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Abstract

3-Ethyl substituted triterpenoids with a fragmented and 5-membered A ring were synthesized from the lupane ketoxime via the Beckmann reaction and intramolecular oxonitrile cyclization, respectively. Transformations of the triterpenic isopropylidene fragment with the formation of C(30) and C(20) modified derivatives were performed by either allylic C(30) oxidation or ozonolytic cleavage of the C(20)-C(29) double bond. Antitumor activity of the synthesized compounds was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,3-diphenyl-tetrazolium bromide test against 7 human cancer cell lines. Methyl 1-cyano-3-ethyl-3-oxo-2,3-seco-2-norlup-20(29)-en-30-al-28-oate (4), methyl 3-[1-bromoethyl]-1-cyano-3-oxo-2,3-seco-2-norlup-20(29)-en-30-al-28-oate (6), and methyl 1-cyano-3-ethyl-2-norlup-1(3),20(29)-dien-30-al-28-oate (9) were selected as compounds with highest cytotoxicity (IC₅₀ 1.38-15.91 μM).

Keywords

betulinic acid A-secotriterpenoids Beckmann fragmentation oxonitrile cyclization cytotoxicity

Native and semisynthetic derivatives of betulinic acid with a fragmented, expanded, or contracted A ring are of interest as either structurally original biological active compounds or as synthetic platforms for their preparation.^1
-3 In nature, triterpenic biosynthetic pathways with a 5-membered A ring formation most often are based on cyclizations of the corresponding A-secotriterpenic precursors.¹ The same approach is successfully used in synthetic organic chemistry to obtain A-pentacyclic triterpenoids, including analogs of natural biologically active products. Thus, the synthesis of natural epiceanotic acid with high anti-HIV-1 properties was carried out on the basis of the 2,3-dicarboxy-2,3-secolupane derivative.⁴

The Grignard alkylation reaction is successfully used in the chemistry of triterpenoids to modify the C(3) atom of the native ring A and to increase the level of biological properties of the natural metabolite betulinic acid.^5

-8 So, 3-ethynyl derivatives of betulinic acid with significant cytotoxic and anti-inflammatory activities were obtained.^5,6 Genet et al^7,8 synthesized a series of betulinic acid derivatives substituted at the C(3) atom by, for example, allyl, methylallyl, cyclopropyl, and aryl radicals via reactions with various Grignard reagents. The C(3) modification of triterpenoids by introducing small substituents was found to lead to the formation of selective TGR5 agonists.

Our group has reported^9

-12 that C(3) methyl substitution by Grignard reaction allows the modification of A-seco- and A-pentacyclic triterpenoids and the synthesis of novel biologically active derivatives. Thus, 2,3-seco-18αH-oleanane 3-methyl-3-ketone combining high virus-inhibitory activities against herpes simplex virus type 1 and HIV-1,⁹ and 2,3-secolupane α-monobromomethyl ketone with a high antitumor activity^10,11 were synthesized. Recently, we also described the synthesis of C(3)-ethyl substituted 18-αH-oleanane derivatives with weak cytotoxicity.¹² Therefore, to analyze comparatively reactivity and cytotoxic potential, we synthesized a series of lupane 3-ethyl substituted derivatives with a 2,3-fragmented and 5-membered ring A including products with a modified isopropylidene fragment. 3-(4,5-Dimethylthiazol-2-yl)-2,3-diphenyl-tetrazolium bromide (MTT) screening for cytotoxic activity, as well as a structure-activity relationship study of the semisynthetic products, were conducted.

3-Ethyl-substituted hydroxyimino ketone 2 was obtained by Grignard reductive alkylation of lupane α-ketoxime 1 with freshly prepared С₂H₅MgBr (Scheme 1). Then, Beckmann fragmentation of the C(2)-C(3) bond of compound 2 in the SOCl₂-CH₂Cl₂ system resulted in 2,3-secotriterpenic 3-ethyl-3-ketone 3. Introduction of a carbonyl substituent into the isopropylidene fragment of 3-ethyl-3-ketone 3 was carried out via either allylic oxidation by the H₂SeO₃-dioxane system with formation of the corresponding C(30) aldehyde 4 or by ozonolytic cleavage of the C(20)-C(29) double bond to 20-methyl-3-ethyldiketone 5. Pyridinium bromide perbromide (C₅H₆Br₃N) was used as a brominating agent for the preparation of α-monobromoethyl ketone 6 on the basis of C(30) aldehyde 4.¹⁰

Scheme 1

Synthesis of 3-ethyl substituted lupane derivatives with a 2,3-fragmented and 5-membered ring A.

