Synthesis, Structure, and Cytotoxic Activities of a Novel Lactam of the Diterpenoid Isosteviol

Article abstract of DOI:10.1007/s10600-020-03171-3

A novel compound lactam of 15β-hydroxymethylisosteviol ethyl ester 3 has been synthesized and structurally characterized by IR, NMR, and HR-MS. Its X-ray crystallographic analysis revealed that the nitrogen is attached to C-13 instead of C-15. The reaction mechanism was discussed, and the title compound was further evaluated against HCT-116, HGC-27, and JEKO-1 cells by the MTT assay. The results demonstrated that compound 3 exhibited better cytotoxic activities than its corresponding precursor isosteviol.

Full text of DOI:10.1007/s10600-020-03171-3

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DOI 10.1007/s10600-020-03171-3
Chemistry of Natural Compounds, Vol. 56, No. 5, September, 2020
SYNTHESIS, STRUCTURE, AND CYTOTOXIC ACTIVITIES
OF A NOVEL LACTAM OF THE DITERPENOID ISOSTEVIOL
1,2*
1
1
1
Cong-Jun Liu,
Jing-Jing Li, Yu-Tao Min,
1
Ling-Li Zhang, Bao-Yu Wang,
1
and Ya-Hao Wang
A novel compound lactam of 15β-hydroxymethylisosteviol ethyl ester 3 has been synthesized and structurally
characterized by IR, NMR, and HR-MS. Its X-ray crystallographic analysis revealed that the nitrogen is
attached to C-13 instead of C-15. The reaction mechanism was discussed, and the title compound was
further evaluated against HCT-116, HGC-27, and JEKO-1 cells by the MTT assay. The results demonstrated
that compound 3 exhibited better cytotoxic activities than its corresponding precursor isosteviol.
Keywords: isosteviol, synthesis, cytotoxic activity.
Isosteviol (ent-16-ketobeyeran-19-oic acid, 1), a metabolite of stevioside originally isolated from the natural
Stevia rebaudiana Bertoni, is a tetracyclic diterpenoid with the beyerane skeleton [1, 2]. In recent years, isosteviol as well as
its derivatives have been shown to exhibit a wide range of biological activities, such as antihyperglycemic [3], antihypertension
[4], and antimicrobial [5] activities, as well as anticancer activity [6]. In particular, when some bioactive structural fragments,
such as exo-methylene cyclopentanone, amino-alcohol, and so on, were bridged onto the isosteviol skeleton, these hybridized
isosteviol derivatives can display promising anticancer activities [7, 8]. The acylamino group is an important structural fragment
of many pharmaceutical molecules [9–11]. Introduction of the acylamino group to terpenoid compounds can enhance their
existing biological properties or lead to new biological capabilities [12]. However, few reports have focused on the relationship
of amide-fused isosteviol structure with anticancer activities. Based on these findings and in continuation of our previous
work, a lactam-fused isosteviol derivative was designed and synthesized by a ring-expansion reaction (Scheme 1), and its
antitumor activity was also evaluated in order to screen out highly active lead compounds with application values.
17
11
13
O
NOH
OH
1
C
D
16
9
B
a
A
3
15
7
5
COOEt
COOH
18
19
1
2
O
NH
CN
OH
CN
OH
O
+
+
+
CN
OH
COOEt
COOEt
COOEt
COOEt
6
4
5
3
a. BF ⋅OEt , toluene, reflux
3
2
Scheme 1
1) College of Chemical Engineering and Food Technology, Zhengzhou Institute of Technology, Zhengzhou, 450044,
Henan, P. R. China, fax: +86 0371 68229079, e-mail: congjunliu@zzut.edu.cn; 2) Postdoctoral Innovation Practice Base,
Zhengzhou Institute of Technology, Zhengzhou, 450044, Henan, P. R. China. Published in Khimiya Prirodnykh Soedinenii,
No. 5, September–October, 2020, pp. 741–744. Original article submitted September 8, 2019.
0009-3130/20/5605-0865 ©2020 Springer Science+Business Media, LLC
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TABLE 1. Comparison of Rearrangement Products of Compound 2 Under Different Catalysts (refluxed, 15 h)
Catalyst–solvent
Yield
BF3·OEt2–toluene
81%
H2SO4–acetone
33%
3
4–5
6
8% ( , 3:1)
54% (
, 3:1)
4–5
4–5
7%
8%
a,b
TABLE 2. Cytotoxic Activities of Compound 3 and Isosteviol in vitro, (IC , μM)
50
Compound
HCT-116
HGC-27
JEKO-1
1
3
102.39 2.38
34.28 1.16
3.91 0.26
118.58 2.84
46.73 2.66
2.03 0.10
132.18 3.13
59.93 2.01
2.74 0.24
Cisplatin
______
a
b
Values are the mean of triplicate of three independent experiments;
SD.
