The Synthesis of Glycyrrhetinic Acid Derivatives Containing A Nitrogen Heterocycle and Their Antiproliferative Effects in Human Leukemia Cells

Abstract

Fifteen novel glycyrrhetinic acid derivatives containing a nitrogen heterocycle at C-30 and with different A-ring substituents were designed and synthesized. All of these derivatives have improved antiproliferative effects against human HL-60 leukemia cells. Compounds with a cyano-enone functionality on the A-ring exhibit greater growth inhibitory effects, compared to those with a 2-hydroxymethylene-3-keto, an isoxazole, or a 2-cyano-3-keto group. N-(2-cyano-3,11-dioxoolean-1,12-dien-30-yl)-4-piperidyl piperidine (9b) was found to be two-fold more potent than methyl 2-cyano-3,11-dioxooleana-1,12- dien-30-oate (CDODO-Me-11).

1. Introduction

18β-Glycyrrhetinic acid (GA, 3β-hydroxy-11-oxo-olean-12-ene-30-oic acid), the aglycone of glycyrrhizic acid isolated from licorice root, has been shown to have antitumor effects [1,2]. To improve its antitumor activities, some structural modifications were previously done. We found that introduction of an alkoxyimino group at position C-3 along with a free C-30 carboxyl group improved the antiproliferative effects of GA [3]. We and other groups have also found that introduction of a 2-cyano-1-en-3-one on the A-ring, modification of ring C by converting the 11-oxo-12-ene to 12-oxo-9(11)-en and/or methyl esterification of the C-30 carboxylic group, significantly improved the cytotoxic activities of GA [4,5,6]. Similar structural modifications have been performed in oleanolic acid and it was found that 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its methyl ester CDDO-Me were potent cytotoxic agents against tumor cells [7,8,9].

To determine the importance of introducing a cyano-enone at the A-ring and methyl esterification at C30, glycyrrhetinic acid derivatives containing a nitrogen heterocycle at C-30 and with some additional modifications of the A-ring were designed and synthesized. The antiproliferative abilities of these compounds were tested in human HL-60 leukemia cells and compared with that of GA, methyl 2-cyano-3,11-dioxo- oleana-1,12-dien-30-oate (CDODO-Me-11) and methyl-2-cyano-3,12-dioxo- oleana- 1,9(11)-dien-30- oate (CDODO-Me-12), which have been found to be potent antileukemia agents [4]. The synthesis of these compounds and their abilities to inhibit growth of HL-60 cells are presented in this communication.

2. Results and Discussion

2.1. Chemistry

Four groups of GA derivatives with a different A-ring structure and a replacement at the C-30 carboxylic group with a nitrogen heterocycle – piperidine, 4-piperidyl piperidine, 4-methyl piperazine, or piperazine – were synthesized starting from GA (Scheme 1). Compounds 1-3 were obtained using a routine method [5,9,10]. A benzyl group instead of a methyl group was used to protect the C-30 carboxylic acid as halogenolysis of the methyl ester with LiI in DMF gave an 11-oxo-13(18)-en by-product in approximately 20% yield [8].

The benzyl group of compound 3 was removed under mild conditions to give the key intermediate 4 in 76% yield, which was used as the precursor for generating the 2-hydroxymethylene-3-keto compounds 5a-5c and the isoxazole 6. Addition of oxalyl chloride to compound 4 in chloroform gave the corresponding acyl chloride. Amides 5a-5c were prepared in 50%–72% yield by condensation between the acyl chloride and the corresponding nitrogen heterocycle.

Using the same procedures employed for obtaining compounds 5a-5c, the isoxazole amides 7a-7d were obtained from isoxazole 6, which was prepared in 85% yield by refluxing compound 4 with hydroxylamine hydrochloride in glacial acetic acid. Under basic conditions compounds 7a-7d were isomerized to the corresponding 2-cyano-3-keto compounds 8a-8d, that were then dehydrogenated using dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in toluene to give the cyanoenone amides 9a-9d in fair to moderate yields.

Scheme 1. Synthetic routes to the target compounds.

Scheme 1. Synthetic routes to the target compounds.

