Stereoselective synthesis of natural product inspired carbohydrate fused pyrano[3,2-: C] quinolones as antiproliferative agents

Article abstract of DOI:10.1039/c7ob03186f

Pyrano[3,2-c]quinolone structural motifs are commonly found in natural products with diverse biological activities. As part of a research programme aimed at developing the efficient synthesis of natural product-like small molecules, we designed and developed the microwave assisted, facile stereoselective synthesis of two series of carbohydrate fused pyrano[3,2-c]quinolone derivatives (n = 23) starting from 2-C-formyl galactal and 2-C-formyl glucal, reacting with various 4-hydroxyquinolones in shorter reaction times (15-20 min). The antiproliferative activity of these synthesized pyrano[3,2-c]quinolones was determined against MCF-7 (breast) and HepG2 (liver) cancer cells. The selected library members displayed low micromolar (3.53-9.68 μM) and selective antiproliferative activity. These findings on carbohydrate fused pyrano[3,2-c]quinolone derivatives are expected to provide new leads for anticancer drug discovery.

Full text of DOI:10.1039/c7ob03186f

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Stereoselective synthesis of natural product inspired carbohydrate
fused pyrano[3,2-c]quinolones as antiproliferative agents
a
a
b
b
a,c
Priti Kumari, Chintam Narayana, Shraddha Dubey, Ashish Gupta and Ram Sagar
aDepartment of Chemistry, School of Natural Sciences, Shiv Nadar University (SNU), NH91, Tehsil-Dadri, Gautam
Buddha Nagar, Uttar Pradesh, 201314, India.
b
Department of Life Sciences, School of Natural Sciences, Shiv Nadar University (SNU), NH91, Tehsil-Dadri,
Gautam Buddha Nagar, Uttar Pradesh, 201314, India.
c
Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, 221005, India.
*
E-mail (Corresponding author): ram.sagar@snu.edu.in; ashish.gupta@snu.edu.in,
Abstract
Pyrano[3,2-c]quinolone structural motifs are commonly found in natural products with diverse
biological activities. As part of a research programme aimed at developing efficient synthesis of
natural products like small molecules, we designed and developed microwave assisted, facile
stereoselective synthesis of two series of carbohydrate fused pyrano[3,2-c]quinolone derivatives
(n = 23) starting from 2-C–formyl galactal and 2-C–formyl glucal, reacting with various 4-
hydroxyquinolones in shorter reaction time (15-20 min). Antiproliferative activity of these
synthesized pyrano[3,2-c]quinolones were determined against MCF-7 (breast) and HepG2 (liver)
cancer cells. The selected library members displayed low micromolar (3.53-9.68 µM) and
selective antiproliferative activity. These findings on carbohydrate fused pyrano[3,2-c]quinolone
derivatives are expected to provide new leads for anticancer drug discovery.
Keywords
Pyrano[3,2-c]quinolone, carbohybrids, microwave assisted synthesis, anticancer, MCF-7 and
HepG2.
1

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Introduction
Natural products and their derivatives have been a rich source of new leads in medicinal
chemistry; therefore preparation of natural product like molecules has been of interest to
medicinal chemists and is an important area of research in modern drug discovery.1 The
syntheses of natural products or natural product-like molecules are often difficult due to the
-3
4
structural complexity with many stereogenic centers and intricate ring systems. As part of a
research programme aimed to develop efficient synthesis of natural products like small
molecules, we identified pyrano[3,2-c]quinolone scaffolds as an important structural motif
5
present in many bioactive natural products. This structural motif is broadly represented by
pyranoquinolone alkaloids, which are generally seen in the plant family Rutaceae, exhibiting
6
diverse biological activities.
Pyranoquinolones and fused pyrano[3,2-c]quinolone motifs are widely present in bioactive
natural products, synthetic products as well as in pharmaceutical agents.7 Pyrano[3,2-
8
c]quinolones are primarily known for their anticancer activity, along with antimalarial,
9
-10
11
12
antibacterial,
antiinflammatory, antifungal, properties as well as inhibitors of calcium
signaling, platelet aggregation, and nitric oxide production. Pyrano[3,2-c]quinolone is a core
structural motif present in many alkaloids possessing significant pharmacological and therapeutic
activities (Figure 1). For instance Huajiaosimuline (A), a potent and selective anticancer agent
towards breast cancer, Zanthosimuline (B), an anticancer agent having activity against multidrug
resistant KB-VI cancer cells, were both isolated from Zanthoxylum simulans. In addition, the
natural product Veprisine (C) possesses antimycobacterial activity; Flindersine (D) isolated from
1
3
14
15
16
1
7
1
8
Toddalia asiatica exhibits antimicrobial and antifungal activity; Haplamine (E) isolated from
1
9
Haplophyllum villosum, displays good anticancer activity (Figure 1). These literature reports
strongly support pyrano[3,2-c]quinolone as a privileged structural motif. We envisioned and
designed a new carbohydrate fused pyrano[3,2-c]quinolone molecule (F) as a hybrid structural
motif in anticipation of getting better anticancer candidates. Further when the natural product-
like (privileged) molecules fused with carbohydrates the resulting hybrid molecules possess
many additional pharmacological properties like better aqueous solubility, improved selectivity,
improved cellular uptake, reduced cellular toxicity etc.20
2

