21.94 (C-
21), 43.20 (C-22), 17.40 (C-23), 40.61 (C-24), 74.95 (C-25),
25.07 (C-26), 25.07 (C-27), 27.27 (C-28), 21.73 (C-29), 17.19 (C-
30), 48.91 (–OCH3).
20IJR)-25-Methoxy-2,3-seco-12β,20-dihydroxydammaran-2,3-
dioc acid (3e). Yield: 42.3%, Mp: 161–163 °C, [α]2D0 + 17.6
(0.10, MeOH); HR-ESI-TOF-MS: m/z 537.3771 [M − H]− (calcd
for C31H53O7, 537.3791); 1H-NMR (400 MHz, pyridine-d5): δ
4.05 (1H, td, J = 9.5, 4.4 Hz, H-12), 3.15 (3H, s, –OCH3), 1.62
(3H, s, CH3), 1.56 (3H, s, CH3), 1.42 (3H, s, CH3), 1.16 (3H, s,
CH3), 1.15 (9H, s, 3 × CH3), 1.07 (3H, s, CH3); 13C-NMR (100
MHz, pyridine-d5): δ 43.57 (C-1), 174.81 (C-2), 182.80 (C-3),
47.50 (C-4), 49.19 (C-5), 18.45 (C-6), 34.93 (C-7), 40.31 (C-8),
42.60 (C-9), 42.43 (C-10), 31.86 (C-11), 71.23 (C-12), 49.72 (C-
13), 52.70 (C-14), 31.80 (C-15), 27.15 (C-16), 51.11 (C-17), 16.08
(C-18), 20.91 (C-19), 73.40 (C-20), 23.65 (C-21), 44.12 (C-22),
19.60 (C-23), 41.51 (C-24), 74.91 (C-25), 25.52 (C-26), 25.52 (C-
27), 28.14 (C-28), 25.34 (C-29), 17.87 (C-30), 49.30 (–OCH3).
20IJR)-25-Methoxy-20-hydroxydammaran-3,12-dione
(3a).
Yield: 20.4%, Mp: 128–131 °C, [α]2D0 + 45.6 (0.10, MeOH); HR-
ESI-TOF-MS: m/z 489.3928 [M + H]+ (calcd for C31H53O4,
1
489.3944); H-NMR (400 MHz, CDCl3): δ 3.18 (3H, s, –OCH3),
1.22 (3H, s, CH3), 1.15 (6H, s, 2 × CH3), 1.10 (3H, s, CH3),
1.06 (3H, s, CH3), 1.03 (3H, s, CH3), 1.00 (3H, s, CH3), 0.81
(3H, s, CH3); 13C-NMR (100 MHz, CDCl3): δ 39.21 (C-1), 33.34
(C-2), 216.90 (C-3), 47.53 (C-4), 55.30 (C-5), 19.80 (C-6), 33.92
(C-7), 39.14 (C-8), 52.70 (C-9), 37.24 (C-10), 40.19 (C-11),
214.21 (C-12), 56.72 (C-13), 54.03 (C-14), 30.97 (C-15), 25.23
(C-16), 44.06 (C-17), 15.63 (C-18), 15.63 (C-19), 73.65 (C-20),
21.98 (C-21), 43.22 (C-22), 17.70 (C-23), 40.56 (C-24), 74.88 (C-
25), 25.13 (C-26), 25.17 (C-27), 26.68 (C-28), 21.21 (C-29),
17.48 (C-30), 49.25 (–OCH3).
20IJR)-25-Methoxy-3β,20-dihydroxydammaran-12-one (3b).
Yield: 13.9%, Mp: 141–143 °C, [α]2D0 + 29.3 (0.10, MeOH); HR-
ESI-TOF-MS: m/z 491.4119 [M + H]+ (calcd for C31H55O4,
491.4100); 1H-NMR (400 MHz, CDCl3): δ 3.20 (1H, dd, J =
11.5, 4.8 Hz, H-3), 3.18 (3H, s, –OCH3), 1.17 (3H, s, CH3), 1.15
(6H, s, 2 × CH3), 0.99 (6H, s, 2 × CH3), 0.92 (3H, s, CH3), 0.80
(6H, s, 2 × CH3); 13C-NMR (100 MHz, CDCl3): δ 39.05 (C-1),
27.24 (C-2), 78.68 (C-3), 39.10 (C-4), 55.80 (C-5), 18.46 (C-6),
34.01 (C-7), 38.54 (C-8), 53.31 (C-9), 37.56 (C-10), 40.25 (C-11),
215.04 (C-12), 56.60 (C-13), 54.58 (C-14), 30.88 (C-15), 25.25
(C-16), 44.06 (C-17), 15.94 (C-18), 15.97 (C-19), 73.60 (C-20),
21.98 (C-21), 43.32 (C-22), 17.70 (C-23), 40.54 (C-24), 74.90 (C-
25), 15.17 (C-26), 25.13 (C-27), 28.13 (C-28), 15.45 (C-29),
17.61 (C-30), 49.24 (–OCH3).
