Conjugates of betulinic acid with 2-(fluoroacyl)cycloalkane-1,3-dione motifs were synthesized by reacting 3-chloro-2-(fluoroacyl)-2-cycloalkene-1-ones with an equivalent amount of betulinic acid aminoalkylamide hydrochlorides in the presence of Et3N in CHCl3. Reduction of betulonic acid conjugates with 2-trifluoroacetylcyclopentane-1,3-dione by NaBH4 in THF produced betulinic acid derivatives containing 2-(1-hydroxy-2,2,2-trifluoroethyl)cyclopentane-1,3-dione. The cytotoxicity of the synthesized compounds was assessed against glioblastoma multiforme (U-87 MG) and breast cancer cell lines (MCF7).
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Chemical compounds produced by living organisms such as bacteria, fungi, higher plants, and animals play important roles in drug discovery [1]. Conjugation of natural and synthetic compounds is one of the most fruitful approaches to designing new biologically active structures [2]. Pentacyclic triterpenoids are a promising class of natural products for this strategy [3, 4]. Their potential for generating antitumor, anti-inflammatory, hepatoprotective, anti-HIV, and other drugs stems from broad profiles of biological activity, availability, and renewable raw-material sources [5,6,7]. Lupane-type triterpenoids are highly interesting because of the high cytotoxicity of betulinic acid and its anti-HIV activity [8]. Cyclic β-triketones are common in nature; exhibit various types of biological activity [9], e.g., antitumor [10]; and form an interesting class of polyfunctional compounds. 2-Acylcycloalkane-1,3-diones are widely used to synthesize various classes of bioactive compounds [11, 12]. Currently, methods for synthesizing F-containing compounds of interest as potential drugs and new plant-protection agents are rapidly developing [13, 14]. Previously, efficient synthetic methods for 2-(fluorobenzoyl)cyclohexane-1,3-diones and 2-perfluoroalkanoylcycloalkane-1,3-diones were developed by us and made such compounds accessible for further research on their reactivity [15]. The goals of the present work were to synthesize new betulinic-acid conjugates with F-containing 2-acylcycloalkane-1,3-diones using a diamine linker and to study their cytotoxicity.
Conjugates of betulinic acid (1) were synthesized using betulonic acid and 2-(fluoroacyl)cycloalkane-1,3-diones as starting materials. Treatment of 1 with an excess of (COCl)2 synthesized its acid chloride (2) [16] that was reacted with polyamines, e.g., putrescine or 2,2′-(ethylenedioxy)bis(ethylamine). Hydrochlorides of betulonic-acid aminoalkylamide derivatives 3a and 3b were prepared by our previously developed method [17] in yields up to 84% by treating acid chloride 2 with a four-fold excess of diamine in CHCl3 in the presence of Et3N followed by work up with HCl solution (5%).
Resulting betulonic-acid derivatives 3a and 3b were reduced by NaBH4 in THF to give 4a and 4b in 90–92% yield. 3-Chloro-2-(fluoroacyl)cycloalken-1-ones 6a–d were prepared by treating the corresponding F-containing cyclic β-triketones 5a–d with an excess of (COCl)2 and were used to acylate the hydrochlorides of 4a and 4b in CHCl3 in the presence of Et3N to synthesize betulinic-acid conjugates 7a–f in 50–82% yield. Reaction of the hydrochlorides of the betulonic-acid aminoalkylamide derivatives (3a and 3b) with 2-trifluoroacetyl-3-chloro-2-cyclopenten-1-one (6c) and reduction by NaBH4 of the obtained betulonic-acid conjugates 8a and 8b synthesized betulinic-acid conjugates 9a and 9b in 94–95% yield. Both the 3-carbonyl of the lupane skeleton and the 2-trifluoroacetyl carbonyl were reduced to hydroxyls.
