Semi-synthesis of some heterocyclic triterpene derivatives on the basis of allobetulin

Journal of Science Hong Duc University, E.2, Vol.7, P (107 - 113), 2016 107 SEMI-SYNTHESIS OF SOME HETEROCYCLIC TRITERPENE DERIVATIVES ON THE BASIS OF ALLOBETULIN Dinh Ngoc Thuc1 Received: 25 January 2016 / Accepted: 4 April 2016 / Published: May 2016 ©Hong Duc University (HDU) and Journal of Science, Hong Duc University Abstract: Semi-synthesized products derived from natural compounds currently play an important role in the research and finding new substances. Allobetulin, obtained through a process of transformation from betulin, is compound extracted from the birch trees, could be used as the starting material for some transformation reactions. Some new derivatives containing heterocyclic moieties have been obtained via unique reactions. The results of our research are satisfactory and could be further investigated. Keywords: Triterpenoids, betulin, allobetulone, condensation, tetrahydroquinoxaline, triazine 1. Introduction Semi-synthesis of natural compounds for the purpose of developing biologically active agents have become the basis of the actively advancing scientific direction of perfect organic synthesis and medical chemistry. Triterpenes, such as betulin1 (the trivial name for lup - 20(29) - ene - 3b, 28 - diol) are abundantly present in birch bark. Betulin and its derivatives possess many interesting biological activities; Therefore, they could be seen as excellent renewable starting materials [1,2,3,4]. Betulin could be converted to the isomeric allobetulin 2 (18α - 19β, 28 - epoxyoleanan - 3β - ol) by Wagner - Meerwein rearrangement reaction in the presence of different acid catalysts [5,6]. In the molecular of betulin 1, the intermolecular reaction of the functional groups presented, such as hydroxyl and alkenes, reacted each other to form ether group in allobetulin 2 in order to lock this site. Oxidation of allobetulin to allobetulone 3, (18α - 19β, 28 - epoxyoleanan - 3 - one) was performed by using various oxidative reagents such as sodium hypochlorite [7], chromium (VI) oxide in sulfuric acid [8], meta-chloroperoxybenzoic acid [9], or FeCl3/SiO2 [10]. In this study, we focus on the reaction of the ring A of the allobetulin 3 in order to synthesize of some new allobetulin - type compounds bearing heterocycles by using different methods. Dinh Ngoc Thuc Faculty of Natural Sciences, Hong Duc University Email: [email protected] () Journal of Science Hong Duc University, E.2, Vol.7, P (107 - 113), 2016 108 2. Materials and methods 2.1. Chemicals and equipment Chemicals were purchased from Sigma Aldrich or Acros Company in Belgium; all chemicals were made in Germany, Belgium and Switzerland. All reactions were carried out in flame-dried glassware, but no special precautions were taken to exclude moisture. Solvents were mostly dried and in some cases were used as received. 