Facile preparation of a symmetric hexavalent oleanolic acid/per-O-methylated α-cyclodextrin conjugate with two conformations in solution and unambiguous NMR analysis

Article abstract of DOI:10.1016/j.tet.2017.03.036

A newly synthesized hexavalent oleanolic acid/per-O-methylated α-cyclodextrin conjugate shows significant differences in NMR spectra with previously synthesized analogs. Characterization of the product by ¹H NMR, ¹³C NMR, COSY, HSQC, HMBC, TOCSY, NOE, and ROESY experiments were performed. Detailed investigations revealed that the compound has two conformations in solution and the ratio of them was 1:1. Further variable-temperature NMR study revealed that the two conformations were stable at temperature range of 273–323?K in CDCl₃ solution.

Full text of DOI:10.1016/j.tet.2017.03.036

Tetrahedron xxx (2017) 1e6
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Facile preparation of a symmetric hexavalent oleanolic acid/per-O-
methylated
a-cyclodextrin conjugate with two conformations in
solution and unambiguous NMR analysis
a
a
b
b
c
a
Zhenyu Tian , Kun Meng , Yanhong Shi , Naixia Zhang , Xuehui Liu , Hongwei Jin ,
d
a
a
a, *
Yongmin Zhang , Lihe Zhang , Demin Zhou , Sulong Xiao
a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, PR China
Department of Analytical Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 200032, PR China
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, PR China
Sorbonne Universit ꢀe s, UPMC Univ Paris 06, Institut Parisien de Chimie Mol ꢀe culaire, CNRS UMR 8232, 4 Place Jussieu, 75005, Paris, France
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 January 2017
Received in revised form
A newly synthesized hexavalent oleanolic acid/per-O-methylated a-cyclodextrin conjugate shows sig-
nificant differences in NMR spectra with previously synthesized analogs. Characterization of the product
1
13
by H NMR, C NMR, COSY, HSQC, HMBC, TOCSY, NOE, and ROESY experiments were performed. Detailed
investigations revealed that the compound has two conformations in solution and the ratio of them was
5
March 2017
Accepted 14 March 2017
Available online xxx
1:1. Further variable-temperature NMR study revealed that the two conformations were stable at tem-
perature range of 273e323 K in CDCl solution.
3
©
2017 Elsevier Ltd. All rights reserved.
Keywords:
Per-O-methylated
Oleanolic acid
a-cyclodextrin
Click chemistry
Conformational exchange
NMR analysis
1. Introduction
activity against a wide range of cancer cell lines in vitro and
inhibited the growth of several cancer in vivo. In our previous
6
Pentacyclic triterpene acids, such as oleanolic acid (OA), betu-
linic acid (BA) and echinocystic acid (EA), and their glycosides are
widely distributed in the plant kingdom. Many compounds of this
study, a series of multivalent pentacyclic triterpene-cyclodextrin
conjugates have been synthesized and a dramatic multivalent ef-
fect for their anti-influenza virus entry activity has been found.
1
7
group are reported to have various interesting biological, phar-
Recently, per-O-methylated cyclodextrins and their derivatives
2
macological, or medicinal activities including antiviral, anti-
have attracted considerable attention due to their improved solu-
tumor,3 anti-inflammation, and so on. However, in many cases,
the potency of these triterpenoids is relatively weak. Therefore, the
semi-synthetic derivatives of pentacyclic triterpene could be of
great value in discovering highly potent bioactive compounds.
Several synthetic derivatives of OA, 2-cyano-3,12-dioxoolean-1,9-
dien-28-oic acid (CDDO), and its C-28 methyl ester (CDDO-Me)
and C-28 imidazole (CDDO-Im), have shown potent antitumor
4
5
bility both in water and in organic solvents. Inclusion complexes of
8
methylated cyclodextrins are usually more stable than the corre-
sponding complexes of unmodified cyclodextrins. As a further step
to our previous work towards the development of novel antiviral
7,9
inhibitors, we would like to synthesize the similar conjugates of
compound a-b (Fig. 1) in which the acetyl or hydroxyl groups of
cyclodextrin were replaced by methyl groups. To our surprise, the
obtained hexavalent oleanolic acid/per-O-methylated -cyclodex-
a-
a
trin conjugate 8 showed significant differences in NMR spectra
with previously synthesized analogs. Herein, we would like to
report the synthesis and unambiguous structure characterization of
this compound with two conformations in solution by one- and
two-dimensional NMR experiments.
*
Corresponding author. State Key Laboratory of Natural and Biomimetic Drugs,
School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Haidian
District, Beijing, 100191, PR China.
E-mail address: slxiao@bjmu.edu.cn (S. Xiao).
http://dx.doi.org/10.1016/j.tet.2017.03.036
0
040-4020/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Tian Z, et al., Facile preparation of a symmetric hexavalent oleanolic acid/per-O-methylated
a-cyclodextrin
conjugate with two conformations in solution and unambiguous NMR analysis, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.03.036

