L. T. Karachurina

Synthesis of Betulinic Acid from Betulin Extract and Study of the Antiviral and Antiulcer Activity of Some Related Terpenoids

3 -Hydroxy-lup-20(29)-en-(28)-oic (betulinic) acid (I) was isolated at the beginning of the 20th century and originally called gratiolone [1]. In the past decade, some special biological properties of this triterpenoid has drawn the attention of researchers. In 1994, betulinic acid was found to exhibit anti-HIV-1 activity in H9 lymphocyte cell culture [2]. The most promising inhibitor of HIV-1 replication is 3-O-(3 ,3 -dimethylsuccinate) of betulinic acid [3]. In 1995, it was established that betulinic acid is capable of selectively inhibiting the growth of malignant human melanoma [4]. In addition, betulinic acid and its derivatives possess antibacterial, antimalarial, antiinflammatory, bile-expelling, and other properties [5 – 7]. Since the content of betulinic acid in plants is very low, isolation of this compound from raw plant material is poorly profitable. A more effective method of obtaining betulinic acid is synthesis from betulin (II) and betulonic acid (III). The first synthesis of betulinic acid was suggested by Ruzicka et al. [8]. This original procedure involved (i) protection of the hydroxy groups of II by acetylation, (ii) selective hydrolysis of the acetyl group at C-28, (iii) oxidation of the oxymethyl group to carboxy group, and (iv) removal of the protective acetyl group at C-3. The total yield of betulinic acid from this process did not exceed 30%. In the late 1990s, betulinic acid was synthesized (I) in two steps by oxidizing betulin (II) with Jones reagent or chromic anhydride in acetic acid, followed by selective oxidation of the resulting betulonic acid (III) with NaBH4 [9, 10], whereby the total yield of the target compound reached up to 60%. We used Jones reagent to oxidize the extract obtained by treating birch bark with aqueous isopropanol in an autoclave at 80°C. The initial extract contained up to 90% of betulin and about 5% of lupeol (IV). Betulonic acid (III) formed as a result of this oxidation process was isolated from the reaction mixture in the form of a potassium salt (V) with a yield of 82%, which allowed us to exclude the stage of intermediate product purification by column chromatography [10]. Upon acidification of an ethanol solution of the potassium salt V with a 5% hydrochloric acid solution, betulonic acid (III) of 95% purity was isolated with a yield of 85%. The physicochemical parameters of the product corresponded to the published data [2, 10, 11]. The total yield of betulonic acid (III) calculated for the initial betulin extract was 67%. Column chromatography of the organic fraction yielded 3-keto-lupeol (VI) with a yield of 1.7%.

Synthesis and Pharmacological Activity of Betulin, Betulinic Acid, and Allobetulin Esters

A series of new triterpene hemisuccinates, hemiphthalates and nicotinates have been obtained with 52 – 95% yields via reactions of monoacetates of betulin, betulinic acid, and allobetulin with succinic and phthalic anhydrides and with nicotinic acid chloroanhydride. 3-O-Acetylbetulin-28-O-hemiphthalate showed the most pronounced antiinflammatory activity comparable with the effect of ortophen (diclofenac). 3-O-Acetylbetulin-28-O-hemisuccinate exhibited the most pronounced antiulcer activity comparable with that of carbenoxolone.

SYNTHESIS AND PHARMACOLOGICAL ACTIVITY OF ACYLATED BETULONIC ACID OXIDES AND 28-OXO-ALLOBETULONE

In recent years, the chemical transformations and biological activity of triterpenoids of the lupane group have received much attention. It was found that betulin acylates possess antitumor properties [1], while lupeol esters with palmitic and linolic acids produce antiarrhythmic action [2]. The most promising inhibitors of HIV replication include 3-O-(3,3-dimethylsuccinate) of betulinic acid and 3-O(3,3-dimethylsuccinate)-28-O-(2,2-dimethylsuccinate) of betulin [3]. Recently, Kashiwada et al. [4] reported on the synthesis of derivatives of 3-alkylamino-3-deoxo-betulinic acid possessing anti-HIV-1 properties [4]. Another promising compound is betulin 3,28-di-O-nicotinate, which shows hepatoprotector, antiulcer, antiinflammatory, wound-healing, anti-HIV, and immunomodulant activity [5]. An analysis of published data suggests that the class of lupane triterpenoids and related compounds containing acyl groups is a promising source of new biologically active substances. Below we report on the first synthesis of acylated oximes based on betulonic acid (Ia), its methyl ester (IIa), and 28-oxo-allobetulone (IIIa). In the first step, boiling 3-oxo-triterpenoids (Ia, IIa, IIIa) with hydroxylamine hydrochloride in anhydrous pyridine led to a quantitative yield of the corresponding 3-oximes (Ib, IIb, IIIb). By acylating these compounds in anhydrous benzene with excess acetic, succinic, and phthalic anhydrides at room temperature in the presence of triethylamine, we obtained acylated oximes (Ic – Ie, IIc – IIe, IIIc – IIIe) with a yield of 64 – 78% after purification of the products by column chromatography (Table 1).

