Synthesis and antitumor activity of cyclopropane derivatives of betulinic and betulonic acids

New cyclopropane derivatives of betulin were synthesized by attachment of dichlorocarbenes or dibromocarbenes to the double bond of betulin diacetate, followed by the deprotection of hydroxyl groups. The betulin cyclopropane derivative was obtained from 20,29-dihydro-20,29-dichloromethylenebetulin by treatment with lithium in tert-butanol. These compounds were converted into the corresponding derivatives of betulonic acid by oxidation with chromium trioxide. The reduction of oxo group with sodium borohydride led to the corresponding derivatives of betulinic acid. 20,29-Dihydro-20,29-dichloromethylenebetulinic acid proved to be the most cytotoxic toward human melanoma of the Colo 38 and Bro lines and human ovarian carcinoma of the CaOv line (IC50 10 microM). 20,29-Dihydro-20,29-dibromomethylenebetulinic acid inhibited the growth of the Bro melanoma cell line and the CaOv carcinoma cell line practically by 50% at a concentration of 10 microM. The other derivatives of betulinic and betulonic acids were active toward all of the three cancer cell lines at concentrations higher than 10 microM. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

Novel semisynthetic derivatives of betulin and betulinic acid with cytotoxic activity

A series of new imidazole carboxylic esters (carbamates) and N-acylimidazole derivatives of betulin and betulinic acid (14–29) have been synthesized. The new compounds were screened for in vitro cytotoxicity activity against human cancer cell lines HepG2, Jurkat and HeLa. A number of compounds have shown IC₅₀ values lower than 2 μM against the cancer cell lines tested and the vast majority has shown a better cytotoxicity profile than betulinic acid, including the betulin derivatives. N-Acylimidazole derivatives 26 and 27 (IC₅₀ 0.8 and 1.7 μM in HepG2 cells) and the C-3 carbamate derivative 16 (IC₅₀ 2.0 μM in HepG2 cells) were the most promising compounds. Based on the observed cytotoxicity, structure–activity relationships have been established.     

Epimerization of hydroxyl group in the lupan series of triterpenes

Two methods of obtaining of 3 alpha-betulinic acid and related compounds from their 3 beta-epimers were studied: the reaction of bimolecular substitution and the stereoselective reduction of 3-ketoderivatives. The substitution of acyloxy by formyloxy group in 3-O-tosyllupeol or of the betulin hydroxyl by benzoyloxy group resulted only in delta 2, 3-elimination products, with none of the expected products of bimolecular substitution being found. The catalytic hydrogenation of betulonic acid over Raney nickel resulted only in reduction of the isopropenyl double bond, whereas the use of 5% Ru/C gave a 60:40 mixture of epimers of dihydrobetulinic acid. Practically the same mixture of betulinic acid epimers was obtained when reducing betulonic acid with L-Selectride. The cytotoxic activity of 3 alpha-betulinic acid increased toward melanoma Bro cells and decreased toward melanoma MS cells.     

Effect of nitrogen-containing derivatives of the plant triterpenes betulin and glycyrrhetic acid on the growth of MT-4, MOLT-4, CEM,and Hep G2 tumor cells

Derivatives of the available plant triterpenes glycyrrhetic acid and betulin (betulin succinates and amides of betulonic and 18β-glycyrrhetic acids containing fragments of long-chain amino acids and a peptide) were synthesized. The inhibitory action of these compounds on the growth of MT-4, MOLT-4, CEM, and Hep G2 tumor cells and their effect on the apoptosis of these cells were studied. It was shown that betulonic acid amides are more effective inhibitors of the tumor cell growth than the corresponding amides of glycyrrhetic acid. It was also found that betulonic acid amides containing fragments of caprylic, pelargonic, and undecanoic acids are more effective inhibitors of tumor cell growth than betulinic acid. The 17-dipeptide derivative of betulonic acid N-{N-[3-oxo-20(29)-lupen-28-oyl]-9-aminononanoyl}-3-amino-3-phenylpropionic acid exhibited the maximum inhibitory activity toward the tumor cells studied. Data on the induction of apoptosis in tumor cells by betulin derivatives at a concentration of 10 μg/ml were obtained by flow cytometry. The amides of betulonic acid proved to be the most effective inducers of apoptosis.     

