Note pubs.acs.org/jnp
Autoxidation Products of Betulonaldehyde Sloan Ayers,* Tamas Benkovics, Jonathan Marshall, Yichen Tan, Neil A. Strotman, and Susanne Kiau Bristol-Myers Squibb Co., Chemical & Synthetic Development, 1 Squibb Drive, New Brunswick, New Jersey 08903, United States S Supporting Information *
ABSTRACT: Three major degradation products resulted from the exposure of betulonaldehyde (1) to air in solution at room temperature. From HRMS and NMR data, the products, which were isolated by preparative supercritical fluid chromatography (SFC), were identified as betulonic acid (2) and C-17 hydroperoxide epimers 3 (β-OOH) and 4 (α-OOH). For 3 and 4, the H-18 multiplet pattern of the isolated products established the configuration at C-17.
B
etulonaldehyde (1, also referred to as betulonal or betulonic aldehyde) is a naturally occurring pentacyclic triterpenoid, found in minor amounts along with the related constituents betulin and betulinic acid.1−7 The betulin triterpenoids are widely distributed throughout the plant kingdom but are usually associated with the genus Betula (birch), where they are present in large abundance.8,9 The betulin triterpenoids have been shown to exhibit important biological activities, most notably antiviral and anticancer, but also antibacterial, antiparasitic, and anti-inflammatory.10−16 Betulinic acid has recently been reported as the starting material for the synthesis of the potent second-generation HIV maturation inhibitor BMS-955176.17 Betulonaldehyde has also been reported to be a useful starting material for the semisynthesis of novel C-2 and C-28 derivatives.18
Figure 1. Overlaid HPLC chromatograms (220 nm) of betulonaldehyde (1) at time = 0 (50 μg injection, blue trace); betulonic acid (2, 10 μg injection, green trace); and betulonaldehyde (1) after 10 days of gentle stirring at room temperature in acetone (50 μg injection, red trace).
HRMS fragments for all three degradants showed a strong m/z 409.34, indicating C29H45O+, which is consistent with the loss of the group attached to C-17. Therefore, compared to the elemental composition of the molecular ions, 3 and 4 were consistent with C-17 hydroperoxides. To confirm the proposed hydroperoxide structures based on HRMS data, compounds 2−4 were isolated by preparative supercritical fluid chromatography (SFC) (Figure S4, Supporting Information), and 1D and 2D NMR data were acquired for 3 and 4 (Figures S5−S14, Supporting Information). When vigorously stirred, after 14 days 1 had been completely degraded according to HPLC, indicating that stirring rate appears to be important for the rate of degradation. The NMR data for 2 (Table 1) were virtually identical to literature data for betulonic acid.19 For degradants 3 and 4, the clearest difference in the NMR data versus 1 was the loss of the aldehyde signal in both the 1H (δH 9.70, Figure 2) and 13C NMR spectra (δC
In a stability study of betulonaldehyde (1), the compound was dissolved in acetone to 70 mg/mL and gently stirred at room temperature for 10 days under air. Three major products were observed by HPLC (Figure 1). By LC-HRMS, product 2 had an elemental composition of C30H46O3, while both 3 and 4 had the formula C29H46O3 (Figures S1−S3, Supporting Information). Compound 2 therefore gains one oxygen compared to 1, while 3 and 4 lose one carbon and gain one oxygen. A fair assumption for compound 2 would be oxidation of the aldehyde to the carboxylic acid (betulonic acid). Indeed, comparison with a betulonic acid standard showed a retention time matching 2 in the HPLC analysis (Figure 1). In-source © 2016 American Chemical Society and American Society of Pharmacognosy
Received: August 8, 2016 Published: September 29, 2016 2758
DOI: 10.1021/acs.jnatprod.6b00735 J. Nat. Prod. 2016, 79, 2758−2761
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Table 1. NMR Spectroscopic Data (600 MHz, CDCl3) for Compounds 2−4 2 position
δC, type
1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16a 16b 17 18 19 20 21a 21b 22a 22b 23 24 25 26 27 28 29E 29Z 30
