distilled. The residue was dissolved in CH Cl and passed over a layer of Al O . Eluent fractions containing product were
2
2
2 3
combined and evaporated in vacuo. The residue was recrystallized from MeCN to afford 8 (1.28 g, 80%). C H NO Br,
20 20
4
+
1
mp 162.5°C. Mass spectrum (API-ES, m/z): 420.062 (M + 1), calcd 420.063. Í NMR spectrum (400 MHz, CDCl , ꢅ, ppm,
3
J/Hz): 2.54–2.68 (2Í, m, Í-5, 6), 3.03–3.20 (3Í, m, Í-5, 6, 13), 3.25 (1Í, dd, J = 16.7, 4.0, Í-13), 3.45–3.51 (2Í, m,
Í-8, 14), 3.81 (3Í, s, ÎÑÍ ), 3.82 (3Í, s, ÎÑÍ ), 4.19 (1Í, d, J = 15.9, Í-8), 5.91 (2Í, s, ÎÑÍ O), 6.57 (1Í, s, Í-4), 6.77 (1Í,
3
3
2
13
s, Í-1), 7.01 (1Í, Í-11). C NMR spectrum (100 MHz, CDCl , ꢅ, ppm): 29.36 (Ñ-5), 37.43 (Ñ-13), 50.93 (Ñ-6), 53.83 (Ñ-8),
3
55.89 (10-ÎÑÍ ), 59.41 (Ñ-14), 60.12 (9-ÎÑÍ ), 100.72 (ÎÑÍ Î), 105.51 (Ñ-1), 108.29 (C-4), 114.61 (Ñ-11), 118.40 (Ñ-12),
3
3
2
120.00 (Ñ-8à), 126.84 (Ñ-12à), 127.54 (Ñ-4à), 130.29 (Ñ-1à), 144.26 (Ñ-9), 145.92 , 146.08 (Ñ-2, 3), 150.65 (Ñ-10).
Tetrahydroberberrubine (11). A suspension of 9–EtOH solvate (1:1, 2.00 g) in MeOH (20 mL) at 0°C was stirred
on a magnetic stirrer, treated in portions with NaBH (0.83 g, 4 eq), and stirred at room temperature for 1.5 h. The solvent was
4
vacuum distilled. The residue was dissolved in CH Cl and passed over a layer of Al O . Eluent fractions containing product
2
2
2 3
were combined and evaporated in vacuo. The residue was recrystallized from MeCN to afford 11 (0.682 g, 78%). The spectral
data agreed with those published [29].
9-Demethyl-9-O-tosyltetrahydroberberine (14). A) A suspension of 13 (230 mg) in MeOH (5 mL) at 20°C was
stirred on a magnetic stirrer, treated in portions with NaBH (68 mg, 4 eq), and stirred at room temperature for 2 h. The solvent
4
was vacuum distilled. The residue was dissolved in CH Cl and passed over a layer of Al O . Eluent fractions containing
2
2
2 3
product were combined and evaporated in vacuo. The residue was recrystallized from MeOH to afford 14 (67 mg, 31%).
B) A solution of 11 (800 mg) in CH Cl (10 mL) was stirred on a magnetic stirrer at room temperature, treated
2
2
dropwise with a solution of p-toluenesulfonyl chloride (705 mg) in CH Cl (10 mL) and NEt (0.51 mL), stirred for 1 h, left
2
2
3
overnight, and evaporated. The residue was chromatographed over a column of silica gel [CH Cl and CH Cl –MeOH
2
2
2
2
(100:1) eluents]. Eluent fractions containing product were combined and evaporated in vacuo. The residue was recrystallized
+
from MeCN to afford 14 (843 mg, 71%). C H NO S, mp 184.3°C. Mass spectrum (API-ES, m/z): 480.149 (M + 1),
26 25
6
1
calcd 480.148. Í NMR spectrum (400 MHz, CDCl , ꢅ, ppm, J/Hz): 2.45 (3Í, s, ÑÍ ), 2.52–2.68 (2Í, m, Í-5, 6), 2.78 (1Í,
3
3
dd, J = 16.3, 11.4, Í-13ax), 3.00–3.12 (2Í, m, Í-5, 6), 3.22 (1Í, dd, J = 16.3, 3.7, Í-13eq), 3.45 (3Í, s, ÎÑÍ ), 3.54 (1Í, dd,
3
J = 11.4, 3.7, Í-14), 3.57 (1Í, d, J = 15.8, Í-8), 4.18 (1Í, d, J = 15.9, Í-8), 5.90 (2Í, s, ÎÑÍ O), 6.56 (1Í, s, Í-4), 6.68 (1Í,
2
s, Í-1), 6.69 (1Í, d, J = 8.2, Í-12), 6.99 (1Í, d, J = 8.2, Í-11), 7.33 (2Í, d, J = 8.2, Í-3ꢀ, 5ꢀ), 7.86 (2Í, d, J = 8.2, Í-2ꢀ, 6ꢀ).
