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Communication

A Regioselective Synthesis of E-Guggulsterone

1
Korea Institute of Science and Technology, 290 Daejeon-dong, Gangneung 210-340, Korea
2
Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, Seoul National University, NS-80, Seoul 151-747, Korea
3
Research Institute of Oceanography, Seoul National University, NS-80, Seoul 151-741, Korea
*
Authors to whom correspondence should be addressed.
Molecules 2011, 16(5), 4165-4171; https://doi.org/10.3390/molecules16054165
Submission received: 18 April 2011 / Revised: 17 May 2011 / Accepted: 18 May 2011 / Published: 20 May 2011

Abstract

:
We have successfully prepared E-guggulsterone from 16,17-epoxy-pregnenolone in 84% yield over two steps via a hydrazine reduction and Oppenhauer oxidation. Additionally, isomerization was induced by heat, light (hν) and acid catalysis to convert E- guggulsterone into the corresponding Z isomer.

1. Introduction

Guggulipid from the resin of the Commiphora mukul tree (guggulu in Sanskrit) [1] has been used as an Asian folk remedy for chronic disorders such as rheumatism, obesity and atherosclerosis since at least 600 BC [2,3]. It has been reported that E- and Z-guggulsterones (Figure 1), two active ingredients in the Commiphora mukul resin [1,4] lower the level of low density lipoprotein cholesterol (LDLc) [5,6,7,8] and triglycerides in mouse. Guggulsterones are also known to have therapeutic effects for the treatment of inflammatory bowel diseases [9] and various cancers [10], and the molecular mechanisms underlying those effects are currently under investigation. Thus, there is a great demand for large amounts of the guggulsterones to further in vitro and in vivo studies. Because this demand has not been met by natural sources, which only provides the compounds in low yield (1.1%), [1,4] we have developed a first regioselective synthesis of guggulsterone.
Figure 1. Chemical structures of E- and Z-guggulsterones.
Figure 1. Chemical structures of E- and Z-guggulsterones.
Molecules 16 04165 g001
In 1964, the first synthesis of guggulsterone was reported by Benn and Dodson [11,12], and a patent by Hamied and co-workers was published in 1991 [13]. In Benn and Dodson’s method, [11,12] the final E- and Z-guggulsterones were prepared from 16-dehydropregnenolone acetate (16-DPA) or 16,17-epoxypregnenolone (2) as starting steroid. Our initial attempts to synthesize guggulsterone were based on this protocol. However, the low yields and long reaction times ultimately led us to abandon this route. Moreover, we sought to investigate a stereoselective preparation of the guggulsterones because this was not detailed in either the papers or the patent.

