Stereoselective synthesis of carbohydrate fused pyrano[3,2-c]pyranones as anticancer agents

Article abstract of DOI:10.1039/c8nj01395k

Pyrano[3,2-c]pyranone is an important structural motif present in many natural products exhibiting diverse biological activities. Two series of carbohydrate fused pyrano[3,2-c]pyranone derivatives (n = 20) were efficiently synthesized starting from 2-C-formyl galactal and 2-C-formyl glucal, reacting with various 4-hydroxycoumarins in a very short reaction time (10 min) under microwave assisted conditions. The anticancer activity of these synthesized pyrano[3,2-c]pyranones was determined in detail through cellular assays against MCF-7 (breast), MDA-MB-231 (breast) and HepG2 (liver) cancer cell lines. The newly synthesized pyrano[3,2-c]pyranones were screened for their cell-viability and anti-proliferative activity against MCF-7, MDA-MB-231 and HepG2 cell lines. Compounds 12, 13 and 14 exhibited high growth inhibitory potencies selectively against MCF-7 cells with half-maximal inhibitory concentration (IC₅₀) values of 19.9, 14.5 and 10.9 μM respectively. Compounds 12, 13, 14, 15 and 19 inhibited the growth of MDA-MB-231 cells (breast) by 43, 44, 37, 31 and 45% respectively. However, no inhibitory effect was observed for these compounds in the human liver cancer cell line (HepG2) and normal cell lines (HEK293, human embryonic kidney cells). Mechanistic studies showed that these compounds alter the cell morphology and cause G2/M arrest in MCF-7. Further studies showed that compounds 12, 13 and 14 significantly inhibited cell migration which was accompanied by altered microtubule distribution. An enhanced accumulation of these compounds in cells was observed as compared to the 4-hydroxycoumarins precursor in the intracellular uptake assay. These findings confirm that carbohydrate fused pyrano[3,2-c]pyranones are better candidates for anticancer activity.

Full text of DOI:10.1039/c8nj01395k

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PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Stereoselective synthesis of carbohydrate fused pyrano[3,2-
c]pyranones as anticancer agents
Received 00th January 20xx,
Accepted 00th January 20xx
Priti Kumaria†, Sonal Guptab†, Chintam Narayanaa‡, Shakeel Ahmadb‡, Shailja Singhb* and Ram
,
Sagara c
*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Abstract
Pyrano[3,2-c]pyranone is an important structural motif present in many natural products exhibiting diverse biological activities. Two series of carbohydrate
fusedpyrano[3,2-c]pyranone derivatives (n = 20) were efficiently synthesized starting from 2-C–formyl galactal and 2-C–formyl glucal, reacting with various
4-hydroxycoumarins in very short reaction time (10 min) under microwave assisted condition. Anticancer activity of these synthesized pyrano[3,2-
c]pyranones were determined in detail through cellular assays against MCF-7 (breast), MDA-MB-231 (breast) and HepG2 (liver) cancer cell lines. The newly
synthesized pyrano[3,2-c]pyranones were screened for their cell-viability and anti-proliferative activity against MCF-7, MDA-MB-231 and HepG2 cell lines.
The compounds 12, 13 and 14 exhibited high growth inhibitory potencies selectively against MCF-7 cell with half maximal inhibitory concentration (IC50)
values 19.9, 14.5 and 10.9 µM respectively. The compounds 12, 13, 14, 15 and 19 inhibited cell growth of MDA-MB-231 cell (breast) by 43, 44, 37, 31 and
45% respectively. However, no inhibitory effect was observed by these compounds in human liver cancer cell line (HepG2) and normal cell lines (HEK293,
human embryonic kidney cells). Mechanistic studies showed that, these compounds alter cell morphology and cause G2/M arrest in MCF-7. Further study
showed compounds 12, 13 and 14 significantly inhibited cell migration which was accompanied by altered microtubule distribution. An enhanced
accumulation of these compounds in cells was observed as compared to 4-hydroxycoumarins precursor in intracellular uptake assay. These findings confirms
that, carbohydrate fused pyrano[3,2-c]pyranone are better candidates for anticancer activity.
USA alone.4 This situation becomes even more problematic with the
development of resistant in breast cancer.5 These alarming situation
Introduction
attracted attention of medicinal chemists and chemical biologists
Cancer is one of the leading cause of death worldwide and as per
to, search for new molecules as chemotherapeutics with better
World Health Organization (WHO) report there will be 26 million
activity and understand mechanistic aspects of resistant at
new cancer cases and 17 million cancer deaths estimated per year
due to cancer by 2030.1,2 Breast cancer is most common type of
molecular level through detailed studies. Therefore, efficient
synthesis of drug-like small molecules has been interest of the
cancer found in women worldwide. In 2012 it was estimated that,
research for medicinal chemists because, they play vital role in new
1,671,149 new breast cancer cases were diagnosed and 521,907
drug development processes.6 These drugs like bioactive
deaths occurred worldwide due to breast cancer.3 Cancer statistics
compounds are broadly interact with enzyme or receptor and
review revealed that, by end of 2017 number of new diagnosed
modulate their function, thus serve as important leads in medicinal
female breast cancer cases would be around 252710, which is 15%
chemistry.7 The incorporation of privileged substructures into novel
of all new cancer cases, and estimated death would occur 40,610 in
core skeletons is an important approach in medicinal chemistry.8
Coumarin derivatives and fused pyrano[3,2-c]pyranones are widely
present in bioactive natural products, synthetic products as well as
in pharmaceutical agents (A-C, Fig. 1).9 Coumarins and pyrano[3,2-
c]pyranones well documented primarily for their anticancer activity,
along with antidiabetic, anti-Alzheimer, anticoagulant, insecticidal,
and antifungal properties.10,11 Coumarin derivatives have been
Corresponding Author:
Email: ram.sagar@snu.edu.in, shailja.jnu@gmail.com,
shown to exhibit growth inhibitory activity in different types of
cancer like breast cancer12 prostate cancer13 and malignant
†equal contribution
melanoma.14 Coumarin–monastrol fused hybrid molecules showed
‡equal contribution
potent inhibitory activity against human breast cancer lines like
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basal type MCF-7 and triple negative MDA-MB-231.12 Most of these transformations reported this type of annulation reaction in 1944.18
This reaction later explored by many research groups for synthesis
of natural products and biologically active molecules.19 The
synthesis of few pyrano[3,2-c]-5(2H)-one was carried out earlier
have faced in term of reaction time, isolated yields and substrate
scope etc. Therefore, there was a need to develop an efficient and
clean synthetic method for library synthesis of pyrano[3,2-
c]pyranones. Herein we are reporting microwave assisted efficient
synthesis of carbohydrate fused pyrano[3,2-c]pyranones and their
detailed anticancer activity. The initial annulation reaction of
coumarin 1 with 2-C-formyl galactal 1a was performed under
microwave heating condition in toluene at 80 °C it doesn’t go to
completion even after 20 min. whereas similar reaction in the
presence of L-proline furnished carbohydrate fused pyrano[3,2-
c]pyranone 11 was obtained in 90% isolated yield with highly
stereoselective and regioselective manner (entry 2, Table 1). The
structure of pyrano[3,2-c]pyranone was established by 1D and 2D
NMR (NOE, COSY and HSQC) experiments (SI) and comparing it with
earlier spectral data.20
coumarin derivatives were shown to suppress cell proliferation by
arresting cell cycle at G1 or G2/M phases thereby inducing
apoptosis. Tomoda and coworkers reported that compounds
bearing pyrano-pyranone skeleton inhibits the DNA synthesis by 79-
91% and tumor cell growth by 93-100%. It has also been observed
that extended conjugation in pyrano-pyranone molecules showed
better activity and these compounds inhibit the tubulin
polymerization.15
The above literature reports strongly support pyrano[3,2-
c]pyranonesas privileged skeleton. When the privileged skeleton
fused with carbohydrates and resulted hybrid molecules have many
additional advantages eg. a)
A better aqueous solubility, b)
improved selectivity, c) improved cellular uptake, d) reduced
cellular toxicityetc.16
OH
O
R
OH
To optimize reaction condition under microwave condition and see
the effect of organocatalyst, we planned and tested a series of
reaction conditions using various organocatalysts and solvent
systems (table 1) under microwave irradiation. To enhance the
reactivity of 2-C-formyl galactal 1a various secondary amine (L-
proline I, pyrrolidine II, piperidine III and morpholine IV) based
organocatalysts were tested. After screening a set of reaction
conditions by using different organocatalysts under microwave
condition, it was found that the annulation of coumarin 1 with 1a in
the presence of pyrrolidine II (0.5 equiv) in toluene at 80 °C affords
11 in 93% isolated yield with excellent diastereoselectivity (table 1,
entry 5). When we carried out the same transformation in the
presence of 0.1 equiv of pyrrolidine II under similar condition
compound 11 was obtained in 91% isolated yield (table 1, entry 14),
we obtained the identical product 11 without the loss of
stereoselectivity, when different organocatalysts (I-IV) were used
for this annulation reaction as observed earlier under thermal
condition.20 The isolated yield was varied with different
organocatalyst without any significant difference in the reaction
time (Table 1, entries 2-14).
OBn
O
O
O
O
O
O
O
O
R1
R2
O
N
MeO
MeO
O
O
O
O
Me
OH
OBn
O
Me
D
A
B
C
Fig. 1. Bioactive pyrano-pyranone molecules (A-C): (A) Calanolide A,
anti-HIV drug; (B) anticancer tricyclic pyrano[3,2-c]pyranone; (C)
Arisugacin A, a selective inhibitor of acetylcholinesterase (D) our
anticancer pyrano[3,2-c]pyranone molecules presented in current
work.
Herein, we report microwave assisted synthesis and anticancer
activity of carbohydrate fused pyrano[3,2-c]pyranones (D) derived
from the hybrid structure of 4-hydroxycoumarin and 2-C-formyl
glycals in good to very good yield in very short reaction time (10
min). Anticancer activity of these newly synthesized fused
pyrano[3,2-c]pyranones were determined by various cellular assays
against MCF-7, MDA-MB-231 and HepG2 cancer cell lines.
Carbohydrate fused pyrano[3,2-c]pyranones 12, 13 and 14
exhibited high growth inhibitory potencies selectively against MCF-
7 with IC50 in the range of 10-20 µM. The cell growth inhibition for
compounds 12, 13, 14, 15 and 19 for MDA-MB-231 cell (breast) was
found 43, 44, 37, 31 and 45% respectively. The efficient microwave
assisted synthesis of these molecules and their detailed anticancer
activity are presented in this paper.
To study solvent effects for this transformation under microwave
condition, we screened toluene, ethyl acetate and acetonitrile in
the presence of L-proline (Table 1, entries 2-4) and 1% acetic acid.
In comparison, toluene with 1% acetic acid was found to be
superior over ethyl acetate and acetonitrile. These three solvent
systems were screened with other organocatalysts (II-IV) and result
showed that, indeed 1% acetic acid in toluene was the best solvent
among others (Table 1).