Materials and methods
The schemes and methods of compounds
PD was derived from laboratory preparation with a purity
>97%. In this study, a series of ketone, enol, pyrazine, pyrim-
idine and azole derivatives were synthesized and the syn-
thetic routes of their preparation are illustrated in
Schemes 1–5. The structural characterizations of these com-
pounds were conducted using 1D-, 2D-NMR and MS (ESI†).
All compounds were successfully obtained in the end by a se-
ries of reactions. It is worth noting that 20 compounds such
as 1c, 1d, 1f–1n, 2b, 2d–2g, and 3a–3e were novel com-
pounds. According to the reported methods,25,26 PD was
added to a solution of hydrochloric acid in dry methanol.
The reactants were kept at 40 °C under ultrasonic reaction
conditions for 30 min to afford 2 and 3.27,28 PD, compounds
2 and 3 were oxidized with pyridine chlorochromate (PCC) in
CH2Cl2 to obtain compound 1, 2a, 2b, 2c, 3a, 3b, and 3c. In
addition, compound 1c was obtained in the same way as
shown in Scheme 1. Further, derivatives 1a, 2d, and 3d were
obtained by introducing air into tert-butyl alcohol solutions
of the 3-oxo derivatives, in the presence of potassium
tert-butoxide. Compounds 1a, 2d, and 3d were then oxidized
by the combined action of hydrogen peroxide and KOH in
refluxing methanol to obtain diacid derivatives 1b, 2e, and
3e. Compound 1 was added to morpholine, stirred and
dissolved; then, sublimed sulfur, ethylenediamine and
o-phenylenediamine were added to obtain compounds 1d and
1f. Compound 1 was added to a glacial acetic acid solution of
phenylhydrazine, and the reactants were heated at 120 °C un-
der reflux conditions for 6 h to yield 1e. In the presence of
methanol, triethylamine and hydroxylamine hydrochloride
20IJR)-25-Methoxy-12β,20-dihydroxydammaran-3-one (3c).
Yield: 34.2%, Mp: 176–179 °C, [α]2D0 + 39.9 (0.07, MeOH); HR-
ESI-TOF-MS: m/z 491.4114 [M + H]+ (calcd for C31H55O4,
1
491.4100); H-NMR (400 MHz, CDCl3): δ 3.51 (1H, td, J = 10.3,
5.1 Hz, H-12), 3.07 (3H, s, –OCH3), 1.05 (9H, s, 3 × CH3), 0.98
(3H, s, CH3), 0.94 (3H, s, CH3), 0.93 (3H, s, CH3), 0.88 (3H, s,
CH3), 0.80 (3H, s, CH3); 13C-NMR (100 MHz, CDCl3): δ 39.86
(C-1), 34.21 (C-2), 217.90 (C-3), 47.51 (C-4), 55.45 (C-5), 19.80
(C-6), 34.21 (C-7), 39.79 (C-8), 49.48 (C-9), 36.94 (C-10), 31.58
(C-11), 70.81 (C-12), 49.26 (C-13), 51.75 (C-14), 31.13 (C-15),
26.44 (C-16), 49.89 (C-17), 16.07 (C-18), 16.07 (C-19), 74.92 (C-
20), 21.98 (C-21), 43.16 (C-22), 17.42 (C-23), 40.61 (C-24),
74.92 (C-25), 25.10 (C-26), 25.10 (C-27), 26.85 (C-28), 21.17 (C-
29), 17.19 (C-30), 48.80 (–OCH3).
20IJR)-25-Methoxy-2,12β,20-trihydroxydammaran-1-en-3-one
(3d). Yield: 62.3%, Mp: 178–181 °C, [α]2D0 + 38.1 (0.07,
MeOH); HR-ESI-TOF-MS: m/z 527.3733 [M + Na]+ (calcd for
C31H52NaO5, 527.3712); 1H-NMR (400 MHz, CDCl3): δ 6.50
(1H, s, H-2), 3.71 (1H, td, J = 9.8, 4.4 Hz, H-12), 3.19 (3H, s,
–OCH3), 1.22 (3H, s, CH3), 1.20 (6H, s, 2 × CH3), 1.16 (6H, s,
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Scheme 1 The synthesis of intermediates. Reagents and conditions: (a) PCC, CH2Cl2, rt, 3 h; (b) CH3OH, HCl, ultrasonic, 1.5 h.
