Article pubs.acs.org/jnp
Left, Right, or Both? On the Configuration of the Phenanthroindolizidine Alkaloid Tylophorine from Tylophora indica Alexander Stoye, Theodor Edmund Peez, and Till Opatz* Institute of Organic Chemistry, Johannes Gutenberg-University, Duesbergweg 10-14, D-55128 Mainz, Germany S Supporting Information *
ABSTRACT: The alkaloid (−)-tylophorine was isolated from a sample of Tylophora indica, and the crude extract was analyzed by HPLC/MSn and chiral HPLC/MS. While the literature states that the naturally occurring form of this alkaloid is the R-enantiomer and that its S-antipode is usually not found in nature, we confirmed the hypothesis of Govindachari and Nagarajan that natural levorotatory tylophorine is indeed a nearly racemic mixture with a slight excess of the R-enantiomer.
T
CHCl3) for (R)-1, ee >98%; −77.6 (c 0.65, CHCl3)11 for (R)1; +62.1 (c 1.0, CHCl3) for (S)-1, ee = 81%.12 In accordance with these results, our own synthesis of (S)-1 via radical cyclization from (S)-proline13 gave a product with [α]22D = +78.9 (c 0.5, CHCl3) and >99% ee. Remarkably, the specific rotation of 1 decreases rapidly during the measurement due to the high light sensitivity of the material, especially in CHCl3 solution, as reported by Nordlander.10
he phenanthroindolizidine alkaloid tylophorine (1, Figure 1) was first isolated by Ratnagiriswaran and Venkatacha-
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Figure 1. Enantiomers of tylophorine.
lam in 1935.1 It constitutes a major active principle of the plant Tylophora indica, belonging to the family of Apocynaceae, and has been shown to be responsible for the pronounced antiinflammatory2 and cytotoxic2b,f,3 effects first described in 1917.4 The elucidation of the molecular structure of 1 was done by Govindachari in 1960,5 while the first synthesis of a racemate was published in 1961.6 In 1974, Govindachari assigned the configuration of the levorotatory natural product with the reported specific rotation of −11.6 (c = 1.07, CHCl3) to be S based on degradation studies as well as by comparison of ECD spectra of 1 with those of antofine and isotylocrebrine, two other phenanthroindolizidines with known absolute configurations.7 However, this initial assignment was revised to R in 1983 by Buckley and Rapoport,8 who reported discrepancies with literature data. In particular, a synthetic sample of (S)-1 prepared by ex-chiral-pool synthesis from (S)-pyroglutamate turned out to be dextrorotatory {[α]23D = +15 (c 0.7, CHCl3)}. Surprisingly, stereoselective syntheses of 19 led to products with significantly higher specific rotation, e.g., +73 (c 0.7, CHCl3) for (S)-1,10 −76 (c 0.1, © 2013 American Chemical Society and American Society of Pharmacognosy
RESULTS AND DISCUSSION
Since we had both the racemate14 and synthetic (S)enantiomer13 of 1 in hand, we isolated the alkaloid from dried leaves (200 g) of T. indica obtained from a commercial supplier for comparison of its enantiomeric composition. The extraction was performed according to a slight modification of Shri’s protocol15 using EtOH containing citric acid (2%). After filtration and concentration in vacuo, the mixture was washed with EtOAc and the alkaloid-containing fraction was obtained by extraction with CHCl3 after adjusting the pH to 9−10 (see Experimental Section).16 The temperature during isolation and concentration was kept below 40 °C to minimize decomposition and the risk of racemization. However, increasing the temperature to 60 °C did not change the observed enantiomeric ratio. Analytical HPLC of the alkaloid fraction revealed several (>20) phenanthrene-based alkaloids as judged by UV spectroscopy (characteristic absorption bands, Figure 3) and MSn experiments (characteristic retro-Diels−Alder fragmentation of ring D; for MSn signals see Supporting Information, Table S3), with 1 being the major constituent (Figure 2). Purification of an analytical sample of 1 was performed by RP-HPLC (Figure 2). The purity was checked by RP-HPLC/MS (Figure 4). Received: December 4, 2012 Published: January 31, 2013 275
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Figure 2. RP-HPLC of the crude extract from T. indica, λ = 286 ± 4 nm. tR (1): 9.3 min. Figure 5. HPLC-ESI mass spectrum of the peak at 2.8 min.