2
Z. Tian et al. / Tetrahedron xxx (2017) 1e6
Fig. 1. Chemical structures of three hexavalent oleanolic acid/
a
-cyclodextrin conjugates.
7
2
. Results and discussion
(6 ꢁ NH), respectively. However, the 1H NMR spectrum of com-
pound 8 consists of two sets of peaks of nearly equal amplitude for
NH, trizolyl-CH, OA-H18, methoxy, etc. For example, the signal of
compound 8 corresponding to the (NH) proton splits into two broad
The synthesis route of the unusual oleanolic acid/per-O-meth-
ylated
Briefly,
a
-cyclodextrin conjugate 8 was summarized in Scheme 1.
-cyclodextrin was converted into hexakis (2,3-di-O-
-CD 4 in 51% yield through a three-step
a
signals appearing at
d 8.23 and 6.75 ppm, and each signal has a
methyl-6-deoxy-6-azide)-
procedure described by Baer et al. N-propargyl 3
2-en-28-amide 7 was prepared according to our previously re-
a
integration value of six (6 ꢁ NH). Similar results were also observed
1
0
13
b
-hydroxyolean-
in the
C NMR spectrum of compound 8 (see Supporting
1
information).
9b
ported method. When compound 4 was reacted with 6.6 equiv of
The structure of 8 was further assigned through detailed anal-
ysis of one- and two-dimensional NMR experiments. The lower
field region spanning 2.5e8.5 ppm of 8 was relatively well-resolved
compound 7 (1.1equiv per N
3
) in the present of CuSO
4
and L-
ꢀ
ascorbic acid under microwave condition at 100 C for 1 h, conju-
gate 8 was separated out by conventional silica gel column chro-
matography in 62% yield.
1
1
1
13
and fully assigned with the aid of He H COSY, He C HSQC and
1
13
1
13
He C HMBC spectra. The chemical shifts of H and C NMR of
compound 8 at 298 K are listed in Table 1. In the aromatic region,
the signals at
The MALDI-TOF mass spectrum for compound 8 showed an
þ
[
4
MþCu]/2 ion at m/z 2156.9030 Da (Calcd for C246
H
384CuN24
O
36
,
d
¼ 7.36 and 7.80 ppm could be assigned to two
1 1 1 13
þ
313.8251); and an [M þ Na þ Cu]/2 ion at m/z 2168.4004 Da
triazole-CHs according to its He H and He C correlation spectra
(Fig. 2A). Additional signals were observed for two amide protons at
(
Calcd for C246
H
384CuN24NaO36, 4336.8149) (SI Fig. 1), indicating
1
13
that it is a hexavalent conjugate. However, compound 8 demon-
6.75 and 8.23 ppm. From the He C HSQC spectrum, two sets of
signals at 5.46 and 5.37 ppm, overlapped with the signals of CD-H
and CD-H , respectively, could be assigned to two H12 of OA. In
addition, compound 8 shows two sets of CD-H signals appearing at
5.36 and 5.01 ppm and two CD-C signals appearing at 100.86 and
1
13
strated complex H and C NMR spectra and significant differences
1
1
were found between compound 8 and its analogs a and b. H NMR
6
spectrum of the compounds a and b in CDCl
3
solution showed only
1
a broad signal at 6.80 and 7.15 ppm with the integration of six
1
ꢀ
ꢀ
Scheme 1. Synthesis of the hexavalent oleanolic acid/per-O-methylated
a
-cyclodextrin conjugate 8. Reagents and condition: (a) Ph
CO , DMF, rt, 1 h; (f) CuSO
3
P, I
2
, DMF, 80 C, overnight; (b) NaN
3
, H
2
O, 60 C,
ꢀ
1
2 h; (c) CH I, NaH, DMF; (d) TBTU, DIEA, THF, rt, overnight; (e) 2-propynylamine, Na
3
2
3
4
, Na-L-ascorbate, THF-H
2
O (1:1, v/v), 100 C, microwave.
Please cite this article in press as: Tian Z, et al., Facile preparation of a symmetric hexavalent oleanolic acid/per-O-methylated
a-cyclodextrin
conjugate with two conformations in solution and unambiguous NMR analysis, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.03.036