Results and Discussion
Chemistry
The structure of pyrano[3,2-c]pyranone was unambiguously
established by 1D and 2D NMR (NOE, COSY and HSQC) experiments
(Supporting Information, SI). The stereochemistry of newly
generated chiral center in compound 12 was confirmed by careful
NOE experiments. A NOE correlation was observed between newly
generated chiral methine proton H-11a (δ 6.10) and predefined
chiral methine proton H-10 (δ 3.87). This suggested that the H-11a
proton is in the same face as the H-10 at the sugar part in
compound 12.
For the efficient synthesis of privileged carbohydrate-fused
pyrano[3,2-c]pyranones, we identified 4-hydroxycoumarin 1 as
substrate which can be coupled with 2-C-formyl galactal 1a17 under
microwave reaction condition and get transformed to the desired
pyrano[2,3-c]pyranones 11 (table 1) through selective C-1,2
nucleophilic addition followed by 6π-electron electrocyclization
reaction i.e. formal [3+3] cycloaddition reaction. Link and co-
woekers first under thermal condition.20 However, some of
2 | J. Name., 2012, 00, 1-3
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Table 1.Reaction optimization using different organocatalysts and solvents
2
3
4
O
OH
1
6
OBn
13
BnO
BnO
11
12
O
H
O
H
See table for
rxn condition
11a
+
10
O 5
OBn O
O
O
BnO
9
8
7
OBn
O
1a
1
11
S. No.
1b
2
Reaction conditionsa
Toluene:AcOH, µW 80 °C, 30 min
Yield (%)
23
I, Toluene:AcOH, µW 80 °C, 10 min
I, EtOAc:AcOH, µW 80 °C, 10 min
90
3
84
4
I, Acetonitrile:AcOH, µW 80 °C, 10 min
II, Toluene:AcOH, µW 80 °C, 10 min
II, EtOAc:AcOH, µW 80 °C, 10 min
II, Acetonitrile:AcOH, µW 80 °C, 10 min
III, Toluene:AcOH, µW 80 °C, 10 min
III, EtOAc:AcOH, µW 80 °C, 10 min
III, Acetonitrile:AcOH, µW 80 °C, 10 min
IV, Toluene:AcOH, µW 80 °C, 10 min
IV, EtOAc:AcOH, µW 80 °C, 10 min
IV, Acetonitrile:AcOH, µW 80 °C, 10 min
II, Toluene:AcOH, µW 80 °C, 10 min
82
5
93
6
77
7
55
8
65
9
60
10
11
12
13
14c
55
70
61
50
91
a0.5 equiv of organocatalyst was used in each cases, bReaction was not completed, c0.1 equiv oforganocatalystwas used.
After having optimized reaction condition, we were interested to positions on 4-hydroxycoumarins neither remarkable affected the
investigate the scope of this transformation with different yield nor the reaction completion time. However R2 (= Br)
substituted 4-hydroxycoumarins under microwave reaction substituted 4-hydroxycoumarin afforded the lesser yield (64%) as
condition. Therefore, various 4-hydroxycoumarins 1-10 were freshly compare to other mono- and di-substituted 4-hydroxycoumarins
prepared in laboratory starting from commercially available (Table 2, entries 5, 9 and 10).
corresponding ortho-hydroxyacetophenone and diethyl carbonate
(Scheme S1, SI).21 The various 4-hydroxycoumarinswere then
The proposed mechanism for this transformation is already
discussed earlier, which involves C-1,2-addition with 4-
coupled with 2-C formyl galactal 1a and 2-C formyl glucal 1b under
hydroxycoumarin and 2-C-formyl galactal followed by dehydration
optimized reaction condition (µW, 80 °C, II, Toluene:AcOH, 10 min).
and β-elimination. An 1-oxatriene intermediate formed after β-
As shown in Table 2 (entries 2-10) 2-C formyl galactal 1a was
elimination which undergo 6-π electron electrocyclization and
furnishes the corresponding pyrano[3,2-c]pyranone.20, 22
successfully coupled with various substituted 4-hydroxycoumarins
2-10 to afford respective carbohydrate fused pyrano[3,2-
c]pyranones 11-20 in good to very good yields with excellent
Further when 4-hydroxycoumarins 1 was coupled with 2-C formyl
stereoselectivity(dr ˃99:1). The substituents at the R1, R2, R3 and R4
glucal 1b under optimized reaction condition it afforded
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unexpectedly, epimeric mixture (1:1) of carbohydrate fused pyrano- is equilibrating with 21b through
Journal Name
a
common 1-oxatriene
pyranone 21a/21b in good yield. The stereoselectivity in this case intermediate in reversible fashion and furnishing a mixture of
could not get restored even using different organocatalysts and epimers 21a/b. The energy calculation was done to understand this
conditions. As mentioned in proposed mechanism that, this type of equilibrium and it was found that, the energy difference between
reaction proceeds through 1-oxatriene intermediate followed by 2-C-formyl galactal derived compound pyrano[3,2-c]pyranones (11a
6π-electron electrocyclization and reversibility of this cyclization =11 vs. 11b) was ΔE = 3.975 kcal/mole, whereas, that of 2-C-formyl
was well studied and reported in the literature.22 Based on glucal derived
literature we can presumed that fused pyrano[3,2-c]pyranone 21a
Table 2. Synthesis of 2-C formyl galactal fused pyrano[3,2-c]pyranones 11-20.
R2
R3
R4
R1
O
R1 OH
OBn
BnO
BnO
R2
R3
H
O
O
H
II, Toluene:AcOH
ꢀW, 80ꢁ120 °C
+
O
OBn O
O
O
BnO
R4
OBn
O
1a
1-10
11-20
Entry
1
R1
H
R2
R3
R4
H
Product a
11
Time (min)
Yield (%)
93
H
H
H
H
H
H
10
10
10
10
10
10
10
10
20
20
2
H
OCH3
Cl
H
12
92
3
H
H
13
78
4
H
CH3
Br
H
14
95
5
H
H
15
64
6
H
-C6H4-
H
H
16
77
7
H
F
H
17
78
8b
9b
10b
H
Cl
H
Cl
OCH3
H
18
76
H
H
OCH3
OCH3
19
80
OCH3
H
20
68
aStereoselectively only one diastereomer was formed in each case(drα:β, >99:1)determined by crude 1H NMR;. bRequired temperature was
120
pyrano[3,2-c]pyranones (21a vs. 21b) was ΔE = 1.240 kcal/mole. 20
4-hydroxycoumarins 2-10 to afford respective pyrano[3,2-
This calculation results demonstrate that 2-C-formyl galactal c]pyranones 22a/b-30a/b in acceptable to good yields as epimeric
derived pyrano[3,2-c]pyrone 11 is thermodynamically more mixture (dr~1:1). Similarly different substituents here at the R1, R2,
favorable than its diastereomer 11b. However, the energy R3 and R4 positions on 4-hydroxycoumarins neither remarkable
difference between 2-C-formyl glucal derived pyrano[3,2- affects the yield nor the reaction completion time when got
c]pyranones (21a vs. 21b) was significantly smaller than that of coupled with 2-C formyl glucal 1b (Table 3, entries 1-10). Thus
galactal case therefore they keep equilibrating between 21a and library of twenty different diverse carbohydrate fused pyrano[3,2-
21b in reversible fashion.20 We tried to isolate one stereoisomer c]pyranone compounds along with ten 4-hydroxycoumarins were
21a through column but it was futile as it again appeared mixture prepared and anticancer activity were determined by various
of epimers 21a/b in NMR. As shown in Table 3 (entries 2-10), 2-C
cellular assays and detailed biological results are discussed below
.
formyl glucal 1b was successfully coupled with various substituted
4 | J. Name., 2012, 00, 1-3
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Table 3. Synthesis of 2-C formyl glucal fused pyrano[3,2-c]pyranones 21a/b-30a/b.
ARTICLE
R2
O
R3
R4
R1
O
R1 OH
OBn
BnO
BnO
R2
R3
H
O
O
H
II, Toluene:AcOH
ꢀW, 80ꢁ120 °C
∗
+
OBn O
O
O
BnO
R4
OBn
O
1b
1-10
21a/b-30a/b
Entry
1
R1
H
H
H
H
H
H
H
H
H
R2
R3
H
H
H
H
H
R4
Product a
21a/21b
22a/22b
23a/23b
24a/24b
25a/25b
26a/26b
27a/27b
28a/28b
29a/29b
30a/30b
Time (min)
Yield (%)
81
H
H
15
15
15
15
15
15
15
20
20
20
2
OCH3
Cl
H
76
3
H
84
4
CH3
Br
H
80
5
H
73
6
-C6H4-
H
H
77
7
F
H
80
8b
9b
10b
Cl
H
Cl
OCH3
H
54
H
OCH3
OCH3
54
OCH3
H
51
aDiastereomeric ratio (dr)was ~1:1 (α:β) determined by crude 1H NMR; bRequired temperature was 120 °C
Biology
2
3
33.4 ±0.65
35.6 ±0.16
23.7 ±0.09
49.7 ±0.17
36.5 ±0.07
29.2 ±0.04
27.8 ±0.11
27.4 ±0.06
06.7 ±0.22
26.2 ±0.06
81.4 ±0.04
77.8 ±0.03
-01.1 ±0.08 17.72 ±0.01
-15.9±0.02 2.29 ±0.02
-13.9 ±0.10 2.94 ±0.01
-45.5 ±0.11 37.37 ±0.04
-24.4 ±0.10 18.79 ±0.01
10.6±0.05 -6.78 ±0.02
01.7±0.43 10.97 ±0.03
-05.7±0.25 13.34 ±0.01
08.0±0.22 3.03±0.00
-12.9±0.26 11.58 ±0.00
-18.0±0.07 43.21 ±0.01
Anticancer potential of all synthesized carbohydrate fused
pyrano[3,2-c]pyranones 11-20 and 21a/b-30a/b (n= 20) along with
different substituted 4-hydroxycoumarin precursors 1-10 (n = 10)
at 50 µM was investigated by MTT assay in MCF-7 (human breast
adeno carcinoma), HepG2 and MDA-MB231 cell lines. The results
showed (Table 4) growth inhibition in all tested compounds in MCF-
7 breast cancer cells with no effect in 25a/b. Compounds 12, 13, 14,
15 and 19 were effective against MDA-MB231 cells (breast) at 20
µM. However, no significant inhibition of HepG2 (Liver heptocellular
carcinoma) cells were observed with tested compounds even at
higher concentration of 100 µM indicating specificity of these
compounds for breast cancer cells.
4
5
6
7
8
9
10
11
12
13
Table 4. Anticancer screening results
Compounds
Growth Inhibition (%)a ±SD
MCF 7
HepG2
MDA-MB231
1
39.6 ±0.12
-29.4±0.04 4.47 ±0.00
-8.2±0.06
44.37 ±0.01
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14
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compounds in this series shown better activity against MDA-MB-
231; iv) glucal fused pyrano[3,2-c]pyranones were not very effective
against used cell lines. None of these compounds shown cell growth
inhibition against HepG2 cells indicating these structural
frameworks has specificity towards breast cancer.
