Synthesis, antiproliferative activity and SAR analysis of (?)-cleistenolide and analogues

Article abstract of DOI:10.1016/j.ejmech.2020.112597

A new, modified total synthesis of (?)-cleistenolide (1) and sixteen new analogues or derivatives was achieved starting from commercially available 1,2-O-isopropylidene-α-D-glucofuranose. The synthesis of 1 proceeds in six steps and 67% overall yield, usi

Full text of DOI:10.1016/j.ejmech.2020.112597

Journal Pre-proof
Synthesis, antiproliferative activity and SAR analysis of (−)-cleistenolide and
analogues
Goran Benedeković, Mirjana Popsavin, Ivana Kovačević, Vesna Kojić, Marko Rodić,
Velimir Popsavin
PII:
S0223-5234(20)30569-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112597
EJMECH 112597
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 May 2020
Revised Date: 11 June 2020
Accepted Date: 15 June 2020
Please cite this article as: G. Benedeković, M. Popsavin, I. Kovačević, V. Kojić, M. Rodić, V. Popsavin,
Synthesis, antiproliferative activity and SAR analysis of (−)-cleistenolide and analogues, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112597.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Elsevier Masson SAS. All rights reserved.

Graphical Abstract
Synthesis, antiproliferative activity and SAR analysis of (−)-cleistenolide and analogues
Goran Benedeković a, Mirjana Popsavin a, Ivana Kovačević a, Vesna Kojić b, Marko Rodić a,
Velimir Popsavin a,c,*
a Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences,
University of Novi Sad, Trg Dositeja Obradovića 3, Novi Sad, Serbia
b Oncology Institute of Vojvodina, Faculty of Medicine, University of Novi Sad, Put Dr Goldmana
4, 21204 Sremska Kamenica, Serbia
c Serbian Academy of Sciences and Arts, Knez Mihajlova 35, 11000 Belgrade, Serbia
O
O
O
O
O
O
O
O
O
isosterism
isosterism
(H → NO2)
(NO2 → Cl)
O
O
O
OAc OAc
OAc OAc
OAc OAc
O2N
Cl
Cleistenolide (1)
IC50 = 36.94 µM (Raji)
5
4
IC50
= 18.54 µM (Raji)
IC50
= 1.85 µM (Raji)
New synthesis of cleistenolide (1) was achieved through 6 steps with a total yield of 67%. Twenty
analogs of natural product 1 were designed, synthesized, and evaluated for their antiproliferative
activity against eight tumour cell lines.
1

Graphical Abstract
Synthesis, antiproliferative activity and SAR analysis of (−)-cleistenolide and analogues
Goran Benedeković a, Mirjana Popsavin a, Ivana Kovačević a, Vesna Kojić b, Marko Rodić a,
Velimir Popsavin a,c,*
a Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences,
University of Novi Sad, Trg Dositeja Obradovića 3, Novi Sad, Serbia
b Oncology Institute of Vojvodina, Faculty of Medicine, University of Novi Sad, Put Dr Goldmana
4, 21204 Sremska Kamenica, Serbia
c Serbian Academy of Sciences and Arts, Knez Mihajlova 35, 11000 Belgrade, Serbia
O
O
O
O
O
O
O
O
O
isosterism
isosterism
(H → NO2)
(NO2 → Cl)
O
O
O
OAc OAc
OAc OAc
OAc OAc
O2N
Cl
Cleistenolide (1)
IC50 = 36.94 µM (Raji)
5
4
IC50
= 18.54 µM (Raji)
IC50
= 1.85 µM (Raji)
New synthesis of cleistenolide (1) was achieved through 6 steps with a total yield of 67%. Twenty
analogues of natural product 1 were designed, synthesized, and evaluated for their antiproliferative
activity against eight tumour cell lines.
1

Synthesis, antiproliferative activity and SAR analysis of (−)-cleistenolide and
analogues
Goran Benedeković a, Mirjana Popsavin a, Ivana Kovačević a, Vesna Kojić b, Marko Rodić a,
Velimir Popsavin a,c,*
a Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences,
University of Novi Sad, Trg Dositeja Obradovića 3, Novi Sad, Serbia
b Oncology Institute of Vojvodina, Faculty of Medicine, University of Novi Sad, Put Dr Goldmana
4, 21204 Sremska Kamenica, Serbia
c Serbian Academy of Sciences and Arts, Knez Mihajlova 35, 11000 Belgrade, Serbia
Abstract
A new, modified total synthesis of (−)-cleistenolide (
was achieved starting from commercially available 1,2-
synthesis of proceeds in six steps and 67% overall yield, using single-carbon atom degradation
of a protected chiral precursor, ( )-selective Wittig olefination, and acid catalyzed δ-lactonization.
A new Lewis acid promoted procedure for one-pot -debenzylation/ -acylation has been
developed to complete the synthesis of natural product and selected analogues. The synthesized
1
) and sixteen new analogues or derivatives
O
-isopropylidene-α- -glucofuranose. The
D
1
Z
O
O
1
compounds were tested in vitro to evaluate their cytotoxicity against K562, HL-60, Jurkat, Raji,
MCF-7, MDA-MB 231, HeLa, A549, and MRC-5 cell lines. All (−)-cleistenolide analogues
exhibited significantly higher cytotoxicity than lead
1 against the majority of cell lines tested.
Most of the synthesized compounds are more active than doxorubicin on at least one malignant
cell line, but were almost completely inactive against normal MRC-5 cells. The structural features
of the tested compounds responsible for their antiproliferative activity have been identified by
preliminary SAR analysis.
Keywords: (−)-cleistenolide, 4,5-dihydropyran-2-ones, α,β-unsaturated-δ-lactones, antitumour
agents, SAR analysis.
2

1. Introduction
(−)-Cleistenolide (4,6-di-
, Fig. 1) is a naturally occurring α,β-unsaturated δ-lactone isolated from Cleistochlamys kirkii
Oliver [1], which shows both antibacterial [1] and antitumour activity [2,3].
O-acetyl-7-O-benzoyl-2,3-dideoxy-D-arabino-hept-2-eno-1,5-lactone,
1
O
O
O
R
O
R
O
O
O
O
R2
R1
O
O
RO
OAc OAc
OR1 OR1
OR OR
R
1 R = R1 = R2 = H
(cleistenolide)
2 R = R1 = H, R2 = OMe
3 R = R1 = Cl, R2 = H
4 R = R2 = H, R1 = Cl
5 R = R2, R1 = NO2
6 R = H, R1 = Bn
14 R = Bz
15 R = 3-MeOBz
16 R = Ac
7 R = OMe, R1 = Bn
8 R = F, R1 = Bn
9 R = NO2, R1 = Bn
10 R = H, R1 = Ac
11 R = OMe, R1 = Ac
12 R = F, R1 = Ac
13 R = NO2, R1 = Ac
Fig. 1. Structures of (−)-cleistenolide (1) and the corresponding analogues/derivatives 2–16.
There are ten reports on the total synthesis of (−)-cleistenolide [4–13] including an approach
recently implemented in our laboratory [3]. Schmidt and co-workers [4] reported the first total
synthesis of cleistenolide that proceeded in six steps and 18% overall yield. Cai and co-workers [5]
reported the most efficient synthesis of (−)-cleistenolide starting from
49% overall yield. Another approach for the synthesis of was reported by Ramesh and Meshram
[6] starting from -mannitol. Reddy and co-workers [7] reported a stereoselective synthesis of (−)-
D-arabinose in eight steps and
1
D
cleistenolide employing the Barbier allylation, MacMillan α-hydroxylation, Stille-Gennari
olefination, and CeCl3-mediated lactonization as key steps. The next stereoselective total synthesis
of (−)-cleistenolide was accomplished starting from
D-(−)-isoascorbic acid [8]. An efficient total
synthesis of (−)-cleistenolide from -mannitol with an overall yield of 23.6% was described by
D
Babu and co-workers [9]. This approach involves a one-C-atom Wittig olefination, a selective
allylic triethylsilyl protection, and a Grubbs ring-closure metathesis reaction as the key steps.
Kumar and co-workers [10] reported a stereoselective synthesis of (−)-cleistenolide in 10 steps and
in an overall yield of 19%. Ghogare and co-workers [11] described an efficient stereoselective
synthesis of (−)-cleistenolide starting from
D-tartaric acid. It is completed within 13 steps with an
overall yield of 7.92%. A concise and efficient total synthesis of cleistenolide has been achieved by
3

Reddy and co-workers starting from D-glucose in six steps and 42.5% overall yield [12]. The last
synthesis of cleistenolide based on a modulable tin based allylation method for the synthesis of
1,2,3-triols is in the presence of BF3·OEt2 as a catalyst was published by Karier and co-workers in
2018 [13].
Cleistochlamys kirkii (Benth) Oliv. (Annonaceae) is an African medicinal plant traditionally
used in Mozambique for the treatment of wound infections, tuberculosis, and rheumatism. Previous
phytochemical studies on this species reported the isolation of (−)-cleistenolide (1) that exhibited
antibacterial activity against susceptible strains of Staphylococcus aureus and Bacillus anthracis
and antifungal activity against Candida albicans [1]. However, despite the structural similarity of
natural product
1 with some antitumour δ-lactones, literature data related to the cytotoxicity of
cleistenolide are limited. So far, only one paper has been published on its cytotoxicity toward a
single malignant cell line [2].
Our preliminary research on antiproliferative properties of (−)-cleistenolide (1) and analogues
[3] showed that they exhibits moderate to strong citotoxicity. Brief SAR study which pointed to
structural features beneficial for potency of analogues (such as the presence of additional functional
groups in the aromatic ring, and the presence of cinnamoyl functionality instead of benzoyl group at
the O-7 position) prompted us to prepare a larger number of analogues and derivatives in order to
perform a more detailed SAR study.
2. Results and discussion
2.1. Chemistry
Synthesis of (−)-cleistenolide is illustrated in Scheme 1. This approach represents a successful
modification of our previous synthesis [3], but the applied reaction conditions have been more or
less altered, which significantly increased the overall yield of the final product. The commercially
available monoacetonide of D-glucose (17) was used as a convenient starting material. Compound
17 was converted to the lactol 19 (80.5% from 2 steps) according to a previously published
procedure [3]. Treatment of 19 with periodic acid in aq EtOAc at room temperature, gave the
corresponding aldehyde (19a) which was further converted into alkene 20 via a (Z)-selective Wittig
reaction [14] with methyl (triphenylphosphoranylidene)acetate (Ph3P=CHCO2Me). The required
intermediate 20 was obtained in excellent yield (92% from 2 steps). The Z-olefin (δ 6.02, J2,3 = 11.8
Hz, H-2) was exclusively formed with no E-product being observed in the reaction mixture.
4

