Chemistry of Natural Compounds, Vol. 50, No. 3, July, 2014 [Russian original No. 3, May–June, 2014]
FIRST CONJUGATES OF THE DITERPENOID ISOSTEVIOL AND GLUCURONIC ACID
O. V. Andreeva, R. R. Sharipova, I. Yu. Strobykina, and V. E. Kataev*
Conjugates of the diterpenoid isosteviol (16-oxo-ent-beyeran-19-oic acid) and glucuronic acid containing two diterpenoid (ent-beyerane) skeletons joined by a 1,6-hexanedicarboxylate spacer and E-D-glucopyranuronoyl moieties were synthesized for the first time. Keywords: isosteviol, diterpenes, diterpenoids, glucuronic acid, glucuronates, beyeranes, conjugates. Glucuronic acid is produced by UDP-glucuronyltransferase and is involved in the metabolism of xenobiotics (including active drug forms) to convert them into water-soluble glucuronides (glucuronosides) [1]. This is responsible for the continuously increasing interest in glucuronides of various biologically active compounds in order to increase their bioavailability and to decrease the general toxicity. Several hundred O-, N-, S-, and C-glucuronides have now been synthesized and studied [2–4]. However, much less attention has been paid to the carboxylate derivatives. Less than 30 amides of glucuronic acid (glucuronamides) were reported. They included branched glycopeptidomimetics [5], glycolipids [6–8] including anionic surfactants that transported hydrophilic drugs through hydrophobic cell membranes [7], oligosaccharides [9], conjugates with chlorin and bacteriochlorin [10], and receptors of chiral carboxylate anions [11]. Esterification (as a rule, by methyl, allyl, and benzyl alcohols) of glucuronic acid is used only to protect the carboxylic acid during glucuronide synthesis [2–4]. Herein we report the first synthesis of more complicated glucuronates that are conjugates of glucuronic acid with the diterpenoid isosteviol (16-oxo-ent-beyeran-19-oic acid) (1). 20
1
O
13
9
5
19
HO
17 11
10
3
16 7
18
O
15
1
i
AcO
Cl O
O
OAc OAc
OAc
vii
O
+ AcO
OAc
CH3
HO
O
O HO
OH
OAc
O
OAc O
6
2
OH
ii O
7 OH
OAc O
AcO
OAc
v
OAc OAc
O
O
AcO
OAc OAc
OAc
5
4
iv
O
O AcO
OH OAc
3
OAc
iii or vi
i. NaBH4, MeOH, yield 100%; ii. C2O2Cl2, CH2Cl2, Py, DMF, 0qC; iii. Ac2O, I2, MeOH, 0qC, 1 h; iv. Ac2O, I2, yield 65%; v. H2O, THF; vi. Ac2O, H2SO4 (cat), 60qC, yield 20%; vii. CH2Cl2, Py, DMF, 0qC
Scheme 1 A. E. Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences, 420088, Kazan, Ul. Arbuzova, 8, e-mail:
[email protected]. Translated from Khimiya Prirodnykh Soedinenii, No. 3, May–June, 2014, pp. 403–406. Original article submitted March 5, 2014. 0009-3130/14/5003-0465
©2014
Springer Science+Business Media New York
465
Isosteviolglucuronate 7 was obtained from 1 by chemically selective and stereospecific reduction of the oxo group by the literature method [12] to give dihydroisosteviol (2) (100% de). 