Figure 6. Chiral HPLC: (a) (S)-1 (synthetic); (b) natural 1; (c) rac-1 (synthetic); (d) co-injection of natural and racemic 1. Figure 3. Purification of the crude alkaloid fraction. 3D view and contour plot (from top to bottom).
data. While the ratio determined by us is even lower, we could confirm their hypothesis. If 1 is biosynthesized in enantioselective fashion, its subsequent racemization could be effected by oxidation to the iminium ion or enamine followed by unselective reduction, and it is possible that the observed scalemic mixture is the result of a process occurring within the living plant or in the dead plant material. On the other hand, a racemization during isolation seems unlikely since changing the conditions for extraction did not affect the enantiomeric ratio. A solution of 1 in CHCl3 is highly light sensitive and produces the corresponding 12,13-dihydro-11H-dibenzo[f,h]pyrrolo[1,2b]isoquinolinium salt (2), which can be identified by its yellow color (Scheme 1).18 No reduction of this achiral oxidation product to rac-1 could be observed during handling of the samples, and a racemization via the formation of 2 would require an external reductant such
Figure 4. Analytical RP-HPLC of purified tylophorine.
The enantiomeric composition of the purified natural alkaloid 1 was determined by chiral HPLC/MS and compared to the racemate and pure (S)-1 (Figure 6). The ratio of (R)- to (S)-1 amounted to 56:44, corresponding to an enantiomeric excess of only 12%. The same ratio was found in several instances during the isolation of 1 on a larger scale by semipreparative HPLC with or without an additional crystallization. The reported specific rotation of natural 15a corresponds to 15% ee, whereas we determined an enantiomeric excess of 10% based on polarimetry (see Experimental Section). Nagarajan mentioned a ∼3:2 (R:S) enantiomeric ratio for the natural alkaloid in a paper on Govindachari’s research,17 albeit without any experimental
Scheme 1. Degradation of Tylophorine to the Isoquinolinium Salt 2
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as sodium borohydride.19 Moreover, 2 could be detected only in minute amounts in the crude extract by HPLC-MS. Besides the enatioselective biosynthesis followed by racemization, the direct enzymatic formation of the observed scalemic mixture could be a possibility. It is tempting to speculate that the methoxy-substitution pattern of the phenanthrene portion may be linked to the absolute configuration at C-13a. As already pointed out by Chemler, (−)-tylocrebrine (3) and (+)-isotylocrebrine (4) possess opposite configuration, while their phenanthrene units have identical substitution if one allows the indolizidine nitrogen to shift.3e The same appears to be true for most other phenathroindolizidines such as antofine (5) or tylophoridicine E (6).16b If an oxygen substituent is present at C-2 and C-7 is free, R-configuration is observed; if C-7 is oxygenated and C-2 is unsubstituted, the S-configurated alkaloid is produced instead. In tylophorine, both C-2 and C-7 carry a methoxy group and a low optical purity is observed (Figure 7). Remarkably, highly similar obervations have been made for the antiproliferative activity of the phenanthroindolizidines.3e
