Beckmann rearrangement within the ring C of oleanolic acid lactone: Synthesis, structural study and reaction mechanism analysis

Article abstract of DOI:10.1016/j.molstruc.2017.01.057

Synthesis, spectral and X-ray analysis of three compounds, i.e. 3β-acetoxy-12-hydroxyimino-18β-oleanan-28,13β-olide (substrate) and 3β-acetoxy-12-nitrile-12,13-seco-15(14?→?13)-abeoolean-14(27)-en-28,13β-olide and 3β-acetoxy-12-oxo-12a-aza-C-homoolean-13(18)-en-28-oic acid (Beckmann rearrangement reaction products) are described. Structural analysis revealed that the oxime group in the ring C in substrate molecule had an E-configuration. The nitrile product with retained lactone group was a result of major transformations within rings C and D of oleanane skeleton. In lactam product free carboxyl group and a double bond in ring D instead of lactone system were formed in Beckmann rearrangement reaction.

Full text of DOI:10.1016/j.molstruc.2017.01.057

Accepted Manuscript
Beckmann rearrangement within the ring C of oleanolic acid lactone: Synthesis,
structural study and reaction mechanism analysis
Anna Froelich, Barbara Bednarczyk-Cwynar, Lucjusz Zaprutko, Andrzej Gzella
PII:
S0022-2860(17)30087-X
10.1016/j.molstruc.2017.01.057
MOLSTR 23362
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 30 September 2016
Revised Date: 15 January 2017
Accepted Date: 18 January 2017
Please cite this article as: A. Froelich, B. Bednarczyk-Cwynar, L. Zaprutko, A. Gzella, Beckmann
rearrangement within the ring C of oleanolic acid lactone: Synthesis, structural study and reaction
mechanism analysis, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.01.057.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Beckmann rearrangement within the ring C of oleanolic acid lactone:
synthesis, structural study and reaction mechanism analysis
Anna Froelich1, Barbara Bednarczyk-Cwynar2, Lucjusz Zaprutko2 and Andrzej Gzella2,*
1Department of Pharmaceutical Technology, Faculty of Pharmacy, Poznan University of
Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland. Tel.: +48 (61) 854 66 55; fax: +48
(61) 854 66 66.
2Department of Organic Chemistry, Faculty of Pharmacy, Poznan University of Medical
Sciences, Grunwaldzka 6, 60-780 Poznan, Poland. Tel.: +48 061 854 66 74; fax: +48 (61) 854
66 80.
*Corresponding Author address: Department of Organic Chemistry, Poznan University of
Medical Sciences, Grunwaldzka 6, 60-780 Poznań, Poland, tel. +48 61 854 66 79, fax: +48
61 854 66 80, e-mail: akgzella@ump.edu.pl
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Highlights
• 3β-Acetoxy-12-hydroxyimino-18β-oleanan-28,13β-olide was synthesized and used as
a substrate in Beckmann rearrangement reaction within ring C of oleanane skeleton.
1
13
• IR, H- and C-NMR spectroscopy and X-ray analysis for Beckmann rearrangement
substrate (oxime) and products (nitrile and lactam) were performed.
• In nitrile unexpected tandem rearrangements within rings C and D leading to the
cleavage of the first one and the contraction of the latter were observed.
• In obtained lactam the hydrolysis of lactone ring and formation of double bond in D
ring were observed.
Keywords
1
Oleanane triterpenoids; H- and 13C-NMR, IR spectroscopy, X-ray analysis; Beckmann
rearrangement; triterpene lactam; triterpene nitrile; 1,2-rearrangement; triterpenoid ring D
contraction
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Abstract
Synthesis, spectral and X-ray analysis of three compounds, i.e. 3β-acetoxy-12-
hydroxyimino-18β-oleanan-28,13β-olide (substrate) and 3β-acetoxy-12-nitrile-12,13-seco-
15(14→13)-abeoolean-14(27)-en-28,13β-olide
and
3β-acetoxy-12-oxo-12a-aza-C-
homoolean-13(18)-en-28-oic acid (Beckmann rearrangement reaction products) are described.
Structural analysis revealed that the oxime group in the ring C in substrate molecule had an E-
configuration. The nitrile product with retained lactone group was a result of major
transformations within rings C and D of oleanane skeleton. In lactam product free carboxyl
group and a double bond in ring D instead of lactone system were formed in Beckmann
rearrangement reaction.
1. Introduction
Oleanolic acid and its derivatives display various interesting biological activities [1-3].
