Xylapyrrosides A and B, two rare sugar-morpholine spiroketal pyrrole-derived alkaloids from Xylaria nigripes: Isolation, complete structure elucidation, and total syntheses

Article abstract of DOI:10.1016/j.tet.2015.06.020

Two new [named xylapyrrosides A (1) and B (2)] along with two known [pollenopyrrosides A (3) and B (=acortatarin A, 4)] naturally occurring spirocyclic pyrrole alkaloids were isolated and identified as minor components from the EtOH extract of the dried mycelia of the edible medicinal fungus Xylaria nigripes. Their structures were established by a combination of interpretation of spectroscopic data and single-crystal X-ray diffraction analyses. The isolates possess a unique tricyclic skeleton comprising a common bicyclic 2-formyl-pyrrole-fused morpholine, with a variable ketohexoside ring. This class of alkaloids is quite rare from natural sources. In this study, the total syntheses of compounds 1, 2 and 4 were successfully achieved by two alternative strategies, and three new analogs [named xylapyrrosides A₁ (1a), A₂ (1b) and B₁ (2a)] were also produced. Notably, the total synthesis of such spiroketal alkaloids with a pyranose ring (e.g.,1) was accomplished for the first time. The absolute configurations of the new isolates can be thereafter unequivocally secured by the total syntheses. The above isolated and synthesized spiro-alkaloids were found to show moderate antioxidant effects by preventing the oxidative stress-induced cytotoxicity of A7r5 rat vascular smooth muscle cells (VSMCs).

Full text of DOI:10.1016/j.tet.2015.06.020

Accepted Manuscript
Xylapyrrosides A and B, Two Rare Sugar-Morpholine Spiroketal Pyrrole-Derived
Alkaloids from Xylaria nigripes: Isolation, Complete Structure Elucidation, and Total
Syntheses
Ming Li, Juan Xiong, Ya Huang, Li-Jun Wang, Yu Tang, Guo-Xun Yang, Xin-Hua Liu,
Bang-Guo Wei, Hui Fan, Yun Zhao, Wen-Zhu Zhai, Jin-Feng Hu
PII:
S0040-4020(15)00875-3
10.1016/j.tet.2015.06.020
TET 26855
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 5 May 2015
Revised Date: 1 June 2015
Accepted Date: 3 June 2015
Please cite this article as: Li M, Xiong J, Huang Y, Wang L-J, Tang Y, Yang G-X, Liu X-H, Wei B-G, Fan
H, Zhao Y, Zhai W-Z, Hu J-F, Xylapyrrosides A and B, Two Rare Sugar-Morpholine Spiroketal Pyrrole-
Derived Alkaloids from Xylaria nigripes: Isolation, Complete Structure Elucidation, and Total Syntheses,
Tetrahedron (2015), doi: 10.1016/j.tet.2015.06.020.
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Xylapyrrosides A and B, Two Rare Sugar-
Morpholine Spiroketal Pyrrole-Derived
Alkaloids from Xylaria nigripes: Isolation,
Complete Structure Elucidation, and Total
Syntheses
Leave this area blank for abstract info.
a
a,*
a
a
a
a
b
Ming Li, Juan Xiong, Ya Huang, Li-Jun Wang, Yu Tang, Guo-Xun Yang, Xin-Hua Liu, Bang-Guo
c,*
d
d
d
a,*
Wei, Hui Fan, Yun Zhao, Wen-Zhu Zhai, and Jin-Feng Hu
a
b
Departments of Natural Products Chemistry and Pharmacology, School of Pharmacy, Fudan University, No.
26 Zhangheng Road, Shanghai 201203, China
Department of Chemistry, Fudan University, No. 220 Handan Road, Shanghai 200032, China
Department of Natural Products for Chemical Genetic Research, Key Laboratory of Brain Functional
Genomics (Ministry of Education), East China Normal University, No. 3663 Zhongshan Road, Shanghai
8
c
d
200062, China

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Xylapyrrosides A and B, Two Rare Sugar-Morpholine Spiroketal Pyrrole-Derived
Alkaloids from Xylaria nigripes: Isolation, Complete Structure Elucidation, and Total
Syntheses
a
a, *
a
a
a
a
b
Ming Li, Juan Xiong, Ya Huang, Li-Jun Wang, Yu Tang, Guo-Xun Yang, Xin-Hua Liu, Bang-Guo
c, *
d
d
d
a, *
Wei, Hui Fan, Yun Zhao, Wen-Zhu Zhai, and Jin-Feng Hu
a
b
Departments of Natural Products Chemistry and Pharmacology, School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, China
c
Department of Chemistry, Fudan University, No. 220 Handan Road, Shanghai 200032, China
d
Department of Natural Products for Chemical Genetic Research, Key Laboratory of Brain Functional Genomics (Ministry of Education), East China Normal
University, No. 3663 Zhongshan Road, Shanghai 200062, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Two new [named xylapyrrosides A (1) and B (2)] along with two known [pollenopyrrosides A
(3) and B (= acortatarin A, 4)] naturally occurring spirocyclic pyrrole alkaloids were isolated
and identified as minor components from the EtOH extract of the dried mycelia of the edible
medicinal fungus Xylaria nigripes. Their structures were established by a combination of
interpretation of spectroscopic data and single-crystal X-ray diffraction analyses. The isolates
possess a unique tricyclic skeleton comprising a common bicyclic 2-formyl-pyrrole-fused
morpholine, with a variable ketohexoside ring. This class of alkaloids is quite rare from natural
sources. In this study, the total syntheses of compounds 1, 2 and 4 were successfully achieved by
two alternative strategies, and three new analogues [named xylapyrrosides A1 (1a), A2 (1b) and
B1 (2a)] were also produced. Notably, the total synthesis of such spiroketal alkaloids with a
pyranose ring (e.g.,1) was accomplished for the first time. The absolute configurations of the
new isolates can be thereafter unequivocally secured by the total syntheses. The above isolated
and synthesized spiro-alkaloids were found to show moderate antioxidant effects by preventing
the oxidative stress-induced cytotoxicity of A7r5 rat vascular smooth muscle cells (VSMCs).
Available online
Keywords:
Xylaria nigripes
Xylapyrrosides A and B
Spiro-alkaloids
Asymmetric synthesis
Antioxidant
2
015 Elsevier Ltd. All rights reserved.
1
1
. Introduction
alkaloids (Figures 1 and 2) contains an unprecedented tricyclic
pyrrole–morpholine–ketohexoside fused framework. Among the
above spiro-alkaloids, capparisines A and B were previously
reported to have no inhibitory effect on human hepatocyte cell
As described previously, pyrrole-containing alkaloids, such as
1
2
discorhabdins and lamellarins with complex structures and
interesting biological activities, have greatly stimulated
interdisciplinary studies by chemists and biologists worldwide.
During the past decade, the number of new pyrrole-related
alkaloids isolated, identified, and synthesized for their medicinal
potential has increased. This includes a very small class of sugar-
morpholine spiroketal pyrrole-derived alkaloids that emerged
only in recent years. Capparisines A and B from the fruits of
3
HL-7702 apoptosis. But acortatarin A was found to have
antioxidant activity by inhibiting reactive oxygen species (ROS)
5,7
production in high glucose-induced mesangial cells.
3
Capparis spinosa, pollenopyrrosides A and B from the bee-
collected Brassica campestris pollen, and acortatarins A and B
from the rhizomes of Acorus tatarinowii, were independently
4
5
reported by three Chinese research groups in 2010. About two
years later, acortatarins A and C were isolated from the crust of
6
whole wheat bread by Peterson, et al. In general, this group of
—
——
*
Corresponding authors. (+86)-21-5198-0172; e-mail:
jfhu@fudan.edu.cn
Figure 1. Structures of naturally-occurring (1–4) spiroketal pyrrole-derived
alkaloids from Xylaria nigripes and their synthesized analogues (1a, 1b, 2a).

2
ACCEPTED MA (a n
N
d
U
sem
S
i
C
-p
R
rep
I
arative HPLC to furnish compounds 1 (4.0 mg), 2
P
T
21.3 mg), 3 (1.2 mg), and 4 (20.1 mg). By comparing their
spectroscopic data and physicochemical properties with those
reported in the literature, the structures 4with absolute
configurations of 4
pollenopyrroside B or acortatarin A
3
(= pollenopyrroside A ), and
4
(=
5,8-12
) were undoubtedly
established as shown in Figure 1. Compound 2 is reported herein
as a natural product for the first time, which is identical in all
9-13
respects with the synthesized 9-epi-acortatarin A.
Xylapyrroside A (1) was obtained as a colorless crystal from
acetone. Its molecular formula was determined to be C H NO
1
2
15
5
+
based on a molecular ion peak at m/z 253.0952 [M] in its HR-
EIMS, implying six degrees of unsaturation. The UV absorption
at 296 nm of 1 indicated the presence of a pyrrole-2-aldehyde
Figure 2. Other previously reported naturally-occurring spiroketal pyrrole-
a
derived alkaloids. The stereochemistries of capparisines A and B reported by
Wang et al. were determined by MoKα (not CuKα) X-ray crystallographic
5
3
b
moiety. In accordance with the above observation, a typical
analysis. The relative and absolute configuration of acortatarin C remains
6
undetermined, and the configurations at C-11 and C-12 were most likely to
proton signal at δ 9.39 (1H, s, H-7) for an aldehyde group and
two mutually-coupling olefinic protons at δ 7.04 (1H, d, J = 4.0,
H-3) and 6.09 (1H, d, J = 4.0 Hz, H-4) were present in the H
6
be S* and R*, respectively, based on related biogenetic considerations.
1
Due to the intriguing structures, this class of alkaloids
attracted considerable interest for organic synthesis soon after
their isolation. So far, several groups have accomplished the total
NMR spectrum (Table 1) of 1.
1
13
a
Table 1. H (400 MHz) and C (100 MHz) NMR data of compound 1.
8-12
8,9
syntheses of acortatarins A and B. Inexplicably, the absolute
configurations of the naturally occurring acortatarins were
initially assigned by both single-crystal X-ray (MoKα) diffraction
δ
H
(J values in Hz)
δ
C
No
b
c
d
Capparisine
b
1
1
1
1
4
,d
5
B
analysis and the modified Mosher’s method, but they were8
2
132.4
eventually revised after stereoselective total syntheses.
Meanwhile, acortatarin A was found to be identical with
3
4
7.04 (d, 4.0)
6.09 (d, 4.0)
6.97 (d, 4.0)
6.05(d, 4.0)
6.90 (d, 4.0)
5.99 (d, 4.0)
6.92 (d, 3.8)
6.01 (d, 3.5)
125.8
106.1
9,10
pollenopyrroside B.
5
6
137.1
58.6
As a part of our continuing interest in novel bioactive
4.86 (d, 16.0)
4.77 (d, 16.0)
4.86 (d, 15.6)
4.74 (d, 15.6)
4.80 (d, 15.6)
4.71 (d, 15.6)
4.82 (d, 15.2)
4.74 (d, 15.7)
1,2,13
alkaloids from nature,
four sugar-morpholine spiroketal
pyrrole-derived alkaloids (1−4, Figure 1) were isolated from the
EtOH extract of the dried mycelia of Xylaria nigripes (Koltz.)
Sacc. (family Xylariaceae), also known by the folklore name of
Wuling Shen in Chinese. X. nigripes is considered to be a
precious medicinal fungus, which is edible and delicious when it
is young. Differing from our previously studied wood-rotting
7
8
9.39 (s)
4.62 (d, 14.0),
9.46 (s)
4.55 (d, 14.0)
9.42 (s)
4.68 (d, 13.9)
9.45 (s)
4.70 (d, 14.4)
180.2
53.5
4.00 (d, 14.0)
3.95 (d, 14.0)
3.99 (d, 13.9)
4.02 (d, 13.8)
9
96.9
36.1
1
0
2.02 (dd,
12.8, 11.6)
1.92 (dd,
1.98 (dd, 12.8,
11.6)
1.90 (dd, 12.8,
5.6)
1.90 (dd, 12.8,
11.6)
2.02 (dd, 12.8,
5.6)
4.14 (ddd,
11.6, 5.6, 2.8)
3.87 (m,
overlapped)
3.87 (dd,
1.91 (dd,
12.6, 11.8)
2.04 (dd,
12.8, 5.3)
4.14 m
14
fungus Fomes fomentarius, the wild X. nigripes has a very
special ecological niche in that it usually grows in the abandoned
nests of the subterranean (ca -2 m) termite Odontotermes
1
2.8, 5.2)
4.10 (ddd,
1.6, 5.2, 2.8)
11
4.06 (m)
68.7
66.0
66.3
1
1
2
3
3.81 (m)
3.85 (m)
3.88 m
15,16
formosanus.
Although the wild fungus is quite rare in nature,
1
3.83 (br d,
3.77 (dd,
overlapped)
3.75 (dd,
3.89 (d, 12.1)
3.81 (d, 12.1)
the mycelia of X. nigripes nowadays can be largely manufactured
through fermentation, and the dried culture filtrate is usually
called Wuling Powder in China. As a popular traditional Chinese
medicine (TCM) used as a nerve tonic, the commercially
available Wuling Capsule (a single herbal formula made from
Wuling Powder) has been used clinically for the treatment of
insomnia, anxiety disorders and depression in China since
1
2.0)
3.78 (br d,
2.0)
overlapped)
3.78 (dd, 12.8,
1.2)
1
overlapped)
a
b
Assignments were made by a combination of 1D and 2D NMR experiments;
in methanol-d ; in acetone-d ; in CDCl3.
4 6
c
d
Additionally, two unequivalent aliphatic methylene protons [δ
.02 (1H, dd, J = 12.8, 11.6 Hz) and 1.92 (1H, dd, J = 12.8, 5.2
2
15,16a,17
1
999.
Nevertheless, studies on the secondary metabolites
Hz), H -10], three heteroatom-bearing methylenes [δ 4.86 and
4
=
1
Hz, H-11); 3.81 (1H, m, H-12)] also appeared in the H NMR
spectrum (Table 1). The C NMR spectrum of 1 exhibited
twelve well-resolved signals (Table 1) classified by DEPT and
HSQC NMR experiments: four methylenes [δ 36.1 (C-10), 53.5
(C-8), 58.6 (C-6) and 66.0 (C-13)], five methines [two
oxygenated at δ 66.3 (C-12), 68.7 (C-11), two olefinic at δ 106.1
2
and their biological properties of this fungus remain
.77 (each 1H, d, J = 16.0 Hz, H -6); 4.62 and 4.00 (each 1H, d, J
14.0 Hz, H -8); 3.83 and 3.78 (each 1H, br d, J = 12.0 Hz, H -
2 2
3)], and two oxymethines [δ 4.10 (1H, ddd, J = 11.6, 5.2, 2.8
16b,17f,18
2
limited.
In the present investigation, the pyrrole-fused
spiroketal architectures are reported from X. nigripes for the first
time. We herein describe their isolation, structural elucidation,
total syntheses, and their anti-oxidant effects. Regarding to the
total syntheses, the reactions proceeded with considerable
diastereoselectivity to give the target compounds in good yields.
1
13
2
2
. Results and discussion
.1 Isolation and structural elucidation
The commercially fermented mycelium (20 kg) of X. nigripes
(
C-4) and 125.8 (C-3), and one formyl carbon at δ 180.2 (C-7)],
and three quaternary carbons [one ketal at δ 96.9 (C-9), and two
olefinic at δ 132.4 (C-2) and 137.1 (C-5)].
was extracted with 75% EtOH (20 L) at room temperature three
times to afford a brown residue (1.3 kg, semi-dry), which was
The above spectroscopic data (in CD OD) closely resembled
3
suspended in H O (2.0 L) and then successfully extracted with
2
those of capparisine B (Figure 2), a sugar-morpholine spiroketal
pyrrole-derived alkaloid previously isolated from the mature
fruits of Capparis spinosa. The H NMR data of compound 1
petroleum ether (3×1.5 L), EtOAc (3×1.5 L), and n-BuOH (3×1.5
L). The EtOAc extract (281.6 g) was separated by repeated
column chromatography (CC) over silica gel, Sephadex LH-20,
3
1