Z. Tian et al. / Tetrahedron xxx (2017) 1e6
3
Table 1
Partial 1H and 13C NMR signal assignments of conjugate 8 at 298 K in CDCl
3
.
Position
3
d
H
(J, Hz)
d
C
Position
d
H
(J, Hz)
d
C
0
3.21 (m)
5.45 (m)
2.62 (br d, 8.8)
6.75 (br t)
4.72 (dd, 6.3, 15.3) and 4.08 (m)
7.36 (s)
5.34 (m)
2.94 (m)
3.48 (m)
3.25 (m)
78.96
123.09
41.80
e
3
3.21 (m)
5.36 (m)
2.76 (br d, 10.6)
8.23 (br t)
4.08 (m) and 3.89 (m)
7.80 (s)
5.01 (br s)
2.86 (dd, 2.8, 10.7)
3.40 (m)
3.26 (m)
4.09 (m)
78.96
122.09
40.85
e
0
0
1
1
a
b
c
2
8
12
18
0
a
0
34.82
124.04
99.54
81.58 [
80.29
81.78
69.50
48.36
b
33.03
126.35
100.86
81.47
80.65
85.61
72.57
52.59
57.96
61.77
0
c
0
CD-1
CD-2
CD-3
CD-4
CD-5
CD-6
OMe (2)
OMe (3)
CD-1
a]
0
0
0
0
0
[a]
CD-2
CD-3
CD-4
CD-5
CD-6
3.95 (m)
4.06 (m)
3.43 (s)
3.57 (s)
5.44 (m) and 4.10 (m)
3.43 (s)
3.50 (s)
[b]
0
OMe (2 )
[b]
[c]
58.04
61.84 [
c]
0
OMe (3 )
[
a-c]
These values could be exchanged each other.
9
9.54 ppm, which is unlike to its analogs a (Fig. 2B) and b (SI Fig. 2)
and one CD-C signals were observed. In
addition, in the 2D-ROESY experiment, strong and negative NOE
cross-peaks (highlighted in blue) were also clearly observed along
the diagonals (Fig. 4), such as the negative NOE signals between
in which only one CD-H
1
1
the higher field region, H18 proton of OA, the characteristic signal of
oleanane-type triterpene, is also displayed as two sets of broad
absorptions at 2.62e2.76 ppm.
It is well known that CDs are shaped like a truncated cone with
internal cavities ranging from 5 to 8 Å, which are capable to form
inclusion complex with a variety of molecules to enhance solubility,
bioavailability, and thermal stability of hydrophobic guest com-
pounds.11 Therefore, we first wonder whether that the split signals
1
two NH protons, two trizolyl-CH, and two glu-H signals, indicating
that there was conformational exchange of compound 8 in solution.
However, no obvious changes were observed in variable-
temperature NMR experiments at temperature range of
3
273e323 K in CDCl solution (SI Fig. 6), suggesting that two con-
formations of 8 were stable below 323 K. This phenomenon of
rotational isomerism has been observed previously for N-
13
of compound 8 are caused by inclusion between OA and
a-CD.
substituted tetrahydroisoquinoline derivatives. In our previously
study, we also found that the two methyl of dimethylamino group
Several studies have reported that pentacyclic triterpene could
form inclusion complexes with different types of cyclodextrin de-
rivatives. But as far as we know, none of them reported that the
formation of inclusion could make the H and C NMR signals split
to duplicate. We also found that no significant proton shifts in H
14
of morphiniques derivatives showed different chemical shift. The
two isomers may arise from the eNHeCOe bond, which was likely
to exhibit partial double-bond character with cis- and trans-iso-
mers. It was likely that the energy barrier for rotation around the
amide bond was large resulting in geometrically and magnetically
1
13
1
NMR spectrum has been observed with the increasing of com-
pound 7 in per-O-methylated
15
a-CD (SI Fig. 3).
non-equivalent N-substituents.
All the information indicated that two kinds of triazole linker
between cyclodextrin scaffold and OA were included in compound
3. Conclusion
8
, which lead us to think that the structure of 8 is a symmetric
hexavalent conjugate in which three arms of OA are linked to the
primary face of -cyclodextrin by 1,4-adduct while the other three
In summary, a hexavalent oleanolic acid/per-O-methylated
cyclodextrin conjugate 8 was synthesized via copper-catalyzed
alkyne-azide cyclodextrin reaction and unambiguously character-
ized by NMR and MALDI-TOF MS. Compared to its analogs, this
product showed complex H and C NMR spectra. Detailed spec-
troscopic studies highlighted that there are two different confor-
mations of this conjugate in solution arising from hindered rotation
around the amide bond, and the population of the two conformers
at room temperature is equal.
a
arms linked by 1,5-adduct alternately (Fig. 3).
However, we are unable to explain what led to produce the
compound in Fig. 3. It is generally believed that the cycloaddition of
alkyne with azide to produce a mixture of 1,4-adduct and 1,5-
adduct in which the formation of one regioisomer is predomi-
nant. The use of copper reagents dramatically improves regiose-
lectivity to afford the 1,4-regioisomer exclusively, as first
1
13
12
demonstrated extensively by Sharpless. To the best of our
knowledge, no example has highlighted that the copper reagent
allows 1,5-adduct. Therefore, other NMR experiments, such as 1D
4. Experimental
1
NOE, ROESY and variable-temperature H NMR, were further con-
4.1. General
ducted to characterize the structure of 8. To our surprise, in the 1D
NOE spectrum of 8 strong negative NOE enhancement was
observed between the two NH signals (SI Fig. 4 and SI Fig. 5). In
a
-cyclodextrin was purchased from Aladdin Chemistry Co. Ltd
(Shanghai, China). Oleanolic acid was kindly supplied by Nanjing
Please cite this article in press as: Tian Z, et al., Facile preparation of a symmetric hexavalent oleanolic acid/per-O-methylated
a-cyclodextrin
conjugate with two conformations in solution and unambiguous NMR analysis, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.03.036

