ISSN 0012�5008, Doklady Chemistry, 2011, Vol. 439, Part 1, pp. 204–208. © Pleiades Publishing, Ltd., 2011.Original Russian Text © I.V. Nechepurenko, U.A. Boyarskikh, N.I. Komarova, M.P. Polovinka, M.L. Filipenko, G.I. Lifshits, N.F. Salakhutdinov, G.A. Tolstikov, 2011, publishedin Doklady Akademii Nauk, 2011, Vol. 439, No. 3, pp. 346–350.

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Currently, atherosclerosis is the most commoncause of death in developed countries of the world.The development of this disease is particularly relatedto the metabolism of lipids, first of all, cholesterol.Therefore, the search for drugs capable of reducing theblood cholesterol level is among the priority tasks ofpharmacology.

Now statins are used most often as hypolipidemicagents. In 2007 the world market of statins was esti�mated at 35 billion dollars [1]. Statins terminate thesynthesis of endogenic cholesterol by inhibiting theHMG CoA reductase in the first stage of cholesterolbiosynthesis [2]. As a result, together with cholesterolbiosynthesis, this also terminates the biosynthesis ofubiquinone, which is involved in oxidative phosphory�lation and in mitochondrial electron transfer chain.The indications to statin therapy must take intoaccount the risk of side effects such as rhabdomyolysis,myositis, and an increase in liver enzyme activity.

In blood plasma, cholesterol mainly occurs withinlow density lipoproteins (LDLs). From the blood,LDLs enter the liver (up to 75%) and other tissueswhere they are bound by LDL receptors (LDLRs) andare transported inside the cells. Thus, membrane LDLreceptors play a key role in regulating the blood cho�lesterol level [3]. One can conclude that the pharma�ceutical compounds that promote the LDLR expres�sion by liver cells would be efficient for reducing theblood plasma cholesterol concentration. The expres�sion level of LDL receptors depends on the intracellu�

lar cholesterol concentration and is mainly regulatedat the transcription level according to the negativefeedback mechanism [4]. Indeed, LDLR gene tran�scription is induced upon binding of the sterol regula�tory element binding proteins (SREBPs) to the SRE�1(sterol regulatory element) sequence of the LDLRgene promoter. The SRE binding protein is in turnactivated in response to the decrease in the cell choles�terol concentration by means of the SCAP protein(SREBP cleavage activating protein), which containsa cholesterol�sensitive domain [5].

The isoquinoline alkaloid berberine is a commonrepresentative of the family of protoberberine alka�loids; most often, it was isolated as chloride 1 or sulfate[6]. Usually, the name “berberine” is used to describethe common properties of this alkaloid system,whereas when describing particular characteristics, itis necessary to indicate what particular salt of the alka�loid is meant, e.g., berberine chloride.

Berberine salts possess a broad range of biologicalactivities: antitumor, antibacterial, antimicrobial, andantifungal [6]. Long�term administration of berberinechloride was found to reduce the overall blood choles�terol concentration by 29% [7]. The hypocholestere�mic action of berberine chloride 1 comes from anincrease in the LDLR expression level; however,unlike statins, the berberine action is related to stabili�zation of the mRNA of the LDLR gene rather than tothe intracellular cholesterol level or activation of theLDLR gene transcription [7, 8]. For some berberinederivatives, the ability to increase the LDLR geneexpression was studied; however, the best results werefound for the proper berberine and its isomer, pseudo�berberine [9, 10].

The purpose of the present work is to preparebromo and iodo berberine derivatives and to studytheir effect on the LDLR gene expression level usingthe human hepatoma cell model HepG2.