78.7 ±0.04
54.2 ±0.14
20.7 ±0.19
24.0 ±0.12
15.3 ±0.27
17.6±0.10
47.0 ±0.11
53.5 ±0.04
03.1 ±0.37
21.5 ±0.06
01.4 ±0.38
-18.1 ±0.29
09.4 ±0.09
18.6 ±0.06
23.7 ±0.03
20.3 ±0.16
25.3±0.11
93.7 ±0.11
-14.9±0.08 37.66 ±0.00
01.2 ±0.48 31.55 ±0.17
04.0±0.17 6.34 ±0.0
07.6 ±0.01 15.49 ±0.00
-15.2±0.23 7.12 ±0.00
-04.4 ±0.22 45.84 ±0.03
04.8±0.51 1.36 ±0.01
-19.1±0.05 -6.02 ±0.0
19.0±0.37 4.66 ±0.00
06.3 ±0.06 -1.53 ±0.00
13.9 ±0.22 5.87 ±0.00
-02.8±0.37 5.85 ±0.12
-03.7 ±0.14 8.12±0.00
02.5 ±0.45 5.54 ±0.01
06.4±0.03 8.53 ±0.00
-00.4 ±0.10 0.58 ±0.00
-08.3±0.11 1.94 ±0.01
07.3±0.37 24.45 ±0.0
15
16
17
Furthermore, the effect of compounds 12, 13 and 14 on cell viability
and survival of cancer cells was determined using different
approaches. The cells were treated with compounds 12, 13 and 14
at 50 µM, which is higher concentration than their respective IC50
value, for 24 and 48 hours. The trypan blue staining showed > 80%
reduction in number of viable cells at this concentration (Fig. 3B (i)
). Additionally, we performed clonogenic assay wherein we treated
cells at 50 µM were plated in fixed number and allowed to grow to
form individual colonies. The survival fraction was calculated with
respect to untreated cells and the results showed < 20% survival of
breast cancer cells in presence of these compounds, which further
confirmed cell growth inhibitory potencies of carbohydrate fused
pyrano[3,2-c]pyranones compounds 12, 13 and 14 (Fig.2B (ii)).
Etoposide, which causes apoptosis in cancer cells, was used as
positive control for these experiments.
18
19
20
21a/b
22a/b
23a/b
24a/b
25a/b
26a/b
27a/b
28a/b
29a/b
30a/b
We further investigated if this antiproliferative activity was the
result of apoptotic mechanisms. MCF-7 cells treated with all three
compounds 12, 13 and 14 at 50 µM showed altered morphology,
cellular shrinkage, chromatin condensation and cell death which are
the characterstics of apoptosis. This phenomenon became more
obvious after 48 hours of treatment as shown in Fig. 3A. Next we
evaluated the effect of these compounds on cell cycle of MCF-7
cells by flow cytometry. The characterstic of cancer cells is their
uncontrollable growth and proliferation capability and cell cycle
arrest is an important target in oncotherapy. Treatment with
compounds 12 and 14 significantly increased the population of cells
in G2/M phase to 59.8% and 65.1% respectively at 50 µM as
compared to 17.1% in control cells. Compound 13 showed
accumulation of 42.4% cells in G2/M phase at lower concentration
of 25 µM (Fig. 3B). Moreover with compound 13 treatment at 50
µM, significant increase in polyploid cells (73.6%) and apoptotic
cells was observed. In addition to this, the population of G1 phase
has drastically decreased in cells treated with 50 µM compounds
12, 13 and 14 to 1.46%, 2.14% and 0.39% respectively as compared
to 65.7% in untreated cells. Taken together, these results suggested
that after 48 hours of treatment, compound 12, 13 and 14 caused
an obvious G2/M phase arrest which is related to reduced cell
viability. MTT assay of most active compounds against MCF-7 (12,
13 and 14) was carried out using normal cell (HEK293, human
embryonic kidney cells) and they were found non-toxic (Fig. S1, SI).
Etoposide
(5µM)
a50 µM, 100 µM and 20 µM concentration used for MCF-7, HepG2 and MDA-MB-231
cells respectively
From the study, it has been shown that carbohydrate fused
pyrano[3,2-c]pyranone compounds 12, 13 and 14 exhibited good
inhibitory effect against MCF 7 cells among all derivatives with an
IC50(μM) of 19.9, 14.6 and 10.9 respectively (Fig. 2A).
The structure-activity relationship (SAR) of these compounds for
anticancer activity can be summarized as follows: i) in the series of
galactal fused pyrano[3,2-c]pyranones, the substitution at C-2 (R2)
in coumarin ring is important for better anticancer activity; ii) 2-
OMe, 2-Cl and 2-CH3 substitution in this series shown better activity
against MCF-7; iii) 2-OMe, 2-Cl, 2-CH3 and 2-Br substituted
6 | J. Name., 2012, 00, 1-3
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Fig. 2. Cytotoxicity of carbohydrate fused pyrano[3,2-c]pyranones. (A) To
calculate half maximal inhibitory concentration (IC50) of compounds 12, 13
or 14, MCF 7 cells were treated with different concentrations (1-100 µM) for
48 hours. The percent growth inhibition was determined by MTT assay. IC50
values were determined by plotting values of percent inhibition against log
concentration of each of these compounds. IC50 values for compounds 12,
13 and 14 were 19.9, 14.6 and 10.98 respectively. The experiments were
performed in triplicates, n=3 and ± SD value was calculated for each data
point. (B) (i) Growth Inhibitory potential of 12, 13 or 14 was evaluated
towards MCF 7 cells at 50 µM. Cells were treated with compounds for 24
and 48 hours, and cell viability was determined by trypan blue exclusion
assay. (ii) Survival fractions obtained by in vitro clonogenic assay showed
significant inhibition in cells treated with these compounds.
treatment of these compounds significantly decreased the
migration of MCF-7 cells in concentration and time dependent
manner (Fig. 4).
Microtubule dynamics are critical prerequisite for mitotic spindle
formation during cell division. Anticancer agents altering this
microtubule dynamics drive the cancer to apoptosis.24 Thus we had
examined microtubulin distribution in cells treated with compounds
12, 13 and 14 by immunofluorescence assay. Fluorescence
microscopic images revealed tubulin aggregation in cells treated
with compound 13. Also, altered microtubule distribution was
observed in elongated protrusions in cells treated with compound
12 and 14 (Fig. 5). Nocodazole, which is used as positive control for
the experiment showed significant tubulin depolymerization. These
findings corroborate with our cell migration results suggesting
impairment of tubulin distribution affects cancer cell migration.
Together our results suggest that carbohydrate fused pyrano[3,2-
c]pyranones 12, 13 and 14 affect breast cancer cell migration and
induce apoptosis via microtubule distribution.
Since pyrano[3,2-c]pyranones 12, 13 and 14 were synthesized with
Fig. 3. Carbohydrate fused pyrano[3,2-c]pyranones induces morphological
changes and modulate cell cycle. (A) MCF 7 cells treated with 12, 13 or 14
for 24 hours and 48 hours showed significant changes in their morphology.
Cellular shrinkage and dead cells were seen after treatment. (B) MCF 7 cells
were treated with 12, 13 or 14 at 25 µM and 50 µM concentrations for 48
hours and a changes in cell cycle phases were analyzed by flow cytometry.
fused carbohydrate precursors, 2-C formyl galactal and 2-C formyl
glucal with the aim to improve their cellular uptake by cancer cells,
we monitored the time dependent intracellular uptake of these
compounds in MCF-7 cells. These compounds having fluorescence
properties due to conjugated double bond in pyrano[3,2-
Etoposide (5 µM), was used as a positive control for the experiments. M1, c]pyranone ring so without adding any fluorescent tag we can
M2, M3 and M4 represent apoptotic cells, G1, S and G2/M phases
observe cellular uptake. Briefly, the cells were grown on coverslips
respectively. The percent of cells in each phase were analyzed from
and treated with of 50 µM of compounds 12, 13 and 14 for 1 h and
histogram FL2 vs cell counts. Representative images are shown from three
4 h. The cells were then visualized for autofluorescence in UV filter
independent experiments.
under fluorescence microscopy. The results indicated higher cellular
uptake of these carbohydrate fused pyrano[3,2-c]pyranones as
Cancerous cells migration is essential for invasion and metastasis
phenotypes.23 Herein we performed wound healing assay to
compared to 4-hydroxycoumarin compound 5 (Fig. S2, SI).
determine ability of compound 12, 13 and 14 to inhibit the
migration of MCF-7 cells. The results showed that in untreated
control, the cells migrated within 48 hours to fill scratched area, but
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Fig. 5. Compounds 12, 13 and 14 regulate microtubules distribution. MCF 7
cells were incubated with compounds for 48 hours, permeabilized, stained
with α tubulin antibody (red), and analyzed by fluorescence microscopy.
Nucleuses are stained with DAPI (blue). Treated cells showed disturbed
microtubule distribution. Nocodazole (1µM), positive control for the
experiment causes microtubule depolymerization in MCF 7 cells. Scale bar =
10 µm.
Conclusions
In summary, we report an efficient microwave assisted synthesis of
carbohydrate fused pyrano[3,2-c]pyranones. The diversely
substituted on 4-hydroxycoumarins were successfully transformed
to corresponding privileged carbohydrate fused pyrano[3,2-
c]pyranones (n=20) within 10 min in good yield. Subsequently, we
demonstrated that three of these carbohydrate fused pyrano[3,2-
Fig. 4. Compounds 12, 13 and 14 regulate Breast Cancer Cell Migration. The
cell migration was analyzed by wound healing method. Confluent monolayer
of MCF 7 cells was scratched using pipette tip and the cells were incubated
with indicated concentrations of these compounds for 24 and 48 hours.
Untreated control closed completely after 48 hours, whereas compounds c]pyranone molecules possess anticancer activity at micromolar
12, 13 and 14 efficiently inhibit cell migration. The images are
levels. Cellular uptake assay indicates that, carbohydrate moiety
representative of three assays. Bar graph (bottom) represents significant cell
fusion to 4-hydroxycoumarin derivatives enhance their uptake by
migration inhibition by these compounds at 25 and 50 µM as compared to
breast cancer cells which in turn results in their enhanced cell
untreated control.
growth inhibitory potential than precursor 4-hydroxycoumarin
compounds. We showed that, these molecules alter cell
morphology and cause G2/M arrest in MCF-7. Moreover these
compounds also affect cell migration and cell cycle via disturbing
microtubule distribution. These findings shed light in considering
these carbohyrate fused pyrano[3,2-c]pyranone molecules for
developing potential anticancer chemotherapeutic agents.
Experimental Section
General Experimental Methods.
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All experiments were performed in an oven-dried apparatus and in product. The crude product was purified by flash column
anhydrous solvents in CEM microwave synthesiser. High resolution chromatography to obtain the pure products 11-20 in good to very
mass spectra obtained from a qudrapole/TOF mass spectrometer good yield (Table 2). The similar reaction protocol was adopted for
with an ESI source. Solvents were distilled by standard distillation the synthesis of compounds 21a/b-30a/b starting from 3,4,6-tri-
O-
procedures and stored in 4Å and 3Å molecular sieves. High- benzyl-2- -formyl D-glucal 1b and respective 4-hydroxycoumarin 1-
C
performance liquid chromatography (HPLC) experiments, to check 10 (Table 3). Compounds 21a/b-30a/b were isolated as inseparable
purity of compounds, were carried out on a Waters Alliance System mixture (1:1) of two epimers. Therefore in 1 H NMR spectra of these
(Milford, MA) consisting of e2695 separation module and a2998 compounds protons were integrated and reported as doubled
photodiode-array detector. The HPLC system was controlled with number of protons. The required temperature for preparation of
EMPOWER software (Waters Corporation, Milford, MA). 1H (400 18-20 and 28a/b-30a/b was 120 °C.