2. Results and Discussion

During the course of our synthetic studies of bioactive compounds, we discovered a stereoselective two-step reaction for the preparation of E-guggulsterone from 16,17-epoxypregnenolone (2) through a hydrazine reduction [11,14] and Oppenhauer oxidation [15,16]. Herein, we report a regioselective method for the preparation of E-guggulsterone and methods for the conversion of E-guggulsterone into the corresponding Z isomer (Scheme 1).
Scheme 1. Regioselective synthesis of E-guggulsterone.
Scheme 1. Regioselective synthesis of E-guggulsterone.
Molecules 16 04165 g002
In the first step, we prepared the cis-diol 3 from steroid 2 using hydrazine monohydrate (98% purity) and 9.0 equiv of KOH at 160 °C for 2 h (91% yield). This stereoselectivity and yield differ dramatically from those reported in the literature for the same reaction. Specifically, Benn and Dodson reported that compound 3 was obtained as a mixture (66% cis-3 and 34% trans-3) in 22% yield when using hydrazine at 195 °C for 5.5 h [11,14]. In 1968, Kessar and Rampal reported the preparation of cis-3 using 80% hydrazine. They obtained a total yield of only 47% as a mixture of cis- and trans-3 [14]. Based on these results, we identified two important factors that affect the regioselectivity and yield of cis-3: the purity of hydrazine monohydrate and the reaction time (reaction times greater than 3 h decrease the stereoselectivity). In the second and final step, the target compound, E-guggulsterone, was prepared through an Oppenhauer oxidation. We investigated the reaction conditions in order to maximize both regioselectivity and yield (Table 1).
Table 1. Optimization of Oppenhauer Oxidation Conditions for the Synthesis of E-Guggulsterone. a Molecules 16 04165 i001
Table 1. Optimization of Oppenhauer Oxidation Conditions for the Synthesis of E-Guggulsterone. a Molecules 16 04165 i001
EntryEquiv of AKetoneSolventTime (h)% Yield bE:Z Ratio c
10.5cyclohexanonetoluene29467:33
21.0cyclohexanonetoluene29686:14
30.5cyclohexanonebenzene292only E
4 d1.0acetonebenzene7no reaction-
50.52-butanonebenzene432only E
a All reactions were carried out on a 1.0 mmol scale of cis-3; b The yields refer to the average isolated yield of three runs; cE: Z ratios were calculated based on 1H-NMR through the integration of peaks at 5.75 ppm and 6.52 ppm, respectively. d This reaction was performed in a sealed tube at 120 °C.
When the reaction was run in toluene using 0.5 equiv of Al(O-i-propyl)3, the desired compound was obtained as a mixture of isomers in 94% yield (Table 1, entry 1). By increasing the Al(O-i-propyl)3 loading from 0.5 equiv to 1.0 equiv, the regioselectivity for E-guggulsterone increased from 67% to 86% and the yield also increased slightly. The choice of solvent also proved to play an important role in the regioselectivity of the reaction; when benzene and 0.5 equiv of Al(O-i-propyl)3 were used, we obtained pure E-guggulsterone in 92% yield (Table 1, entry 3). On the other hand, when we changed the from cyclohexanone to acetone, the reaction did not proceed at all (Table 1, entry 4). When 2-butanone was used as an oxidant in the presence of 1.0 equiv of Al(O-i-propyl)3 at 80 °C for 4 h, E-guggulsterone was obtained as a single isomer in poor yield (Table 1, entry 5). Based on these results, we concluded that the most important variable for the preparation of pure E-guggulsterone was the reaction temperature. Next, we examined the isomerization of E-guggulsterone to Z-guggulsterone under various reaction conditions such as heat, light (hν), and acid catalysis. The results of the isomerization reactions are summarized in Table 2.
Table 2. Reaction Conditions for Isomerization from E- to Z-Guggulsterone. a Molecules 16 04165 i002
Table 2. Reaction Conditions for Isomerization from E- to Z-Guggulsterone. a Molecules 16 04165 i002
EntryDriving forceSolvent/Temp.Time (h)E(1a):Z(1b) Ratio b
1heattoluene/110 °C295:5
2heatmesitylene/170 °C265:35
3heattoluene/sealed tube,140 °C245:55
4light cMeOH/25 °C1250:50
5p-TsOHbenzene/80 °C140:60
62 N-HClacetonitrile/36 °C1860:40
a All reactions were carried out on a 0.5 mmol scale; b The yield refer to the average isolated yield of three runs; c Light source was a 300 W-tungsten lamp.
Heating compound 1a for 2 h at reflux (high temperature) gave 5-35% yields of the Z-isomer 1b, (entries 1-2). When the reaction was carried out at 140 °C using a sealed tube, the yield of the Z-isomer was increased to 55% (Table 2, entry 3). In the light-induced isomerization (Table 2, entry 4), which contained 1.0 mol % of methylene blue as a photosensitizer, the E-isomer dissolved in CH2Cl2 was converted to the Z-isomer 1b in the 50% yield after 12 h at 25 °C. On the other hand, the use of acid catalysts showed that conversion to Z-guggulsterone 1b was higher when using p-toluenesulfonic acid as compared to 2N HCl, which is condition that mimics the human stomach (Table 2, entries 5 and 6). Interestingly, E- and Z- guggulsterones were very stable during the isomerization reaction and did not generate side products. They were also easily purified by chromatography on silica gel with hexane/EtOAc (v/v = 5/4).