RO
RO
BnO
BnO
OH
O
O
b
c
OH
OH
O
BnO
O
OBn OBn
O
BnO
19
RO
17 R = H
18 R = Bn
19a
a
d
O
O
MeO2C
OH
f
e
O
O
BnO
BnO
BzO
OBn OBn
OBn
OBn
OAc OAc
1
21
20
Scheme 1. Reagents and conditions: (a) and (b) reference [3], 80.5% from 2 steps; (c) H5IO6, EtOAc, H2O, rt, 2 h; (d)
Ph3P=CHCO2Me, MeOH, 0 °C for 1 h, then rt for 22 h, 92% from 19; (e) TsOH, CH2Cl2, rt, 96 h, 94%; (f) FeCl3,
BzOH, Bz2O, rt, 24 h, then AcBr, rt, 2 h, 96%.
When the first steps of the sequence were performed without isolation and purification of
intermediates 19 and 19a, the desired olefin 20 was obtained in an overall yield of 75%. Using the
known procedure (TsOH, CH2Cl2, rt), the olefin 20 was converted to α,β-unsaturated δ-lactone 21
in 94% yield. Compound 21 was finally converted to the target
1 (96%) using a new and efficient
one-pot procedure that involves an initial treatment of 21 with Bz2O/BzOH/FeCl3 reagent system,
followed by subsequent acylation with AcBr. Both reactions were carried out in the presence of
catalytic amount of FeCl3 (27 and 54 molar %, respectively). This procedure provided the target
natural product
good agreement with the literature reports [4–13], except for the optical rotation. Namely, we
observed in repeated measurements a value of [α]D24 = −212.2 (
0.5, CHCl3), while Nkunya et al.
[1] reported [α]D = −63.5 ( 0.7, CHCl3) for the isolated natural product. Nearest values for
synthetic sample 0.48, CH2Cl2); Rao et al. [10]
: Schmidt et al. [4] reported [α]D24 = −164.6 (
reported [α]D25 = −160.6 (
1, CHCl3). The other published values are even more different and
1
in 96% yield. For melting point, HRMS, IR, 1H and 13C NMR data we found a
c
c
1
c
c
belong to the interval from −118 to −153.1 [5–9, 12–13]. So far we have no explanation for these
disagreements.
This is the most efficient synthesis of (−)-cleistenolide achieved so far. It is completed in six
steps and 67% overall yield. A similar overall yield (66% from 6 steps) was obtained when the
reaction sequence was performed without isolation and purification of intermediates 19 and 19a
.
Although this new approach is not based on the concepts of green chemistry, its high efficiency in
5

terms of total yield (67%) and number of synthetic steps (6) results in a significant reduction in the
consumption of environmentally harmful chemicals and organic solvents.
The synthesis of isosteres
4
and
5
is shown in Scheme 2. The rationale for the preparation of
and (Fig. 1), with additional
these analogues stems from the fact that (−)-cleistenolide analogues
2
3
substituents in the aromatic rings, exhibit antitumour activity against several human malignant cell
lines [3].
The synthesis of
benzoic acid, in the presence of 1,1′-carbonyldiimidazole (CDI) as a catalyst. Subsequent treatment
with acetic anhydride in pyridine, gave an acceptable yield of desired isostere (31%). The first
step of this one-pot procedure is based on the esterification method adopted from the literature [15].
Compound is a novel cleistenolide analogue aimed for biological testing.
4 begins with the selective esterification of the triol 22 [3] with 4-chloro-
4
4
Recently reported method for the selective esterification of carboxylic acids based on the
Garegg-Samuelsson reaction [15], was adopted for the preparation of analogue . Thus, 4-nitro-
benzoic acid reacted with 22 in the presence of I2/Ph3P reagent system. As in previous case, the
esterification reaction was regioselective. However, the intermediary 7- -monoester was not
isolated or purified, but was in crude form treated with acetic anhydride in pyridine to give the
5
O
isostere
5
(35% from 2 steps) ready for biological testing.
O
O
O
O
O
O
f
O
a
d
O
O
O
(From 22)
(From 21)
RO
AcO
BnO
O
BnO
OAc OAc
OBn OBn
OR OR
Cl
21 R = Bn
22 R = H
23
25
4
(From 22)
e
g
c
(From 22)
b
O
O
O
O
O
O
O
O
O
O
AcO
HO
HO
O
OAc OAc
OBn OBn
OAc OAc
16
HO
O2N
24
26
5
Scheme 2. Reagents and conditions: (a) (i) 4-chlorobenzoic acid, CDI, CH2Cl2, DMF, rt, 24 h, (ii) Ac2O, Py, rt, 24 h,
31%; (b) (i) 4-nitrobenzoic acid, I2, Ph3P, CH2Cl2, Py, rt, 22 h, (ii) Ac2O, Py, rt, 24 h, 35%; (c) AcCl, Py, CH2Cl2, rt, 3.5
h, 75%; (d) Ac2O, AcOH, ZnCl2, rt, 2.5 h, 86%, or Ac2O, AcOH, FeCl3, rt, 5 h, 91%; (e) Amberlyst-15, MeOH, 37 °C,
24 h, 67%; (f) 6 M aq HCl, MeCN, rt, 24 h, 50%; (g) FeCl3, CH2Cl2, rt, 22 h, 94%.
6

Finally, standard acetylation conditions (Ac2O/Py) were applied to convert the triol 22 into the
corresponding tri- -acetyl derivative 16 (75%) also aimed for biological testing.
O
In order to obtain a convenient intermediate for the preparation of cinnamic (−)-cleistenolide
bioisosteres, the synthesis of alcohol 24 is planned (Scheme 2). Using the reported procedure [17]
for selective debenzylation of primary benzyl groups in mono- and disaccharide derivatives
(ZnCl2/Ac2O/HOAc), the tri-O-benzyl ether 21 was converted to the 7-O-acetyl derivative 23 in
86% yield. Our slight modification of this procedure (replacement of ZnCl2 with FeCl3) provided
the desired intermediate 23 in somewhat higher yield (91%). An additional advantage of this new
O
-debenzylation process is the fact that it requires the use of far smaller amounts of the catalyst (0.5
molar equivalents of FeCl3) than the literature procedure (7.5 molar equivalents of ZnCl2 [17]).
Using the adapted literature procedure for transesterification of aromatic acetates by the action of
Amberlyst-15 in methanol [18], the 7-O-acetyl derivative 23 was transformed into the primary
alcohol 24 (67%), which represent an important intermediate for further work.
In an attempt to obtain a better yield of required intermediate 24, we further examined the
method of its preparation by hydrolysis of 23. However, the reaction of 23 with 6 M aq HCl in
acetonitrile, instead of the expected alcohol 24, yielded the bicyclic lactone 25, as a product of the
intramolecular Michael cyclization of intermediate 24. The absence of a double bond in structure 25
and the presence of signals for CH2-2 and CH-3 functions in the 1H NMR spectrum indicate that
Michael cyclization occurred in intermediate 23. The large coupling constant (J6.7a = 10.7 Hz)
indicates the trans diaxial arrangement of H-6 and H-7a, as well as the C-7 being part of a six-
membered ring. The stereochemistry of the intramolecular Michael cyclization (from the α-side)
was confirmed by remote W-coupling between H-3 and H-5 proton signals (4J3.5 = 1.6 Hz).
Since the compound 25 contains [3.3.1] bicyclic core, which is present in some recently
discovered natural products [19], we decided to evaluate the in vitro antitumor activity of both 25
and the corresponding debenzylated derivative (26). Di-O-benzyl derivative 25 was converted to the
required diol 26 after treatment with FeCl3 in dichloromethane. The desired product 26 was thus
obtained in 94% yield.
Esters of 24 with cinnamic acid and derivatives were prepared by using the Steglich protocol
[19] based on the DCC/DMAP reagent system (Table 1).
7

Table 1. Preparation of 7-O-cinnamoyl derivatives 6–9.
O
O
O
O
O
DCC, CH2Cl2
DMAP, rt, 1 h
O
+
OH
O
HO
R
OBn OBn
OBn
OBn
R
6 − 9
24
Cinnamic acid
Entry Cinnamic acid
General structure
Products
% Yield
1
2
3
4
Cinnamic acid (R = H)
6 (R = H)
73
69
84
67
O
O
O
4-Methoxycinnamic acid (R = OMe)
4-Fluorocinnamic acid (R = F)
4-Nitrocinnamic acid (R = NO2)
7 (R = OMe)
8 (R = F)
O
OBn OBn
R
9 (R = NO2)
As can be seen from the table, the highest yield (84%) was obtained in the reaction of
esterification with 4-fluorocinnamic acid (entry 3). The yields of the remaining three reactions are
in the range of 67–73% (entries 1, 2 and 4). The resulting esters represent potential synthetic
6–9
intermediates for the preparation of the corresponding cinnamic (–)-cleistenolide bioisosteres. In
addition, we planned to test the obtained esters in the culture of selected malignant cells, to study
the effect of altering lipophilicity on the antiproliferative activity of analogues.
Both
acetyl bromide in the presence of zinc(II)-triflate as a catalyst (Scheme 3). For this purpose, we
adapted the procedure [21] for the conversion of -benzyl- to -benzoyl functions by replacing
8 and 9 were converted to the corresponding diacetates 12 and 13 after treatment with
O
O
benzoyl bromide with acetyl bromide. This method provided the targets 12 and 13 in 58 and 62%
yield, respectively. This procedure has not yet been used to prepare the remaining two cinnamates
from Fig. 1 (10 and 11). Derivative 10 was obtained using our previously published method [3].
Compound
involves its initial transesterification, followed by esterification with 4-nitrocinnamic acid under the
Steglich conditions. This procedure provided the target in 47% yield (from 2 steps).
9 was alternatively prepared starting from 23, using a two-step sequence, which
9
Methoxy derivative 11 (Scheme 3) was alternatively obtained using the one-pot process which
commenced with the selective esterification of the primary hydroxyl group of 22 using the I2/Ph3P
reagent system [16]. Subsequent acetylation of the resulting diol (not shown in Scheme 3) gave
desired 11 in 64% yield.
8