2.2. HL-60 cell growth inhibition activity

The abilities of compounds 5a-5c, 7a-7d, 8a-8d and 9a-9d to inhibit growth of HL-60 cells were determined and compared to that of GA, CDODO-Me-11 and CDODO-Me-12 (Table 1). All of these new synthetic compounds had GI₅₀ values in the 0.5–20 μM range, which were much lower than that of GA (>40 μM). The activity order among these compounds was cyano-enone amides 9 > 2-hydroxy- methylene-3-keto compounds 5 > isoxazoles 7 and 2-cyano-3-keto compounds 8. Since compounds 8a-8d have decreased activities comparing to that of compounds 9a-9d, it suggests that a double bond between C-1 and C-2 is required for maintaining high activity in these cyano-enone amides. Compounds 9a, 9c and 9d have activities similar to that of CDODO-Me-11. The antiproliferative activity of N-(2-cyano-3,11-dioxoolean-1,12-dien-30-yl)-4-piperidyl piperidine (9b) is two-fold more than that of CDODO-Me-11, indicating that the methyl ester could be replaced to further improve the antiproliferative activities of these compounds. Although 9b has lower activity than that of CDODO-Me-12, which has a 12-oxo-9(11)-en structure in the C-ring, the simple synthesis of 9b compared to that of CDODO-Me-12 makes this compound potentially more useful. The antitumor effects of compound 9b in additional cell lines are worthy of further study.

Table 1. The antiproliferative activities of target compounds in HL-60 cells.

Compd.	R	IG50(μM)*	Compd.	R	IG50(μM)*
5a		5.5 ± 0.2	8c		17.9 ± 1.0
5b		3.3 ± 0.3	8d		11.9 ± 0.9
5c		6.1 ± 0.3	9a		1.4 ± 0.4
7a		1.7 ± 0.4	9b		0.8 ± 0.1
7b		7.7 ± 0.1	9c		1.2 ± 0.2
7c		7.9 ± 0.2	9d		1.7 ± 0.1
7d		8.2 ± 0.2	GA		>40
8a		8.6 ± 0.9	CDODO-Me-11		1.5 ± 0.1
8b		7.5 ± 0.7	CDODO-Me-12		0.4 ± 0.1

* The IG₅₀ values were obtained in HL-60 cells after treatments with various concentrations of these compounds for 3 days. The data shown are the mean plus standard deviation (SD) of three independent experiments.

Table 1. The antiproliferative activities of target compounds in HL-60 cells.

Compd.	R	IG50(μM)*	Compd.	R	IG50(μM)*
5a		5.5 ± 0.2	8c		17.9 ± 1.0
5b		3.3 ± 0.3	8d		11.9 ± 0.9
5c		6.1 ± 0.3	9a		1.4 ± 0.4
7a		1.7 ± 0.4	9b		0.8 ± 0.1
7b		7.7 ± 0.1	9c		1.2 ± 0.2
7c		7.9 ± 0.2	9d		1.7 ± 0.1
7d		8.2 ± 0.2	GA		>40
8a		8.6 ± 0.9	CDODO-Me-11		1.5 ± 0.1
8b		7.5 ± 0.7	CDODO-Me-12		0.4 ± 0.1

* The IG₅₀ values were obtained in HL-60 cells after treatments with various concentrations of these compounds for 3 days. The data shown are the mean plus standard deviation (SD) of three independent experiments.

3. Experimental

3.1. General

GA (purity over 98%) was purchased from Shanghai Haokang Chemicals Co. Ltd., China. Other reagents were bought from commercial suppliers in analytic grade and used without further purification, unless noted otherwise. ¹H-NMR and ¹³C-NMR spectra were recorded on a Bruker ARX-300 instrument with tetramethylsilane as an internal standard. Infra-red (IR) spectra were recorded on a Bruker IR-27G spectrometer. Mass spectra (MS) were determined on a Finnigan MAT/USA spectrometer (LC-MS). HRMS spectra were obtained on a Bruker micrOTOF-Q in an ESI mode. The melting points were determined on an electrically heated X4 digital visual melting point apparatus and are uncorrected. TLC plates (Alugram silica gel G/UV254) were purchased from Macherey-Nagel GmbH & Co.

3,11-Dioxoolean-12-en-30-oic acid (1). Jones’ reagent (15 mL) was added to a solution of GA (10.0 g, 21.2 mmol) in THF (35 mL). The solution was stirred at 0 ºC for 1 h and then was poured into H₂O (100 mL). The precipitate was filtered and dried to afford compound 2 (9.5 g, 95.6% yield) as a white solid. ¹H- NMR (CDCl₃) δ (ppm): 5.76 (1H, s, H-12), , 2.97–2.94 (1H, m, H-1), 2.63–2.61 (1H, m, H-1), 2.45 (1H, s, H-9), 1.38, 1.27, 1.23, 1.17, 1.10, 1.07, 0.86 (s, CH₃×7); LC-MS: 469.4 [M+H]⁺, 591.3 [M+Na]⁺.