Fig. 1. Molecular structure of compound 3.
Compound 2 has been synthesized in our previous work [13]. Under acid condition, compound 2 underwent Beckmann
rearrangement and fragmentation (Table 1). Treatment of compound 2 with BF ·OEt in refluxing toluene can result in a
3
2
Beckmann rearrangement, yielding the D-ring expanded lactam 3 in 81% yield. In its IR and NMR spectra, the broad absorption
–1
band at ν 3371 cm is assigned for N-H, whose proton signal is displayed as a singlet peak at δ 6.29 ppm. The stretching
H
–1
vibration of C=O, whose carbon signal appears at δ 177.03 ppm, is observed as a strong band at ν 1644 cm . In accord with
the molecular formula C H NO , there was an [M + Na] peak at m/z 414.2622 in the HR-MS. The stereo-structure of
C
+
23 37
4
compound 3 was further confirmed by X-ray crystallographic analysis (Fig. 1), indicating the nitrogen is attached to C-13
instead of C-15. When H SO was used as catalyst, Beckmann fragmentation occurred readily to afford the olefinic nitriles 4
2
4
and 5 with a yield of 54%, which could not be separated by the chromatography method due to the isomers having very
–1
similar physical properties and polarities. Their IR spectrum shows the weak absorption band of -CN group at ν 2230 cm . In
1
their H NMR spectra, resonances are distinctly observed at δ 5.53, 5.15 ppm, demonstrating the formation of the CH=C
H
group. The peak integral area at δ 5.53 and 5.15 ppm is 1 and 0.37, respectively, which indicates a 3:1 mixture of olefinic nitriles
H
4 and 5. Moreover, the by-product 6 was also characterized by IR, NMR, and HR-ESI-MS. According to some related
literature [14, 15], the mechanism of the rearrangement and fragmentation reaction can be tentatively shown in Fig. 2.
866

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N
N
OH
R
HOR
- R
N
N
NH
+
OH
R
+
H
O
OH
O
OH
OH
OH
OH
COOEt
COOEt
COOEt
2
3
Intermediate Y
H
1
2
+
N
OHR
+
+
-H
H
CN
OH
CN
OH
OH
CN
OH
+
Intermediate X
COOEt
COOEt
COOEt
4
5
6
+
+
H
O
O
+
+
CN
OH
-H
N
C
OH
CN
CN
COOEt
Intermediate Z
COOEt
Fig. 2. Possible reaction mechanism.
Firstly, compound 2 was attacked by acid to furnish the intermediate X with positive charge on the oxygen atom due to the
electron deficiency of acid. In the next stage, intramolecular migration of C-13 (with its pair of electrons) anti to the departing
hydroxyl function occurred intermediate Y with the electron-deficient carbon atom formed by partial ionization of the
oxygen-nitrogen bond as the migration terminus. The resulting imine derivatives may then rearrange to the lactam 3. Meanwhile,
a competitive process was proceeding as fragmentation to furnish the intermediate Z with a stable carbonium ion. As the H-12
and H-14 went, the corresponding fission products 4 and 5 were generated with olefin-nitrile character. The formation of
compound 6 from intramolecular cyclization of the intermediate Z further proves the rationality of the reaction mechanism.
20
The title compound 3 and isosteviol 1 ([α] –110.2° (c 1.0, CH Cl )) were further utilized to investigate their cytotoxic
D
2
2
activities against three human cancer cell lines: colon carcinoma (HCT-116), gastric carcinoma (HGC-27), mantle cell lymphoma
(JEKO-1). Cisplatin, employed in the treatment of human cancers as one of the most effective chemotherapeutic drugs, was
used as the positive control. The in vitro antiproliferative activities were evaluated by the standard MTT assay. The IC ,
50
summarized in Table 2, expresses the inhibitory activity of tumor cell growth. The obtained results showed that the IC values
50
of compounds 1 and 3 against HCT-116 were lower than HGC-27 and JEKO-1, which demonstrated that HCT-116 was the
most sensitive cell line to the tested compounds. More importantly, the title compound 3 exhibited better cytotoxic activities
than its corresponding precursor 1, which indicated that the introduction of the lactam subunit could enhance the antitumor
activities.
EXPERIMENTAL
General. All chemicals were used as received unless otherwise noted. Reagent grade solvents were redistilled prior
to use. Chromatography was performed on silica gel (100–200 mesh). All melting points were measured with a Beijing Keyi
XT5 apparatus, and the temperature was not corrected. Optical rotations were determined on a INESA WZZ-2B polarimeter.