CHEMISTRY

LDLR Up�Regulatory Activity of Berberine and Its Bromo and Iodo Derivatives

in Human Liver HepG2 CellsI. V. Nechepurenkoa, U. A. Boyarskikhb, N. I. Komarovaa, M. P. Polovinkaa,

M. L. Filipenkob, G. I. Lifshitsb, N. F. Salakhutdinova, and Academician G. A. Tolstikova

Received March 16, 2011

DOI: 10.1134/S0012500811070093

a Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences,pr. Akademika Lavrent’eva 9, Novosibirsk, 630090 Russia

b Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences,pr. Akademika Lavrent’eva 8, Novosibirsk, 630090 Russia

DOKLADY CHEMISTRY Vol. 439 Part 1 2011

LDLR UP�REGULATORY ACTIVITY OF BERBERINE 205

RESULTS AND DISCUSSION

Preparation of Bromo and Iodo Berberine Derivatives

Methods of direct introduction of halogens into theberberine molecule have not been described previ�ously. A method for the preparation of 12�bromober�berine bromide 2 by the reaction of dihydroberberinewith 10 equivalents of bromine involving the aromati�zation step was reported [11]. Here, we showed that in

the bromination of berberine chloride with elementalbromine with heating in acetic acid, complete conver�sion is achieved by using 5 equivalents of bromine.12�Bromoberberine salts, monobromide 2 and tribro�mide 3, were isolated as the reaction products in31% and 47% yields, respectively (Scheme 1). Com�pounds 2 and 3 have identical 1H NMR spectra andHPLC patterns.

Scheme 1.

NCH3O

OCH3

OO

Cl

1

4

567

89

12 13 1a

4a13a

12a11

10 8a

2

3

NCH3O

OCH3

OO

Br

Br3

NCH3O

OCH3

OO

Br

Br

+5 equiv. Br2

t°

1 2 3

A study of iodination of berberine chloride 1showed that on treatment with reagents such as ICl,I2 + NaIO3 + H2SO4, HgO + AcOH + KI + I2, KI +CAN, KI + H2O2 + H2SO4, or KI + AcOOH [12], noreaction took place, and the initial compound 1 was

recovered unchanged. However, on treatment of ber�berine chloride 1 with the HgO + TFA/ KI + I2, iodina�tion into position 4 takes place to give compound 4(Scheme 2).1 The reacting species in this reaction is thehighly electrophilic mercury trifluoroacetate.

Scheme 2.

NCH3O

OCH3

OO

Cl

4

NCH3O

OCH3

OO

HgOCOCF3

CF3COO

NCH3O

OCH3

OO

I

I

HgO + TFAKI, I2

1 4

Biological Activity of Berberine Chloride and Its Bromo and Iodo Derivatives

We studied the effect of compounds 1–4 in con�centrations of 10, 20, and 30 μg/mL on the expressionlevel (up�regulation) of the LDLR gene in theHepG2 line cells (figure); the expression level wasmeasured relative to the blank sample containing nocompounds 1–4. It was shown that compounds 1–3up�regulate the expression of the LDLR gene.

The most pronounced up�regulation of the LDLRexpression was found for berberine chloride 1. Indeed,culturing of HepG2 cells in the presence of com�pound 1 (20 μg/mL) led to a 2.56�fold increase in theamount of the LDLR gene mRNA in the cells. Furtherincrease in the concentration of compound 1 to

30 μg/mL was not accompanied by a significantchange in the amount of the LDLR gene mRNA.According to published data, compound 1 in 5 μg/mLconcentration induces a 2.2�fold increase in theLDLR gene expression [7].

However, modification of the berberine structureby introducing a halogen atom and replacing thecounterion, resulted in a decrease in the berberineability to up�regulate the LDLR gene expression. Forbromo derivatives 2 and 3, the most pronounced up�regulation of the LDLR mRNA expression in theHepG2 cells (1.77� and 1.43�fold, respectively) wasachieved when 30 μg/mL of the compounds was used.The addition of berberine iodide 4 (10–30 μg/mL) did

1CAN is cerium ammonium nitrate, TFA is trifluoroacetic acid.

206

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NECHEPURENKO et al.

not result in significant change in the LDLR geneexpression level.