Among many different pharmacological features, hepatoprotective and anti-inflammatory
effects of unmodified oleanolic acid and some of its derivatives seem to be the most important
ones [4]. It is noteworthy that investigations concerning bardoxolone methyl ester, which was
considered as a very promising antioxidative and anti-inflammatory agent for chronic kidney
disease in diabetes patients treatment, reached the third stage clinical trials, even though it
ended in failure [5]. However, oleanolic acid derivatives show also many other biological
properties, i.e. anti-proliferative [6], antiviral [7] and antidiabetic [8], to name just a few. In
several studies [9-11] this class of compounds was reported to interact with biological
membranes, changing their permeability.
This paper is a part of ongoing research aiming at the introduction of lactam system in
pentacyclic triterpenoids of oleanane group in order to obtain a novel class of compounds
with interesting biological properties. It is commonly known that the activity of N-
dodecylcaprolactam (Azone®), one of the most popular and effective transepidermal transport
promoters, is associated with the presence of seven-membered lactam ring in its molecule
[12]. Therefore, introducing it in the oleanane triterpenoids gives the possibility to enhance
their ability to interact with biological membranes. The results of in vitro experiments
performed for azacyclic oleanane derivatives are described by Zaprutko et al. [13]. Some of
the analyzed compounds showed activity exceeding the activity of Azone® used as a reference
absorption enhancer.
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In this paper we present the synthesis and spectral and X-ray analysis of the structures
of 3β-acetoxy-12-hydroxyimino-18β-oleanan-28,13β-olide (5) as the substrate and nitrile (6)
and lactam (7) as products of Beckmann rearrangement reaction within the ring C of oleanane
skeleton.
2. Results and discussion
2.1. Synthesis of compounds 2 – 4
The starting material in our experiments, oleanolic acid (1), was isolated from by-
product residue obtained during the industrial production of mistletoe (Viscum album) extract
[14]. The purity of the obtained triterpene was proved on the basis of spectral data which were
in accordance with the literature spectral characteristics [15]. At this point it is worth noting
that the spatial structure of oleanolic acid was solved by Froelich and Gzella [16]. The
transformations of the mentioned triterpene are presented in Scheme 1.
Within 18β-oleanan-28,13β-olid derivative with hydroxyl group in C-12 position
(Scheme 1, comp. 3) can be obtained with the use of ozone or KMnO4 [17,18].
Oxidation of 3-acetoxyoleanolic acid (2) with the mixture of KMnO4 and CuSO4 [17] or
ozone [18] requires the application of complicated and time-consuming chemical procedures
or ozone generator. Moreover, as a result a mixture of products is obtained. As we present in
this paper, similar transformation can also be performed with the application of m-CPBA in
chloroform. By-products formed in this reaction weres removed by the application of
extraction with ethyl acetate that allowed us to avoid time-consuming column
chromatography.
In the first step of the synthetic route C-3 hydroxyl group of oleanolic acid (1) was acylated to
acetyloleanolic acid (2) with 10-fold excess of acetic anhydride [19]. After that the latter was
subjected to the action of m-CPBA in chloroform to give 12-hydroxylactone (3). The
secondary hydroxyl group in the obtained 12-hydroxy-28,13β-lactone (3) was oxidized with
Jones reagent with application of our method [20] to give the corresponding 12-ketone (4).
The results of spectral studies of both compounds were in accordance with the literature data
[21].
2.2. Synthesis and spectral characterization of compounds 5 – 7
The resulting ketone (4) was subjected to the action of hydroxylamine hydrochloride
in ethanol to form oxime (5). In the last step of the chemical transformations 5 was subjected
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to Beckmann rearrangement with POCl3 in dried pyridine to give two products (6 and 7)
showing different polarity which was proved with TLC analysis.
Scheme 1. Chemical transformations of oleanolic acid leading to oxime (5), nitrile (6) and
lactam (7). Reactions and conditions: (a) (CH3CO)2O, pyridine, rt; 97.5%; (b) m-CPBA,
CHCl3, 60.6%; (c) Jones reagent, acetone, rt.; 90.6%; (d) NH2OH·HCl, C2H5OH, reflux;
95.4%; (e) POCl3, pyridine, rt; 75.7% (for 6); 20.4% (for 7).
The structures of 5 - 7 were analyzed with IR, 1H- and 13C-NMR spectroscopy and X-
ray crystallography.