3
3
and capparisine B are found to be identical wh
A
en
C
us
C
in
E
g
P
th
T
e s
ED MANUSCRIPT
am
e
NMR solvent (CDCl ) (Table 1), indicating both should have the
3
same planar structure with the same relative configuration. The
framework of 1 was further confirmed by detailed analyses of 2D
NMR spectra (COSY, HSQC, HMBC and NOESY) (Figure 3).
25
However, the optical rotation value ([α]D −189 (MeOH)) of
compound 1 was found to be quite different from that of
2
5
capparisine B ([α]
+38 (MeOH)) with a (9R*,11R*,12S*)
D
3
configuration (Figure 2), suggesting that the two compounds are
enantiomers of each other. The absolute configuration
(
(
9S,11S,12R) of 1 was finally confirmed by single-crystal X-ray
CuKα) diffraction analysis (Figure 4 and Supplementary data).
Scheme 1. Retrosynthetic analysis for the spiro-alkaloids.
In our first synthetic route, the synthesis of 10 commenced
with the commercially available (R)-2,2-dimethyl-1,3-dioxolane-
4
-carbaldehyde (9) which was converted to allylic alcohol 10 as
an inseparable diastereomeric mixture (dr = 5:3, 86% yield) by
2
0
reaction with allylmagnesium chloride (a Grignard reagent).
After the alcohol 10 was protected as its tert-butyldimethylsilyl
TBS) ether (11) in 88% yield, epoxidation of 11 with m-
chloroperoxybenzoic acid (m-CPBA) gave 12 (70% yield).
(
Figure 3. Key COSY, HMBC and NOE correlations of 1.
21
Subsequent N-alkylation of the substituted pyrrole 7 via
opening of the epoxide in 12 with KOH/MeOH afforded 13 in a
22
satisfactory yield (60%). Oxidation of the alcohol 13 with Dess-
Martin periodinane (DMP) furnished the ketone 14 in 92% yield.
The
precursor
14
underwent
an
intramolecular
10,23
spiroketalization
in the presence of moderate acidic
conditions to deliver a mixture of compounds 1, 2 and 4, together
with another three new diastereoisomers (1a, 1b and 2a) (Figure
1
and Scheme 2). Separation of the above six diastereoisomeric
spiro-alkaloids was successfully achieved by repeated
chromatography on silica gel and followed by semi-preparative
HPLC (Figure 5).
Figure 4. X-ray crystal structure of 1.
Xylapyrrosides A1 (1a) and A2 (1b) were both found by
HREIMS to have the same molecular formula (C H NO ) as 1.
2
.2 Total synthesis
12
15
5
1
13
They also exhibited quite similar H and C NMR data to those
of 1, and only slight differences around the deoxyketohexose
moieties (from C-10 to C-13) could be observed. Further detailed
comparison of their COSY and HMBC spectroscopic data (See
Supplementary data) with those of 1 revealed that these three
compounds possess the same planar structure (Scheme 2) but
different relative configurations that can be easily deduced by the
observed coupling constants and the NOE correlations. For
Since 2011, total syntheses of such sugar-morpholine[6,5]-
spiroketal pyrrole-derived alkaloids with a furanose ring, e.g.,
acortatarin B, pollenopyrroside B/ acortatarin A and its 9-epimer
(
2, 9-epi-acortatarin A), have been carried out by several research
9-13
groups. However, the total synthesis of such structures with a
pyranose ring (e.g., 1) has so far not been achieved. Only in a
total synthetic work towards the synthesis of acortatarin A, a
presumed [6,6]-spiro compound was mentioned as a minor
13
compound 1a, the large coupling constants (JH-10,11 = 11.6 Hz; JH-
impure byproduct. Therefore, we sought a process to synthesize
such spiro-alkaloids with a pyranose ring to provide enough
material for further in vivo bioactivity assays such as anti-
depression evaluation.
=
10.7 Hz) indicated both H-11 (δ 3.89) and H-12 (δ 3.50)
12,13
took axial orientations (i.e., the hydroxyl groups were in opposite
orientations and both took equatorial positions). Clear NOE
correlations of H-10ax (δ 1.67)/H -8 (δ 4.50, 4.04), H-10 /H-12
ax
2
ax
From a retrosynthetic perspective, the [6,6]- (e.g., 1) and [6,5]-
e.g., 2) spiroketals could be anticipated and synthesized using
(
δ 3.50), H-10eq (δ 2.23)/H-11ax (δ 3.89), and H-11 / H-13 (δ
ax ax
(
3
.42) were then observed. In a similar way, the relative
the same strategy (Scheme 1), because they would all rely on the
spiroketalization of the intermediate 5. This precursor was
envisioned to be available from a protected 2-formyl-5-
hydroxymethyl-pyrrole (7) by N-alkylation with the epoxide 6,
which in turn could be generated from the commercially
configuration of compound 1b was determined as depicted in
Scheme 2. The absolute configuration of 1a was finally
unambiguously established as (9R,11R,12R) by CuKα X-ray
crystallographic analysis (Figure 6 and Supplementary data).
8,19
available aldehyde 8.

4
Considering that the chiral center at C-12 of the above six
ACCEPTED MANUSCRIPT
synthesized spiro-alkaloids was introduced by the initial
chemical (R)-9, it is reasonable that the absolute configuration at
C-12 should be definitively assigned as R (Scheme 2). Therefore,
the absolute configuration at C-11 of both 1b and 2a could be
easily determined to be R based on the above analysis of their
relative configurations. However, due to the shortage of single-
crystal data, determination of the absolute configuration at C-9 in
1
b and 2a seems to be challenging. Interestingly, after careful
analysis of the optical rotations of compounds 1, 1a, 2−4, and
3-5
their diastereoisomers reported in the literature, we reasoned
that the absolute configuration at the spiro-center (C-9) would be
the key factor to influence the sign (positive or minus) of the [α]D
values. In general, such sugar-morpholine spiroketal pyrrole-
derived alkaloids with a 9R configuration all exhibit positive [α]
values (1a: +132; 3: +65; 4: +255; capparisine B : +38), while
D
3
minus values were observed only for those compounds with a 9S
configuration (1: [α]D −189; 2: [α] −187). Accordingly, the
D
absolute configuration at C-9 in 1b ([α]D −241) and 2a ([α]D
+
225) could be assigned as 9S and 9R (Figure 1), respectively.
The above synthetic route could efficiently afford the target
Scheme 2. Route1: Synthesis of 1, 2, 4, and related spiro-alkaloids (1a, 1b,
a).
compounds (only six steps from the commercially available 9),
along with three new analogues (1a, 1b, 2a) for potential
structure-activity relationship (SAR) studies. In Scheme 2, TBS
was employed to protect the secondary hydroxyl group in 10,
yielding an inseparable epimeric mixture of 11 (Schemes 2 and
2
0.20
0.15
3
). Interestingly, when a more rigid protective group (e.g.,
0
.10
benzyl) was used instead of TBS, an optical pure product 15
could be obtained as the major product that can be easily
separated from its epimer by silica gel CC (Scheme 3). Thus, we
0.05
0.00
explored
a second synthetic strategy, which successfully
0
.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
?
?
furnished the [6,6]-spiroketal 1 (Scheme 4) or [6,5]-spiroketals 2
and 4 (Scheme 5) with much higher steroselectivity.
Figure 5. HPLC-PDA profiles of the six synthesized spiro-alkaloids
separated on a Fluophase PFP column (7.7 × 250 mm, 5 µm, Thermo): 1 (t
R
=
=
2
2
2
5.1 min), 1a (t
7.0 min), and 4 (t
99 nm. The elution program consisted of a linear gradient of methanol in
R R R R
= 29.5 min), 1b (t = 19.4 min), 2 (t = 26.3 min), 2a (t
R
= 20.1 min). The PDA detector measured absorbance at
water from 25% to 35% in 35 min, then followed by an isocratic elution with
1
00% methanol for 15 min (flow rate: 2.0 mL/min).
Scheme 3. Different protected groups on the secondary hydroxyl of 10.
After cleavage of the acetonide by 60% (v/v) TFA, the diol 16
was furnished in 88% yield, and different protective groups were
then employed to construct [6,6]- and [6,5]-spiroketals. As
shown in Scheme 4, the primary hydroxyl group in 16 was
protected with TBS (17, 85% yield) and the secondary hydroxyl
group was subsequently converted to the benzyl ether 18 (90%
yield), which underwent a few steps to proceed the target [6,6]-
spiroketal (Scheme 4). In contrast, the [6,5]-spiroketals were
constructed by protecting the primary hydroxyl in 16 with benzyl
ether (24, 75% yield) and the secondary hydroxyl with TBS ether
(
25, 82% yield) followed by a series of reactions (Scheme 5).
Figure 6. X-ray crystal structure of 1a.
Actually, subsequent epoxidation of terminal olefins provided 19
and 26 each in 75% yield. Ring opening of the epoxides in the
presence of cerous chloride heptahydrate and sodium iodide gave
20 and 27 each in 80% yield, and then the oxidation of both
with DMP furnished ketones 21 and 28 each in 90% yield.
Xylapyrroside B (2a) was also assigned a molecular formula
1
of C H NO as determined by HR-EIMS, identical with those of
12
15
5
1
13
24
compounds 1−4. The H and C NMR spectra of 2a resembled
8-12
those of 2 and 4, in which the deoxyhexose moiety existed as a
furanose ring instead of a pyranose ring in 1. This was supported
by the observation of a key HMBC correlation between H-12 (δ
Treatment of α-iodo ketones and pyrrolederivative 7 with K CO
2 3
25
in DMF afforded 22 and 29 each in 87% yield. Compound 22
experienced an intramolecular spiroketalization under acidic
condition (4N HCl) together with the cleavage of THP and TBS
groups, giving 23 as the sole product in a 90% yield, which was
then treated with TiCl in CH Cl to afford 1 (70%) (Scheme 4).
Interestingly, the same treatment on compound 29 resulted in a
pair of C-9 epimers (30, dr = 2:1) that finally afforded 2 (37%)
4
.14) and C-9 (δ 103.6), as well as the lack of correlation from
H -13 (δ 3.83 and 3.78) to C-9 (See Supplementary data).
Detailed comparison of the NMR data of 2a with those of 4
suggested that the only difference was the orientation of the
hydroxy group at C-11; thus 2a was assumed to be the 11-epimer
of 4.
2
4 2 2

5
and 4 (32%) (Scheme 5). In our opinion,
A
t
C
he
C
la
E
rg
P
e
TED
ric MANUSCRIPT
s
te
hindrance, which may be explained by considering the two
possible transition states during the acid catalyzed
26,27
spiroketalisation of 29,
was unfavorable for the production of
9
R-epimer of 23 by spirocyclization and hence compound 3
could not be obtained. This phenomenon coincided in both
synthetic strategies (Schemes 2 and 4), which was also consistent
12
with the previous synthetic work by Teranishi et al.
Scheme 5. Route 2: Stereoselective synthesis of 2 and 4.
Figure 7. Effects of tested compounds on cell viability of tBHP-stimulated
Scheme 4. Route 2: Stereoselective synthesis of 1.
A7r5 cells. Data were generated from at least three independent experiments,
and eachperformed in triplicate. p< 0.05 vs unstimulated cells, p< 0.05 vs
#
*
tBHP-stimulated cells.
2
.3 Antioxidant evaluation
Considering that acortatarin A (4) was previously reported to
3. Conclusions
In this study, four diastereoisomeric alkaloids (1−4) with a
unique pyrrole-fused morpholine spiroketal architecture, were
isolated and identified from the commercially fermented product
(Wuling Powder) of X. nigripes for the first time. The concise
and efficient total syntheses of 1, 2, 4 and their analogues were
achieved by two alternative strategies. Compared with the known
5,7
have antioxidant activity, the obtained spiro-alkaloids (except
the mass-limited 3) were also evaluated for their antioxidant
effects on preventing the oxidative stress-induced cytotoxicity of
A7r5 rat vascular smooth muscle cells (VSMCs). Indeed,
oxidative stress, generated by excessive reactive oxygen species
8-12
(
ROS), is an important trigger of VSMC apoptosis and has been
procedures,
our synthetic procedures possess the following
28
implicated in the pathogenesis of cardiovascular disorders. As
advantages: lower-cost starting material, simple preparation
process and higher total yield. Particularly, the total synthesis
towards the spiro-alkaloids with a pyranose ring (in the case of 1,
1a and 1b) was reported herein for the first time. The successful
total syntheses could not only unambiguously confirm the
absolute configurations of the new isolates, but also afford
enough samples for future biological screenings. In fact, in order
to support the traditional application of Wuling Powder in
shown in Figure 7, tert-butyl hydroperoxide (tBHP), a lipid-
29
soluble source of peroxide radicals employed in this study,
caused 40.4% cell death of VSMCs at a concentration of 200 µM.
Interestingly, this oxidative stress-induced cytotoxicity was
found to be remarkably attenuated by pretreating VSMCs with
the spiro-alkaloids and an antioxidant flavonoid (+)-catechin
30
hydrate (positive control). Compared with the tBHP-group, all
the tested compounds at 100 µM exhibited significant preventive
effects against the tBHP-induced cytotoxicity in VSMCs with
viability in the range of 85.3−103.8%. Among them, compounds
15,16a,17
treating anxiety disorders and depression,
the well-
established mouse behavioral despair tests (i.e., the tail
31
32
suspension test and the forced swim test ) will be used for
evaluating the anti-depressant activity of the enriched spiro-
alkaloids 1, 2 and 4. In the present study, the spiro-alkaloids 4
and 1b could significantly inhibit the tBHP-induced apoptosis in
VSMCs, which would provide new insights into deeper
exploration of the unique spiro-alkaloids in drug discovery of
cardiovascular diseases, and future studies on the mode of the
antioxidant action are needed.
4
and 1b were found to show the most potent anti-oxidative
stress activities, i.e., they both could significantly suppress the
tBHP-induced apoptosis in VSMCs at all concentrations tested
(25, 50, and 100 µM). Moreover, the antioxidant effects of all the
spiro-alkaloids were dose-dependent and reached the maximum
at 100 µM.
4
4
. Experimental Section
.1 General information