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Fig. 1. Naturally occurring and bioactive pyrano[3,2-c]quinolone molecules (A-E): (A)
Huajiaosimuline, selective anticancer agent (breast); (B) Zanthosimuline, anticancer against
multidrug resistant KB-VI cancer cells; (C) Veprisine, antimycobacterial activity; (D)
Flindersine, antimicrobial and antifungal activity; (E) Haplamine, anticancer activity (F) Our
anticancer pyrano[3,2-c]quinolone molecules presented in current work.
Considering the biological relevance of pyrano[3,2-c]quinolone structural motif, several methods
2
1-31
have emerged for their preparation and their derivatives over the past few decades.
These
22
23
include multistep synthesis and one-pot synthesis. Hsung et al developed a formal [3+3]
cycloaddition reaction for the construction of pyrano[3,2-c]quinolones, but it requires 48 h
continuous refluxing in toluene. Few other similar methods for the preparation of
pyranoquinolones skelton are documented which mainly involve reaction between 4-
hydroxyquinolin-2-one and various 1,3-dielectrophiles like α,β-unsaturated aldehyde, β-
ketoester, diethylmalonate, arylidenemalononitrile and arylidenecyanoacetate under thermal
reflux condition. Lee et al reported quite a few similar methods for preparation of
pyranoquinolones using Iodine, InCl3, and Yb(OTf)3. All the above reported conditions
required longer reaction time (12-24 h) and reported yields were moderate. A greener approach
has been reported with few examples, where a similar structural motif can be constructed in 4-6
h in moderate to good yield by refluxing suitable substrates in water.30 Recently Hajra et al
reported synthesis of pyrano[3,2-c]quinolin-2-ones catalyzed by Brønsted acidic ionic liquids
BAILs), which required 6 h continuous refluxing at 110 °C.31 The synthesis of some acetal
fused pyrano[3,2-c]pyran-5(2H)-one derivatives was carried out earlier, which required 3-6 h
continuous heating at 80 °C. We envisioned developing a method to construct a diverse and
new carbohydrate fused pyrano[3,2-c]quinolones library with shorter reaction time and high
yielding under microwave condition.
2
4
2
5
26
27
28
29
23
(
3
2
3