In 5 the results of spectral analyses confirmed the presence of 28,13β-lactone system,
oxime group in position 12 of ring C as well as acetoxy group in C-3 position in ring A. The
13
signals observed in C-NMR spectrum at 179.45 and 90.76 ppm for C-28 and C-13 atoms
indicated the presence of lactone system, while the signals at 171.03 and 80.64 ppm were
assigned to C-31 and C-3 atoms of acetoxy group. In IR spectrum two absorption bands at
carbonyl region at 1770 and 1730 cm-1 were observed. The first can be attributed to the
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lactone system and the other one to the acetoxy function [22]. As far as oxime group is
concerned, in 1H-NMR spectrum singlet signal with intensity of 1H at 7.60 ppm was found. It
13
was assigned to the hydrogen atom of OH group. In C-NMR spectrum a signal from C-12
atom was found at 156.75 ppm.
In 6 the presence of lactone and acetoxy group in C-3 position were confirmed with the
use of 13C-NMR analysis. The signals observed at 178.20 and 96.73 ppm for atoms C-28 and
C-13, respectively, indicate the presence of 28,13β-lactone system. The conclusions
concerning the acetoxy group were made based on the signals found at 170.67 and 79.98 ppm
assigned to atoms C-31 and C-3. In the IR spectrum recorded for 6 two absorption bands at
1775 and 1725 cm-1 were found which was similar to the IR spectrum obtained for 5. The first
one can be attributed to C=O group within 28,13β-lactone system and the other one to the
13
acetoxy carbonyl [22]. Moreover, for 6 C-NMR spectrum showed resonances at δ 121.37,
13.49 and 50.62 ppm for C-12, C-11 and C-9 of the cyanomethyl group in C-9 position while
at δ 150.74 and 116.41 ppm for the new double bond C14=C27. The presence of the double
bond between the mentioned atoms indicate structural transformations resulting in the
excluding of C-14 atom from ring D. In IR spectrum two absorption bands typical for seco-
nitriles were observed. The first one located at 3060 cm-1 was the result of stretching
vibrations of hydrogens linked to atom C-27 of the C14=C27 double bond. The other one was
situated at 2210 cm-1 and indicated the presence of the triple bond within the nitrile group.
In the case of lactam 7 the results of spectral IR, 1H- and 13C-NMR analyses allowed
to confirm the formation of lactam system and hydrolysis of 28,13β-lactone ring which led to
the regeneration of carboxyl group at C-17 position and formation of new C13=C18 double
bond. In the IR spectrum of 7 two absorption bands at 3435 cm-1 and 3330 cm-1 can be
observed. First of them was attributed to the OH group within the COOH function and the
1
other one arose from the NH of lactam system. In H-NMR spectrum of 7 only a signal as a
doublet situated at 6.69 ppm from the lactam proton was observed. The presence of C13=C18
double bond was confirmed by the signals situated at 135.08 and 134.07 ppm assigned to
13
mentioned atoms. In C-NMR spectrum of lactam 7 three characteristic signals for carbonyl
C were observed at 180.13, 177.39 and 170.96 ppm. First of them was assigned to C-28 atom,
the second one to C-12 and the last one to C-31 atom. Moreover, in the IR spectrum three
carbonyl absorption bands were observed at 1730, 1715 and 1655 cm-1. First of them can be
assigned to the acetoxy group [22], the second one to the carboxyl function [17] and last one
to the lactam system [22].
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2.2. X-ray analysis of 5 – 7
X-ray structures of oxime 5, nitrile 6 and lactam 7 together with the atom numbering
schemes are shown in Fig. 1 – 3. The crystal data and refinement parameters are summarized
in Table 1.
Figure 1. The molecular structure of 5, showing the atomic labelling scheme. Displacement
ellipsoids are drawn at the 50% probability level.
Figure 2. The molecular structure of 6, showing the atomic labelling scheme. Displacement
ellipsoids are drawn at the 50% probability level.
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Figure 3. The molecular structure of 7, showing the atomic labelling scheme. Displacement
ellipsoids are drawn at the 50% probability level.