The structures of 4a, 4b, 7a–f, 9a, and 9b were established and confirmed using IR, PMR, 13C NMR, and 19F NMR spectroscopy and elemental analysis. PMR spectra of the hydrochlorides of betulinic-acid aminoalkylamide derivatives 4a and 4b showed resonances for NH3+ at δ 8.08 and 8.24 ppm, respectively, and for the amide proton as a broad singlet at δ 7.68 (4a) or a triplet with J = 5.8 Hz at δ 7.70 (4b). PMR spectra of betulinic-acid conjugates 7a–f were characterized by amide proton resonances as triplets at δ 5.74–6.28 ppm (J = 5.9–6.2 Hz) (7b, c, e, f) or in several instances as broad singlets at δ 5.77–5.79 ppm (7a and 7d) and resonances for NH protons in H-bonds with the carbonyl of the side fluoroacyl group as broad singlets at δ 10.09–11.66 ppm (7a and 7c–f) or a triplet at δ 11.56 ppm with J = 5.2 Hz (7b). PMR spectra of conjugates 9a and 9b were characterized by amide proton resonances as broad singlets at δ 5.84–6.12 ppm and resonances of NH protons in H-bonds with hydroxyls as broad singlets at 6.89–7.08 ppm. Thus, reduction of the trifluoroacetyl carbonyl to an alcohol produced a strong-field shift of the resonance for the NH bonded to the cyclopentane ring as compared with the analogous NH proton in conjugates 7a–f.
Cytotoxicity in vitro of the hydrochlorides of betulinic-acid aminoalkylamide derivatives 4a and 4b, betulinic-acid conjugates 7a–f, and 9a and 9b was assayed using U-87 MG glioblastoma multiforme, MCF7 breast cancer, and hTERT immortalized human fibroblast cell lines and the MTT assay [18]. Table 1 shows that synthesized derivatives 7e and 9a exhibited moderate cytotoxicity against U-87 MG cell line; conjugate 9b, against MCF7 cell line. Compounds 7a–d and 7f had no cytotoxic activity. In general, the cytotoxicity of the synthesized compounds (7a–f and 9a and 9b) was less than the starting hydrochlorides of aminoalkyl derivatives 4a and 4b.
Experimental
PMR, 19F NMR, and 13C NMR spectra were taken in CDCl3 or DMSO-d6 solutions at 293 K on a Bruker-Biospin Avance 500 spectrometer at operating frequencies 500, 470, and 125 MHz, respectively, using a 5-mm probe (QNP) with a Z-gradient. Residual solvent resonances were used as internal standards for PMR [δH 7.26 ppm, CD(H)Cl3; 2.50 ppm, DMSO-d6] and 13C NMR spectra (δC 77.16 ppm, CDCl3; 39.52 ppm, DMSO-d6). An external standard of α,α,α-trifluorotoluene (δ 63 ppm, 19F) was used. IR spectra were taken from KBr pellets on a PerkinElmer Spectrum 100 FT-IR spectrometer. Melting points were determined on a Boetius apparatus. Elemental analysis was performed on a Eurovector EA 3000 CHNS-O analyzer. The course of reactions and purity of products were monitored by TLC on Silufol UV-254 plates (EtOAc–petroleum ether). Column chromatography used silica gel (70–230 mesh) with elution by EtOAc–petroleum ether. Elemental analyses of all compounds agreed with those calculated.
Hydrochlorides of betulonic-acid aminoalkylamide derivatives 3a and 3b [17], 2-(fluorobenzoyl)cyclohexane-1,3-diones 5a and 5b [19], 2-trifluoroacetylcycloalkane-1,3-diones 5c and 5d [20, 21], and 2-(fluoroacyl)-3-chloro-2-cycloalken1-ones 6a–d [21,22,23] were prepared using published methods. The physicochemical characteristics of 3a, 5a–d, and 6a–d corresponded to those in the literature.