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Herein we have developed microwave assisted facile stereoselective synthesis of carbohydrate
fused pyrano[3,2-c]quinolones (F) derived from the hybrid structure of 4-hydroxyquinolone and
2
-C-formylglycals in good to very good yield in very short reaction time (15-20 min).
Antiproliferative activity of these synthesized fused pyrano[3,2-c]quinolones were determined by
cellular assays against MCF-7, and Hep G2 cancer cell lines. The preliminary screening results
show that selected carbohydrate fused pyrano[3,2-c]quinolones exhibited high growth inhibitory
potencies with IC50 in the range of 3.53-9.68 µM. The microwave assisted efficient synthesis of
these pyrano[3,2-c]quinolones along with their preliminary antiproliferative activity are
presented in this paper.
Results and Discussion
For the efficient synthesis of carbohydrate-fused pyrano[3,2-c]quinolones hybrids, we identified
4
-hydroxyquinolone 1 as substrate which can be coupled with 2-C-formyl galactal 1a33 under
microwave reaction condition and get transformed to the desired pyrano[2,3-c]quinolone hybrid
1 (table 1) through selective C-1,2 nucleophilic addition followed by 6π-electron
1
electrocyclization reaction i.e. formal [3+3] cycloaddition reaction. Although some earlier
reports have been described the synthetic methods for preparation of pyranoquinolone alkaloids,
34
these have limited substrate scope. Later improved synthetic methods have been reported for
2
3-
the synthesis of pyranoquinolone natural products and related biologically active molecules.
3
1,35
However, some of transformations have faced challenges in terms of reaction time, isolated
yield and substrate scope etc. Therefore, there was a need to develop an efficient synthetic
approach to this transformation reaction for better yields and shorter reaction time, which is an
essential aspect in privileged library synthesis. In continuation of our work on efficient synthesis
of natural product like molecules, we have developed microwave assisted efficient synthesis of
carbohydrate fused pyrano[3,2-c]quinolones. A library of twenty two compounds was prepared
using developed protocol and their preliminary anticancer activity was studied.
The initial reaction of 4-hydroxyquinolone 1 with 2-C-formyl galactal 1a was performed under
thermal condition (80 °C) in toluene (3 h) but no product was seen, whereas using pyrrolidine as
organocatalyst furnished stereoselective product 12 after 3 h in poor yield (entry 1, table 1).
When a similar reaction was undertaken in microwave heating condition at 80 °C in the presence
of pyrrolidine, carbohydrate fused pyrano[3,2-c]quinolone 12 was obtained in better isolated
yield (66%) in highly stereoselective and regioselective manner (entry 2, table 1). However in
the absence of pyrrolidine, the above reaction does not go to completion even after 20 min. This
indicates that secondary amine based organocatalyst (pyrrolidine) is required for this
transformation, not only to accelerate the reaction but also to induce the regioselectivity. The
structure of new pyrano[3,2-c]quinolone 12 was unambiguously established by 1D and 2D NMR
(HSQC, COSY and NOE) experiments (SI). The stereochemistry of the newly generated chiral
center was confirmed by a NOE correlation between the H-11a methine proton (new chiral
4

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center) and predefined chiral protons (H-10). This suggested that the H-11a proton is in the same
face as the H-10 proton at the sugar part in compound 12.
Table 1. Reaction optimization using different organocatalysts and solvents
S. No.
Reaction conditionsa
Yield (%)b
1
2
3
4
5
Pyrrolidine, Toluene:AcOH, ∆ 80 °C, 3 h
Pyrrolidine, Toluene:AcOH, µW 80 °C, 15 min
Pyrrolidine, EtOAc:AcOH, µW 80 °C, 15 min
Pyrrolidine, Benzene:AcOH, µW 80 °C, 15 min
Pyrrolidine, Xylene:AcOH, µW 80 °C, 15 min
40
66
71
60
41
6
7
8
9
Pyrrolidine, Ethanol:AcOH, µW 80 °C, 20 min
Pyrrolidine, Water:AcOH, µW 80 °C, 20 min
Pyrrolidine, PEG-200:AcOH, µW 80 °C, 20 min
Piperidine, Toluene:AcOH, µW 80 °C, 15 min
40
NR
NR
35
1
1
1
1
1
1
1
0
1
2
3
4
5
6
Piperidine, EtOAc:AcOH, µW 80 °C, 15 min
Piperidine, Benzene:AcOH, µW 80 °C, 15 min
Piperidine, Xylene:AcOH, µW 80 °C, 15 min
Proline, Toluene:AcOH, µW 80 °C, 15 min
Proline, EtOAc:AcOH, µW 80 °C, 15 min
Proline, Benzene:AcOH, µW 80 °C, 15 min
Proline, Xylene:AcOH, µW 80 °C, 15 min
52
41
42
50
55
45
42
68
c
1
7
Pyrrolidine, EtOAc:AcOH, µW 80 °C, 15 min
a
b
0
.5 equiv of organocatalyst was used in each cases, Isolated yield.diastereomeric ratio (dr) was determined by
crude H NMR ; dr (α:β ˃99:1), 0.1 equiv of organocatalyst was used in this cases. NR = No reaction,
1
c
To optimize the reaction condition and see the effect of reagent on stereoselectivity, we planned
and tested a series of reaction conditions using various organocatalysts and solvent systems
(
table 1). To enhance the reactivity of 2-C-formyl galactal 1a various secondary amine-based
organocatalysts were tested. After screening a set of reaction conditions by using different
organocatalysts under microwave conditions, it was found that the condensation of 4-
hydroxyquinolone 1 with 2-C-formyl galactal 1a in the presence of pyrrolidine (0.5 equiv) in
ethyl acetate at 80 °C affords 12 in 71% isolated yield with excellent diastereoselectivity (entry
3
, table 1). When we carried out the same transformation in the presence of 0.1 equiv of
pyrrolidine under similar conditions, compound 12 was obtained in 68% isolated yield (entry 17,
table 1); we obtained the identical product 12 without the loss of stereoselectivity, when
5