Table 1. Crystal data, data collection and structure refinement for oxime 5, nitrile 6 and
lactam 7
Compound
oxime 5
nitrile 6
lactam 7
Chemical formula
Formula weight
Temperature [K]
Wavelength [Å]
Crystal system
Space group
C
32
H
49NO
5
C
32
H
47NO
4
C
32
H
49NO
5
527.72
100(2)
0.71073
monoclinic
P21
509.71
100(2)
0.71073
Triclinic
P1
527.72
100(2)
0.71073
orthorhombic
P212121
a = 8.9322(5) Å
b = 9.3534(4 )Å
c = 10.4867(5) Å
a = 8.14950 (10)
Å
b = 14.3252(3) Å
c = 25.2958(5) Å
a = 7.4690(10) Å
b = 15.288(3) Å
c = 12.943(3) Å
Unit cell parameters
α
β
γ
= 102.119(4)°
= 109.856(4)°
= 111.838(4)°
β
= 104.78(3)°
Volume [Å3]
1429.0(5)
2 (Z’=1)
1.226
705.88(6)
1 (Z’=1)
1.199
2953.11(9)
4 (Z’=1)
1.187
Z
Calculated density [Mg m-3]
Absorption coefficient [mm-1] 0.081
0.077
0.079
F(000)
576
278
1152
Crystal dimensions [mm]
0.58 x 0.22 x 0.15 0.50 x 0.20 x 0.10 0.40 x 0.10 x 0.10
θ
Range for data collection [°]
3.12 ≤ ≤ 36.85 2.54 ≤ ≤ 29.05° 2.63 ≤ ≤ 29.12
θ
θ
θ
h: -12 → 11
k: -25 → 25
l: -21 → 21
25270 / 11246
h: -12 → 12
k: -12 → 12
l: -14 → 14
17497 / 6666
h: -10 → 10
k: -19 → 15
l: -23 → 33
12843 / 6488
Max./Min. indices
Reflections collected/unique
Observed reflections [I ≥
2σ(Ι)]
10294
6318
5573
Number of parameters
Completeness to θfull = 25.00°
Goodness of fit on F2
355
100%
1.038
341
100%
1.086
359
99.9%
0.986
0.0395
0.1026
0.53; -0.27
0.0292
0.0807
0.27; -0.16
0.0329
0.0764
0.22; -0.19
R[I ≥ 2
wR(F2)
Largest diff. peak and hole
σ(Ι)]
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X-ray analysis revealed that 28,13β-lactone ring was retained only in the product of
Beckmann fragmentation (nitrile 6, Fig. 2). In the molecule of lactam 7 carboxyl group in C17
position and an additional C13=C18 double bond [1.3386(18) Å] in the ring D are observed.
The carboxyl group is oriented axially with respect to the ring E with C28=O4 bond revealing
conformation halfway between anticlinal and antiperiplanar [torsion angle C18–C17–C28–
O4: -150.40(13)°] with the respect to C17‒C18 bond.
X-ray analysis showed that in the molecule of oxime 5 hydroxyimino group in
position C(12) of the C ring displays E configuration [torsion angle O3–N1–C12–C13: -
176.48(7)°].
Structural investigation of nitrile 6, the product of Beckmann fragmentation, showed
that the compound was a result of major structural transformations within rings C and D of
the original molecule. The C12–C13 bond was cleaved which resulted in opening of the ring
C and formation of β-oriented cyanomethyl group connected to C9 atom of ring B. This group
revealed conformation halfway between anticlinal and antiperiplanar (+ac/+ap) [torsion angle
C8–C9–C11–C12: 141.46(9)°] with the respect to C8–C9 bond. The double bond formed
along with the nitrile group is located between C14 and C27 atoms [bond length:
1.3337(15) Å]. In the original molecule the first was quaternary carbon atom, while the latter
was methyl group. Moreover, the original six-membered D ring was transformed into five-
membered one which was a result of the cleavage of C14–C15 bond and formation of new
C13–C15 bond.
The molecule contains two rigid parts. One of them consists of rings A and B, whereas
the other one consists of rings D, E and γ-lactone moiety. These two parts are separated by
two single bonds, i.e. C8–C14 [1.5481(14) Å] and C14–C13 [1.5323(14) Å]. Spatial
arrangement of the mentioned systems can be described in terms of mutual orientation of two
trans-fused systems, i.e. A – B and E – γ-lactone. The dihedral angle between the least squares
planes of these two systems assumes 77.39(3)°.
The C14=C27 double bond is anticlinal (+ac) with respect to C(8)–C(9) bond [torsion
angle C(9)–C(8)–C(14)–C(27): 119.35(11)°] and synclinal (+sc) with respect to C(13)–C(18)
bond [torsion angle C(18)–C(13)–C(14)–C(27): 58.44(13)°].
In the molecule of 7 seven-membered lactam ring with the N atom connecting C12
and C13 was observed (Fig. 3).
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In the molecules of compounds 5 – 7 six-membered saturated rings A, B (compounds 5
– 7), D (compound 5) and E (compounds 5 and 6) reveal chair conformation. The named
conformation is also observed in the ring C of oxime 5 and in the ring E of lactam 7, each of
the mentioned rings containing one Csp2 atom. In the molecule of nitrile 6 five-membered
ring D displays envelope conformation {puckering parameters [23]: Q = 0.5739(13) Å,
145.12(14)°}. In the molecule of lactam 7 the ring D with C13=C18 double bond reveals half-
chair conformation [puckering parameters: Q = 0.4194(14) Å, = 51.54(19)°, = 143.5(2)°].
