Synthesis and antitumor activity of a novel series of helicid-pyrrolidone derivatives

Article abstract of DOI:10.1007/s10600-015-1216-9

This paper reports the synthesis of a new series of helicid-pyrrolidone derivatives (4a-4d, 5a-5d, and 6a-6d), which were tested for antitumor activity against human skov3 cell. In this study, two compounds (4b and 6c) demonstrate high antitumor activity

Full text of DOI:10.1007/s10600-015-1216-9

DOI 10.1007/s10600-015-1216-9
Chemistry of Natural Compounds, Vol. 51, No. 1, January, 2015
SYNTHESIS AND ANTITUMOR ACTIVITY OF A NOVEL SERIES
OF HELICID-PYRROLIDONE DERIVATIVES
Li-Juan Jiang, Shi-Ming Lv, Chao Cheng,
Lin Dong, Ying Li, and Shu-Fan Yin*
This paper reports the synthesis of a new series of helicid–pyrrolidone derivatives (4a–4d, 5a–5d, and
6a–6d), which were tested for antitumor activity against human skov3 cell. In this study, two compounds
(4b and 6c) demonstrate high antitumor activity against this cell line having IC values of 0.22 and 6.5 ꢀM,
50
respectively, which are lower than those of an inhibitor of heat shock protein 90 (17AAG), currently under
clinical evaluation for cancer treatment.
Keywords: antitumor, helicid, pyrrolidone.
Currently, people are increasingly suffering from various diseases, especially cancer. Cancer is a major disease, and
according to the World Health Organization, it is considered to be the fourth leading cause of death. Developing effective
cancer treatment has been a primary goal of medicine for the last two decades, and chemotherapy is considered to be an
effective approach for combating cancer. Although there are a large number of anticancer drugs for clinical use, advancing
novel compounds with higher activity that are also active against mutant cells has remained a high priority ꢁ1ꢂ.
The chaperone heat shock protein 90 (Hsp90) is emerging as an important target for antitumor drugs because of its
effect on proteins, which plays an important role in maintaining transformation and increasing the survival and growth potential
of cancer cells. The most well-known inhibitor of Hsp90, 17-(allylamino)-17-demethoxygeldanamycin (17AAG), is currently
in phase II clinical trials for the treatment of cancer in the United States and United Kingdom and has shown early evidence of
therapeutic activity when administered alone or in combination with docetaxel ꢁ2, 3ꢂ.
Small and simple heterocyclic structures often have surprisingly complex biological properties. For example,
pyrrolidone derivatives represented by the general formula 1 have a neural differentiation induction effect, making them
useful as antitumor agents, nootropic agents, etc. ꢁ4ꢂ for treating nervous diseases. The natural product helicid [4-formylphenyl-
ꢃ-D-allopyranoside, C H O ] (2), originally extracted from the fruit of Helicia nilagirica Bedd., exhibits various biological
13 16
7
activities on the central nervous system, such as hypnotic, anti-inflammatory, and anticonvulsant activity, owing to the rare
allopyranoside moiety ꢁ5ꢂ. Using the principle of combining active groups, we incorporated the pyrrolidone active group into
the helicid compounds to form new helicid–pyrrolidone derivatives.
In this paper, we report several new helicid–pyrrolidone derivatives and describe their chemical synthesis. All new
compounds, 17AAG, and 2 were tested for antitumor activity against human hepatoma carcinoma cell line 7402, and their
IC values are reported.
50
R
1
R
R
2
O
N
3
R
1
College of Chemistry, Sichuan University, 29 Wangjiang Road, 610064, Chengdu, Sichuan Province, P. R. China,
fax: 86 028 85503392, e-mail: chuandayouji217@163.com. Published in Khimiya Prirodnykh Soedinenii, No. 1, January–February,
2015, pp. 106–110. Original article submitted June 4, 2013.
0009-3130/15/5101-0121 02015 Springer Science+Business Media New York
121

TABLE 1. In vitro Activity of Compounds 4a–4d, 5a–5d, and 6a–6d (IC , ꢀM) against Human Hepatoma Carcinoma Cell
50
Line 7402
Compound
Compound
IC50, ꢀM*
IC50, ꢀM*
16.5
33.6
62.5
62.5
> 100
> 100
> 100
> 100
6.2
17AAG
5b
5c
5d
6a
6b
6c
6d
2
4a
4b
4c
4d
5a
0.22
> 100
> 100
> 100
> 100
______
*IC values represent the drug concentration required to inhibit cancer cell replication by 50%. The compounds were tested
50
2
up to 625 ꢀM. IC values were calculated by probit analysis (P < 0.05, ꢄ test).