O
O
a
O
O
O
O
O
O
OBn OBn
OAc OAc
R
R
8 R = F
9 R = NO2
12 R = F
13 R = NO2
O
O
O
O
O
b
O
OH
+
O
HO
MeO
OAc OAc
OH OH
MeO
4-Methoxycinnamic acid
11
22
Scheme 3. Reagents and conditions: (a) AcBr, Zn(OTf)2, CH2Cl2, rt, 22 h, 58% of 12, 62% of 13; (b) (i) I2, Ph3P, Py,
CH2Cl2, rt, 22 h, (ii) Ac2O, Py, rt, 24 h, 64%.
In an attempt to obtain a higher yield of analogue 13, we examined an alternative method of its
preparation starting with the transesterification of 23 in the presence of toluene-4-sulfonic acid as a
catalyst (Scheme 4).
O
O
O
a
O
O
O
+
OMe
HO
HO
AcO
OBn OBn
OBn
OBn
OBn OBn
24
23a
23
b
O
O
O
O
O
O
OMe
OMe
O
O
OAc OAc
OBn OBn
O2N
O2N
27
23b
c
+
O
O
O
O
O
O
O
O
OAc OAc
OBn OBn
O2N
O2N
13
9
Scheme 4. Reagents and conditions: (a) TsOH, MeOH, rt, 24 h; (b) 4-nitrocinnamic acid, DCC, CH2Cl2, DMAP, rt, 2 h;
(c) (i) AcBr, Zn(OTf)2, CH2Cl2, rt, 24 h, 21% of 27, 18% of 13, (ii) flash column chromatography
.
9

Accordingly, treatment of 23 with TsOH in methanol gave an inseparable mixture of the
expected unsaturated alcohol 24 and the 3- -methyl derivative 23a resulting from Michael addition
O
of methanol to the, α,β-unsaturated enone system. The crude mixture was subjected to subsequent
esterification with 4-nitrocinnamic acid, under the Steglich conditions [20]. A mixture of the
corresponding esters was obtained which, again, could not be separated by column chromatography.
The mixture was reacted with Zn(OTf)2/Ac2O reagent system, in dichloromethane, to afford a low
yield of 27 (21%), accompanied with a similar amount of unsaturated ester 13 (18%). Both products
were readily separated by flash chromatography and fully characterized by spectroscopic methods
(IR, HRMS, 1H and 13C NMR).
The structure and stereochemistry of 27 has been confirmed by X-ray crystallographic analysis
and the corresponding ORTEP presentation is shown in Fig. 2.
Fig. 2. ORTEP presentation of compound 27. H-atoms are omitted for clarity.
As shown in Fig. 2, the newly introduced C-3 methoxy group is α-oriented, while the
conformation of six-membered lactone ring (O1–C1–C2–C3–C4–C5) can be practically described
as boat (BC2,C5), since asymmetry parameters [22] ΔCs(C2) = ΔCs(C5) = 7.2(4)°, and ΔCs(O1–
C1) = ΔCs(C3–C4) = 6.0(6)° are in accordance with approximate mirror planes passing through C2
and C5 atoms, as well as centres of O1–C1 and C3–C4 bonds, both perpendicular to ring mean
plane. Rigorous ring puckering analysis [23, 24] revealed that the exact conformation deviates from
O1
perfect BC2,C5 boat towards twist boat O1TC2 by 15% along BC2,C5
Based on angle
vector [25], main ring substituents are classified as: axial (O4,
80.8(3)°), and pseudoequatorial (O3, = 58.2(3)°).
→
T
C2
pseudorotational pathway.
δ
, subtended between Cremer-Pople ring mean plane normal and substituent bond
δ
= 21.3(2)°), equatorial (C6,
δ =
δ
10

2.2. Antiproliferative activities and SAR analysis
Antiproliferative activities of synthesized compounds were evaluated in vitro against eight
human tumour cell lines (myelogenous leukaemia K562, promyelocytic leukaemia HL-60, Jurkat T
cells leukaemia, Raji Burkitt's lymphoma, estrogen receptor positive (ER+) breast adenocarcinoma
MCF-7, estrogen receptor negative (ER−) breast adenocarcinoma MDA-MB-231, cervix carcinoma
HeLa, alveolar basal adenocarcinoma A549) and one normal cell line (foetal lung fibroblasts MRC-
5) by using MTT assay [26]. Natural product cleistenolide (1) and commercial antitumour drug
doxorubicin (DOX) were used as positive controls. The results are presented in Table 2.
Table 2. In vitro cytotoxicity (−)-cleistenolide (1) and analogues/derivatives (2–26).
IC50 (µM) a
Compounds
K562
7.65 b
2.69 b
1.02 b
3.69
7.64
1.59
0.43
1.01
1.27
0.28 b
6.96
3.46
2.34
3.25
4.65
7.54
0.88
1.42
8.10
2.92
2.38
0.25
HL-60
1.21 b
0.89 b
36.21 b
32.56
1.11
Jurkat
14.22 b
8.31 b
6.25 b
24.36
7.45
4.96
10.33
4.16
Raji
36.94 b
25.64 b
11.24 b
1.85
18.54
1.33
0.69
MCF-7
26.07 b
4.71 b
0.62 b
2.64
5.32
2.57
1.11
3.01
MDA-MB 231
2.25 b
7.46 b
0.02 b
1.85
4.12
4.97
3.26
3.58
HeLa
7.32 b
1.95 b
14.02 b
11.02
4.65
9.51
1.08
7.31
2.65
0.21
5.93
1.68
1.84
15.47
7.64
2.64
5.35
4.80
8.22
1.17
0.67
0.07
A549
16.34 b
11.02 b
4.23 b
15.66
8.64
MRC-5
>100 b
91.34 b
45.68 b
˃100
>100
>100
>100
>100
>100
˃100 b
78.03
˃100
˃100
˃100
˃100
˃100
>100
>100
>100
>100
˃100
0.10
1 (cleistenolide)
2
3
4
5
6
7
8
0.61
1.57
5.40
13.25
4.25
2.47
12.12
5.24
2.53
5.71
0.14
1.66
1.55
9
24.59 b
10.02
1.89
0.25 b
2.59
11.21 b
10.01
2.31
15.98 b
9.31
1.46 b
22.79
34.38
6.93
2.02 b
1.40
10
11
12
13
14
15
16
23
24
25
26
27
DOX
5.32
0.58
4.26
1.40
2.36
30.25
3.87
18.85
17.63
0.21
3.34
5.12
2.12
2.26
4.03
8.25
6.21
29.65
9.23
10.18
3.48
1.64
3.11
0.92
2.75
1.08
2.69
10.05
1.82
0.01
0.60
8.12
5.64
47.21
21.25
24.57
14.66
12.28
1.08
1.65
0.12
0.03
52.32
1.58
7.48
6.42
4.79
21.54
18.21
12.64
15.04
12.87
6.24
1.15
0.18
4.91
0.16
31.50
28.61
0.09
2.98
0.20
a IC50 is the concentration of compound required to inhibit the cell growth by 50% compared to an untreated control. Values are means of three
independent experiments. Coefficients of variation were less than 10%.
b Taken from reference [3].
The results in Table 2 show that all investigated compounds but one (25) were more active than
lead
which 10 showed similar potency as commercial drug DOX. Only three compounds were more
potent than natural product against promyelocytic leukaemia cells (HL-60), but two ( and
1, in the culture of K562 cells. Three compounds exhibited submicromolar activities, among
1
2
6)
displayed better activity than DOX. Although none of the derivatives were more active than DOX
toward Jurkat cells, many showed prominent activities; 75% of the investigated compounds were
more active than lead
analogue 13 and 3- -methyl derivative 27 with submicromolar IC50 values (0.25, 0.58, and 0.12
μM) being 24.5–118.5 fold more active than . All synthesized compounds were more active than
1. Especially stand out 7-O-cinnamoyl ester 10, its 4-nitro-cinnamoyl
O
1
11

lead
1 toward Raji cells and twelve compounds were more active than DOX in this cell culture. It
should be noted that four analogues showed submicromolar activities, and among them 4,6-dibenzyl
derivative 24 showed remarkable potency of 0.01 μM, which is the best activity recorded in the
synthesized series (298-fold more active than DOX and 3694-fold more active than lead
culture of ER− breast adenocarcinoma, MDA-MB-231, six analogues are more active than
all but one were more potent against ER+ breast adenocarcinoma MCF-7 cells. Trichlorobenzoyl
analogue and bicyclic di- -benzyl derivative 25 showed submicromolar activities against both
ER+ and ER− breast adenocarcinoma cell lines. Compound 25 exhibited similar activity as DOX and
124-fold higher activity than lead toward MCF7 cells, while was 5-fold more active than DOX
and 132-fold more active than against MDA-MB 231 cells. Fourteen compounds were more
active than (−)-cleistenolide in HeLa cell culture, and the highest potencies toward these cells were
demonstrated by 7- -cinnamoyl derivative 10 and 3- -methyl derivative 27 with submicromolar
activities recorded (IC50 0.21 and 0.67μM; 35-fold and 11-fold more active than respectively).
A549 cancer cells were sensitive to all synthesized compounds, of which 90% showed better
activity than lead , while eight were more active than DOX. The highest potency in the culture of
A549 cell was recorded after treatment with analogue 27 (IC50 0.18 μM), which demonstrated 27-
fold better activity than DOX and over 90-fold higher potency than
1
). In the
1
, while
3
O
1
3
1
O
O
1
1
1
.
Overall, eighteen of twenty cleistenolide analogues showed higher potency than DOX against
at least one human tumour cell line (often two), and in average all compounds were more active
than lead
1 against six (of eight) tumour cell lines. Further, it is important to highlight that, in
contrast to commercial drug doxorubicin, seventeen of twenty synthesized compounds were
completely non-toxic toward normal foetal fibroblasts (MRC-5) while three of them showed very
modest activity in the culture of these cells (
μM). In addition, the most of tested compounds (18 of 21) showed good to excellent selectivity for
malignant cell lines with a selectivity index (SI) in a range of 4 to 24000. Only three compounds (
, and 11) displayed SI outside this range (see, Table S4 in the Supplementary Data for details).
2: IC50 91.34 μM; 3: IC50 45.68 μM; 11: IC50 78.03
2
,
3
These results suggest potential selectivity of synthesized compounds toward tumour cells but this
should be further verified by investigating toxicity toward a wider panel of normal cells.
In order to correlate the structures of synthesized compounds with their antiproliferative
activities, we first considered the influence of additional substituents in the aromatic ring (Fig. 3a).
We compared activity of synthesized (−)-cleistenolide (control in this analysis) with analogues
2, 3, 4, 5 and activity of 4-chlorobenzoyl analogue 4 (control) with 2,4,6-tri-chloro-benzoyl
12

derivative 3. The data clearly showed that the presence of a substituent in the benzene ring leads to
an increase in activity toward most cell lines. We believe that a crucial structural feature of the
newly introduced functional groups is their ability to act as proton acceptors when establishing H-
bonds with the receptor. That was also true for tri-chloro
3 vs. monochloro derivative 4. Next, we
studied impact of C-7 substituent on cytotoxicity (Fig. 3b).
Fig. 3. Contributions of selected structural features to the
cytotoxic activities: (a) influence of substituents in the 7-O-
benzoyl residue; (b) influence of an acid moiety at the C-7
position; (c) influence of the lactone ring rigidity. A negative
Δ log IC50 value means that the analogue is more active than
the control.
13