Benzyl 3,11-dioxoolean-12-en-30-oate (2). BnBr (1.29 mL, 10.9 mmol) was added to a solution of 1 (5.0 g, 10.7 mmol) in DMF (50 mL). The mixture was stirred at 100 ºC for 3 h, cooled to room temperature and then poured into water (100 mL). The precipitate was filtered, washed with water to pH 7 and dried to give 2 (4.8 g, 80.9% yield) as a white solid. ¹H-NMR (CDCl₃) δ (ppm): 7.40–7.31 (5H, m, -CH₂Ph), 5.58 (1H, s, H-12), 5.21 (1H, d, J = 12.3 Hz, -CH₂Ph), 5.08 (1H, d, J = 12.3 Hz, -CH₂Ph), 2.97–2.94 (1H, m, H-1), 2.42 (1H, s, H-9), 2.37–2.33 (1H, m, H-1), 1.40, 1.36, 1.27, 1.16, 1.15, 1.06, 0.75 (s, CH₃×7); ¹³C-NMR (CDCl₃): δ (ppm) 216.9 (C-3), 199.1 (C-11), 176.7 (C-30), 170. 2 (C-13), 136.0 (C-12), 128.5 (Ph), 128.3 (Ph), 128.2 (Ph), 128.1 (Ph), 101.1 (C-2); LC-MS: 559.4 [M+H]⁺, 581.4 [M+Na]⁺.

Benzyl 2-hydroxymethylene-3,11-dioxoolean-12-en-30-oate (3). NaH (3.84 g, 160 mmol) was added slowly to a solution of 2 (5.6 g, 10.1 mmol) in ethyl formate (100 mL). The mixture was stirred at r.t. for 10 min and methanol was added until no bubbles were generated. The mixture was diluted with a mixture of CH₂Cl₂ and Et₂O (1:2) and washed three times with 5% aqueous HCl solution. The organic layer was evaporated in vacuum and purified on a silica gel column with cyclohexane-acetone (v/v) = 20:1 to give 3 (4.7 g, 80.0% yield) as a white solid. ¹H-NMR (CDCl₃) δ (ppm): 14.89 (s, 1H, OH), 8.61 (s, 1H, =CH-OH), 7.40-7.34 (m, 5H, -CH₂Ph), 5.60 (s, 1H, H-12), 5.21 (d, 1H, J = 12.3 Hz, -CH₂Ph), 5.07 (d, 1H, J = 12.3 Hz, -CH₂Ph), 3.45 (d, 1H, J = 14.7 Hz, H-1), 2.44 (s, 1H, H-9), 1.35, 1.19, 1.16, 1.15, 1.13, 1.12, 0.75 (s, CH₃×7); ¹³C-NMR (CDCl₃): δ (ppm) 199.7 (C-11), 189.8 (C-3), 189.1 (C=CH-OH), 176.4 (C-30), 170.1 (C-13), 128.9 (Ph), 128.8 (Ph), 128.6 (Ph), 128.5 (-Ph), 128.3 (C-12), 106.1 (C-2), 66.4 (-CH₂Ph), 59.8 (C-9); LC-MS: 585.3 [M-H]^-.

2-Hydroxymethylene-3,11-dioxoolean-12-en-30-oic acid (4). 10% Pd/C (0.14 g) was added to a solution of 3 (0.76 g, 1.3 mmol) in THF (20 mL). The mixture was stirred at r.t. for 8 hr and filtered to remove Pd/C. The filtrate was concentrated in vacuum and the residue was purified on a silica gel column with petroleum ether : acetone (v/v) = 20:1 to give 4 (0.49 g, 76.3% yield) as a pink solid. ¹H-NMR (CDCl₃) δ (ppm): 14.89 (s, 1H, =CH-OH), 8.64 (s, 1H, =CH-OH), 5.79 (s, 1H, H-12), 3.47 (d, 1H, J = 15.0 Hz, H-1), 2.46 (s, 1H, H-9), 1.39, 1.25, 1.21, 1.19, 1.15, 1.14, 0.88 (s, CH₃×7); ¹³C-NMR (CDCl₃): δ (ppm) 200.0 (C-11), 189.8 (C-3), 189.2 (C=CH-OH), 182.7 (C-30), 170.3 (C-13), 128.8 (C-12), 106.1 (C-2), 59.9 (C-9); LC-MS: 495.4 [M-H]^-.