MHz), 13C (100 MHz) NMR spectra was recorded with a Bruker AMX-
400 MHz instrument. 1H and 13C chemical shifts are referenced to (8R,9R,10R,11aS)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-
1
the solvents residual signals (CDCl3: δ 7.26 for H NMR and δ 77.16 8,9,10,11a-tetrahydro-6H-pyrano[3',2':5,6]pyrano[3,2-
for 13C NMR) and reported in parts per million (ppm) at 25 °C. c]benzopyran-6-one (11). Sticky solid, Rf
=
0.56 (3:7,
Coupling constants are expressed in hertz (Hz). Reactions were Ethylacetate:Hexane),1H NMR (400 MHz, CDCl3): δ 7.86 (d,
= 7.2
monitored by thin-layer chromatography carried out on 0.25 mm E. Hz, 1H, ArH), 7.61 (s, 1H, ArH), 7.52 (t, = 14.8 Hz,1H), 7.39-7.28
Merck silica gel plates (60F-254), Spots were visualized by (m, 16H, ArH), 7.03 (s, 1H, H-7), 6.09 (s, 1H, H-11a), 4.94 (d,
J
J
J
= 11.6
phosphomolybdic acid and 10% H2SO4 in ethanol. 4- Hz, 1H, CH2Ph), 4.82 (d,
hydroxycoumarins (1-10), 2- -formyl galactal 1a and 2- -formyl Hz,1H, CH2Ph), 4.62 (d,
glucal 1b were freshly prepared in laboratory. Organocatalysts (I-IV) 1H, CH2Ph), 4.41 (d,
J
= 12 Hz, 1H, CH2Ph), 4.67 (d, J = 11.6
C
C
J
= 12 Hz, 1H, CH2Ph), 4.47 (d, J = 11.6 Hz,
J
J
= 11.6 Hz, 1H, CH2Ph), 4.15 (s, 1H, H-8), 3.98
used in this paper were purchased from sigma-aldrich.
(m, 1H, H-9), 3.86 (t,
= 11.6 Hz, 1H, H-10), 3.62 (dd,
J
= 6.0 Hz and
J
= 9.6 Hz, 1H, H-13a), 3.51 (dd,
J
= 6.4 Hz,
J
= 9.6 Hz, 1H, H-13b).13
C
NMR (100 MHz, CDCl3): δ 160.7 (C-6), 157.2, 156.2, 152.7 (ArqC),
138.1, 138.0, 137.6, 137.5, 132.2, 128.6, 128.5, 128.4, 128.2, 128.0,
127.9, 127.7, 127.5, 127.4, 126.7, 124.2, 122.8 (ArC), 116.7, 114.5,
112.6 (C-7), 99.6, 96.9 (C-11a), 79.4 (C-8), 76.3 (C-9), 75.2 (C-10),
74.4,73.6, 71.7 (3 × CH2Ph), 68.7 (C-13). HRMS (ESI), m/z calcd for
C37H32O7[M+H]+ 589.2226; Found: 589.2221.
General Experimental Procedure for reaction optimization: To a
10 mL of microwave vial 3,4,6-tri-O-benzyl-2-C-formyl D-galactal 1a
(100 mg, 0.225 mmol) and 4-hydroxycoumarin 1 (43.74 mg, 0.270
mmol) was added different combination of organocatalysts (I-IV,
0.112 mmol) in 2 mL of various solvents (viz. 1% acetic acid in
toluene, EtOAc and Acetonitrile) and heated at 80°C (100W) for 10
min under microwave condition. The completion of reaction was
monitored by TLC (Rf= 0.56, 3 : 7 = Ethylacetate: Hexane, v/v). After
completion, reaction was quenched by adding 5 mL of NaHCO3
solution and reaction mixturewas extracted with Ethylacetate (3 × 5
mL). The combined organic layer was dried over anhydrous Na2SO4
and combined organic layer was concentrated in vacuum to get
crude product. The crude product was purified by flash column
chromatography which furnished the designed product 11 in good
yield (93%) as sticky solid. Different organocatalysts and solvent
combinations furnished same product 11 in different yield
percentage (Table 1).
(8R,9R,10R,11aS)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2-
methoxy-8,9,10,11a-tetrahydro
6H-pyrano[3′2′:5,6]pyrano[3,2-
c]benzopyran-6-one (12):Off white amorphous solid, Rf = 0.51 (7:3,
Ethylacetate: Hexane), 1H NMR (400 MHz, CDCl3): δ 7.39-7.27 (m,
15H, ArH), 7.24 (s, 1H, ArH), 7.22 (s, 1H, ArH), 7.09 (dd,
and = 8.8 Hz, 1H), 7.03 (d, = 2.0 Hz, 1H, H-7), 6.10 (s, 1H, H-11a),
= 11.6 Hz, 1H, CH2Ph), 4.82 (d, = 12.4 Hz, 1H, CH2Ph),
= 12.4 Hz, 1H, CH2Ph), 4.64 (d, = 9.6 Hz, 1H, CH2Ph), 4.48
= 11.6 Hz,1H, CH2Ph), 4.15 (brs,
= 1.6 Hz ,1H, H-9), 3.88 (s, 1H, H-10), 3.83 (s, 3H,
= 6.4 Hz and = 9.6 Hz, 1H, H-13a), 3.49 (dd,
= 9.6, 1H, H-13b). 13C NMR (100 MHz, CDCl3): δ 160.8
J = 3.2 Hz
J
J
4.94 (d,
4.69 (d,
J
J
J
J
(d,
1H, H-8), 3.98 (d,
OCH3), 3.63 (dd,
6.0 Hz and
J = 12.0 Hz, 1H, CH2Ph), 4.41 (d, J
J
J
J
J =
J
(C-6), 156.0, 147.3, 138.1, 137.5 (ArqC), 128.6, 128.5,128.4, 128.2,
128.0, 127.9, 127.7, 127.6, 127.5, 127.4, 126.6, 120.8 (ArC), 117.9,
114.8, 112.8 (C-7), 104.0, 99.7, 96.9 (C-11a), 79.3 (C-8), 76.3 (C-9),
75.2 (C-10), 74.3, 73.6, 71.7 (3 × CH2Ph), 68.8 (C-13), 55.9 (OCH3).
HRMS(ESI), calcd, m/z C38H34O8, [M+K]+ 657.1885;Found: 657.1948.
(8R,9R,10R,11aS)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2-
General experimental procedure for synthesis of compounds 11-
20 and 21a/b-30a/b.
A mixture of 3,4,6-tri-O-benzyl-2-C-formyl D-galactal 1a (200 mg,
0.45 mmol), various 4-hydroxycoumarins 1-10 (0.54 mmol) and
pyrrolidine II (8 mg, 0.224 mmol) was heated in microwave vial
using 2 mL of toluene: AcOH (1:0.01) at 80°C (100W) for 10 min
under microwave condition. The progress of reaction was
chloro-8,9,10,11a-tetrahydro
c]benzopyran-6-one (13): Yellow amorphous solid, Rf = 0.62 (3:7,
Ethylacetate : Hexane),1H NMR (400 MHz, CDCl3): δ 7.83 (d,
= 2.0
= 2.0 Hz, 1H, H-3), 7.37-7.23
= 1.6 Hz, 1H, H-7), 6.08 (s, 1H, H-11a), 4.93
6H-pyrano[3′2′:5,6]pyrano[3,2-
J
Hz, 1H, H-1), 7.45 (dd,
(m ,16H, ArH), 7.00 (d,
J = 8.8 Hz and J
monitored by TLC (3:7
= Ethylacetate: Hexane,v/v). After
J
completion, reaction mixture was quenched by adding 5 mL of
NaHCO3 solution and reaction mixture was extracted with
Ethylacetate (3 × 5 mL). The combined organic layer was dried over
anhydrous Na2SO4 and concentrated in vacuum to get crude
(d,
11.8 Hz, 1H, CH2Ph), 4.62 (d,
11.6 Hz, 1H, CH2Ph), 4.41 (d,
J
= 11.6 Hz, 1H, CH2Ph), 4.80 (d,
J
= 12.4 Hz, 1H, CH2Ph), 4.66 (d,
J
=
J
= 12.0 Hz, 1H, CH2Ph), 4.48 (d,
J
=
J
= 12.0 Hz, 1H, CH2Ph), 4.15 (brs, 1H,
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H-8), 4.01 (brs, 1H, H-9), 3.86 (t,
J
= 6.0 Hz, 1H, H-10), 3.61 (dd,
J
=
137.6, 137.5, 135.0 (ArqC), 128.7, 128.6, 128.4, 128.2, 128.0,127.8,
6.0 Hz and
J
= 9.2 Hz, 1H, H-13a), 3.51 (dd,
J
= 6.4 Hz and = 9.6 Hz, 127.7,127.4, 127.1, 126.2, 124.2, 122.8, 122.6 (ArC), 118.2, 112.7
J
1H, H-13b).13C NMR (100 MHz, CDCl3): δ 160.1 (C-6), 156.0, 151.0, (C-7), 109.8, 99.3, 97.0 (C-11a), 79.4 (C-8), 76.2 (C-9), 75.2 (C-10),
138.0, 137.5, 137.4 (Arq C), 132.1, 129.8, 128.6, 128.4, 128.2, 128.0, 74.4, 73.6, 71.7 (3 × CH2Ph), 68.7 (C-13) HRMS(ESI), m/z calcd. for
127.9, 127.7, 127.6, 127.4, 122.2 (ArC), 118.1, 115.6, 112.3 (C-7), C41H34O7, [M + Na]+ 661.2197; Found : 661.2222.
