Effective Synthesis of a Novel Tetrahydrofuran Containing Triterpenoid : 5 ′ ( Z )-Benzylidene-tetrahydrofurano [ 3 , 2-b ] lup-20 ( 29 )-en-28-oate

The title compound 5′(Z)-benzylidene-tetrahydrofurano[3,2-b]lup-20(29)-en-28-oate was synthesized with high chemo-, regio-, and stereoselectivity by 5-exo-dig cycloisomerization of methyl 2α-phenylpropynyl-3-oxolup-20(29)-en-28-oate with use of KN(SiMe3)2-DME. The novel betulinic acid derivative was fully characterized by conventional analytical methods and all proton and carbon signals have been completely assigned by 2D-NMR experiments.


Introduction
The available plant metabolite, that is betulinic acid and its semi-synthetic derivatives, represent an important class of biologically active substances, which are in high demand in medicinal chemistry and pharmacological studies [1][2][3][4].In the synthesis of numerous derivatives of betulinic acid, directed at enhancing its biological potential, particular emphasis is focused on the approaches aimed at constructing of various types of heterocyclic fragments at triterpenoid core [5].The ketone carbonyl at C-3 of betulinic acid was utilized in syntheses of various fused heterocycles at the 2,3-position of the lupane skeleton including isoxazole, pyrazine, benzopyrazine, pyridine, indole, and pyrazole rings [5][6][7][8][9].These triterpenoid derivatives modified with heterocyclic rings attached to the A-ring of the triterpene have shown antitumor, anti-inflammatory and leishmanicidal activities.In this group of heterocyclic ring-substituted triterpenoids, betulinic acid analogues containing furan or tetrahydrofuran rings are little-known compounds.At the same time, polysubstituted furans, tetrahydrofurans and their precursors, 2-alkylidenetetrahydrofurans, are present in numerous natural products or used as important synthetic building blocks in the synthesis of promising biologically active substances [10][11][12][13].Recently, we developed an efficient method for the synthesis of 2-propargyl 3-oxo-triterpene acid derivatives [14].The resulting triterpene compounds containing a 4-pentyn-1-one structural unit in ring A have been successfully used in the anionic 5-exo-dig cycloisomerization induced by a strong base, KN(SiMe 3 ) 2 -DME [15].The heterocyclization of these compounds also was performed in the presence of Au(I) + phosphine complexes [16].In the continuation of our studies, this article describes the preparation of new [3,2-b] tetrahydrofuran-fused lupane triterpenoid 6-That is, 5 (Z)-benzylidene-tetrahydrofurano [3,2-b]lup-20(29)-en-28-oate, by employing the 5-exo dig heterocyclization of accessible 2-phenylpropynyl derivative of betulinic acid.