50
OH
OAc
1
O
3
3
O1
3'
a
3'
b
HO
HO
AcO
AcO
O
O
1'
CHO
CHO
HO5
AcO5
1'
5'
5'
2
3
OH
H COOC
3
S
3''
HOOC
R
O 1''
S
H COOC
3
S
HO
HO
c
d
5
3
O
HO 5''
4'
1'
N
N
1
O
N
O
R
O
R
4a - d
5a - d
6a - d
4a, 5a, 6a: R = n-C H ; 4b, 5b, 6b: R = iso-C H ; 4c, 5c, 6c: R = cyclohexyl; 4d, 5d, 6d: R = Bn
3
7
4 9
a. Ac O, TEA, DMF, 96%; b. p-thiocresol, RNH , maleic anhydride, 50ꢅC, 110ꢅC, 45%–56%;
2
2
c. K CO CH I, r.t., 91%–94%; d. NH , MeOH, r.t., 87%–90%
2
3,
3
3
Scheme 1.
Helicid–pyrrolidone derivatives 4a–4d, 5a–5d, and 6a–6d and intermediate 3 were synthesized by the method shown
in Scheme 1. Compound 3 is an important intermediate and is obtained by the reaction of 4-formylphenyl-ꢃ-D-allopyranoside
(2) with acetic anhydride in the presence of triethylamine (TEA) in dimethylformamide (DMF), followed by reduction at room
temperature, in good yield (96%, step a). Then, helicid–pyrrolidone derivatives 4a–4d were synthesized through a
four-component (primary amine, maleic anhydride, p-thiocresol, and intermediate 3) condensation reaction (step b).
Helicid–pyrrolidone derivatives 5a–5d were obtained in good yield by esterification of (4a–4d) in which propan-2-one used
as a sovlent under alkaline conditions at room temperature (step c). Transesterification of helicid–pyrrolidone derivatives
5a–5d in the presence of NH and CH OH at room temperature gave new compounds 6a–6d (step d).
3
3
In preliminary screening, compounds 4a–4d, 5a–5d, and 6a–6d were evaluated for their antitumor activity against
skov3 cell. The test compounds were dissolved in DMSO and evaluated using five concentrations, the highest being 625 ꢀM
(remaining concentrations prepared as fivefold dilutions: 125, 62.5, 25, and 5 ꢀM). Table 1 reports the results obtained expressed
as log, considering half maximal inhibitory concentration IC .
50
All new compounds were tested to determine their antitumor activity against human skov3 cell with IC values.
50
Considering the results shown in Table 1, 4b and 6c emerged as analogs having higher antitumor activity than 17AAG.
Therefore, the development of related helicid–pyrrolidone derivatives merits further attention. The results of pharmacokinetic
and toxicological studies on these agents will be published elsewhere.
EXPERIMENTAL
1
General. Melting points were recorded with a micromelting point tester and are reported uncorrected. H and
13
C NMR spectra were obtained in DMSO-d solutions on a Bruker AVII-400 spectrometer operating at 400 and 100 MHz,
6
122

respectively. The chemical shift values are given in units with reference to internal standard Me Si, and signals are designated
4
as s (singlet), br.s (broad singlet), d (doublet), t (triplet), q (quadruplet), qu (quintuplet), h (heptuplet), dt (double triplet), dd
(doublet of doublets), ddd (double doublet of doublets), or m (multiplet) with coupling constants given in Hertz (Hz). Infrared
spectra (as KBr disks) were recorded using a PerkinElmer 16PC-FT spectrometer. Kieselgel 60F254 (0.25 mm) silica gel and
TLC aluminum sheets were used for routine monitoring of reaction samples and for confirming the homogeneity of analytical
samples. Silica gel column chromatography was carried out using GF254 (300–400 mesh) silica gel. High-resolution mass
spectra were recorded on a Bruker Daltonics ESI-BioTOF Q spectrometer.
Starting Materials. All reagents were commercially available and were used without further purification unless
ꢆ ꢅ
otherwise indicated. Toluene was dried by distillation from sodium and stored over activated molecular sieves (4A), and DMF
was dried by distillation from magnesium sulfate prior to use.
Synthesis of (2R,3R,4R,5R,6S)-2-(Acetoxymethyl)-6-(4-formylphenoxy)tetrahydro-2H-pyran-3,4,5-triyl
Triacetate (3). A solution of 4-formylphenyl-ꢃ-D-allopyranoside 2 (2.84 g, 10 mmol) was dissolved in anhydrous DMF
(8 mL); then acetic anhydride (5 equiv) and triethylamine (4.2 equiv) were added under stirring at 0ꢅC; the resulting mixture
was stirred for 2 h at 0ꢅC, then for 12 h at room temperature. The reaction mixture was diluted with water (50 mL). The
resulting crystals were collected by filtration, washed with water (10 mL), and dried under vacuum to give 4.37 g (96%) of 3
(white crystals).