Hence, we used the natural lactone
1 as control and cinnamoyl derivatives as analogues (10, 11,
12
, 13, and 27). We observed that 4-substitued cinnamates were more active than benzoyl control
toward all tumour cell lines but HL-60 and MDA-MB231, while non-substituted cinnamoyl
derivative 10 was more active toward all cell lines with the exception HL-60. Final SAR analysis
(Fig. 3f) showed that system rigidity is beneficial for antitumour activity of this type of compounds
(bicyclic 25 was more active toward majority of cell lines than the ring-open analogue 24) and that
presence of benzyl ether at C-4 and C-6 is not crucial since bicyclic di-O-benzyl analogue (25) was
more active toward just 50% of cell lines than corresponding diol (26).
We further explored the effects of different groups in the para position of 7-O-cinnamoyl
derivatives, the influence of C-4 and C-6 substituents, as well as the impact of different ester groups
at C-4, C-6 and C-7 positions. However, none of these structural changes showed any clear
structure-activity relationship (see Supplementary data).
To determine whether antiproliferative potency is associated with water solubility, lipophilicity,
or other molecular parameters, brief ADME calculations were performed using the SwissADME
web tool (http://www.svissadme.ch/). The obtained results (Table S5 in the Supplementary data)
show that the influence of applied physicochemical parameters is very different depending on the
type of tumour cells evaluated. This indicates that the synthesized compounds do not act on the
same molecular target. Finally, the results obtained show that all compounds (except 15) meet
Lipinski's "rule five", which indicates their potential bioavailability.
3. Conclusions
A new, efficient synthesis of (−)-cleistenolide (
lactone, has been achieved. The synthesis was accomplished in six steps and in a total yield of 67%
starting from a commercially available -glucose derivative, and represents the most efficient route
to this natural product so far. Key features of our route to included: the efficient utilization of a
single-carbon atom degradation of protected chiral precursor, a ( )-selective Wittig olefination,
followed by an acid catalyzed δ-lactonization. A new Lewis acid promoted procedure for one-pot
-debenzylation/ -acylation has been developed to complete the synthesis of natural product . A
similar methodology was then applied to prepare sixteen new and four known [3] analogues of
1), a naturally occurring biologically active δ-
D
1
Z
O
O
1
1
.
The antiproliferative activities of the synthesized compounds were evaluated in vitro against a panel
of eight human tumour cell lines, as well as against one normal cell line. All newly synthesized
analogues exhibited higher cytotoxicities than the lead
1 against the majority of cell lines tested.
14

Furthermore, eighteen of twenty evaluated compounds showed higher potency than the commercial
drug doxorubicin (DOX) against at least one human tumour cell line. In contrast to DOX, seventeen
of twenty synthesized compounds were completely non-toxic toward normal MRC-5 cells, while
only three compounds exhibited weak activities. SAR analysis was used to identify the structural
features of the tested compounds responsible for their antitumor activity. Introduction of additional
substituents into the aromatic ring, replacement of benzoyl group at O-7 with the cinnamoyl group,
and introduction of conformational constraints by construction of the [3,3,1] bicyclic ring system,
caused an increase in activity toward the most cells tested. Other structural changes did not show
clear structure-activity relationships. Taken together the antitumour activity profile, and reasonable
ADME properties it is assumed that these pyranone based molecules are promising candidates as
novel anticancer agents. However, further structural refinement and more detailed biological
characterizations are required.
4. Experimental section
4.1. General experimental procedures
Melting points were determined on a Hot Stage Microscope Nagema PHMK 05 and were not
corrected. Optical rotations were measured on an Autopol IV (Rudolph Research) polarimeter at
room temperature. NMR spectra were recorded on a Bruker AC 250 E or a Bruker Avance III 400
MHz instrument and chemical shifts are expressed in ppm downfield from TMS. IR spectra were
recorded with an FTIR Nexus 670 spectrophotometer (Thermo-Nicolet). High resolution mass
spectra (ESI) were acquired on a Agilent Technologies 1200 series instrument equipped with
Zorbax Eclipse Plus C18 (100 mm × 2.1 mm i.d. 1.8 µm) column and DAD detector (190–450 nm)
in combination with a 6210 time-of-flight LC/MS instrument (ESI) in the positive ion mode. Flash
column chromatography was performed using Kieselgel 60 (0.040–0.063, E. Merck). All organic
extracts were dried with anhydrous Na2SO4. Organic solutions were concentrated in a rotary
evaporator under diminished pressure at a bath temperature below 35 °C. The purities of final
products were established by HPLC-HRMS and were found to be >95% pure.
4.2. Methyl (2Z)-4,6,7-tri-O-benzyl-2,3-dideoxy-D-arabino-hept-2-enoate (20)
Procedure A: To a solution of 19 (2.3 g, 5.11 mmol) in EtOAc (40 mL) and H2O (40 mL) was
added H5IO6 (2.327 g, 10.21 mmol) and the resulting solution was stirred at room temperature for 2
h. The mixture was poured to 10% NaCl (80 mL) and extracted with EtOAc (3 × 40 mL). The
combined organic phases were first washed with 10% NaCl (100 mL), then dried and evaporated.
15

The residue was dried under high vacuum for 12 h and the resulting crude compound 19a was
dissolved in absolute MeOH (200 mL) and cooled to 0 °C. Stabilized ylide Ph3P=CHCO2Me (3 g,
8.98 mmol) was added, and after 1 h the cooling bath was removed and the reaction continued at
room temperature for 4 h. A new portion of Ph3P=CHCO2Me (2.12 g, 6.35 mmol) was then added
and stirring continued for an additional 18 h. The reaction mixture was evaporated and the residue
was purified by flash chromatography (1:1 light petroleum/Et2O) to afford pure 20 (2.235 g, 92%
from 19) as a colourless syrup.
Procedure B: A solution of 18 (0.600 g, 1.22 mmol) in 50% aq TFA (10 mL) was stirred at
room temperature for 2.5 h, then evaporated by co-distillation with toluene to remove the traces of
acid and water. To a solution of remaining crude 19 in 1:1 EtOAc/H2O (8 mL) was added H5IO6
(0.557 g, 2.45 mmol) and the resulting solution was stirred at room temperature for 2.5 h. The
mixture was poured in 10% aq NaCl (20 mL) and extracted with EtOAc (3 × 30 mL). Organic
phase was separated, dried and evaporated, and the residue was dried in high vacuum for 2 h. To a
cooled (0 °C) solution of crude 19a in absolute MeOH (60 mL) was added Ph3P=CHCO2Me (0.560
g, 1.83 mmol). The mixture was stirred at 0 °C for 1 h and at room temperature for 2 h. A new
portion of reagent (0.337 g, 1.35 mmol) was added and stirring at room temperature was continued
for the next 20 h. The mixture was evaporated and the residue purified on a column of flash silica
(2:1 light petroleum/Et2O) to give pure product 20 (0.437 g, 75% from 18) as a colourless syrup.
The physical and spectroscopic data of the synthesized intermediate 20 are identical to the
values previously reported by us [3].
4.3. 4,6,7-Tri-O-benzyl-2,3-dideoxy-D-arabino-hept-2-eno-1,5-lactone (21)
Lactone 21 was prepared according to the procedure published by us [3]. Its IR, 1H and 13C
NMR spectra, as well as HRMS spectral data are identical to the reported values.
4.4. Cleistenolide (4,6-Di-O-acetyl-7-O-benzoyl-2,3-dideoxy-D-arabino-hept-2-eno-1,5-lactone, 1)
To a solution containing compound 21 (0.05 g, 0.11 mmol), Bz2O (0.031 g, 0.14 mmol) and
BzOH (0.067 g, 0.55 mmol) in dry CH2Cl2 (10 mL) was added while stirring at room temperature
FeCl3 (0.005 g, 0.03 mmol). After 2 hours, a new portion of Bz2O (0.031 g, 0.14 mmol) was added
and the reaction continued for another 20 h at room temperature. A new portion of FeCl3 (0.005 g,
0.03 mmol) was then added to the solution, stirring was continued for 2 hours, after which AcBr
(0.16 mL, 2.2 mmol) was finally added. After the next 2 hours the mixture was distributed between
16

CH2Cl2 (10 mL) and 10% NaHCO3 (60 mL). The organic phase was separated and the aqueous
phase was extracted with CH2Cl2 (2 × 15 mL). The combined organic phases were washed with
10% NaCl (30 mL), dried and evaporated. The residue was purified by flash chromatography (1:2
light petroleum/Et2O) to give pure
mp 133−134 °C, [α]D = −212.2 (
−164.6 ( 0.48, CH2Cl2). Recrystallization from Et2O/hexane gave white needles, mp 138 °C, Rf =
1
(0.039 g, 96%) in the form of a white solid, mp 135 °C, lit. [4]
c
0.5, CHCl3) or [α]D = −212.4 ( 0.5, CH2Cl2), lit. [4] [α]D =
c
c
0.36 (3:7 light petroleum/Et2O).
The spectroscopic data (IR, 1H and 13C NMR spectra, as well as HRMS data) of synthesized
natural product
1 were identical in all respects to the reported values [1,9]; see Table S3 in the
Supplementary data.
4.5. 4,6-Di-O-Acetyl-7-O-(4-chlorobenzoyl)-2,3-dideoxy-D-arabino-hept-2-eno-1,5-lactone (4)
To stirred solution of 4-chlorobenzoic acid (0.047 g, 0.30 mmol) in a 7:3 mixture of
CH2Cl2/DMF (5 mL) was added CDI (0.056 g, 0.35 mmol), and after 2 h crystalline compound 22
[3] (0.050 g, 0.29 mmol) is added. After additional 22 h, anhydrous Py (3 mL) was first added to the
mixture, followed by the addition of Ac2O (6 mL). The reaction solution was stirred at room
temperature for another 24 h, then evaporated, and the residue was purified twice by flash column
chromatography (first with 3:2 Et2O/light petroleum, then with 49:1 CH2Cl2/Me2CO). Pure
4
(0.035
g, 31%) was obtained as a colourless glass, [α]D = −242.4 (c 0.5, CHCl3), Rf = 0.34 (7:3 Et2O/light
petroleum).
IR (film): νmax 1747 (C=O), 1173 (C-O-C).
1H NMR (400 MHz, CDCl3): δ 1.99 and 2.09 (2 × s, 3 H each, 2 × CH3CO), 4.30 (dd, 1 H,
J7a,7b = 12.7, J6,7a = 4.1 Hz, H-7a), 4.71 (dd, 1 H, J7a,7b = 12.5, J6,7b = 2.3 Hz, H-7b), 4.87 (dd, 1 H,
J4,5 = 3.0, J5,6 = 9.6 Hz, H-5), 5.42 (m, 1 H, J6,7b = 2.8, J6,7a = 3.8, J5,6 = 9.6 Hz, H-6), 5.61 (dd, 1 H,
J4,5 = 2.7, J3,4 = 6.2 Hz, H-4), 6.33 (d, 1 H, J2,3 = 9.6 Hz, H-2), 7.14 (dd, 1 H, J2,3 = 9.6, J3,4 = 6.1
Hz, H-3), 7.44 and 7.93 (2 × d, 2 H, Ph).
13C NMR (100 MHz, CDCl3): δ 20.6 and 20.7 (2 ×
75.3 (C-5), 125.6 (C-2), 126.8, 129.1, 131.3, 140.6 (Ph), 139.7 (C-3), 161.1 (C-1), 164.6
OC6H4Cl), 169.1 and 170.4 (2 × OCH3).
CH3CO), 60.7 (C-4), 61.3 (C-7), 67.6 (C-6),
(C
C
HRMS: m/z 419.0499 (M++Na), calcd for C18H17ClO8Na: 419.0504.
17