39.8, CH2 34.3, CH2 218.4, 47.5, 55.2, 19.8,
C C CH CH2
33.8, CH2 40.8, 50.1, 37.1, 21.6,
C CH C CH2
25.7, CH2 38.7, CH 42.7, C 29.9, CH2 32.3, CH2 56.6, 49.4, 47.1, 150.5, 30.8,
C CH CH C CH2
37.2, CH2 26.9, 21.2, 16.2, 16.0, 14.8, 182.5, 110.0,
CH3 CH3 CH3 CH3 CH3 C CH2
19.6, CH3
1.38, 1.89, 2.40, 2.48,
1.32, 1.44, 1.47, 1.42,
3 δH, (J in Hz)
δC, type
m ddd (12.5,7.5,4.5) ddd (15.6,7.5,4.3) ddd (15.6,9.6,7.8)
39.8, CH2
m m m m
1.38, m 1.30, 1.46, 1.05, 1.72, 2.22,
m m m m td (12.2,2.6)
1.21, 1.54, 1.42, 2.28,
br dt (13.3,2.7) m m dt (12.9,2.8)
1.63, t (11.4) 3.01, td (10.7,4.8) 1.43, 2.00, 1.48, 1.98, 1.07, 1.01, 0.92, 0.97, 0.99,
m m m m s s s s s
4.61, br s 4.74, br s 1.69, s
34.3, CH2 218.8, 47.5, 55.2, 19.9,
C C CH CH2
33.9, CH2 40.9, 50.1, 37.1, 21.7,
C CH C CH2
25.4, CH2 37.0, CH 42.2, C 27.1, CH2 27.5, CH2 91.6, 49.3, 48.1, 150.1, 29.7,
C CH CH C CH2
32.4, CH2 26.8, 21.2, 16.2, 16.1, 14.1,
CH3 CH3 CH3 CH3 CH3
109.9, CH2 19.3, CH3
206.8, Figure 3). A new broad downfield 1H signal was observed for 3 and 4 (δH 7.51 and 7.74, respectively, Figure 2), and a new 13C signal was observed for both as well (δC 91.6 and 93.4, respectively, Figure 3). HMBC correlations established the new 13C NMR signal as C-17, indicating direct attachment to oxygen. Therefore, this signal is technically not “new”, but shifted significantly downfield as compared to C-17 in 1, which has a chemical shift of δC 60.0. These data therefore agree with the HRMS data, confirming hydroperoxide attachment to C-17. The C-17 chemical shifts are consistent with a carbon attached to a hydroperoxy group as opposed to a hydroxy group, which should not be as far downfield.20 To determine the epimer assignments, the coupling for H-18 was examined; H-18 for 3 was a partially obscured doublet of doublets (appearing as a triplet, J = 11.7 Hz), indicating axial− axial relationships between H-18 and H-13 and between H-18
1.37, 1.88, 2.41, 2.48,
1.31, 1.45, 1.49, 1.45,
4 δH, (J in Hz)
δC, type
m m ddd (15.7,7.7,4.4) ddd (15.7,9.6,7.7)
39.9, CH2
m m m m
1.36, m 1.25, 1.39, 0.97, 1.63, 1.90,
m m m m m
1.07, 1.83, 1.34, 2.23,
m td (13.3,4.3) m ddd (14.1,4.1,2.5)
1.67, t (11.7) 2.62, td (10.9,5.9) 1.39, 2.03, 1.25, 2.18, 1.06, 1.01, 0.91, 1.05, 0.94,
m m m ddd (13.3,8.7,1.2) s s s s s
4.59, br s 4.70, d (1.6) 1.66, s
34.2, CH2 218.5, 47.0, 55.1, 19.8,
C C CH CH2
33.4, CH2 40.7, 50.7, 37.1, 22.1,
C CH C CH2
27.0, CH2 44.4, CH 40.9, C 28.3, CH2 27.5, CH2 93.4, 47.0, 53.8, 150.4, 29.0,
C CH CH C CH2
33.6, CH2 27.0, 21.1, 16.6, 15.5, 14.7,
CH3 CH3 CH3 CH3 CH3
109.2, CH2 21.1, CH3
δH, (J in Hz) 1.41, m 1.90, m 2.45, m
1.35, m 1.46, m 1.39, m 1.47, m 1.41, m 1.25, 1.49, 0.95, 1.71, 1.27,
m m m m m
1.27, 1.40, 1.73, 2.01,
m m m td (13.1,4.9)
1.59, dd (11.2,3.6) 2.35, td (8.7,4.1) 1.65, 1.73, 1.65, 1.87, 1.06, 1.01, 0.91, 0.96, 0.93,
m m m m s s s s s
4.64, m 4.76, m 1.71, s
and H-19. The H-18 signal for 4 was a doublet of doublets (J = 11.2 and 3.6 Hz), indicating one axial−axial relationship (H-18 and H-13) and one axial−equatorial relationship (H-18 and H19; see Figure 4). Compound 3 was therefore assigned as the C-17 β-OOH isomer, and 4 was assigned as the C-17 α-OOH isomer. Bar et al.21 have previously reported the C-17 hydroperoxide epimers of the naturally occurring analogue betulinaldehyde (βOH at C-3 instead of ketone as in 1). The C-17 chemical shifts for 3 and 4 reported in our work are identical to the C-17 chemical shifts reported for the betulinaldehyde C-17 hydroperoxide epimers. The authors had also reported betulinaldehyde as one of the known compounds isolated; it is therefore possible that the C-17 hydroperoxide epimers reported in their work were autoxidation artifacts of betulinaldehyde and not naturally occurring compounds. 2759
DOI: 10.1021/acs.jnatprod.6b00735 J. Nat. Prod. 2016, 79, 2758−2761
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Figure 2. Comparison of downfield region of the 1H NMR spectra of 1, 3, and 4 in CDCl3 showing the loss of the aldehyde signal in 3 and 4 (dashed box) and the new broad signal in 3 and 4 (arrows).