100.4, 96.9 (C-11a), 79.3 (C-8), 76.4 (C-9), 75.2 (C-10), 74.4, 73.6,
(8R,9R,10R,11aS)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-3-
71.7 (3 × CH2Ph), 68.6 (C-13). HRMS (ESI), m/z calcd. for C37H31ClO7,
[M+Na]+ 645.1651; Found:645.1724.
fluoro-8,9,10,11a-tetrahydro
c]benzopyran-6-one (17): Off white solid, Rf = 0.75 (3:7 Ethyl
acetate : Hexane).1H NMR (400 MHz, CDCl3):δ 7.77 (dd,
= 6.0 Hz
and = 6.4 Hz, 1H, H-4), 7.31-7.17 (m, 15H,ArH), 6.95-6.91 (m, 3H,
ArH), 6.00 (s, 1H, H-7), 4.86 (d,
6H-pyrano[3',2':5,6]pyrano[3,2-
(8R,9R,10R,11aS)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2-
J
methyl-8,9,10,11a-tetrahydro
6H-pyrano[3′2′:5,6]pyrano[3,2-
J
c]benzopyran-6-one (14):Yellow amorphous solid, Rf = 0.57(7:3,
Ethylacetate:Hexane).1H NMR (400 MHz, CDCl3): δ 7.65 (s, 1H, ArH),
7.39-7.18 (m, 17H, ArH), 7.02 (s, 1H, H-7), 6.08 (s, 1H, H-11a), 4.94
J
= 11.6 Hz, 1H, CH2Ph), 4.73 (d,
J
=
=
12.4 Hz, 1H, CH2Ph), 4.59 (d,
12.4 Hz, 1H, CH2Ph), 4.40 (d,
J
= 11.6 Hz, 1H, CH2Ph), 4.54 (d, J
J
= 11.2 Hz, 1H, CH2Ph), 4.33 (d, J = 12
(d,
= 11.6 Hz, 1H, CH2Ph), 4.62 (d,
12.0 Hz, 1H, CH2Ph), 4.41 (d, = 12.0 Hz, 1H, CH2Ph), 4.15 (brs, 1H,
H-8), 4.00 (d, = 2.0 Hz, 1H, H-9), 3.86 (t, = 6.4 Hz, 1H, H-10), 3.63
(dd, = 6.0 Hz and = 9.6 Hz, 1H, H-13a), 3.53 (dd, = 6.4 Hz and J =
J
= 12.0 Hz, 1H, CH2Ph), 4.82 (d,
J = 12.4 Hz, 1H, CH2Ph), 4.68 (d,
Hz, 1H, CH2Ph), 4.07(s, 1H, H-8), 3.91 (s, 1H, H-9), 3.78 (t,
1H, H-10), 3.54 (dd, = 6.4 Hz and = 9.2 Hz, 1H, H-13a), 3.42 (dd,
= 6.4 Hz and
= 9.4 Hz, 1H, H-13b).13C NMR (100 MHz, CDCl3): δ
J = 6.0 Hz,
J
J
= 12.0 Hz, 1H, CH2Ph), 4.47 (d,
J =
J
J
J
J
J
J
J
160.4 (C-6), 156.9, 153.9, 137.0, 137.5, 137.4 (ArqC), 128.6, 128.4,
128.0, 127.9, 127.7, 127.4, 126.9, 126.6, 124.7, 124.6 (ArC), 112.5,
112.3, 111.2 (C-7), 104.4, 104.1, 98.7, 96.8 (C-11a), 79.3 (C-8), 76.2
(C-9), 75.2 (C-10), 74.4, 73.6, 71.7 (3 × CH2Ph), 68.7 (C-13).
HRMS(ESI), m/z calcd. for C37H31FO7, [M+Na]+ for 629.1946; Found:
629.1971.
J
J
J
9.6 Hz, 1H, H-13b), 2.38 (s, 3H, CH3). 13C NMR (100MHz, CDCl3): δ
160.8 (C-6), 156.2, 150.9, 140.8, 138.1, 137.6, 137.5 (ArqC), 133.9,
133.2, 128.6, 128.5, 128.4, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6,
127.4, 126.9, 126.4, 122.3 (ArC), 116.4, 114.1, 112.7 (C-7), 99.5,
96.9 (C-11a), 79.3 (C-8), 76.3 (C-9), 75.1 (C-10), 74.4, 73.6, 71.7 (3 ×
CH2Ph), 68.6 (C-13), 20.8 (CH3), HRMS(ESI), m/z calcd. For C38H34O7,
[M +Na]+ 625.2197; Found : 625.2253.
(8R,9R,10R,11aS)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2,4-
dichloro-8,9,10,11a-tetrahydro
6H-pyrano[3',2':5,6]pyrano[3,2-
(8R,9R,10R,11aS)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2-
c]benzopyran-6-one (18): Yellow amorphous solid. Rf = 0.78 (3:7,
Ethylacetate:Hexane), 1H NMR (400 MHz, CDCl3): δ 7.74 (s, 1H, H-1),
7.55 (s, 1H, H-4), 7.37-7.25 (m, 14H ArH), 6.99 (s, 1H, H-7), 6.08 (s,
bromo-8,9,10,11a-tetrahydro
6H-pyrano[3′2′:5,6]pyrano[3,2-
c]benzopyran-6-one (15): Light yellow amorphous solid, Rf = 0.71
1
(3:7, Ethylacetate:Hexane), H NMR (400 MHz, CDCl3): δ 7.98 (d,
2.0 Hz, 1H, ArH), 7.59 (dd, = 8.8 Hz and = 2.0 Hz, 1H, ArH), 7.38-
= 8.4 Hz, 1H, ArH), 7.00 (s, 1H, H-7),
= 11.6Hz, 1H, CH2Ph), 4.80 (d, = 12.4
=12.4 Hz, 1H, CH2Ph), 4.62 (d, = 12.4 Hz,
J =
1H, H-11a), 4.93 (d,
CH2Ph), 4.68 (d,
CH2Ph), 4.47 (d,
4.15 (s, 1H, H-8), 3.98 (d,
3.59 (dd, = 6.4 Hz and = 8.8 Hz, 1H, H-13a), 3.51 (dd,
= 8.8 Hz, 1H, H-13b), C NMR(100 MHz, CDCl3): δ 159.0 (C-6)
J
= 11.6 Hz, 1H, CH2Ph), 4.80 (d,
= 10 Hz, 1H,CH2Ph), 4.62 (d,
= 12 Hz, 1H, CH2Ph), 4.41 (d, = 12 Hz, 1H, CH2Ph),
= 9.2Hz, 1H, H-9), 3.85 (brs, 1H, H-10),
= 6 Hz and
J
= 12.4 Hz, 1H,
J
J
J
J = 12.4 Hz, 1H,
7.28 (m,15H, ArH), 7.18 (d,
6.09 (s, 1H, H-11a), 4.94 (d,
J
J
J
J
J
J
Hz, 1H, CH2Ph), 4.67 (d,
1H, CH2Ph), 4.48 (d, = 12.0 Hz,1H, CH2Ph), 4.41 (d,
CH2Ph), 4.16 (brs, 1H, H-8), 4.01 (s, 1H, H-9), 3.86 (t,
H-10), 3.62 (dd, = 6.4 Hz and
Hz and
= 9.6 Hz, 1H, H-13b), 13C NMR (100MHz, CDCl3): δ 160.0 (C-
J
J
J
J
J
J
J
J
= 11.6 Hz, 1H,
= 12.0 Hz, 1H,
13
J
154.5, 137.9, 137.5, 137.3 (ArqC), 128.6, 128.5, 128.4, 128.2, 128.0,
127.9, 127.8, 127.6, 127.4, 126.9, 122.5, 120.8 (ArC), 116.6, 112.1
(C-7), 100.9, 96.9 (C-11a), 79.2 (C-8), 76.4 (C-9), 75.3 (C-10), 74.5,
73.6, 71.8 (3 × CH2Ph), 68.5 (C-13). HRMS(ESI), calcd. m/z, for
J
J = 9.2Hz, 1H, H-13a), 3.51 (dd, J = 6.0
J
6), 154.9, 151.4, 138.0, 137.5, 137.4, 134.9 (ArqC), 128.6, 128.4,
128.2, 128.0, 127.9, 127.7, 127.6, 127.4, 125.2 (ArC), 118.4, 117.1,
116.0, 112.3 (C-7), 100.4, 96.9 (C-11a), 79.3 (C-8), 76.4 (C-9), 75.2
(C-10), 74.4, 73.6, 71.7 (3 × CH2Ph), 68.5 (C-13). HRMS (ESI), m/z,
calcd.for C37H31BrO7, [M+Na]+ 689.1145; Found: 689.1167.
C
37H30Cl2O7, [M + Na]+,679.1261; Found: 679.1305.
(8R,9R,10R, 11aS)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-3,4-
dimethoxy-8,9,10,11a-tetrahydro 6H-pyrano[3',2':5,6]pyrano[3,2-
c]benzopyran-6-one(19): Yellow amorphous solid, Rf = 0.5 (3:7 Ethyl
(8R,9R,10R,
8,9,10,11a–terahydro6H-2,3-benzo[g]pyrano[3′,2′:5,6]pyrano[3,2-
c]benzopyran-6-one (16): Amorphous solid, Rf 0.62 (3:7
Ethylacetate : Hexane).1HNMR (400 MHz, CDCl3):δ 8.53 (s,1H, ArH),
7.83 (d, = 8.8 Hz, 2H, ArH), 7.67-7.63 (m, 3H, ArH), 7.40-7.25 (m,
15H, ArH), 7.08 (s, 1H, H-7), 6.13 (s, 1H, 11a), 4.96 (d, = 11.6 Hz,
11aS)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-
acetate :Hexane), 1H NMR (400 MHz, CDCl3): δ 7.56 (d,
1H, H-2), 7.39-7.27 (m, 15H, ArH), 7.00(d, = 1.6 Hz,1H), 6.87 (d,
8.8 Hz, 1H,H-1), 6.04 (s, 1H, H-11a), 4.94 (d, = 11.6 Hz, 1H, CH2Ph),
J = 8.8 Hz,
=
J
J =
J
J
4.82 (d,
4.61 (d,
4.41 (d,
J
J
= 12.4 Hz, 1H, CH2Ph), 4.66 (d,
= 12.4 Hz, 1H, CH2Ph), 4.47 (d,
J
J
= 12.0 Hz ,1H, CH2Ph),
= 12.0 Hz, 1H, CH2Ph),
J
1H, CH2Ph), 4.84 (d,
CH2Ph), 4.64 (d, = 12.8 Hz, 1H, CH2Ph), 4.48 (d,
CH2Ph), 4.41 (d,
H-9), 3.87 (t, = 11.6 Hz, 1H, H-10), 3.64 (dd,
Hz, 1H, H-13a), 3.52 (dd, = 6.4 Hz and
= 9.2 Hz, 1H, H-13b).13C
J
= 12 Hz, 1H, CH2Ph), 4.69 (d,
J
= 12.0 Hz, 1H,
J
= 12.0 Hz, 1H, CH2Ph), 4.13 (s, 1H, H-8), 4.00 (d,
J
= 2.0 Hz,
= 12.0 Hz,
= 9.6 Hz,1H, H-13a), 3.51 (dd, J =
= 9.6 Hz, 1H, H-13b). 13C NMR (100 MHz, CDCl3): δ
J
J
= 12.0Hz, 1H,
1H, H-9), 3.96 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.84 (t,
1H, H-10), 3.62 (dd, = 6.0 Hz and
6.4 Hz and
J
J
= 11.6 Hz, 1H, CH2Ph), 4.17 (s, 1H, H-8), 4.01(s, 1H,
= 6.4 Hz and = 8.8
J
J
J
J
J
J
J
J
159.4, 155.5, 154.8, 145.8, 137.1, 136.5, 135.0 (ArqC), 127.5, 127.4,
127.2, 126.9, 126.8, 126.3, 125.94, 124.51, 116.9, 111.7 (C-7),
NMR (100 MHz, CDCl3): δ 160.6 (C-6), 157.2, 150.2, 138.1, 138.1,
10 | J. Name., 2012, 00, 1-3
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107.9, 107.4, 96.5, 95.8 (C-11a), 78.3 (C-8), 75.2 (C-9), 74.1 (C-10), 118.1, 117.9, 111.3, 104.3, 104.1, 100.3, 99.8, 99.2, 96.2, 81.9, 80.9,
73.3, 72.5, 70.7 (3 × CH2Ph), 67.7 (C-13), 60.5 (OCH3), 55.3 (OCH3). 78.7, 78.5, 76.5, 75.1, 73.5, 73.4, 73.3, 73.0, 71.5, 70.2, 68.8, 68.6,
HRMS(ESI), calcd. m/z, C39H36O9, [M+H]+ 649.2439; Found
:
56.0, 55.9 (2 × OCH3). HRMS(ESI), calcd. m/z, C38H34O8, [M + H]+
,
649.2424.