28,13β-Lactone rings in compounds 5 and 6 reveal envelope conformation [puckering
parameters for oxime 5: Q = 0.4903(10) Å, = 71.18(10)°; for nitrile 6: Q = 0.5744(13) Å,
= 73.82(12)°]. Seven-membered lactam ring in the molecule of 7 has chair conformation
[puckering parameters: Q(2) = 0.4822(13) Å, Q(3) = 0.6863(13) Å, (2) = 320.76(16)°, (3)
= 279.33(11)°], despite the presence of two Csp2 atoms (C-12 and C-13).
In the molecules of all of the investigated compounds planar acetoxy group in C(3)
position has orientation with carbonyl groups displaying conformation halfway between
ϕ =
θ
ϕ
ϕ
ϕ
ϕ
ϕ
β
synclinal and anticlinal with the respect to C2−C3 bond (+sc/+ac) [torsion angle C2–
C3···C31–O2 for oxime 5: 94.16(2)°; for nitrile 6: 83.37(1)°; for lactam 7: 78.15(13)°] and
similar to synperiplanar with the respect to O1−C3 bond [torsion angle C3–O1–C31–O2
oxime 5: 17.35(15)°; for nitrile 6: 4.17(16)°; for lactam 7: 4.3(2)°].
In the crystal lattices of all of the investigated compounds molecules related by
translation along the a axis are linked into chains through O–H···O, N–H···O and/or C–H···O
hydrogen bonds. In the crystal of oxime 5 molecules are connected by O3–H3···O4i hydrogen
bonds [Table 2, Fig. 4] involving hydroxyimino group and carbonyl oxygen atom of lactone
system. In the crystal lattice of nitrile 6 molecules are connected by C18–H18B···O2i
hydrogen bonds (Table 3, Fig. 5) with carbonyl oxygen of acetoxy group acting as a proton
acceptor. In the crystal of lactam 7 molecules are linked through N1–H1···O4i and O5–
H5···O3ii hydrogen bonds (Table 4, Fig. 6) involving amide group of lactam system and
carboxyl function. The motifs observed in the latter can be described in terms of graph set
notation [24-26] as first order chains C(7), C(9) and more interesting second order rings
2
R2 (8) (Fig. 7). Additionally, C25–H25A···O2ii hydrogen bonds with carbonyl oxygen atom
as a proton acceptor is observed.
Table 2. Hydrogen bond geometry in the crystal lattice of oxime 5
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d(D–H)
(Å)
d(H···A)
(Å)
d(D····A)
(Å)
Angle (D−H···A)
Hydrogen bond
(°)
O3–H3···O4i
0.89(2)
1.91(2)
2.758 (12)
159.1(17)
Symmetry code: (i) 1+x, y, z
Figure. 4. The hydrogen bonding (dashed lines) in the crystal structure of oxime 5. The H
atoms not involved in hydrogen bonds have been omitted for clarity.
Table 3. Hydrogen bond geometry in the crystal lattice of nitrile 6.
d(D–H)
(Å)
d(H···A)
(Å)
d(D····A)
(Å)
Angle (D−H···A)
Hydrogen bond
(°)
0.97
0.98
0.98
2.23
2.18
2.46
2.9145(14)
2.9346(15)
3.4072(14)
127
133
162
C7−H7A···O4
C9−H9···O4
C18−H18···O2i
Symmetry code: (i) 1+x, y, z.
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Figure. 5. The hydrogen bonding (dotted lines) in the crystal structure of nitrile 6. The H
atoms not involved in hydrogen bonds have been omitted for clarity.
Table 4. Hydrogen bond geometry in the crystal lattice of lactam 7.
d(D–H)
(Å)
d(H···A)
(Å)
d(D····A)
(Å)
Angle (D−H···A)
Hydrogen bond
(°)
N1–H1···O4i
O5–H5···O3ii
0.832(16)
0.94(2)
0.96
2.213(16)
1.62(2)
2.56
2.9707 (14)
2.5503(13)
3.4498(18)
151.4(14)
166.9(19)
155
C25–H25A···O2ii
Symmetry codes: (i) 1+x, y, z; (ii) -1+x, y, z.
Figure. 6. The hydrogen bonding (dashed and dotted lines) in the crystal structure of lactam 7.
The H atoms not involved in hydrogen bonds have been omitted for clarity.