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piperidine and proline were used as organocatalysts. The isolated yield varied with different
organocatalyst without any significant difference in the reaction time (Table 1, entries 9-16).
To study solvent effects for this transformation under microwave condition, we screened 1%
acetic acid in toluene, ethyl acetate, benzene, xylene, ethanol, water and poly(ethylene glycol)-
200 (PEG-200) in the presence of pyrrolidine (Table 1, entries 2–8) keeping other reaction
parameters constant. In comparison, 1% acetic acid in ethyl acetate was found to be superior
over toluene, benzene, xylene and ethanol. No reaction was observed when water and PEG-200
used as solvent on these substrates (Table 1, entry 7-8). When the same reaction was carried out
using 1% citric acid or tartaric acid, as an alternative of acetic acid, in ethyl acetate as solvent
only 10-15% conversion was noticed even after prolonged (25 min) heating under microwave
conditions. The four solvent systems viz. ethyl acetate, toluene, benzene and xylene were
screened with other organocatalysts (piperidine and proline) and result showed that, indeed 1%
acetic acid in ethyl acetate was the best solvent among others (Table 1, entry 9-16).
After having optimized reaction conditions, we were interested in investigating the scope of this
formal [3+3] cycloaddition reaction with different substituted 4-hydroxyquinolones. Therefore,
various 4-hydroxyquinolones 1-11 were freshly prepared in laboratory starting from the
commercially available corresponding substituted anilines, reacting with Meldrum’s acid
followed by Eaton’s reagent (Scheme S1, SI).36 The diverse 4-hydroxyquinolone derivatives
were then treated with with 2-C formyl galactal 1a and 2-C formyl glucal 1b under optimized
reaction conditions (µW, 80 °C, EtOAc:AcOH, pyrrolidine) in different sets of reactions. As
shown in scheme 1, 2-C formyl galactal 1a was successfully coupled with various substituted 4-
hydroxyquinolones 1-11 to afford the respective carbohydrate fused pyrano[3,2-c]quinolones 12-
2
2 in good to very good yields with excellent stereoselectivity (dr ˃ 99:1). The electron donating
1
2
3
4
and electron withdrawing groups substituents at the R , R , R and R positions on 4-
hydroxyquinolones significantly affected neither the yield nor the reaction completion time.
However unsubstituted 4-hydroxyquinolone afforded good yield (71%) as compare to other
mono- and di-substituted 4-hydroxyquinolones (Scheme 1).
Scheme 1. Synthesis of 2-C formyl galactal fused pyrano[3,2-c]quinolones 12-22.
6

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OPh
OBn
OBn
H
OBn
H
H
O
O
O
O
O
O
F
NH
NH
NH
BnO
BnO
BnO
OBn
O
OBn
O
OBn
O
12 (71%)
13 (68%)
14 (69%)
CF3
OBn
OBn
OBn
H
O
H
H
O
O
O
O
O
NO2
BnO
OCF3
NH
NH
NH
BnO
BnO
OBn
O
OBn
O
OBn
O
15 (65%)
16 (63%)
17 (66%)
The mechanism of this formal [3+3] cycloaddition reaction can be proposed as shown in Figure
. Pyrrolidine attacks the 2-C-formyl galactal 1a to form an intermediate I, which undergoes
3
dehydration to form the iminium ion intermediate II. The resulting iminium ion (II) is a better
electrophile than 1a and undergoes selective nucleophilic C-1,2-addition with 4-
hydroxyquinolone to furnish III. Intermediate III rearranges to IV, which transforms into 1-
oxatriene intermediate V through β-elimination. Finally, V undergoes 6π-electron
electrocyclization to afford the corresponding pyrano[3,2-c]quinolone with excellent
diastereoselectivity. There is evidence for reversibility of 6π-electron electrocyclization of ring
closure of 1-oxatrienes reported in the literature.37 Therefore, we can postulate that
thermodynamically more stable diastereoisomers are formed and the final step equilibrium is
shifted toward the product side.
7