Effect of nitrogen-containing derivatives of the plant triterpenes betulin and glycyrrhetic acid on the growth of MT-4, MOLT-4, CEM, and Hep G2 tumor cells

Derivatives of the available plant triterpenes glycyrrhetic acid and betulin (betulin succinates and amides of betulonic and 18beta-glycyrrhetic acids containing fragments of long-chain amino acids and a peptide) were synthesized. The inhibitory action of these compounds on the growth of MT-4, MOLT-4, CEM. and Hep G2 tumor cells and their effect on the apoptosis of these cells were studied. It was shown that betulonic acid amides are more effective inhibitors of the tumor cell growth than the corresponding amides of glycyrrhetic acid. It was also found that betulonic acid amides containing fragments of caprylic, pelargonic, and undecanoic acids are more effective inhibitors of tumor cell growth than betulinic acid. The 17-dipeptide derivative of betulonic acid N-{N-[3-oxo-20(29)-lupen-28-oyl]-9-aminononanoyl}-3-amino-3-phenylpropionic acid exhibited the maximum inhibitory activity toward the tumor cells studied. Data on the induction of apoptosis in tumor cells by betulin derivatives at a concentration of 10 microg/ml were obtained by flow cytometry. The amides of betulonic acid proved to be the most effective inducers of apoptosis.     

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 Antiviral Activity of Hydrazides and Substituted Benzalhydrazides of Betulinic Acid and Its Derivatives

New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N"-benzalhydrazides, were synthesized. Their antiviral activities toward viruses of influenza A virus, herpes simplex type I virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the highest activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid 3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus. Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under study displayed a low antiviral activity toward enterovirus ECHO6.     

Selective inhibition of the interaction of C1q with immunoglobulins and the classical pathway of complement activation by steroids and triterpenoids sulfates

Since undesirable activation of the complement system through the classical pathway is associated with tissue damage and other pathologic proinflammatory consequences at ischemia/reperfusion injury, autoimmune diseases, and rejection of allo- and xenografts, creation of selective inhibitors of the classical pathway leaving the alternative pathway intact is of great importance. Classical pathway is triggered by binding of its recognizing unit, protein C1q, to a number of targets like antibodies, pentraxins, apoptotic cells, and others. In order to obtain inhibitors blocking the first step of the classical cascade, synthesis of sulfates of steroids (Delta(5)-3beta-hydroxycholenic, Delta(5)-3beta-hydroxyetiocholenic, deoxycholic, and cholic acids) and triterpenoids (betulin, 20,29-dihydro-20,29-dichloromethylenbetulin, betulinic, ursolic, and oleanolic acids) has been performed. Testing of the compounds in classical pathway inhibition assay has displayed derivatives of triterpenoid betulin (betulin disulfate and betulinic acid sulfate) to be the most potent inhibitors. Further studies of the two compounds established that their activity to inhibit the classical pathway had been due to their capability to block the interaction of C1q with antibodies. Betulin disulfate and betulinic acid sulfate have shown weak inhibition of the alternative route of activation, what makes them promising inhibitors for the selective suppression of the classical complement pathway at the earliest possible level as well as perspective agents for blocking the interaction of C1q with its other targets.     