619.2326; Found: 619.2363.
(8R,9R,10R,11aS)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-1,3-
(8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2-chloro-
dimethoxy-8,9,10,11a-tetrahydro 6H-pyrano[3',2':5,6]pyrano[3,2- 8,9,10,11a-tetrahydro-6H-pyrano[3',2':5,6]pyrano[3,2-
c]benzopyran-6-one (20): Light yellow amorphous solid, Rf = 0.43 c]benzopyran-6-one (23a/b): Yellow amorphous solid, Rf = 0.71
1
(3:7, Ethylacetate:Hexane), H NMR (400 MHz,CDCl3): δ 7.64 (s, 1H, (3:7, Ethylacetate:Hexane), 1H NMR (400 MHz, CDCl3): δ 7.89 (s,
ArH), 7.40-7.32 (m, 13H, ArH), 7.13-7.00 (m, 2H, ArH), 6.48 (brs, 1H, 2H), 7.50-7.18 (m, 34H, ArH), 6.93 (s, 1H, ArH), 6.83 (s, 1H), 6.63 (s,
ArH), 6.28 (d,
= 12.0 Hz, 1H, CH2Ph), 4.83 (d,
11.6 Hz, 1H, CH2Ph), 4.69 (d,
Hz, 1H, CH2Ph), 4.45 (d,
J
= 13.6 Hz, 1H), 6.04 (d,
J
= 9.2 Hz, 1H, H-2), 5.01 (d,
J
1H), 6.34 (s, 1H), 6.01 (s, 1H), 4.93 (d,
(d, = 11.6 Hz, 2H, CH2Ph), 4.66 (d, = 12.0 Hz, 2H, CH2Ph), 4.56 (d,
= 15.8 Hz, 1H, CH2Ph), 4.42 (d,
J = 10.8 Hz, 2H,CH2Ph), 4.73
J
= 10.8 Hz, 1H, CH2Ph), 4.75 (d,
J
=
J
J
J
J
= 11.6 Hz, 1H, CH2Ph), 4.63 (d,
J
= 13.2 = 9.6 Hz, 2H, CH2Ph), 4.50 (d,
J
J
=
J
= 12.4 Hz, 1H, CH2Ph), 4.30 (s, 1H, H-8), 12.4 Hz, 1H, CH2Ph), 4.36 (d,
J
= 12.8 Hz, 1H, CH2Ph), 4.23 (d, J = 9.6
4.17 (m, 1H, H-9), 4.00 (s, 1H, H-10), 3.88 (s, 3H, OCH3),3.86 (s, 3H, Hz, 2H), 3.98-3.65 (m, 8H).13C NMR (100 MHz, CDCl3): δ 160.0,
OCH3), 3.61(m, 1H, H-13a), 3.20 (m,1H, 13b).13C NMR(100 MHz, 158.4, 154.9, 151.7, 151.0, 140.9, 137.9, 137.7, 137.4, 137.3,137.1,
CDCl3): δ 167.7, 166.0, 163.4, 158.1, 157.5, 157.2, 138.7, 138.4, 132.8, 132.2,131.5, 130.0, 129.9, 128.6, 128.5, 128.4, 128.1, 128.0,
138.0, 137.7, 136.1 (ArqC), 128.5, 128.4, 128.1, 128.0, 128.3, 128.1, 127.9, 127.8, 127.6, 127.3, 126.9, 122.8, 122.4, 122.4, 122.3, 122.2,
128.0, 128.0, 127.90, 127.8, 127.6, 127.5, 127.3, 127.1, 126.8 (ArC), 118.3, 118.2, 115.5, 110.9, 108.5, 100.9, 100.4, 99.4, 97.2, 96.2,
112.9 (C-7), 106.07, 100.5, 95.1, 95.0 (C-11a), 93.3, 93.1, 76.2 (C-8), 81.9, 80.9, 78.6, 78.4, 76.6, 75.9, 75.2, 73.6, 73.4, 73.1, 71.6, 70.3,
75.0 (C-9), 74.0 (C-10), 72.9, 72.1, 71.6 (3×CH2Ph), 68.2 (C-13), 56.2 68.8, 68.4. HRMS(ESI), calcd. m/z, for
(OCH3), 55.7 (OCH3). HRMS(ESI), calcd., m/z C39H36O9, [M+H]+ 645.1651; Found: 645.1693.
649.2439; Found: 649.2476.
C ,
37H31ClO7, [M+ Na]+
(8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2-methyl-
(8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-8,9,10,11a- 8,9,10,11a-tetrahydro-6H-pyrano[3',2':5,6]pyrano[3,2-
tetrahydro-6H-pyrano[3',2':5,6]pyrano[3,2-c]benzopyran-6-one
c]benzopyran-6-one (24a/b): Yellow amorphous solid, Rf = 0.59(3:7,
(21a/b): Off white solid, Rf = 0.56 (3:7,Ethylacetate:Hexane),1HNMR Ethylacetate:Hexane),1H NMR (400 MHz, CDCl3): δ 7.69 (s,2H), 7.35-
(400 MHz,CDCl3): δ 7.87-7.85 (m,4H, ArH), 7.52-7.12 (m, 34H, ArH ), 7.16 (m, 33H ArH), 6.68 (s, 1H), 6.32 (s, 1H), 6.27 (s, 1H), 5.72 (s,
6.89 (s, 1H, H-7 of 21a), 6.80 (s, 1H, H-7 of 21b), 6.56 (s, 1H, H-11a 1H), 5.66 (s, 1H), 4.95 (d,
of 21b), 5.95 (s, 1H, H-11a of 21a), 4.88 (d, = 10.8 Hz, 2H, CH2Ph,), Hz, 2H, CH2Ph), 4.90 (d,
= 12.0 Hz, 2H, CH2Ph), 1H, CH2Ph), 4.64 (d, = 11.4 Hz, 1H, CH2Ph), 4.60 (d,
= 11.2Hz, 2H, CH2Ph), 4.44 CH2Ph), 4.54 (d, = 11.2 Hz, 1H, CH2Ph), 4.53 (d,
CH2Ph), 3.96-3.692 (m, 10H), 2.40 (s, 3H, CH3), 2.38 (s, 3H, CH3).13
NMR (100 MHz, CDCl3): δ 160.9 (C-4), 160.5, 159.6,159.6, 156.2,
J
= 10.8 Hz, 2H, CH2Ph), 4.92 (d,
= 11.6 Hz, 2H, CH2Ph), 4.67 (d, = 8.4 Hz,
= 12.4 Hz, 2H,
= 10.6 Hz, 1H,
J = 10.8
J
J
J
4.66 (d,
4.56 (d,
J
= 10.8 Hz, 2H, CH2Ph), 4.60 (d,
= 3.2 Hz, 1H, CH2Ph), 4.50 (d,
= 12.0Hz, 1H, CH2Ph), 4.30 (d,
= 8.0 Hz, 2H), 3.92-3.57 (m, 8 H), 13
J
J
J
J
J
J
J
(d,
J
= 11.6 Hz, 1H, CH2Ph), 4.36 (d,
J
J
C
= 11.6 Hz,1H, CH2Ph) 4.16 (d,
J
C
NMR (100 MHz,CDCl3): δ 160.5, 159.5, 156.1, 153.4, 152.8, 138.0, 156.0,151.6, 137.9, 137.7, 137.4, 137.0, 136.8, 135.4, 134.0, 133.4,
137.7, 137.5, 137.4, 137.2,135.4, 132.9, 132.3, 131.7, 128.6, 128.4, 132.8, 128.6, 128.5,128.4, 128.3, 128.2, 128.1, 128.0, 128.0, 127.9,
128.1, 127.9, 127.7, 127.6, 127.4, 127.1, 127.0, 124.3, 124.2, 123.4, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 123.0, 122.5, 122.3, 116.6,
122.9, 121.4, 116.9, 116.7, 114.4, 111.2, 109.0, 100.3, 99.7, 99.2, 116.5, 116.1, 111.3, 109.4, 99.1, 97.2, 96.2, 96.1, 82.0, 80.9, 79.2,
97.2, 96.2, 82.0, 80.9, 78.7, 78.5, 76.6, 75.8, 75.1, 73.5, 73.3, 73.1, 79.1, 75.8, 75.1, 73.5, 73.0, 72.7, 72.5, 71.56, 70.5, 70.3, 68.5, 67.9,
71.5, 70.2, 68.8, 68.6. HRMS(ESI) calcd, for m/z, C37H32O7 [M + H]+ 22.7, 20.8 (2 × CH3). HRMS(ESI), m/z, C38H34O7[M+H]+ 603.2377;
589.2226; Found:589.2221.
Found : 603.2388.