Scheme 2 The synthesis of oxidation products of intermediate 1. Reagents and conditions: (a) O2, t-BuOK, t-BuOH, 40 °C, 2 h; (b) H2O2, KOH,
CH3OH, reflux, 100 min; (c) CH3OH, HCl, ultrasonic, 1.5 h.
were added to PD-2, and after 24 h, 1g was obtained. Com-
pound 1 was dissolved in dimethylformamide, followed by
the addition of ethyl formate and sodium hydride; after 3 h
of reaction, compound 1h was obtained. Hydroxylamine hy-
drochloride or hydrazine dihydrochloride was added to the
ethanol solution of 1h, and refluxed at 85 °C for 2 h to obtain
compound 1i and 1j, respectively. Compound 1i was
dissolved in methanol at 0 °C, and then diethyl ether and so-
dium methoxide were added and the temperature was gradu-
ally raised to room temperature, and reaction was carried out
for 2 h to obtain compound 1k. Dimethylformamide acetal,
sodium methoxide and triethylamine were added to the
methanol solution of compound 1, and refluxed for 24 h to
obtain compound 1l. Interestingly, when guanidine hydro-
chloride or acetyl hydrochloride was added to 1l, 1m and 1n
were obtained, respectively. Moreover, the synthesis method
of the compounds 2g and 2f are similar to that of compound
1d. All products were purified from the reaction mixture by
silica gel column chromatography.
Cytotoxicity assay for human cancer cell and normal cell
The MTT assay was used to test the ability of each compound
to inhibit the proliferation of the human lung cancer A549
cells, human breast cancer MCF-7 cells, human glioma can-
cer U87 cells, human cervical cancer HeLa cells, human ovar-
ian cancer 8901 cells and one normal cell line IOSE144. In
addition, all of the cell lines were obtained from the Cell
Bank of Chinese Academy of Sciences (Shanghai, China). In
short, 5 × 103 cells per well were seeded into 96-well plates;
24 h later, the cells were treated with serial dilutions of the
compounds (0–100 μM) for another 48 h. Then 10 μL of the
MTT solution (5 mg mL−1) was added to each well, and the
tumor cells were incubated at 37 °C in a humidified atmo-
sphere of 5% CO2 for 4 h. At the end of the incubation pe-
riod, the growth medium was removed and replaced with 100
μL DMSO (Sigma Chemical Co., Ltd, USA) at room tempera-
ture. After agitating for 10 min using a vortex mixer, and the
absorbance was determined at 492 nm using a Bio-Rad
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Scheme 3 The synthesis of pyrazine, pyrimidine and azole compounds of intermediate 1. Reagents and conditions: (a) ethylenediamine,
morpholine, sulfur, reflux, 5 h; (b) phenylhydrazine, AcOH, reflux, 6 h; (c) o-phenylenediamine, morpholine, sulfur, reflux, 5 h; (d) MeOH,
NH2OH·HCl, Et3N, rt, 24 h; (e) HCOOEt, NaH, DMF, rt, 4 h; (f) EtOH, NH2OH·HCl, reflux, 2 h; (g) CH3OH, CH3ONa, EtOH, 0 °C-rt, 2 h; (h) EtOH,
H2NNH2·2HCl, reflux, 2 h; (i) N,N-dimethylformamide dimethyl acetal, TEA, toluene, reflux, 24 h; (j) guanidine hydrochloride, NaOMe, EtOH, reflux,
12 h; (k) ethanimidamide hydrochloride, NaOMe, EtOH, reflux, 12 h.
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Scheme 4 The synthesis of oxidation products and pyrazine compounds of intermediate 2. Reagents and conditions: (a) PCC, CH2Cl2, rt, 3 h; (b)
Ethylenediamine, morpholine, sulfur, reflux, 8 h; (c) O2, t-BuOK, t-BuOH, 40 °C, 2 h; (d) H2O2, KOH, MeOH, reflux, 100 min.
Scheme 5 The synthesis of oxidation products of intermediate 3. Reagents and conditions: (a) PCC, CH2Cl2, rt, 3 h; (b) O2, t-BuOK, t-BuOH, 40
°C, 2 h; (c) H2O2, KOH, MeOH, reflux, 100 min.
(model 550) microplate reader to calculate the 50% inhibi- Acknowledgements
tion concentration (IC50). Results of cytotoxicity studies were
expressed by the IC50 values (Table 1). PD and 5-Fluorouracil
were used as the positive control. It was clear that all of the
obtained compounds showed different cytotoxic activities
and lower toxicity for IOSE144 cells (normal cell).
This study was supported by the Technology Platform of Indus-
trialization Chromatographic Preparation for Standard Extract
of Traditional Chinese Medicine (2010ZX09401-304-105B),
Liaoning (FGW) Engineering Technology Research Center for
industrial chromatographic preparation of natural innovative
drugs materials (2017-1007) and National Nature Science Foun-
dation of China (Grant No. 81703389). We are grateful to the
Analytical Center of Shenyang Pharmaceutical University for
identification of the measurements of NMR, HR-ESI-TOF-MS.
Conflicts of interest
All authors have no conflict of interest to declare.
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