4.6. 4,6-Di-O-acetyl-2,3-dideoxy-7-O-(4-nitrobenzoyl)-D-arabino-hept-2-eno-1,5-lactone (5)
To a stirred solution of I2 (0.109 g, 0.43 mmol) in anhydrous CH2Cl2 (7 mL) was added Ph3P
(0.113 g, 0.43 mmol) whereby the solution turns brown-yellow. Pyridine is then added (0.12 mL,
9.32 mmol) whereby the colour of the solution changes to light yellow. After the next 5 minutes to
the solution was added 4-nitrobenzoic acid (0.072 g, 0.43 mmol) followed by the compound 22
(0.049 g, 0.28 mmol). Stirring at room temperature was continued for an additional 22 h and then
Ac2O (7 mL) and Py (1 mL, 77.36 mmol) were successively added to the solution. The stirring at
room temperature was continued for additional 24 h and then the mixture was evaporated by co-
distillation with toluene. The residue was suspended in H2O (80 mL) and extracted with CH2Cl2 (3
× 20 mL). The combined organic phases are dried and evaporated and the residue was purified by
flash column chromatography (3:1 Et2O/light petroleum) to yield pure
5 (0.040 g, 35%) as a
colourless solid, which crystallizes from the mixture CH2Cl2/hexane in the form of white powder,
mp (190 °C transition to wax) 196–198 °C, [α]D = −187.4 (c 0.5, CHCl3), Rf = 0.44 (3:1 Et2O/light
petroleum).
IR (KBr): νmax 1750 (C=O, lactone), 1735 (C=O, ester).
1H NMR (400 MHz, CDCl3): δ 2.06 and 2.10 (2 × s, 3 H each, 2 × CH3CO), 4.58 (dd, 1 H,
J7a,7b = 12.4, J6,7a = 4.9 Hz, H-7a), 4.77 (dd, 1 H, J4,5 = 2.5, J5,6 = 9.6 Hz, H-5), 4.95 (dd, 1 H, J7a,7b
= 12.4, J6,7b = 2.1 Hz, H-7b), 5.43 (dd, 1 H, J4,5 = 2.6, J3,4 = 6.0 Hz, H-4), 5.54 (ddd, 1 H, J6,7b = 2.3,
J6,7a = 4.8, J5,6 = 9.4 Hz, H-6), 6.29 (d, 1 H, J2,3 = 9.7 Hz, H-2), 7.00 (dd, 1 H, J2,3 = 9.7, J3,4 = 6.1
Hz, H-3), 8.18 and 8.29 (2 × d, 4 H, J = 8.6 Hz, Ph).
13C NMR (100 MHz, CDCl3): δ 20.4 and 20.6 (2 ×
75.5 (C-5), 125.3 (C-2), 123.6, 130.8, 134.8, 150.7 (Ph), 139.7 (C-3), 160.9 (C-1), 164.1
OC6H4NO2), 169.4 and 169.8 (2 × OCH3).
CH3CO), 59.6 (C-4), 63.2 (C-7), 67.4 (C-6),
(C
C
HRMS: m/z 446.0484 (M++K), calcd for C18H17NO10K: 446.0490.
4.7. 4,6,7-Tri-O-acetyl-2,3-dideoxy- -arabino-hept-2-eno-1,5-lactone (16
D
)
To a stirred solution of 22 (0.04 g, 0.23 mmol) in a mixture of anhydrous Py (1.5 mL) and
CH2Cl2 (1.5 mL) was added slowly AcCl (0.11 mL, 1.38 mmol). After stirring at room temperature
for 3.5 h, the solution was co-distilled with toluene until complete removal of Py and the resulting
residue was purified by flash column chromatography (19:1 CH2Cl2/EtOAc), to give pure 16 (0.052
18

g, 75%) in the form of colourless syrup, [α]D = −204.8 (c 0.5, CHCl3), Rf = 0.33 (7:3 Et2O/light
petroleum).
IR (film): νmax 1744 (C=O).
1H NMR (400 MHz, CDCl3): δ 2.03, 2.05, 2.06 (3 × s, 3 H each, 3 × CH3CO), 4.23 (dd, 1 H,
J7a,7b = 12.5, J6,7a = 4.1 Hz, H-7a), 4.67 (br d, 1 H, J7a,7b = 12.5, H-7b), 4.72 (dd, 1 H, J4,5 = 2.5, J5,6
= 9.5 Hz, H-5), 5.31–5.40 (m, 2 H, H-4 and H-6), 6.25 (d, 1H, J2,3 = 9.7 Hz, H-2), 6.97 (dd, 1 H, J2,3
= 9.7, J3,4 = 6.1 Hz, H-3).
13C NMR (100 MHz, CDCl3): δ 20.4, 20.6, 20.61 (3 ×
CH3CO), 59.7 (C-4), 61.5 (C-7), 67.4
(C-6), 75.2 (C-5), 125.3 (C-2), 139.7 (C-3), 161.1 (C-1), 169.4, 169.8, 170.4 (3 × COCH3).
HRMS: m/z 323.0740 (M++Na), calcd for C18H16O8Na: 323.0743.
4.8. 7-O-Acetyl-4,6-di-O-benzyl-2,3-dideoxy-D-arabino-hept-2-eno-1,5-lactone (23)
Procedure A: To a stirred solution of 21 (0.15 g, 0.34 mmol) in a mixture of Ac2O (2 mL) and
AcOH (2 mL) was added ZnCl2 (0.345 g, 2.53 mmol). The mixture was stirred at room temperature
for 2.5 h, then poured into 10% NaHCO3 (80 mL) and extracted with CH2Cl2 (3 × 20 mL). The
organic phases were combined, dried and evaporated and the residue was purified on a column of
flash silica (1:2 EtOAc/light petroleum). Pure 23 (0.115 g, 86%) was obtained as colourless glassy
solid.
Procedure B: To a stirred solution of 21 (0.4 g, 0.9 mmol) in anhydrous CH2Cl2 (15 mL) were
successively added AcOH (0.62 mL, 10.8 mmol), Ac2O (0.76 mL, 8.1 mmol) and FeCl3 (0.075 g,
0.45 mmol) in five equal portions during 45 min. The mixture was stirred at room temperature for 5
h, then poured into 2% aq NaHCO3 (150 mL) and extracted with CH2Cl2 (3 × 30 mL). The
combined organic layers were dried and evaporated and the residue was purified by flash column
chromatography (1:2 EtOAc/light petroleum) to afford pure 23 (0.325 g, 91%) as colourless glassy
solid. Recrystallization from ether gave white needles, mp 64 °C (phase change at 35 °C), [α]D =
−168.6 (c 0.5, CHCl3), Rf = 0.43 (2:3 EtOAc/light petroleum).
IR (KBr): νmax 1737 (C=O).
1H NMR (400 MHz, CDCl3): δ 2.10 (s, 3 H, CH3CO), 4.19–4.28 (m, 3 H, H-4, H-6 and H-7a),
4.46–4.52 (m, 2 H, CH2Ph and H-5), 4.56 and 4.62 (2 × d, 2 H, Jgem = 11.5 Hz, CH2Ph at C-4), 4.80
19

(d, 1 H, Jgem = 10.9 Hz, CH2Ph at C-6), 4.87 (dd, 1 H, J6,7b = 2.6, J7a,7b = 12.6 Hz, H-7b), 6.21 (d, 1
H, J2,3 = 9.8 Hz, H-2), 7.00 (dd, 1 H, J2,3 = 9.8, J3,4 = 5.7 Hz, H-3), 7.24–7.40 (m, 10 H, Ph).
13C NMR (100 MHz, CDCl3): δ 20.9 (
72.4 ( H2Ph at C-6), 74.0 (C-6), 77.7 (C-5), 124.4 (C-2), 127.8, 127.9, 128.0, 128.2, 128.5, 128.6,
CH3CO), 60.9 (C-7), 65.2 (C-4), 71.3 (CH2Ph at C-4),
C
137.5, 137.53 (2 × Ph), 143.0 (C-3), 162.3 (C-1) and 170.8 (CH3CO).
LRMS (ESI+): m/z 397 (M++H).
Anal. Found: C, 69.43; H, 6.05. Calcd for C23H24O6: C, 69.68; H, 6.10.
4.9. 4,6-Di-O-benzyl-2,3-dideoxy- -arabino-hept-2-eno-1,5-lactone (24
D
)
Suspension containing compound 23 (0.2 g, 0.5 mmol), acidic ion-exchange resin A15 (0.5 g,
2.35 mmol of H+) in dry MeOH (10 mL) was vigorously stirred at 37 °C for 24 h. The resign was
separated by filtration and washed with dry MeOH. Combined organic solutions were evaporated
and the residue was purified by flash column chromatography (1:1 EtOAc/light petroleum). Pure 24
(0.12 g, 67%) was obtained as a colourless glassy solid that crystallized from Et2O/light petroleum
in the form of colourless needles, mp 88 °C, [α]D = −281.6 (c 0.5, CHCl3), Rf = 0.25 (1:1
EtOAc/light petroleum).
IR (KBr): νmax 3504 (OH), 1716 (C=O).
1H NMR (400 MHz, CDCl3): δ 2.05 (bs, 1 H, OH), 3.94 (dd, 1 H, J6,7a = 2.6, J7a,7b = 12.3 Hz,
H-7a), 4.01 (dd, 1 H, J6,7a = 2.8, J7a,7b = 12.3 Hz, H-7b), 4.07 (dt, 1 H, J6,7b = 2.7, J6,7a = 2.6, J5,6
=
9.5 Hz, H-6), 4.26 (dd, 1 H, J4,5 = 2.5, J3,4 = 5.7 Hz, H-4), 4.50–4.64 (m, 4 H, CH2Ph and H-5), 4.74
(d, 1 H, Jgem = 11.3 Hz, CH2Ph), 6.20 (d, 1 H, J2,3 = 9.8 Hz, H-2), 7.01 (dd, 1 H, J2,3 = 9.8, J3,4 = 5.7
Hz, H-3), 7.25–7.41 (m, 10 H, Ph).
13C NMR (100 MHz, CDCl3): δ 59.3 (C-7), 65.2 (C-4), 71.0 and 72.1 (2 ×
CH2Ph), 76.0 (C-6),
77.4 (C-5), 124.3 (C-2), 127.6, 127.62, 127.9, 128.0, 128.4, 128.5, 137.4, 137.6 (Ph), 143.0 (C-3),
162.5 (C-1).
HRMS: m/z 377.1366 (M++Na), calcd for C21H22O5Na: 377.1365.
4.10. (3R,4R,5R,6R)-4,6-dibenzyloxy-8,9-dioxabicyclo[3.3.1]nonan-1-one (25
)
20