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2.3. Possible reaction mechanism
The structures of substrate 5 and products 6 and 7 of Beckmann rearrangement
reaction obtained from X-ray analysis suggest the possible mechanism of the reaction
(Scheme 2). The transformation of oxime 5 into nitrile 6 and lactam 7 leads to the formation
of nitrilium ion (5-2) which is a result of the dissociation of water molecule from the unstable
intermediate (5-1), cleavage of C12–C13 bond and formation of a new one, i.e. C13–N1. The
nitrilium ion undergoes two alternative reactions leading to intermediates 5-3 and 5-5. The
first one is the result of the cleavage of C13–N1 bond and the formation of cyanomethyl
group comprising of C11, C12 and N1 atoms. The other one is the result of the addition of
water molecule to carbocation in C-12 position. Intermediate (5-3) is a carbocation with a
positive charge localized on C13 atom. Atoms C13+, C18 and O3 cannot be arranged in plane
which seems to be the reason for 1,2-rearrangement within D ring with the contraction of the
latter. The C13−C14 bond is cleaved and C13−C15 bond is formed. 1,2-Rearrangement
within the ring D is followed by the migration of the positive charge from C13 to C14
[intermediate (5-4)]. Subsequent dissociation of proton from C27 atom and formation of
C14=C27 double bond result in formation of the final product 6. Intermediate (5-5) undergoes
two further structural transformations which seem to be independent processes. The first one
is proton dissociation from oxonium group in C-12 position, the other one is a hydrolysis of
28,13β-lactone system. As a result of the latter the formation of carboxyl group and C13=C18
double bond in D ring are observed (comp. 7). The reaction route presented in Scheme 2 is
based on the assumption that elimination reaction follows commonly accepted E1 mechanism.
It is noteworthy that lactone system avoided the hydrolysis process in the reaction leading to
nitrile 6. The possible reason for this phenomenon is associated with the presence of the
positive charge localized at first on C13 [intermediate (5-3)] and then on C14 [intermediate
(5-4)]. The positive charge may stabilize C13–O3 bond.
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Scheme 2. Possible mechanism of transformations leading to nitrile 6 and lactam 7
3. Conclusions
The molecular structures of 3β-acetoxy-12-hydroxyimino-18β-oleanan-28,13β-olide
(5) and the products of Beckmann rearrangement, identified as 3β-acetoxy-12-nitrile-12,13-
seco-15(14→13)-abeoolean-14(27)-en-28,13β-olide (6) and 3β-acetoxy-12-oxo-12a-aza-C-
1
13
homoolean-13(18)-en-28-oic acid (7), were elucidated with IR, H- and C-NMR methods
and X-ray analysis. It was revealed that the oxime group in molecule of 5 displayed E
configuration. Transformation of six-membered isocyclic ring C into seven-membered
azacyclic one in the molecule of 7 in the course of Beckmann rearrangement was
accompanied by the hydrolysis of lactone ring and formation of C13=C18 double bond. In the
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molecule of nitrile 6 major structural transformations in comparison with the molecule of
original compound 5 were observed. Ring C cleavage and nitrile function formation, typical
for Beckmann fragmentation course, were followed by 1,2-rearrangement. As a result, in
molecule of 6 five-membered ring D is observed instead of six-membered one. Double bond
is located between C14 and C27. To the best of our knowledge, transformations leading to the
ring D of oleanane skeleton contraction have never been reported in the literature before.
4. Experimental
4.1. General Experimental Procedures:
The thin layer chromatography (TLC) analyses which were applied in order to
evaluate the reactions progress and the purity of compounds were performed on HPTLC
aluminium sheets (HPLC Alufolien Kieselgel 60 F245) with benzene and ethyl acetate as a
mobile phase. The chromatograms were visualized by spraying them with 10% ethanol
solution of sulfuric acid and then keeping the plates in the temperature of about 110oC for a
few minutes. The column chromatography was performed using Kieselgel 60; 0.063 – 0.200
mm (70 – 230 mesh). Melting points were determined with open capillary method in Büchi
apparatus and were uncorrected. IR spectra were recorded using Specord IR–75
1
13
spectrophotometer, for 0.5% mixtures of tested compounds and KBr. The H-NMR and C-
NMR spectra were recorded on Bruker 600 MHz spectrometer operating at room temperature
1
in deuterated chloroform as a solvent. For H-NMR spectra TMS was used as an internal
standard. The multiplicities of signals in 1H NMR spectra were marked as follows: s – singlet,
dd – doublet of doublets, t – triplet, m – multiplet. Mass spectra (MS) were recorded using
AMD 402 spectrophotometer with electroionisation.