4
Z. Tian et al. / Tetrahedron xxx (2017) 1e6
, 298 K) spectra of compounds 8 (A) and a (B), with the 1D 1H and C NMR spectra along the top and the side, respectively. The frame
13
Fig. 2. A portion of the 400 MHz HSQC (CDCl
corresponds to correlation
3
1
1
13
1 1
JCH CD-C and CD-H . Unlike compound a, compound 8 displayed two sets of H and C signals, although the frequencies for some atoms could not be
measured accurately due to lower resolution.
Please cite this article in press as: Tian Z, et al., Facile preparation of a symmetric hexavalent oleanolic acid/per-O-methylated
a-cyclodextrin
conjugate with two conformations in solution and unambiguous NMR analysis, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.03.036

Z. Tian et al. / Tetrahedron xxx (2017) 1e6
5
Fig. 3. Another possible structure of conjugate 8.
Zelang Medical Technology Co., Ltd (Nanjing, China). All reactions
4.2. Hexakis [6-deoxy-6-[4-N-(3b-hydroxyolean-12-en-28-oate)
were carried out under an inert atmosphere with dry solvents
under anhydrous conditions unless otherwise mentioned. TLC
was run on silica gel 60 F254 plates, and the spots were detected
aminomethyl-1H-1,2,3-triazol- 1-yl]-2,3-di-O-methyl]-a-
cyclodextrin (8)
by staining with H
containing Ce(NH
24.0 g) in 6% H SO
chromatography was performed using 200e300
2
SO
(NO
(500 mL) followed by heating. Flash column
M mesh silica
4
in ethanol (10%, v/v) or a yellow solution
To a solution of hexakis (2,3-di-O-methyl-6-deoxy-6-azide)-
cyclodextrin 4 (58 mg, 0.044 mmol) and N-propargyl 3 -hydrox-
yolean-12-en-28-amide 7 (146.9 mg, 0.30 mmol) in 50% of THF
and H O (15 mL) was added CuSO (4.7 mg, 0.03 mmol) and so-
a-
4
)
2
3
)
6
(0.5 g) and (NH Mo 24$4H
4
)
6
7
O
2
O
b
(
2
4
m
2
4
gel 60 (Qingdao Haiyang Chemical Co. Ltd, Qingdao, China).
Microwave-assisted CuAAC reactions were performed using a
dium ascorbate (58.8 mg, 0.30 mmol). The resulting solution was
heated in a microwave reactor at 100 C until the azide was
completely consumed as determined by thin-layer chromatog-
ꢀ
1
13
CEM Discover SP microwaver reactor (USA). H NMR, C NMR
and variable temperature NMR spectra were recorded using a
Brucker 400 MHz spectrometer; the residual solvent protons
were used to reference the chemical shift in ppm. ESI-HRMS and
MALDI-TOF-MS were obtained with an APEX IV FT_MS (7.0 T)
spectrometer (Bruker) and an AB Sciex Triple TOF 4600 spec-
raphy (TLC). Then the reaction mixture was extracted with CH
(20 mL ꢁ 3). The CH Cl fraction was dried over Na SO , filtered,
evaporated, and purified by silica gel column chromatography
using CH Cl /CH OH (20:1e15:1 v/v) as eluent to give the purified
white product in 62% yield. R
2 2
Cl
2
2
2
4
2
2
3
1
f
¼ 0.35 (CH
2
Cl
2
/CH