A possibility was studied of intramolecular nitrile-anionic cyclization of 2,3-secotriterpenic derivatives 3 to 6 under conditions of basic catalysis. When 3-ethyl-3-ketone 3 or 20-methyl-3-ethyldiketone 5 was boiled in a t-BuOK-t-BuOH mixture, reactions proceeded via the oxo-nitrile mechanism with formation of the corresponding A-pentacyclic 3-ethyl α,β-alkenenitriles 8 and 7 with 44% to 45% yields. It should be noted that under the same conditions, both 3-ethyl-3-ketone 4 and α-bromoethyl ketone 6 with a C(30) aldehyde group produced a multicomponent mixture of products that could not be separated by column chromatography (CC). In this regard, the C(30) aldehyde derivative 9 was prepared by direct introduction of the carbonyl moiety into the structure of α,β-alkenenitrile 8. It should be noted that direct ozonolytic cleavage of the C(20)-C(29) double bond of α,β-alkenenitrile 8 did not lead to the formation of the corresponding C(20) methyl ketone 7. As a result of the reaction (from the Thin-layer chromatography (TLC) data), a mixture of highly polar products was formed, as was in the previously described case with C(3) unsubstituted alkenenitrile 10 ¹³ (Figure 1).

Figure 1

Previously synthesized A-seco- and A-pentacyclotriterpenoids 10¹⁴ and 11 to 13.¹⁰

Cytotoxic screening was conducted and resulted—by the MTT assay with a panel of 7 human tumor cell lines—in the selection of triterpenoids 4, 6, and 9 with high cytotoxic activity (IC₅₀ 1.38-15.91 μM) among the synthesized compounds (Table 1).

Table 1

Cytotoxic Activities of Compounds 2 to 9 and 11 to 13 Against Human Tumor Cell Lines.

	IC₅₀, μM (mean ± SD)
	HEp-2	HCT 116	MS	RD TE32	A549	MCF-7	MDA-MB-231
2	>100	82.73 ± 10.05	74.08 ± 28.10	>100	>100	>100	>100
3	>100	>100	>100	>100	>100	>100	>100
4	15.91 ± 2.33	5.83 ± 1.22	7.07 ± 0.94	5.41 ± 2.37	7.93 ± 0.27	5.71 ± 0.76	7.48 ± 0.08
5	>100	>100	>100	>100	>100	88.18 ± 25.02	>100
6	11.86 ± 1.86	5.34 ± 0.16	7.32 ± 1.02	2.96 ± 1.40	7.18 ± 0.57	5.01 ± 0.29	6.72 ± 0.73
7	>100	>100	>100	>100	>100	>100	>100
8	>100	>100	>100	>100	>100	>100	>100
9	6.41 ± 0.11	2.73 ± 0.12	4.50 ± 0.06	1.38 ± 0.23	5.28 ± 0.38	4.88 ± 0.25	3.66 ± 0.46
11	52.14 ± 6.43	7.30 ± 0.86	5.48 ± 0.54	5.04 ± 0.47	15.36 ± 0.57	6.65 ± 0.28	ND
12	5.28 ± 0.90	10.35 ± 0.73	3.41 ± 0.31	3.84 ± 0.74	25.44 ± 1.66	6.34 ± 1.02	15.23 ± 5.31
13	17.39 ± 0.94	7.09 ± 0.68	8.59 ± 0.73	21.82 ± 1.54	36.03 ± 8.22	27.47 ± 2.90	ND
DOX^a	1.78 ± 0.31	1.96 ± 0.19	1.29 ± 0.16	1.27 ± 0.03	2.04 ± 0.22	0.14 ± 0.03	0.34 ± 0.03

^aDoxorubicin—reference drug.

