L. R. Nigmatullina

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 Antiviral Activity of Betulonic Acid Amides and Conjugates with Amino Acids

Betulonic acid amides with aliphatic and heterocyclic amines and with L-amino acids were synthesized by the acid chloride method. Betulonic acid amide and L-methionine derivatives of betulonic acid and its 3-oxime effectively inhibit the influenza A virus. Betulonic acid octadecylamide is active against the herpes simplex Type 1 virus. The conjugate of betulonic acid 3-oxime with L-methionine is also active toward HIV-1. The tested compounds mainly show no activity toward the ECHO6 virus, which is devoid of a coat.

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 Antiviral Activity of Lupane Triterpenoids and Their Derivatives

It was reported that derivatives of lupane triterpenoids (lupeol, betulin, betulinic acid) exhibit antiviral activity, in particular, with respect to human immunodeficiency virus (HIV) [1], herpes simplex virus (HSV), and Epstein – Barr virus (EBV) [2 – 7]. Previously, we have also studied the anti-HIV properties of betulin 3,28-di-O-nicotinate [8] and the antiviral activity of some betulin oximes [9] and 28-oxo-allobetulone [10]. In continuation of our investigations of the structure – antiviral activity relationship in the series of lupane triterpenoids, we have synthesized a series of new derivatives and studied their activity in comparison to that of the known compounds. Lupeol 3-O-nicotinate (II) was obtained with 94% yield by acylating lupeol (I) with nicotinic acid chloroanhydride in a mixture of anhydrous pyridine and tributylamine. 2-Furfurylidene methylbetulonate (IV) was synthesized via interaction of methylbetulonate (III) with furfurol in an alcohol solution of alkali (after purification by column chromatography, the product yield was 80%). The proposed structures of compounds II and IV were confirmed by NMR data (see experimental part below). N-formylamine (V) was obtained with a 62% yield using the Leuckart reaction between methylbetulonate (III) and formamide in formic acid. The C NMR spectrum of this compound displays a shift of the signal from the C3 nuclei toward stronger field ( = 56.3 ppm) and reveals a signal due to C1 atom at = 164.1 ppm. In the H NMR spectra, the signals from ptotons of the formyl group is manifested by a singlet at = 8.15 ppm and the NH proton, by a broad signal at = 8.00 ppm.

Synthesis and antiviral activity of ureides and carbamates of betulinic acid and its derivatives

Ureides and carbamates of betulinic acid and its derivatives were prepared in good yields by interaction of betulinic acid, betulonic acid, and betulonic acid 3-oxime with amines, amino acids, and alcohols. Ureides of betulonic acid containingL-Val and L-Met residues were found to be effective against herpes simplex type 1 virus.

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 Ketals of Methyl 3-Oxo-lup-20(29)-en-28-oate

Ketals were synthesized by reacting methylbetulonate and ethylenediol.

Synthesis and hepatoprotector activity of 2-arylidene methylbetulonate derivatives

Betulinic acid (I), representing triterpenoids o f the lupane group, was isolated as long ago as in the beginning of the 20th century [ 1 ]. However, it was not until recent years that this compound drew the attention of pharmacists by showing a broad spectrum of biological activity, including antitumor and antiviral properties [2 8]. Below we report on the synthesis of new 2-arylidene derivatives of betulonic acid methyl ether (Ilia IIIg) obtained by transformation of the triterpenoid A ring. We have also studied the hepatoprotector activity of compounds Illb and llIg. Previously we have demonstrated this type of activity in some betulin esters [9]. Compounds Ilia IIIg were synthesized using the interaction of methylbetulonate (II) with aromatic aldehydes in alcoholic alkali solutions, the products being obtained at a 81 9 0 % yield. The proposed structures of the synthesized compounds were confirmed by data of 1R, UV, and NMR spectroscopies (Tables 1 and 2) and by agreement with the data published for betulonic acid [6, 10, 11], The IR spectra of compounds I l i a IIIg contained, besides the absorption bands characteristic of betulonic acid, the bands at 1610 1600 cm I belonging to vibrations in the aromatic rings. The 13C NMR spectra exhibit additional signals due to carbon atoms of the aromatic substituents (in the region of ~ = 110.5 159.3 ppm), while the signal of C-2 carbon of the aglycon shifts toward weaker fields (from 8 = 33.6ppm for betulonic acid to 131.9137.1 for compounds Ilia IIIg). In the IH NMR spectrum, the proton sig-

THE SYNTHESIS AND THE ANTI-INFLAMMATORY AND ANTIULCER ACTIVITIES OF A NUMBER OF 2-SUBSTITUTED DERIVATIVES OF BETULONIC ACID, METHYLBETULONE, AND LUPENONE

In previous reports, we have described the synthesis and hepatoprotective activity of a variety of esters – 2-arylidene derivatives of betuline, lupol, and methylbetulonate [1, 2]. The present report presents data on the synthesis and study of the anti-inflammatory and antiulcer activities of 2-methylideneureido derivatives of betulonic acid, methylbetulonate, and lupenone. 2-Hydroxymethylene derivatives (IIa – IIc) were prepared by reaction of betulonic acid (Ia), methylbetulonate (Ib), and lupenone (Ic) with ethyl formiate in the presence of sodium methylate, with yields of 89 – 95%. Interaction of these compounds with urea and thiourea leads to the formation of 2-methylideneureido (IIIa – IIIc) and thioureido (IIId – IIIf) derivatives. The structures of these compounds were confirmed by IR, UV, and NMR spectra (see Tables 1, 2) and by comparison with published data for lupane triterpenoids [3 – 5]. The IR spectra of compounds IIa – IIc included, along with absorption peaks typical of these triterpenoids, bands in the region 3160 – 3150 cm –1 , due to the appearance of OH groups on the hydroxymethylene fragment [6]. In the 13 C NMR spectrum, the C-3 signal showed strongfield shifts compared with the signals in the initial 3-ketotriterpenoids, and also contained new signals corresponding to the C-1 atoms at 188.8 – 188.9 ppm, while the 1 H NMR spectra acquired weakfield signals at 8.52 – 8.60 ppm and 14.65 – 14.85 ppm corresponding to protons in =CH–OH and =CH–OH hydroxymethyl substituents. Analogous signals were also seen in the spectra of the formyl derivative of oleanolic acid [7]. The 13 C NMR spectra of compounds IIIa – IIIf showed resonating C-1 carbon atoms at 139.9 – 140.0 and 141.5 – 141.7 ppm, along with signals from the carbon atoms in the ureide fragments C=O and C=S at 155.3 – 155.5 and 182.4 – 182.6 ppm respectively. In addition, the 1 H NMR spectra of 2-methylideneureido (IIIa – IIIc) and thioureido (IIId – IIIf) derivatives showed interaction of the protons of these fragments in the form of doublets at 7.17 – 7.25 and 7.65 – 7.70 ppm, and 11.35 – 11.45 and 11.68 – 11.75 ppm.

Synthesis of Methyl [3,2-c]-Pyrazol-lup-20(29)-en-28-oate

Reaction of 2-hydroxymethylenebetulonic acid with hydrazine hydrate produced methyl-[3,2-c]-pyrazol-lup- 20(29)-en-28-oate.