3
OH ¼ 10:1);
H
0
trometer, respectively. Chemical shifts are given in
coupling constants (J) in hertz. The assignment of the H and
d
(ppm) and
NMR (400 MHz, CDCl
CH ), 7.36 (s, 6H, trizolyl-CH), 6.75 (br s, 6H, NH), 5.46e5.35 (m,
3
):
d
8.23 (br s, 6H, NH ), 7.80 (s, 6H, trizolyl-
1
13
0
C
signals was based on 2D (COSY, HSQC, HMBC, and NOESY) NMR
techniques. The HPLC analysis was performed with an Agilent
HPLC 1260 system (Agilent, Palo Alto, CA, USA). The chromato-
graphic separation was carried out using a Waters 250 ꢁ 4.6 mm,
24H, H12, H12
J ¼ 6.3, 15.3 Hz, 6H, NHCHb'), 4.10e3.87 (m, 48H, NHCHa', CD-H6a'
CD-H , CD-H , NHCH ), 3.57 (s, 18H, OMe(3)), 3.50 (s,
, 2ꢁCD-H
18H, OMe(3 )), 3.48 (m, 6H, CD-H ), 3.43 (s, 36H, OMe(2),
), 3.29e3.17 (m, 24H, H , H , CD-H
), 2.96e2.91 (m, 6H, CD-H
), 2.86 (dd, J ¼ 2.8, 10.7 Hz, 6H,
), 2.77 (br d, J ¼ 10.6 Hz, 6H, H18 ), 2.62 (br d, J ¼ 8.8 Hz, 6H,
1 1
, CD-H6b', CD-H ), 5.01 (s, 6H, CD-H ), 4.72 (dd,
0 0
,
0
5
5
0
6
2
3
0
5
m
m C18 column and eluted with CH
3
OHeH
2
O (95:5) at a flow
OMe(2 )), 3.40 (m, 6H, CD-H
3
0
3
3
0
4
,
rate of 1.0 mL/min. The purity of compound 8 was 96.92% (SI
Fig. 7).
CD-H
CD-H
4
0
2
0
0
2
Compounds 2e4 and 6e7 were synthesized according to the
H
18), 1.17, 1.04, 0.99, 0.96, 0.94, 0.91, 0.89, 0.87 (8ꢁs, each 18H,
48ꢁCH ), 0.81 (s, 36H, 12ꢁCH ), 0.79 (s, 54H, 18ꢁCH ), 2.00e1.03
(m, 254H, other aliphatic ring protons), 0.75e0.66 (m, 12H, H
9
b,10
literature (Scheme 1).
The structure of those compounds was
3
3
3
confirmed by H NMR, 13C NMR and ESI-HRMS (Supporting
1
,
5
information).7
H
5
), 0.50 (s, 18H, 6ꢁCH
13
0
3 3
); C NMR (100 MHz, CDCl ): d 178.01,
Please cite this article in press as: Tian Z, et al., Facile preparation of a symmetric hexavalent oleanolic acid/per-O-methylated
a-cyclodextrin
conjugate with two conformations in solution and unambiguous NMR analysis, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.03.036

6
Z. Tian et al. / Tetrahedron xxx (2017) 1e6
Fig. 4. 2D-ROESY spectrum of compound 8 in CDCl
3
at 298 K. Strong, negative exchange cross-peaks (blue) as well as other positive NOE signals (red) are visible.
177.45, 145.58, 144.38, 144.32, 143.76, 126.35, 124.04, 123.09,
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2017.03.036.
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4. Labidalle S, Min ZY, Reynet A, et al. Tetrahedron. 1988;44:1159e1169.
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Please cite this article in press as: Tian Z, et al., Facile preparation of a symmetric hexavalent oleanolic acid/per-O-methylated
a-cyclodextrin
conjugate with two conformations in solution and unambiguous NMR analysis, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.03.036