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O
O
H
O
PO
PO
PO
PO
PO
H O
2
N
OH
O
N
H
PO
OH
N
CH COO
OP
H
OP
H
H O
OP
H
3
2
1
a
I
II
OP
OP
OP
OP
OP
N
O
H
OP
OP
OP
OP
OP
OP
O
H
O
O
O
O
O
OH
O
OP
N
H
N
N
H
O
6
-electron
-elimination
N
H
O
electrocycli-
zation
N
H
O
N
H
IV
O
N
H
III
1
-Oxatriene (V)
Fig 2. Proposed reaction mechanism.
Further when 4-hydroxyquinolone 1 was reacted with 2-C formyl glucal 1b under optimized
microwave reaction conditions (µW, 80 °C, EtOAc:AcOH, pyrrolidine) it afforded unexpectedly,
an epimeric mixture (1:1) of carbohydrate fused pyrano[3,2-c]quinolone 23a/b in good yield
(70%). The stereoselectivity in this case could not get restored even using different
organocatalysts and different reaction conditions. As mentioned in the proposed mechanism that,
this type of reaction proceeds through a 1-oxatriene intermediate followed by 6π-electron
electrocyclization and reversibility of this cyclization was well studied and reported in the
literature.37 Based on the literature we can presume that fused pyrano[3,2-c]quinolone 23a is
equilibrating with 23b through a common 1-oxatriene intermediate in reversible fashion and
furnishing a mixture of epimers 23a/b. We tried to isolate one stereoisomer 23a through a
column but it again becomes a mixture of epimers 23a/b (NMR and TLC). This further
confirmed that both isomers equilibrated in reversible fashion. As shown in scheme 2, 2-C
formyl glucal 1b was successfully coupled with various other substituted 4-hydroxyquinolones
1
-11 to afford the respective pyrano[3,2-c]quinolones 23a/b-33a/b in acceptable to good yields
1
2
3
4
as epimeric mixture (dr~1:1). Similarly different substituents here at the R , R , R and R
position on 4-hydroxyquinolone remarkable affects neither the yield nor the reaction completion
time when coupled with 2-C formyl glucal 1b (scheme 2). Thus a library of twenty two different
diverse carbohydrate fused pyrano[3,2-c]quinolone derivatives were prepared and preliminary
anticancer activity was examined in-vitro through cellular assays using MCF-7 and HepG2
cancer cell lines and the bioactivity results are discussed below.
8