As seen from Scheme 1, the active compounds contain the C(30) modified isopropylidene fragment in their structure which can act as a typical Michael acceptor in nonspecific interactions with proteins and, probably, reflects their toxicity.¹⁵ At the same time, the sensitivity of HEp-2, HCT 116, and A549 cells exposed to the action of 3-ethyl substituted derivatives 4 and 6 has increased markedly as compared with the 3-methyl substituted analogs 11 and 12 ¹⁰ (Figure 1). Moreover, the cytotoxic effects of 3-ethyl-substituted α,β-alkenenitrile 9 (IC₅₀ 1.38-6.41 μM) in some cases were almost 20-fold higher than those of the 3-methyl substituted analog 13.¹⁰ Thus, the performed study showed that elongation of the C(3) alkyl chain led to a rise in the antitumor level of the semisynthetic triterpenoids with a fragmented and, especially, 5-membered ring A.

Experimental

General

Infrared spectroscopy (IR) spectra of the compounds dissolved in CHCl₃ were recorded on a Bruker 66/S IFS Fourier spectrometer (Bruker, Germany). The ¹H and ¹³C Nuclear magnetic resonance (NMR) spectra of the compounds dissolved in CDCl₃ were recorded on a Bruker AVANCE II spectrometer (Bruker BioSpin GmbH, Germany) at 400 and 100 MHz, respectively. Chemical shifts (δ) were expressed in parts per million (ppm) relative to Tetramethylsilane (TMS) as an internal standard. Optical rotation was measured on a Perkin Elmer 341 polarimeter (Perkin Elmer, United States) using sodium D light for CHCl₃ solutions. Mass spectra (MS) of compounds 3 to 5, 8, and 9 were determined on an Agilent 6890N/5975B chromatograph (Agilent Technologies, United States) equipped with a DB-35ms capillary column (4 m × 0.25 mm, 0.25 µm; 70 eV electron impact). Melting points were measured using an OptiMelt MPA100 (Stanford Research Systems, United States) instrument at a heating rate of 1°C/min. Column chromatography was carried out on a “Macherey-Nagel” (60-200 μm); eluent light petroleum/ethyl acetate. The reactions were monitored by TLC using Sorbfil plates (Russia). The solvents were purified and dried in accord with standard procedures.

Synthesis of Compound (2)

Compound 1 (2.5 mmol) in small portions was added to a solution of freshly prepared C₂H₅MgBr (5.0 mmol) in dry Et₂O (10 mL), and then Tetrahydrofuran (THF) (4 mL), as well as Et₂O (6 mL), was slowly added dropwise. The reaction mixture was stirred under heating for 1 hour, and then the mixture was cooled to room temperature. Ice water (15 mL), as well as a mix of HCl:H₂O (1:1, 10 mL), was added dropwise; after that, the resulting mixture was stirred for 1 hour until the precipitate was completely dissolved. The reaction product was extracted with ethyl acetate (3 × 20 mL). The organic layer was separated, washed with a saturated solution of NaHSO₃, then with a solution of 5% NaHCO₃ and a small amount of water. The organic layer was dried (MgSO₄), filtered, and evaporated to dryness. The residue was subjected to silica gel CC (eluent: light petroleum/ethyl acetate 10:1) to yield product 2.

Methyl 3-Ethyl-3β-Hydroxy-2-Hydroxyimino-Lup-20(29)-en-28-oate (2)

Yield: 42%.

R _f : 0.6 (chloroform-methanol 20:1).

MP: 101.4°С (light petroleum/ethyl acetate 10:1).

[α]_D: +22.2 (с 0.9; CHCl₃).

IR ν (CHCl₃) cm^–1: 1642 (C= N), 1726 (СООСН₃), 3330 and 3423 (ОН).