(8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2-methoxy- (8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2-bromo-
8,9,10,11a-tetrahydro-6H-pyrano[3',2':5,6]pyrano[3,2- 8,9,10,11a-tetrahydro-6H-pyrano[3',2':5,6]pyrano[3,2-
c]benzopyran-6-one (22a/b): Yellow amorphous solid, Rf = 0.71 c]benzopyran-6-one (25a/b): Off white amorphous solid, Rf = 0.81
(3:7, Ethylacetate:Hexane),1H NMR (400 MHz, CDCl3):δ 7.41-7.15 (3:7, Ethylacetate: Hexane), 1H NMR (400 MHz,CDCl3): δ 7.95-7.94
(m, 36H, ArH), 6.94 (s,1H), 6.85 (s,1H), 6.61 (s, 1H), 6.01 (s, 1H), 4.92 (m, 2H), 7.54 - 7.47 (m, 3H ), 7.33-7.05 (m, 29H, ArH), 6.82 (d,
(m, 2H, CH2Ph), 4.70 (d, = 11.6 Hz, 1H, CH2Ph), 4.65 (d, = 12.0 Hz, Hz, 1H), 6.73 (d, = 1.2 Hz,1H), 6.77 (d, = 1.2 Hz, 1H), 6.52 (s, 1H),
1H, CH2Ph), 4.59 (brs, 2H, CH2Ph), 4.53 (d, = 11.6 Hz,3H, CH2Ph), 6.23 (d, = 6.0 Hz, 1H), 5.90 (s, 1H), 4.87 (d, = 9.6 Hz, 1H, CH2Ph),
= 12.4 Hz, 1H, CH2Ph), 4.82 (d, = 12.0 Hz, 2H, CH2Ph), 4.62 (d, = 12.0 Hz, 1H, CH2Ph),
= 8.8 Hz, 1H), 3.98 (m,2H), 4.57 (d, = 9.2 Hz, 1H, CH2Ph), 4.54 (d, = 10.2 Hz, 1H, CH2Ph), 4.50
= 9.8 Hz, 2H, CH2Ph), 4.45 (d, = 9.2 Hz, 1H, CH2Ph), 4.38 (d,
(100 MHz, CDCl3): δ 160.7, 160.6, 159.3, 156.1, 156.0, 155.9, 148.0, 11.6 Hz, 1H, CH2Ph), 4.30 (d, = 12.4 Hz, 1H, CH2Ph), 4.25 (d,
147.3 , 137.9, 137.7, 137.6, 137.4, 137.3,137.1, 128.5, 128.4, 128.3, 11.6Hz, 1H, CH2Ph), 4.11(d,
= 12.0 Hz, 2H), 3.89-3.53 (m, 8H), 13
J = 8.0
J
J
J
J
J
J
J
4.47 (d,
4.33 (d,
J
= 12.4 Hz, 1H, CH2Ph), 4.39 (d,
= 11.2 Hz,1H, CH2Ph), 4.21 (d,
J
J
J
J
J
J
J
3.85 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.80-3.58 (m, 7H).13C NMR (d,
J
J
J
=
=
C
J
J
J
128.1, 128.0, 127.9, 127.8, 127.7, 126.9, 123.0, 121.8, 121.3, 121.0, NMR (100 MHz,CDCl3): δ 159.9, 159.8, 158.2, 154.8, 152.1, 151.5,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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137.9, 137.7, 137.4, 137.3, 137.1, 135.6, 135.0, 128.6, 128.4, 128.1, MHz,CDCl3): δ 158.8, 154.4, 153.6, 147.1, 137.9, 137.3, 137.2,
128.0, 127.9, 127.8, 127.7, 127.5, 127.3, 125.9, 125.4, 122.4, 132.7, 132.1, 131.4, 129.6, 128.7, 128.6, 128.4, 128.1, 128.0, 127.9,
122.3, 118.6, 118.4, 117.2, 117.1, 116.0, 110.9, 100.9, 100.4, 99.4, 127.8, 127.5, 127.3, 122.6, 122.0, 121.4, 120.9, 120.4, 116.5, 110.7,
97.2, 96.2, 81.9, 80.9, 78.6, 78.4,76.6, 75.2, 73.6, 73.4, 73.1, 71.6, 99.6, 97.1, 96.3, 81.8, 81.0, 78.5, 78.4, 75.9, 75.2, 73.7, 73.6, 73.4,
71.6, 70.3, 68.7, 68.4. HRMS(ESI) m/z, calcd. forC37H31BrO7, [M + 73.2, 71.6, 70.4, 68.7, 68.4, HRMS(ESI), calcd. m/z for C37H30Cl2O7,
Na]+ , 689.1145; Found : 689.1197.
[M + Na]+ 679.1261; Found: 679.1264.
(8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-
(8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-3,4-
8,9,10,11a-terahydro-6H-2,3-benzo[g]pyrano[3′,2′:5,6]pyrano[3,2-
dimethoxy-8,9,10,11a-tetrahydro-6H-pyrano[3',2':5,6]pyrano[3,2-
c]benzopyran-6-one (26a/b): Light yellow amorphous solid, Rf
0.53 (3:7, Ethylacetate:Hexane), 1H NMR (400 MHz, CDCl3): δ 8.52 0.62(3:7,Ethylacetate:Hexane).1H NMR (400 MHz, CDCl3):δ8.09 (d,
(s, 2H), 7.88-7.84 (m, 4H, ArH), 7.69-7.59 (m, 7H, ArH), 7.48-7.30 (m, = 7.6Hz 1H, H-8), 7.62 (s, 1H), 7.64-7.60 (m, 2H), 7.40-7.27(m, 25H,
26H, ArH), 7.18 (s, 2H), 6.98 (s, 1H), 6.90 (s, 1H), 6.73 (s, 1H), 6.64 (s, ArH), 7.15 (s, 3H), 6.90(d, = 8.8 Hz, 2H), 6.83(s, 1H), 6.55(s, 1H),
1H), 6.39 (s,1H), 6.33 (s,1H), 4.95 (d, = 10.4Hz, 2H, CH2Ph), 4.73 (d, 6.33(s, 1H), 5.95(s, 1H),4.97-4.88(m, 2H, CH2Ph), 4.69(d, = 11.2 Hz,
= 12 Hz, 1H, CH2Ph), 4.66 (d, = 10.4 Hz, 2H, CH2Ph), 4.60 (d, 2H, CH2Ph), 4.65(d, = 9.5 Hz,2H,CH2Ph), 4.61-4.58(m, 1H, CH2Ph),
13.6 Hz, 2H, CH2Ph), 4.55 (d, = 7.6 Hz, 2H, CH2Ph), 4.50 (d, = 11.6 4.53(d, = 12.0Hz, 2H, CH2Ph), 4.49(d, = 8.0 Hz, 2H, CH2Ph), 4.44(d,
Hz, 2H, CH2Ph), 4.42 (d, = 12 Hz, 1H, CH2Ph), 4.36 (m, 1H), 4.22 (s, = 11.6 Hz, 1H, CH2Ph), 4.18(d, = 9.2 Hz, 2H), 3.98(s, 6H, 2 × OCH3
=
c]benzopyran-6-one(29a/b): Yellow amorphous solid, Rf =
J
J
J
J
J
J
J
=
J
J
J
J
J
J
J
J
1H), 4.01-3.63 (m, 8 H).13C NMR(100 MHz,CDCl3): δ 160.7, 160.4, ), 3.94(s, 6H, 2 × OCH3 ), 3.83 - 3.57(m, 8H).13C NMR(100 MHz,
157.1, 156.9, 156.5,156.1, 156.0, 155.0, 149.6, 138.0,137.9, 137.8, CDCl3):δ 160.5,160.3, 159.9, 158.9, 157.3,156.5, 156.2, 156.0,
137.7, 137.4, 135.5, 135.4,134.8, 128.6, 128.5, 128.4, 128.3, 128.0, 155.6, 138.1, 138.0, 137.7,137.3, 136.1, 128.5, 128.4, 128.3, 128.0,
127.9, 127.8, 127.6, 127.5, 127.4, 127.3, 127.1, 126.6, 124.2, 123.5, 127.9, 127.7, 127.6, 125.8, 123.2, 120.2, 118.7, 118.1, 111.4, 108.8,
122.9, 122.6, 122.5,118.2, 111.3, 110.0, 109.4, 109.2, 101.9,99.3, 99.1, 98.1, 97.2, 96.1, 81.9, 80.9, 78.7, 78.5, 75.7, 75.1, 73.3, 73.0,
97.2, 97.1,96.3, 96.1,82.0,80.9, 78.7, 78.6, 75.8, 75.2, 73.7, 73.5, 72.3, 71.5, 70.1, 68.9, 68.6, 68.2, 67.7, 61.6, 56.4. HRMS (ESI),
73.4, 73.1, 70.8, 70.5, 70.3, 69.5,68.9. HRMS(ESI), calcd. m/z, for calcd.,m/z for C39H36O9, [M+H]+ 649.2439; Found : 649.2424.
C
41H34O7, [M + Na]+661.2197; Found : 661.2232.
(8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-1,3-
dimethoxy-8,9,10,11a-tetrahydro-6H-pyrano[3',2':5,6]pyrano[3,2-
c]benzopyran-6-one (30a/b): Yellow amorphous solid Rf = 0.50 (3:7,
(8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2-fluoro-
8,9,10,11a-tetrahydro-6H-pyrano[3',2':5,6]pyrano[3,2-
c]benzopyran-6-one (27a/b): Yellow amorphous solid, Rf = 0.65(3:7, Ethylacetate:Hexane), 1H NMR (400 MHz, CDCl3): δ 7.42-7.16 (m,
Ethylacetate: Hexane), 1H NMR (400 MHz, CDCl3): δ 7.87-7.82 (m, 30H, ArH), 7.17-7.16 (m, 2H), 6.88 (s, 1H), 6.80 (s, 1H), 6.48 (s,1H),
3H), 7.39- 7.21 (m, 21H,ArH), 7.12 (s, 5H, ArH), 7.00-6.98 (m, 6H, 6.43 (s, 1H, ArH), 6.29 (m, 1H, ArH), 5.93 (s, 1H, H-11a), 4.91 (dd, J =
ArH), 6.85 (s, 1H), 6.77 (s, 1H), 6.55 (s,1H), 6.26 (d,
5.94 (s, 1H), 4.87 (d, = 11.2 Hz, 2H, CH2Ph), 4.68 (s, 1H, CH2Ph), 4.61(d,
= 12.0 Hz, 2H, CH2Ph), 4.56 (d, = 12.8 Hz, 2H, CH2Ph), (d,
= 8.0 Hz, 2H, CH2Ph), 4.44 (d, = 12.8 Hz, 1H, CH2Ph), 4.35 2H, CH2Ph), 4.16 (s, 2H), 4.01 (m, 2H), 3.90-3.84 (m, 6H, 2 × OCH3),
= 11.6 Hz, 1H, CH2Ph), 4.16 (d,
3.77 - 3.72(m, 6H, 2 × OCH3), 3.77 - 3.59 (m, 8H). 13C NMR (100
J
= 6.8 Hz, 1H), 12 Hz and
J
= 11.2 Hz, 2H, CH2Ph), 4.67(d,
J
= 12.0 Hz, 1H, CH2Ph),
= 12.4 Hz, 2H, CH2Ph), 4.51
= 12.0 Hz,
J
J
= 8.4 Hz, 2H, CH2Ph), 4.56 (d, J
4.63 (d,
4.50 (d,
J
J
J
= 11.2 Hz, 2H, CH2Ph), 4.38 (s, 1H, CH2Ph,), 4.34 (d,
J
J
J
(d,
J
= 12.0 Hz, 1H, CH2Ph), 4.30 (d,
J
J
= 7.6 Hz, 2H), 3.94-3.57 (m, 8H). 13C NMR(100 MHz, CDCl3): δ 160.5, MHz, CDCl3): δ 164.0, 156.9, 145.4, 138.2, 138.0, 137.8, 137.6,
160.3, 160.2, 159.2, 156.2, 155.8, 154.6, 138.2, 138.0, 137.7, 137.4, 137.3, 137.0, 128.5, 128.4, 128.2, 128.0, 127.9, 127.8, 123.5, 98.8,
137.1, 137.2, 128.6, 128.4, 128.0, 127.9, 127.4, 126.9, 125.4, 124.8, 96.0, 93.4, 82.0, 80.9, 78.8, 75.0, 73.4, 73.1, 72.8, 71.4, 69.9, 69.1,
122.6, 121.3, 112.6, 111.0, 104.2, 99.3, 98.8, 97.1, 96.2, 81.9, 80.9, 63.9, 56.3, 55.7. HRMS(ESI), calcd, m/z,for C39H36O9, [M + H]+
78.6, 78.4, 75.8, 75.2, 73.7, 73.5, 73.3, 73.1, 71.6, 70.2, 68.8, 68.5. 649.2439 Found: 649.2474.
HRMS(ESI), calcd., m/z C37H31FO7 [M+ Na]+ , 629.1946; Found :
Cell Culture
629.1967.