6
1
9
U
10
20
11
Optical rotations were measured on a
A
J
C
AS
C
C
E
O
P
P
T
-1
E
0
D
20 MA[α
N
]
−
S
1
C
11
R
(c
I
1.0, MeOH); lit. : [α] −85 (c 0.2, MeOH); lit. :
D
P
T
D
23
polarimeter. UV and IR spectra were recorded on a Shimadzu
UV-2550 and an Avatar 360 ESP FTIR spectrometer,
respectively. NMR spectra were recorded on a Varian Mercury
Plus 400 MHz and Bruker Avance III 400 or 600 MHz
spectrometers. Chemical shifts are expressed in δ (ppm) and
referenced to the residual solvent signals. ESI-MS were measured
on Waters UPLC H ClassSQD and Agilent 1100 series mass
spectrometers; EI-MS was obtained from an Agilent 5975c mass
spectrometer. HR-ESI-MS and HR-EI-MS were measured on
Bruker Daltonics micrOTOF-QII or Waters Gct Premier mass
spectrometers. Semi-preparative HPLC was performed on a
Waters e2695 system coupled with a 2998 Photodiode Array
Detector (PDA) and a 2424 Evaporating Light Scattering
Detector (ELSD), and a Fluophase PFP column (7.7 × 250 nm, 5
µm, Thermo). Column chromatography (CC) was performed
using silica gel (200-300 mesh, Kang-Bi-Nuo Silysia Chemical
Ltd., Yantai, China) and Sephadex LH-20 (GE Healthcare Bio-
Sciences AB, Uppsala, Sweden). Silica gel-precoated plates
[α] −73 (c 0.05, MeOH)]; UV (MeOH) λ (log ε) 254 (0.36),
D
max
-
1
1
296 (1.36) nm; IR (film) ν : 3336, 1644, 1452, 1046 cm ; H
max
1
3
8
and C NMR data (in CD OD) see lit. ; (+) ESIMS m/z 254 [M +
3
+
+
H] ; HREIMS m/z 253.0951 [M] (calcd for C H NO ,
1
2
15
5
253.0950, ∆ = 0.4 ppm).
4
22
Pollenopyrroside A (3): colorless gum; [α]
MeOH) [lit. : [α]
data (in CD OD) are identical with those of literature; (+)
ESIMS m/z 254 [M+H] .
+65 (c 0.01,
13
D
4
20
1
+126 (c 0.08, MeOH)]; H and C NMR
D
5
3
+
5
5,8-12
Pollenopyrroside B (= acortatarin A,
4): white powder;
8
22
5
27
[α] +255 (c 0.1, MeOH) [lit. : [α] +178 (c 0.4, MeOH); lit. :
D
D
27
9
19
[α]
+191 (c 0.27, MeOH); lit. : [α]
+200 (c 0.4, MeOH);
D
11 23
D
10
27
lit. : [α]
+195 (c 0.15, MeOH); lit. : [α]
+185 (c 0.15,
D
D
12
24
MeOH); lit. : [α]
(log ε) 254 (0.58), 296 (2.01) nm; IR (film) ν : 3326, 1644,
1452, 1035 cm ; H and C NMR (in CD OD) data see lit. ; (+)
ESIMS m/z 254 [M + H] ; HREIMS m/z 253.0949 [M] (calcd
for C H NO , 253.0950, ∆ = −0.4 ppm).
+190 (c 0.28, MeOH)]; UV (MeOH) λmax
D
max
-
1
1
13
5,8
3
+
+
(GF254, 0.25 mm, Kang-Bi-Nuo Silysia Chemical Ltd., Yantai,
1
2
15
5
China) were used for TLC detection. Spots were visualized using
UV light (254 nm) and 15% H SO -EtOH. All reactions were
4
1
.3 Synthesis
-((R)-2,2-Dimethyl-1,3-dioxolan-4-yl)but-3-en-1-ol (10): To a
2
4
carried out under an atmosphere of nitrogen or argon unless
otherwise specified. All solvents used for column
chromatography were of analytical grade (Shanghai Titan Chem
Co. Ltd, Shanghai, PR China), and solvents used for HPLC were
of HPLC grade (Jiangsu Hanbon Sci. & Tech. Co. Ltd., PR
China). All commercially available reagents including the
substrates were used as received.
o
cooled (-78 C) solution of commercial (R)-2,2-dimethyl-1,3-
dioxolane-4-carbaldehyde 9 (5.0 g, 38.5 mmol) in anhydrous
THF (100 mL), compound 17 (351 mg, 1.0 mmol) was treated
with a solution of allylmagnesium chloride in THF (30 mL, 51
mmol, 1.7 M). After being stirred for 3h, the mixture was
quenched with a saturated NH Cl aqueous solution and warmed
4
to room temperature. Then the mixture was extracted with EtOAc
4
.2 Isolation of compounds 1−4
(3 × 50 mL) and the combined organic layers were washed with
The commercially fermented mycelia of X. nigripes, also
brine. Dried, filtrated and concentrated, the residue was purified
by flash chromatography on silica gel (PE/EtOAc = 10/1) to give
an inseparable epimer mixture of 10a and 10b (5.68 g, 86%; dr =
called Wuling Powder, were produced by Zhejiang Jolly
Pharmaceutical Company (Deqing County in Zhejiang Province,
PR China). In September 2008, the crude samples were dried on
site and then shipped to the laboratory, where they were
lyophilized upon arrival. A 20 kg aliquot was extracted with 75%
EtOH (3 × 20 L) at room temperature to afford a brown residue
2
2
5:3) as a pale yellow oil. [α]
= –25.2 (c 0.029, CHCl ); IR
3
-1 1
D
(film) νmax: 3402, 2909, 2345, 2328, 1397, 1052, 663 cm ; H
NMR (400 MHz, CDCl ) data of 10a (major product): δ 5.85
3
(m), 5.18 (m), 5.13 (m), 4.03 (m), 3.94 (m), 3.78 (m), 2.34 (m),
2.23 (m), 1.44 (s), 1.37 (s) ppm; 10b (minor product): δ5.85 (m),
5.15 (m), 4.03 (m), 3.76 (m), 3.60 (m), 2.23 (m), 2.18 (m), 1.45
(1.3 kg), which was suspended in H O (2.0 L) and then
2
successively extracted with petroleum ether (PE, 3×1.5 L),
EtOAc (3×1.5 L) and n-BuOH (3×1.5 L). The EtOAc-soluble
fraction (281.6 g) was subjected to silica gel CC (PE/EtOAc,
from 20/1 to 2/1, v/v; CH Cl /MeOH, from 20/1 to 2/1, v/v) to
give fifteen fractions (Fr. 1-15). Fr. 11 (3.95 g) was separated by
a silica gel column (CH Cl /MeOH, from 50/1 to 30/1, v/v) to
13
(s), 1.38 (s) ppm; C NMR (100 MHz, CDCl ) data of 10a: δ
3
134.0, 118.3, 109.1, 78.0, 70.3, 65.2, 37.6, 26.5, 25.2 ppm; 10b:
δ 134.0, 117.9, 109.4, 78.5, 71.6, 66.0, 38.2, 26.6, 25.3 ppm;
2
2
+
HREIMS m/z 172.1097 [M] (calcd for C H O , 172.1099, ∆ =
9
16
3
2
2
−1.2 ppm).
give six sub-fractions (Fr.11A-11F). Fr. 11B (200 mg) was
repeatedly chromatographed on silica gel (CH Cl /MeOH 50:1,
v/v) followed by gel permeation chromatography (GPC) on
Tert-Butyl((1-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)but-3-en-1-
yl)oxy)dimethylsilane (11): 2,6-Lutidine (5.3 ml, 45.3 mmol)
was dropped to a stirred solution of alcohol 10 (3.9 g, 22.7
mmol) in dry CH Cl (50 mL) at 0 °C, then TBSOTf (7.8 mL,
2
2
Sephadex LH-20 (MeOH) to furnish compound 4 (20.1 mg). Fr.
2
2
1
1C (140 mg) was first subjected to CC over silica gel
3
4.1 mmol) was dropped and the reaction was stirred 2 h. The
(
CH Cl /MeOH 50:1, v/v) and was finally refined by GPC on
2
2
mixture was quenched with water and separated. The aqueous
layer was extracted with EtOAc (3 × 50 mL) and the combined
organic layers were washed with brine. Dried, filtrated and
concentrated, the residue was purified by flash chromatography
Sephadex LH-20 (MeOH) to afford compound 1 (4.0 mg).
Compounds 2 (21.3 mg) and 3 (1.2 mg) were isolated and
purified from Fr. 11D (550 mg) by semi-preparative HPLC on a
Fluophase PFP column (MeOH/H O, from 25:75 to 35:65 in 40
2
on silica gel (PE/EtOAc = 40/1) to give an inseparable epimer
min, v/v; flow rate: 1.5 mL/min; 2: t = 34.6 min, 3: t = 33.0
R
R
22
mixture of 11a and 11b (dr = 5:4). Colorless oil; [α] = +24.7
D
min).
(
c 0.049, CHCl ); IR (film) νmax: 2942, 2368, 2334, 1441, 1265,
3
-
1 1
Xylapyrroside A (= ent-capparisine B, 1): colorless crystal
1090, 821,646 cm ; H NMR (400 MHz, CDCl ) data of 11a: δ
3
22
(
acetone); [α] −189 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
5.84 (m), 5.09 (m), 5.05 (m), 3.96 (m), 3.81 (m), 2.29 (m), 1.39
(s), 1.33 (s), 0.88 (s), 0.07 (s), 0.06 (s); 11b: δ 5.83 (m), 5.04 (m),
4.05 (dd, J = 13.1, 6.6 Hz), 3.96 (m), 3.74 (m), 2.27 (m), 2.10
(ddd, J = 14.0, 7.5. 7.0 Hz), 1.41 (s), 1.34 (s), 0.88 (s), 0.07 (s),
D
2
54 (0.62), 296 (2.11) nm; IR (film) νmax: 3408, 1660, 1474, 1036
-1 1 13
cm ; H and C NMR data see Table 1; (+) ESIMS m/z 254 [M
+
+
+
H] ; HREIMS m/z 253.0952 [M] (calcd for C H NO ,
12 15 5
13
2
53.0950, ∆ = 0.8 ppm).
0.05 (s) ppm; C NMR (100 MHz, CDCl ) data of 11a: δ 134.1,
3
8
-12
117.5, 108.8, 77.8, 72.1, 66.1, 39.2, 26.7, 25.9×3, 25.5, 18.1, -
4.2, -4.5; 11b: δ 135.1, 117.1, 109.1, 78.4, 73.1, 65.4, 37.5,
Xylapyrroside B (= 9-epi-acotatarin A, 2): colorless gum;
[
22
8
27
9
α] −187 (c 0.1, MeOH) [lit. : [α] −58 (c 0.04, MeOH); lit. :
D D