acetonitrile) were purchased from Fisher Scientific (Germany). Dried leaves of Tylophora indica collected in India were purchased from Oskar Tropitzsch e. K. (Marktredwitz, Germany). NMR spectra were recorded with a Bruker ARX-400 spectrometer. The spectra were measured in CDCl3, and the chemical shifts were referenced to the residual solvent signal (CDCl3: δH = 7.26 ppm). All procedures for isolation were carried out under exclusion of light using amber glassware. Analytical and preparative HPLC on a small scale were carried out on a Knauer Smartline HPLC system (Berlin, Germany) in high pressure gradient mode equipped with two pumps, a Knauer Smartline K-1050 (water) and a Knauer K-1001 (acetonitrile or methanol), each pump equipped with a 10 mL pump head. The system was connected to a K-2800 diode array detector. The size of the injection loop was 20 μL, the total flow rate 1.5 mL·min−1. An ACE3-C18 column (Advanced Chromatography Technologies, 125 × 4.6 mm, 3 μm) was used. The elution was performed with a gradient of H2O (30 mM NH4OAc) and MeCN. Preparative RP-HPLC was carried out on a Knauer Smartline HPLC- system (high pressure gradient), equipped with two K-1800 pumps (pump head size: each 100 mL), a diode array detector (S-2600), and a 2.5 mL injection loop. The elution was performed using an ACE5-C18 column (Advanced Chromatography Technologies, 125 × 21.2 mm, 5 μm) with a gradient of H2O + 0.1% v/v HCO2H and MeOH at 21 °C and a total flow rate of 35 mL·min−1 (for gradient profile see Supporting Information). Chiral HPLC-MS was carried out on an Agilent 1200 system (binary pump, column oven, autosampler, DAD), using a Chiralpak AD-H column (250 × 4.6 mm, 5 μm) and a mixture of MeOH/EtOH (v/v = 80:20) as eluent, applying a total flow rate of 0.90 mL·min−1 and a temperature of 40 °C. RP-HPLC/MS was perfomed using an Ascentis Express C18 column (50 × 2.1, core−shell, 2.7 μm) with a gradient of H2O (15 mM NH4HCO3) and MeCN at 50 °C and a total flow rate of 0.50−1.0 mL·min−1 (for gradient profile, see Supporting Information). RP-HPLC/MS3 experiments were perfomed using two Ascentis Express C18 columns connected in series (50 + 30 × 2.1 mm, core− shell, 2.7 μm) with a gradient of H2O (15 mM NH4HCO3) and MeCN at a temperature of 50 °C and a total flow rate of 0.60 mL·min−1 (for gradient profile, see Supporting Information). The mass spectra were recorded using positive electrospray ionization. Optical rotation was measured on a Perkin-Elmer polarimeter at wavelengths of 546 and 578 nm (Hg lamp). The data were extrapolated to a wavelength of λ = 589 nm using the Drude equation.23 Extraction of Tylophorine from Tylophora indica. For the extraction of the alkaloids from the plant material, we used Shri’s protocol with slight modifications.15 Dried and crushed leaves of T. indica (200 g) were extracted under N2, in a 2.5 L bottle, with a 95:5 v/v mixture of EtOH (containing 1% ethyl methyl ketone) and H2O containing citric acid (2%). The extraction was carried out in three portions (each 1.5 L) by ultrasonication for 15 min at 33 °C, followed by shaking at room temperature for 60 min at 200 rpm. After filtration, the dark but clear solution was concentrated under reduced pressure, while the temperature was kept below 40 °C, yielding a brown syrup. An equal volume of H2O was added, and the extract was washed three times with twice the volume of EtOAc. After the aqueous layer was basified to pH = 9−10 (NaOH, 3 mol·L−1), the alkaloids were extracted three times with an equal volume of CHCl3. The combined organic extracts were succesively washed with H2O, then dried over anhydrous Na2SO4, before the solvent was removed under reduced pressure (T = 40 °C), furnishing a brown-colored crude extract (407 mg, ∼0.2%), mainly consisting of phenanthrene-based alkaloids. Analytical Purification. Half of the crude extract (203 mg) obtained by the procedure above was suspended in a mixture of EtOH/CHCl3/DMF (8:5:2, v/v/v, total volume 7.5 mL). The resulting slurry (10 μL) was diluted with EtOH (10 μL) and purified by analytical HPLC (no prior filtration, Figure 2). Chiral HPLC/MS. The solvent of the tylophorine-containing fraction purified by analytical HPLC was evaporated by using a moderate flow of N2. The residue was dissolved in EtOH (300 μL), filtered, and analyzed by chiral HPLC-MS (injection volume: 100 μL).