Synthesis of Compounds 4a–4d.Asolution of succinic anhydride (0.39 g, 4 mmol), RNH (4 mmol), and p-thiocresol
2
(0.50 g, 4 mmol) in toluene (10 mL) was stirred at 50ꢅC for 15 min, and 3 (1.84 g, 4 mmol) was added. Then the resulting
mixture was stirred for 14 h at 110ꢅC. The reaction mixture was diluted with a large amount of water and extracted with ethyl
acetate. The organic layer was purified by column chromatography on silica gel using chloroform–methanol–petroleum ether
5:1:4 as the eluent to give 1.34 g (45–56%) of 4a–4d.
5-Oxo-1-propyl-3-(p-tolylthio)-2-(4-(((2S,3R,4R,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-
1
pyran-2-yl)oxy)phenyl)pyrrolidine-3-carboxylic Acid (4a). Yellow powder. Yield 47%, mp 130–134ꢅC. H NMR (400 MHz,
DMSO-d , , ppm, J/Hz): 6.98–7.20 (8H, m, ArH), 5.63 (1H, dd, J = J = 2.7, CH), 5.53 (1H, d, J = 8.3, CH), 5.28 (1H, s,
6
12
13
CH), 5.03 (1H, dd, J = 2.9, J = 8.2, CH), 4.98 (1H, dd, J = 2.9, J = 10.2, CH), 4.30–4.45 (1H, m, 1/2CH ), 4.16–4.26
12
13
12
13
2
(2H, m, CH + 1/2CH ), 3.40–3.48 (1H, m, 1/2CH ), 2.95–3.08 (2H, m, CH ), 2.37–2.46 (1H, m, 1/2CH ), 2.25 (3H, s, CH ),
2
2
2
2
3
2.16 (3H, d, J = 1.9, CH ), 2.02 (3H, s, CH ), 2.00 (3H, s, CH ), 1.99 (3H, s, CH ), 1.23–1.40 (2H, m, CH ), 0.72 (3H, t, J = 7.3,
3
3
3
3
2
13
CH ). C NMR (100 MHz, DMSO-d , , ppm): 11.07, 19.85, 20.32, 20.44, 20.63, 22.34, 40.27, 40.86, 41.90, 58.76, 61.83,
3
6
65.89, 67.35, 68.78, 69.90, 95.68, 115.83, 128.34, 129.20, 130.47, 134.66, 137.87, 156.29, 169.10, 169.93, 169.98, 170.02,
–1
171.75. IR (KBr, ꢇ, cm ): 3437, 2965, 1754, 1669, 1611, 1511, 1412, 1374, 1227, 1091, 1045, 949, 908, 814, 599, 510.
+
+
HR-MS (ESI): calcd for C H O NS [M + H] 716.2377, found 716.2392; [M + K] 754.1936, found 754.1882.
35 41 13
1-Isobutyl-5-oxo-3-(p-tolylthio)-2-(4-(((2S,3R,4R,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-
–1
pyran-2-yl)oxy)phenyl)pyrrolidine-3-carboxylic Acid (4b). Yellow powder, Yield 51%, mp 134–138ꢅC. IR (KBr, ꢇ, cm ):
1
3437, 2961, 1755, 1667, 1610, 1510, 1414, 1372, 1226, 1091, 1045, 949, 907, 814, 598, 509. H NMR (400 MHz, DMSO-d ,
6
, ppm, J/Hz): 6.90–7.31 (8H, m, ArH), 5.63 (1H, dd, J = J = 2.7, CH), 5.53 (1H, d, J = 7.8, CH), 5.30 (1H, s, CH,), 4.95–5.06
12
13
(2H, m, 2CH), 4.35–4.45 (1H, m, 1/2CH ), 4.15–4.26 (2H, m, CH + 1/2CH ), 3.40–3.57 (1H, m, 1/2CH ), 3.03–3.27 (2H, m,
2
2
2
CH ), 2.30–2.44 (1H, m, 1/2CH ), 2.24 (3H, s, CH ), 2.16 (3H, d, J = 2.9, CH ), 2.02 (3H, s, CH ), 2.00 (3H, s, CH ), 1.99
2
2
3
3
3
3
13
(3H, s, CH ), 1.64–1.75 (1H, m, CH), 0.75 (3H, d, J = 6.6, CH ), 0.72 (3H, d, J = 6.6, CH ). C NMR (100 MHz, DMSO-d ,
3
3
3
6
, ppm): 19.83, 20.15, 20.31, 20.43, 20.63, 25.94, 40.81, 47.71, 58.76, 61.86, 65.93, 67.38, 67.97, 68.79, 69.93, 95.74, 115.87,
128.68, 129.09, 129.72, 130.61, 134.49, 134.55, 137.62, 156.24, 169.07, 169.90, 169.95, 169.98, 172.13. HR-MS (ESI):
+
+
calcd for C H O NS [M + H] 730.2533, found 730.2534; [M + K] 768.2092, found 768.2020.