To a stirred solution of 23 (0.15 g, 0.38 mmol) in CH3CN (10 mL) was added 6 M aq HCl (1
mL). The mixture was stirred at room temperature for 24 h, then poured into 1% aq NaHCO3 (60
mL) and extracted with EtOAc (3 × 15 mL). The organic phases were combined, dried and
evaporated, and the residue was purified by flash column chromatography (2:1 light
petroleum/EtOAc) whereby pure 25 (0.067 g, 50%) was obtained as a colourless glassy solid, [α]D
= +64.6 (c 0.5, CHCl3), Rf = 0.33 (3:2 light petroleum/EtOAc).
IR (film): νmax 1744 (C=O).
1H NMR (400 MHz, CDCl3): 2.78 (dd, 1 H, J2a,3 = 5.1, J2a,2b = 19.4 Hz, H-2a), 2.91 (dd, 1 H,
J2b,3 = 1.6, J2a,2b = 19.4 Hz, H-2b), 3.55 (dd, 1 H, J7a,7b = 12.4, J6,7a = 10.8 Hz, H-7a), 3.87 (dd, 1 H,
J3,4 = 1.6, J4,5 = 4.6 Hz, H-4), 3.92 (dd, 1 H, J7a,7b = 12.4, J6,7b = 6.0 Hz, H-7b), 4.08 (ddd, 1 H, J6,7b
= 6.0, J6,7a = 10.7, J5,6 = 2.5 Hz, H-6), 4.20 (m, 1 H, J2a,3 = 5.1, J2b,3 = 1.6, J3,5 = 1.6, J3,4 = 1.6 Hz,
H-3), 4.50–4.69 (4 × d, 1 H each, Jgem = 12.1, Jgem = 11.9 Hz, 2 × CH2Ph), 4.76 (m, 1 H, J3,5 = 1.6,
J4,5 = 4.6, J5,6 = 2.5 Hz, H-5), 7.28–7.42 (m, 10 H, Ph).
13C NMR (100 MHz, CDCl3): 34.8 (C-2), 59.2 (C-7), 66.2 (C-3), 69.7 (C-6), 71.1 and 71.4 (2 ×
C
H2Ph), 71.6 (C-4), 72.9 (C-5), 127.8, 127.9, 128.0, 128.2, 128.4, 128.6, 136.8, 137.5 (2 × Ph),
167.9 (C-1).
HRMS (ESI): m/z 377.1361 (M++Na), calcd for C21H22O5Na: 377.1365.
4.11. (3R,4R,5R,6R)-4,6-dihydroxy-8,9-dioxabicyclo[3.3.1]nonan-1-one (26
)
To a stirred solution of 25 (0.13 g, 0.37 mmol) in anhydrous CH2Cl2 (13 mL) were added two
identical portions of FeCl3 (each 0.06 g, 0.37 mmol) in the interval of 1 h. The suspension was
stirred at room temperature for 20 h, and evaporated. The residue was purified on two columns of
flash silica (99:1 EtOAc/MeOH) whereupon pure product 26 (0.060 g, 94%) was obtained as
colourless crystals. Crystallization from Me2CO/hexane gave colourless crystals, mp 149 °C, [α]D =
+19.2 (c 0.5, Me2CO), Rf = 0.28 (97:1 EtOAc/MeOH).
IR (KBr): νmax 3413 (OH), 1728 (C=O).
1H NMR (400 MHz, DMSO-d6): 2.63 (dd, 1 H, J2a,3 = 1.3, J2a,2b = 19.1 Hz, H-2a), 2.99 (dd, 1
H, J2b,3 = 5.1, J2a,2b = 19.1 Hz, H-2b), 3.20 (pseudo t, 1 H,
6.0 Hz, J7a,7b = 12.1 Hz, H-7b), 3.87 (m, 1 H, H-3), 4.00–4.08 (bs, 2 H, H-6 and H-4), 4.39 (m, 1 H,
H-5), 5.20 (d, 1 H, = 5.1 Hz, OH), 4.61 (d, 1 H, = 4.2 Hz, OH).
J = 11.9 Hz, H-7a), 3.64 (dd, 1 H, J6,7b =
J
J
21

13C NMR (100 MHz, DMSO-d6): 34.8 (C-2), 60.7 (C-7), 62.0 (C-6), 64.5 (C-4), 68.4 (C-3),
77.7 (C-5), 169.0 (C-1).
HRMS (ESI): m/z 197.0424 (M++Na), calcd for C7H10O5Na: 197.0426.
4.12. General procedure for the Steglich esterification protocol: Preparation of 6–9
To a solution of 24 (1 equiv) in anhydrous CH2Cl2 (0.02–0.03 M) were successively added
cinnamic acid or 4-substituted cinnamic acid (2 equiv), DCC (2.1 equiv) and DMAP (2 equiv). The
mixture was stirred at room temperature for 1 h, then poured into 10% aq NaCl (for
aq NaHCO3 (for and ) and extracted with CH2Cl2. The combined organic phases were washed
with 10% NaCl, dried and evaporated, and the residues purified by flash column chromatography
(7:3 cyclohexane/EtOAc for 6, 8, and , or 3:1 light petroleum/EtOAc for ). All analytical samples
6 and 7) or 1%
8
9
9
7
have been prepared by crystallization from CH2Cl2/hexane.
4.12.1. 7-O-((2'E)-Cinnamoyl)-4,6-di-O-benzyl-2,3-dideoxy-
D
-arabino-hept-2-eno-1,5-lactone (
6)
Yield 73%. White powder, mp 110 °C, [α]D = −214 (
petroleum).
c 0.5, CHCl3), Rf = 0.39 (3:7 EtOAc/light
IR (KBr): νmax 1728 (C=O, lactone), 1708 (C=O, cinnamate).
1H NMR (400 MHz, CDCl3): δ 4.24–4.32, (m, 2 H, H-2 and H-4), 4.41 (dd, 1 H, J6,7a = 3.3,
J7a,7b = 12.4 Hz, H-7a), 4.50–4.66 (m, 4 H, CH2Ph and H-5), 4.88 (d, 1 H, Jgem = 10.9 Hz, CH2Ph),
5.03 (dd, 1 H, J6,7b = 2.3, J7a,7b = 12.4 Hz, H-7b), 6.23 (d, 1 H, J2,3 = 9.8 Hz, H-2), 6.49 (d, 1 H, J2′,3′
= 16.0 Hz, H-2′), 7.01 (dd, 1 H, J2,3 = 9.7, J3,4 = 5.7 Hz, H-3), 7.27–7.57 (m, 15 H, 3 × Ph), 7.74 (d,
1 H, J2′,3′ = 16.0 Hz, H-3′).
13C NMR (100 MHz, CDCl3): δ 60.8 (C-7), 65.1 (C-4), 71.3 and 72.3 (2 ×
CH2Ph), 74.1 (C-6),
77.7 (C-5), 117.6 (C-2′), 124.4 (C-2), 127.7, 127.9, 128.1, 128.4, 128.5, 129.8, 130.3, 134.2, 137.4,
137.44 (Ph), 142.9 (C-3), 145.2 (C-3′), 162.2 (C-1), 166.6 (C-1′).
LRMS (ESI+): m/z 485 (M+H).
Anal. Found: C, 74.10; H, 5.91. Calcd for C30H28O6: C, 74.34; H, 5.82.
4.12.2. 7-O-((2'E)-4''-Methoxycinnamoyl)-4,6-di-O-benzyl-2,3-dideoxy-D-arabino-hept-2-eno-1,5-
lactone (
7
)
22

Yield 69%. White powder, mp 99 °C, [α]D = −116 (c 0.5, CHCl3), Rf = 0.26 (7:3 light
petroleum/EtOAc).
IR (KBr): νmax 1735 (C=O, lactone), 1709 (C=O, cinnamate).
1H NMR (400 MHz, CDCl3): δ 3.86 (s, 3 H, OMe), 4.26–4.31 (m, 2 H, H-6 and H-4), 4.40 (dd,
1 H, J6,7a = 3.3, J7a,7b = 12.4 Hz, H-7a), 4.50–4.65 (m, 4 H, CH2Ph and H-5), 4.88 (d, 1 H, Jgem
10.9 Hz, CH2Ph), 5.01 (dd, 1 H, J6,7b = 2.3, J7a,7b = 12.4 Hz, H-7b), 6.22 (d, 1 H, J2,3 = 9.8 Hz, H-2),
6.36 (d d, 1 H, J2′,3′ = 16.0 Hz, H-2′), 6.92 (d, 2 H, = 8.8 Hz, H-3″ and H-5″), 7.00 (dd, 1 H, J2,3
=
J
=
9.8, J3,4 = 5.7 Hz, H-3), 7.25–7.39 (m, 10 H, 2 × CH2Ph), 7.49 (d, 2 H,
7.69 (d, 1 H, J2′,3′ = 16.0 Hz, H-3′).
J = 8.8 Hz, H-2″ and H-6″),
13C NMR (100 MHz, CDCl3): δ 55.3 (OMe), 60.7 (C-7), 65.2 (C-4), 71.3 and 72.3 (2 ×
H2Ph), 74.1 (C-6), 77.7 (C-5), 114.3 (C-3″ and C-5″), 115.1 (C-2′), 124.4 (C-2), 127.0 (C-1″),
C
127.8, 127.9, 128.0, 128.1, 128.4, 128.5, 129.5, 129.7 (C-2″ and C-6″), 137.4 137.5 (2 × CH2Ph),
142.9 (C-3), 144.9 (C-3′), 161.4 (C-4″), 162.3 (C-1), 166.9 (C-1′).
Anal. Found: C, 72.07; H, 6.11. Calcd for C30H28O6: C, 72.36; H, 5.88.
4.12.3. 7-O-((2'E)-4''-Fluorocinnamoyl)-4,6-di-O-benzyl-2,3-dideoxy-
lactone (
D-arabino-hept-2-eno-1,5-
8
)
Yield 84%. White powder, mp 118 °C, [α]D = −174 (c 0.5, CHCl3), Rf = 0.36 (3:7 EtOAc/light
petroleum).
IR (KBr): νmax 1720 (C=O, lactone), 1710 (C=O, cinnamate).
1H NMR (400 MHz, CDCl3): δ 4.25–4.35 (m, 2 H, H-4 and H-6), 4.41 (dd, 1 H, J6,7a = 3.2,
J7a,7b = 12.3 Hz, H-7a), 4.49–4.65 (m, 4 H, CH2Ph and H-5), 4.87 (d, 1 H, Jgem = 10.9 Hz, CH2Ph),
5.01 (dd, 1 H, J6,7b = 2.3, J7a,7b = 12.4 Hz , H-7b), 6.23 (d, 1 H, J2,3 = 9.8 Hz, H-2), 6.41 (d, J2′,3′
=
16.0 Hz, H-2′), 7.01 (dd, 1 H, J2,3 = 9.8, J3,4=5.7 Hz, H-3), 7.07–7.57 (m, 14 H, Ar), 7.69 (d, 1 H,
J2′,3′ = 16.0 Hz, H-3′).
13C NMR (100 MHz, CDCl3): δ 61.2 (C-7), 65.5 (C-4), 71.7 and 72.7 (2 ×
78.1 (C-5), 116.2 and 116.4 (C-3″, J3″,F = 22 Hz), 117.77 and 117.8 (C-2′, J2′,F = 2.3 Hz), 124.8 (C-
2), 128.0, 128.3, 128.5, 128.7, 128.8, 128.9, 130.3 and 130.4 (C-2″), 130.88 and 130.9 (C-1″, J1″,F
CH2Ph), 74.4 (C-6),
=
23