4.2. Materials
4.2.1. 3β-Acetoxy-12α-hydroxyoleanan-28,13β-olide (3): a saturated solution of
acetyloleanolic acid (2) (499 mg, 1 mmol) and m-CPBA (345 mg, 2 mmol) in chloroform was
left in darkness at room temperature for two days. The resulting mixture was washed with 5%
solutions: FeSO4, Na2CO3, HCl (5 x 10 mL) and next with water (5 x 10 mL). Organic
solution was dried with MgSO4 and the solvent was removed with the evaporator. The
obtained yellowish, partly crystalline residue was grinded and extracted with ethyl acetate (5
x 10 mL). The extract was rejected and the remaining white residue was dried and crystallized
to give the title compound 3: C32H50O5, mol. mass 514.75; yield: 312 mg (60.6 %); 297 – 300
15

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o
oC (EtOH) (lit. m.p. 301 – 303 C), white needles,; Rf: 0.77 (4:1), 0.31 (9:1), dark-yellow
spot; spectral data in accordance with literature data [27].
4.2.2. 3β-Acetoxy-12-oxo-18β-oleanan-28,13β-olide (4): the synthesis was performed
on the basis of literature protocol [20] starting from 1 mmol (515 mg) of hydroxylactone 3 to
give the title compound 4: C32H48O5; mol. mass: 512.73; yield: 464 mg (90.6 %); m.p.: 287 –
290 oC (EtOH; decomp), lit. mp.: 287 – 289 oC [21]; Rf: 0.87 (4:1), 0.67 (9:1), yellow-brown
spot; spectral data in accordance with literature data [21].
4.2.3. 3β-Acetoxy-12-hydroxyimino-18β-oleanan-28,13β-olide (5): Hydroxylamine
hydrochloride (695 mg, 10 mmol) and sodium acetate (1.31 g, 16 mmol) were added to a
saturated, hot solution of an appropriate triterpenic ketone (1.02 g, 2 mmol) in ethanol (90 ml)
and the obtained mixture was refluxed 90 min, cooled and poured into a 5-fold volume of
water. The resulting white precipitate was filtered off, washed with water, dried (yield: 1.00 g,
o
95.4%) and crystallized from ethanol (small, colourless needles); m.p.: 228 – 231 C
(decomp.); Rf: 0.79 (4:1), 0.54 (9:1), purple-grey spot. IR (ν, cm-1): 3355 (OH, oxime); 1770
(C=O, lacton); 1730 (C=O, CH3COO); 1H NMR (δ, ppm): 7.60 (1H, s, oxime); 4.48 (1H, dd,
J = 5.1 and 11.1, C(3)-Hα); 2.75 (1H, m, C(18)-Hβ); 2.05 (3H, s, CH3COO); 1.20; 0.97; 0.97;
13
0.95; 0.91; 0.86; 0.86 (7 x 3H, 7 x s, CH3 groups); C NMR (δ, ppm): 179.42 [Cq, C-28];
171.03 [Cq, CH3COO]; 156.75 [Cq, C-12]; 90.76 [Cq, C-13]; 80.56 [CH, C-3]; 43.64; 43.30;
42.19; 37.80; 36.98; 31.65 (6 x Cq), 55.03 [CH, C-5]; 48.77 [CH, C-9]; 44.50 [CH, C-18];
38.30; 37.48; 34.31; 32.91; 27.48; 25.93; 23.49; 20.69; 19.03; 17.45 [10 x CH2]; 33.26; 27.88;
23.76; 21.27; 18.81; 18.32; 16.38; 15.72 [8 x CH3]; MS (m/z): 527.7 (0.1%) M+•.
4.2.4. 3β-Acetoxy-12-nitrile-12,13-seco-15(14→13)-abeoolean-14(27)-en-28,13β-
olide (6) and 3β-acetoxy-12-oxo-12a-aza-C-homoolean-13(18)-en-28-oic acid (7): POCl3
(0.28 ml, 0.46 g, 3 mmol) was added dropwise to a stirred and cooled solution of oxime 5
(528 mg, 1 mmol) in dried pyridine (12 ml). The resulting solution was left at room
temperature overnight. The brownish mixture was then poured into 5-fold volume of water
with ice and acidified with HCl. The obtained light-brown precipitate was filtered off, washed
with water, dried (502 mg) and subjected to the column chromatography on the silicagel
(benzene with ethyl acetate, 4:1 and next ethanol with THF, 1:1).