2-{2-[2-(3-Oxolup-20(29)-en-28-amido)ethoxy]ethoxy}ethane-1-ammonium Chloride (3b). C36H61ClN2O4, yield 62%, mp 127–132°C. IR spectrum (KBr, ν, cm–1): 1640, 1705. 1H NMR spectrum (500 MHz, CDCl3, δ, ppm, J/Hz): 0.80–2.16 (21H, m, CH, CH2), 0.90 (3H, s, CH3), 0.95 (3H, s, CH3), 0.96 (3H, s, CH3), 1.00 (3H, s, CH3), 1.05 (3H, s, CH3), 1.66 (3H, s, CH3), 2.35–2.55 (3H, m, CH, CH2), 2.94–3.89 (13H, m, H-19, 6CH2), 4.57 (1H, br.s, H-29), 4.71 (1H, br.s, H-29), 6.62 (1H, t, J = 5.8, NH), 8.25 (3H, s, NH3+). 13C NMR spectrum (125 MHz, DMSO-d6, δ, ppm): 14.7, 16.1, 16.2, 19.6, 19.8, 21.2, 21.6, 25.7, 26.8, 29.5, 31.0, 33.7, 33.9, 34.3, 37.1, 37.8, 38.5, 39.3, 39.8, 40.8, 42.6, 46.8, 47.4, 50.1, 50.2, 55.1, 55.8, 67.1, 70.3, 70.4, 70.5, 77.4, 109.6, 151.0, 176.9, 218.2.
Preparation of Betulinic-acid Aminoalkylamide Hydrochlorides (4a and 4b). A solution of 3a or 3b (1 mmol) in anhydrous THF (10 mL) was treated with NaBH4 (4 mmol), stirred for 4 h, diluted with HCl solution (30 mL, 10%) cooled to 0°C, and evaporated (THF) at reduced pressure. Hydrochlorides of 4a and 4b were isolated as colorless powders by filtration and drying at reduced pressure.
4-{2-[3β-Hydroxylup-20(29)-en-28-amido]}butane-1-ammonium Chloride (4a). C34H59ClN2O2, yield 90%, mp 253–257°C (dec.). IR spectrum (KBr, ν, cm–1): 1635. 1H NMR spectrum (500 MHz, DMSO-d6, δ, ppm, J/Hz): 0.58–1.80 (27H, m, CH, CH2), 0.63 (3H, s, CH3), 0.75 (3H, s, CH3), 0.83 (3H, s, CH3), 0.85 (3H, s, CH3), 0.89 (3H, s, CH3), 1.61 (3H, s, CH3), 2.14 (1H, d, J = 12.1), 2.52–2.58 (1H, m), 2.63–3.10 (5H, m), 4.52 (1H, br.s, H-29), 4.63 (1H, br.s, H-29), 7.68 (1H, br.s, NH), 8.08 (3H, br.s, NH3+). 13C NMR spectrum (125 MHz, DMSO-d6, δ, ppm): 14.3, 15.8, 15.9, 17.9, 19.0, 20.5, 24.3, 25.2, 26.2, 27.1, 28.1, 28.9, 30.3, 32.4, 33.9, 36.6, 36.7, 37.6, 37.7, 38.3, 38.4, 38.4, 40.2, 41.8, 46.1, 49.6, 50.1, 54.8, 54.9, 76.7, 109.1, 150.9, 175.5.
2-{2-[2-(3β-Hydroxylup-20(29)-en-28-amido)ethoxy]ethoxy}ethane-1-ammonium Chloride (4b). C36H63ClN2O4, yield 92%, mp 144–148°C. IR spectrum (KBr, ν, cm–1): 1635. 1H NMR spectrum (500 MHz, DMSO-d6, δ, ppm, J/Hz): 0.57–1.81 (23H, m, CH, CH2), 0.64 (3H, s, CH3), 0.75 (3H, s, CH3), 0.83 (3H, s, CH3), 0.85 (3H, s, CH3), 0.89 (3H, s, CH3), 1.61 (3H, s, CH3), 2.13 (1H, d, J = 12.1), 2.50–2.56 (1H, m), 2.82–3.65 (13H, m), 4.52 (1H, br.s, H-29), 4.64 (1H, br.s, H-29), 7.70 (1H, t, J = 5.8, NH), 8.24 (3H, br.s, NH3+). 13C NMR spectrum (125 MHz, DMSO-d6, δ, ppm): 14.3, 15.8, 16.0, 18.0, 19.1, 20.6, 25.2, 27.2, 28.1, 28.9, 30.3, 32.4, 34.0, 36.7, 36.7, 37.6, 38.2, 38.3, 38.4, 38.5, 40.3, 41.9, 46.2, 49.6, 50.1, 54.9, 55.0, 66.6, 69.1, 69.4, 69.6, 76.8, 109.2, 150.9, 175.7.