7
2
3
5.8×3, 26.5, 25.3, 18.2, –4.4, –4.6 ppm; (+
A
)
C
HR
C
E
E
SI
P
M
T
S
E
m
D
/z MA1H
N
),
U
6.
S
30
C
(d
R
,
I
J
P
= 3.9 Hz, 1H), 5.28 (d, J = 11.2 Hz, 1H, CH N),
a
T
+
09.1859 [M+Na] (calcd for C H O SiNa, 309.1862, ∆ = −0.9
4.60 (d, J = 11.2 Hz, 1H, CH N), 4.60-4.32 (m, 3H), 4.24-4.10
15
30
3
b
ppm).
(m, 1H), 4.00 (m, 2H), 3.80 (m, 2H), 3.50 (m, 1H), 2.88-2.82 (m,
1
6
H), 2.75-2.60 (m, 1H), 1.83-1.48 (m, 6H), 1.36-1.32 (2×CH3,
H), 0.85 (s, 9H; C(CH ) ), 0.10-0.09 (Si(CH ), 3H), 0.06-0.04
Tert-Butyl(1-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-(oxiran-2-
yl)ethoxy)dimethylsilane (12): To a cooled solution (0 °C) of
the ether 11 (3.0 g, 10.5 mmol) in dry CH Cl (30 mL) was added
m-CPBA (2.17 g, 12.6 mmol) in one portion. After stirring for
overnight at r.t, the reaction was quenched with a solution of
3
3
3
13
(Si(CH ) , 3H) ppm; C NMR (100 MHz, CDCl ): δ 202.1,
3
2
3
2
2
2
1
1
7
6
5
2
1
02.0, 201.7, 201.6, 179.8 (3C) 178.3, 138.9, 138.8 (2C), 138.7,
32.5 (3C), 131.1, 123.7 (2C), 123.6, 122.3, 111.8, 111.7 (2C),
11.6, 109.2 (3C), 107.9, 97.2, 97.1, 97.0, 95.9, 78.6, 78.5, 77.3,
7.2, 76.0, 75.9, 75.6, 75.3, 68.9, 68.7, 67.8, 67.7, 66.6, 65.2,
5.0, 64.9, 62.5 (2C), 62.4, 62.3, 59.3 (2C), 59.2 (2C); 55.2 (2C),
5.1, 53.8, 45.3, 45.2, 43.0, 42.8, 30.1 (3C), 28.9, 26.3 (2C),
6.2, 25.7 (7C), 25.1 (3C), 24.9, 24.8, 24.3, 19.4, 19.3, 19.2,
saturated NaHSO aqueous solution and stirred for 10 min. Then
3
the mixture was separated and the aqueous layer was extracted
with CH Cl (3× 50 mL). The combined organic layers were
2
2
washed with saturated aqueous NaHCO3 solution and brine.
Dried, filtrated and concentrated, the residue was purified by
flash chromatography on silica gel (PE/EtOAc = 20/1) to give 12
9.1, 17.9 (2C), 17.3 (2C), –4.5 (2C), –4.9 (2C), –5.0 (4C) ppm;
+
HREIMS m/z 509.2811 [M] (calcd for C H NO Si, 509.2809,
∆ = 0.4 ppm).
26
43
7
(
2.2 g, 70%; containing four inseparable diastereomers) as
22
colorless oil. [α] = +60 (c 0.015, CHCl ); IR (film) νmax: 2920,
2
D
3
-
1
1
854, 2372, 1386, 1265, 1073, 838 cm ; H NMR (400 MHz,
Xylapyrrosides A (1), B (2) and their analogues (Method 1):
To compound 14 (3.9 g, 7.7 mmol) in THF (50 mL) was dropped
a solution of 4N HCl (7.8 mL, 30.8mmol) at 0 °C. After stirring
for 4h, the mixture was carefully neutralized with a saturated
aqueous NaHCO solution and extracted with EtOAc (4 × 50
mL). The combined organic extracts were washed with brine,
CDCl ): δ 4.14-3.69 (m, 4H), 3.15-3.04 (m, 1H), 2.83-2.76 (m,
3
1
1
6
1
6
3
2
H), 2.55-2.46 (m, 1H), 1.84-1.54 (m, 2H), 1.41-1.40 (Me, 3H),
.35-1.34 (Me, 3H), 0.90 [Si-C-(CH ) , 9H], 0.12-0.07 [Si(CH ) ,
3
3
3 2
13
H] ppm; C NMR (100 MHz, CDCl ): δ 109.2, 109.1, 109.0,
3
3
08.9; 78.3, 78.3, 78.3, 78.2; 71.5, 70.9, 70.7, 70.7; 66.5, 66.3,
5.4, 65.1; 49.5, 49.3, 48.8, 48.6; 47.9, 47.5, 46.7, 46.6; 38.0,
7.6, 36.3, 35.5; 26.7, 26.5, 26.4, 26.3; 25.7×6, 25.6×6; 25.3,
dried over MgSO , filtrated and concentrated. Analysis of the
4
product by HPLC with a Fluophase PFP column (a linear
gradient of MeOH in water from 25% to 35% over 35 min, then
followed by isocratic elution with 100% methanol; flow rate: 2.0
mL/min) clearly revealed the presence of six compounds (Figure
5.1, 25.0, 24.3; 18.0, 18.0, 17.9, 17.9; –4.4, –4.5, –4.7, –5.0
+
ppm; HREIMS m/z 302.1911 [M] (calcd for C H O Si,
15
30
4
3
02.1913, ∆ = −0.7 ppm).
6
, detection by PDA at 299 nm), which were isolated by repeated
1
-(4-((tert-Butyldimethylsilyl)oxy)-4-((R)-2,2-dimethyl-1,3-
chromatography on silica gel and semi-preparative HPLC with
the Fluophase PFP column. The synthesized xylappyrosides A (1)
and B (2), and pollenopyrroside B (4) showed physical and
spectroscopic characteristics equivalent to the natural products.
dioxolan-4-yl)-2-hydroxybutyl)-5-(((tetrahydro-2H-pyran-2-
yl)oxy)methyl)-1H-pyrrole-2-carbaldehyde (13): To
suspension of KOH (37 mg, 0.66 mmol) in dry MeOH (5 mL)
was added pyrrole 7 (0.55 g, 2.63 mmol; for preparation
procedurs, see Supplementary data) under N atmosphere at r.t.
a
2
2
Xylapyrroside A (1a): colorless crystal (acetone); [α] +132
1 D
2
Then a solution of epoxide 12 (1.0 g, 3.3 mmol) in MeOH was
dropped and the reaction was refluxed for 48h. After cooling, the
mixture was quenched with water and extracted with EtOAc (4×
(c 0.1, MeOH); UV ( MeOH) λmax (log ε) 254 (0.59), 295 (2.15)
nm; IR (film) νmax: 3375, 1633, 1402, 1024 cm ; H NMR (400
-1 1
MHz, CD OD): δ 9.38 (s, H-7), 7.04 (d, J = 4.0 Hz, H-3), 6.09
3
5
0 mL). The combined organic layers were washed with brine,
(d, J = 4.0 Hz, H-4), 4.86 (d, J = 15.8 Hz, H-6 ), 4.78 (d, J = 15.8
a
dried over MgSO , filtered and concentrated. The crude was
Hz, H-6 ), 4.50 (d, J = 14.0 Hz, H-8 ), 4.04 (d, J = 14.0 Hz, H-
4
b
a
purified by chromatography on silica gel (PE/EtOAc = 8/1) to
8b), 3.89 (ddd, J = 11.2, 8.8, 5.2 Hz, H-11), 3.73 (dd, J = 10.4, 5.2
give 13 (0.93 g, 55%; containing eight inseparable diastereomers)
Hz, H-13 ), 3.50 (ddd, J = 10.4, 8.8, 5.2 Hz, H-12), 3.42 (dd, J =
a
22
as a pale yellow oil. [α] = +10.5 (c 0.1, CHCl ); IR (film) νmax:
10.4, 10.4 Hz, H-13 ), 2.23 (dd, J = 13.2, 5.2 Hz, H-10 ), 1.67
D
3
b
a
-
1
1
13
3
254, 2904, 2849, 1654, 1189, 1008, 767 cm ; H NMR (400
(dd, J = 13.2, 11.2 Hz, H-10b); C NMR (100 MHz, CD OD): δ
3
MHz, CDCl ): δ 9.50–9.49 (CHO, 1H), 6.92 (d, J = 3.9 Hz, 1H),
132.4 (C-2), 126.0 (C-3), 106.2 (C-4), 136.9 (C-5), 58.2 (C-6),
3
6
3
0
5
.29 (d, J = 3.8 Hz, 1H), 4.91-4.51 (m, 4H), 4.29-3.68 (m, 6H),
180.2 (C-7), 53.1 (C-8), 96.7 (C-9), 41.2 (C-10), 72.3 (C-11),
1
.62-3.06 (m, 2H), 1.88-1.53 (m, 8H), 1.41-1.33 (2×CH , 6H),
70.0 (C-12), 65.2 (C-13); H NMR (400 MHz, Acetone-d ): δ
3
6
.87 (C(CH ) , 9H), 0.14-0.05 (Si(CH ) , 6H) ppm; HREIMS m/z
9.46 (s, H-7), 6.97 (d, J = 4.0 Hz, H-3), 6.06 (d, J = 4.0 Hz, H-4),
4.86 (d, J = 15.6 Hz, H-6 ), 4.76 (d, J = 15.6 Hz, H-6 ), 4.49 (d, J
3
3
3 2
+
11.2968 [M] (calcd for C H NO Si, 511.2965, ∆ = 0.6 ppm).
26
45
7
a
b
=
14.0 Hz, H-8 ), 4.01 (d, J = 14.0 Hz, H-8 ), 3.87 (ddd, J = 11.6,
a
b
1
-(4-((tert-Butyldimethylsilyl)oxy)-4-((R)-2,2-dimethyl-1,3-
8
.8, 5.6 Hz, H-11), 3.68 (dd, J = 10.8, 5.2 Hz, H-13 ), 3.51 (ddd,
a
dioxolan-4-yl)-2-oxobutyl)-5-(((tetrahydro-2H-pyran-2-
yl)oxy)methyl)-1H-pyrrole-2-carbaldehyde (14): To a cooled
solution (0 °C) of alcohol 13 (2.4 g, 4.7 mmol) in dry CH Cl (30
mL) was added DMP (3.98 g, 9.4 mmol) in one portion and
stirred for 3h. Then the reaction was carefully quenched with a
solution of mixture (V/V = 50: 50) saturated aqueous NaHCO3
and Na S O solution until the mixture turned a clear solution.
Then the resulting mixture was separated and the the aqueous
layer was extracted with CH Cl (2× 50 mL). The combined
organic layers were washed with brine, dried over MgSO4,
filtrated and concentrated. The residue was purified by flash
chromatography on silica gel (PE/EtOAc = 8/1) to obtain the
ketone 14 (2.2 g, 92%; containing four inseparable
J = 10.4, 8.8, 5.2 Hz, H-12), 3.38 (dd, J = 10.8, 10.4 Hz, H-13b),
2
1
4
.20 (dd, J = 13.2, 5.6 Hz, H-10 ), 1.68 (dd, J = 13.2, 11.6 Hz, H-
0 ); H NMR (400 MHz, CDCl ): δ 9.43 (s, H-7), 6.90 (d, J =
.0 Hz, H-3), 5.99 (d, J = 4.0 Hz, H-4), 4.79 (d, J = 15.6 Hz, H-
a
2
2
1
b
3
6a), 4.74 (d, J = 15.6 Hz, H-6 ), 4.59 (d, J = 14.4 Hz, H-8 ), 4.03
b
a
(d, J = 14.0 Hz, H-8 ), 4.00 (ddd, overlapped, H-11), 3.80 (dd, J
b
2
2
3
=
10.8, 5.2 Hz, H-13 ), 3.62 (ddd, J = 10.4, 8.8, 5.2 Hz, H-12),
a
3
1
.43 (t-like, J = 10.4 Hz, H-13 ), 2.24 (dd, J = 12.8, 5.2 Hz, H-
0 ), 1.70 (dd, J = 12.8, 11.2 Hz, H-10 ); (+) ESIMS m/z 254
b
2
2
a
b
+
+
[
M+H] ; HREIMS m/z 253.0949 [M] (calcd for C H NO ,
12 15 5
2
53.0950, ∆ = −0.4 ppm).
22
Xylapyrroside A2 (1b): white powder; [α]
MeOH); UV ( MeOH) λmax (log ε) 254 (0.50), 296 (1.86) nm; IR
(film) νmax: 3419, 1633, 1447, 1025 cm ; H NMR (400 MHz,
−241 (c 0.1,
D
22
diastereomers) as a colorless oil. [α] = +13.3 (c 0.07, CHCl );
IR (film) νmax: 3354, 2920, 2843, 2357, 1654, 1068, 663 cm ; H
D
3
-
1
1
-1 1
NMR (400 MHz, CDCl ): δ 9.46 (s, 1H), 6.91 (d, J = 3.9 Hz,
CD OD): δ 9.38 (s, H-7), 7.03 (d, J = 4.4 Hz, H-3), 6.08 (d, J =
3
3