3. Experimental

3.1. General

All reactions were performed in oven- and flame-dried glassware under nitrogen atmosphere. Air and moisture sensitive reagents and solvents were transferred via syringes or cannula, and they were introduced into the reaction vessel through a rubber septum. Chemicals obtained from commercial sources were used without further purification. Flash column chromatography was carried out on silica gel (230–400 mesh). Analytical thin-layer chromatography (TLC) was performed with silica gel 60 F254. TLC plates were visualized with UV light and 5% ammonium dimolybdate or p-anisaldehyde in ethanol with heat. NMR spectra (300 MHz for 1H and 75 MHz for 13C) were recorded in CDCl3 on a Bruker Avance III 400 MHz NMR spectrometer and chemical shifts (δ) were expressed in ppm downfield from the internal tetramethylsilane or with reference to residual CHCl3. The purity of compounds was assessed by HPLC/MS spectra, which were recorded on a Finnigan LTQ LC/MS system. Optical rotations were measured on a Rudolph Research Autopol Model III polarimeter.
5,17(20)-(cis)-Pregnadiene-3β,16α-diol (3): To a suspension of 16α,17α-epoxypregnenolone (3.31 g, 10.0 mmol) in diethylene glycol (25 mL, 99%) was added KOH (5.0 g, 89.0 mmol) and hydrazine monohydrate (9.7 mL, 200 mmol) at room temperature. After the mixture was heated at 120 °C for 1 h, the condenser was removed and the reaction temperature of 160 °C maintained for 2 h. The reaction was monitored by thin-layer chromatography. After being completely reacted, it was cooled, poured into water (30 mL) and extracted with CHCl3 (3 ° 40 mL). The combined organic layer was washed with brine, dried over MgSO4, filtered, and evaporated under reduced pressure to give the crude product. The crude compound was purified by recrystalization from hot ethyl acetate to obtain 3 as a white solid (2.88 g, 91%). 1H-NMR: δ 5.60 (q, 1H, J = 7.1 Hz), 5.36 (d, 1H, J = 5.3 Hz), 4.44 (s, 1H), 3.54 (m, 1H), 2.30−0.91 (m, 19H), 1.74 (d, 3H, J = 7.1 Hz), 1.03 (s, 3H), 0.89 (s, 3H); 13C-NMR: δ 155.7, 141.2, 121.9, 120.0, 74.8, 72.1, 53.1, 50.5, 44.6, 42.7, 37.6, 37.5, 37.0, 35.6, 32.0, 31.2, 21.5, 19.8, 17.7, 13.7; mp = 192−194 °C; [α]D −79.2° (c=1.0, EtOH); HRMS (EI): calcd for C21H32O2 316.2402, found 316.2402.