Preparation of Betulinic-acid Conjugates 7a–f. A solution of 3-chloro-2-(fluoroacyl)-2-cycloalken-1-ones (6a–d, 1 mmol) in CHCl3 (10 mL) was treated with a solution of the hydrochloride of betulinic-acid aminoalkylamide derivative 4a or 4b in CHCl3 (10 mL) and dropwise with Et3N (2 mmol), stirred for 30 min, and evaporated at reduced pressure. Conjugates 7a–f were isolated as colorless powders in 50–82% yield by column chromatography over silica gel.
Betulinic Acid N-{4-[3-Oxo-2-(3-fluorobenzoyl)cyclohex-1-enylamino]butyl}amide (7a). C47H67FN2O4, yield 66%, mp 135–139°C. IR spectrum (KBr, ν, cm–1): 1580, 1640. 1H NMR spectrum (500 MHz, CDCl3, δ, ppm, J/Hz): 0.64–1.96 (27H, m, CH, CH2), 0.72 (3H, s, CH3), 0.78 (3H, s, CH3), 0.90 (3H, s, CH3), 0.94 (3H, s, CH3), 0.95 (3H, s, CH3), 1.66 (3H, s, CH3), 2.04 (2H, qt, J = 6.2, CH2), 2.36 (2H, t, J = 6.2, CH2), 2.42 (1H, td, J = 12.6, 3.8), 2.67 (2H, t, J = 6.2, CH2), 3.05–3.50 (6H, m, H-3, 19, 2CH2), 4.58 (1H, br.s, H-29), 4.72 (1H, br.s, H-29), 5.79 (1H, br.s, NH), 7.02–7.13 (2H, m, Harom), 7.15–7.22 (1H, m, Harom), 7.24–7.30 (1H, m, Harom), 11.54 (1H, br.s, NH). 13C NMR spectrum (125 MHz, CDCl3, δ, ppm, J/Hz): 14.8, 15.5, 16.3, 18.4, 19.6, 20.0, 21.0, 25.7, 26.6, 27.1, 27.4, 27.5, 28.1, 29.6, 29.8, 31.0, 33.8, 34.5, 37.3, 37.4, 37.9, 38.3, 38.6, 38.8, 38.9, 40.9, 42.6, 43.3, 46.9, 50.2, 50.7, 55.5, 55.8, 79.1, 108.2, 109.5, 114.4 (d, J = 22), 116.8 (d, J = 21), 123.2, 129.3 (d, J = 8), 145.2 (d, J = 7), 151.0, 162.4 (d, J = 245), 172.6. 19F NMR spectrum (470 MHz, CDCl3, δ, ppm): –113.75 – (–113.87) (m, F).
Betulinic Acid N-[2-(2-{2-[3-Oxo-2-(3-fluorobenzoyl)cyclohex-1-enylamino]ethoxy}ethoxy)ethyl]amide (7b). C49H71FN2O6, yield 60%, mp 110–115°C. IR spectrum (KBr, ν, cm–1): 1580, 1640. 1H NMR spectrum (500 MHz, CDCl3, δ, ppm, J/Hz): 0.65–1.96 (23H, m, CH, CH2), 0.74 (3H, s, CH3), 0.80 (3H, s, CH3), 0.92 (3H, s, CH3), 0.93 (3H, s, CH3), 0.95 (3H, s, CH3), 1.66 (3H, s, CH3), 2.04 (2H, qt, J = 6.2, CH2), 2.36 (2H, t, J = 6.4, CH2), 2.38–2.52 (1H, m), 2.69 (2H, t, J = 6.2, CH2), 3.10 (1H, td, J = 11.1, 3.7, H-19), 3.16 (1H, dd, J = 11.4, 4.8), 3.29–3.77 (12H, m, 6CH2), 4.57 (1H, br.s, H-29), 4.71 (1H, br.s, H-29), 6.28 (1H, t, J = 5.8, 1H, NH), 6.98–7.14 (2H, m Harom), 7.14–7.20 (1H, m, Harom), 7.25–7.31 (1H, m, Harom), 11.56 (1H, t, J = 5.2, NH). 13C NMR spectrum (125 MHz, CDCl3, δ, ppm, J/Hz): 14.8, 15.5, 16.2, 16.3, 18.5, 19.6, 20.0, 21.1, 25.7, 27.3, 27.5, 28.1, 29.5, 31.0, 33.6, 34.6, 37.3, 37.4, 37.8, 38.4, 38.8, 39.0, 39.1, 40.9, 42.6, 43.6, 46.9, 50.3, 50.7, 55.5, 55.7, 69.0, 70.2, 70.3, 70.9, 79.1, 108.5, 109.4, 114.4 (d, J = 22), 116.8 (d, J = 21), 123.1, 129.3 (d, J = 8.0), 145.3, 151.2, 162.4 (d, J = 245), 172.5, 176.4, 194.2, 196.7. 19F NMR spectrum (470 MHz, CDCl3, δ, ppm): –113.64 – (–113.82) (m, F).