Synthesis of triterpenoid triazine derivatives from allobetulone and betulonic acid with biological activities

The synthetic transformation and modification of natural products with the aim to improve the biological properties is an area of current interest. The triterpenoids betulin and betulinic acid are very abundant in nature and now are commercially available. In our study, starting from betulin and betulinic acid, we obtained allobetulone and betulonic acid in a few synthetic steps. The ketone function at the A-ring was used as the starting point for the synthesis of a series of 1,2,4-triazine-fused triterpenoids. The alkylation and Liebeskind–Srogl coupling were used for further substitution of 1,2,4-triazines, and the intramolecular hetero Diels–Alder reaction leads to interesting fused thienopyridine derivatives. All new compounds were tested for their cytostatic activities against murine leukemia L1210, human cervix carcinoma HeLa and human lymphoblast CEM tumor cells. The results show that some triterpenoid triazine betulonic acid derivatives have a promising cytostatic activity in vitro and could be used as potential leads for the development of new type of anti-cancer agents. Several compounds were also endowed with anti-HCMV activity in the low micromolar range.     

Determination of betulinic acid in mouse blood, tumor and tissue homogenates by liquid chromatography–electrospray mass spectrometry

A rapid and sensitive high-performance liquid chromatography–electrospray MS method has been developed to determine tissue distribution of betulinic acid in mice. The method involved deproteinization of these samples with 2.5 volumes (v/w) of acetonitrile–ethanol (1:1) and then 5 μl aliquots of the supernatant were injected onto a C₁₈ reversed-phase column coupled with an electrospray MS system. The mobile phase employed isocratic elution with 80% acetonitrile for 10 min; the flow-rate was 0.7 ml/min. The column effluent was analyzed by selected ion monitoring for the negative pseudo-molecular ion of betulinic acid [M−H]⁻ at m/z 455. The limit of detection for betulinic acid in biological samples by this method was approximately 1.4 pg and the coefficients of variation of the assay (intra- and inter-day) were generally low (below 9.1%). When athymic mice bearing human melanoma were treated with betulinic acid (500 mg/kg, i.p.), distribution was as follows: tumor, 452.2±261.2 μg/g; liver, 233.9±80.3 μg/g; lung, 74.8±63.7 μg/g; kidney, 95.8±122.8 μg/g; blood, 1.8±0.5 μg/ml. No interference was noted due to endogenous substances. These methods of analysis should be of value in future studies related to the development and characterization of betulinic acid.     

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 containing L-Val and L-Met residues were found to be effective against herpes simplex type 1 virus. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Synthesis and antiviral activity of hydrazides and substituted benzalhydrazides of betulinic acid and its derivatives

New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N'-benzalhydrazides, were synthesized. Their antiviral activities toward of influenza A virus, herpes simplex type I virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the highest activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid 3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus. Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under study displayed a low antiviral activity toward enterovirus ECHO6.     

Synthesis of 3-O-acyl/3-benzylidene/3-hydrazone/3-hydrazine/17-carboxyacryloyl ester derivatives of betulinic acid as anti-angiogenic agents

New 3-O-acyl, 3-benzylidene, 3-hydrazone, 3-hydrazine, 17-carboxyacryloyl ester derivatives of betulinic acid (2-6, 8-11, 13, 17, 18, 21, and 22) were synthesized and evaluated in vitro for anti-angiogenic activity on endothelial cell cytotoxicity, specificity, and tube-formation ability. All derivatives reported here showed IC(50)<4 microg/mL. Compounds 3, 9, 10, 17, 21, and 22 have shown better cytotoxicity (IC(50)<1.2 microg/mL) than betulinic acid (1) and improved endothelial cell specificity (ECS>10) in some cases. Compounds 10, 17, and 18 have shown 20%, 32%, and 48% reduction in TLS, respectively, and were found better than betulinic acid (1). We have shown that 20,29-dihydrobetulinic acid derivatives have better anti-angiogenic activity as compared to betulinic acid or its other derivatives.     