36 43 13
1-Cyclohexyl-5-oxo-3-(p-tolylthio)-2-(4-(((2S,3R,4R,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)- tetrahydro-2H-
–1
pyran-2-yl)oxy)phenyl)pyrrolidine-3-carboxylic Acid (4c). Yellow powder. Yield 45%, mp 158–162ꢅC. IR (KBr, ꢇ, cm ):
1
3433, 2934, 1755, 1660, 1609, 1510, 1414, 1373, 1225, 1091, 1044, 948, 906, 813, 596, 509. H NMR (400 MHz, DMSO-d ,
6
, ppm, J/Hz): 6.80–7.41 (8H, m, ArH), 5.63 (1H, dd, J = J = 2.8, CH), 5.53 (1H, d, J = 8.2, CH), 5.38 (1H, s, CH), 4.94–5.06
12
13
(2H, m, CH ), 4.36–4.44 (1H, m, 1/2CH ), 4.12–4.28 (2H, m, CH + 1/2CH ), 3.50–3.64 (1H, m, CH), 2.80–3.00 (2H, m,
2
2
2
CH ), 2.25 (3H, s, CH ), 2.23 (3H, s, CH ), 2.16 (3H, d, J = 3.9, CH ), 2.02 (3H, s, CH ), 1.99 (3H,s, CH ), 1.98 (3H, s, CH ),
2
3
3
3
3
3
3
13
0.79–1.74 (10H, m, 5CH ). C NMR (100 MHz, DMSO-d , , ppm): 20.34, 20.44, 20.63, 24.93, 25.15, 31.22, 40.95, 51.87,
2
6
58.76, 59.91, 60.81, 65.90, 66.21, 67.34, 68.83, 69.88, 95.50, 115.53, 128.92, 133.63, 134.28, 134.36, 137.19, 155.80, 155.90,
+
+
169.11, 169.95, 170.00, 171.53. HR-MS (ESI): calcd for C H O NS [M + H] 756.2690, found 756.2684; [M + K]
38 45 13
794.2249, found 794.2169.
123

1-Benzyl-5-oxo-3-(p-tolylthio)-2-(4-(((2S,3R,4R,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-
–1
2-yl)oxy)phenyl)pyrrolidine-3-carboxylic Acid (4d). Yellow powder, Yield 56%, mp 158–162ꢅC. IR (KBr, ꢇ, cm ): 3427,
1
2925, 1754, 1663, 1609, 1510, 1412, 1373, 1226, 1091, 1045, 948, 909, 814, 703, 599, 508. H NMR (400 MHz, DMSO-d ,
6
, ppm, J/Hz): 7.00–7.25 (13H, m, ArH), 5.64 (1H, dd, J = J = 2.8, CH), 5.53 (dd, J = 4.9, J = 8.1, CH), 5.08 (1H, s,
12
13
12
13
CH), 4.98–5.06 (2H, m, 2CH), 4.85 (1H, d, J = 15.5, 1/2CH ), 4.36–4.44 (1H, m, 1/2CH ), 4.14–4.28 (2H, m, CH + 1/2CH ),
2
2
2
3.50 (1H, d, J = 15.6, 1/2CH ), 3.15 (1H, d, J = 15.6, 1/2CH ), 2.25 (3H, s, CH ), 2.16 (3H, d, J = 1.8, CH ), 2.02 (3H, s, CH ),
2
2
3
3
3
13
2.01 (3H, s, CH ), 1.99 (3H, s, CH ). C NMR (100 MHz, DMSO-d , , ppm): 20.34, 20.47, 20.64, 40.82, 42.05, 43.81,
3
3
6
58.80, 61.84, 65.90, 67.36, 68.79, 69.91, 95.69, 115.84, 127.04, 127.22, 128.21, 128.41, 129.17, 127.96, 134.63, 136.22,
+
137.73, 156.28, 169.11, 169.94, 170.02, 172.33. HR-MS (ESI): calcd for C H O NS [M + H] 764.2377, found 764.2365;
39 41 13
+
[M + K] 802.1936, found 802.1730.
Synthesis of Compounds 5a–5d. Compound 4 (1 mmol) was dissolved in propan-2-one (5 mL), and K CO (1.00 g,
2
3
7 mmol) and CH I (280 mg, 2 mmol) were added. The mixture was stirred for 24 h at room temperature. The solvent was
3
evaporated, and the residue was purified by flash chromatography (silica gel, propan-2-one–petroleum ether, 2:3, v/v). A
yellow powder was obtained: 5a 91% yield, 5b, 5c, and 5d (92% yield).