Betulinic Acid N-{4-[3-Oxo-2-(4-fluorobenzoyl)cyclohex-1-enylamino]butyl}amide (7c). C47H67FN2O4, yield 82%, mp 151–155°C. IR spectrum (KBr, ν, cm–1): 1580, 1640. 1H NMR spectrum (500 MHz, CDCl3, δ, ppm, J/Hz): 0.65–1.96 (27H, m, CH, CH2), 0.73 (3H, s, CH3), 0.78 (3H, s, CH3), 0.91 (3H, s, CH3), 0.94 (3H, s, CH3), 0.95 (3H, s, CH3), 2.04 (2H, qt, J = 6.2, CH2), 2.36 (2H, t, J = 6.4, CH2), 2.42 (1H, td, J = 12.4, 3.5), 2.68 (2H, t, J = 6.3, CH2), 3.07–3.50 (6H, m, H-3, 19, 2CH2), 4.58 (1H, br.s, H-29), 4.72 (1H, br.s, H-29), 5.74 (1H, t, J = 6.2, NH), 7.00 (2H, t, J = 8.6, Harom), 7.46 (2H, t, J = 6.8, Harom), 11.46 (1H, br.s, NH). 13C NMR spectrum (125 MHz, CDCl3, δ, ppm, J/Hz): 14.8, 15.5, 16.3, 18.4, 19.6, 20.0, 21.1, 25.7, 26.6, 27.1, 27.5, 27.5, 28.1, 29.6, 31.0, 33.9, 34.5, 37.3, 37.5, 37.9, 38.3, 38.6, 38.8, 39.0, 40.9, 42.6, 43.2, 46.9, 50.3, 50.7, 55.5, 55.8, 79.1, 108.2, 109.6, 114.8 (d, J = 22), 130.1 (d, J = 9), 138.9, 151.0, 164.1 (d, J = 250), 172.3, 176.6, 194.3, 196.7. 19F NMR spectrum (470 MHz, CDCl3, δ, ppm): –110.16 – (–110.28) (m, F).
Betulinic Acid N-{4-[3-Oxo-2-(trifluoroacetyl)cyclopent-1-enylamino]butyl}amide (7d). C41H61F3N2O4, yield 58%, mp 146–150°C. IR spectrum (KBr, ν, cm–1): 1595, 1635. 1H NMR spectrum (500 MHz, CDCl3, δ, ppm, J/Hz): 0.64–2.00 (27H, m, CH, CH2), 0.73 (3H, s, CH3), 0.78 (3H, s, CH3), 0.88 (3H, s, CH3), 0.94 (3H, s, CH3), 0.95 (3H, s, CH3), 1.66 (3H, s, CH3), 2.40 (1H, td, J = 12.3, 3.6), 2.50–2.53 (2H, m, CH2), 2.78–2.89 (2H, m, CH2), 3.07–3.57 (6H, m, H-3, 19, 2CH2), 4.58 (1H, br.s, H-29), 4.71 (1H, br.s, H-29), 5.77 (1H, br.s, NH), 10.09 (1H, br.s, NH). 13C NMR spectrum (125 MHz, CDCl3, δ, ppm, J/Hz): 14.8, 15.4, 16.2, 16.3, 18.4, 19.6, 21.1, 24.9, 25.7, 26.7, 27.4, 27.5, 28.1, 29.6, 31.0, 33.5, 33.9, 34.5, 37.3, 37.9, 38.1, 38.6, 38.8, 39.0, 40.9, 42.6, 44.3, 46.9, 50.2, 50.7, 55.5, 55.8, 79.1, 106.4, 109.6, 116.6 (d, J = 287), 150.9, 176.3 (d, J = 38), 176.8, 184.1, 196.2. 19F NMR spectrum (470 MHz, CDCl3, δ, ppm): –75.71 (s, CF3).