1,2,3,4-Tetra-O-acetyl-E-D-glucuronic acid (5) was converted to the acid chloride (6) (Scheme 1). We tested several known methods for preparing 5. These were a) a single-pot acetylation of 3 by acetic anhydride in the presence of I2 with subsequent work up with MeOH [13]; b) acetylation of D-glucuronic acid by acetic anhydride in the presence of I2, isolation of anhydride 4, and its subsequent hydrolysis in THF [14, 15]; and c) acetylation of D-glucuronic acid by acetic anhydride with an H2SO4 (conc.) catalyst [8]. In the first method, the yields of tetraacetylated 5 were insignificant with the main product being its methyl ester. In the second method, a mixture of 5 anomers was obtained in 60% yield. The third method turned out to be the most effective [8], producing a mixture of 5 anomers in 90% yield. Compound 5 that was obtained in 20% yield by recrystallization from Et2O–CHCl3 was reacted with oxalylchloride and transformed into acid chloride 6, which was reacted in situ with 2 (Scheme 1). Glucuronate 7 did not form. Apparently, steric hindrance between the C-1 acetate of 6 and the C-13 methyl of 2 prevented the molecules from approaching each other. In order to circumvent this unfavorable steric effect, we decided to move the reaction center away from the bulky ent-beyerane skeleton. For this, isosteviol chloride (8) was reacted with ethyleneglycol to synthesize ketoalcohol 9 (Scheme 2), which reacted with 6 prepared in situ by analogy with previous work [15] to afford glucuronates 10 in 20% yield (after chromatography over silica gel) (Scheme 2). O O i
1
ii
O
O
iii
O O
O
O
O
O
Cl
O AcO
OH
8
OAc 10
9 AcO
OAc
i. SOCl2, CH2Cl2; ii. HO(CH2)2OH, CH2Cl2, yield 48%; iii. 6 chloride in situ, CH2Cl2, Py, DMF, 0°C, yield 20%
Scheme 2 Next, we synthesized dinuclear glucuronate 14 in which two molecules of 10 were covalently linked. For this, diacid 11 that was obtained by the published method [16, 17] was reacted with ethyleneglycol to afford diol 12 in 98% yield (Scheme 3). Diol 12 did not react with 6 under the same conditions that were used to prepare 10. Glucuronates 13 and 14 were found only in trace quantities. The reaction of 12 directly with 5 in the presence of dicyclohexylcarbodiimide (DCC) was successful. Column chromatography over silica gel isolated 13 and 14 in yields of 12 and 23% (Scheme 4). Only one glucuronic-acid conjugate with a terpenoid, steviol (13-hydroxy-ent-caur-16-en-19-oic acid, an isosteviol structural isomer), has been reported [18]. Our seminal syntheses of dinuclear glucuronates containing two diterpenoids and two E-D-glucopyranuronoyl groups set a new direction in chemical modifications of glucuronic acid. HO HO OH
O
Cl
O
O O
O
O
Cl 6
O
ii, iii
O
i O O
OH 2
O
O
O O
OH 11
O
OH
i. CH2Cl2, reflux, yield 50%; ii. SOCl2, CH2Cl2; iii. HO(CH2)2OH, CH2Cl2, yield 98%
Scheme 3
466
O 12
O
AcO
OAc
AcO
OAc O O
HO Cl
12 +
O
O
O
OAc
O
O
O
O
O
O
O
O
i
AcO
OAc
+
OH OH
12 +
O
O
OAc
O
ii
AcO
OAc
O O
O
OH
O
O
O
O
O
O O
AcO 13
AcO
O O
O
AcO
OAc
OAc OAc
14
AcO
OAc
i. CH2Cl2, DMAP / Py, 0qC; ii. CH3CN, DMAP, DCC, yields: 12% (13), 23% (14)