Figure 4. Overlaid 1H NMR spectra of 3 (red) and 4 (blue) with H-18 circled for each.
would be similarly vulnerable. It would therefore be prudent to exercise caution when working with isolated triterpenoids containing a C-28 aldehyde in solution (as well as other aldehydes) due to the possible instability to atmospheric oxygen. There are many examples of biologically active peroxides,28,29 both synthetic and natural, so stable C-17 triterpenoid hydroperoxides could potentially find utility as a new class of therapeutic agents.
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EXPERIMENTAL SECTION
General Experimental Procedures. Solvents for isolation were purchased from Sigma-Aldrich (St. Louis, MO, USA). NMR solvents were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Optical rotations were measured on a PerkinElmer 241 polarimeter. NMR spectra were acquired on a Bruker Avance III 600 (Billerica, MA, USA) in acetone-d6 and CDCl3 and were referenced to residual protonated solvent resonances (δH 2.05 and 7.26, respectively, for 1H, and δC 29.9 and 77.2, respectively, for 13C) and processed with TopSpin v3.5 software. LC-HRMS data were obtained on a Waters Acquity/Thermo Q-Exactive Orbitrap LC-HRMS with an ESI source and a YMC Pack-Pro C18 column (150 × 4.6 mm, 3 μm), using Xcalibur v2.2 software. Preparative SFC was performed using a Berger Multigram II system with a PrincetonSFC CN column (250 × 21.2 mm, 5 μm), using SFC ProNTo software v. 1.5.305.15. Analytical HPLC was performed on a Shimadzu Process-VP system using the same column as for LC-HRMS. Betulonaldehyde (1) and betulonic acid (2) were obtained from an internal supply at BMS. HPLC and LC-MS. For analytical LC work, compound 1 was dissolved in acetone to 70 mg/mL and gently stirred for 10 days at room temperature. The solution was diluted 70:1 in acetonitrile for analytical HPLC and LC-HRMS. The mobile phase was (A) 100% water with 0.05% TFA and (B) 100% acetonitrile with 0.05% TFA. The gradient was 75% → 100% B from 0 to 5 min, then hold at 100% B to 13 min, with a 1.0 mL/min flow rate. Analytical injection volume was 50 μL, and LC-HRMS injection volume was 25 μL. Degradation and Isolation. For isolation, compound 1 (1.00 g) was dissolved in 14.3 mL of acetone. This solution was vigorously stirred for 14 days at room temperature, at which time 1 was completely degraded according to HPLC. For preparative SFC, the initial solution was diluted 1:1 with 2-propanol, and 25 injections were made at 1 mL each. The mobile phase was isocratic 85:15 supercritical CO2/2-propanol at 75 mL/min, 40 °C, and 100 bar outlet pressure. Compound 2 was collected from 2.63 to 3.12 min (176.4 mg), 3 from
Figure 3. Comparison of the downfield region of the 13C NMR spectra of 1, 3, and 4 in CDCl3, showing the loss of the aldehyde signal in 3 and 4 (dashed box) and the appearance of the new C-17 signal (solid box).