Betulinic Acid N-[2-(2-{2-[3-Oxo-2-(trifluoroacetyl)cyclopent-1-enylamino]ethoxy}ethoxy)ethyl]amide (7e). C43H65F3N2O6, yield 60%, mp 82–86°C. IR spectrum (KBr, ν, cm–1): 1600, 1635. 1H NMR spectrum (500 MHz, CDCl3, δ, ppm, J/Hz): 0.65–2.00 (23H, m, CH, CH2) 0.74 (3H, s, CH3), 0.80 (3H, s, CH3), 0.92 (3H, s, CH3), 0.95 (6H, s, 2CH3), 1.66 (3H, s, CH3), 2.43 (1H, td, J = 12.2, 3.5), 2.50–2.53 (2H, m, CH2), 2.80–2.83 (2H, m, CH2), 3.11 (1H, td, J = 11.1, 4.0, H-19), 3.17 (1H, dd, J = 11.4, 4.8), 3.34–3.76 (12H, m), 4.57 (1H, br.s, H-29), 4.72 (1H, br.s, H-29), 6.17 (1H, t, J = 5.9, NH), 10.25 (1H, br.s, NH). 13C NMR spectrum (125 MHz, CDCl3, δ, ppm, J/Hz): 14.8, 15.5, 16.2, 16.3, 18.4, 19.6, 21.1, 25.1, 25.7, 27.5, 28.1, 29.5, 31.0, 33.5, 33.7, 34.6, 37.3, 37.9, 38.4, 38.8, 39.0, 39.1, 40.9, 42.6, 44.6, 46.9, 50.2, 50.7, 55.5, 55.8, 68.8, 70.2, 70.3, 70.9, 79.1, 106.6, 109.4, 116.6 (d, J = 288), 151.2, 176.0 (d, J = 39), 176.5, 184.2, 196.3. 19F NMR spectrum (470 MHz, CDCl3, δ, ppm): –75.59 (s, CF3).
Betulinic Acid N-{4-[5,5-Dimethyl-3-oxo-2-(trifluoroacetyl)-cyclohex-1-enylamino]butyl}amide (7f). C44H67F3N2O4, yield 50%, mp 139–143°C. IR spectrum (KBr, ν, cm–1): 1590, 1650. 1H NMR spectrum (500 MHz, CDCl3, δ, ppm, J/Hz): 0.65–1.96 (27H, m, CH, CH2), 0.74 (3H, s, CH3), 0.79 (3H, s, CH3), 0.89 (3H, s, CH3), 0.95 (3H, s, CH3), 0.96 (3H, s, CH3), 1.10 (3H, s, CH3), 1.10 (3H, s, CH3), 1.67 (3H, s, CH3), 2.33 (2H, s, CH2), 2.35–2.46 (1H, m), 2.53 (2H, s, CH2), 3.07–3.52 (6H, m, H-3, 19, 2CH2), 4.58 (1H, br.s, H-29), 4.72 (1H, s, H-29), 5.78 (1H, t, J = 6.1, NH), 11.66 (1H, br.s, NH). 13C NMR spectrum (125 MHz, CDCl3, δ, ppm, J/Hz): 14.8, 15.5, 16.2, 16.3, 18.4, 19.6, 21.1, 25.7, 26.4, 27.4, 27.5, 28.1, 28.4, 28.5, 29.6, 31.0, 31.1, 33.9, 34.5, 37.3, 37.9, 38.2, 38.6, 38.8, 39.0, 40.8, 40.9, 42.6, 43.8, 46.9, 50.2, 50.7, 51.3, 55.5, 55.8, 79.1, 105.1, 109.6, 117.5 (d, J = 287), 150.9, 173.7, 176.8, 179.8 (d, J = 36), 192.6. 19F NMR spectrum (470 MHz, CDCl3, δ, ppm): –72.41 (s, CF3).