8
4
.4 Hz, H-4), 4.89 (d, J = 16.0 Hz, H-6 ), 4.8
A
2 (
C
d,
C
J
E
=
P
16
T
.0
E
H
D
z, MAla
N
yer
U
w
S
as
C
e
R
xt
I
racted with EtOAc (4 × 30 mL) and the combined
P
T
a
H-6 ), 4.60 (d, J = 14.0 Hz, H-8 ), 4.16 (dd, J = 12.2, 1.6 Hz, H-
organic layers were washed with brine, dried over MgSO ,
b
a
4
1
1
1
3 ), 3.95 (d, J = 14.0 Hz, H-8 ), 3.91 (m, H-11), 3.61 (br d, J =
filtrated and concentrated. The residue was purified by flash
chromatography on silica gel (PE/EtOAc = 2/1) to produce diol
a
b
2.2 Hz, H-13 ), 3.57 (m, H-12),2.20 (dd, J = 14.6, 4.4 Hz, H-
b
13
22
0 ), 1.92 (dd, J = 14.6, 2.8 Hz, H-10 ); C NMR (100 MHz,
16 (0.75 g, 88%) as a colorless oil. [α] = –5.6 (c 0.1, CHCl );
a
b
D
3
-
1 1
CD OD): δ 132.4 (C-2), 125.8 (C-3), 106.1 (C-4), 136.9 (C-5),
IR (film) νmax: 3324, 2888, 1651, 1467, 1359, 1113, 842 cm ; H
3
5
8.6 (C-6), 180.2 (C-7), 53.2 (C-8), 95.3 (C-9), 34.6 (C-10), 68.6
NMR (400 MHz, CDCl ): δ 7.41–7.29 (m, 5H), 5.92 (ddt, J =
3
1
(C-11), 68.0 (C-12), 62.4 (C-13); H NMR (400 MHz Acetone-
17.2, 10.2, 7.1 Hz, 1H), 5.19 (dq, J = 17.2, 1.5 Hz, 1H), 5.14 (br
d, J = 10.2 Hz, 1H), 4.65 (d, J = 11.4 Hz, 1H), 4.55 (d, J = 11.4
,
d6): δ 9.46 (s, H-7), 6.97 (d, J = 4.0 Hz, H-3), 6.06 (d, J = 4.0 Hz,
H-4), 4.92 (d, J = 16.0 Hz, H-6 ), 4.84 (d, J = 16.0 Hz, H-6 ),
1
3
Hz, 1H), 3.88–3.65 (m, 5H), 3.56 (m, 1H), 2.45 (m, 2H) ppm; C
a
b
4
3
.57 (d, J = 14.0 Hz, H-8 ), 4.15 (dd, J = 12.0, 1.2 Hz, H-13 ),
NMR (100 MHz, CDCl ): δ 138.1, 134.4, 128.4 (2C), 127.9,
a
a
3
.93 (d, J = 14.0 Hz, H-8 ), 3.85 (m, H-11), 3.60 (br d, J = 12.0
127.8 (2C), 117.6, 79.9, 72.7, 72.3, 63.5, 34.9 ppm; (+)
b
+
Hz, H-13 ), 3.56 (m, H-12), 2.27 (dd, J = 14.4, 4.0 Hz, H-10 ),
1
HRESIMS m/z 245.1157 [M+Na] (calcd for C H O Na,
b
a
13 18
3
1
.90 (dd, J = 14.4, 3.0 Hz, H-10 ); H NMR (400 MHz, CDCl ):
245.1154, ∆ = 1.2 ppm).
b
3
δ 9.41 (s, H-7), 6.90 (d, J = 4.0 Hz, H-3), 6.00 (d, J = 4.0 Hz, H-
), 4.87 (d, J = 15.6 Hz, H-6 ), 4.82 (d, J = 15.6 Hz, H-6 ), 4.66
(
2R,3S)-3-(Benzyloxy)-1-((tert-butyldimethylsilyl)oxy)hex-5-
4
a
b
en-2-ol (17): To a stirred solution of diol 16 (6.0 g, 27 mmol)
and dimethylaminopyridine (0.82 g, 6.75 mmol) in dry CH Cl
(
d, J = 14.0 Hz, H-8 ), 4.14 (br d, J = 11.6 Hz, H-13 ), 3.99 (d, J
a
a
2
2
=
14.0 Hz, H-8 ), 3.95 (m, H-11), 3.70 (br d, J = 11.6 Hz, H-13 ),
b b
(100 mL) was continuously added triethylamine (7.5 mL, 81
3
1
2
.72 (m, H-12), 2.27 (dd, J = 14.8, 4.0 Hz, H-10 ), 1.94 (dd, J =
a
+
mmol) and tert-butyldimethylsilyl chloride (4.95 g, 32.4 mmol)
at 0 °C. After stirring for overnight at r.t., the reaction was
quenched with water and separated. The aqueous layer was
extracted with CH Cl (3× 100 mL) and the combined organic
4.8, 2.4 Hz, H-10 ); (+) ESIMS m/z 254 [M+H] ; HREIMS m/z
b
+
53.0952 [M] (calcd for C H NO , 253.0950, ∆ = 0.8 ppm).
12
15
5
22
Xylapyrroside B1 (2a): colorless gum; [α]
+225 (c 0.1,
D
2
2
MeOH); UV (MeOH) λmax (log ε) 255 (0.85), 296 (2.54) nm; IR
layers were washed with a solution of 1M KHSO and brine.
4
-
1
1
(film) νmax: 3364, 1644, 1468, 1035 cm ; H NMR (600 MHz,
Dried, filtrated and concentrated, the residual oil was purified by
column chromatography on silica gel (PE/EtOAc = 8/1) to
CD OD): δ 9.39 (s, H-7), 7.04 (d, J = 4.1 Hz, H-3), 6.08 (d, J =
3
22
4
.1 Hz, H-4), 5.00 (d, J = 15.6 Hz, H-6 ), 4.83 (d, J = 15.6 Hz, H-
generate 17 (7.7 g, 85%) as a colorless oil. [α] = –12.3 (c 0.05,
a
D
6b), 4.75 (d, J = 14.0 Hz, H-8 ), 4.53 (ddd, J = 6.9, 4.2, 2.9 Hz,
CHCl ); IR (film) νmax: 3301, 2915, 2849, 2367, 1249, 1079, 832
a
3
-
1
1
H-11), 4.27 (d, J = 14.0 Hz, H-8 ), 4.14 (ddd, J = 6.8, 4.4, 4.2
cm ; H NMR (400 MHz, CDCl ): δ7.37–7.29 (m, 5H), 5.94
b
3
Hz, H-12), 3.83 (dd, J = 12.0, 4.4 Hz, H-13 ), 3.78 (dd, J = 12.0,
(ddt, J = 17.2, 10.2, 7.1 Hz, 1H), 5.17 (br d, J = 17.2 Hz, 1H),
5.10 (br d, J = 10.2 Hz, 1H), 4.65 (d, J = 11.6 Hz, 1H), 4.53 (d, J
= 11.6 Hz, 1H), 3.77 (m, 1H), 3.70 (m, 2H), 3.53 (m, 1H), 2.47
a
6
1
2
5
6
2
.8 Hz, H-13 ), 2.56 (dd, J = 14.5, 6.8 Hz, H-10 ), 2.08 (dd, J =
b
a
13
4.5, 2.9 Hz, H-10b); C NMR (150 MHz, CD OD): δ 132.4 (C-
3
13
), 126.1 (C-3), 106.1 (C-4), 137.5 (C-5), 59.2 (C-6), 180.2 (C-7),
2.7 (C-8), 103.6 (C-9), 47.5 (C-10), 72.1 (C-11), 84.6 (C-12),
(m, 2H), 0.91 (s, 9H), 0.08 (s, 6H) ppm; C NMR (100 MHz,
CDCl ): δ 138.3, 134.8, 128.4 (2C), 127.9 (2C), 127.7, 117.3,
3
+
1.8 (C-13); (+) ESIMS m/z 254 [M+H] ; HREIMS m/z
80.5, 73.8, 72.8, 64.0, 35.6, 25.8 (3C), 18.0, –4.4, –4.6 ppm; (+)
+
+
53.0951 [M] (calcd for C H NO , 253.0950, ∆ = 0.4 ppm).
HRESIMS m/z 359.2019 [M+Na] (calcd for C H O SiNa,
12
15
5
19 32
3
3
59.2018, ∆ = 0.3 ppm).
(R)-4-((S)-1-(Benzyloxy)but-3-en-1-yl)-2,2-dimethyl-1,3-
dioxolane (15): To a stirred solution of alcohol 10 (5.0 g, 29.1
mmol) in anhydrous DMF (50 mL) at 0 °C was added sodium
hydride (2.55 g, 64.0 mmol) in two portions and stirred for 30
min, then benzyl bromide (5.2 mL, 43.6 mmol) was added
dropwise at the same temperature. After being stirred for 1h at 0-
(((2R,3S)-2,3-Bis(benzyloxy)hex-5-en-1-yl)oxy)(tert-butyl)
dimethylsilane (18): To a stirred solution of alcohol 17 (7.5 g,
22.3 mmol) in anhydrous DMF (80 mL) was added sodium
hydride (1.82 g, 44.6 mmol) in three portions at 0 °C and stirred
for 30 min. Then benzyl bromide (4.13 mL, 26.8 mmol) was
added dropwise and the reaction was stirred for 1h at 0-5 °C. The
resulting mixture was poured into ice water. The aqueous layer
was extracted with EtOAc (3× 150 mL) and the combined
organic layers were washed with water, brine, dried over MgSO4,
filtrated and concentrated. The residual oil was purified by
5
°C, the mixture was poured into crushed ice and stirred until
the ice disappear. The resulting mixture was extracted with
EtOAc (3 × 100 mL) and the combined organic layers were
washed with brine, dried over MgSO , filtrated and concentrated.
4
The residue was purified by flash chromatography on silica gel
(
9
PE/EtOAc = 40/1) to give 15 (6.1 g, 80%) and epi-15 (0.7 g,
column chromatography on silica gel (PE/EtOAc = 40/1) to
22
22
.2%). 15 (major product): [α]
= +14.6 (c 0.1, CHCl ); IR
afford 18 (8.5 g, 90%) as a colorless oil. [α] = –29.1 (c 0.06,
D
3
D
-
1 1
(film) νmax: 2975, 1463, 1254, 1210, 909, 734, 684 cm ; H NMR
CHCl ); IR (film) νmax: 2931, 2854, 2359, 2334, 1095, 832, 695
3
-
1
1
(400 MHz, CDCl ): δ 7.45–7.29 (m, 5H), 5.97 (ddt, J = 17.2,
cm ; H NMR (400 MHz, CDCl ): δ 7.37–7.28 (m, 10H), 5.90
3
3
1
1
1
0.1, 7.1 Hz, 1H), 5.22 (dq, J = 17.2, 1.5 Hz, 1H), 5.17 (br d, J =
0.2 Hz, 1H), 4.72 (d, J = 11.4 Hz, 1H), 4.65 (d, J = 11.4 Hz,
H), 4.17 (ddd, J = 6.4, 6.3, 5.9 Hz, 1H), 4.10 (dd, J = 8.1, 6.3
(ddt, J = 17.2, 10.1, 7.1 Hz, 1H), 5.12 (br d, J = 17.2 Hz, 1H),
5.06 (br d, J = 10.1 Hz, 1H), 4.76 (d, J = 11.8 Hz, 1H), 4.65 (d, J
= 11.8 Hz, 1H),4.60 (s, 2H), 3.86 (dd, J = 10.9, 4.2 Hz, 1H), 3.79
(dd, J = 10.9, 5.3 Hz, 1H), 3.68 (m, 1H), 3.60 (ddd, J = 5.6, 5.3,
Hz, 1H), 3.96 (dd, J = 8.1, 6.4 Hz, 1H), 3.64 (dd, J = 10.8, 5.9
Hz, 1H), 2.50 (m, 1H), 2.41 (m, 1H), 1.49 (s, 3H), 1.42 (s, 3H)
ppm; C NMR (100 MHz, CDCl ): δ 138.4, 134.2, 128.3 (2C),
1
3
4.2 Hz, 1H), 2.45 (m, 2H), 0.92 (s, 9H), 0.07 (s, 6H) ppm;
C
13
NMR (100 MHz, CDCl ): δ138.8, 138.6, 135.4, 128.3 (4C),
3
3
1
2
27.8, 127.6 (2C), 117.5, 109.0, 78.9, 77.2, 72.5, 66.4, 35.6, 26.7,
5.4 ppm; (+) HRESIMS m/z 285.1456 [M+Na] (calcd for
127.9 (2C), 127.8 (2C), 127.5, 127.4, 116.9, 80.9, 78.5, 72.7,
+
72.2, 62.8, 35.0, 25.9×3, 18.3, –5.3, –5.4 ppm; (+) HRESIMS m/z
+
C H O Na, 285.1461, ∆ = −1.7 ppm).
449.2487 [M+Na] (calcd for C H O SiNa, 449.2488, ∆ = −0.2
16
22
3
26 38
3
ppm).
(2R,3S)-3-(Benzyloxy)hex-5-ene-1,2-diol (16): To a solution of
1
5 (1.0 g, 3.8 mmol) in CH Cl (10 mL) was dropped 60%
((2R,3S)-2,3-Bis(benzyloxy)-4-(oxiran-2-yl)butoxy)(tert-
butyl)dimethylsilane (19): To a stirred solution of the TBS
protected compound 18 (4.6 g,10.7 mmol) in dry CH Cl (50mL)
2
2
○
trifluoroacetic acid (5 mL) at 0 C and stirred for 2h. Then the
reaction was carefully quenched with saturated aqueous NaHCO3
solution and the solvent was removed in vacuo. The aqueous
2
2
was added m-CPBA (3.2 g, 12.8 mmol) in one portion at 0 °C.