3.2. General procedure for the preparation of 2-hydroxymethylene-30-amides 5a-5c

To a solution of 4 (2.34 g, 4 mmol) in chloroform (10 mL), oxalyl chloride (2 mL) was added. The mixture was stirred at r.t. for 1 hr and excess oxalyl chloride was removed by co-evaporation with hexane (three times). The obtained solid was dissolved in chloroform (30 mL) and an appropriate nitrogen heterocycle (6 mmol) was added. The mixture was stirred at r.t. for 5 min and washed three times with 5% aqueous HCl solution. The organic layer was dried over anhydrous MgSO₄. After MgSO₄ was removed by filtration, the filtrate was concentrated in vacuum and the residue was purified on a silica gel column with chloroform-methanol (v/v) = 50:1 to give a white solid. The Rf values were determined using TLC plates with chloroform-methanol (v/v) = 10:1.

N-(2-Hydroxymethylene-3,11-dioxoolean-12-en-30-yl)-piperidine (5a). Crystallization of the white solid from hexane and acetone (10:1) afforded white needles (1.6 g, 2.9 mmol). Yield: 72%; R_f = 0.4; m.p. 178–181 ºC; IR (KBr): 3425, 2927, 2867, 1612, 1446, 1417, 1382, 1362 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 14.89 (d, 1H, J = 3.0 Hz, =CH-OH), 8.64 (d, 1H, J = 3.0 Hz, =CH-OH), 5.80 (s, 1H, H-12), 3.65-3.53 (m, 4H), 3.48 (d, 1H, J = 14.1 Hz, H-1), 2.45 (s, 1H, H-9), 1.38, 1.23, 1.21, 1.18, 1.15, 1.14, 0.85 (s, CH₃×7); ¹³C-NMR (CDCl₃): δ (ppm) 200.4 (C-11), 189.8 (C-3), 189.2 (CH=CH-OH), 174.0 (C-30), 171.4 (C-13), 129.8 (C-12), 106.1 (C-2), 59.9 (C-9); LC-MS: 562.6 [M-H]^-; HRMS: m/z, calcd. for C₃₆H₅₂NO₄ (M-H) 562.3890. Found: 562.3882.

N-(2-Hydroxymethylene-3,11-dioxoolean-12-en-30-yl)-4-piperidyl piperidine (5b). Crystallization of the white solid from hexane and acetone (10:1) afforded white needles (1.8 g, 2.8 mmol). Yield: 70%; R_f = 0.3; m.p. 210–212 ºC; IR (KBr): 3426.1, 2947.7, 2650.1, 2527.8, 1633.5, 1456.8 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 14.89 (d, 1H, J = 3.0 Hz, =CH-OH), 8.63 (d, 1H, J = 3.0 Hz, =CH-OH), 5.73 (s, 1H), 4.54 (br, 2H), 3.45 (d, 1H, J = 14.7 Hz, H-1), 3.06-2.70 (m, 10H), 2.44 (s, 1H, H-9), 1.38, 1.23, 1.21, 1.17, 1.15, 1.13, 0.84 (s, CH₃×7); LC-MS: 665.4 [M+NH₄]⁺; HRMS: m/z, calcd. for C₄₁H₆₃N₂O₄ (M+H) 647.4782. Found: 647.4783.

N-(2-Hydroxymethylene-3,11-dioxoolean-12-en-30-yl)-4-methyl piperazine (5c). Crystallization of the white solid from hexane and acetone (5:1) afforded white needles (1.6 g, 2.7 mmol). Yield: 68%; R_f = 0.2; m.p. 155–157 ºC; IR (KBr): 3439, 2951, 2587, 1636, 1462, 1406, 1384 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 14.90 (d, 1H, J = 3.0 Hz, =CH-OH), 8.64 (d, 1H, J = 3.0 Hz, =CH-OH), 5.80 (s, 1H), 3.67 (br, 4H), 3.49 (d, 1H, J = 14.7 Hz, H-1), 2.50–2.38 (m, 5H), 2.33 (s, 3H, N-CH₃), 1.38, 1.23, 1.21, 1.18, 1.15, 1.14, 0.85 (s, CH₃×7); LC-MS: 577.4 [M-H]^-; HRMS: m/z, calcd. for C₃₆H₅₄N₂O₄ (M-H) 577.3999. Found: 577. 3990.