Gallic acid ester derivatives induce apoptosis and cell adhesion inhibition in melanoma cells: The relationship between free radical generation, glutathione depletion and cell death

Malignant melanoma is a lethal disease, and the incidence and mortality associated with it are increasing worldwide. It has a significant tendency to develop both metastasis and resistance to chemotherapy. The tumor cells show abnormal redox regulation, and although the molecular mechanisms involved are not well characterized, they seem to be related to oxidative stress. In a previous study, we showed the antitumoral properties of gallic acid ester derivatives in leukemia cells. Here, we show the effect of octyl, decyl, dodecyl and tetradecyl gallates on B16F10 cells, a melanoma cell line. All compounds induced cytotoxic effects, and the IC₅₀ values obtained were between 7 μM and 17 μM after 48 h of incubation. Cell death occurred through apoptosis, as demonstrated by the genomic DNA fragmentation pattern. The gallates were able to induce significant production of free radicals, deplete both glutathione and ATP, activate NF-κB and promote the inhibition of cell adhesion under the experimental conditions. The glutathione depletion induced by these compounds was related to the inhibition of γ-glutamylcysteine synthase activity. These results suggest that gallates induce tumoral cell death through apoptosis as a consequence of oxidative stress, though they use different mechanisms to do so. These findings are important since melanoma cells are resistant to death because of their high level of antioxidant defense, adhesion capability and propensity to metastasize.     

黄龙山白桦桦木醇与桦木酸含量研究

为了阐明黄龙山白桦不同生境、不同径级、不同器官桦木醇与桦木酸含量,系统采集了陕北黄土高原黄龙山林场阳坡、阴坡、林缘、孤立木4种生境下的白桦不同径级植株的树皮、树枝和树叶样品,采用超声波辅助提取法提取样品,用高效液相色谱法测定了不同生境、不同径级白桦各器官桦木醇、桦木酸含量。结果表明：（1）不同生境下白桦各器官桦木醇、桦木酸含量均存在显著差异（P＜0.05）,白桦树皮中桦木醇与桦木酸含量、白桦树枝中桦木酸含量在4种生境下均表现为阴坡＞孤立木＞林缘＞阳坡,白桦树叶中桦木醇与桦木酸含量及树枝中桦木醇含量均表现为阴坡＞林缘＞孤立木＞阳坡,总体来看阴坡生境下白桦各器官桦木醇与桦木酸含量都是最高的。（2）在白桦各器官中,桦木醇、桦木酸含量均表现为树皮＞树枝＞树叶。（3）不同生境下白桦各器官桦木醇、桦木酸含量随着胸径的增大均先增大后减小,均在第Ⅱ径级即胸径10.1~20 cm达到最大。研究认为,阴坡生境有利于白桦各器官积累桦木醇、桦木酸;白桦各器官中桦木醇含量较高,桦木酸含量比较低,而以桦木酸为原料的生物制剂已进入临床应用阶段,因此,以桦木醇为原料制取桦木酸是解决桦木酸在天然植物中含量低、提取困难的有效途径;对白桦各器官的采收应注意把握时机,于白桦生长到胸径10.1~20 cm时为最佳采收期。     

Biotransformation of betulinic and betulonic acids by fungi

Betulinic acid (1), a triterpenoid found in many plant species, has attracted attention due to its important pharmacological properties, such as anti-cancer and anti-HIV activities. The closely related, betulonic acid (2) also has similar properties. In order to obtain derivatives potentially useful for detailed pharmacological studies, both compounds were submitted to incubations with selected microorganisms. In this work, both were individually metabolized by the fungi Arthrobotrys, Chaetophoma and Dematium, isolated from the bark of Platanus orientalis as well as with Colletotrichum, obtained from corn leaves; such fungal transformations are quite rare in the scientific literature. Biotransformations with Arthrobotrys converted betulonic acid (2) into 3-oxo-7beta-hydroxylup-20(29)-en-28-oic acid (3), 3-oxo-7beta,15alpha-dihydroxylup-20(29)-en-28-oic acid (4) and 3-oxo-7beta,30-dihydroxylup-20(29)-en-28-oic acid (5); Colletotrichum converted betulinic acid (1) into 3-oxo-15alpha-hydroxylup-20(29)-en-28-oic (6) acid whereas betulonic acid (2) was converted into the same product and 3-oxo-7beta,15alpha-dihydroxylup-20(29)-en-28-oic acid (4); Chaetophoma converted betulonic acid (2) into 3-oxo-25-hydroxylup-20(29)-en-28-oic acid (7) and both Chaetophoma and Dematium converted betulinic acid (1) into betulonic acid (2). Those fungi, therefore, are useful for mild, selective oxidations of lupane substrates at positions C-3, C-7, C-15, C-25 and C-30.     