1
13
The IR spectra were recorded as KBr pellets on a Thermo Nicolet (IR200) spectrometer. H and C NMR spectra were
collected with a Bruker DPX-400 spectrometer at 400 and 100 MHz, respectively, with TMS as internal standard.
High-resolution mass spectra (HR-MS) were obtained with a Waters Micromass Q-Tof Micro™ instrument by the ESI technique.
Synthesis of Compound 2. The solution of stevioside in 10% H SO solution was stirred for 6 h at 60°C. The
2
4
precipitate was filtered and crystallized with acetone to give the isosteviol 1. Isosteviol 1, KOH, and CH CH Br were added
3
2
in DMSO. After stirring the solution for 5 h at 40°C, a precipitate came out as excess water was added. The precipitate was
filtered and crystallized with ethyl acetate to give the ethyl ent-16-oxobeyeran-19-oate. To a solution of Na in ethanol, 37%
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formaldehyde aqueous solution and ethyl ent-16-oxobeyeran-19-oate were added. After stirring for 8 h at 55°C, the mixture
was concentrated under vacuum. Excess water was added, and a white solid separated out. The precipitate was filtered and
crystallized with ethyl acetate to give the ethyl ent-15α-hydroxymethyl-16β-hydroxybeyeran-19-oate. ent-15α-Hydroxymethyl-
16β-hydroxybeyeran-19-oate was dissolved in CH Cl and then TCC/Silica gel was added. After stirring for 0.5 h at room
2
2
temperature, the reaction mixture was filtered and washed with CH Cl . Finally, the combined organic phase was washed,
2
2
dried, concentrated, and purified by column chromatography on silica to give the ethyl ent-15α-hydroxymethyl-16-oxobeyeran-
19-oate. A mixture of ethyl ent-15α-hydroxymethyl-16-oxobeyeran-19-oate and hydroxylamine hydrochloride in C H OH
2
5
was stirred in the presence of NaHCO under refluxing condition for 3 h; then the reaction mixture was concentrated under
3
vacuum and extracted with CH Cl and H O. Finally, the organic layer was washed, dried, and concentrated under vacuum to
2
2
2
give target compound 2.
Synthesis of Compounds 3–6. To a solution of compound 2 (3.9 g, 10 mmol) in toluene (50 mL), 48% BF ·OEt (1 mL)
3
2
was added dropwise, and the mixture was refluxed for 15 h under nitrogen atmosphere. After completion of the reaction, water
(100 mL) was added to the mixture, which was next neutralized with NaHCO , and the organic phase was separated and dried
3
over Na SO . After evaporation in vacuo, the crude product was purified by column chromatography to give compounds 3–6.
2
4
20
–1
Compound 3, mp 109.7–111.8°C; [α] –39.8° (c 1.0, CH Cl ). IR (KBr, ν, cm ): 3371 (NH), 1720 (COO), 1644
(CONH). H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 6.29 (1H, s, NH), 4.13–4.08 (2H, m, 19-(O)OCH ), 4.02 (1H, dd,
D
2
2
1
3
2
J = 10.0, 4.2, CH -15), 3.67 (1H, t, J = 9.4, H-15), 2.81 (1H, dd, J = 8.0, 4.0, CH -15), 2.16 (1H, d, J = 13.6, H-3), 1.90–0.85
2
2
(17H, m, isosteviol skeleton), 1.26 (3H, t, J = 7.2, 19-(O)OCH CH ), 1.18 (3H, s, H-18), 0.99 (3H, s, H-17), 0.81 (3H, s, H-20).
2
3
13
C NMR (100 MHz, CDCl , δ, ppm): 177.30 (C, C-19), 177.03 (C, C-16), 62.13 (CH , 19-(O)OCH ), 60.39 (C, C-13), 59.31
3
2
2
(CH , 15-CH ), 57.65 (CH, C-5), 51.45 (CH, C-15), 47.04 (CH, C-9), 44.52 (CH , C-14), 43.65 (C, C-4), 40.11 (CH , C-1),
2
2
2
2
40.00 (CH , C-12), 38.53 (C, C-8), 38.29 (C, C-10), 37.84 (CH , C-3), 36.81 (CH , C-7), 28.71 (CH , C-18), 28.56 (CH , C-17),
2
2
2
3
3
20.10 (CH , C-6), 18.87 (CH , C-2), 17.75 (CH , C-11), 14.19 (CH , C-20), 14.19 (CH , 19-(O)OCH CH ). HR-ESI-MS
2
2
2
3
3
2
3
+
m/z 414.2622 [M + Na] (calcd for C H NO Na, 414.2620).