Isoxazolo[4, 5-b]olean-11-oxo 12-en-30-oic acid (6). A mixture of compound 4 (0.89 g, 0.9 mmol), NH₂OH^.HCl (0.36 g, 5.1 mol), and anhydrous sodium acetate (0.04 g, 0.49 mmol) were refluxed for 1.5 h in acetic acid (20 mL), cooled, and poured into ice-water. The precipitate was filtered, dried, and purified on a silica gel column with petroleum ether-acetone (v/v) = 20:1 to give 6 (0.74 g, 85.0% yield) as a white solid. ¹H-NMR (CDCl₃) δ (ppm): 12.07 (brs, 1H, -COOH), 8.03 (s, 1H, -CH=N-), 5.77 (s, 1H, H-12), 3.64 (d, 1H, J = 15.3 Hz, H-1), 2.56 (s, 1H, H-9), 1.41, 1.27, 1.25, 1.21, 1.18, 1.09, 0.86 (s, CH₃×7); LC-MS: 492.4 [M-H]^-.

3.3. General procedure for the preparation of isoxazolo[4,5-b]olean-11-oxo-12-en-30-amides 7a-7d

Oxalyl chloride (2 mL) was added to a solution of 6 (0.98 g, 2 mmol) in chloroform (10 mL). The mixture was stirred at r.t. for 1 hr and excess oxalyl chloride was removed by evaporation with hexane (three times). The obtained solid was dissolved in 30 mL chloroform and a nitrogen heterocycle (6 mmol) was added. The mixture was stirred at r.t. for 5 min and concentrated under vacuum. The residue was purified on a silica gel column with chloroform-methanol (v/v) = 50:1 to give a white solid. The Rf values were determined by TLC plates with chloroform-methanol (v/v) = 10:1.

N-(Isoxazolo[4,5-b]olean-11-oxo-12-en-30-yl)-piperidine (7a). Crystallization of the white solid from hexane afforded an amorphous solid (0.67 g, 60% yield). m.p. 148–151 ºC; R_f = 0.4; IR (KBr): 3429, 2929, 2853, 1628, 1462, 1411, 1383 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 7.99 (s, 1H, -CH=N-), 5.81 (s, 1H, H-12), 3.65 (d, 1H, J = 15.7 Hz, H-1), 3.59 (br, 4H), 2.54 (s, 1H, H-9), 2.05 (m, 4H), 1.42, 1.38, 1.32, 1.24, 1.22, 1.18, 0.88 (s, CH₃×7); ¹³C-NMR (CDCl₃): δ (ppm) 199.3 (C-11), 173.6 (C-30), 172.2 (C-13), 170.4 (C-3), 150.3 (C-C=N), 128.6 (C-12), 109.1 (C-2); LC-MS: 559.4 [M-H]^-; HRMS: m/z, calcd. for C₃₆H₅₁N₂O₃ (M-H) 559.3894. Found: 559.3903.

N-(Isoxazolo[4,5-b]olean-11-oxo-12-en-30-yl)-4-piperidyl piperidine (7b). Crystallization of the white solid from hexane afforded an amorphous solid (0.86 g, 67% yield). m.p. 158–162 ºC; R_f = 0.2; IR (KBr): 3439, 2932, 2853, 1724, 1630, 1456, 1413, 1383 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 7.99 (s, 1H, -CH=N-), 5.79 (s, 1H, H-12), 4.51 (br, 2H), 3.65 (d, 1H, J = 15.7 Hz, H-1), 2.56 (s, 1H, H-9), 1.38, 1.32, 1.24, 1.22, 1.18, 1.10, 0.83 (s, CH₃×6); ¹³C-NMR (CDCl₃): δ (ppm) 200.7 (C-11), 173.6 (C-30),172.2 (C-13), 170.3 (C-3), 150.4 (C-C=N), 128.6 (C-12), 109.1 (C-2); LC-MS: 644.4 [M+H]⁺; HRMS: m/z, calcd. for C₄₁H₆₂N₃O₃ (M+H) 644.4785. Found: 643.4717.