Derivatives 8a and 8b were prepared as before [17]. The physicochemical characteristics of 8a agreed with those in the literature.
Betulonic Acid N-{2-[2-(2-(3-Oxo-2-(trifluoroacetyl)cyclopent-1-enylamino)ethoxy)ethoxy]ethyl}amide (8b). C43H63F3N2O6, yield 85%, mp 86–90°C. IR spectrum (KBr, ν, cm–1): 1595, 1635, 1705. 1H NMR spectrum (500 MHz, CDCl3, δ, ppm, J/Hz): 0.81–1.99 (21H, m, CH, CH2), 0.90 (3H, s, CH3), 0.95 (3H, s, CH3), 0.96 (3H, s, CH3), 1.00 (3H, s, CH3), 1.04 (3H, s, CH3), 1.66 (3H, s, CH3), 2.35–2.52 (5H, m), 2.72–2.89 (2H, m, CH2), 3.11 (1H, td, J = 11.0, 4.1, H-19), 3.32–3.80 (12H, m, 6CH2), 4.57 (1H, br.s, H-29), 4.71 (1H, br.s, H-29), 6.17 (1H, t, J = 5.7, NH), 10.25 (1H, br.s, NH). 13C NMR spectrum (125 MHz, CDCl3, δ, ppm, J/Hz): 14.7, 16.0, 16.1, 19.6, 21.1, 21.6, 25.0, 25.7, 26.7, 29.5, 31.0, 33.5, 33.6, 33.8, 34.3, 37.0, 37.9, 38.4, 39.1, 39.7, 40.8, 42.6, 44.6, 46.9, 47.4, 50.1, 50.1, 55.1, 55.7, 68.8, 70.2, 70.3, 70.9, 77.4, 106.6, 109.5, 116.6 (q, J = 288), 151.1, 176.0 (q, J = 37), 176.4, 184.2, 196.3, 218.3. 19F NMR spectrum (470 MHz, CDCl3, δ, ppm): –75.69 (s, CF3).
Preparation of Derivatives 9a and 9b. A solution of 8a or 8b (1 mmol) in THF (15 mL) was treated with NaBH4 (4 mmol), stirred for 16 h at room temperature, and evaporated at reduced pressure. The residue was diluted with HCl solution (5%, 60 mL) cooled to 0°C. The aqueous fraction was extracted with CHCl3 (3 × 20 mL). The combined organic fractions were dried over anhydrous Na2SO4 and filtered. The solvent was evaporated at reduced pressure to afford 9a and 9b as colorless powders.
Betulinic Acid N-{4-[3-Oxo-2-(1-hydroxy-2,2,2-trifluoroethyl)cyclopent-1-enylamino]butyl}amide (9a). C41H63F3N2O4, yield 94%, mp 171–175°C. IR spectrum (KBr, ν, cm–1): 1590, 1635. 1H NMR spectrum (500 MHz, CDCl3, δ, ppm, J/Hz): 0.65–2.00 (27H, m, CH, CH2), 0.74 (3H, s, CH3), 0.80 (3H, s, CH3), 0.90 (3H, s, CH3), 0.95 (3H, s, CH3), 0.96 (3H, s, CH3), 1.67 (3H, s, CH3), 2.35–2.45 (3H, m), 2.60–2.62 (2H, m, CH2), 3.08 (1H, td, J = 11.6, 5.8, H-19), 3.13–3.41 (5H, m, H-3, 2CH2), 4.58 (1H, br.s, H-29), 4.72 (1H, br.s, H-29), 4.84–5.05 (1H, m, CF3CHOH), 5.84 (1H, br.s, NH), 6.89 (1H, br.s, NH). 13C NMR spectrum (125 MHz, CDCl3, δ, ppm, J/Hz): 14.8, 15.5, 16.2, 16.3, 18.4, 19.6, 21.0, 25.0, 25.7, 26.8, 27.5, 27.8, 28.1, 29.6, 31.0, 32.9, 33.9, 34.5, 37.3, 37.9, 38.5, 38.6, 38.9, 39.0, 40.9, 42.6, 44.2, 46.8, 50.2, 50.7, 55.5, 55.8, 66.5 (q, J = 34), 79.1, 104.0, 109.6, 125.7 (d, J = 283), 150.9, 176.3, 177.1, 201.8. 19F NMR spectrum (470 MHz, CDCl3, δ, ppm): –75.02 – (–75.20) (m, CF3).