Synthesis,modification, and antimicrobial activity of the <Emphasis Type="Italic">N</Emphasis>-methylpiperazinyl amides of triterpenic acids

N-methylpyperazinyl amides of betulinic, platanic, glycyrrhetic, oleanolic, ursolic, and moronic acids were synthesized and modified. Betulin and betulonic acid showed antimicrobial activity against Staphylococcus aureus at a concentration of 90 mg/ml, and betulin manifested a bacteriostatic effect against Klebsiella pneumoniae at a concentration of 60 mg/ml. Among the studied N-methylpyperazinyl amides, the highest activity against S. aureus was observed for a betulonic acid derivative.     

Synthesis and cytotoxic activity of 3-O-acyl/3-hydrazine /2-bromo/20,29-dibromo betulinic acid derivatives

A series of 3-O-acyl, 3-hydrazine, 2-bromo, and 20,29-dibromo betulinic acid derivatives (1-27) have been synthesized and screened for in vitro cytotoxic activity on human cancer cell lines MOLT-4, JurkatE6.1, CEM.CM3, BRISTOL8, U937, DU145, PA-1, A549, and L132. A number of compounds have shown ED(50)<1 microg/mL against the cancer cell lines tested and have shown better cytotoxicity than betulinic acid. Compounds 13, 19, 20, 23, and 27 were the best derivatives and were selected as lead molecules for further development. The structure-activity relationship has been described.     

Synthesis of a-secomethylenamino- and substituted amidoximotriterpenoids

Development of the functionalization of triterpenoids to A-secoamidoximes, A-secomethylenamines, and branched 3-(3-aminopropylamino)-3-(3-aminopropoxy)amidoximes is illustrated by the betulonic acid ketoxime. An effective way to get of the derivatives of 20,29-dihydrolupanes using diborane is suggested. The antiviral and antituberculosis activity data of some compounds are presented.     

Synthesis and structure-activity relationship study of cytotoxic germanicane- and lupane-type 3beta-O-monodesmosidic saponins starting from betulin

Germanicane-type triterpenes allobetulin (3) and 28-oxoallobetulin (4) can be obtained by the Wagner-Meerwein rearrangement of the more available lupane-type triterpenes betulin (1) and betulinic acid (2), respectively. The medical uses of betulinic acid (2) and its derivatives are limited because of their poor hydrosolubility and pharmacokinetics properties. In order to overcome this major problem, we synthesized and studied the in vitro anticancer activity of a series of 3beta-O-monodesmosidic saponins derived from betulin (14-16), betulinic acid (20-22), allobetulin (23-28) and 28-oxoallobetulin (29-34) based on six different natural sugar residues (d-glucose, l-rhamnose, d-arabinose, d-galactose, d-mannose and d-xylose). This structure-activity relationship study confirmed that betulinic acid saponins are generally better in vitro anticancer agents than those derived from betulin with the exception of betulin 3beta-O-alpha-d-mannopyranoside (15) which exerted a potent cytotoxic activity against lung carcinoma (A-549) and colorectal adenocarcinoma (DLD-1) human cell lines with IC(50) ranging from 7.3 to 10.1mumol/L. Furthermore, although the synthesis of novel germanicane-type saponins was carried out with success, the bioactivity measured for these glycosides was not as high as we anticipated since only the 3beta-O-beta-d-glucopyranoside and 3beta-O-beta-d-galactopyranoside of allobetulin (23,24) showed moderate anticancer activity (IC(50) 30-40 micromol/L).