Autoxidation Products of Betulonaldehyde

Article abstract of DOI:10.1021/acs.jnatprod.6b00735

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 a

Full text of DOI:10.1021/acs.jnatprod.6b00735

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.
etulonaldehyde (1, also referred to as betulonal or
B
betulonic aldehyde) is a naturally occurring pentacyclic
triterpenoid, found in minor amounts along with the related
1
−7
constituents betulin and betulinic acid.
The betulin
triterpenoids are widely distributed throughout the plant
kingdom but are usually associated with the genus Betula
8
,9
(
birch), where they are present in large abundance. The
betulin triterpenoids have been shown to exhibit important
biological activities, most notably antiviral and anticancer, but
10−16
also antibacterial, antiparasitic, and anti-inflammatory.
Betulinic acid has recently been reported as the starting
material for the synthesis of the potent second-generation HIV
17
maturation inhibitor BMS-955176. Betulonaldehyde has also
been reported to be a useful starting material for the
semisynthesis of novel C-2 and C-28 derivatives.
Figure 1. Overlaid HPLC chromatograms (220 nm) of betulonalde-
hyde (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).
18
HRMS fragments for all three degradants showed a strong m/z
+
4
09.34, indicating C H O , which is consistent with the loss
29
45
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, Support-
ing Information), and 1D and 2D NMR data were acquired for
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 C H O , while both 3 and 4
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
3
0
46
3
had the formula C H O (Figures S1−S3, Supporting
2
9
46
3
19
betulonic acid. For degradants 3 and 4, the clearest difference
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
in the NMR data versus 1 was the loss of the aldehyde signal in
1
13
both the H (δ 9.70, Figure 2) and C NMR spectra (δC
H
Received: August 8, 2016
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00735
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Table 1. NMR Spectroscopic Data (600 MHz, CDCl ) for Compounds 2−4
3
2
3
4
position
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
1.41, m
1
1
2
2
3
4
5
6
6
7
7
8
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
3
a
39.8, CH2
1.38, m
39.8, CH2
1.37, m
39.9, CH2
b
a
1.89, ddd (12.5,7.5,4.5)
2.40, ddd (15.6,7.5,4.3)
2.48, ddd (15.6,9.6,7.8)
1.88, m
1.90, m
2.45, m
34.3, CH2
34.3, CH2
2.41, ddd (15.7,7.7,4.4)
2.48, ddd (15.7,9.6,7.7)
34.2, CH2
b
218.4, C
47.5, C
218.8, C
47.5, C
218.5, C
47.0, C
55.2, CH
19.8, CH2
1.32, m
1.44, m
1.47, m
1.42, m
55.2, CH
19.9, CH2
1.31, m
1.45, m
1.49, m
1.45, m
55.1, CH
19.8, CH2
1.35, m
1.46, m
a
b
a
33.8, CH2
33.9, CH2
33.4, CH2
1.39, m
1.47, m
b
40.8, C
40.9, C
40.7, C
50.1, CH
37.1, C
1.38, m
50.1, CH
37.1, C
1.36, m
50.7, CH
37.1, C
1.41, m
0
1a
1b
2a
2b
3
21.6, CH2
1.30, m
21.7, CH2
1.25, m
1.39, m
0.97, m
1.63, m
1.90, m
22.1, CH2
1.25, m
1.49, m
0.95, m
1.71, m
1.27, m
1.46, m
25.7, CH2
1.05, m
25.4, CH2
27.0, CH2
1.72, m
38.7, CH
42.7, C
2.22, td (12.2,2.6)
37.0, CH
42.2, C
44.4, CH
40.9, C
4
5a
5b
6a
6b
7
29.9, CH2
1.21, br dt (13.3,2.7)
1.54, m
27.1, CH2
1.07, m
28.3, CH2
1.27, m
1.40, m
1.73, m
1.83, td (13.3,4.3)
1.34, m
32.3, CH2
1.42, m
27.5, CH2
27.5, CH2
2.28, dt (12.9,2.8)
2.23, ddd (14.1,4.1,2.5)
2.01, td (13.1,4.9)
56.6, C
49.4, CH
47.1, CH
150.5, C
30.8, CH2
91.6, C
49.3, CH
48.1, CH
150.1, C
29.7, CH2
93.4, C
47.0, CH
53.8, CH
150.4, C
29.0, CH2
8
1.63, t (11.4)
1.67, t (11.7)
1.59, dd (11.2,3.6)
2.35, td (8.7,4.1)
9
3.01, td (10.7,4.8)
2.62, td (10.9,5.9)
0
1a
1b
2a
2b
3
1.43, m
2.00, m
1.48, m
1.98, m
1.07, s
1.01, s
0.92, s
0.97, s
0.99, s
1.39, m
1.65, m
1.73, m
1.65, m
1.87, m
1.06, s
1.01, s
0.91, s
0.96, s
0.93, s
2.03, m
37.2, CH2
32.4, CH2
1.25, m
33.6, CH2
2.18, ddd (13.3,8.7,1.2)
1.06, s
26.9, CH3
21.2, CH3
16.2, CH3
16.0, CH3
14.8, CH3
182.5, C
26.8, CH3
21.2, CH3
16.2, CH3
16.1, CH3
14.1, CH3
27.0, CH3
21.1, CH3
16.6, CH3
15.5, CH3
14.7, CH3
4
1.01, s
5
0.91, s
6
1.05, s
7
0.94, s
8
9E
9Z
0
110.0, CH2
4.61, br s
4.74, br s
1.69, s
109.9, CH2
19.3, CH3