Betulinic Acid N-[2-(2-{2-[3-Oxo-2-(1-hydroxy-2,2,2-trifluoroethyl)cyclopent-1-enylamino]ethoxy} ethoxy)ethyl]amide (9b). C43H67F3N2O6, yield 95%, mp 118–121°C. IR spectrum (KBr, ν, cm–1): 1590, 1635. 1H NMR spectrum (500 MHz, CDCl3, δ, ppm, J/Hz): 0.65–2.00 (23H, m, CH, CH2), 0.74 (3H, s, CH3), 0.80 (3H, s, CH3), 0.90 (3H, s, CH3), 0.95 (6H, s, 2CH3), 2.38 (1H, td, J = 13.3, 3.3), 2.47 (2H, br.s, CH2), 2.65 (2H, br.s, CH2), 3.07 (1H, td, J = 11.7, 4.3, H-19), 3.17 (1H, dd, J = 11.2, 4.8, H-3), 3.29–3.70 (12H, m), 4.58 (1H, br.s, H-29), 4.72 (1H, br.s, H-29), 4.87–5.14 (1H, m, CF3CHOH), 6.12 (1H, br.s, NH), 7.08 (1H, br.s, NH). 13C NMR spectrum (125 MHz, CDCl3, δ, ppm, J/Hz): 14.8, 15.4, 15.5, 16.2, 16.3, 18.4, 19.6, 21.0, 25.3, 25.7, 27.5, 28.1, 29.5, 31.0, 32.7, 33.7, 34.5, 37.3, 37.9, 38.5, 38.8, 39.0, 39.2, 40.9, 42.6, 44.2, 46.9, 50.2, 50.7, 55.5, 55.9, 66.0, 66.2 (d, J = 33), 69.8, 70.3, 70.3, 71.1, 71.1, 79.1, 104.9, 109.6, 125.6 (d, J = 283), 151.0, 177.0, 177.3, 200.7. 19F NMR spectrum (470 MHz, CDCl3, δ, ppm): –78.41 – (–78.61) (m, CF3).
Cytotoxicity of 4a, 4b, 7a–f, 9a, and 9b. Cultures of breast cancer MCF7 (ATCC HTB-22) and U-87 MG glioblastoma multiforme cells (ATCC HTB-14) were obtained commercially (ATCC, USA). The healthy control was immortalized human fibroblasts (hTERT) from A. G. Shilov (FRC ICG, SB, RAS). Cytotoxicity of the tested compounds was assayed using the MTT assay and the standard procedure [18]. Cells were inoculated into 96-well plates (5,000 cells per well) and incubated at 37°C in 5% CO2 for attachment. Medium in wells was replaced after 24 h with fresh medium containing the tested compounds in DMSO (1 vol%) and incubated for 72 h. Optical density was measured in a plate spectrophotometer as usual. All compounds were tested at concentrations of 10, 25, 50, and 100 μM using the required controls, i.e., negative, DMSO (solvent), and positive, doxorubicin (standard cytostatic). Each experiment was performed independently in triplicate with three tests in each. Results were reported as mean inhibitory concentration IC50 ± SEM.
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Acknowledgment
The work was financially supported by the National Academy of Sciences of Belarus and the Siberian Branch, Russian Academy of Sciences (Grant X15CO-001, Project 0302-2015-0003).
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Translated from Khimiya Prirodnykh Soedinenii, No. 6, November–December, 2018, pp. 936–940.
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Khlebnikova, T.S., Piven’, Y.A., Lakhvich, F.A. et al. Synthesis and Cytotoxicity of Conjugates of Betulinic Acid and F-Containing 2-Acylcycloalkane-1,3-Diones. Chem Nat Compd 54, 1100–1105 (2018). https://doi.org/10.1007/s10600-018-2565-y
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DOI: https://doi.org/10.1007/s10600-018-2565-y