Scheme 4 EXPERIMENTAL PMR spectra were recorded on an Avance-400 spectrometer (Bruker, Germany). MALDI mass spectra in the range m/z 400-3000 were obtained in an UltraFlex III TOF/TOF time-of-flight mass spectrometer (Bruker Daltonik GmbH, Germany). Data were processed using the flexAnalysis 3.0 program (Bruker Daltonik GmbH, Germany). The matrix was p-nitroaniline. Optical rotation was measured on a PerkinElmer 341 polarimeter (USA). IR spectra in the range 400–4000 cm–1 were recorded from films on a Vector 22 Fourier spectrometer (Bruker, Germany). Melting points were determined on a Boetius melting-point apparatus. The course of reactions and purity of products were monitored using TLC on Sorbfil PTSKh-AF-A plates (Krasnodar, Russia) with detection by H2SO4 (5%) with subsequent heating. Compounds were isolated by flash chromatography over KSK silica gel (0.063–0.125 mm, KhromLab Ltd., Russia) using petroleum ether–EtOAc (10:1–1:1) eluent. Isosteviol (1) was obtained by the literature method [19] from the sweetener Sweta (Stevian Biotechnology Corp., Malaysia). Compounds 2 [12], 5 [8], 8 [20], 9 [21], and 11 [16] were prepared by the published methods. The physical constants agreed with those in the literature. Compound 5 was dried before use by refluxing in benzene with a Dean–Stark trap and subsequently stored in vacuo. We used commercial D-glucuronic acid (98%, Fluka AG, Switzerland), ethyleneglycol (99%, Alfa Aesar, USA), oxalylchloride (98%, Acros Organics, Belgium), and thionylchloride (imp., Vekton, Russia). 2-(1c,2c,3c,4c-Tetra-O-acetyl-E-D-glucopyranuronoyloxy)-ethyl-16-oxo-ent-beyeran-19-oate (10). Compound 5 ° molecular sieves (0.3 g, 0.8 mmol) was dissolved in anhydrous CH2Cl2 (30 mL), cooled (ice bath) in the presence of 4-A under Ar, treated with freshly distilled DMF (0.09 mL, 1.2 mmol) and oxalylchloride (0.1 mL, 1.2 mmol), stirred with cooling for 1 h and at room temperature for 1 h, cooled to 0°C, and treated dropwise with a solution of 9 (0.2 g, 0.55 mmol) in a mixture of Py (0.2 mL) and CH2Cl2 (5 mL). The resulting mixture was stirred with cooling for 1 h and at room temperature for 20 h, 20 –29.5q (ñ 1.0, washed with H2O, and chromatographed over silica gel to afford light-brown 10. Yield 0.08 g (20%), [D]D –1 1 CHCl3). IR spectrum (Q, cm ): 1217 (C–O), 1710, 1762 (C=O). Í NMR spectrum (400 MHz, ÑDÑl3, G, ppm, J/Hz): 0.70– 2.20 (19Í, m, ent-beyerane), 0.71 (3Í, s, H3-20), 0.97 (3Í, s, H3-1 7), 1.20 (3Í, s, Í3-18), 2.024 (3H, s, CH3C(O)), 2.030 (3H, s, CH3C(O)), 2.032 (3H, s, CH3C(O)), 2.10 (3H, s, CH3C(O)), 2.61 (1H, dd, J = 18.6, 3.8, Í-15), 4.15–4.33 (4Í, m, -OCH2CH2O-), 4.34–4.42 (1Í, m, Í-5c), 5.11–5.17 (1H, m, H-2c), 5.21–5.35 (2H, m, H-3c, 4c), 5.75 (1H, d, J = 7.76, H-1c). Mass spectrum: m/z 729.32 [M + Na]+, calcd 729.31 [M + Na]+. Ñ36Í50Î14.
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Bis[19-nor-4D(2-hydroxyethyloxycarbonyl)-ent-beyeran-16-yl]-1,6-hexanedicarboxylate (12). A solution of diacid 11 (0.4 g, 0.5 mmol) in anhydrous CH2Cl2 (5 mL) was treated with SOCl2 (2 mL, 27.5 mmol) and held for 2 h at 60°C. The solvent was distilled under Ar at reduced pressure. The residue was dissolved in anhydrous CH2Cl2 (20 mL), stirred, treated dropwise under Ar over 15 min with ethyleneglycol (3 mL, 53.8 mmol), stirred at room temperature for 20 h. The organic layer was separated, washed with H2O (3 u 10 mL), and