9
After being stirred for overnight at r.t., the react
A
io
C
n w
C
a
E
s q
P
u
T
en
E
ch
D
ed MA26
N
.4
U
, 1
S
9.
C
1,
R
7.
I
8, –5.3, –5.3 ppm; (+) HRESIMS m/z 591.1408
P
T
+
with saturated aqueous NaHSO solution and stirred for another
[M+Na] (calcd for C H IO SiNa, 591.1404, ∆ = 0.7 ppm).
3
26 37
4
1
0min. The resulting mixture was separated and the aqueous
1
2
-((4S,5R)-4,5-Bis(benzyloxy)-6-((tert-butyldimethylsilyl)oxy)-
-oxohexyl)-5-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-
layer was extracted with CH Cl (3 × 100 mL). The combined
organic layers were washed with saturated aqueous NaHCO3
solution and brine. Dried over MgSO , filtrated and concentrated,
the residue was purified by flash chromatography on silica gel
2
2
pyrrole-2-carbaldehyde (22): To a solution of potassium
carbonate (0.48 g. 3.5 mmol) and compound 7 (0.73 g, 3.5 mmol)
in DMF (20 mL) was treated with a solution of ketone 21 (2 g,
4
(PE/EtOAc = 20/1) to give the corresponding epoxide as an
3
.5 mmol) in DMF (5 mL). After stirring for 6 h, the resulting
inseparable epimeric mixture of 19a and 19b (3.6 g, 75%; dr =
2
2
MHz, CDCl ) data assignable to 19a: δ 7.37-7.29 (m), 4.73 (d, J
=
22
mixture was poured into water and extracted with EtOAc (3 × 50
mL). The combined organic layers were washed with brine, dried
over MgSO , filtrated and concentrated. The residual oil was
purified by column chromatography on silica gel (PE/EtOAc =
1
:1). Colorless oil; [α] = +10.9 (c 0.05, CHCl ); IR (film) νmax:
D 3
-1 1
926, 2854, 1380, 1256, 1210, 1106, 778 cm ; H NMR (400
4
3
11.4 Hz), 4.57 (d, J = 11.4 Hz), 4.58-4.48 (m), 4.00 (m), 3.79
22
0/1) to give 22 (1.9 g, 87%, dr = 2:1) as a colorless oil. [α]
=
D
(
(
ddd, J = 9.6, 3.1, 3.0 Hz), 3.50 (d, J = 5.2 Hz), 3.07 (m), 2.79
dd, J = 4.7, 4.2 Hz), 2.51 (dd, J = 5.2, 2.7 Hz), 1.89 (ddd, J =
+
1
27.6 (c 0.033, CHCl ); IR (film) νmax: 3339, 2936, 2849, 1654,
3
-1 1
386, 1024, 832 cm ; H NMR (400 MHz, MHz, CD OD): 9.44
3
1
(
4
4.3, 9.6, 4.4 Hz), 1.57 (ddd, J = 14.3, 7.3, 3.0 Hz), 0.89 (s), 0.07
s), 0.06 (s) ppm; 19b: δ 7.37–7.29 (m), 4.66 (d, J = 11.6 Hz),
.58–4.48 (m), 4.50 (d, J = 12.4 Hz), 4.00 (m), 3.70 (m), 3.53 (d,
J = 5.2 Hz), 3.07 (m), 2.79 (dd, J = 4.6, 4.4 Hz), 2.42 (dd, J =
(s, 0.5H), 9.43 (s, 0.5H), 7.37-7.25 (m, 10H), 7.03 (d, J = 4.0 Hz,
1
5
H), 6.33 (dd, J = 4.0 Hz, 0.5H), 6.32 (dd, J = 4.0 Hz, 0.5H),
.31 (d, J = 16.4, Hz, 1H), 5.27 (d, J = 16.4 Hz, 1H), 4.65-4.40
(m, 7H), 4.12 (m, 1H), 4.06 (m, 1H), 3.79 (m, 1H), 3.54-3.43 (m,
5
1
.0, 2.7 Hz), 1.99 (ddd, J = 14.3, 7.6, 5.1 Hz), 1.65 (ddd, J =
13
3H), 2.91-2.73 (m, 2H), 1.69-1.33 (m, 6H), 0.91 (s, 9H), 0.09–
4.3, 6.1, 3.9 Hz), 0.90 (s), 0.08 (s), 0.07 (s) ppm; C NMR (100
13
0
2
1
.06 (Si(CH ) , 6H) ppm; C NMR (100 MHz, CD OD): δ205.3,
3 2 3
MHz, CDCl ): δ138.6, 138.5, 138.2, 128.3 (2C) 127.9, 127.8,
1
7
ppm;(+) HRESIMS m/z 465.2428 [M+Na] (calcd for
C H O SiNa, 465.2437, ∆ = −1.9 ppm).
3
05.3, 181.5, 140.9, 139.9, 139.6, 134.3, 129.4, 129.2, 128.9,
28.7, 128.5, 125.4, 112.9, 112.8, 98.9, 98.8, 78.1, 78.0, 74.4,
27.7 (2C), 127.6, 78.6, 78.4, 73.3, 73.2, 73.1, 72.8, 72.2, 71.8,
1.7, 50.3, 50.2, 47.9, 47.0, 34.1, 33.6, 25.9, 18.2, –4.5, –4.8
+
74.0, 73.9, 73.7, 73.7, 72. 8, 63.6, 63.5, 60.6, 56.4, 41.9, 31.4,
2
6
6.4, 20.5, 20.4, 19.0, –4.4, –4.4 ppm; (+) HRESIMS m/z
26
38
4
+
72.3348 [M+Na] (calcd for C H NO SiNa, 672.3333, ∆ = 2.2
37
51
7
(4S,5R)-4,5-Bis(benzyloxy)-6-((tert-butyldimethylsilyl)oxy)-1-
ppm).
iodohexan-2-ol (20): To a solution of epoxide 19 (2.0 g, 4.5
mmol) and cerium chloride heptahydrate (2 g, 5.4 mmol) in
(
[
2S,4S,5R)-4,5-Bis(benzyloxy)-1',3,4,4',5,6-hexahydrospiro
pyran-2,3'-pyrrolo[2,1-c][1,4]oxazine]-6'-carbaldehyde (23):
CH CN (20 mL) was added sodium iodide (0.81 g, 5.4 mmol).
3
To ketone 22 (0.8 g, 1.2 mmol) in THF (10 mL) was treated with
N HCl (1.3 mL, 4.9 mmol) at 0 °C. After being stirred for 4 h,
the reaction was carefully neutralized with a saturated aqueous
NaHCO solution and the resulting mixture was extracted with
After stirring for 5 h, the mixture was concentrated in vacuo to
give crude product, which was purified by flash chromatography
on silica gel (PE/EtOAc = 15/1) to give 20 (2.1 g, 80%; dr = 2:1)
4
2
2
3
as colorless oil. [α] = +12.5 (c 0.075, CHCl ); IR (film) νmax:
3
D
3
-
1
1
EtOAc (4 × 50 mL). The combined organic extracts were washed
with brine, dried over MgSO , filtrated and concentrated. The
residue was purified by flash chromatography on silica gel
331, 2926, 2849, 1463, 1260, 1057, 827 cm ; H NMR (400
4
MHz, CDCl ): δ7.46-7.30 (m, 10H), 4.81-4.68 (m, 2H), 4.61-
3
4
=
.52 (m, 2H), 4.13 (ddd, J = 6.0, 5.8, 2.4 Hz, 0.34H), 4.09 (ddd, J
5.2, 5.0, 3.2 Hz, 0.66H), 4.00-3.87 (m, 1H), 3.86-3.72 (m, 2H),
.57-3.52 (m, 1H), 3.31-3.16 (m, 2H), 2.03-1.73 (m, 2H), 0.96-
(PE/EtOAc = 8:1) to obtain 23 (0.53 g, 86%) as a colorless oil.
22
[
α] = +48.8 (c 0.025, CHCl ); IR (film) νmax: 3408, 2915, 2843,
D
3
3
0
1
7
4
-1 1
13
1652, 1095, 1051, 684cm ; H NMR (400 MHz, CDCl
3
): δ 9.45
.94 (9H), 0.14-0.10 (6H) ppm; C NMR (100 MHz, CD OD): δ
3
(s, 1H), 7.45-7.30 (m, 10H), 6.91 (d, J = 4.1 Hz, 1H), 5.98 (d, J =
40.1, 139.7, 129.4, 129.3, 129.0, 128.9, 128.8, 128.7, 128.6,
9.4, 74.8, 74.4, 74.0, 73.2, 68.9, 38.9, 38.5, 26.4, 19.1, 14.9, –
4
.1 Hz, 1H), 4.77 (d, J = 16.0 Hz, 1H), 4.78-4.68 (m, 4H), 4.56
+
(d, J = 12.1 Hz, 1H), 4.55 (d, J = 12.1 Hz, 1H), 4.02 (d, J = 14.0
Hz, 1H), 3.98 (ddd, J = 12.4, 4.8, 2.8 Hz, 1H), 3.95 (dd, J = 12.4,
2
.3, –4.4, –5.1 ppm; (+) HRESIMS m/z 593.1562 [M+Na] (calcd
for C H IO SiNa, 593.1565, ∆ = −0.5 ppm).
26
39
4
.4 Hz, 1H), 3.74 (br s, 1H), 3.56 (dd, J = 12.4, 0.8 Hz, 1H), 2.29
(
4S,5R)-4,5-Bis(benzyloxy)-6-((tert-butyldimethylsilyl)oxy)-1-
(dd, J = 12.4, 12.2 Hz, 1H), 2.07 (dd, J = 12.4, 4.8 Hz, 1H) ppm;
C NMR (100 MHz, CDCl ): δ 178.7, 138.4, 138.3, 134.2,
3
13
iodohexan-2-one (21): Alcohol 20 (2.0 g, 3.5 mmol) was
dissolved in dry CH Cl (20 mL) at 0 °C then, DMP (2.98 g, 7.0
131.1, 128.4, 128.4, 127.8, 127.7, 127.6, 127.4, 123.8, 104.6,
95.7, 72.8, 71.3, 71.0, 70.2, 62.5, 57.7, 52.4, 33.6 ppm; (+)
2
2
mmol) was added in one portion and the mixture was stirred for
h. A mixture of saturated aqueous NaHCO3 and Na S O
+
3
HRESIMS m/z 456.1762 [M+Na] (calcd for C H NO Na,
2
2
3
26 27
5
solution (V/V = 50:50) was carefully dropped and the resulting
mixture was stirred until turned to clear. The organic layer was
separated and the aqueous layer was extracted with CH Cl (2 ×
456.1781, ∆ = -4.2 ppm).
Xylapyrroside A (1) (Method 2): According to a known
2
2
8
procedure, a solution of compound 23 (0.25 g, 0.6 mmol) in
1
00 mL). The combined organic layers were washed with brine,
CH Cl (30 mL) was carefully treated with TiCl at -78 °C for 40
2
2
4
dried over MgSO , filtrated and concentrated to give crude
4
h. Then the reaction was quenched with saturated aqueous
aldehyde, which was purified by flash chromatography on silica
NaHCO solution and allowed to warm to room temperature.
3
gel (PE/EtOAc = 8/1) to produce ketone 21 (1.8 g, 90%) as a
2
2
After removing solvent, the resulting mixture was extracted with
EtOAc (4 × 20 mL) and the combined organic layers were
washed with brine. Dried, filtrated and concentrated, the residue
was purified by flash chromatography on silica gel
colorless oil. [α] = +9.9 (c 0.139, CHCl ); IR (film) νmax: 3441,
2
D
3
-
1
1
942, 2843, 1720, 1375, 1095, 843 cm ; H NMR (400 MHz,
CD OD): δ 7.40-7.28 (m, 10H), 4.72 (d, J = 11.8 Hz, 1H), 4.63
3
(d, J = 11.8 Hz, 1H), 4.59 (s, 2H), 4.19 (m, 1H), 4.09 (m, 1H),
(
CH Cl /MeOH = 40/1) to give xylapyrroside A (1, 0.1 g, 70%)
2 2
3
6
3
.91 (s, 2H), 3.79 (dd, J = 10.6, 5.2 Hz, 1H), 3.65 (dd, J = 10.6,
.1Hz, 1H), 3.09 (dd, J = 16.6, 8.2 Hz, 1H), 2.99 (dd, J = 16.6,
as an off-white solid. The spectral data of the synthetic 1 were
identical to the reported data of natural product.
13
.7 Hz, 1H), 0.93 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H) ppm;
C
NMR (100 MHz, CD OD): δ 203.7, 139.9, 139.6, 129.4, 129.3,
(2R,3S)-1,3-Bis(benzyloxy)hex-5-en-2-ol (24): Compound 16 (7
3
1
29.1, 129.0, 128.8, 128.7, 81.9, 77.2, 73.7, 73.6, 63.3, 41.7,
g, 3.1 mmol) in THF (20 mL) was treated with NaH (1.89 g, 4.7