Synthesis and Antiinflammatory Activity of New Acylated Betulin Derivatives

The content of betulin, a triterpenoid of the lupane group, in the birch bark varies from 10 to 40% depending on the particular plant species, location, and growth conditions [1]. Betulin can be readily converted into allobetulin and betulinic acid. As is known, betulin (Ig), as well as allobetulin (IId), betulinic acid (IIId), and their derivatives, are low-toxicity substances possessing a wide spectrum of biological activity [2 – 5] and antioxidant properties [6]. These substances can be used as additives to improve existing products and create new products in pharmacy, cosmetics, and food industry. It was found that some derivatives of the lupane triterpenoids (in particular, those with ester and amide bonds) exhibit antiviral activity [7, 8]. Some drugs based on other triterpenoids contain the analogous residues of bioactive acids [9]. Recently [10, 11], we found highly active substances among betulin acylates: betulin dinicotinate, bis-hemisuccinate, bis-hemiphthalate, etc., were shown to possess hepatoprotector and anti-HIV-1 activity. Therefore, the synthesis and characterization of new biologically active substances in the series of acylates of these triterpenoids is of practical importance. Below we present data on a series of new esters of betulin, allobetulin, and betulinic acid. The interaction of initial triterpenoids Ig, IId, and IIId with chloroanhydrides of 2-(4-chlorophenyl)-3-methylbutyric, tetramethylcyclopropanecarboxylic, and permethric acids (representing semiproducts in the synthesis of pyrethroids) in a mixture of pyridine with tributylamine led to acylates Ia – If, IIa – IIc, and IIIa – IIIc (see scheme). 3-O-Acylates of allobetulin (IIa – IIc) and betulinic acid (IIIa – IIIc) were obtained with a yield of 55 – 87% at room temperature for a triterpenoid to chloroanhydride ratio of 1 : 1.5 (Table 1). Selective esterification performed under the same conditions led to 28-monosubstituted esters Ib, Id, If with a yield of 80 – 85%. In the presence of a threefold excess of chloroanhydrides over betulin, heating the reaction mixture up to 60 – 70°C led to the formation of 3,28-disubstituted betulin esters Ia, Ic, Ie with a yield of 75 – 80%. The structures of the synthesized acyl derivatives were established by NMR spectroscopy in comparison with the data published for analogous compounds [10, 12, 13]. The formation of ester bonds was confirmed by 2 – 4 ppm low-field shifts of the signals from C3 atoms (for compounds Ia, Ic, Ie, IIa – IIc, IIa – IIIc) and from C28 atoms (for compounds Ia – If) and by the appearance of signals from ester

Synthesis and Pharmacological Activity of Betulin Dinicotinate

The assignment of NMR resonances of lupane triterpenoids was refined by the example of 3O,28O-dinicotinoylbetulin, obtained by acylation of betulin. Hepatoprotective, antiulcer, antiinflammatory, reparative, and anti-HIV activities were found for the compound. In addition, it was demonstrated to have immunomodulatory activity, for the first time detected among lupane triterpenoids.