¹H NMR (400 MHz, CDCl₃): δ 0.66 (3Н, t, J = 7.5 Hz, H-33), 0.69, 0.71, 0.85, 0.88, 0.94 (15H, 5s, CH₃ × 5), 1.62 (3Н, s, H-30), 1.83 and 3.30 (2H, 2d, J = 12.0 Hz, H-1), 2.93 (1H, td, J = 10.9, 4.4 Hz, Н-19), 3.61 (3H, s, CООСH₃), 4.54 and 4.67 (2H, 2s, H-29).

¹³C NMR (100 MHz, CDCl₃): δ 7.36, 14.84, 15.75, 16.85, 18.89, 18.96, 19.38, 21.26, 23.96, 25.40, 26.68, 29.68, 29.73, 30.62, 32.18, 34.32, 36.95, 38.26, 41.16, 41.36, 42.52, 44.87, 46.96, 49.54, 50.48, 51.25, 53.14, 56.56, 77.20, 109.66 (C-29), 150.42 (C-20), 161.68 (C-2), 176.63 (C-28).

Synthesis of Compound (3)

Compound 2 (1.1 mmol) was dissolved in anhydrous CH₂Cl₂ (50 mL) with stirring and treated with SOCl₂ (3.4 mmol). The reaction mixture was then stirred for 30 minutes at room temperature. After completion of the reaction (as indicated by TLC), the solvent was removed under reduced pressure. The residue was washed with CH₂Cl₂ and then the solvent was evaporated. The procedure was repeated twice. The crude product was purified by silica gel CC (eluent: light petroleum/ethyl acetate 15:1).

Methyl 1-Cyano-3-Ethyl-3-Oxo-2,3-Seco-2-Norlup-20(29)-en-28-oate (3)

Yield: 54%.

R _f : 0.7 (chloroform-methanol 20:1).

MP: 135.1°С (light petroleum/ethyl acetate 15:1).

[α]_D: +28.3 (с 0.4; CHCl₃).

IR ν (CHCl₃) cm^–1: 1704 (C=O), 1724 (СООСН₃), 2239 (C≡N).

¹H NMR (400 MHz, CDCl₃): δ 0.86, 0.89, 0.97, 1.10, 1.17 (15H, 5s, CH₃ × 5), 1.02 (3Н, t, J = 7.2 Hz, H-33), 1.62 (3Н, s, H-30), 2.31 and 2.48 (2H, 2d, J = 18.5 Hz, H-1), 2.49 and 2.68 (2Н, 2dq, J = 17.7, 7.2 Hz, H-32), 2.93 (1H, td, J = 10.6, 4.6 Hz, Н-19), 3.59 (3H, s, CООСH₃), 4.55 and 4.67 (2H, 2s, H-29).

¹³C NMR (100 MHz, CDCl₃): δ 8.71, 14.64, 15.66, 18.80, 19.21, 21.59, 21.76, 23.86, 24.13, 25.47, 29.50, 29.64, 30.33, 30.48, 31.89, 33.28, 36.80, 38.25, 40.52, 42.42, 42.80, 45.23, 46.91, 48.79, 49.23, 51.21, 52.89, 56.52, 109.82 (C-29), 118.54 (C-2), 150.23 (C-20), 176.54 (C-28), 216.78 (C-3).

GC-MS (m/z): 509.4 (M⁺).

General Procedure for the Preparation of Compounds 4 and 9

Compound 3 or 8 (3.0 mmol) was dissolved in 30 mL of 1,4-dioxane, whereupon H₂SeO₃ (9.0 mmol) was added. The reaction mixture was refluxed for 15 minutes. Completeness of the reaction was monitored by TLC. After cooling at room temperature, the precipitated Se was filtered off, and 1,4-dioxane was evaporated. The residue was diluted with water and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed consistently with solution of 5% NaHCO₃ and with water, and then dried (MgSO₄), filtered, and evaporated to dryness. The residue was subjected to silica gel CC to yield compound 4 (eluent: light petroleum/ethyl acetate 10:1) or 9 (eluent: light petroleum/ethyl acetate 20:1).

Methyl 1-Cyano-3-Ethyl-3-Oxo-2,3-Seco-2-Norlup-20(29)-en-30-al-28-oate (4)

Yield: 30%.