(2R,3R,4R,5R,6S)-2-(Acetoxymethyl)-6-(4-(3-(methoxycarbonyl)-5-oxo-1-propyl-3-(p-tolylthio)pyrrolidin-2-
–1
yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl Triacetate (5a). Yellow powder. Yield 94%, mp 68–72ꢅC. IR (KBr, ꢇ, cm ):
1
1754, 1698, 1610, 1510, 1412, 1373, 1225, 1091, 1044, 948, 909, 816, 715, 599, 508. H NMR (400 MHz, DMSO-d , , ppm,
6
J/Hz): 7.29 (2H, d, J = 7.6, ArH), 7.12–7.20 (6H, m, ArH), 5.63 (1H, dd, J = J = 2.7, CH), 5.56 (1H, d, J = 8.2, CH), 5.22
12
13
(1H, s, CH), 5.03 (dd, J = 2.9, J = 8.1, CH), 4.99 (1H, dd, J = 2.7, J = 10.3, CH), 4.35–4.42 (1H, m, 1/2CH ), 4.16–4.26
12
13
12
13
2
(2H, m, CH + 1/2CH ), 3.62 (3H, s, CH ), 3.40–3.54 (1H, m, 1/2CH ), 2.94 (1H, d, J = 18.0, 1/2CH ), 2.85 (1H, d, J = 18.0,
2
3
2
2
1/2CH ), 2.40–2.48 (1H, m, 1/2CH ), 2.29 (3H, s, CH ), 2.16 (3H, s, CH ), 2.02 (3H, s, CH ), 2.01 (3H, s, CH ), 1.99 (3H, s,
2
2
3
3
3
3
13
CH ), 1.22–1.38 (2H, m, CH ), 0.68 (3H, t, J = 7.3, CH ). C NMR (100 MHz, DMSO-d , , ppm): 10.77, 19.58, 20.32,
3
2
3
6
20.45, 20.70, 40.35, 41.28, 41.72, 52.95, 58.45, 61.82, 65.88, 67.33, 68.77, 69.93, 95.58, 116.04, 125.91, 128.48, 129.84,
+
135.75, 139.68, 156.64, 169.10, 169.94, 169.99, 170.03, 170.74, 171.42. HR-MS (ESI): calcd for C H O NS [M + H]
730.2533, found 730.2545; [M + Na] 752.2353, found 752.2374.
36 43 13
+
(2R,3R,4R,5R,6S)-2-(Acetoxymethyl)-6-(4-(1-isobutyl-3-((methylperoxy)methyl)-5-oxo-3-(p-tolylthio)pyrrolidin-
–1
2-yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl Triacetate (5b). Yellow powder. Yield 92%, mp 74–78ꢅC. IR (KBr, ꢇ, cm ):
1
1754, 1698, 1610, 1374, 1225,1091, 1044, 948, 909, 816, 713, 599, 509. H NMR (400 MHz, DMSO-d , , ppm, J/Hz):
6
7.13–7.32 (8H, m, ArH), 5.63 (1H, dd, J = J = 2.7, CH), 5.55 (1H, d, J = 8.2, CH), 5.20 (1H, s, CH), 5.05 (1H, dd, J = 2.8,
12
13
12
J
= 8.1, CH), 4.99 (dd, J = 2.7, J = 10.3, CH), 4.35–4.45 (1H, m, 1/2CH ), 4.15–4.26 (2H, m, CH + 1/2CH ), 3.61 (3H,
13
12 13 2 2
s, CH ), 3.40–3.47 (1H, m, 1/2CH ), 2.96 (1H, d, J = 16.9, 1/2CH ), 2.87 (1H, d, J = 16.9, 1/2CH ), 2.22–2.32 (1H, m, 1/2CH ),
3
2
2
2
2
2.29 (3H, s, CH ), 2.16 (3H, s, CH ), 2.02 (3H, s, CH ), 2.00 (3H, s, CH ), 1.99 (3H, s, CH ), 1.64–1.80 (1H, m, CH), 0.73
3
3
3
3
3
13
(3H, d, J = 6.6, CH ), 0.66 (3H, d, J = 6.6, CH ). C NMR (100 MHz, DMSO-d , , ppm): 19.32, 19.80, 20.32, 20.45, 20.70,
3
3
6
25.71, 41.28, 47.37, 52.97, 58.54, 61.80, 65.86, 66.51, 67.32, 68.77, 69.93, 95.57, 116.14, 125.81, 128.25, 129.85, 135.69,
+
139.70, 156.67, 169.10, 169.93, 169.99, 170.03, 170.95. HR-MS (ESI): calcd for C H O NS [M + H] 744.2690, found
37 45 13
+
744.2706; [M + Na] 766.2509, found 766.2530.
(2R,3R,4R,5R,6S)-2-(Acetoxymethyl)-6-(4-(1-cyclohexyl-3-((methylperoxy)methyl)-5-oxo-3-(p-
tolylthio)pyrrolidin-2-yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl Triacetate (5c). Yellow powder. Yield 94%, mp 80–84ꢅC.