E-Guggulsterone (1a): To a suspension of 3 (2.0 g, 6.3 mmol) in benzene (60 mL) was added cyclohexanone (6.6 mL, 63.0 mmol), followed by addition of Al(O-isopropyl)3 (650 mg, 3.2 mmol) at the room temperature. The reaction mixture was warmed at 80 °C for 2 h. After that, the mixture was cooled to the room temperature, added 10% H2SO4 (4 mL), and vigorously stirred for 10 min. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 ° 35 mL). The combined extract was washed with water, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure to give the crude product. The crude compound was purified by column chromatography on silica gel using hexane/ethyl acetate (v/v = 5/4) as eluent to give 1a as a white solid (1.62 g, 91%). 1H-NMR: δ 6.52 (q, 1H, J = 7.5 Hz), 5.75 (s, 1H), 2.50−1.08 (m, 19H), 1.86 (d, 3H, J = 7.5 Hz), 1.24 (s, 3H), 1.08 (s, 3H); 13C-NMR: δ 206.0, 199.6, 170.6, 147.8, 129.9, 124.5, 53.8, 49.9, 43.5, 39.0, 38.2, 36.4, 35.9, 34.7, 34.3, 32.9, 32.2, 21.1, 17.9, 17.7, 13.6; mp = 168−171 °C; [α]D −34.5° (c=1.0, EtOH); HRMS (FAB): calcd for C21H28O2 [M+H]+ 313.4601, found 313.2168.
Z-Guggulsterone (1b): (a) Photoreaction method: Methylene blue (1 mg) was added as a photosensitizer to a solution of 1a (1.0 g, 3.2 mmol) in CH2Cl2 (50 mL) at room temperature. The mixture was irradiated with 300W-tungsten lamp in water bath for 6 h. After that, solvent was removed under reduced pressure at the room temperature. The residue was purified by chromatography on silica gel with hexane/ethyl acetate (v/v = 5/4) to afford 1b as a white solid (433 mg, 43%).1H-NMR: δ 5.73 (s and m, 2H), 2.43−0.75 (m,19H), 2.08 (d, 3H), 1.22 (s, 3H), 0.96 (s, 3H); 13C-NMR: δ 208.2, 199.6, 170.7, 148.2, 130.9, 124.5, 54.0, 49.4, 43.4, 39.7, 39.1, 35.9, 35.0, 34.3, 33.0, 32.2, 21.0, 19.9, 17.7, 14.5;mp = 191−193 °C; [α]D −54.8° (c=1.0, EtOH); HRMS (FAB): calcd for C21H28O2 [M+H]+ 313.4601, found 313.2168. (b) Sealed-tube method: In a dried sealed tube, 1a (200 mg, 0.64 mmol) was dissolved in toluene (20 mL) and then the tube was completely sealed with flame. The mixture was reacted at 160 °C for 2 h. After cooling to room temperature, solvent was removed under reduced pressure, and the residue was purified by chromatography on silica gel with hexane/ethyl acetate (v/v = 5/4) to obtain 1b as a white solid (117 mg, 59%) and recovered 1a (81 mg). (c) Acid-catalyzed method: To a solution of 1a (1.0 g, 3.2 mmol) in benzene (50 mL) was added p–toluenesulfonic acid (61.0 mg, 0.32 mmol) at the room temperature. The resulting mixture was heated at 80 °C for 1 h. After that, the reaction mixture was cooled to room temperature and solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel with hexane/ethyl acetate (v/v = 5/4) to obtain 1b as a white solid (644 mg, 64%) and recovered 1a (350 mg).