R _f value 0.4 (light petroleum/ethyl acetate 7:3).

MP: 159.2°С (light petroleum/ethyl acetate 10:1).

[α]_D: +20.8 (с 0.3; CHCl₃).

IR ν (CHCl₃) cm^–1: 1694 (C=O), 1723 (СООСН₃), 2239 (C≡N).

¹H NMR (400 MHz, CDCl₃): δ 0.84, 0.86, 0.93, 1.09, 1.15 (15Н, 5s, CH₃ × 5), 1.02 (3Н, t, J = 7.2 Hz, H-33), 2.29 and 2.43 (2H, 2d, J = 18.1 Hz, H-1), 2.48 and 2.67 (2Н, 2dq, J = 17.5, 7.2 Hz, H-32), 3.26 (1H, td, J = 11.0, 4.9 Hz, Н-19), 3.60 (3H, s, CООСH₃), 5.85 and 6.20 (2H, s, H-29), 9.46 (3Н, s, H-30).

¹³C NMR (100 MHz, CDCl₃): δ 8.69, 14.56, 15.62, 18.78, 21.51, 21.71, 22.63, 24.04 (2C), 27.12, 29.43, 29.58, 29.65 (2C), 30.36, 31.72, 33.28, 36.70, 38.11, 40.46, 42.38, 42.74, 45.08, 48.87, 51.32, 52.82, 56.64, 118.49 (C-2), 133.71 (C-29), 156.29 (C-20), 176.32 (C-28), 194.75 (C-30), 216.79 (C-3).

GC-MS (m/z): 523.4 (M⁺).

Methyl 1-Cyano-3-Ethyl-2-Norlup-1(3),20(29)-Dien-30-al-28-oate (9)

Yield: 42%.

R _f : 0.6 (light petroleum/ethyl acetate 7:3).

MP: 68.9°С (light petroleum/ethyl acetate 20:1).

[α]_D: +14.5 (с 1.1; CHCl₃).

IR ν (CHCl₃) cm^–1: 1693 (C=O), 1725 (СООСН₃), 2205 (C≡N).

¹H NMR (400 MHz, CDCl₃): δ 0.93, 1.04, 1.08 (9H, 3s, CH₃ × 3), 0.94 (6Н, s, 2CH₃), 1.12 (3Н, t, J = 7.7 Hz, H-33), 2.27 (2Н, dq, J = 1.3, 7.7 Hz, H-32) 3.28 (1H, td, J = 11.1, 4.9 Hz, Н-19), 3.68 (3H, s, CООСH₃), 5.89 and 6.26 (2H, 2c, H-29), 9.51 (3Н, s, H-30).

¹³C NMR (100 MHz, CDCl₃): δ 13.72, 14.73, 17.32 (2C), 18.80, 20.48, 20.96, 22.17, 26.76, 27.19, 29.66, 31.77, 32.14, 34.99, 36.84, 38.09, 42.32, 42.82, 46.98, 47.32, 50.24, 50.68, 51.29 (2C), 56.51, 62.53, 118.37 (C-2), 120.42 (C-1), 134.28 (C-29), 155.89 (C-20), 172.55 (C-3), 176.47 (C-28), 194.89 (C-30).

GC-MS (m/z): 505.4 (M⁺).

Synthesis of Compound (5)

A stream of a dry ozone/air mix at atmospheric pressure and at −60°C was bubbled through a solution of compound 3 (1 mmol) in CH₂Cl₂ (50 mL). After 2 hours, the reaction mixture was warmed to room temperature, and the solvent evaporated under reduced pressure. The residue was subjected to chromatographic purification (eluent: light petroleum/ethyl acetate 7:1) to yield 3-ethyl-20-methyldiketone 5. The course of reaction was monitored and the purity of the compound obtained was checked by TLC.

Methyl 1-Cyano-3,20-Dioxo-3-Ethyl-2,3-Seco-2,29-Dinorlup-28-oate (5)

Yield: 42%.

R _f : 0.4 (light petroleum/ethyl acetate 7:3).