12.0 Hz), 137.78, 137.8 (Ph), 143.3 (C-3), 144.2 (C-3′), 162.6 (C-1), 163.0 and 165.5 (C-4″, J4″,F =
252 Hz), 166.8 (C-1′).
Anal. Found: C, 71.38; H, 5.50. Calcd for C30H27FO6: C, 71.70; H, 5.42.
4.12.4. 7-O-((2'E)-4''-Nitrocinnamoyl)-4,6-di-O-benzyl-2,3-dideoxy-
lactone (
D-arabino-hept-2-eno-1,5-
9
)
Yield 67%. White powder, mp 159 °C, [α]D = −158.2 (c 0.5, CHCl3), Rf = 0.29 (3:7
EtOAc/light petroleum).
IR (KBr): νmax 1721 (C=O).
1H NMR (400 MHz, CDCl3): δ 4.24–4.33 (m, 2 H, H-6 and H-4), 4.43 (dd, 1 H, J6,7a = 3.2,
J7a,7b = 12.4 Hz, H-7a), 4.50–4.65 (m, 4 H, CH2Ph and H-5), 4.85 (d, 1 H, Jgem = 10.9 Hz, CH2Ph),
5.01 (dd, 1 H, J6,7b = 2.3, J7a,7b = 12.4 Hz, H-7b), 6.23 (d, 1 H, J2,3 = 9.8 Hz, H-2), 6.59 (d, J2′,3′
=
16.0 Hz, H-2′), 7.02 (dd, 1 H, J2,3 = 9.8, J3,4 = 5.7 Hz, H-3), 7.25–7.39 (m, 10 H, 2 × CH2C6H5),
7.74 and 8.26 (2 × d, 2 H each, C6H4, cinnamate), 7.74 (d, 1 H, J2′,3′ = 16.0 Hz, H-3′).
13C NMR (100 MHz, CDCl3): δ 61.4 (C-7), 65.1 (C-4), 71.4 and 72.5 (2 ×
CH2Ph), 74.1 (C-6),
77.8 (C-5), 122.0 (C-2′), 124.2 (H-2‴ and H-6‴), 124.5 (C-2), 127.8, 128.0, 128.1, 128.2, 128.5,
128.6, 137.4 (2 × Ph), 128.7 (H-3″ and H-5″), 140.4 (C-1″), 143.0 (C-3′), 148.6 (C-4″), 162.2 (C-1),
165.7 (C-1′).
HRMS: m/z 552.1612 (M++Na), calcd for C30H27NO8Na: 552.1634.
Anal. Found: C, 67.63; H, 4.88; N, 2.67. Calcd for C30H27NO8: C, 68.04; H, 5.14; N, 2.65.
4.13. Alternative procedure for the preparation of
9
To a solution of compound 23 (0.15 g, 0.38 mmol) in dry MeOH (10 mL) which is stirred and
heated at 35−37 °C, an acidic ion exchange resign (A15) was added (0.5 g, 2.35 mmol of H+). After
24 hours, the solid phase was filtered off and the precipitate was washed with dry MeOH. The
combined liquid phases were evaporated and the residue was dried in high vacuum overnight.
Crude 24 was dissolved in dry CH2Cl2 (15 mL), and to this solution were successively added 4-
nitrocinnamic acid (0.164 g, 0.85 mmol), DCC (0.187 g, 0.89 mmol) and DMAP (0.103 g, 0.85
mmol) while stirring at room temperature. After 2 h, the reaction solution was poured into 10% aq
NaCl (70 mL) and extracted with CH2Cl2 (3 × 15 mL). The organic phases were combined, dried
24

and evaporated. The residue was purified twice by flash column chromatography (3:7 EtOAc/light
petroleum) to afford pure (0.095 g, 47%) which crystallizes from CH2Cl2/hexane in the form of
white powder, mp 159 °C, [α]D = −158.2 ( 0.5, CHCl3), Rf = 0.29 (3:7 EtOAc/light petroleum).
Spectral data of such prepared are identical to those shown above.
9
c
9
4.14. 7-O-((2'E)-4''-Fluorocinnamoyl)-4,6-di-O-acetyl-2,3-dideoxy-D-arabino-hept-2-eno-1,5-
lactone (12
)
To a stirred solution of compound
8 (0.150 g, 0.3 mmol) in anhydrous CH2Cl2 (17 mL), AcBr
(0.3 mL, 3.6 mmol) and two equal portions of Zn(OTf)2 (0.034 g each, 0.09 mmol) were added
successively, at an interval of 1 h. After stirring at room temperature for 20 h, the reaction mixture
was poured into 1% NaHCO3 (75 mL) and extracted with CH2Cl2 (3 × 15 mL). The organic phase
(white emulsion) is dried and evaporated, and the residue purified twice on a column of flash silica
(5:1 toluene/EtOAc), whereby pure 12 (0.07 g, 58%) was obtained as a white solid.
Recrystallization from CH2Cl2/hexane gave colourless needles, mp 170 °C, [α]D = −203.2 (c 0.5,
CHCl3), Rf = 0.45 (17:3 toluene/Me2CO).
IR (KBr): νmax 1741 (C=O).
1H NMR (400 MHz, CDCl3): δ 2.07 and 2.10, (2 × s, 3 H each, 2 × CH3CO), 4.43 (dd, 1 H,
J6,7a = 4.2, J7a,7b = 12.5 Hz, H-7a), 4.76–4.86 (m, 2 H, J4,5 = 2.7, J6,7b=2.3 Hz, H-5 and H-7b), 5.42
(dd, 1 H, J4,5 = 2.7, J3,4 = 6.2 Hz, H-4), 5.45 (ddd, 1 H, J6,7a = 4.1, J6,7b = 2.3 Hz, J5,6 = 9.6 Hz, H-6),
6.29 (d, 1 H, J2,3 = 9.6 Hz, H-2), 6.38 (d, J2′,3′ = 16.0 Hz, H-2′), 7.01 (dd, 1 H, J2,3 = 9.6, J3,4 = 6.1
Hz, H-3), 7.09 (m, 2 H, H-3″ and H-5″), 7.54 (m, 2 H, H-2″ and H-6″), 7.68 (d, 1 H, J2′,3′ = 16.0 Hz,
H-3′).
13C NMR (100 MHz, CDCl3): δ 20.4 and 20.6 (2 ×
CH3CO), 59.7(C-4), 61.5 ( C-7), 67.6 (C-6),
75.3 (C-5), 116.0 and 116.2 (J3″,F = 22 Hz, C-3″), 116.9 and 116.9 (J2′,F = 2.2 Hz, C-2′), 125.3 (C-
2), 130.0 and 130.1 (J2″,F = 8.5 Hz, C-2″ and C-6″), 130.37 and 130.4 (J1″,F = 8.5 Hz, C-1″), 139.7
(C-3), 144.4 (C-3′), 161.1 (C-1), 162.8 and 165.2 (J4″,F = 252 Hz, C-4″), 166.2 (C-1′), 169.4 and
169.9 (2 ×
COCH3).
HRMS: m/z 429.0954 (M++Na), calcd for C20H19FO8Na: 429.0962.
4.15. 7-O-((2'E)-4''-Nitrocinnamoyl)-4,6-di-O-acetyl-2,3-dideoxy-D-arabino-hept-2-eno-1,5-lactone
(13
)
25