Synthesis of 3-O-Acetylbetulinic And Betulonic Aldehydes According to Svern and the Pharmacological Activity of Related Oximes

As is known, triterpenoids of the lupane group exhibit a wide spectrum of biological activity [1 – 3]. Of considerable interest are lupane aldehydes, the derivatives of which were reported to possess anti-HIV and antimelanoma properties [4, 5]. Previously, compounds of the lupane group were oxidized by reagents containing Cr(VI): CrO3 in acetone [6], pyridinium bichromate in a DMF – H2O mixture, and pyridinium chlorochromate in CH2Cl2 [4, 5]. The carbonyl products were obtained with a yield of not greater than 80%, sometimes in the form of difficultly separable mixtures of acids and deeper oxidation products [7]. Recently, Evers, et al. [8] demonstrated that a convenient method for the synthesis of 3 -[(dimethyl-tert-butylsilyl)oxy]betulinic aldehyde is offered by the oxidation reaction according to Svern. In continuation of our previous investigations devoted to the transformation of lupane triterpenoids [9 – 11], we have synthesized 3-O-acetylbetulinic aldehyde (III) and betulonic aldehyde (IV) by oxidizing betulin (I) and its 3-O-acetate (II) with “activated” DMSO according to Svern. It was established that the oxidation of triterpenoid I by one or two molar equivalents of the oxidizing reagent proceeds in a nonselective manner, leaving unreacted betulin, with the formation of betulinic and betulonic (IV) aldehydes. The complete conversion of betulin (I) into betulonic aldehyde (IV) requires using four molar equivalents of activated DMSO; the yield of product IV in this case amounts to 93% (Table 1). The oxidation of 3-O-acetylbetulin (II) at –30°C with one molar equivalent of activated DMSO to 3-O-acetylbetulinic aldehyde (III) proceeds with a very low conversion of the initial compound. The complete conversion of compound II requires using two molar equivalents of the Svern reagent; the yield of product III in this case amounts to 95%. The oxidation process conducted at –10°C led to only a 50% conversion of the initial reagents, while the reaction at 0°C yielded no aldehydes at all. The proposed structures of the target aldehydes III and IV were confirmed by the results of IR and C and H NMR measurements (Tables 1 and 2) and by agreement with the published data [5, 6]. The NMR spectra of the synthesized compounds contained no signals of alcohol hydroxy groups, but displayed signals from the aldehyde (206.4 ppm in C and 9.64 ppm in H NMR) and keto (217.6 ppm in C NMR) groups. Aldehydes III and IV were used for the synthesis of related oximes (V – VIII). 3-O-Acetyl-28-oxime-betulin (V) and 3,28-dioxime-betulin (VI) were obtained with a yield of 85 – 92% by boiling aldehydes III and IV with hydroxylamine hydrochloride in pyridine. Saponification of the acetate group of oxime V with a 5% KOH solution in methanol led to 3 -hydroxy-28-oxime-betulin (VII). Dehydration of oxime VI in acetic anhydride led to 3-oximebetulonic acid nitrile (VIII) with a yield of 87%. It was established that compounds VI – VIII possess pronounced antiviral properties with respect to the type A strain of influenza virus. This result is in discrepancy with the data reported in [13], according to which betulin was ineffective against the influenza virus. In our tests, compound VI was also capable of suppressing the growth of ECHO-6 and herpes simplex virus (HSV), but to a lower extent than it was with the type A influenza virus (Table 3). Experiments on the models of mucous membrane damage induced by indomethacin and acetylsalicylic acid (aspirin) showed that compound III produces an antiulcer effect

SYNTHESIS AND ANTIARRHYTHMIC ACTIVITY OF (1,3-DIMETHYL-5-NITRO-5-HEXAHYDROPYRIMIDINYL)- PROPIONIC ACID METHYL ESTER

Abstract(1,3-Dimethyl-5-nitro-5-hexahydropyrimidinyl)propionic acid methyl ester (I) was obtained with a 83% yield using a Mannich type reaction of 4-nitrobutanoic acid methyl ester with excess formalin and methylamine. It was found that compound I possesses low toxicity and shows antiarrhythmic activity on models of arrhythmia induced by intravenous injections of calcium chloride and aconitine in rats.