–1
1
IR (KBr, ꢇ, cm ): 2857, 1754, 1694, 1610, 1510, 1412, 1373, 1224, 1091, 1044, 949, 909, 815, 715, 598, 509. H NMR
(400 MHz, DMSO-d , , ppm, J/Hz): 6.80–7.40 (8H, m, ArH), 5.63 (1H, dd, J = J = 2.8, CH), 5.57 (1H, d, J = 8.2, CH),
6
12
13
5.24 (1H, s, CH), 4.92–5.05 (2H, m, 2CH), 4.32–4.46 (1H, m, 1/2CH ), 4.12–4.28 (2H, m, CH + 1/2CH ), 3.55–3.64 (1H, m,
2
2
CH), 3.59 (3H, s, CH ), 2.97 (1H, d, J = 16.4, 1/2CH ), 2.80 (1H, d, J = 16.4, 1/2CH ), 2.23 (3H, s, CH ), 2.16 (3H, d, J = 3.9,
3
2
2
3
13
CH ), 2.03 (3H, s, CH ), 2.01 (3H, s, CH ), 1.99 (3H, s, CH ), 0.80–1.74 (10H, m, 5CH ). C NMR (100 MHz, DMSO-d ,
3
3
3
3
2
6
, ppm): 20.14, 20.32, 20.45, 20.70, 25.02, 25.11, 26.41, 29.51, 30.85, 40.60, 41.28, 51.83, 53.05, 59.91, 60.81, 64.69, 65.88,
67.32, 68.83, 69.93, 95.34, 115.88, 125.91, 129.78, 130.13, 130.97, 135.53, 136.08, 139.54, 156.40, 169.09, 169.93, 170.01,
+
+
170.14, 171.32. HR-MS (ESI): calcd for C H O NS [M + H] 770.2846, found 770.2866; [M + Na] 792.2666, found
39 47 13
792.2679.
(2R,3R,4R,5R,6S)-2-(Acetoxymethyl)-6-(4-(1-benzyl-3-((methylperoxy)methyl)-5-oxo-3-(p-tolylthio)pyrrolidin-
–1
2-yl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl Triacetate (5d). Yellow powder. Yield 91%, mp 72–76ꢅC. IR (KBr, ꢇ, cm ):
1
1754, 1601, 1510, 1412, 1373, 1225, 1091, 1045, 948, 910, 816, 704, 600, 508. H NMR (400 MHz, DMSO-d , , ppm, J/Hz):
6
7.10–7.35 (11H, m, ArH), 6.95 (2H, d, J = 3.2, ArH), 5.64 (1H, dd, J = J = 2.8, CH), 5.56 (1H, dd, J = 2.9, J = 8.2, CH),
12
13
12
13
5.04 (1H, dd, J = 2.9, J = 8.1, CH), 5.02 (1H, dd, J = 2.7, J = 10.3, CH), 4.92 (d, J = 16.1, CH, 1/2CH ), 4.89 (1H, s,
12
13
12
13
2
124

CH), 4.38–4.45 (1H, m, 1/2CH ), 4.16–4.24 (2H, m, CH + 1/2CH ), 3.51 (3H, s, CH ), 3.50 (1H, d, J = 14.1, 1/2CH ), 3.01
2
2
3
2
(1H, d, J = 18.1, 1/2CH ), 2.94 (1H, d, J = 18.0, 1/2CH ), 2.29 (3H, s, CH ), 2.16 (3H, d, J = 2.0, CH ), 2.03 (3H, s, CH ), 2.02
2
2
3
3
3
13
(3H, s, CH ), 2.00 (3H, s, CH ). C NMR (100 MHz, DMSO-d , , ppm): 20.33, 20.47, 20.70, 40.15, 41.28, 43.62, 52.88,
3
3
6
58.53, 61.82, 65.75, 65.88, 67.35, 68.77, 69.94, 95.58, 116.20, 125.70, 127.30, 127.46, 127.87, 128.53, 129.87, 135.78,
+
139.75, 156.78, 169.10, 169.93, 170.01, 170.77, 171.22. HR-MS (ESI): calcd for C H O NS [M + H] 778.2533, found
40 43 13
+
778.2509; [M + K] 816.2092, found 816.2025.
Synthesis of Compounds 6a–6d. Ammonia water (10 mL, 130 mmol) was heated and the ammonia dissolved in
CH OH (60 mL); an NH -methanol solution was obtained. Compound 5a (0.5 mmol) was dissolved in the NH -methanol
3
3
3
solution (5 mL) and stirred at room temperature. The resulting crystals were collected by filtration and purified by flash
chromatography (silica gel, chloroform–methanol–petroleum ether 5:1:2, v/v). A yellow powder was obtained 6a, 6c, 6d
(87% yield), and 6b (89% yield).
4-((Methylperoxy)methyl)-1-propyl-4-(p-tolylthio)-5-(4-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)- tetrahydro-2H-pyran-2-yl)oxy)phenyl)pyrrolidin-2-one (6a). Yellow powder. Yield 89%, mp 88–92ꢅC.