Figure 7. Structural similarities of phenathroindolizidine alkaloids.
In any case, tylophorine belongs to the group of natural products found as scalemic mixtures,20 while phenanthroindolizidines with an unsymmetrical oxygenation pattern in the phenanthrene moiety such as antofine (5)11,21 and ficuseptine C11,22 do not show similar discrepancies between the optical rotation of synthetic and natural material, while they should suffer the same abiotic redox processes affecting their configurational integrity.
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EXPERIMENTAL SECTION
General Experimental Procedures. Chemicals were purchased from Fisher Scientific or Sigma Aldrich and used without further purification. LC/MS solvents (Optima LC-MS, water, methanol, and 277
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Semipreparative Purification. Half of the crude extract (203 mg) was suspended in a mixture of EtOH/CHCl3/DMF (8:5:2 v/v/v, total volume 7.5 mL), yielding a brown slurry, before filtering the mixture by using a syringe tip filter (PTFE, 0.2 μM, 13 mm diameter). The filtrate contained only traces of tylophorine and was discarded. After the residue in the syringe filter was redissolved in CHCl3 (7 mL), the solvent was evaporated under reduced pressure, yielding 25.9 mg of a yellow powder. Recrystallization from MeOH afforded tylophorine (13.6 mg, 0.0136% w/w) as a pale yellow powder, which still contained several impurities. The recrystallized product was dissolved in CHCl3 (2 mL) and purified by preparative HPLC, yielding the title compound as a nearly colorless solid (1.41 mg, 0.0014% w/w). During the measurement of the optical rotation, partial degradation of 1 occurred due to its sensitivity to light in CHCl3 solution. After polarimetry, the sample contained 20% of the yellow achiral isoquinolinium salt (2), as judged by peak integration at λ = 286 ± 8 nm taking into account the published extinction coefficients for both compounds.18,24 Thus, the observed value of [α]31D = −6.5 needs to be corrected to [α]31D = −8.1, corresponding to an enantiomeric excess of 10%. Tylophorine (1): nearly colorless solid; [α]31D = −8.1 (c 0.08, CDCl3); 1H NMR (400 MHz, CDCl3) δ (ppm) 7.84 (s, 1H, Aryl-H), 7.83 (s, 1H, Aryl-H), 7.30 (s, 1H, Aryl-H), 7.11 (s, 1H, Aryl-H), 4.80 (d, 2J = 14.7 Hz, 1H, Ha-9), 4.12 (s, 3H, OCH3), 4.12 (s, 3H, OCH3), 4.06 (s, 3H, OCH3), 4.05 (s, 3H, OCH3), 3.67−3.52 (m, 1H, Hb-9), 3.50−3.38 (m, 1H, CH2), 3.44 (dd, J = 16.1 Hz, 3.6 Hz, CH2), 3.17 (mc, 1H, CH2), 2.86−2.72 (m, 2H, CH2), 2.41−2.30 (m, 2H, CH2), 2.23−1.86 (m, 4H, 2 × CH2); ESI-MS (pos.) m/z 394.2 [M + H]+, calcd 394.2.
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ASSOCIATED CONTENT
S Supporting Information *
HPLC data, mass spectra, and HPLC-MSn data of the crude extract, HPLC-MS data of the purified extract, and 1H NMR spectrum of 1. These data are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: (49) 6131-3924443. Fax: (49) 6131-3922338. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. J. C. Liermann (Mainz) for NMR spectroscopy and Prof. Paul Margaretha (Hamburg) for helpful discussions. REFERENCES
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