N-(Isoxazolo[4,5-b]olean-11-oxo -12-en-30-yl)-4-methyl piperazine (7c). Crystallization of the white solid from hexane afforded an amorphous solid (0.58 g, 51% yield). m.p. 157–159 ºC; R_f = 0.2; IR (KBr): 3436, 2939, 2790, 1722, 1633, 1460, 1410.8, 1383.5 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 7.99 (1H, s, -CH=N-), 5.81(s, 1H, H-12), 3.74 (br, 4H), 3.65 (d, 1H, J = 15.7 Hz, H-1), 2.53 (s, 3H, N-CH₃), 2.41 (br, 4H), 1.38, 1.32, 1.24, 1.22, 1.18, 1.10, 0.85 (s, CH₃×7); LC-MS: 576.5 [M+H]⁺, 598.5 [M+Na]⁺; HRMS: m/z, calcd. for C₃₆H₅₄N₃O₃ (M+H) 576.4159. Found: 576.4153.

N-(Isoxazolo[4,5-b]olean-11-oxo-12-en-30-yl)-piperazine (7d). Crystallization of the white solid from hexane afforded an amorphous solid (0.26 g, 50% yield). m.p. 216–218 ºC; R_f = 0.1; IR (KBr): 3427, 2968, 2866, 2467, 1725, 1637, 1459, 1409 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 7.99 (1H, s, -CH=N-), 5.80 (s, 1H, H-12), 3.68 (m, 4H), 3.65 (d, 1H, J = 15.3 Hz, H-1), 2.91 (m, 4H), 2.54 (s, 1H, H-9), 1.38, 1.32, 1.24, 1.22, 1.18, 1.10, 0.85 (s, CH₃×7); LC-MS: 562.3 [M+H]⁺; HRMS: m/z, calcd. for C₃₅H₅₂N₃O₃ (M+H) 562.4003. Found: 562.3999.

3.4. General procedure for the preparation of 2-cyano-3,11-dioxoolean-12-en-30-amides 8a-8d

NaOCH₃ (7.25 g, 134 mmol) was added to a solution of isoxazolo[4,5-b]-olean-11-oxo-12-en- 30-amide (3.9 mmol) in MeOH (60 mL) and Et₂O (125 mL). The mixture was stirred at r.t. for 8 hr and was extracted with a mixture of CH₂Cl₂ and Et₂O (1:2). The extract was washed three times with 5% aqueous HCl solution and the acidic washings were re-extracted with a mixture of CH₂Cl₂ and Et₂O (1:2). The combined organic layers were evaporated in vacuum and the precipitate was purified on a silica gel column with chloroform-methanol (v/v) = 50:1 to give a white solid. The Rf values were determined by TLC plates with chloroform-methanol (v/v) = 10:1.

N-(2-Cyano-3,11-dioxoolean-12-en-30-yl)-piperridine (8a). Crystallization of the white solid from hexane and EtOAc (10:1) afforded an amorphous solid (1.81 g, 83% yield). m.p. 163–165 ºC; R_f = 0.4; IR (KBr): 3434, 2931, 2855, 2359, 2204, 1722, 1629 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 5.77 (s, 1H, H-12), 3.89 (m, 1H, H-2), 3.57 (br, 4H), 3.32 (m, 1H, H-1), 2.37 (s, 1H, H-9), 2.08 (m, 6H), 1.39, 1.35, 1.34, 1.22, 1.21, 1.10, 0.83 (s, CH₃×7); ¹³C-NMR (CDCl₃): δ (ppm) 207.4 (C-3), 197.8(C-11), 173.5(C-30), 171.3(C-13), 128.3 (C-12), 117.1(-CN), 79.6 (C-2), 61.3 (C-9); LC-MS: 559.4 [M-H]^-; HRMS: m/z, calcd. for C₃₆H₅₃N₂O₃ (M-H) 559.3894. Found: 559.3898.

N-(2-Cyano-3,11-dioxoolean-12-en-30-yl)-4-piperidyl piperidine (8b). Crystallization of the white solid from hexane and EtOAc (10:1) afforded an amorphous solid (1.91 g, 76% yield). m.p. 189–191 ºC; R_f = 0.3; IR (KBr): 3423, 2948, 2647, 2528, 2202, 1627, 1457, 1417 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 5.74 (s, 1H, H-12), 4.53 (br, 2H), 3.96 (m, 1H, H-2), 3.55 (m, 1H, H-1), 2.88-2.76 (br, 8H), 2.44-2.38 (m,4H), 2.36 (s, 1H, H-9),1.37, 1.35, 1.22, 1.18, 1.16, 1.11, 0.82 (s, CH₃×7); ¹³C-NMR (CDCl₃): δ (ppm) 205.2 (C-3), 198.6 (C-11), 173.8 (C-30), 170.8 (C-13), 128.1 (C-12), 117.1 (-CN), 79.5 (C-2), 63.1 (C-9); LC-MS: 644.7 [M+H]⁺; HRMS: m/z, calcd. for C₄₁H₆₂N₃O₃ (M+H) 644.4785. Found: 644.4789.