–1
1
IR (KBr, ꢇ, cm ): 3414, 2927, 1731, 1677, 1610, 1510, 1415, 1232, 1177, 1081, 1039, 897, 816, 691, 574, 508. H NMR
(400 MHz, DMSO-d , , ppm, J/Hz): 7.22 (2H, d, J = 7.5, ArH), 7.17–7.26 (4H, s, ArH), 7.07 (2H, d, J = 8.2, ArH), 5.17 (1H,
6
s, CH), 5.15 (1H, d, J = 7.8, CH), 5.10 (1H, d, J = 6.6, OH), 4.96 (1H, d, J = 2.9, OH), 4.67 (1H, d, J = 7.4, OH), 4.53 (1H, dd,
J
= 5.6, J = 11.0, OH), 3.93 (1H, m, 1/2CH ), 3.65–3.75 (2H, m, CH + 1/2CH ), 3.62 (3H, s, CH ), 3.40–3.52 (4H, m,
12
13 2 2 3
3CH + 1/2CH ), 2.93 (1H, d, J = 17.0, 1/2CH ), 2.87 (1H, d, J = 17.0, 1/2CH ), 2.42 (1H, m, 1/2CH ), 2.28 (3H, s, CH ),
2
2
2
2
3
13
1.18–1.40 (2H, m, CH ), 0.68 (3H, t, J = 7.4, CH ). C NMR (100 MHz, DMSO-d , , ppm): 10.78, 19.59, 20.71, 40.41,
2
3
6
41.70, 52.95, 58.59, 60.88, 66.09, 66.96, 70.18, 71.45, 74.62, 98.32, 98.49, 115.85, 126.01, 127.29, 129.83, 135.73, 139.62,
+
+
158.08, 158.14, 170.72, 171.52. HR-MS (ESI): calcd for C H O NS [M + H] 562.2111, found 562.2108; [M + Na]
28 35
9
584.1930, found 584.1931.
1-Isobutyl-4-((methylperoxy)methyl)-4-(p-tolylthio)-5-(4-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)- tetrahydro-2H-pyran-2-yl)oxy)phenyl)pyrrolidin-2-one (6b). Yellow powder. Yield 90%, mp 94–98ꢅC.
–1
IR (KBr, ꢇ, cm ): 3419, 2957, 2925, 1732, 1678, 1610, 1510, 1414, 1240, 1177, 1081, 1040, 898, 816, 689, 574, 508.
1
H NMR (400 MHz, DMSO-d , , ppm, J/Hz): 7.07–7.30 (6H, m, ArH), 7.06 (2H, d, J = 8.1, ArH), 5.18 (1H, s, CH), 5.16 (1H,
6
d, J = 13.2, CH), 5.10 (1H, d, J = 6.6, OH), 4.96 (1H, d, J = 3.2, OH), 4.66 (1H, d, J = 7.4, OH), 4.52 (1H, dd, J = 5.2, J = 9.8,
12
13
OH), 3.93 (1H, m, 1/2CH ), 3.64–3.76 (2H, m, CH + 1/2CH ), 3.62 (3H, s, CH ), 3.38–3.52 (4H, m, 3CH + 1/2CH ), 2.96
2
2
3
2
(1H, d, J = 16.9, 1/2CH ), 2.89 (1H, d, J = 16.9, 1/2CH ), 2.29 (3H, s, CH ), 1.66–1.78 (1H, m, CH), 0.73 (3H, d, J = 6.6, CH ),
2
2
3
3
13
0.66 (3H, d, J = 6.6, CH ). C NMR (100 MHz, DMSO-d , , ppm): 19.31, 19.82, 20.71, 25.71, 39.96, 40.31, 47.35, 52.97,
3
6
58.62, 60.89, 66.67, 66.96, 70.18, 71.44, 74.62, 98.31, 98.49, 115.95, 116.44, 125.92, 127.09, 129.85, 135.69, 139.64, 158.12,
+
+
158.19, 170.94, 171.57. HR-MS (ESI): calcd for C H O NS [M + H] 576.2267, found 576.2269; [M + Na] 598.2087,
29 37
9
found 598.2095.
1-Cyclohexyl-4-((methylperoxy)methyl)-4-(p-tolylthio)-5-(4-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)pyrrolidin-2-one (6c). Yellow powder. Yield 90%, mp 106–110ꢅC.