13
C NMR spectrum (100 MHz, CDCl , ꢅ, ppm): 23.92 (CH ), 31.76 (Ñ-5), 38.37 (Ñ-13), 53.32 (Ñ-6), 56.34 (Ñ-8), 57.68
3
3
(ÎÑÍ ), 61.63 (Ñ-14), 103.03 (ÎÑÍ Î), 107.69 (Ñ-1), 110.69 (C-4), 112.94 (Ñ-12), 129.88 (Ñ-11), 130.03, 130.30 (Ñ-12à,
3
2
4à), 130.65 (C-2ꢀ, 6ꢀ), 131.54 (C-3ꢀ, 5ꢀ), 132.88, 132.89 (Ñ-8à, 1a), 136.92 (Ñ-1ꢀ), 137.73 (Ñ-4ꢀ), 146.99 (Ñ-9), 148.22, 148.40
(Ñ-2, 3), 152.07 (Ñ-10).
Cell Cultivation. HepG2 cells were cultivated at 37°C and 5% CO in 25-mL vials with DMEM/F12 medium
2
containing FBS (10%), glutamine (0.03%), and kanamycin at a concentration of 100 ꢂg/mL. Cells in the exponential growth
phase were seeded 24 h before the experiment in wells of a 24-well plate and cultivated in DMEM/F12 medium containing
lipoprotein-deficient serum (LPDS, 10%, Sigma) and glutamine (0.03%) up to an 80% density monolayer. Then, the growth
medium was replaced by DMEM/F12 without serum with added 1–16 at concentrations of 20 ꢂg/mL. Medium in the control
sample was replaced by DMEM/F12 without serum. Cells were incubated with drugs for 12 h, after which the growth medium
was removed. Cells were lysed by TRIzol reagent (Invitrogen) for subsequent mRNA isolation.
Assay of LDLR Gene mRNAby Real-time RT-PCR. Total cellular RNAwas isolated according to the manufacturer’s
recommendations. cDNA was synthesized by reverse transcriptase (RT) at 42°C for 45 min in a reaction mixture (20 ꢂL)
containing Tris-HCl (10 mM, pH 8.5), MgCl (5 mM), DTT (10 mM), KCl (100 mM), dNTP (0.2 mM), Stat-9 primer
2
(10 ng/ꢂL), RNA polymerase (MoMLV, 200 U), and total RNA (200 ng to 1 ꢂg). The real-time PCR method determined in
each of the cDNA samples the relative amount of cDNA of the LDLR gene. Primer F: 5ꢀ-ACTGCAAGGACAAATCTGACGA-5ꢀ
and R: 5ꢀ-CCTCGCAGAGTGTCACATTAAC-5ꢀ was used for amplification. cDNA samples were amplified in a reaction
mixture (20 ꢂL) containing Tris-HCl (10 mM, pH 8.9), MgCl (2.5 mM), KCl (55 mM), dNTP (0.2 mM), Taq polymerase
2
(0.5 U), and SYBR Green I (Invitrogen) at a 1:25,000 dilution in addition to the corresponding pair of F and R primers (each
300 nM) and cDNA sample. The results were normalized relative to a coefficient that was calculated as the average geometric
amount of mRNA for POLR2A, GAPDH, RPL32, and ACTB genes.
Laboratory Animals. The experiments used white male laboratory mice (20–35 g) from the Experimental Animal
Nursery at the Institute of Cytology and Genetics, SB, RAS, and were kept under standard conditions. Experiments with
animals were conducted according to the European Convention for the Protection of VertebrateAnimals Used for Experimental
and Other Scientific Purposes (Strasburg, 1986). Each group consisted of 10 animals.
920