1
0
mmol) at 0 °C for 30 min, then benzyl brom
A
id
C
e (
C
3.
E
77
P
m
T
L
E
, 3
D
.1 MA7.
N
25
U
(m
S
),
C
4.
R
71IP(d
T
, J = 12.0 Hz), 4.68 (d, J = 12.0 Hz), 4.65 (d, J =
mmol) was added dropwise at the same temperature and the
reaction was stirred for 3h. The mixture was poured into ice
water, and extracted with EtOAc (3 × 200 mL). The combined
organic layers were washed with brine, dried over MgSO4,
filtrated and concentrated under reduced pressure. The residue
was purified by flash chromatography on silica gel (PE/EtOAc =
11.2 Hz), 4.56 (d, J = 11.2 Hz), 3.94 (ddd, J = 10.1, 2.8, 2.6 Hz),
3.76 (m), 3.64 (m), 3.51 (m), 3.30 (dd, J = 10.1, 5.6 Hz), 3.24
(dd, J = 10.1, 5.6 Hz), 1.92 (ddd, J = 14.0, 10.4, 2.8 Hz), 1.64
(ddd, J = 14.4, 9.7, 2.3 Hz), 0.92 (s), 0.08 (s), 0.07 (s) ppm; 27b:
δ 7.38-7.25 (m), 4.65 (d, J = 11.6 Hz), 4.52 (s), 4.47 (d, J = 11.6
Hz), 4.12 (ddd, J = 6.1, 5.9, 2.8 Hz), 3.76 (m), 3.74 (m), 3.61
(m), 3.18 (dd, J = 10.4, 4.0 Hz), 2.98 (dd, J = 10.4, 5.2 Hz), 1.84
22
5
0
1
1
1
/1) to give 24 (7.0 g, 75%) as a colorless oil. [α] = +21.2 (c
.07, CHCl ); IR (film) νmax: 3435, 2936, 2849, 1452, 1079,
030, 750 cm ; H NMR (400 MHz, CDCl ): δ 7.41-7.29 (m,
0H), 5.96 (ddt, J = 17.2, 10.1, 7.1 Hz, 1H), 5.18 (dq-like, J =
7.2, 2.0 Hz, 1H), 5.13 (dq-like, J = 10.1, 2.0 Hz, 1H), 4.65 (d, J
D
(ddd, J = 14.4, 8.5, 6.0 Hz), 1.74 (ddd, J = 14.4, 6.8, 4.0 Hz),
0.93 (s), 0.11 (s), 0.09 (s) ppm; C NMR (100 MHz, CD OD)
data of 27a: δ 140.1, 140.0, 129.4, 129.3, 129.0, 128.7, 128.6,
82.6, 77.8, 73.9, 73.7, 68.9, 63.8, 39.2, 26.4, 19.1, 15.0, −5.2
ppm; 27b: δ 139.7, 139.6, 129.4, 129.3, 129.0, 128.8, 128.7,
78.7, 74.4, 72.9, 72.6, 68.9, 63.8, 38.1, 26.4, 19.1, 14.9, −4.4
3
-
1
1
13
3
3
=
(
11.4 Hz, 1H), 4.57 (s, 2H), 4.54 (d, J = 11.4 Hz, 1H), 3.92
ddd, J = 6.5, 6.4, 3.5 Hz, 1H), 3.69 (dd, J = 9.6, 3.5 Hz, 1H),
+
3
.63 (dd, J = 9.6, 6.5 Hz, 1H), 3.60 (m, 1H), 2.47 (m, 2H) ppm;
ppm.(+) HRESIMS m/z 593.1542 [M+Na] (calcd for
C H IO SiNa, 593.1560, ∆ = -3.0 ppm).
26 39
1
3
C NMR (100 MHz, CDCl ): δ 138.3, 137.9, 134.6, 128.4 (2C),
3
4
1
7
28.3 (2C), 127.8 (4C), 127.7, 127.6, 117.3, 79.1, 73.4, 72.2+,
(4S,5R)-4,6-Bis(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)-1-
iodohexan-2-one (28): The title compound was furnished from
alcohol 27 (1.4 g, 2.5 mmol) according to the above procedure.
1.5, 71.0, 34.7 ppm; (+) HR-ESIMS m/z 335.1614 [M+Na]
(calcd for C H O Na, 335.1623, ∆ = -2.7 ppm).
20 24 3
2
2
(((2R,3S)-1,3-Bis(benzyloxy)hex-5-en-2-yl)oxy)(tert-
Product 28 (1.28 g, 90%) was obtained as a colorless oil. [α]
=
D
butyl)dimethylsilane (25): The title compound was synthesized
from alcohol 24 (3.7 g,11.8 mmol) through the above procedure.
+46.0 (c 0.03, CHCl ); IR (film) νmax: 3397, 2926, 2367, 1384,
3
-
1
1
1090, 805, 657 cm ; H NMR (400 MHz, CD OD): δ7.36-7.27
3
22
Product 25 (4.1 g, 82%) was obtained as colorless oil. [α]
=
(m, 10H), 4.62 (d, J = 11.2 Hz, 1H), 4.52 (s, 2H), 4.52 (d, J =
11.2 Hz, 1H), 4.12-4.04 (m, 2H), 3.90 (s, 2H), 3.51 (dd, J = 10.1,
5.6 Hz, 1H), 3.48 (dd, J = 10.1, 5.2 Hz, 1H), 3.05 (dd, J = 16.6,
8.2 Hz, 1H), 2.93 (dd, J = 16.6, 3.7 Hz, 1H), 0.92 (s, 9H), 0.10 (s,
D
+
51.2 (c 0.025, CHCl ); IR (film) νmax: 3065, 2923, 2849, 1463,
3
-1 1
1
254, 816, 728 cm ; H NMR (400 MHz, CDCl ): δ7.37-7.28
3
(m, 10H), 5.90 (ddt, J = 17.2, 10.1, 7.1 Hz, 1H), 5.12 (br d, J =
13
1
1
7.2 Hz, 1H), 5.06 (br d, J = 10.1 Hz, 1H), 4.63 (d, J = 11.6 Hz,
H), 4.57 (d, J = 11.6 Hz, 1H), 4.55 (d, J = 12.1 Hz, 1H), 4.51 (d,
3H), 0.08 (s, 3H) ppm; C NMR (100 MHz, CD OD): δ 203.9,
3
139.7, 139.5, 129.4 (2C), 129.3 (2C), 129.1 (2C), 129.0 (2C),
128.7, 128.6, 78.6, 74.3, 73.9, 73.7, 72.7, 41.1, 26.4 (3C), 19.0,
J = 12.1 Hz, 1H), 3.94 (dd, J = 9.6, 4.6 Hz, 1H), 3.61 (dd, J =
+
9
2
.7, 4.1 Hz, 1H), 3.60 (m, 1H), 3.56 (dd, J = 9.7, 5.5 Hz, 1H),
7.7, −4.4 (2C) ppm; (+) HRESIMS m/z 591.1415 [M+Na] (calcd
13
.38 (m, 2H), 0.91 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H) ppm;
C
for C H O SiNa, 591.1398, ∆ = 2.9 ppm).
26
37
4
NMR (100 MHz, CDCl ): δ 138.8, 138.4, 135.6, 128.3 (2C),
3
1
2
-((4S,5R)-4,6-Bis(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)-
-oxohexyl)-5-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-
1
7
28.2 (2C), 127.8 (2C), 127.7 (2C), 127.5, 127.4, 116.8, 80.4,
3.5, 73.4, 72.6, 72.0, 35.3, 25.9 (3C), 18.2, –4.4, –4.7 ppm; (+)
+
pyrrole-2-carbaldehyde (29): The title compound was
synthesized from ketone 28 (1.1 g, 1.9 mmol) using the above
procedure. Product 29 (1.1 g, 87%; dr = 1:1) was obtained as a
HRESIMS m/z 449.2489 [M+Na] (calcd for C H O SiNa,
26
38
3
4
49.2488, ∆ = 0.2 ppm).
22
(((2R,3S)-1,3-Bis(benzyloxy)-4-(oxiran-2-yl)butan-2-
colorless oil. [α] = +3.6 (c 0.05, CHCl ); IR (film) νmax: 3408,
D 3
-1 1
yl)oxy)(tert-butyl)dimethylsilane (26): The title compound was
prepared from compound 25 (3.9 g,9.2 mmol) according to the
above procedure. Product 26 (3.0 g, 75%) was obtained as an
inseparable mixture of epimers 26a and 26b (dr = 5:2). Colorless
2926, 2843, 1665, 1446, 1386, 1024 cm ; H NMR (400 MHz,
CD OD): δ 9.44 (s, 0.5H), 9.43 (s, 0.5H), 7.37-7.25 (m, 10H),
7.03 (d, J = 4.0 Hz, 1H), 6.33 (dd, J = 4.0 Hz, 0.5H), 6.32 (dd, J
= 4.0 Hz, 0.5H), 5.37-5.18 (m, 2H), 4.72-4.40 (m, 7H), 4.26-3.61
(m, 3H), 3.53-3.44 (m, 3H), 2.97-2.73 (m, 2H), 1.75-1.37 (m,
3
2
2
oil; [α] = −18.9 (c 0.037, CHCl ); IR (film) νmax: 2947, 2854,
D
3
-
1
1
13
1
382, 1249, 1073, 1041, 800 cm ; H NMR (400 MHz, CDCl )
6H), 0.91 (SiC(CH ) , 9H), 0.09-0.06 (Si(CH ) , 6H) ppm;
C
3
3 3
3 2
data of 26a: δ 7.36-7.28 (m), 4.74 (d, J = 12.0 Hz), 4.67 (d, J =
NMR (100 MHz, CD OD): δ 205.3, 205.1, 181.5, 141.0, 139.9,
3
9
3
.6 Hz), 4.64 (d, J = 9.6 Hz), 4.59 (d, J = 12.0 Hz), 3.81 (m),
.71 (m), 3.58 (m), 2.99 (m), 2.76 (dd, J = 4.8, 4.0 Hz), 2.49 (dd,
139.6, 134.3, 134.2, 129.4, 129.3, 129.2, 129.0, 128.9, 128.9,
128.7, 128.5, 125.4, 112.9, 112.8, 98.9, 98.7, 78.5, 78.0, 74.4,
74.1, 74.0, 73.7, 73.6, 72.8, 60.6, 56.4, 42.0, 31.4, 26.4, 20.6,
20.5, 19.0, –4.3, –4.4, –5.2 ppm; (+) HRESIMS m/z 672.3323
J = 4.8, 2.4 Hz), 1.80 (m), 1.65 (ddd, J = 14.3, 7.2, 3.6 Hz), 0.90
s), 0.06 (s), 0.05 (s) ppm; 26b: δ 7.36-7.28 (m), 4.78 (d, J = 12.0
(
+
Hz), 4.60 (d, J = 12.0 Hz), 4.58 (d, J = 9.6 Hz), 4.56 (d, J = 9.6
Hz), 3.81 (m), 3.71 (m), 3.63 (m), 2.89 (m), 2.64 (dd, J = 4.8, 4.4
Hz), 2.39 (dd, J = 5.0, 2.8 Hz), 1.83 (m), 1.65 (ddd, J = 14.3, 7.2,
[M+Na] (calcd for C H NO SiNa, 672.3332, ∆ = -1.5 ppm).
37
51
7
(
4S,5R)-4-(Benzyloxy)-5-((benzyloxy)methyl)-1',4,4',5-
tetrahydro-3H-spiro[furan-2,3'-pyrrolo[2,1-c][1,4]oxazine]-
'-carbaldehyde (30): The title compound was prepared from
13
3.6 Hz), 0.90 (s), 0.06 (s), 0.05 (s) ppm; C NMR (100 MHz,
CDCl ): δ 138.6, 138.4, 138.2, 128.3, 128.3, 127.8, 127.8, 127.7,
6
3
ketone 29 (0.9 g, 1.6 mmol) according to the above procedure.
1
7
27.6, 127.6, 127.5, 78.6, 78.4, 73.3, 73.2, 73.1, 72.7, 72.2, 71.8,
Product 30 (0.53 g, 86%) was obtained as a pair of epimers (30a
1.7, 50.2, 50.1, 47.8, 47.0, 34.1, 33.6, 25.9, 18.1, −4.5, −4.8
22
+
and 30b, dr = 2:1). Colorless oil; [α]
D
= +33.6 (c 0.05, CHCl );
3
ppm; (+) HRESIMS m/z 465.2448 [M+Na] (calcd for
C H O SiNa, 465.2437, ∆ = 2.4 ppm).
-1 1
IR (film) νmax: 3463, 2920, 2849, 2361, 1656, 1035, 684 cm ; H
26
38
4
NMR (400 MHz, CDCl ) data of 30a: δ9.46 (s), 7.38-7.28 (m),
3
(
4S,5R)-4,6-Bis(benzyloxy)-5-((tert-butyldimethylsilyl)oxy)-1-
6.92 (d, J = 4.0 Hz), 6.01 (d, J = 4.0 Hz), 5.03 (d, J = 15.6 Hz),
4.86 (d, J = 15.6 Hz), 4.68-4.38 (m), 4.37 (dd, J = 8.4, 4.4 Hz),
4.27 (d, J = 14.0 Hz), 4.10 (ddd, J = 7.9, 3.9, 2.0 Hz), 3.53 (dd, J
= 10.8, 4.4 Hz), 3.49 (dd, J = 10.8, 4.4 Hz), 2.31 (dd, J = 14.2,
1.9 Hz), 2.20 (dd, J = 14.2, 8.0 Hz) ppm; 30b: δ 9.44 (s), 7.38–
7.28 (m), 6.91 (d, J = 4.0 Hz), 6.00 (d, J = 4.0 Hz), 5.01 (d, J =
15.6 Hz), 4.78 (d, J = 15.6 Hz), 4.68-4.38 (m), 4.22 (d, J = 14.0
iodohexan-2-ol (27): The title compound was prepared from
epoxide 26 (1.6 g, 3.6 mmol) according to the above procedure.
Product 27 (1.7 g, 80%; dr = 3:1) was obtained as an inseparable
mixture of epimers 27a and 27b. Colorless oil; [α] = +25.3 (c
0
1
22
D
.03, CHCl ); IR (film) νmax: 3413, 2936, 2854, 2361, 1380,
3
-1 1
030, 673 cm ; H NMR (400 MHz, CD OD) data of 27a: δ7.38-
3

1
1
Hz), 3.82 (dd, J = 10.4, 4.8 Hz), 3.77 (dd, J = 1
A
0.
dd, J = 14.0, 1.8 Hz), 2.16 (dd, J = 14.0, 6.8 Hz) ppm; C NMR
100 MHz, CDCl ) data of 30a: δ 178.7, 137.8, 135.2, 131.1,
C
4,
C
7.6
E
H
P
z),
T
E
2.
D
47 MAcu
N
ltu
U
re
S
d
C
inRI
D
P
M
T
EM containing 10% FBS, 100 U/mL
1
3
(
(
penicillin, and 100 µg/mL streptomycin in 5% CO at 37°C. Cell
viability was determined by conventional MTT assay. Catechin
2
2
9
3
1
7
1
7
28.5, 127.9, 127.7, 127.6, 124.2, 104.8, 103.3, 84.8, 78.7, 73.5,
1.7, 70.0, 57.9, 51.0, 42.4 ppm; 30b: δ 178.7, 138.0, 135.2,
31.0, 128.4, 127.9, 127.7, 127.6, 124.1, 104.7, 102.8, 83.0, 78.7,
3.4, 71.8, 69.4, 58.4, 51.6, 42.1ppm; (+) HRESIMS m/z
hydrate (purity ≥ 98%, Beyotime Biotechology, China) was used
30a
as a positive control. In brief, A7r5 cells in 96-well plates were
incubated with different concentrations of compounds (25, 50
and 100 µM) or catechin hydrate (50 and 100 µM) for 4 h, then
stimulated with 200 µM of tert-Butyl hydroperoxide (tBHP,
purity ≥ 99%, Sigma-Aldrich) for 12 h. Cells were then incubated
with MTT solution (0.5 mg/mL) in culture medium. After
incubation at 37°C for 4 h, the culture medium containing MTT
was removed. DMSO was then added into each well, and the
absorbance at 570 nm was measured using a microplate reader
+
4
56.1787 [M+Na] (calcd for C H NO Na, 456.1781, ∆ = 1.3
26
27
5
ppm).
Xylapyrroside B (2) and acortatarin A (4) (Method 2):
19
According to a known procedure, a solution of compound 30
0.52 g, 1.2 mmol) in CH Cl (50 mL) was carefully treated with
(
2
2
TiCl at -78 °C for 40 h. Then the reaction was quenched with
aqueous saturated NaHCO solution and extracted with EtOAc (4
×
4
(M1000, TECAN, Austria GmbH, Austria). Values were
3
expressed as percentage of cell survival. Absorbance from tBHP-
untreated cells was set at 100% (control group).
30 mL). The combined organic extracts were washed with
brine, dried over MgSO , filtrated and evaporated. The residue
4
was purified by flash chromatography on silica gel
Acknowledgments
(
4
CH Cl /MeOH = 40/1) to afford compounds 2 (0.1 g, 32%) and
(0.11 g, 37%) as an off-white solid and exhibited physical and
2 2
This work was supported by NSFC grants (No. 81273401,
8
1202420, 21472021), grants from the Ph.D. Programs
spectroscopic characteristics agreed to the natural products.
Foundation of Ministry of Education (MOE) of China (No.
20120071110049, 20120071120049), and the National Basic
Research Program of China (973 Program, Grant No.
2013CB530700). We also thank Zhejiang Jolly Pharmaceutical
Company (ZJPC, Deqing County, China) for providing Wuling
Powder and Ms. Jian Chen from ZJPC for the sample
identification.
X-ray Crystal Data. Colorless crystals of 1 and 1a were both
obtained in acetone. Crystal data were obtained on a Bruker
APEX Duo CCD detector employing graphite monochromated
Copper-Kα radiation (λ = 1.54178 Å) at 140(2) K and operating
in the φ/ω scan mode. The crystal structure was solved by direct
method using the program SHELXS-97 and subsequent Fourier
difference techniques, and was finally refined anisotropically by
full-matrix least-squares on F2 using SHELXL-97. All non-
hydrogen atoms were refined anisotropically. The hydrogen atom
positions were geometrically idealized and allowed to ride on
their parent atoms. Crystallographic data of 1 and 1a have been
deposited at the Cambridge Crystallographic Data Centre (1:
deposition No. CCDC-1029230; 1a: deposition No. CCDC-
Supplementary data
Copies of NMR spectra, and X-ray crystal structure data are
available as Supplementary data.
References and notes
1
2
3
4
.
.
.
.
Hu, J.-F.; Fan, H.; Xiong, J.; Wu, S.-B. Chem. Rev. 2011, 111, 5465–
491.
Fan, H.; Peng, J.-N.; Hamann, M. T.; Hu, J.-F. Chem. Rev. 2008, 108,
64–287.
1
029231). Copies of these data can be obtained free of charge via
5
www.ccdc.cam.ac.uk/conts/retrieving.html or from the
Cambridge Crystallographic Data Centre, 12, Union Road,
Cambridge CB21EZ, UK. [fax: (+44) 1223-336-033; or email:
deposit@ccdc.cam.ac.uk].
2
Yang, T.; Wang, C.-H.; Chou, G.-X.; Wu, T.; Cheng, X.-M.; Wang, Z.-
T. Food Chem. 2010, 123, 705–710.
Guo, J.-L.; Feng, Z.-M.; Yang, Y.-J.; Zhang, Z.-W.; Zhang, P.-C.
Chem. Pharm. Bull. 2010, 58, 983–985.
Xylapyrroside A (1): C H NO , M = 253.25, orthorhombic, a
=
9
12
15
5
7.03200(10), b = 8.45490(10), c = 19.6634(4) Å, α = 90.00, β =
3
5.
Tong, X.-G.; Zhou, L.-L.; Wang, Y.-H.; Xia, C.-F.; Wang, Y.; Liang,
M.; Hou, F.-F.; Cheng, Y.-X. Org. Lett. 2010, 12, 1844–1847.
Jiang, D.-S.; Peterson, D. G. Food Chem. 2013, 141, 1345–1353.
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0.00, γ = 90.00 deg., V = 1169.08(3) Å , T = 140(2) K, space
group P2 2 2 , Z = 4, 4329 reflections measured, 1986
1
1 1
6
7
.
.
independent reflections (Rint = 0.0274). The final R1 values were
.0312 (I >2σ (I)). The final R1 values were 0.0318 (all data).
0
The final wR values were 0.0807 (all data). The goodness fit on
2
8
9
.
.
Sudhakar, G.; Kadam, V. D.; Bayya, S.; Pranitha, G.; Jagadeesh, B.
Org. Lett. 2011, 13, 5452–5455.
F2 was 1.041. Flack parameter = 0.19(18). Crystallographic data
of 1 have been deposited at the Cambridge Crystallographic Data
Centre (deposition No. CCDC-1029230).
Wurst, J. M.; Verano, A. L.; Tan, D. S. Org. Lett. 2012, 14, 4442–
4
445.
Xylapyrroside A (1a): C H NO , M = 253.25, orthorhombic,
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Synlett 2012, 23, 855–858.
1
12 15
5
a = 7.10980(10), b = 8.64620(10), c = 18.8598(4) Å, α = 90.00, β
3
=
90.00, γ = 90.00 deg., V = 1159.36(3) Å , T = 140(2) K, space
11. Borrero, N. V.; Aponick, A. J. Org. Chem. 2012, 77, 8410–8416.
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group P2 2 2 , Z = 4, 7512 reflections measured, 2153
1
1 1
independent reflections (Rint = 0.0463). The final R1 values were
.0288 (I >2σ(I)). The final R1 values were 0.0290 (all data). The
0
13. Tang, Y.; Fu, Y.; Xiong, J.; Li, M.; Ma, G.-L.; Yang, G.-X.; Wei, B.-
G.; Zhao, Y.; Zhang, H.-Y.; Hu, J.-F. J. Nat. Prod. 2013, 76, 1475–
1484.
final wR values were 0.0745 (all data). The goodness fit on F2
2
was 1.050. Flack parameter = –0.07(15). Crystallographic data of
1
a have been deposited at the Cambridge Crystallographic Data
14. Zang, Y.; Xiong, J.; Zhai, W.-Z.; Cao, L.; Zhang, S.-P.; Tang, Y.;
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.4 Anti-oxidative stress assay
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ACCEPTED MANUSCRIPT
Supplementary Material for
Xylapyrrosides A and B, Two Rare Sugar-Morpholine Spiroketal Pyrrole-Derived Alkaloids
from Xylaria nigripes: Isolation, Complete Structure Elucidation, and Total Syntheses
a
a, *
a
a
a
a
b
Ming Li, Juan Xiong, Ya Huang, Li-Jun Wang, Yu Tang, Guo-Xun Yang, Xin-Hua Liu,
c, *
d
d
d
a, *
Bang-Guo Wei, Hui Fan, Yun Zhao, Wen-Zhu Zhai, and Jin-Feng Hu
a
b
Departments of Natural Products Chemistry and Pharmacology, School of Pharmacy, Fudan University, No. 826
Zhangheng Road, Shanghai 201203, PR China
c
Department of Chemistry, Fudan University, No. 220 Handan Road, Shanghai 200032, PR China
d
Department of Natural Products for Chemical Genetic Research, Key Laboratory of Brain Functional Genomics
(Ministry of Education), East China Normal University, No. 3663 Zhongshan Road, Shanghai 200062, PR China
S1