To a stirred solution of compound
9 (0.125 g, 0.24 mmol) in anhydrous CH2Cl2 (13 mL), AcBr
(0.21 mL, 2.83 mmol) and two equal portions of Zn(OTf)2 (0.018 g each, 0.05 mmol) were added
successively, at an interval of 1 h. After stirring at room temperature for 20 h, the reaction mixture
was poured into 1% NaHCO3 (75 mL) and extracted with CH2Cl2 (3 × 15 mL). The organic phase
(white emulsion) is dried and evaporated, and the residue purified twice on two columns of flash
silica (first with 2:3 EtOAc/light petroleum, then with 9:1 toluene/Me2CO). Pure 13 (0.077 g, 62%)
was obtained as a pale yellow solid. Recrystallization from CH2Cl2/hexane gave pale yellow
crystals, mp 145 °C, [α]D = −183.2 (
c 0.5, CHCl3), Rf = 0.43 (17:3 toluene/Me2CO).
IR (KBr): νmax 1742 (C=O).
1H NMR (400 MHz, CDCl3): δ 2.07 and 2.09 (2 × s, 3 H each, 2 × CH3CO), 4.44 (dd, 1 H, J6,7a
= 4.3, J7a,7b = 12.6 Hz, H-7a), 4.81 (dd, 1 H, J4,5 = 2.7, J5,6 = 9.6 Hz, H-5), 4.83 (dd, 1 H, J6,7b = 2.3,
J7a,7b = 12.6 Hz, H-7b), 5.42 (dd, 1 H, J4,5 = 2.7, J3,4 = 6.2 Hz, H-4), 5.45 (ddd, 1 H, J6,7a = 4.1, J5,6
9.5, J6,7b = 2.3 Hz, H-6), 6.29 (d, 1 H, J2,3 = 9.7 Hz, H-2), 6.58, d, J2′,3′ = 16.0 Hz, H-2′), 7.01 (dd, 1
H, J2,3 = 9.7, J3,4 = 6.1 Hz, H-3), 7.69 and 8.25 (2 × d, 4 H, Ph, cinnamate), 7.74 (d, 1 H, J2′,3′ = 16.0
Hz, H-3′).
=
13C NMR (100 MHz, CDCl3): δ 20.4 and 20.6 (2 ×
CH3CO), 59.6 (C-4), 62.0 ( C-7), 67.4 (C-
6), 75.3 (C-5), 121.4 (C-2′), 124.1 (C-3″ and C-5″), 125.2 (C-2), 128.7 (C-2″ and C-6″), 140.2 (C-
1″), 139.7 (C-3), 142.7 (C-3′), 148.6 (C-4″), 161.0 (C-1), 165.4 (C-1′), 169.4 and 169.8 (2 ×
CH3
C
O).
HRMS: m/z 456.0904 (M++Na), calcd for C20H19NO10Na: 456.0906
4.16. 7-O-((2'E)-4''-Methoxycinnamoyl)-4,6-di-O-acetyl-2,3-dideoxy-D-arabino-hept-2-eno-1,5-
lactone (11
)
To a stirred solution of I2 (0.109 g, 0.43 mmol) in anhydrous CH2Cl2 (7 mL) was added Ph3P
(0.113 g, 0.43 mmol) whereby the solution turns brown-yellow. Pyridine is then added (0.2 mL,
15.53 mmol) whereby the colour of the solution changes to light yellow. To the solution was added
4-methoxycinnamic acid (0.077 g, 0.43 mmol) followed by the compound 22 (0.049 g, 0.28 mmol)
which was added to the solution after the next 10 min. Stirring at room temperature was continued
for an additional 22 h and then Ac2O (7 mL) and Py (1 mL, 77.36 mmol) were successively added
to the solution. The stirring at room temperature was continued for an additional 24 h and then the
mixture was evaporated by co-distillation with toluene. The residue was suspended in H2O (80 mL)
26

and extracted with CH2Cl2 (3 × 20 mL). The combined organic phases are dried and evaporated and
the obtained residue was purified by flash column chromatography (3:1 Et2O/light petroleum) to
yield pure 11 (0.075 g, 64%) as a colourless syrup, [α]D = −198.4 (c 0.5, CHCl3), Rf =0.30 (2:1
Et2O/light petroleum).
IR (film): νmax 1747 (C=O).
1H NMR (400 MHz, CDCl3): δ 2.07 and 2.09 (2 × s, 3 H each, 2 × CH3CO), 3.85 (s, 3 H,
OMe), 4.41 (dd, 1 H, J7a,7b = 12.6, J6,7a = 4.1 Hz, H-7a), 4.80 (dd, 1 H, J4,5 = 2.7, J5,6 = 9.5 Hz, H-5),
4.82 (dd, 1 H, J6,7b = 2.4, J7a,7b = 12.6 Hz, H-7b), 5.42 (dd, 1 H, J4,5 = 2.7, J3,4 = 6.1 Hz, H-4), 5.44
(ddd, 1 H, J6,7b = 2.3, J6,7a = 4.0, J5,6 = 9.6 Hz, H-6), 6.27 (d, 1 H, J2,3 = 9.6 Hz, H-2), 6.36 (d, 1 H,
J2′,3′ = 15.9 Hz, H-2′), 6.92 and 7.49 (2 × d, 4 H, Ph), 7.01 (dd, 1 H, J2,3 = 9.7, J3,4 = 6.1 Hz, H-3),
7.67 (d, 1 H, J2′,3′ = 15.9 Hz, H-3′).
13C NMR (100 MHz, CDCl3): δ 20.4 and 20.7 (2 ×
7), 67.7 (C-6), 75.35 (C-5), 114.4 (C-2′), 125.3 (C-2), 114.6, 126.9, 129.9 and 161.6 (Ph), 139.7 (C-
3), 145.4 (C-3′), 161.2 (C-1), 166.7 (C O cinnamate), 169.5 and 169.9 (2 × OCH3).
CH3CO), 55.4 (OMe), 59.8 (C-4), 61.3 (C-
=
C
HRMS: m/z 441.1173 (M++Na), calcd for C21H22O9Na: 441.1162.
4.17. 7-O-((2'E)-4''-Nitrocinnamoyl)-4,6-di-O-acetyl-2,3-dideoxy-
13) and 4,6-Di-O-acetyl-2-deoxy-3-O-methyl-7-O-((2'E)-4''-nitrocinnamoyl)-
lactone (27
D
-arabino-hept-2-eno-1,5-lactone
(
D-gluco-heptano-1,5-
)
To a solution of 23 (0.2 g, 0.5 mmol) in absolute MeOH (10 mL) was added TsOH (0.096 g,
0.5 mmol) and the resulting mixture was stirred vigorously at room temperature for 24 h. The
solution was neutralized with solid NaHCO3 (0.05 g, 0.6 mmol) and evaporated, to give an
inseparable mixture composed of crude 24 and 3-O-methyl derivative 23a. The mixture was
separated by flash column chromatography (1:1 EtOAc/light petroleum) with difficulties, to give
small amount of 23a sufficient for partial characterization purposes. 1H NMR (400 MHz, CDCl3): δ
2.68 (dd, 1 H, J2a,3 = 2.9, J2a,2b = 17.6 Hz, H-2a), 2.88 (dd, 1 H, J2b,3 = 5.1, J2a,2b = 17.6 Hz, H-2b),
3.78 (m, 1 H, H-3), 3.87–4.04 (m, 3 H, 2 × H-7 and H-6), 4.43 (m, 1 H, H-4), 4.46, 4.50, 4.60 and
4.70 (4 × d, 4 H, Jgem = 11.6 Hz, 2 × CH2C6H5), 4.76 (dd, 1 H, J = 1.9, J = 9.2 Hz, H-5), 7.25–7.40
(m, 10 H, 2 × CH2C6H5). 13C NMR (100 MHz, CDCl3): δ 32.2 (C-2), 56.8 (OCH3), 59.7 (C-7), 71.0
(C-4), 71.8 and 72.2 (2 × CH2Ph), 73.3 (C-3), 75.7 (C-5), 76.2 (C-6), 127.6, 127.7, 128.0, 128.04,
128.1, 128.4, 128.45, 128.55, 128.58, 128.6, 128.7, 137.8 (2 × Ph), 168.8 (C-1). The mixture of 24
27

and 23a was dissolved in anhydrous CH2Cl2 (20 mL), to the solution was successively added 4-
nitrocinnamic acid (0.205 g, 1.06 mmol), DCC (0.234 g, 1.11 mmol) and DMAP (0.129 g, 1.06
mmol) and the resulting solution was stirred at room temperature for 2 h. The reaction solution was
poured into 10% aq NaCl (70 mL) and extracted with CH2Cl2 (3 × 15 mL). The combined organic
phases are dried and evaporated and the residue was purified by flash column chromatography (3:7
EtOAc/light petroleum) to give the mixture
9 and 3-O-methyl derivative 23b. To a stirred solution
of mixture and 23b (0.21 g, 0.42 mmol) in anhydrous CH2Cl2 (21 mL) were successively added,
9
while stirring at room temperature AcBr (0.38 mL, 5 mmol) and four equal portions of Zn(OTf)2
(each, 0.017 g, 0.05 mmol) at a time interval of 1 h, and the reaction was continued at room
temperature for the additional 20 h. The reaction solution was evaporated and the residue purified
twice by flash column chromatography (41:9 light petroleum/Me2CO). Pure pyranone 13 (0.04 g,
18%) was first eluted in the form of a bright yellow solid. Recrystallization from CH2Cl2/hexane
gave pale yellow crystals, mp 145 °C, [α]D = −183.2 (c 0.5, CHCl3), Rf = 0.43 (17:3
toluene/Me2CO). 3- -Methyl derivative 27 (0.06 g, 21%) was then eluted as a syrup, which after
O
crystallization from CH2Cl2/hexane gave pale yellow crystals, mp 135 °C, [α]D = +2.4 (c 0.5,
CHCl3), Rf = 0.37 (17:3 toluene/Me2CO).
Spectral data for 27
:
IR (KBr): νmax 1738 (C=O, lactone), 1719 (C=O, ester).
1H NMR (400 MHz, CDCl3): δ 2.08 (s, 3 H, CH3CO2 at C-4), 2.13 (s, 3 H, CH3CO2 at C-6),
2.81 (d, 2 H, J2,3 = 9.7 Hz, H-2), 3.49 (s, 3 H, OCH3), 3.75 (m, 1 H, H-3), 4.40 (dd, 1 H, J6,7a = 4.5,
J7a,7b = 12.5 Hz, H-7a), 4.78 (dd, 1 H, J6,7b = 2.4, J7a,7b = 12.5 Hz, H-7b), 4.94 (dd, 1 H, J4,5 = 1.9,
J5,6 = 9.7 Hz, H-5), 5.29 (dd, 1 H, J4,5 = 2.0, J3,4 = 3.8 Hz, H-4), 5.33 (ddd, 1 H, J6,7a = 4.5, J5,6
=
9.8, J6,7b = 2.4 Hz, H-6), 6.60 (d, 1 H, J2′,3′ = 16.1 Hz, H-2′), 7.71 (d, 2 H, J2″,3″ = 8.8 Hz, H-2″ and
H-6″), 7.75 (d, 1 H, J2′,3′ = 16.1 Hz, H-3′), 8.27 (d, 2 H, J2″,3″ = 8.8 Hz, H-3″ and H-5″).
13C NMR (100 MHz, CDCl3): δ 20.7 and 20.73 (2 ×
CH3CO), 32.0 (C-2), 57.3 (OCH3), 62.4
(C-7), 63.7 (C-4), 67.4 ( C-7), 73.4 (C-3), 73.9 (C-5), 121.6 (C-2′), 124.1 (C-3″ and C-5″), 128.8
(C-2′ and C-6″), 140.3 (C-4″), 142.6 (C-3′), 148.6 (C-1″), 165.5 (C-1′), 167.0 (C-1), 169.57 and
169.6 (2 × COCH3).
HRMS: m/z 504.0906 (M++K), calcd for C21H23NO11K: 504.0908.
4.4. X-ray Crystal Structure Analysis
28