Compound 6 (nitrile) was obtained from benzene-ethyl acetate fractions (400 mg,
75.7%) and crystallized from ethyl acetate (small, colourless needles); m.p.: 249 – 251 oC; Rf:
0.68 (4:1), 0.36 (9:1), yellow-brown spot. IR (ν, cm-1): 3060 [C(14)=C(27)]; 2210 (C≡N);
1
1775 (C=O, lacton); 1725 (C=O, CH3COO); H NMR (δ, ppm): 5.21 [2H, s, C(27)-H2]; 4.56
16

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[1H, dd, J = 4.5 and 11.7, (C3)-Hα]; 2.45 [1H, t, J = 5.1, C(18)-Hβ]; 2.05 (3H, s, CH3COO);
1.06; 1.01; 0.94; 0.92; 0.89; 0.87 (6 x 3H, 6 x s, C-26, C-30, C-25, C-23, C-29, C-24 methyl
13
groups); C NMR (δ, ppm): 178.20 [Cq, C-28]; 170.67 [Cq, CH3COO]; 150.74 [Cq, C-14];
121.37 [Cq, C-12]; 96.73 [Cq, C-13]; 48.99 [Cq, C-17]; 45.40 [Cq, C-8]; 38.80 [Cq, C-10];
37.67 [Cq, C-4]; 30.93 [Cq, C-20]; 116.41 [CH2, C-27]; 13.49 [CH2, C-11]; 79.98 [CH, C-3];
53.70 [CH, C-5]; 53.35 [CH, C-18]; 50.62 [CH, C-9]; 38.80; 36.66; 35.45; 34.71; 33.49;
23.68, 23.21; 20.10; 18.18 [9 x CH2]; 32.83; 27.93; 24.26; 21.27; 21.20; 16.43; 16.09 [7 x
CH3; C-30, C-23, C-29, C-26, CH3COO, C-24, C-25]; MS (m/z): 509.4 (12.5%) M+•.
Compound 7 (lactam) was obtained from ethanol-THF fractions (108 mg, 20.4%) and
o
crystallized from ethyl acetate (small, colourless needles); m.p.: 278 – 281 C; Rf: 0.21
(AcOEt), pink-brown spot. IR (ν, cm-1): 3435 (O-H, COOH); 3330 (N-H, lactam); 1730
(C=O, CH3COO); 1715 (C=O, COOH); 1655 (C=O, lactam); 1H NMR (δ, ppm): 7.19 (1H, s,
N-H, lactam); 4.51 [1H, dd J = 4.5 and 11.7, C(3)-Hα]; 2.04 (3H, s, CH3COO); 1.13; 1.11,
0.95; 0.87; 0.86; 0.84; 0.83 (7 x 3H; 7 x s; C-27, C-26, C-30, C-23, C-25, C-29, C-24 methyl
13
groups); C NMR (δ, ppm): 180.13, 177.39; 170.85; 135.03; 134.07; 48.21; 46.63; 44.88;
38.60; 37.75; 32.57; [11 x Cq; C-28, C-12, CH3COO, C-13, C-18, C-17, C-14, C-8, C-10, C-
4, C-20]; 80.28; 54.75; 49.17 [3 x CH, C-3, C-5, C-9]; 40.98; 38.69; 36.21; 35.83; 34.88;
33.05; 32.87; 29.96; 23.36; 18.08 [10 x CH2, C-19, C-1, C-21, C-16, C-15, C-11, C-22, C-7,
C-2, C-6]; 31.76; 27.94; 24.44; 21.90; 21.23; 19.02; 16.63; 16.08 [8 x CH3, C-30, C-23, C-29,
C-27, CH3COO, C-26, C-24, C-25]; MS (m/z): 527.3 (40.2%) M+•.
4.3. X-ray analysis
Single-crystal X-ray data were collected on Oxford Diffraction KM4 Sapphire
(compound 5) and Oxford Diffraction (Varian Inc.) Xcalibur A (compounds 6 and 7)
diffractometers [28,29] both using graphite-monochromated MoKα radiation. Intensity data
were corrected for the Lorentz and polarization effects. The structure was solved by the direct
methods using the program SHELXS-97 [30], and refinement was done against F2 for all data
using SHELXL-97 [31]. All non-hydrogen atoms were refined anisotropically. The positions
of the H atoms bonded to N and O atoms were obtained from difference Fourier map and
were refined freely. The remaining H atoms were positioned geometrically and were refined
using a riding model, with C–H = 0.96 Å (CH3), 0.97 Å (CH2), 0.98 Å (Csp3H), 0.93 Å
(Csp2H) and Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C) for methyl H atoms. The methyl groups were
refined as rigid groups, which were allowed to rotate. Software used to prepare materials for
17

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publication was WINGX [32] and PLATON [33]. The molecular illustrations were drawn
using ORTEP-3 for Windows [32].
The deposition numbers CCDC953656 for oxime 5, CCDC953658 for nitrile 6 and
CCDC953657 for lactam 7 contain supplementary crystallographic data for this paper. These
data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html, (or from
the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax:
+44 1223 336033).
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