Results
In the synthesis of the target triterpenoid 6, C-2 propargyl derivative of betulinic acid 5 was used as the starting compound, which was obtained in several stages from betulin by the method previously developed by our research group [14] (Scheme 1).The key stage of the scheme was α alkylation with propargyl bromide of potassium enoxytriethylborate generated from methylbetulonate 2 under the action of KN(SiMe 3 ) 2 -Et 3 B. Stereoselective reduction of the keto group in the propynyl derivative of betulonic acid 3 using NaBH 4 modified with CeCl 3, produced methylbetulinate 4 in good yield [14] (Scheme 1).In the synthesis of the target triterpenoid 6, C-2 propargyl derivative of betulinic acid 5 was used as the starting compound, which was obtained in several stages from betulin by the method previously developed by our research group [14] (Scheme 1).The key stage of the scheme was α alkylation with propargyl bromide of potassium enoxytriethylborate generated from methylbetulonate 2 under the action of KN(SiMe3)2-Et3B.Stereoselective reduction of the keto group in the propynyl derivative of betulonic acid 3 using NaBH4 modified with CeCl3, produced methylbetulinate 4 in good yield [14] (Scheme 1).The starting compound 4 was transformed into triterpenoid 5 by Sonogashira cross-coupling in the presence of PdCl2(PPh3)2, CuI and Et3N (Scheme 2).Triterpenoid 5 heterocyclization was carried out under the action of KN(SiMe3)2 in DME.The reaction proceeded at room temperature and in a short period of time yielded a single product, that is target triterpenoid 6, with a yield of 82% ( 1 Hand 13 C-NMR spectra).It is interesting to note that only 5-exo dig cyclization occurred and stereoisomerically pure compound (Z-5) was found.We did not detect pyran derivatives derived from 6-endo cyclization and stereoisomer (E-5) even in the trace amounts.Exocyclic enol ethers are known to easily undergo hydrolysis [16,17].The triterpenoid 6 obtained by us showed protolytic stability during long-term storage (12 months) in an inert atmosphere at a temperature of ±5 °C.However, it was easily hydrolyzed to give phenylaceton-3β-hydroxylup-20(29)-en-28-oate 7 in chloroform-d within 6 h, producing a mixture of compounds 6 and 7 in 60:40 ratio ( 1 H-and 13 С-NMR).During the purification of triterpenoid 6 by the method of column chromatography on SiO2, an analytically pure sample of the compound was isolated in 32% yield along with its hydrolysis product, that is triterpenoid 7 in 51% yield.
The structure of the resulting compound was defined using one-dimensional ( 1 H, 13 C) and twodimensional (COSY, NOESY, HSQC, HMBC) NMR spectroscopy.The starting compound 4 was transformed into triterpenoid 5 by Sonogashira cross-coupling in the presence of PdCl 2 (PPh 3 ) 2 , CuI and Et 3 N (Scheme 2).Triterpenoid 5 heterocyclization was carried out under the action of KN(SiMe 3 ) 2 in DME.The reaction proceeded at room temperature and in a short period of time yielded a single product, that is target triterpenoid 6, with a yield of 82% ( 1 H-and 13 C-NMR spectra).It is interesting to note that only 5-exo dig cyclization occurred and stereoisomerically pure compound (Z-5) was found.We did not detect pyran derivatives derived from 6-endo cyclization and stereoisomer (E-5) even in the trace amounts.Exocyclic enol ethers are known to easily undergo hydrolysis [16,17].The triterpenoid 6 obtained by us showed protolytic stability during long-term storage (12 months) in an inert atmosphere at a temperature of ±5 • C.However, it was easily hydrolyzed to give phenylaceton-3β-hydroxylup-20(29)-en-28-oate 7 in chloroform-d within 6 h, producing a mixture of compounds 6 and 7 in 60:40 ratio ( 1 H-and 13 C-NMR).During the purification of triterpenoid 6 by the method of column chromatography on SiO 2 , an analytically pure sample of the compound was isolated in 32% yield along with its hydrolysis product, that is triterpenoid 7 in 51% yield.
In the synthesis of the target triterpenoid 6, C-2 propargyl derivative of betulinic acid 5 was used as the starting compound, which was obtained in several stages from betulin by the method previously developed by our research group [14] (Scheme 1).The key stage of the scheme was α alkylation with propargyl bromide of potassium enoxytriethylborate generated from methylbetulonate 2 under the action of KN(SiMe3)2-Et3B.Stereoselective reduction of the keto group in the propynyl derivative of betulonic acid 3 using NaBH4 modified with CeCl3, produced methylbetulinate 4 in good yield [14] (Scheme 1).The starting compound 4 was transformed into triterpenoid 5 by Sonogashira cross-coupling in the presence of PdCl2(PPh3)2, CuI and Et3N (Scheme 2).Triterpenoid 5 heterocyclization was carried out under the action of KN(SiMe3)2 in DME.The reaction proceeded at room temperature and in a short period of time yielded a single product, that is target triterpenoid 6, with a yield of 82% ( 1 Hand 13 C-NMR spectra).It is interesting to note that only 5-exo dig cyclization occurred and stereoisomerically pure compound (Z-5) was found.We did not detect pyran derivatives derived from 6-endo cyclization and stereoisomer (E-5) even in the trace amounts.Exocyclic enol ethers are known to easily undergo hydrolysis [16,17].The triterpenoid 6 obtained by us showed protolytic stability during long-term storage (12 months) in an inert atmosphere at a temperature of ±5 °C.However, it was easily hydrolyzed to give phenylaceton-3β-hydroxylup-20(29)-en-28-oate 7 in chloroform-d within 6 h, producing a mixture of compounds 6 and 7 in 60:40 ratio ( 1 H-and 13 С-NMR).During the purification of triterpenoid 6 by the method of column chromatography on SiO2, an analytically pure sample of the compound was isolated in 32% yield along with its hydrolysis product, that is triterpenoid 7 in 51% yield.
The structure of the resulting compound was defined using one-dimensional ( 1 H, 13 C) and twodimensional (COSY, NOESY, HSQC, HMBC) NMR spectroscopy.The structure of the resulting compound was defined using one-dimensional ( 1 H, 13 C) and two-dimensional (COSY, NOESY, HSQC, HMBC) NMR spectroscopy.
The 13 C-NMR spectrum of compound 6, exhibited no signals for the acetylene group and the 3-OH carbon atom, indicating that these functional groups in the initial compound 5 were involved in the intramolecular cyclization.Along with the characteristic signal of the quaternary carbon atom C-20 (150.6 ppm), a new signal of the quaternary carbon atom (DEPT, HSQC) was registered in the region of 151.3 ppm, which is related to the carbon atom C-5 .The signals of carbon atoms C-6 and C-3 resonated in the range of 97.7 and 96.7 ppm.In the 1 H-NMR spectrum, along with the proton signals at C-29, a new singlet signal of the vinylidene proton H-6 was present in the region of 5.27 ppm.Methylene protons H-4 resonated in the region of 2.66 and 2.41 ppm.The obtained spectral data allowed us to conclude that the structure of compound 6 contains a trisubstituted double bond and a tetrahydrofuran ring.The stereochemistry of Z-5 triterpenoid was defined applying two-dimensional NMR correlation spectra.In the 1 H-NOESY spectrum of compound Z-5, there were cross-peaks between the signals of protons H-4 , H-6 and H-2.