1H-NMR and 13C-NMR spectra were recorded on a Bruker 300 (operating respectively at 300MHz and 75 MHz respectively) Bruker 400 Advance (operating respectively at 400 MHz and 100 MHz respectably). Infrared spectra were measured and processed on a Bruker Alpha-T FT-IR spectrometer with universal sampling module coupled to OPUS software. All samples were applied neat unless stated otherwise. Melting points were determined with a Reichert Thermovar with microscope, and are uncorrected. Low resolution mass spectra were recorded on a Hewlett-Packard 5989A mass spectrometer (EI or CI mode), coupled with an HP Apollo 900 series. HRMS (EIMS) data were acquired on a Kratos MS50TC with ionization energy of 70eV at 150-250 °C (as required), coupled to a MASSPEC II data analyzing system. These data were measured with a resolution of 10000. 2.2. General procedures 2.2.1. Preparation of starting materials 1, 2, 3 A solution of betulin 1 (10 g, 22.59 mmol) and p-toluenesulfonic acid, (5 g, 29.0 mmol) was dissolved in dichloromethane (500 ml) in a 1000 ml round bottomed flask. The reaction mixture was heated under refluxed with boiling chip for 15 hours. After reaction, the solvent was evaporated and the crude mixture was washed on a Buchner funnel with water (3x300ml) and cold methanol (2x100ml) to remove any catalyst traces and the resulting compound then was dried in low pressure desiccators. 9.3 gram of white solid obtained, was found to be (NMR) essentially pure allobetulin 2 and used for further synthesis without purification. In a 500 mL two-neck round-bottomed flask was placed oxalyl chloride (3.96 ml, 45.2 mmol) in 180 ml of dry DCM, stirred while cooling to - 78oC under inert (Ar) atmosphere. Dimethyl sulfoxide (6.55 ml, 90 mmol) was added and the mixture was stirred for 10 minutes. A solution of 10g of allobetulin 2 in 120 ml of dry DCM was added and stirring was continued for 15 minutes. 12.96 ml of Et3N was added and stirring was continued for 15 minutes. After that the mixture was warmed to 0oC, and checked by TLC to show complete reaction. The reaction finished after 2 hours. Then 300 ml of H2O was added to the mixture, stirring was continued for 15 minutes, and was separated by funnel to get DCM layer. This DCM layer was washed by water (2 x 300ml). The organic layer was dried with MgSO4, filtered and concentrated. The crude mixture was separated by chromatography with solvent Journal of Science Hong Duc University, E.2, Vol.7, P (107 - 113), 2016 109 mixture of n-heptane: EtOAc = 9:1 to get allobetulone3. Yield: 90%; Mp: 226-228 oC, 1H-NMR (CDCl3, 300MHz, δppm): 3.79 (d, J=7.8, 1H, H-28), 3.53 (s, 1H, H-19), 3.46 (d, J=7.8, 1H, H-28), 2.45 (m, 2H, H-2), 1.94 (m, 1H, H-19), 1.64 -1.09 (21H- complex CH, CH2), 1.07 (s, 3H), 1.03 (s, 3H), 1.01 (s, 3H), 0.93 (s, 9H), 0.79 (s, 3H), (all s 7x3H, 23-, 24-, 25-, 26-, 27-, 29-, 30- Me). 13C-NMR (CDCl3, 75 MHz, δ ppm): 218.2 (C-3), 87.9 (C-19), 71.2 (H-28), 54.9, 50.4, 47.3, 46.7, 41.4, 40.7, 40.5, 39.8, 36.9, 36.7, 36.2, 34.2, 34.1, 33.1, 32.6, 28.8, 26.7, 26.4, 26.2, 24.5, 21.5, 20.9, 19.6, 16.3, 15.5, 13.4. 