It was shown [7] using the hepatoma cell modelHepG2 that, for increasing the LDLR gene expressionupon the action of berberine, of the key importanceare the presence of the ARE motif (AU rich element)and the UCAU repeat in the 5'�untranslated region ofthe LDLR mRNA. Hence, the mechanism of ber�berine action is attributable to the increase in the sta�bility of the mRNA of the LDLR gene. These resultssuggest that berberine binds to the mRNA sequence ofthe LDLR gene and, hence, changes its accessibilityfor RNA�binding proteins, which regulate the mRNAdegradation rate in the cell. The introduction of ahalogen atom into the berberine molecule (com�pounds 2–4) might prevent binding to mRNA in thisregion. The bulky electronegative iodine atom (com�pound 4) has the most pronounced effect, resulting inthe absence of binding.

Thus, we demonstrated that the introduction ofbromine atom into a berberine molecule decreases itsability to up�regulate the LDLR gene expression inHepG2 cells as compared with berberine chloride; theintroduction of a iodine atom completely eliminatesthis activity.

EXPERIMENTAL

Melting points were determined on a MetierToledo FP 900 instrument; UV spectra wererecorded on an HP 8453 UV Vis instrument inEtOH (c = 10–4 mol/L), IR spectra were measured ona VECTOR 22 spectrophotometer in KBr. Mass spec�tra were run on a Bruker micrOTOF�Q hybrid qua�drupole time�of�flight mass spectrometer. The operat�

ing parameters of the mass detection were as follows: elec�trospray ionization at atmospheric pressure (API�ES).Positive ion scanning in the range m/z = 100–3000.Drying gas (nitrogen) flow rate of 4 L/min and tem�perature of 220°C; sprayer pressure of 1.0 bar. The 1Hand 13C NMR spectra were recorded on a BrukerAM�400 radiospectrometer (400.13 and 100.61 MHz)for 5–10% solutions of the compounds in DMSO�d6.The solvent signal was used as the reference. Columnchromatography was carried out on basic aluminumoxide LL40/250.

HPLC was performed on a Milicrom A�02 micro�column chromatograph (manufactured by theEcoNova company, Novosibirsk) using a standardchromatographic column (2 × 75 mm) packed with areverse phase sorbent (ProntoSIL 120�5�C18, particlesize 5 μm, BISCHOFF, Germany). Gradient elutionwith the 0.1% TFA–methanol system (0 to 100% ofmethanol in 25 min) and simultaneous multiwavedetection at six wavelengths (220, 240, 260, 280, 320,360 nm) were used. The temperature was 35°C,the pressure was 30–36 atm, and the flow rate was150 μL/min.

Berberine chloride was isolated from the roots ofBerberis sibirica vegetating in the Onguday Region,Altay Republic (Russia), mp 204°C. The spectralcharacteristics of berberine chloride correspond topublished data [13], the elemental analysis data corre�spond to the calculated values for berberine chloridemonohydrate.

12�Bromoberberine bromides 2 and 3. Berberinechloride monohydrate (5.58 g, 14.3 mmol) was dis�solved on heating in acetic acid (160 mL). At 120°Cand magnetic stirring, a solution of Br2 (12.00 g,

3.5

0.5

00 10

3.0

2.5

2.0

1.5

1.0

20 30 0 10 20 30 0 10 20 30 0 10 20 30

1.00

1.73

2.562.63

1.00 1.00 1.00

1.161.20

1.77

1.201.12

1.43

1.020.94 0.98

Concentration, µg/mL

Exp

ress

ion

leve

l of t

he

LD

LR

mR

NA 1

23

4

Expression level of the LDLR gene in the HepG2 cells depending on the concentration of compounds 1–4.