MP: 152.1°С (light petroleum/ethyl acetate 7:1).

[α]_D: −8.6 (с 0.1; CHCl₃).

IR ν (CHCl₃) cm^–1: 1707 (2C=O), 1723 (СООСН₃), 2238 (C≡N).

¹H NMR (400 MHz, CDCl₃): δ 0.84, 0.88, 1.00, 1.10, 1.16 (15H, 5s, CH₃ × 5), 1.02 (3Н, t, J = 7.2 Hz, H-33), 2.10 (3Н, s, H-30), 2.32 and 2.45 (2H, 2d, J = 18.0 Hz, H-1), 2.49 and 2.68 (2Н, 2dq, J = 17.7, 7.2 Hz, H-32), 3.18 (1H, td, J = 10.9, 4.3 Hz, Н-19), 3.60 (3H, s, CООСH₃).

¹³C NMR (100 MHz, CDCl₃): δ 8.74, 14.68, 15.60, 18.80, 21.60, 21.76, 23.82, 24.17, 27.12, 28.21, 29.52, 29.71, 29.76, 30.34, 31.25, 33.19, 36.50, 37.40, 40.46, 42.45, 42.63, 45.15, 48.76, 49.17, 51.26, 51.40, 52.94, 56.45, 118.36 (C-2), 176.43 (C-28), 211.74 (C-20), 216.83 (C-3).

GC-MS (m/z): 511.4 (M⁺).

Synthesis of Compound (6)

Compound 4 (0.4 mmol) was dissolved in glacial acetic acid (30 mL) and treated with C₅H₆Br₃N (0.4 mmol). The reaction mixture was stirred at room temperature for 6 hours. Completeness of the reaction was observed by TLC analysis. The residue was diluted with water and extracted with ethyl acetate (3 × 20 mL). The organic layer was washed with a solution of 5% NaHCO₃, water, and then dried over anhydrous MgSO₄. The solvent was evaporated and the residue fractionated by silica gel CC, eluting with a mixture of light petroleum and ethyl acetate (10:1), to afford bromoderivative 6.

Methyl 3-[1-Bromoethyl]-1-Cyano-3-Oxo-2,3-Seco-2-Norlup-20(29)-en-30-al-28-oate (6)

Yield: 50%.

R _f : 0.3 (light petroleum/ethyl acetate 7:3).

MP: 176.4°С (light petroleum/ethyl acetate 10:1).

[α]_D: +45.7 (с 0.2; CHCl₃).

IR ν (CHCl₃) cm^–1: 1691 (C=O), 1718 (СООСН₃), 2238 (C≡N).

¹H NMR (400 MHz, CDCl₃): δ 0.85, 0.91, 0.92, 1.17, 1.43 (15H, 5s, CH₃ × 5), 1.68 (3Н, d, J = 6.6 Hz, H-33), 2.52 and 2.60 (2H, 2d, J = 18.0 Hz, H-1), 3.27 (1H, td, J = 11.1, 4.9 Hz, Н-19), 3.60 (3H, s, CООСH₃), 4.97 (2H, q, J = 6.6 Hz, H-32), 5.86 and 6.21 (2H, 2s, H-29), 9.47 (3Н, s, H-30).

¹³C NMR (100 MHz, CDCl₃): δ 14.48, 15.81, 18.96, 21.35, 21.81, 22.19, 22.40, 25.87, 27.22, 29.56, 29.68, 29.98, 31.70, 32.22, 33.04, 36.70, 38.16, 40.29, 40.52, 42.62, 42.73, 45.28, 46.06, 50.74, 51.37, 54.30, 56.64, 118.52 (C-2), 133.66 (C-29), 156.36 (C-20), 176.31 (C-28), 194.74 (C-30), 208.48 (C-3).

