Synthetic Derivatives of Natural Alkaloid Harmine

The indole alkaloid harmine was extracted from underground part of Peganum harmala L. With the purpose of obtaining the new biological active derivatives on base of alkaloid harmine the chemical modiﬁcation was carried out. The p-toluolsulfochlorid, p-toluolsulfoacid, hydrochloric, sulfuric, nitric acids, dioxide selenium and phthalic anhydride have been chosen as modiﬁers. For the ﬁrst time quaternary ammonium salts, derivatives of N-oxide and N (2)-oxyharminiumphthalate harmine are synthesized. The structure of the synthesized compounds is determined by methods of the spectral analysis and X-ray analysis. Antimicrobic and phagocytosis stimulating activities of isolated alkaloids and their derivatives are investigated.


Introduction
Among the renewed sources of biologically active substances the plants, traditionally used in folk and official medicine, have been taken a special place. In flora of Kazakhstan there is totally over 100 species of alkaloid plants having a wide area of distribution in territory of Republic. There are particular groups of plants, which are the most widespread or dominate over greater territories and thereof, are of interest for the chemical and pharmacological study. Such plants which belong to species of Peganaceae harmala family, are taking extensive covers in natural flora of region in Kazakhstan`s territory and are being a source of indole and chinasolinic alkaloids. They have been widely applied for treatment of cardiovascular diseases and also can be used as antitumor, antibacterial, anticholinesterase, antitussive and psychotropic drugs [1][2][3][4][5][6][7].
In a number of indole the alkaloids with tricyclic structure (carbolinic atom) of harmine and its derivatives takes important place and shows the expressed biological activity, and are accessible and convenient for synthesis of new physiologically active substances and effective medical drugs with a wide spectrum of action.

Experimental
The control of reactions has been carried out by method of thin-layer chromatography on plates of «Sorbfil АF-А». Reagents of qualification «ch» have been used for work. Melting points of individual compounds has been measured on "Boetius". UFspectra have been taken with spectrophotometer Helios-b V-4.60 in an interval of wavelength 190-500 nm. IR-spectra have been taken with Fourier spectrophotometer Vector -22 in KBr tablets. 1 Н and 13 С NMR -spectra have been obtained on spectrometers Bruker АС-300 [working frequencies 400.13 ( 1 Н) and 125.76 MHz ( 13 С)] and Bruker DRХ 500 [working frequencies 500.13 ( 1 Н) and 125.76 MHz ( 13 С)] for 5% solutions of CD 3 OD or (CD 3 ) 2 SO. X-ray experiment have been carried out with diffractometer Bruker P4 (Mo Kα-radiation with graphite monochromator, 2θ/θ-scanning in the field of 2θ <52°).
High-performance liquid chromatography (HPLC) of alkaloids derivatives has been carried out on "Hewlett-Packard Agilent 1100 Ser." with a column in length 4,6х150 mm, filled with phase Sorbax C 18 -CB with the size of particles 5μм. Mobile phase has been acetonitrile. Detection has been carried out at wave length 224 nm.
X-ray research of compound 5*. For experiment the crystal sample of salt compound 5 with size 0.50 0.50 0.10 mm has been selected. Monoclinic crystals: a = 6.7389 (7), b = 17.9250(9), c = 11.3023(8) Å, β = 104.475(7)°, V = 1321.9(2) Å 3 , spatial group P2 1 , Z = 4, C 13 H 13 N 2 O+ 1/2 SO 4 -2 + H 2 O, d cal = 1.403 g/sm 3 , μ = 0.180 mm -1 . Intensity 2684 of independent reflections has been measured. The amendment of absorption has not been entered. Structure has been decoded with direct method on program SIR2002 [11]. Specification of structure parameters has been carried out with method of the least squares in full matrix anisotropic approach on program SHELXL-97 [12]. Parameters of the majority of atoms Н have been counted in each cycle of specification on coordinates of corresponding carbon atoms (model "rider"). Atoms Н at N2 and N9 and also in molecules of water have been localized from difference synthesis, their parameters have been specified isotropically. Final structure specification have been carried on F 2 up to wR 2 = 0.0972, S = 1.04, specified 384 parameters (R = 0.0357 для 2434 F > 4σ).
Synthesis of N-oxide harmine (6): 0.05 g (0.022 millimole) of harmine (1) has been dissolved in 12 ml of methanol and 0.0783 g (0.705 millimole) of dioxide selenium have been added into the obtained solution. The reactionary composition has been mixed for 4 days, residue has been filtered, solvent has been distilled off. Crystal substance (6)

Synthesis of N(2)-hydroxy harmine chloride (7)
: 0.11 g (0.48 millimole) N-oxide harmine (6) has been dissolved in 7 ml of methanol and 0.02 ml (0.72 millimole) hydrochloric acid have been added into the obtained solution. The reactionary composition has been mixed at room temperature (20-25°C) for 22 hours and steamed. Residue has been washed by chloroform with water and dried up. Light yellow crystal substance (7)  The spatial structure of molecule of compound (5) according to X-ray analysis is showed in figure  1. In an independent part of an elementary cell there were 2 molecules of compound (5), one molecule of SO 4 -2 and two solvate molecules of H 2 O. Most close analogue on structure of compound (5) is hydrochloride harmine (4) which structure has been studied by method of X-ray analysis in work [8]. The geometry of all three molecules (5) (in our case two independent) within the limits of 3s has coincided and close to corresponding average sizes [9]. The analysis of intermolecular correlations has been carried out by program PLATON [10]. Molecules of compound (5) are connected by hydrogen bonds with molecules SO 4 -2 and with solvate molecules of water (figure 2). Parameters of hydrogen bonds are showed in the Table 1. While a molecule of compound (5) are put in a pile «side-to-side with shift» due to p…p-correlations with interplane distance 3.36 Å and distance between centers of pentamerous cycles of the next molecules in a pile 3.424 (2) and 3.3.440 (2) Å.  The easy way of reaction and high yield of N-oxide harmine have been found which opens a wide opportunities of synthesis of new biologically active compounds and development of effective medical drugs with a wide spectrum of action on their basis.
The reactions of N-oxide harmine with anhydrides of dicarboxylic acids, hydrochloric and nitric acids were investigated and various derivatives of harmine with the expressed biological activity have been synthesized on their basis for the first time.

Conclusions
The investigations of antimicrobic activity of harmine (1) and its derivatives in relation to strains of gram-positive bacteria of Staphylococcus aureus, Bacillus subtilis, gram-negative strains of Escherichia coli, Pseudomonas aeruginosa and yeasty mushroom Candida albicans by a method of diffusion in agar (lunules) have been carried out in laboratory of bioscreening of JSC "IRPH "Phytochemistry". Gentamycin (for bacteria) and nystatin (for yeasty mushroom Candida albicans) have been used for comparison. Thus it was determined that alkaloid harmine has moderatel antimicrobic action concerning strains of grampositive bacteria of Staphylococcus aureus, Bacillus subtilis and fungicidal activity in relation to yeasty mushroom Candida albicans.