23 37
–1
4
1
Compounds 4–5, IR (KBr, ν, cm ): 3373 (OH), 2230 (CN), 1720 (COO), 1677 (C=C). H NMR (400 MHz, CDCl ,
3
δ, ppm, J/Hz): 5.53 (1H, s, H-12, for 4), 5.15 (0.37H, s, H-14, for 5), 4.16–4.06 (3H, m, 19-(O)OCH , for 4 and 5), 3.86–3.76
2
(3H, m, H-15 and CH -15, for 4 and 5), 3.39 (0.47H, dd, J = 8.9, 4.9, CH -15, for 5), 3.13 (1H, dd, J = 8.2, 4.4, CH -15,
2
2
2
for 4), 2.48–0.86 (23H, m, isosteviol skeleton, for 4 and 5), 1.69 (1H, s, H-17, for 5), 1.67 (3H, s, H-17, for 4), 1.27 (4H, t,
J = 7.2, 19-(O)OCH CH , for 4 and 5), 1.17 (3H, s, H-18, for 4), 1.16 (1H, s, H-18, for 5), 0.91 (3H, s, H-20, for 4), 0.88 (1H,
2
3
13
s, H-20, for 5). C NMR (100 MHz, CDCl , δ, ppm) for 4: 177.01 (C, C-19), 127.34 (C, C-13), 123.26 (C, CN), 122.55 (CH,
3
C-12), 61.28 (CH , 19-(O)OCH ), 60.21 (CH , CH -15), 57.78 (CH, C-5), 54.35 (CH, C-9), 43.51 (C, C-4), 39.76 (CH , C-1),
2
2
2
2
2
39.62 (CH , C-14), 37.83 (CH , C-3), 37.70 (CH, C-15), 37.17 (C, C-10), 36.91 (CH , C-7), 29.71 (CH , C-18), 28.81 (C, C-8),
2
2
2
3
23.21 (CH , C-17), 22.86 (CH , C-11), 20.02 (CH , C-6), 19.00 (CH , C-2), 14.15 (CH , C-20), 13.79 (CH , 19-(O)OCH CH ).
3
2
2
2
3
3
2
3
+
HR-ESI-MS m/z 396.2519 [M + Na] (calcd for C H NO Na, 396.2515).
Compound 6, mp 128.6–129.4°C. IR (KBr, ν, cm ): 2236 (CN), 1719 (COO), 1092 (C–O–C). H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 4.39 (1H, t, J = 12.1, H-15), 4.15–4.07 (2H, m, 19-(O)OCH ), 3.89 (1H, dd, J = 11.8, 6.0, H-16), 2.83
23 35
3
–1
1
3
2
(1H, dd, J = 12.5, 6.0, H-16), 2.49–0.86 (17H, m, isosteviol skeleton), 2.16 (1H, d, J = 13.6, H-3), 1.29 (3H, t, J = 7.2,
13
19-(O)OCH CH ), 1.19 (3H, s, H-18), 1.11 (3H, s, H-17), 0.99 (3H, s, H-20). C NMR (100 MHz, CDCl , δ, ppm): 177.27
2
3
3
(C, C-19), 121.21 (C, CN), 71.32 (C, C-13), 65.13 (CH , C-16), 60.40 (CH , 19-(O)OCH ), 57.93 (CH, C-5), 57.61 (CH, C-9),
2
2
2
53.83 (CH , C-14), 44.27 (C, C-4), 43.69 (CH , C-1), 40.54 (CH , C-12), 38.89 (C, C-10), 38.09 (CH , C-3), 37.60 (CH , C-7),
2
2
2
2
2
37.37 (CH, C-15), 36.82 (C, C-8), 29.58 (CH , C-18), 28.48 (CH , C-17), 21.92 (CH , C-6), 20.63 (CH , C-11), 19.19 (CH ,
3
3
2
2
2
+
C-2), 14.23 (CH , C-20), 13.92 (CH , 19-(O)OCH CH ). HR-ESI-MS m/z 396.2515 [M + Na] (calcd for C H NO Na,
3
3
2
3
23 35
3
396.2515).
X-Ray Crystallographic Analysis of Compound 3. Colorless crystals of the title compound were grown at room
temperature from ethanol by the slow evaporation technique. X-ray diffraction data of the title compound were collected on a
°
Bruker SMART APEXII CCD area-detector diffractometer with Mo Ka (λ = 0.71073 A) radiation at 300(2) K. The collected
data were reduced using the program SAINT. The structure was solved by direct methods and refined by the full-matrix
2
least-squares method on all F data using the SHELX-97 (Sheldrick, 2008) and SHELXL-97 (Sheldrick, 2008) programs,
respectively. Crystallographic data (excluding structure factors) have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication numbers CCDC 1951641 (compound 3). Copies of the data can be obtained, free of
charge, on applications to CCDC, 12 Union Road, Cambridge CB2 1EZ. UK (fax: +44 (0) 1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk or http://www. ccdc.cam.ac.uk).
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