N-(2-Cyano-3,11-dioxoolean-12-en-30-yl)-4-methyl piperazine (8c). Crystallization of the white solid from hexane and EtOAc (10:1) afforded an amorphous solid (1.50 g, 67% yield). m.p. 197–199 ºC; Rf = 0.2; IR (KBr): 3425, 2947, 2792, 2202, 1719, 1633, 1460, 1410, 1383 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 5.78 (s, 1H, H-12), 3.97 (m, 1H, H-2), 3.80 (br, 4H), 3.59 (m, 1H, H-1), 2.55 (s, 3H, N-CH₃), 2.54 (s, 1H, H-9), 1.43, 1.38, 1.35, 1.18, 1.17, 1.15, 0.83 (s, CH₃×7); ¹³C-NMR (CDCl₃): δ (ppm) 205.2 (C-3), 198.6 (C-11), 173.9 (C-30), 170.7 (C-13), 128.2 (C-12), 117.1(-CN), 60.6 (C-9); LC-MS: 576.4 [M+H]⁺; HRMS: m/z, calcd. for C₃₆H₅₄N₃O₃ (M+H) 576.4159. Found: 576.4160.

N-(2-cyano-3,11-dioxoolean-12-en-30-yl)-piperazine (8d). Crystallization of the white solid from hexane and EtOAc (10:1) afforded an amorphous solid (0.88 g, 60% yield). m.p. 260–263 ºC; R_f = 0.1; IR (KBr): 3423, 2950, 2466, 2203, 1718, 1633, 1459 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 5.77 (s, 1H, H-12), 3.97 (m, 1H, H-2), 3.71 (br, 4H), 3.62 (m, 1H, H-1), 2.95 (br, 4H), 2.41 (s, 1H, H-9), 1.43, 1.38, 1.35, 1.23, 1.18, 1.09, 0.83 (s, CH₃×7); LC-MS: 562.6 [M+H]⁺; HRMS: m/z, calcd. for C₃₅H₅₂N₃O₃ (M+H) 562.4003. Found: 562.4009.

3.5. General procedure for the preparation of 2-cyano-3,11-dioxoolean-1,12-dien-30-amide derivatives 9a-9d

A mixture of 2-cyano-3,11-dioxoolean-12-en-30-amide derivative (3 mmol) and DDQ (98%) (0.77 g, 3.32 mmol) in dry toluene (80 mL) was heated under 80 ºC for 30 min. After insoluble material was removed by filtration, the filtrate was evaporated under vacuum to give a brown solid. The solid was purified on a silica gel column with chloroform-methanol (v/v) = 50:1. The R_f values were determined by TLC plates with chloroform-methanol (v/v) = 10:1.

N-(2-Cyano-3,11-dioxoolean-1,12-dien-30-yl)-piperidine (9a). Crystallization of the white solid from hexane and EtOAc (10:1) afforded an amorphous solid (0.94 g, 56% yield). m.p. 187–190 ºC; R_f = 0.4; IR (KBr): 3428, 2933, 2856, 2233, 1687, 1626, 1462 1412 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 8.41 (s, 1H, H-1), 5.74 (s, 1H, H-12), 3.57 (br, 4H), 2.61 (s, 1H, H-9), 1.51, 1.37, 1.29, 1.21, 1.20, 1.15, 0.85 (s, CH₃×7); ¹³C-NMR (CDCl₃): δ (ppm) 197.6 (C-3), 197.5 (C-11), 176.1 (C-30), 172.5 (C-13), 168.5 (C-1), 123.4 (C-12), 114.8 (-CN), 113.3 (C-2), 53.4 (C9); LC-MS: 557.4 [M-H]^-, 593.5 [M+Cl]^-; HRMS: m/z, calcd. for C₃₆H₅₀N₂O₃ (M-H) 557.3737. Found: 5578.3733.