2.2.2. Semi-synthesis of triterpene derivatives 4, 5, 6 A solution of allobetulone 3 (4 gram, 9.08 mmol) and t-BuOK 45.4 mmol, (5.2 gr) in t-BuOH (125 ml) was vigorously stirred at room temperature with the provision of efficient access of oxygen to the reaction mixture (with balloon). The TLC was checked every hour with solvent mixture of n-heptane: EtOAc = 9: 1. The reaction finished after 5hrs. After the reaction finished, the mixture was diluted with MeOBut (400 ml) and neutralized with 1 M HCl (400 ml) on cooling to 0ºC. The organic layer was separated, washed successively with water (2x400 ml) and a saturated solution of NaCl (200 ml), dried over Na2SO4. The crude mixture was purified by column chromatography with solvent mixture of heptane:EtOAc =9:1 to obtain 2-oxoallobetulone 4. Yield: 87%; Mp: 236-238 oC1H-NMR (CDCl3, 300MHz, δ ppm): 6.45 (s, 1H, H-1), 5.92 (s, 1H, OH-2), 3.79 (d, J=7.8, 1H, H-28), 3.53 (s, 1H, H-19), 3.44 (d, J=7.8, 1H, H-28), 1.71-1.27 (20H- complex CH, CH2), 1.21 (s, 3H), 1.15 (s, 3H), 1.11 (s,3H), 1.04 (s, 3H), 0.94 (s, 6H), 0.81(s, 3H), (all s 7x3H, 23-, 24-, 25-, 26-, 27-, 29-, 30- Me). 13C-NMR (CDCl3, 75 MHz, δppm): 201.2 (C-3), 143.8 (C-2), 129 (C-1), 87.8 (C-19), 71.2(C-28), 54.2, 46.7, 46.1, 44.0, 41.4, 41.0, 38.6, 36.6, 36.2, 34.2, 33.5, 32.6, 31.2, 28.7, 27.0, 26.3, 26.2, 24.5, 21.5, 21.2, 20.5, 18.6, 16.2, 13.3. In a 50 mL round-bottomed flask was 2-oxoallobetulone 4 (400 mg, 0.88 mmol) and 1, 2-diamino cyclohexane (0.16 ml, 1.32 mmol) in AcOH (15 ml). The mixture was stirred at 80oC. The TLC was checked with solvent mixture ofheptan: EtOAc = 8:2 every hour to confirm the complete reaction. The reaction was finished after 18hrs. Then the solvent was evaporated. The residue was purified by column chromatography with solvent mixture ofheptan: EtOAc = 8:2 to obtain (5, 6, 7, 8-tetrahydroquinoxalino) allobetulin 5. Yield: 58%; Mp: 265-267 0C; 1H-NMR (CDCl3, 300MHz, δ ppm): 3.79 (d, J=7.7, 1H, H-28), 3.56 (s, 1H, H-19), 3.46 (d, J=7.7, 1H, H-28), 3.01 (d, J=16.3, 1H, H-1α), 2.88 (m, 4H, H2’,H5’)2.41 (d=16.3, 1H, H-1β),1.89 (m, 4H, H3’, H4’), 1.70 -1.09 (22H- complex CH, CH2), 1.27 (s, 3H), 1.25 (s, 3H), 1.04 (s, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.84 (s, 3H), 0.81 (s,3H) (all s 7x3H, 23-, 24-, 25-, 26-, 27-, 29-, 30- Me). 13C-NMR (CDCl3, 75 MHz, δ ppm): 155.8 (C-3), 150.0 (C-2), 148.9 (C-6’), 146.6 (C-1’), 87.9 (C-19), 71.2 (C-28), 53.3, 49.4, 48.6,46.7, 41.5, 40.7, 40.4, 39.0, 36.9, 36.7, 36.3, 34.3, 33.0, 32.7, 31.7, 31.6, 31.5, 28.8, 26.4, 26.2, 24.5, 23.9, 22.9, 22.8, 21.4, 20.0, 16.4, 15.3, 13.5. HRMS: Calculated for C36H54N2O: Journal of Science Hong Duc University, E.2, Vol.7, P (107 - 113), 2016 110 530.42361. Found: 530.42455. A solution of 2-oxoallobetulone 4 (1.203 mmol, 0.5471 g) and thiosemicarbazide (1.564 mmol, 0.143 g) in 50 ml ethanol was vigorously stirred at reflux for 30 mins, after that 1.564 mmol of K2CO3 (0.216g) was added and stirred at reflux. The TLC was checked with solvent n-heptane: EtOAc = 8:2. The reaction finished after 20 hours. Then the reaction mixture was diluted with 50 ml of water and acidified with acetic acid till pH = 4.0. The resulting orange compound was immediately precipitated, filtered and washed with water. Triazine was recrystallized from ethanol to get (3-thio-1,2,4-triazino) allobetulin (6) Yield: 83%; Mp: 227-229 oC; 1H-NMR (CDCl3, 300MHz, δ ppm): 3.81 (d, J=7.8, 1H, H-28), 3.57 (s, 1H, H-19), 3.49 (d, J=7.5, 1H, H-28),3.0 (d, J=16.4, 1H, H-1), 2.24 (d, J=16.3, 1H, H-1), 1.7-1.4 (21H- complex CH, CH2), 1.36 (s, 3H), 1.32 (s, 3H), 1.03 (s, 3H), 0.95 (s, 6H), 0.82(s, 3H), 0.81(s, 3H), (all s 7x3H, 23-, 24-, 25-, 26-, 27-, 29-, 30- Me). 