ACCEPTED MANUSCRIPT
Table of Contents
1
. X-ray crystallographic data for compounds 1, 1a and 4..................................S3
2
. Synthetic procedures towards compound 7......................................................S5
3
. Copies of Spectra .................................................................................................S7
S2

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1
. X-ray crystallographic data for compounds 1, 1a and 4.
Table S1. X-ray Crystallographic Data for 1 and 1a.
Xylapyrroside A (1)
Xylapyrroside A (1a)
1
Empirical formula
Formula weight
Temperature
Wavelength
C H NO
C H NO5
1
2
15
5
12 15
253.25
253.25
140(2) K
140(2) K
1.54178 Å
1.54178 Å
Orthorhombic
P2(1)2(1)2(1)
Crystal system
space group
Orthorhombic
P2(1)2(1)2(1)
Unit cell dimensions
a (alpha)
7.03200(10) Å (90 deg)
8.45490(10) Å (90 deg)
19.6634(4) Å (90 deg)
7.10980 (10) Å (90 deg)
8.64620(10) Å (90 deg)
18.8598(4) Å (90 deg)
b (beta)
c (gamma)
3
3
Volume
1169.08(3) Å
1159.36(3) Å
3
3
Z, Calculated density
Absorption coefficient
F(000)
4, 1.439 mg/m
4, 1.451 mg/m
-
1
-1
0.952 mm
536
0.960 mm
536
Crystal size
0.10 x 0.05 x 0.04 mm
0.35 x 0.16 x 0.05 mm
4.69 to 69.63 deg.
Theta range for data collection 4.50 to 69.94 deg.
-
-
8<=h<=8, -10<=k<=8,
23<=l<=18
-8<=h<=8, -7<=k<=10,
-22<=l<=22
Limiting indices
Reflections collected / unique
Completeness to theta = 69.94
Absorption correction
4329 / 1986 [R(int) = 0.0274]
96.6 %
7512 / 2153 [R(int) = 0.0463]
99.1 %
Semi-empirical from equivalents Semi-empirical from equivalents
Max. and min. transmission
Refinement method
0.9629 and 0.9108
Full-matrix least-squares on F
1986 / 0 / 165
0.9536 and 0.7300
Full-matrix least-squares on F
2153 / 0 / 166
2
2
Data / restraints / parameters
Goodness-of-fit on F^2
1.041
1.050
Final R indices [I > 2sigma(I)]
R indices (all data)
R = 0.0312, wR = 0.0798
R
R
1
= 0.0288, wR
= 0.0290, wR
2
2
= 0.0741
= 0.0745
1
2
R = 0.0318, wR = 0.0807
1
1
2
Absolute structure parameter
Largest diff. peak and hole
0.19(18)
-0.07(15)
0.263 and -0.198 e. Å
-
3
-3
0.141 and -0.214 e. Å
Colorless crystals of 1 and 1a were both obtained in acetone. Crystal data were
obtained on a Bruker APEX Duo CCD detector employing graphite monochromated
Copper-Kα radiation (λ=1.54178 Å) at 140(2) K and operating in the φ/ω scan mode.
The crystal structure was solved by direct method using the program SHELXS-97 and
subsequent Fourier difference techniques, and was finally refined anisotropically by
full-matrix least-squares on F2 using SHELXL-97. All non-hydrogen atoms were
refined anisotropically. The hydrogen atom positions were geometrically idealized and
allowed to ride on their parent atoms. Crystallographic data of 1 and 1a have been
deposited at the Cambridge Crystallographic Data Centre (1: deposition No.
CCDC-1029230; 1a: deposition No. CCDC-1029231). Copies of 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 CB21EZ, UK.
[
fax: (+44) 1223-336-033; or email: deposit@ccdc.cam.ac.uk].
S3

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Table S2. X-ray Crystallographic Data for 4.
Acortatarin A (4)
C H NO
253.25
Empirical formula
Formula weight
Temperature
12
15
5
173(2) K
Wavelength
0.71073 Å
Crystal system
space group
Orthorhombic
P2(1)2(1)2(1)
Unit cell dimensions
a (alpha)
b (beta)
c (gamma)
Volume
7.1019(3) Å (90 deg)
8.6521(3) Å (90 deg)
18.8596(7) Å (90 deg)
3
1158.85(8) Å
4, 1.452 mg/m
3
Z, Calculated density
F(000)
536
Reflections collected / unique
Data Completeness (theta = 25.01)
Max. and min. transmission
Parameters
2046 / 1214
1.68/1.00
0.960 and 0.945
163
Goodness-of-fit on F^2
R (reflections)
1.076
R = 0.0252, wR = 0.0636
1
2
Figure S1. X-ray crystal structure of acortatarin A (4).
Colorless crystals of 4 were obtained in MeOH. Crystal data were obtained on a
Bruker-AXS SMART APEX II CCD detector employing graphite monochromated
Mo-Kα radiation (λ=0.71073 Å) at 173(2) K and operating in the φ/ω scan mode. The
crystal structure was solved by direct method using the program SHELXS-97 and
subsequent Fourier difference techniques, and was finally refined anisotropically by
full-matrix least-squares on F2 using SHELXL-97. All non-hydrogen atoms were
refined anisotropically. The hydrogen atom positions were geometrically idealized and
allowed to ride on their parent atoms. Crystallographic data for 4 have been deposited at
the Cambridge Crystallographic Data Centre (deposition No. CCDC-1029232). Copies
of
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 CB21EZ, UK. [fax: (+44) 1223-336-033; or
email: deposit@ccdc.cam.ac.uk].
S4

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2
1
. Synthetic procedures towards compound 7
H-Pyrrole-2,5-dicarbaldehyde pre-7a
OHC
N
H
CHO
pre-7a
The 1H-pyrrole-2,5-dicarbaldehyde (pre-7a) was prepared by known method
[
2
1
Knizhnikov, et al. Russian J. Org. Chem. 2007, 43, 855–860]. IR (film): ν 3424,
max
-1 1
909, 2849, 1682, 1660, 1424, 1276, 1167, 794 cm ; H NMR (400 MHz, CDCl ): δ
3
13
0.33 (br s, 1H), 9.80 (s, 2H), 7.03 (d, J = 1.2 Hz, 2H) ppm; C NMR (100 MHz,
+
CDCl ): δ 181.2, 135.5, 119.5 ppm; HR-EIMS m/z 123.0321 [M] (calcd for C H NO ,
3
6
5
2
1
23.0320, Δ = 0.8 ppm).
5
-(Hydroxymethyl)-1H-pyrrole-2-carbaldehyde pre-7b
NaBH4
MeOH
HO
OHC
N
H
CHO
N
H
CHO
pre-7a
pre-7b
To a solution of dialdehyde pre-7a (6.0 g, 48.8 mmol) in dry methanol (100 mL) at 0
○
C was added sodium borohydride (0.46 g, 12.2 mmol) in one portion. After being
stirred for 30 min, the reaction was quenched with ice water and concentrated in
vacuum. The resulting aqueous was extracted with EtOAc (3 × 100 mL) and the
combined organic layers were washed with brine, dried, filtrated and concentrated. The
residue was purified by flash chromatography on silica gel (PE/EtOAc = 4/1) to give
mono-aldehyde pre-7b (5.92 g, 98%) as a white amorphous powder. IR (film): ν
max
-1 1
3
1
304, 2915, 2849, 1495, 1364, 1200, 1063, 756 cm ; H NMR (400 MHz, CDCl ): δ
3
0.61 (br s, 1H, D O-exchangeable), 9.38 (s, 1H), 6.96 (dd, J = 3.7, 2.6 Hz, 1H), 6.22
2
(
dd, J = 3.7, 2.2 Hz, 1H), 4.81 (d, J = 4.2 Hz, 2H), 3.46 (t, J = 4.2 Hz, 1H,
1
3
D O-exchangeable) ppm; C NMR (100 MHz, CDCl ): δ 179.2, 141.8, 132.2, 123.4,
1
2
3
+
09.0, 57.9 ppm; HR-EIMS m/z 125.0478 [M] (calcd for C H NO , 125.0477, Δ = 0.8
6 7 2
ppm).
5
-(((Tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-pyrrole-2-carbaldehyde 7
To a solution of pre-7b (1.5 g, 12.1 mmol) and PPTS (54 mg, 0.24 mmol) in CH Cl2
2
(40 mL) was treated with DHP (1.08 g, 13.1 mmol). After stirring for overnight, the
reaction was diluted with saturated aqueous NaHCO solution. The mixture was
3
separated and the aqueous layer was extracted with CH Cl (2 × 50 mL). The combined
2
2
S5

ACCEPTED MANUSCRIPT
organic layers were washed with brine. Dried, filtrated and concentrated, the residue
was purified by flash chromatography on silica gel (PE/EtOAc = 5/1) to give 7 (2.45 g,
9
8
4%, dr = 1:1) as a colorless oil. IR (film): ν 3254, 2942, 1614, 1391, 1178, 1068,
max
-1 1
05 cm ; H NMR (400 MHz, CDCl ) : δ 9.71 (br s), 9.47 (s), 6.91 (d, J = 3.5 Hz), 6.90
3
(
4
d, J = 3.5 Hz), 6.22 (d, J = 3.5 Hz), 6.21 (d, J = 3.5 Hz), 4.76 (d, J = 13.5 Hz), 4.69 (m),
.62 (d, J = 13.5 Hz), 3.91 (m), 3.57 (m), 1.87-1.59 (m, 6H) ppm; C NMR (100 MHz,
1
3
CDCl ): δ 180.3, 178.9, 139.3, 137.9, 134.1, 132.6, 123.1, 121.6, 111.2, 109.8, 100.3,
3
9
2
8.8, 64.1, 63.6, 62.7, 62.1, 31.8, 30.4, 26.6, 25.2, 20.9, 19.4 ppm; (+) HR-ESIMS m/z
+
32.0944 [M+Na] (calcd for C H NO Na, 232.0948, Δ = −1.3 ppm).
1
1
15
3
S6

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1
Figure S1. H NMR spectrum of 1 in CD OD
3
S7

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1
Figure S2. H NMR spectrum of 1 in CD COCD3
3
S8

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1
Figure S3. H NMR spectrum of 1 in CDCl
3
S9

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13
Figure S4. C NMR spectrum of 1 in CD OD
3
S10

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Figure S5. DEPT-135 spectrum of 1 in CD OD
3
1
1
Figure S6. H- H COSY spectrum of 1 in CD OD
3
S11

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Figure S7. HSQC spectrum of 1 in CD OD
3
Figure S8. HMBC spectrum of 1 in CD OD
3
S12

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Figure S9. HMBC spectrum of 1 in CD OD-expansion
3
Figure S10. NOESY spectrum of 1 in CD OD
3
S13

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Figure S11. HR-EIMS of 1
S14

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1
Figure S12. H NMR spectrum of 2 in CD OD
3
S15

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13
Figure S13. C NMR spectrum of 2 in CD OD
3
S16