Materials and Methods
The starting compounds and reagents were purchased from standard commercial suppliers and used without any further purification.Betulonic acid was obtained from betulin according to known procedures [18].IR spectra were obtained with use of a Vertex 70v spectrometer (Bruker, Karlsruhe, Germany) (solutions in CHCl 3 ). 1 H-and 13 C-NMR spectra were recorded on a Bruker Avance-500 instrument (500.13 ( 1 H) and 125.78 MHz ( 13 C)) or on a Bruker Avance-400 instrument (400.13 ( 1 H) and 100.62 MHz ( 13 C)) in CDCl 3 with Me 4 Si as the internal standard.Mass spectra of new compounds were recorded on an LCMS-2010 EV (Shimadzu, Kyoto, Japan) spectrometer of the UfIC RAS Center for Collective Use "Chemistry".Elemental analysis was carried out on a 1106 analyzer (Carlo Erba, Milan, Italy).TLC was carried out on Sorbfil plates (Sorbpolimer, Krasnodar, Russia) in hexane-EtOAc (from 10:1 to 2:1) or in CHCl 3 -MeOH (20:1); spots were visualized with anisaldehyde.Silica gel L (KSKG grade, 50-160 µm) was employed for column chromatography.Starting triterpenoid 4 was prepared as previously reported [15].NMR spectra of all new compounds are in Supplementary Materials.data allowed us to conclude that the structure of compound 6 contains a trisubstituted double bond and a tetrahydrofuran ring.The stereochemistry of Z-5 triterpenoid was defined applying twodimensional NMR correlation spectra.In the 1 H-NOESY spectrum of compound Z-5, there were cross-peaks between the signals of protons H-4′, H-6′ and H-2.

Materials and Methods
The starting compounds and reagents were purchased from standard commercial suppliers and used without any further purification.Betulonic acid was obtained from betulin according to known procedures [18].IR spectra were obtained with use of a Vertex 70v spectrometer (Bruker, Karlsruhe, Germany) (solutions in CHCl3). 1 H-and 13 C-NMR spectra were recorded on a Bruker Avance-500 instrument (500.13 ( 1 H) and 125.78 MHz ( 13 C)) or on a Bruker Avance-400 instrument (400.13 ( 1 H) and 100.62 MHz ( 13 C)) in CDCl3 with Me4Si as the internal standard.Mass spectra of new compounds were recorded on an LCMS-2010 EV (Shimadzu, Kyoto, Japan) spectrometer of the UfIC RAS Center for Collective Use "Chemistry".Elemental analysis was carried out on a 1106 analyzer (Carlo Erba, Milan, Italy).TLC was carried out on Sorbfil plates (Sorbpolimer, Krasnodar, Russia) in hexane-EtOAc (from 10:1 to 2:1) or in CHCl3-MeOH (20:1); spots were visualized with anisaldehyde.Silica gel L (KSKG grade, 50-160 μm) was employed for column chromatography.Starting triterpenoid 4 was prepared as previously reported [15].NMR spectra of all new compounds are in Supplementary Materials.

Conclusions
Thus, we have presented an economical and chemoselective scheme for production of new 2alkylidenetetrahydrofuran-fused pentacyclic triterpenoid using a base promoted 5-exo-dig cycloisomerization of 2-alkynyl derivative of betulinic acid.

Conclusions
Thus, we have presented an economical and chemoselective scheme for production of new 2alkylidenetetrahydrofuran-fused pentacyclic triterpenoid using a base promoted 5-exo-dig cycloisomerization of 2-alkynyl derivative of betulinic acid.