4.59, br s
4.70, d (1.6)
1.66, s
109.2, CH2
21.1, CH3
4.64, m
4.76, m
1.71, s
19.6, CH3
2
06.8, Figure 3). A new broad downfield 1H signal was
observed for 3 and 4 (δ 7.51 and 7.74, respectively, Figure 2),
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 H-
H
13
and a new C signal was observed for both as well (δ 91.6 and
C
93.4, respectively, Figure 3).
1
9; see Figure 4). Compound 3 was therefore assigned as the
HMBC correlations established the new 13C NMR signal as
C-17 β-OOH isomer, and 4 was assigned as the C-17 α-OOH
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
isomer.
21
Bar et al. have previously reported the C-17 hydroperoxide
epimers of the naturally occurring analogue betulinaldehyde (β-
OH 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 hydro-
peroxide epimers. The authors had also reported betulinalde-
hyde 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.
shift of δ 60.0. These data therefore agree with the HRMS
C
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
20
should not be as far downfield.
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
B
DOI: 10.1021/acs.jnatprod.6b00735
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
1
Figure 2. Comparison of downfield region of the H NMR spectra of
, 3, and 4 in CDCl showing the loss of the aldehyde signal in 3 and 4
1
3
1
(
dashed box) and the new broad signal in 3 and 4 (arrows).
Figure 4. Overlaid H 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
28,29
peroxides,
both synthetic and natural, so stable C-17
triterpenoid hydroperoxides could potentially find utility as a
new class of therapeutic agents.
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-d and CDCl and were referenced to
6 3
residual protonated solvent resonances (δ 2.05 and 7.26, respectively,
H
1
13
for H, and δ 29.9 and 77.2, respectively, for C) and processed with
C
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.
Figure 3. Comparison of the downfield region of the 13C NMR
spectra of 1, 3, and 4 in CDCl , showing the loss of the aldehyde signal
3
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. 2−26
2
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
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
2
7
betulin, 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
CO /2-propanol at 75 mL/min, 40 °C, and 100 bar outlet pressure.
2
Compound 2 was collected from 2.63 to 3.12 min (176.4 mg), 3 from
C
DOI: 10.1021/acs.jnatprod.6b00735
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
3
.77 to 4.01 min (119.4 mg), and 4 from 4.12 to 4.38 min (62.0 mg).
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1127−1155.
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).
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1
13
Betulonic acid (2): amorphous, white solid; H and C NMR, see
+
Table 1; HRESIMS m/z [M + H] 455.351 94 (calcd for C H O +
H, 455.351 97, Δ 0.07 ppm).
30
46
3
2
8-Norlup-20(29)-en-3-one-17β-hydroperoxide (3): amorphous,
25
1
13
colorless solid; [α] +60.0 (c 0.19, CHCl ); H and C NMR, see
Table 1; HRESIMS m/z [M + H] 443.352 08 (calcd for C H O +
H, 443.351 97, Δ 0.2 ppm).
D
3
+
29
46
3
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8-Norlup-20(29)-en-3-one-17α-hydroperoxide (4): amorphous,
(
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25
1
13
colorless solid; [α] +39.4 (c 0.54, CHCl ); H and C NMR, see
Table 1; HRESIMS m/z [M + H] 443.351 17 (calcd for C H O +
H, 443.351 97, Δ 1.8 ppm).
D
3
(21) Bar, F. M. A.; Zaghloul, A. M.; Bachawal, S. V.; Sylvester, P. W.;
+
29
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ASSOCIATED CONTENT
■
*
S
Supporting Information
Chem. Prod. Res. Dev. 1978, 17, 260−264.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00735.
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(
AUTHOR INFORMATION
■
*
Corresponding Author
Tel: +1 732 227 5821. E-mail: david.ayers@bms.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors wish to thank Drs. C. Pathirana and S. A. Miller for
helpful discussions.
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DOI: 10.1021/acs.jnatprod.6b00735
J. Nat. Prod. XXXX, XXX, XXX−XXX