4. Conclusions

In conclusion, we have successfully prepared E-guggulsterone in 84% yield over two steps from 16,17-epoxy-pregnenolone via hydrazine reduction and Oppenhauer oxidation. Additionally, by using heat, light (hν), and acid catalysts to induce isomerization, we also easily converted E-guggulsterone into its corresponding Z isomer.

Acknowledgments

We are grateful to members of our laboratory. This work was supported by the Marine Biotechnology Program, Ministry of Land, Transport and Maritime Affairs (MLTM) and in part the BK21 program from the Ministry of Education, Science and Technology (MEST), Korea.

References and Notes

  1. Patil, V.D.; Nayak, U.R.; Dev, S. Chemistry of Ayurvedic crude drugs. Tetrahedron 1972, 28, 2341–2352. [Google Scholar] [CrossRef]
  2. Satyavati, G.V. Gum guggul (Commiphora mukul) - the success story of an ancient insight leading to a modern discovery. Indian J. Med. Res. 1988, 87, 327–335. [Google Scholar]
  3. Dev, S. Ethno therapeutics and modern drug development: The potential of auerveda. Curr. Sci. 1997, 73, 909–928. [Google Scholar]
  4. Mesrob, B.; Nesbitt, C.; Misra, R.; Pandey, R.C. High-performance liquid chromatographic method for fingerprinting and quantitative determination of E- and Z-guggulsterones in Commiphora mukul resin and its products. J. Chromatogr. B 1998, 720, 189–196. [Google Scholar]
  5. Nityanand, S.; Kapoor, N.K. Cholesterol lowering activity of the various fractions of guggul. Indian J. Exp. Biol. 1973, 11, 395–398. [Google Scholar]
  6. Nityanand, S.; Srivastava, J.S.; Asthana, O.P. Clinical trials with gugulipid: A new hypolipidaemic agent. J. Assoc. Physicians India 1989, 37, 323–328. [Google Scholar]
  7. Singh, R.B.; Niaz, M.A.; Ghosh, S. Hypolipidemic and antioxidant effects of Commiphora mukul as an adjunct to dietary therapy in patients with hypercholesterolemia. Cardiovasc. Drugs Ther. 1994, 8, 659–664. [Google Scholar] [CrossRef]
  8. Wang, X.; Greilberger, J.; Ledinski, G.; Kager, G.; Paigen, B.; Jurgens, G. The hypolipidemic natural product Commiphora mukul and its component guggulsterone inhibit oxidative modification of LDL. Atherosclerosis 2004, 172, 239–246. [Google Scholar]
  9. Cheon, J.H.; Kim, J.S.; Kim, J.M.; Kim, N.; Jung, H.C.; Song, S.S. Plant sterol guggulsterone inhibits nuclear factor-kappaB signaling in intestinal epithelial cells by blocking IkappaB kinase and ameliorates acute murine colitis. Inflamm. Bowel Dis. 2006, 12, 1152–1161. [Google Scholar]
  10. Singh, S.V.; Choi, S.; Zeng, Y.; Hahm, E.R.; Xiao, D. Guggulsterone-induced apoptosis in human prostate cancer cells is caused by reactive oxygen intermediate-dependent activation of c-Jun NH2-terminal kinase. Cancer Res. 2007, 67, 7439–7449. [Google Scholar]
  11. Benn, W.R.; Dodson, R.M. The Synthesis and Stereochemistry of Isomeric 16-Hydroxy-17(20)-pregnenes. J. Org. Chem. 1964, 29, 1142–1148. [Google Scholar] [CrossRef]
  12. Owsley, E.; Chiang, J.Y.L. Guggulsterone antagonizes farnesoid X receptor induction of bile salt export pump but activates pregnane X receptor to inhibit cholesterol 7alpha-hydroxylase gene. Biochem. Bioph. Res. Commun. 2003, 304, 191–195. [Google Scholar]
  13. Hamied, Y.K. A Process for the preparation of pharmacologically active synthetic Z and E steroisomeric mixture of guggulsterones. Eur. Patent 0447706 A1.
  14. Kesser, S.V.; Rampal, A.L. Synthetic studies in steroidal sapogenins and alkaloids. III. Synthesis and stereochemistry of isomeric 16-hydroxy- and 16-oxo-5,17(20)-pregnadien-3-ols. Tetrahedron 1968, 24, 887–892. [Google Scholar]
  15. Djerassi, C. Oppenauer oxidation. Org. React. 1951, VI, 207–272. [Google Scholar]
  16. de Graauw, C.F.; Peters, J.A.; van Bekkum, H.; Huskens, J. Meerwein-Ponndorf-Verley Reductions and Oppenauer Oxidations: An Integrated Approach. Synthesis 1994, 10, 1007–1017. [Google Scholar]
  • Sample Availability: E-Guggulsterone is available from the authors.

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Ham, J.; Chin, J.; Kang, H. A Regioselective Synthesis of E-Guggulsterone. Molecules 2011, 16, 4165-4171. https://doi.org/10.3390/molecules16054165

AMA Style

Ham J, Chin J, Kang H. A Regioselective Synthesis of E-Guggulsterone. Molecules. 2011; 16(5):4165-4171. https://doi.org/10.3390/molecules16054165

Chicago/Turabian Style

Ham, Jungyeob, Jungwook Chin, and Heonjoong Kang. 2011. "A Regioselective Synthesis of E-Guggulsterone" Molecules 16, no. 5: 4165-4171. https://doi.org/10.3390/molecules16054165

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