The autoxidation of aldehydes has been well studied.22−26 The mechanism involves the classic initiation−propagation− termination series of steps; however, this case appears to be somewhat unusual in that one of the major pathways terminates with the hydroperoxides. Exposure to light was not found to play a part in catalyzing the degradation, as this experiment was repeated in the dark with practically identical results. The experiment was also repeated with betulonic acid (2), which did not degrade to 3 and 4. It is possible that betulonic acid may be a Baeyer−Villiger product formed from the reaction of a peroxy acid intermediate with 1; however, the C-17 formate ester, which should be the other theoretical Baeyer−Villiger product, was not detected. Reaction of oxygen with the isopropylene functionality (C-29/C-20/C30) has been reported for betulin,27 but was not observed in this work. While betulonaldehyde is clearly susceptible to autoxidation in solution, it is not clear if other C-28 triterpenoid aldehydes 2760
DOI: 10.1021/acs.jnatprod.6b00735 J. Nat. Prod. 2016, 79, 2758−2761
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3.77 to 4.01 min (119.4 mg), and 4 from 4.12 to 4.38 min (62.0 mg). A second pass was required for compound 3 using the same SFC conditions. The first-pass isolate was brought up in 16 mL of 1:1 acetone/2-propanol, and 15 injections were made at 1 mL each. Compound 3 was collected from 3.71 to 3.97 min (57.7 mg). A second pass was also required for compound 4, again using the same SFC conditions. The first-pass isolate was brought up in 12 mL of 1:1 acetone/2-propanol, and 11 injections were made at 1 mL each. Compound 4 was collected from 3.97 to 4.33 min (32.9 mg). Betulonic acid (2): amorphous, white solid; 1H and 13C NMR, see Table 1; HRESIMS m/z [M + H]+ 455.351 94 (calcd for C30H46O3 + H, 455.351 97, Δ 0.07 ppm). 28-Norlup-20(29)-en-3-one-17β-hydroperoxide (3): amorphous, colorless solid; [α]25D +60.0 (c 0.19, CHCl3); 1H and 13C NMR, see Table 1; HRESIMS m/z [M + H]+ 443.352 08 (calcd for C29H46O3 + H, 443.351 97, Δ 0.2 ppm). 28-Norlup-20(29)-en-3-one-17α-hydroperoxide (4): amorphous, colorless solid; [α]25D +39.4 (c 0.54, CHCl3); 1H and 13C NMR, see Table 1; HRESIMS m/z [M + H]+ 443.351 17 (calcd for C29H46O3 + H, 443.351 97, Δ 1.8 ppm).
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(15) Periasamy, G.; Teketelew, G.; Gebrelibanos, M.; Sintayehu, B.; Gebrehiwot, M.; Karim, A.; Geremedhin, G. Arch. Appl. Sci. Res. 2014, 6, 47−58. (16) Zhang, D.-M.; Xu, H.-G.; Wang, L.; Li, Y.-J.; Sun, P.-H.; Wu, X.M.; Wang, G.-J.; Chen, W.-M.; Ye, W. C. Med. Res. Rev. 2015, 35, 1127−1155. (17) Regueiro-Ren, A.; Liu, Z.; Chen, Y.; Sin, N.; Sit, S.-Y.; Swidorski, J. J.; Chen, J.; Venables, B. L.; Zhu, J.; Nowicka-Sans, B.; Protack, T.; Lin, Z.; Terry, B.; Samanta, H.; Zhang, S.; Li, Z.; Beno, B. R.; Huang, X. S.; Rahematpura, S.; Parker, D. D.; Haskell, R.; Jenkins, S.; Santone, K. S.; Cockett, M. I.; Krystal, M.; Meanwell, N. A.; Hanumegowda, U.; Dicker, I. B. ACS Med. Chem. Lett. 2016, 7, 568−572. (18) Kurbatov, E. S.; Kostrub, V. V.; Kurbatov, S. V. Russ. J. Bioorg. Chem. 2012, 38, 740−742. (19) Ito, J.; Chang, F.-R.; Wang, H.-K.; Park, Y. K.; Ikegaki, M.; Kilgore, N.; Lee, K.-H. J. Nat. Prod. 2001, 64, 1278−1281. (20) Lee, C.-K. J. Nat. Prod. 1998, 61, 375−376. (21) Bar, F. M. A.; Zaghloul, A. M.; Bachawal, S. V.; Sylvester, P. W.; Ahmad, K. F.; El Sayed, K. A. J. Nat. Prod. 2008, 71, 1787−1790. (22) McNesby, J. R.; Heller, C. A. Chem. Rev. 1954, 54, 325−346. (23) Zaikov, G. E.; Howard, J. A.; Ingold, K. U. Can. J. Chem. 1969, 47, 3017−3029. (24) Hendriks, C. F.; van Beek, H. C. A.; Heertjes, P. M. Ind. Eng. Chem. Prod. Res. Dev. 1978, 17, 260−264. (25) Larkin, D. R. J. Org. Chem. 1990, 55, 1563−1568. (26) Marteau, C.; Ruyffelaere, F.; Aubry, J. M.; Penverne, C.; Favier, D.; Nardello-Rataj, V. Tetrahedron 2013, 69, 2268−2275. (27) Galeron, M.-A.; Volkman, J. K.; Rontani, J.-F. Org. Geochem. 2016, 91, 31−42. (28) Dembitsky, V. M. Eur. J. Med. Chem. 2008, 43, 223−251. (29) Dembitsky, V. M. J. Mol. Genet. Med. 2015, 9, 163.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00735. 1D and 2D NMR spectra for 3 and 4; HRMS spectra for compounds 2−4 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1 732 227 5821. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors wish to thank Drs. C. Pathirana and S. A. Miller for helpful discussions.
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REFERENCES
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