DOKLADY CHEMISTRY Vol. 439 Part 1 2011

LDLR UP�REGULATORY ACTIVITY OF BERBERINE 207

75 mmol) in acetic acid (10 mL) was added dropwise.The reaction mixture was stirred at reflux at the sametemperature for 4 h and cooled. The precipitate wasfiltered off and washed with 2 × 20 mL of a 10% solu�tion of Na2S2O3, 2 × 20 mL of a 10% solution ofNaHCO3, and 2 × 20 mL of a 20% solution of KBr.The precipitate was dried in a desiccator and refluxedwith methanol (200 mL) for 30 min. The hot solutionwas filtered to give 4.60 g (49%) of the orange precipi�tate of tribromide 3. Cooling of the filtrate yielded2.30 g (32%) of the brown precipitate of bromide 2.

Compound 2: mp 190°C (decomp.).

For C20H17Br2NO4 anal. calcd. (%): C, 48.51; H,3.46; N, 2.83; Br (total), 32.27; Br (ionic), 16.14.

Found (%): C, 48.73; H, 3.74; N, 3.22; Br (total),32.70; Br (ionic), 17.53.

Compound 3: mp 179°C (decomp.).

For C20H17Br4NO4 anal. calcd. (%): C, 36.68; H,2.62; N, 2.14; Br, 48.80.

Found (%): C, 36.58; H, 2.54; N, 1.82; Br, 49.15.

Compounds 2 and 3.

UV (EtOH, λmax, nm, logε): 220 (4.11); 266 (3.84);361 (3.71); 413 (3.44).

IR, cm–1: 1587, 1494, 1319, 1227, 1034.

MS (m/z): 414.042. Calculated: 414.034

(С20H17BrNO ).1H NMR (400 MHz, DMSO�d6, δ, ppm; J, Hz):

3.21 (t, 2H, J = 6, H�5), 4.10 (s, 3H, OCH3), 4.11 (s,3H, OCH3), 4.96 (t, 2H, J = 6, H�6), 6.17 (s, 2H,OCH2O), 7.09 (s, 1H, H�4), 7.89 (s, 1H, H�1), 8.47(s, 1H, H�13), 8.50 (s, 1H, H�11), 9.98 (C, 1H, H�8).

13C NMR (100 MHz, DMSO�d6; δ, ppm): 26.26(C�5), 55.31 (C�6), 57.62 (10�OCH3), 62.14 (9�OCH3), 102.20 (OCH2O), 105.95 (C�1), 108.43 (C�4), 115.06 (C�12), 118.52 (C�13), 120.03 (C�1a),121.97 (C�8a), 129.94 (C�11), 131.15, 131.24 (C�12a,C�4a), 138.84 (C�13a), 144.00 (C�9), 146.72 (C�8),147.84 (C�2), 150.21, 150.34 (C�3, C�10).

4�Iodoberberine iodide 4. Yellow HgO (5.85 g,27 mmol) was dissolved in trifluoroacetic acid(100 mL), berberine chloride monohydrate (3.72 g,9.5 mmol) was added, and the mixture was purgedwith argon and refluxed for 3 h. The solvent was dis�tilled off in vacuum, the dark precipitate containingberberine trifluoroacetate was dissolved in methanol(200 mL), and a suspension of KI (5.64 g, 34 mmol) inmethanol (20 mL) (a yellow precipitate was formed)and I2 (8.64 g, 34 mmol) were added. The reactionmixture was stirred at 80°C for 13 h. After cooling toroom temperature, the excess iodine was decomposedwith a 10% solution of Na2S2O3 (60 mL). The precip�

+

4

itate was filtered off and separated by column chroma�tography on basic alumina repeated many times (elu�tion with methylene chloride–methanol from 100 : 0to 100 : 1) to give totally 3.54 g of fractions enrichedwith the product. The fractions were combined andrefluxed with methanol (50 mL) three times for 1 h togive 2.15 g of compound 4 (yield 37%), mp. 221.5°C.

For C20H17I2NO4 · H2O anal. calcd. (%): C, 39.56;H, 3.15, N, 2.31; I, 41.80.