1
H NMR (400 MHz, DMSO-d , , ppm, J/Hz): 6.98–7.42 (8H, m, ArH), 5.20 (1H, s, CH), 5.15 (1H, t, J = 7.8, OH), 5.11 (1H,
6
d, J = 6.7, OH), 4.95 (1H, d, J = 3.1, OH), 4.67 (1H, d, J = 7.3, OH), 4.53 (1H, dd, J = 5.3, J = 10.7, OH), 3.93 (1H, m,
12
13
1/2CH ), 3.61–3.80 (2H, m, CH + 1/2CH ), 3.59 (3H, s, CH ), 3.35–3.54 (3H, m, 3CH), 2.99 (1H, d, J = 16.2, 1/2CH ), 2.79
2
2
3
2
13
(1H, d, J = 16.3, 1/2CH ), 2.30 (3H, s, CH ), 0.80–1.70 (10H, m, 5CH ). C NMR (100 MHz, DMSO-d , , ppm): 20.70,
2
3
2
6
24.69, 25.02, 29.51, 30.87, 40.66, 51.79, 53.06, 59.95, 60.86, 64.84, 66.92, 70.20, 71.45, 74.61, 98.31, 98.63, 115.61, 126.00,
–1
129.80, 135.54, 139.51, 157.95, 158.06, 170.17, 171.43. IR (KBr, ꢇ, cm ): 3427, 2929, 2856, 1732, 1673, 1610, 1510, 1411,
+
1230, 1177, 1081, 1040, 896, 815, 688, 578, 509. HR-MS (ESI): calcd for C H O NS [M + H] 602.2424, found 602.2421;
[M + Na] 624.2243, found 624.2239.
31 39
9
+
1-Benzyl-4-((methylperoxy)methyl)-4-(p-tolylthio)-5-(4-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)pyrrolidin-2-one (6d). Yellow powder. Yield 87%, mp 92–96ꢅC.
–1
1
IR (KBr, ꢇ, cm ): 3417, 2927, 1731, 1680, 1609, 1510, 1412, 1243, 1177, 1080, 1038, 888, 816, 704, 608, 507. H NMR
(400 MHz, DMSO-d , , ppm, J/Hz): 7.12–7.34 (9H, m, ArH), 7.08 (2H, d, J = 7.6, ArH), 6.95 (2H, d, J = 7.0, ArH), 5.17 (1H,
6
d, J = 7.9, CH), 5.11 (1H, d, J = 6.7, OH), 4.97 (1H, d, J = 3.7, OH), 4.92 (1H, dd, J = 4.5, J = 15.3, 1/2CH ), 4.85 (1H, s,
12
13
2
CH), 4.67 (1H, d, J = 7.4, OH), 4.53 (1H, t, J = 5.6, OH), 3.94 (1H, m, 1/2CH ), 3.61–3.80 (2H, m, CH + 1/2CH ), 3.51 (3H,
2
2
13
s, CH ), 3.40–3.58 (4H, m, 3CH + 1/2CH ), 2.98 (2H, s, CH ), 2.32 (3H, s, CH ). C NMR (100 MHz, DMSO-d , , ppm):
3
2
2
3
6
20.71, 40.19, 43.56, 52.87, 54.87, 58.65, 60.88, 65.85, 66.95, 70.19, 71.46, 74.68, 98.44, 98.50, 116.03, 125.82, 126.66,
125

+
127.28, 127.47, 128.55, 129.86, 135.76, 139.69, 158.27, 170.76, 171.32. HR-MS (ESI): calcd for C H O NS [M + H]
610.2111, found 610.2101; [M + Na] 632.1930, found 632.1937.
32 35
9
+
Cells and Culture. Human skov3 cell line was obtained from State Key of Biotherapy, Sichuan University.
Logarithmically growing skov3 cells were incubated with 0.05% trypsin at 37ꢅC for about 4 min until cells were nonadherent
and formed a single cell suspension. Trypsin activity was neutralized by adding a 20-fold excess of serum-containing medium.
Cells were cultured at 37ꢅC in an atmosphere of air and 5% CO .
2
AntitumorActivity. Antitumor activity of the compounds was determined by their positive action on cultured tumor
cells. Logarithmically growing cells were plated in 0.1 mL aliquots in 96-well microtiter plates. Cells were plated at an initial
density of about 50 000 cells/mL and allowed to acclimatize for 24 h. Cell suspensions were treated with various dilutions of
compounds in triplicate, mixed well, and allowed to incubate for 72 h at 37ꢅC in an atmosphere of air and 5% CO . To the cell
2
suspension was added 10 ꢀL of CCK-8 reagent, the mixture was incubated for 4 h at 37ꢅC in an atmosphere of air and 5% CO ,
2
and absorbance at 450 nm was read by the microplate reader. Control plates with serial dilutions of cell types were counted as
a control for the assay.
ACKNOWLEDGMENT
The authors are thankful to theAnalytical & Testing Center of Sichuan University, P. R. China for providing analytical
data, and Mr. Yang (State Key of Biotherapy, Sichuan University) for completing the antitumor activity test.
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1.
2.
3.
4.
5.
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L. H. Pearl and C. Prodromou, Annu. Rev. Biochem., 75, 271 (2006).
A. Koshi and S. Masakazu, Wo97/32579, 1997.
D. Ye, L. Fu, B. Liang, S. F. Yin, and Y. Li, Chin. J. Org. Chem., 11, 1768 (2009).
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