General Procedure for the Preparation of Compounds 7 and 8

Compound 3 or 5 (0.4 mmol) was dissolved in t-BuOH (30 mL) in the presence of t-BuOK (1.2 mmol). The mixture was refluxed for 2 hours and then cooled at room temperature. After completion of the reaction (as indicated by TLC), the resulting mixture was diluted with 5% HCl and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with a solution of 5% NaHCO₃ and water, dried (MgSO₄), filtered, and evaporated to dryness. The residue was subjected to silica gel CC to yield product 7 (eluent: light petroleum/ethyl acetate 15:1) or 8 (eluent: light petroleum/ethyl acetate 20:1).

Methyl 1-Cyano-3-Ethyl-20-Oxo-2,29-Dinorlup-1(3)-en-28-oate (7)

Yield: 44%.

R _f : 0.5 (light petroleum/ethyl acetate 7:3).

MP: 138.2°С (light petroleum/ethyl acetate 15:1).

[α]_D: −6.2 (с 0.2; CHCl₃).

IR ν (CHCl₃) cm^–1: 1713 (C=O), 1726 (СООСН₃), 2205 (C≡N).

¹H NMR (400 MHz, CDCl₃): δ 0.87, 0.88, 0.95, 0.98, 1.03 (15H, 5s, CH₃ × 5), 1.06 (3Н, t, J = 7.2 Hz, H-33), 2.09 (3Н, s, H-30), 2.21 (2H, q, J = 7.2 Hz, H-32), 3.16 (1H, td, J = 11.0, 4.5 Hz, Н-19), 3.61 (3H, s, CООСH₃).

¹³C NMR (100 MHz, CDCl₃): δ 13.73, 14.90, 17.36, 18.87, 20.51, 20.99, 26.68, 27.18, 28.42, 29.68, 29.81, 30.22, 31.23, 34.93, 36.72, 37.30, 38.18, 42.33, 42.72, 47.15, 47.33, 49.57, 50.72, 51.18, 51.37, 56.35, 62.48, 118.28 (C-2), 120.45 (C-1), 172.40 (C-3), 176.47 (C-28), 211.96 (C-20).

Methyl 1-Cyano-3-Ethyl-2-Norlup-1(3),20(29)-Dien-28-oate (8)

Yield: 45%.

R _f : 0.7 (light petroleum/ethyl acetate 7:3).

MP: 93.5°С (light petroleum/ethyl acetate 20:1).

[α]_D: +27.0 (с 0.3; CHCl₃).

IR ν (CHCl₃) cm^–1: 1593 (C=C), 1727 (СООСН₃), 2205 (C≡N).

¹H NMR (400 MHz, CDCl₃): δ 0.88, 0.89, 0.93, 0.99, 1.04 (15H, 5s, CH₃ × 5), 1.07 (3Н, t, J = 7.6 Hz, H-33), 1.61 (3Н, s, H-30), 2.22 (2Н, dq, J = 1.6, 7.6 Hz, H-32), 2.91 (1H, td, J = 10.9, 4.8 Hz, Н-19), 3.61 (3H, s, CООСH₃), 4.55 and 4.66 (2H, 2s, H-29).

¹³C NMR (100 MHz, CDCl₃): δ 13.73, 14.81, 17.35, 17.38, 18.85, 19.27, 20.48, 20.96, 22.24, 25.05, 27.21, 29.74, 30.61, 32.37, 35.01, 37.03, 38.17, 42.41, 42.90, 47.07, 47.23, 47.32, 49.58, 50.76, 51.21, 56.42, 62.55, 109.93 (C-29), 118.40 (C-2), 120.55 (C-1), 150.06 (C-20), 172.45 (C-3), 176.61 (C-28).

GC-MS (m/z): 491.4 (M⁺).

3-(4,5-Dimethylthiazol-2-yl)-2,3-Diphenyl-Tetrazolium Bromide Assay

Seven human tumor cell lines: larynx carcinoma (HEp-2), colorectal carcinoma (HCT 116), melanoma (MS), rhabdomyosarcoma (RD), nonsmall cell lung carcinoma (A549), estrogen-dependent breast adenocarcinoma (MCF-7), and breast adenocarcinoma (MDA-MB-231) obtained from “N.N. Blokhin National Medical Research Center of Oncology” of the Health Ministry of Russia (Moscow, Russia) were used to assess the cytotoxic potential of compounds 2 to 9. The HEp-2, RD, A549, MCF-7, and MDA-MB-231 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM), while the HCT 116 and MS cells were cultured in Roswell Park Memorial Institute medium (RPMI) 1640. Both media were supplemented with 146 mM glutamine, 10% (v/v) heat-inactivated fetal calf serum, and 1% gentamicin.