Synthesis and Antiarrhythmic Activity of N-(2-Hydroxyethyl)cytisine Hydrochloride and 3-(2-Hydroxyethyl)-1,5-dinitro-3-azabicyclo-[3.3.1]non-6-ene Hydrochloride

Synthetic analogs of natural 3-azabicyclo[3.3.1]nonanes have drawn the attention of researchers as potential drugs [1 – 3]. This study was aimed at the synthesis of 3-(2-hydroxyethyl)-1,5-dinitro-3-azabicyclo[3.3.1]non-6-ene (II) and N-(2-hydroxyethyl)cytosine (IV) and evaluation of the antiarrhythmic activity of these compounds in the form of hydrochlorides. The synthesis of compound II was based on the reduction of 1,3-dinitrobenzene with sodium borohydride to 1,3-dinitrocyclohexene disodium salt (I). This intermediate compound was introduced (without isolation from the reaction mixture) into the Mannich condensation reaction with formaldehyde and monoethanolamine, which led to the formation of target compound II (isolated with a 70% yield) [4].

Antiinflammatory and Antiulcer Properties of Betulin bis-Hemiphthalate

Triterpenoids of the lupane group (betulin and derivatives) were reported to possess hepatoprotector, bile-expelling, antimicrobial, antifungal, and antitumor properties [1 – 3]. Recently, betulonic acid derivatives were identified as a new class of potential selective HIV inhibitors [4]. An analysis of the published data shows that the best prospects in the search for highly active agents among lupane derivatives are related to compounds with ester and amide bonds. For this reason, we have synthesized a series of esters, including bis-cinnamate, bis-hemisuccinate, and bis-hemiphthalate of betulin and studied their hepatoprotector activity [5]. Recently, we also studied the pharmacological properties of betulin 3,28-di-O-nicotinate [6]. The aim of this study was to characterize betulin bis-hemiphthalate (I) with respect to antiinflammatory and antiulcer properties.

Synthesis and pharmacological properties 4-methyl-1-(methylsulfinylmethyl)-7-thiabicyclo[3.3.1]non-3-en-2-one-7-oxide

Some representatives of the class of sulfur-containing organic compounds, including derivatives of sulfones [1], dialkyldisulfides [2], and 3-thiabicyclohexanecarboxylic acid [3], were reported to possess anti-inflammatory activity. Biological activity is also inherent in 1,2,5-thiadiazole and its condensed derivatives, which act upon central nervous system and influence tissue metabolism [4]. In continuation of our investigations into new bicyclic bis-sulfoxides, we have synthesized 4-methyl-1-(methylsulfinylmethyl)-7-thiabicyclo[3.3.1]non-3-en-2-one-7-oxide (I) and studied the anti-inflammatory and antiarrhythmic properties of this compound. The synthesis I was conducted according to the scheme depicted below. In the first step, we obtained 4-methyl-1(methylthiomethyl)-7-thiabicyclo[3.3.1]non-3-en-2-one (II) via alkylthiomethylation of propanone with a mixture of formaldehyde, methylmercaptan, and sodium sulfide in an alkaline medium [5]. In the second step, compound II was oxidized in a 28 – 30% aqueous hydrogen peroxide solution at 20°C, and the target compound I was obtained with a yield of 95%. The purity and the proposed structures of the synthesized compounds were confirmed by the results of elemental analyses and by the data of IR, H NMR, and C NMR spectroscopy (Tables 1 and 2). The IR spectra of compounds I and II exhibit absorption bands at 1656, 1618, 1624, and 1654 cm – 1 characteristic of the stretching vibrations of the C=O and C=C bonds [6]. In the C NMR spectrum of compound I, the signals from carbon atoms C (48.43 and 48.54 ppm), C (56.75 and 56.06 ppm), C (62.62 and 61.52 ppm), and C (36.08 and 35.79 ppm) are shifted toward lower fields as compared to the analogous signals in the spectrum of compound II (Table 2). In addition, almost all signals in the C NMR spectrum of compound I are doubled, which is indicative of the existence of two isomers. The spectra were assigned separately to each isomer. The appearance of a diastereomer pair is related to the presence of a chiral carbon atoms C and a pyramidal sulfoxide sulfur atom in the aliphatic substituent, which make possible the formation of two -diastereomers with R*R* and R*S* centers [7]. The H C NMR spectrum of compound I displays clearly distinguished signals due to the greater isomer and shows the signals from diastereotopic protons at C in the form of two doublets with a geminal spin – spin coupling constant of 13.3 Hz and a relative chemical shift = 0.61 ppm. For the second diastereomer, this difference is below 0.29 ppm. Based on the published data [7], the diastereomer with a greater difference of the chemical shifts is identified as the erythro isomer (Ia), while the component with a smaller value is identified as the threo isomer (Ib). Both isomers are