13C-NMR (CDCl3, 75 MHz, δ ppm): 180.9 (C-31), 173.1 (C-3), 145.6 (C-2), 87.9 (C-19), 71.2(C-28), 53.0, 48.7, 46.7, 44.8,41.4, 41.1, 40.8, 40.4, 36.9, 36.6, 36.2, 34.2, 32.7, 32.6, 30.9, 28.7, 26.3, 26.2, 26.1, 24.5, 24.2, 21.6, 20.0, 16.3, 15.3, 13.4. HRMS: C31H47N3OS, calculated: 509.3440, found: 509.3436. 3. Results and discussion Synthesis of starting materials from betulin has been described previously [3,4] and went through three optimized steps as presented in Scheme 1 of these many methods described we found the most convenient way to rearrange betulin to allobetulin in multigram quantity to be by using p-toluenesulfonic acid as an acid catalyst in dichloromethane at reflux condition. Swern oxidation [11] was then applied to oxidize allobetulin2 to allobetulone3 in order to avoid chromium reagents or other less environmentally friendly reagents. Reagents and conditions: (a) p-toluenesulfonic acid, dichloromethane, reflux; (b) oxalyl chloride, dimethyl sulfoxide, triethylamine, dichloromethane, -78oC-0oC Scheme 1. Synthesis of allobetulone from betulin Allobetulone can be oxidized to the 2-oxoallobetulone 4 (here shown in the enol form) by potassium tert-butoxide in tertbutanol (Scheme 2) [12]. In this reaction, oxygen resource could be used either oxygen in air or pure oxygen (from balloon). The obtained product is used to prepare fused heterocyclic ring compounds. The products could be ether 2- Journal of Science Hong Duc University, E.2, Vol.7, P (107 - 113), 2016 111 hydroxyl-3-ketone derivative (4) and 2,3-diketone derivative one (4’), however, we found that only compound 4 was obtained. The product structure was confirmed by 1H-NMR spectroscopic method through the presence of signals of the proton H-1 at 6.45 ppm (s, 1H, H- 1) and of OH proton at 5.92 (s, 1H, OH-2). Scheme 2. Synthesis of 2-oxoallobetulone Condensation of 2-oxoallobetulone with 1,2-diaminocyclohexane afforded heterocyclic (tetrahydroquinoxalino) allobetulin 5. This reaction is very interesting because oxidation happened in situ and the product is aromatized to the heterocyclic compound (Scheme 3). Reagents and conditions: (a) 1,2-diaminocyclohexane, acetic acid, 80oC; (b) thiosemicarbazide, K2CO3, EtOH, reflux Scheme 3. Synthesis of heterocylic derivatives 5, 6 The condensation of 2-oxoallobetulone 4 with thiosemicarbazide in ethanol with K2CO3 afforded the desired allobetulin derivative having 1,2,4 - triazine moiety 6 (Scheme 3) [13]. The absolute configuration of compound 6 was optimized by theoretical calculation using the Chem - 3D program (Molecular Mechanics, MM2 force field, Figure 1). Figure 1. 3D-Structure of compound 6 (Molecular Mechanics, MM2 force field) Journal of Science Hong Duc University, E.2, Vol.7, P (107 - 113), 2016 112 The structure of this compound was confirmed by 2D-NMR HMBC (measure by Bruker 400 Advance) analysis through the coupling of both C2 with C1 hydrogen and C3 with C23 hydrogen. According to HMBC the signals of the two protons on the C-1 at 3.0 ppm and 2.24 ppm have correlations with C2 at 145.6 ppm and the signal of the proton on the C23 at 1.32 ppm has a correlation with C3 at 173.1 ppm (Figure 2). Figure 2. HMBC of compound 6 4. Conclusion Based on the betulin, interesting transformations to valuable starting materials, which