Synthesis of Tetrahydroberberine N , N -Derived O -Acetamides

: The reaction of berberine derivatives containing at the O -9 position N , N -disubstituted acetamide fragments with sodium borohydride in methanol at 0 ◦ C leads to a mild reduction of the “C” cycle with the formation of corresponding tetrahydroberberine derivatives with moderate to good yields.


Introduction
The isoquinoline alkaloid berberine (berberine chloride, sulfate) is known to have a wide range of diverse biological activities.Currently, research on its hypolipidaemic [1][2][3], anti-inflammatory and antioxidant [4,5], and anti-cancer [6,7] activities is being actively conducted.Works on such types of berberine activity as anti-epileptic [8,9], antidepressant [10,11], and antiallergic [12] are being developed.Berberine chloride contains in its structure an aromatic positively charged nitrogen atom; such a salt has low solubility and, as a result, low bioavailability.In order to increase the bioavailability of berberine, its water-soluble compositions are being developed [13], and complexes of berberine with Ag or Au nanoparticles [14,15], natural polymers such as chitosan [16,17], peptides [18], or hyaluronic acid [19] are used.A number of berberine derivatives have been found with activities exceeding that of the initial alkaloid, such as hypolipidemic [20], hypoglycemic [21], antibacterial [22], and antiviral [23].
The most common modification of berberine chloride involves its demethylation at the O-9 position to form the alkaloid berberubine 1, and further obtaining derivatives at this position by means of alkylation or acylation [20,24], O-9-arylation [25], or C-9 arylation [26].Thus, according to this scheme, we previously synthesized aromatic acetamides 2 by reacting berberubine 1 with bromoacetic acid amides in the presence of a base (Scheme 1) [27,28].A separate direction of berberine modification is the synthetic production of berberine-like molecules [29].Another popular branch of the chemical modification of berberine is the reduction of its isoquinolinium system.The resulting dihydro or tetrahydro derivatives are less stable than the original berberines, but have much greater solubility and often have good bioactivity.Tetrahydroberberine itself has a pronounced lipid-lowering effect [30], although there is some evidence of hepatotoxicity [31].Among tetrahydroberberine derivatives, examples with good lipid-lowering [32][33][34], antiproliferative [35], and antibacterial (antifungal) activity [36] were found.Unusual examples of activity have also been found.For example, we have shown that tetrahydroberberrubine polyfluoroaromatic sulfonates are inhibitors of tyrosyl-DNA phosphodiesterase 1 (Tdp1), an important enzyme of the DNA repair system [37].
The aim of the present work was to synthesize new N,N-disubstituted O-acetamide derivatives of tetrahydroberberine.

Synthesis of 3
The synthesis of acetamides 2 by the substitution of berberubine 1 with seven bromoacetamides in the presence of potassium carbonate was performed by our group (Scheme 1) [27,28].Compound 2 contains in its structure an aromatic heterocyclic ring (cycle "C") in the isoquinolinium system, which is reduced by the action of various reducing agents to form dihydro or tetrahydro derivatives.We have shown that the reaction of compounds 2a-g with 4 mol equivalents of sodium borohydride in methanol at 0 °C resulted in a mild reduction of the "C" cycle to produce tetrahydroberberine derivatives of 3a-g (Scheme 1).The reaction conditions used are similar to those in which our group previously carried out the reduction of compound 1 and some of its derivatives [32].The reduction proceeds non-stereoselectively, and the products are a mixture of diastereoisomers according to position 13a.The products were purified using column chromatography, followed by hexane re-precipitation from isopropyl alcohol.The best yields were achieved for compounds containing a dibutylamide fragment (3c, 96%), a diethylamide fragment (3b, 87%), and a piperidinamide fragment (3f, 80%).The yields of the remaining compounds 3a,d,e were 59-66%, which is probably due to their greater solubility in the hexane-isopropyl alcohol system.To the best of our knowledge, compounds 3a-g have not been previously described in the literature.

Spectral Data of 3
The structures of amides 3 were characterized using spectral data.IR spectra exhibited vibrations in the range 1645-1698 cm −1 that corresponded to the vibrational frequency of tertiary amides.Mass spectra contained peaks (m/z) corresponding to the [M-H] + positively charged molecular fragment.Among the fragmentation peaks, there is a fragment with m/z 324.1, which corresponds to the tetrahydroberberrubine cation-radical C19H18NO4, [M-H] + .
The 1 H NMR and 13 C NMR spectra of compound 3 showed characteristic resonances for the tetrahydroberberine skeleton.In order to analyze these resonances, we used the example of compound 3b for which standard one-dimensional and two-dimensional NMR experiments (COSY, NOESY, HSQC, HMBC) were recorded.When considering the 1 H- 13 C heteronuclear correlation (HSQC) spectrum (Figure S8 in Supplementary

Synthesis of 3
The synthesis of acetamides 2 by the substitution of berberubine 1 with seven bromoacetamides in the presence of potassium carbonate was performed by our group (Scheme 1) [27,28].Compound 2 contains in its structure an aromatic heterocyclic ring (cycle "C") in the isoquinolinium system, which is reduced by the action of various reducing agents to form dihydro or tetrahydro derivatives.We have shown that the reaction of compounds 2a-g with 4 mol equivalents of sodium borohydride in methanol at 0 • C resulted in a mild reduction of the "C" cycle to produce tetrahydroberberine derivatives of 3a-g (Scheme 1).The reaction conditions used are similar to those in which our group previously carried out the reduction of compound 1 and some of its derivatives [32].The reduction proceeds non-stereoselectively, and the products are a mixture of diastereoisomers according to position 13a.The products were purified using column chromatography, followed by hexane re-precipitation from isopropyl alcohol.The best yields were achieved for compounds containing a dibutylamide fragment (3c, 96%), a diethylamide fragment (3b, 87%), and a piperidinamide fragment (3f, 80%).The yields of the remaining compounds 3a,d,e were 59-66%, which is probably due to their greater solubility in the hexane-isopropyl alcohol system.To the best of our knowledge, compounds 3a-g have not been previously described in the literature.