Found (%):C, 39.35; H, 2.82, N, 1.97; I, 42.41.

UV (MeOH, λmax, nm): 232, 260, 346.

IR, cm–1: 1595, 1498, 1456, 1360, 1269, 1041.

MS (m/z): 462.021. Calculated: 462.020

(C20H17INO ).1H NMR (400 MHz, DMSO�d6, δ, ppm, J, Hz):

3.22 (t, 2H, J = 6, H�5), 4.07 (s, 3H, OCH3), 4.10 (s,3H, OCH3), 4.94 (t, 2H, J = 6, H�6), 6.26 (s, 2H,OCH2O), 7.81 (s, 1H, H�1), 8.00 (d, 1H, J = 9, H�12), 8.21 (d, 1H, J = 9, H�11), 8.95 (s, 1H, H�13),9.94 (s, 1H, H�8).

13C NMR (100 MHz, DMSO�d6, δ, ppm):30.83 (C�5), 55.49 (C�6), 57.39 (10�OCH3), 62.29(9�OCH3), 78.64 (C�4), 102.07 (OCH2O), 105.82(C�1), 121.14 (C�13), 121.93, 122.16 (C�1a, C�8a),124.00 (C�12), 127.02 (C�11), 132.98, 133.11 (C�12a,C�4a), 137.13 (C�13a), 143.97 (C�9), 145.67 (C�8),146.37 (C�2), 150.94 (C�10), 152.32 (C�3).

Cell Culturing

The HepG2 line cells were cultured at 37°C in air(5% CO2) in 25�mL vials in the DMEM/F12 mediumcontaining 10% FBS, 0.03% of glutamine and kana�mycin (100 μg/mL). Twenty four hours before theexperiment the cells in the exponential growth phasewere inoculated in the wells of a 24�well plate and cul�tured in the DMEM/F12 medium containing 10%LPDS (lipoprotein deficient serum, Sigma) and0.03% of glutamine up to 80% monolayer density.Then the culture medium was replaced by serum�freeDMEM/F12 with addition of compounds 1–4 (10,20, or 30 μg/mL). In the blank sample, the mediumwas replaced by serum�free DMEM/F12. The cellswere incubated with the sample for 12 h, the culturemedium was removed, and the cells were lysed withTrizol (Invitrogen) for the subsequent isolation ofmRNA.

Determination of the Amount of mRNA of the LDLRGene by Real�Time RT�PCR

The total cellular RNA was isolated according tothe manufacturer recommendations. cDNA was syn�

+

4

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NECHEPURENKO et al.

thesised by reverse transcription (RT) at 42°C for45 min in 20 μL of the reaction mixture containing10 mM Tris�HCl (pH 8.5), 5 mM MgCl2, 10 mMDTT, 100 mM KCl, 0.2 mM dNTP, Stat�9 primer(10 ng/μL), 200U RNA polymerase (MoMLV), and200 ng to 1 μg of the total RNA. By real�time PCR, therelative amount of cDNA of the LDLR gene wasdetermined in each of the obtained cDNA samples.The primers used for amplification were F: 5'�ACTG�CAAGGACAAATCTGACGA�5' and R: 5'�CCTCG�CAGAGTGTCACATTAAC�5'. Amplification of thecDNA samples was performed in 20 μL of the reactionmixture containing 10 mM Tris�HCl (pH 8.9),2.5 mM MgCl2, 55 mM KCl, 0.2 mM dNTP, 0.5 UTaq polymerase, and SybrGreen I (in 1 : 25 000 dilu�tion) (Invitrogen), and also the corresponding pair ofF and R primers (300 nM each) and a cDNA sample.The obtained results were normalized to the normal�ization factor calculated as the geometric mean of theamounts of mRNA of POLR2A, GAPDH, RPL32,and ACTB genes.

ACKNOWLEDGMENTS

This work was supported by the Siberian Branch ofthe RAS (the program “Fundamental Sciences forMedicine,” project no. 5.34).

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