were used to synthesize a series of new heterocyclic products, have been carried out. The structures of these compounds were confirmed by modern spectroscopic methods. This research direction could be continuous and the obtained products could be investigated for further applications. Acknowledgment: The author would like to thank Prof. Dr. WimDehaen - University of Leuven for valuable advice, the Vietnamese Government and University of Leuven - Belgium for financial support. Journal of Science Hong Duc University, E.2, Vol.7, P (107 - 113), 2016 113 References [1] Dehelean, C.A.a, CîntǎPînzaru, S.b,Peev, C.I.a,Soica, C.a,Antal, D.S. (2007), Characterization of birch tree leaves, buds and bark dry extracts with antitumor activity. Journal of optoelectronics and advanced materials. Volume 9, Issue 3, March, P 783 - 787. [2] Shesadri T. R., Vedanthan T.N.C (1971), Chemical examination of the barks and heartwoods of Betula species of American origin. Phytochemistry. 10, 897 - 898. [3] Kovac B.E., Duric K, Kalodera Z., Sofic E., Identification and isolation of pharmacologically active terpenes in Betulae cortex, Betulapendula Roth, Betulaceae Bosnian. Journal of Basic Medical Science. 2009, 9(1), 32 - 38. [4] Dehaen, W; Mashentseva, A.A. and Seitembetov, T.S, (2011), Allobetulin and Its Derivatives: Synthesis and BiologicalActivity. Molecules. 16, 2443 - 2466. [5] Green B.; Bentley M.D; Chung B.Y.; Lynch N.G.; Jensen B.L. (2007), Allobetulin and its derivatives: Synthesis and biological activity. J. Chem. Educ. 84, 1985 - 1987. [6] Li T.S.; Wang J. X.; Zheng X. J. (1998), Simple synthesis of allobetulin, 28- oxyallobetulin and related biomarkers from betulin and betulinic acid catalysed by solid acids. J. Chem. Soc. Perkin Trans. 3957 - 3965. [7] Flekhter, O. B.; Ashavina, O. Y.; Smirnova, I. E.; Baltina, L. A.; Galin, F. Z.; Kabal’nova, N. N.; Tolstikov, G. A. (2004), Selective oxidation of triterpene alcohols by sodium hypochlorite. Chem. Nat. Comp. 40, 141 - 143. [8] Kim D. S. H. L.; Chem Z.; Nguyen Van T.; Pezzuto J.M.; Quit S.; Lu Z.Z. (1997), A Concise Semi-Synthetic Approach to Betulinic Acid from Betulin. Synth.Commun. 27, 1607 - 1612. [9] Tori M; Maisuda R.; Sono M.; Kohama Y.; Asakawa Y. (1988), The reaction of lupane and friedo-oleanane type triterpeneswith m-chloroperbenzoicacid. Bull. Chem. Soc. Jpn.61, 2103 - 2108. [10] Thibeault B.; Legault J.; Bouchard J.; Pichette A. (2007), Useful approach to access germanicanes from betulin. Tetrahedron Lett. 48, 8416 - 8419. [11] Flekhter, O. B.; Ashavina O. Y.; Boreko E. I.; Karachurina, L. T.; Pavlova, N. I.; Kabalnova N. N.; Savinova O. V.; Galin F. Z.; Nikolaeva S. N. Zarudii F. S.; Baltina L. A.; Tolstikov G. A. (2002). Synthesis and local anesthetic activity of 1, 2- disubstituted imidazo [1, 2-a] benzimidazoles. Khim.Pharm.Zh. 36, 21 - 24. [12] A. V. Korovin, and A. V. (2001), Tkachev. Synthesis of quinoxalines fused with triterpenes, ursolic acid and betulin derivatives. Russ. Chem. Bull. 50, 304 - 310. [13] Nagai S.; UedaT.; NagatsuA.; MurakamiN.; SakakibaraJ. (1997), Synthesis and central nervous system stimulant activity of camphor-1, 2, 4-triazines fused with 1, 2, 4-triazole, tetrazole and 1, 2, 4-triazine. Heterocycles. 44, 117 - 120.