Spectral Data of 3
The structures of amides 3 were characterized using spectral data.IR spectra exhibited vibrations in the range 1645-1698 cm −1 that corresponded to the vibrational frequency of tertiary amides.Mass spectra contained peaks (m/z) corresponding to the [M-H] + positively charged molecular fragment.Among the fragmentation peaks, there is a fragment with m/z 324.1, which corresponds to the tetrahydroberberrubine cation-radical C 19 H 18 NO 4 , [M-H] + .
The 1 H NMR and 13 C NMR spectra of compound 3 showed characteristic resonances for the tetrahydroberberine skeleton.In order to analyze these resonances, we used the example of compound 3b for which standard one-dimensional and two-dimensional NMR experiments (COSY, NOESY, HSQC, HMBC) were recorded.When considering the 1 H- 13 C heteronuclear correlation (HSQC) spectrum (Figure S8 in Supplementary Materials), we determined the correspondence of signals from carbon atoms and protons.Thus, a multi-plet signal from the proton at δ H 3.46-3.52ppm corresponds to the signal at δ C 59.32 ppm (C13a), multiplet signals at δ H 2.73-2.81and 3.12-3.20ppm (H13) correspond to the signal at δ C 36.09 ppm (C13), and doublet signals at δ H 3.55 and 4.29 ppm (H8) correspond to the signal at δ C 53.62 ppm (C8), which is typical for the signals of ring C in tetrahydroberberine systems.
The 1 H NMR spectra of 3 exhibited resonances for methylene protons of OCH 2 CON as an AB system with chemical shifts δ H 4.20-4.80ppm.The resonances of chemically identical protons of the alkyl substituents in the amide were nonequivalent.This was indicative of the hindered rotation that is characteristic of tertiary amides.The resonances of the carbon atoms of alkyl substituents in the amide, e.g., dibutylamide 3c, were also nonequivalent in the 13 C NMR spectra (Figure S12 in Supplementary Materials).The corresponding chemical shifts were δ C 45. 38  This was consistent with data in the literature for analogous amides [28,38].

General
Berberine chloride hydrate was purchased from TCI company (Tokyo, Japan), and the basic substance content was 81%.Commercially available organic and inorganic chemicals (reagent grade) from Khimservis Company (Staraya Kupavna, Moscow Oblast, Russia) were used without additional purification.Solvents from Khimservis Company (Staraya Kupavna, Moscow Oblast, Russia) were distilled prior to use.Column chromatography was performed on silica gel manufactured by Macherey-Nagel, fraction 63-200 µm.Berberrubine 1 was synthesized as the solvate with one EtOH molecule according to the procedure in the literature [27].Berberrubine acetamides bromides 2a-g were prepared following a previously reported procedure [27,28].

Instrumentation and Analysis
The spectral and analytical studies of the products were carried out at the Multi-access Chemical Service Center of the Siberian Branch of the Russian Academy of Sciences.The UV spectra were recorded on a HP 8453 UV-Vis spectrophotometer in EtOH (c = 10 −4 mol/L).The IR spectra were measured on a Vector 22 FTIR spectrometer in KBr pellets.Melting points (mp) were obtained with a Metler Toledo FP 900 instrument and Kofler stage.Elemental analyses were from Carlo Erba 1106.High-resolution mass spectra were obtained on a DFS-Thermo-Scientific spectrometer in a full scan mode (15-500 m/z, 70 eV electron-impact ionization, direct sample introduction).HPLC analyses were carried out on a Econova (Novosibirsk, Russia) "Milichrome A-02" HPLC system using ProntoSIL-120-5-C18AQ reversed-phase sorbent (particle size 5 µm, column 75 × 2 mm) at 35 • C, 3.0-3.6MPa, and a flow rate of 150 µL/min with elution by a linear gradient of solvents from 100% A to 100% B over 25 min (solvent A, 0.1% TFA in H 2 O; solvent B, MeOH) and simultaneous multiwave detection at six wavelengths (220, 240, 260, 280, 320, and 360 nm).The 1 H and 13 C NMR spectra of 5-10% solutions of compounds in CDCl 3 or DMSO-d6 were recorded on Bruker (Billerica, MA, USA) AV-400, DRX-500, and AV-600 spectrometers.Solvent signals (δ H 7.24 and δ C 76.90 ppm for CDCl 3 or δ H 2.50 and δ C 39.52 ppm for DMSO-d6) were used as internal references.The numbering of carbon and hydrogen atoms in the spectra of compounds is shown on Scheme 1 and Figure S2.The assignments of the signals in the 1 H and 13 C NMR spectra marked with an asterisk (or a double asterisk) can be interchanged.

General Procedure for Tetrahydroderivative 3a-g Synthesis
At a temperature of 0 • C and stirring on a magnetic stirrer, 4 equivalents of sodium borohydride were added portion by portion to a suspension of 0.6-1.5 mmol (1 eq.) of a derivative of berberine bromide 2 in 10 mL of methanol.The mixture was stirred for 30 min while cooling and then for 4 h at room temperature until the starting substance disappeared (TLC, SiO 2 plates, methylene chloride-methanol 10:1).The reaction mixture was evaporated and purified using column chromatography on silica gel or aluminum oxide for 3a.The eluent is methylene chloride-methanol, 100:2, 100:4.The fractions containing product 3 were combined and dissolved by heating in 5 mL of isopropyl alcohol, and the precipitate was deposited with the addition of 10 mL of hexane.