N-(2-Cyano-3,11-dioxoolean-1,12-dien-30-yl)-4-piperidyl piperidine (9b). Crystallization of the white solid from hexane and EtOAc (10:1) afforded an amorphous solid (0.98 g, 51% yield). m.p. 218–220 ºC; R_f = 0.3; IR (KBr): 3431, 2949, 2524, 2233, 1684, 1627, 1416 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 8.40 (s, 1H, H-1), 5.73 (s, 1H, H-12), 4.58 (br, 2H), 3.14 (m, 1H), 2.85-2.78 (m, 4H), 2.61 (s, 1H, H-9), 2.27 (br, 4H), 1.51, 1.37, 1.31, 1.20, 1.19, 1.15, 0.83 (s, CH₃×7); ¹³C-NMR (CDCl₃): δ (ppm) 197.6 (C-3), 197.5 (C-11), 176.4 (C-30), 172.4 (C-13), 168.2 (C-1), 123.5 (C-12), 114.9 (-CN), 113.3(C-2); LC-MS: 642.6 [M+H]⁺; HRMS: m/z, calcd. for C₄₁H₆₀N₃O₃ (M+H⁺) 642.4629. Found: 642.4626.

N-(2-Cyano-3,11-dioxoolean-1,12-dien-30-yl)-4-methyl piperazine (9c). Crystallization of the white solid from hexane and EtOAc (10:1) afforded an amorphous solid (0.74 g, 43% yield). m.p. 185–187 ºC; R_f = 0.3; IR (KBr): 3438, 2950, 2680, 1719, 1685, 1633, 1461, 1408, 1384 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 8.40 (s, 1H, H-1), 5.73 (s, 1H, H-12), 3.81 (br, 4H), 2.64 (br, 4H), 2.47 (s, 3H, -NCH₃), 1.51, 1.38, 1.30, 1.21, 1.20, 1.15, 0.74 (s, CH₃×7); LC-MS: 574.5[M+H]⁺; HRMS: m/z, calcd. for C₃₆H₅₂N₃O₃ (M+H) 574.4003. Found: 574.3997.

N-(2-Cyano-3,11-dioxoolean-1,12-dien-30-yl)-piperazine (9d). Crystallization of the white solid from hexane and EtOAc (10:1) afforded an amorphous solid (0.59 g, 40% yield). m.p. 201–203 ºC; R_f = 0.1; IR (KBr): 3426, 2951, 2468, 2233, 1721, 1686, 1638, 1459, 1384 cm^-1; ¹H-NMR (CDCl₃): δ (ppm) 8.39 (s, 1H, H-1), 5.72 (s, 1H, H-12), 3.65 (br, 4H), 2.91 (br, 4H), 2.61 (s, 1H, H-9), 1.51, 1.49, 1.37, 1.30, 1.21, 1.20, 1.15, 0.74 (s, CH₃×7); ¹³C-NMR (CDCl₃): δ (ppm) 197.3 (C-3), 197.2 (C-11), 176.1 (C-30), 172.1 (C-13), 167.9 (C-1), 123.2 (C-12), 114.6 (-CN), 113.1 (C-2); LC-MS: 560.5 [M+H]⁺; HRMS: m/z, calcd. for C₃₅H₅₀N₃O₃ (M+H) 560.3846. Found: 560.3853.

3.6. Cell culture

HL-60 cells were cultured in RPMI 1640. The media were supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L-glutamine, and 10% (v/v) heat-inactivated fetal bovine serum.

3.7. Cell growth inhibition assay

All compounds were dissolved in DMSO. A stock solution of 20 mmol/L of each compound was prepared in DMSO and stored in aliquots at -20 ºC. A working solution was diluted with ethanol and fresh medium before assaying. The final concentration of ethanol in the medium was less than 1% and the final concentration of DMSO was less than 0.1%. Cells were seeded at a density of 4 × 10⁴ cells/mL in 24 well plates with various concentrations of the tested compounds and incubated for 3 days. Total cell number in each group was determined using a hemocytometer. The cell growth inhibitory ability was expressed as the ratio of the cell number in the groups treated with the compounds to that of cells treated with DMSO and/or ethanol. The concentration (GI₅₀) which inhibited half of cell growth was calculated.

4. Conclusions

Fifteen novel glycyrrhetinic acid derivatives containing a nitrogen heterocycle at C-30 and with different substituents on the A-ring were designed and synthesized. The antiproliferative effects of these compounds were determined in HL-60 cells. The results reveal that: 1) introduction of the cyano-enone moiety into the A-ring of GA significantly improves the antiproliferative activities in leukemia cells and 2) N-(2-cyano-3,11-dioxoolean-1,12-dien-30-yl)-4-piperidyl piperidine (9b) is the most active compound among these novel GA derivatives with a 11-oxo-12-en structure.