To assess cytotoxic activity of compounds, the cells were placed at a density of 10⁶ cells/mL into a 96-well plate and cultured for 24 hours at 37°C and 5% CO₂. Stock solutions (10 mM) of tested compounds were prepared in Dimethyl Sulfoxide (DMSO) and then diluted in an appropriate culture medium. Then, the cells were subsequently exposed to a range of drug concentrations (1.6-100 µM). Doxorubicin (Tocris bioscience, United Kingdom) was used as a reference drug. After 72 hours incubation at 37°C and 5% CO₂; the number of viable cells was measured using 0.5 mg/mL MTT for 4 hours at 37°C.¹⁶ The formed formazan was dissolved in 100 µL of DMSO and then absorbance was measured spectrophotometrically on a FLUOstar Optima microplate reader (BMG Labtech, Germany) at 544 nm using DMSO as a blank.

Cytotoxicity of the compounds was expressed as an IC₅₀ value (a concentration which reduces absorbance in treated cells by 50% as compared with untreated cells), calculated by means of GraphPad Prism 6.0 (GraphPad Prism Software Inc., United States). The results are presented as the mean ± SD of the replicates from 3 independent experiments.¹⁶

Supplemental Material

Supplementary material - Supplemental material for Synthesis, Cyclization, and Cytotoxic Activity of 2,3-Secolupane Triterpenoids With an Ethylketone Fragment

Supplemental material, Supplementary material, for Synthesis, Cyclization, and Cytotoxic Activity of 2,3-Secolupane Triterpenoids With an Ethylketone Fragment by Anastasia V. Konysheva, Daria V. Eroshenko and Victoria V. Grishko in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the Russian Science Foundation (Project Nr. 16-13-10245).

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Supplementary Material

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Synthesis,Cyclization,and Cytotoxic Activity of 2,3-Secolupane Triterpenoids With an Ethylketone Fragment

Abstract

Keywords

Experimental

General

Synthesis of Compound (2)

Methyl 3-Ethyl-3β-Hydroxy-2-Hydroxyimino-Lup-20(29)-en-28-oate (2)

Synthesis of Compound (3)

Methyl 1-Cyano-3-Ethyl-3-Oxo-2,3-Seco-2-Norlup-20(29)-en-28-oate (3)

General Procedure for the Preparation of Compounds 4 and 9

Methyl 1-Cyano-3-Ethyl-3-Oxo-2,3-Seco-2-Norlup-20(29)-en-30-al-28-oate (4)

Methyl 1-Cyano-3-Ethyl-2-Norlup-1(3),20(29)-Dien-30-al-28-oate (9)

Synthesis of Compound (5)

Methyl 1-Cyano-3,20-Dioxo-3-Ethyl-2,3-Seco-2,29-Dinorlup-28-oate (5)

Synthesis of Compound (6)

Methyl 3-[1-Bromoethyl]-1-Cyano-3-Oxo-2,3-Seco-2-Norlup-20(29)-en-30-al-28-oate (6)

General Procedure for the Preparation of Compounds 7 and 8

Methyl 1-Cyano-3-Ethyl-20-Oxo-2,29-Dinorlup-1(3)-en-28-oate (7)

Methyl 1-Cyano-3-Ethyl-2-Norlup-1(3),20(29)-Dien-28-oate (8)

3-(4,5-Dimethylthiazol-2-yl)-2,3-Diphenyl-Tetrazolium Bromide Assay

Supplemental Material

Supplementary material - Supplemental material for Synthesis, Cyclization, and Cytotoxic Activity of 2,3-Secolupane Triterpenoids With an Ethylketone Fragment

Footnotes

Declaration of Conflicting Interests

Funding

References

Supplementary Material