The human breast cancer cell line MCF-7, MDA-MB-231 and liver
(8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2,4-
cancer cell line HepG2 and normal human embryonic kidney cells
dichloro-8,9,10,11a-tetrahydro-6H-pyrano[3',2':5,6]pyrano[3,2-
HEK293 were obtained from the American Type Culture Collection
c]benzopyran-6-one (28a/b):Yellow amorphous solid, Rf = 0.71 (3:7,
Ethylacetate:Hexane), 1H NMR (400MHz,CDCl3): δ 7.80 (d,
J
= 2.4Hz,
= 8.8 Hz, 1H), 7.45-7.29 (m,
25H, ArH), 7.15-7.13 (m, 3H), 6.90 (s, 1H), 6.80 (s, 1H), 6.60 (s, 1H),
(ATCC, USA).The cells were grown in Dulbecco's Modified Eagle's
Medium (DMEM) (Gibco, St. Louis, USA) containing with L-
glutamine, supplemented with 10% heat inactivated FBS (Fetal
Bovine Serum, Gibco, USA) and 1% penicillin-streptomycin (Gibco
USA)in humidified CO2 incubator at 37ºC. The cells were maintained
as monolayer in 25 cm2 culture flasks (T-25) and the medium was
changed 10-12 hours prior to experiment. The cells used for each
experiment were of less than 10 passage number.
2H), 7.59 (d, J = 8.8 Hz, 2H), 7.53 (d, J
6.31 (s, 1H), 5.99 (s, 1H), 4.95 (d,
J
= 10.4 Hz, 1H, CH2Ph), 4.90 (d,
J
=
=
=
=
12.4 Hz
,
2H, CH2Ph), 4.71 (d,
J
J
J
= 12.0 Hz, 1H, CH2Ph), 4.59 (d,
= 10.4 Hz, 2H, CH2Ph), 4.47 (d,
= 12.0 Hz, 1H, CH2Ph), 4.33 (d,
J
12.8 Hz, 2H, CH2Ph), 4.52 (d,
J
11.6 Hz, 2H, CH2Ph), 4.39 (d,
J
11.6 Hz, 1H, CH2Ph), 4.18 (m, 2H), 3.94-3.62 (m, 8H).13C NMR (100
12 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C8NJ01395K
Journal Name
MTT assay
ARTICLE
Cell cycle and morphology analysis
MCF-7 cells were seeded in 6-well plates at a density of 3 × 105 cells
per well in complete DMEM, and treated with compounds 12, 13
and 14 at 50 μM concentrations for 48 hours. The untreated cells
were taken as negative control. Etoposide treated cells (5 μM) were
taken as positive control for the experiments. The morphological
changes were monitored at 24 hours and 48 hours and the imaging
was done using an inverted light microscope (Olympus, PA, USA).
For cell cycle analysis, the cells were seeded in 6-well plates at 3 ×
105 cells in complete media and treated with these compounds at
25 μM and 50 μM for 48 hours. Following incubation, the adherent
cells were washed with incomplete media (without FBS), detached
by trypsinization and centrifuged at 800 × g for 5 minutes. The
floating cells in each well were collected from supernatant by
centrifuging at 1000 × g for 10 minutes. The cells were washed
twice with PBS and re-suspended in PBS-chilled methanol solution
(1:9 ratio) at 4 °C overnight. The cells were then washed with PBS,
and treated with 20μg/mL RNase (Sigma-Aldrich, St. Louis, MO,
USA), for 30 minutes at 37 °C. Before analysis, the cells were stained
with propidium iodide (10μg/mL, Sigma-Aldrich, USA) and
incubated at room temperature in dark for 20 minutes. The cells
were finally re-suspended in 200μl of PBS and cell cycle phases of
10000 cells was analyzed by flow cytometer (Becton Dickinson, CA,
USA).The population of cells in each phase was determined by using
the BD Diva software.
MTT cell viability assay was performed as per standard protocols
(MTT Cell Proliferation Assay ATCC® 30-1010K). Briefly, MCF-7,
MDA-MB-231 and HepG2 cells were trypsinized (Trypsin-EDTA,
Invitrogen) and seeded at a density of 10,000 cells per well in
complete media in 96-well flat-bottomed plate, followed by
incubation at 37 °C for 20-24 hours. The cells were then treated
with all synthesized carbohydrate fused pyrano[3,2-c]pyranones (n=
20) along with different substituted 4-hydroxycoumarin precursors
(n = 10) for 48 hours at different concentrations.. Following
incubation, 10µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (Sigma-Merck, USA) at
a
concentration of 5mg/mL in phosphate buffer saline( PBS) was
added to each well and incubated for 2-3 hours until a purple
precipitate is visible. The formazan crystals formed were dissolved
by adding 100 μL DMSO and the plate was incubated for 15 minutes
with gentle shaking. The cell viability was evaluated by measuring
absorbance at 570nm and a reference wavelength of 630nm by
using MicroplateReader (Varioskan, ThermoFishcer Scientific). IC50
values were determined by plotting graph with percent growth
inhibition vs concentrations of compounds. Similar method was
followed for MTT assay against normal cell line (HEK 293).
Cell Proliferation Assay
Wound Healing assay
MCF-7 cells were seeded in 6 well plates at a density of ∼3 X 105
cells per well and allowed to attach for 24 hours. Subsequently the
cells were treated with compounds 12, 13 and 14at 50µM
concentration and further incubated for 48 hours. For trypan blue
staining, 1 part of 0.4% trypan blue solution (Sigma-Merck, USA) is
mixed with 1 part of diluted cell suspension and incubated for 2-4
minutes at room temperature. The total numbers of viable cells
(unstained) were counted by hemocytometer (Rohem, India) at 10
X.
MCF-7 cells were seeded in 6-well plates at a density of ∼4 X 105
cells per well. After 24 hours of seeding, the cells were treated with
25µM and 50 µM concentrations of compounds. The cells were
scratched through the center of the plate using microtip and
imaged at different time points, 24 h and 48 h. The wound healing
was monitored by counting five fields for each treatment and
distance of wound was measured using Catymage software.
Immunofluorescence assay
Clonogenic Assay for cell survival
MCF-7 cells were seeded on glass cover slips in complete DMEM
media and allowed to grow for one cycle. The cells were incubated
with 50 μM of compounds 12, 13 and 14 along with nocodazole
(positive control) and incubated for 48 hours. The cells were
washed with cold 1X PBS and fixed with chilled methanol for 30
minutes. Cells were then blocked in 5% bovine serum albumin (BSA)
in PBS and then immunostained with tubulin α polyclonal rabbit
antibodies (Immunotag) for 1 hour at a dilution of 1:500, followed
by incubation in Alexa 594 anti-rabbit secondary antibody at room
temperature for 1 hour. The cover slips were washed with PBS and
mounted with antifade reagent with DAPI and visualized under
fluorescence microscope (Olympus, USA) at 40X for tubulin staining.
MCF-7 cells were seeded in 6 well plates at a density of ∼3 X 105
cells per well and allowed to attach for 24 hours. The cells were
treated with compounds 12, 13 and 14 at 50 µM concentration and
further incubated for 24 hours. Following incubation, the adherent
cells were washed with incomplete media (without FBS), detached
by trypsinization and centrifuged at 800 × g for 5 minutes. The
pellet was further resuspended in complete DMEM media and
single cell suspensions were obtained by pipetting. The number of
cells in each compound are counted carefully using
a
hemocytometer (Rohem, India) and diluted such that appropriate
cell numbers are seeded into petri dishes (in duplicates). The plating
efficiency was calculated by counting the number of colonies
obtained per plate with respect to number of cells plated. The
percent survival was determined for each test condition with
respect to control.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 13
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DOI: 10.1039/C8NJ01395K
ARTICLE
Journal Name
Uptake Assay
9. (a) M. L.Li, H. Liu, X. H. Xue, L. Li, H. Chunyan, W L. Wang, Z.
Chen, L. Wang, L. Gang. J. Med. Chem.2008, 51, 1432. (b) T.
Sunazuka, M. Handa, K. Nagai, T. Shirahta, Y. Harigaya, K.
Otoguro, KuwajimaI, S. Omura, Org. Lett.,2002, 4, 367.
MCF-7 cells were seeded on glass cover slips in complete DMEM
media and allowed to grow for one cycle. The cells were treated
with 50 μM concentration of compounds 12, 13, 14 and 5 for 0-4
hours. The coverslips were washed with 1X PBS and put on glass
slides. Autofluorescence was observed by fluorescence microscopy
(Olympus, USA) at 60X objective in UV filter.
10. (a) T. Ma, L. Liu, H. Xue, LiL, C. Han, L. Wang, Z. Chen, G. Liu, J.
Med. Chem.2008, 51,1432. (b) H-S. Kang, K-R Kim, E-M.Jun, S-
H.Park, T-S. Lee, J. W.Suh, J-P. Kim, Bioorg. Med. Chem.
Lett.2008, 18, 4047. (c) S. Mo, S. Wang, G. Zhou, Y. Yang, Y. Li,
X.Chem, J. Shi, J. Nat. Prod.2004, 67, 823.
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Conflicts of interest
369.(b) L-W.Jin, D. H. Hua, F-S. Shie,I. MaezawaI, B. Sopher, G.
Authors declare no conflict of interest
M. Martin, J. Mol. Neurosci.2002, 19, 57.
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Acknowledgements
Authors are thankful to the Shiv Nadar University (SNU) and
Jawaharlal Nehru University (JNU), New Delhi for providing required
lab facilities to conduct this research. PK and CN are thankful to
Council of Scientific and Industrial Research (CSIR), India for senior
research fellowships. RS are thankful to the Department of Science
and technology (DST) India for financial support (DST-
EMR/2014/000320). SS is a recipient of the IYBA Award from
Department of Biotechnology (DBT). Funding from UPE II GRANT
and DST PURSE, JNU is acknowledged. SG acknowledges University
Grant Commission (UGC) for postdoctoral research fellowship. SA
acknowledges India Council of Medical Research for providing JRF
fellowship.
13. A. Maucher, M. Kager, E. von, J Cancer Res. Clin. Oncol.1993,
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Supplementary data
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Supplementary data associated with this article can be found, in the
online version, at http://.
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14 | J. Name., 2012, 00, 1-3
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Page 15 of 15
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ01395K
F
Cl
O
OCH3
CH3
OBn
OCH3
OCH3
H3CO
OCH3
OBn
H
OBn
OBn
OBn
OBn
OBn
OBn
H
O
OCH3
OCH3
O
O
H
O
H
H
H
H
O O
O
H
O
O
O
O
O
O
O
H
Cl
BnO
O
O
∗
O
O
O
O
O
O
BnO
O
BnO
BnO
BnO
BnO
OBn
BnO
Cl
Cl
O
OBn
OBn
O
OBn
O
BnO
Cl
OBn
O
OBn
O
CH3
OBn
O
OBn
OBn
O
OBn
OBn
O
OBn
OBn
O
H
OBn
H
O
H
O
O
∗
O
O
H
O
H
O
O
O
O
O
O
∗
BnO
O
∗
∗
O
BnO
O
OBn
O
BnO
O
O
BnO
OBn
O
BnO
BnO
OBn
O
OBn
O
OBn
O
OBn
O
Preliminary screening (n = 20)
Untreated cells
Treated cells
OCH3
OBn
H
O
O
O
BnO
OBn
H
O
Cl
OBn
O
O
O
BnO
OBn
O
CH3
OBn
H
O
O
O
BnO
OBn
O
Carbohydrate fused
pyrano[3,2-c]pyranones