dried over MgSO4. The solvent was removed at reduced pressure to 20 –70.4q (ñ 1.0, CHCl ). IR spectrum (Q, cm–1): afford 12 as an amorphous powder. Yield 0.44 g (98%), mp 65–67qÑ, [D]D 3 1 1724 (C=O), 3465 (ÎÍ). Í NMR spectrum (400 MHz, ÑDÑI3, G, ppm, J/Hz): 0.70–2.20 (48Í, m, ent-beyerane, spacer (ÑÍ2)4), 0.73 (6Í, s, H3-20, 20c), 0.90 (6Í, s, H3-17, 17c), 1.19 (6Í, s, Í3-18, 18c), 2.17 (2Í, d, J = 14.6, Íeq-3, 3c), 2.30 (4H, t, J = 7.3, 16, 16c-OC(O)CH2), 3.77–3.85 (4Í, m, -CH2OH, -CcH2OH), 4.10–4.24 (4Í, m, 19, 19c-(O)OCH2), 4.72 (2Í, dd, J = 10.5, 4.2, Í-16, 16c). Mass spectrum: m/z 889.58 [M + Na]+, calcd 889.58 [M + Na]+. Ñ52Í82Î10. General Method for Preparing 13 and 14. A solution of 5 (0.1 g, 0.28 mmol) in anhydrous MeCN (7 mL) was treated with DMAP (0.001 g, 0.008 mmol) and 12 (0.11 g, 0.13 mmol), stirred until dissolved completely, treated with 4-A° molecular sieves and DCC (0.065 g, 0.31 mmol), stirred for 20 h at room temperature, treated again with DCC (0.02 g), and stirred for 24 h. Chromatography over silica gel afforded light-brown sticky products. O-{19-nor-4D[(1s,2s,3s,4s-Tetra-O-acetyl-E-D-glucopyranuronoyloxy)ethoxycarbonyl]-ent-beyeran-16-yl}-Oc[19c-nor-4cD (2-hydroxyethyloxycarbonyl)-ent-beyeran-16c-yl]-1,6-hexanedicarboxylate (13). Yield 0.02 g (12%), –1 1 [D]20 D –26.4q (ñ 0.8, CHCl3). IR spectrum (Q, cm ): 1219 (C–O), 1726, 1763 (C=O), 3130 (ÎÍ). Í NMR spectrum (400 MHz, ÑDÑI3, G, ppm, J/Hz): 0.8–2.2 (48Í, m, ent-beyerane, spacer (ÑÍ2)4), 0.69 (3Í, s, H3-20), 0.72 (3Í, s, H3-20c), 0.90 (3Í, s, H3-17), 0.91 (3Í, s, H3-17c), 1.16 (3Í, s, Í3-18), 1.19 (3Í, s, Í3-18c), 2.02 (3H, s, CH3C(O)), 2.035 (3H, s, CH3C(O)), 2.039 (3H, s, CH3C(O)), 2.10 (3H, s, CH3C(O)), 2.27–2.35 (4H, m, 16, 16c-OC(O)CH2), 3.81 (2Í, dd, J = 9.95, 4.97, CH2OH), 4.10-4.30 (6Í, m, 19-(O)OCH2CH2O-, 19c-(O)OCH2), 4.32–4.42 (1Í, m, Í-5c), 4.68–4.76 (2Í, m, H-16, 16c), 5.11–5.17 (1H, m, H-2c), 5.22 (1H, t, J = 9.5, H-4c), 5.40 (1H, t, J = 9.3, H-3c), 5.89 (1H, d, J = 7.8, H-1c). Mass spectrum: m/z 1233.7 [M + Na]+, calcd 1233.7 [M + Na]+. Ñ66Í98Î20. Bis{19-nor-4D[(1s,2s,3s,4s-tetra-O-acetyl-E-D-glucopyranuronoyloxy)ethoxycarbonyl]-ent-beyeran-16-yl}-1,620 –23.4q (ñ 1.0, CHCl ). IR spectrum (Q, cm–1): 1218 (C–O), 1726, hexanedicarboxylate (14). Yield 0.05 g (23%), [D]D 3 1 1761(C=O). Í NMR spectrum (400 MHz, ÑDÑI3, G, ppm, J/Hz): 0.65–2.2 (48Í, m, ent-beyerane, spacer (ÑÍ2)4), 0.69 (6Í, s, H3-20, 20c), 0.90 (6Í, s, H3-17, 17c), 1.16 (6Í, s, Í3-18, 18c), 2.016 (6H, s, 2CH3C(O)), 2.017 (6H, s, 2CH3C(O)), 2.032 (6H, s, 2CH3 C(O)), 2.10 (6H, s, 2CH3C(O)), 2.32 (4H, t, J = 7.3, 16, 16c-OC(O)CH2), 4.09–4.30 (8Í, m, two -OCH2CH2O-), 4.32–4.40 (2Í, m, Í-5c, 5cc), 4.71 (2Í, dd, J = 10.6, 4.3, H-16, 16c), 5.11–5.16 (2H, m, H-2c, 2cc), 5.22 (2H, t, J = 9.5, H-4c, 4cc), 5.40 (2H, t, J = 9.3, H-3c, 3cc), 5.88 (2H, d, J = 7.8, H-1c, 1cc). Mass spectrum: m/z 1577.76 [M + Na]+, calcd 1577.73 [M + Na]+. Ñ80Í114Î30.
ACKNOWLEDGMENT The work was supported financially by the RFBR (Grant 13-03-00054) and RAS Presidium Program No. 8.
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