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R2
R1
R3
O
R1 OH
OBn
BnO
BnO
R2
H
Pyrrolidine,
EtOAc:AcOH
µW, 80 °C
O
O
H
R4
+
R3
NH
OBn O
N
O
BnO
H
R4
OBn
O
1
b
1-11
23a/b-33a/b
Scheme 2. Synthesis of 2-C formyl glucal fused pyrano[3,2-c]quinolones 23a/b-33a/b.
When N-protected 4-hydroxyquinolone 34 was reacted with 2-C-formyl galactal 1a under
optimized reaction conditions it furnished carbohydrate fused N-methyl pyrano[3,2-c]quinolones
3
5 in 71% isolated yield (Scheme 3).
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OH
O
OBn
BnO
BnO
H
Pyrrolidine,
EtOAc:AcOH
µW, 80 °C,
O
O
H
+
N
N
O
OBn O
20 min
BnO
CH3
CH3
34
OBn
O
1
a
35 (71%)
Scheme 3. Synthesis of N-methyl pyrano[3,2-c]quinolones 35.
Antiproliferative activity of all synthesized carbohydrate-fused pyrano[3,2-c]quinolones 12-22
and 23a/b-33a/b (n = 22) was investigated at 25 µM concentration by cell viability assays
against MCF-7 and HepG2 cell lines using doxorubicin (25 µM) as positive control (Table 2).
The results showed moderate activity with all tested compounds in MCF-7 (breast cancer cells).
The better activity with compounds 19, 24a/b, 30a/b and 33a/b was observed with 54.8%,
4
7.1%, 18.8% and 42.3% cell viability respectively. However anticancer activity with
compounds 15, 30a/b and 33a/b against HepG2 cells was observed with cell viability of 37.6%,
9.8% and 36.7% respectively (Table 2). These active compounds were then further tested at
4
different lower concentration 1 µM, 5 µM, 10 µM and 20 µM respectively (SI) against both cell
lines in order to find out IC50 of the active compounds.
Table 2. Anticancer screening results
Compound a
Cell Viability (%) ±SD
MCF-7
HepG2
1
1
1
1
1
1
1
1
2
2
2
2
3
4
5
6
7
8
9
0
1
2
80.4 ±1.13
85.3 ±1.06
77.1 ±1.62
77.5 ±2.82
77.0 ±1.41
70.2 ±1.06
86.1 ±2.12
54.8 ±2.61
97.3 ±0.91
77.4 ±1.20
70.9 ±1.90
84.3 ±5.86
47.1 ±2.96
82.1 ±3.53
95.4 ±4.87
96.1 ±3.60
93.6±2.40
81.9 ±3.81
99.4±3.81
37.6 ±2.40
89.9 ±3.25
91.3 ±2.19
92.7 ±1.62
84.6±2.47
97.8±3.04
92.5±2.68
94.2±1.34
94.4 ±2.26
93.8 ±2.33
97.6 ±3.39
82.0 ±1.34
96.9 ±2.89
2
2
2
2
2
3a/b
4a/b
5a/b
6a/b
7a/b
1
0

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s
2
2
3
3
3
3
8a/b
9a/b
0a/b
1a/b
2a/b
3a/b
99.5 ±2.12
98.9 ±1.55
18.8 ±1.83
76.8±2.96
73.0±2.26
42.3±1.62
15.3 ±0.77
80.6 ±3.32
88.6 ±1.90
49.8 ±1.76
89.4 ±3.25
74.4 ±3.32
36.7 ±3.60
15.2 ±0.84
Doxorubicin
a
2
5 µM concentration of each compounds used for MCF-7 and HepG2 cells.
From the study, it has been found that carbohydrate-fused pyrano[3,2-c]quinolone compounds 19
and 24a/b exhibited growth inhibitory potencies selectively against MCF-7 cell, whereas
compound 15 shown inhibitory potency selective against HepG2 cells. The compounds 30a/b
and 33a/b show high growth inhibition against MCF-7 cell line with half maximal inhibitory
concentration (IC50) values 9.68 and 5.33 µM respectively, whereas IC50 for compound 33a/b
against Hep G2 was 3.53 µM (Fig 3).
Fig. 3. Antiproliferative activity of carbohydrate fused pyrano[3,2-c]quinolones. To calculate half
maximal inhibitory concentration (IC50) of compounds 30a/b and 33a/b, MCF-7 or HepG2 cells were
treated with different concentrations (1, 5, 10, 20 and 25 µM) for 48 h. The percent cell viability was
determined by cell viability assay. IC50 values were determined by plotting values of percent cell viability
against concentration of each of these compounds. (a-b) IC50 values for compounds 30a/b and 33a/b
against MCF-7 cells were 9.68, and 5.33 µM respectively. (c) IC50 values for compounds 33a/b against
HepG2 cells was 3.53 µM. The experiments were performed in triplicates, n=3 and ± SD value was
calculated for each data point.
Conclusion
In summary, we have developed a new and efficient method for the synthesis of a natural product
inspired library of carbohydrate-fused pyrano[3,2-c]quinolones hybrids. The method involves
microwave assisted facile synthesis of carbohydrate-fused pyrano[3,2-c]quinolones, starting
from freshly prepared diverse 4-hydroxyquinolones and 2-C-formyl glycals in good to very good
yields. The method is endowed with several unique merits including simple reaction conditions,
shorter reaction time, broad substrate scope and no metal catalysis. The method has successfully
1
1

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applied to diverse substituted 4-hydroxyquinolones bearing electron donating and withdrawing
groups and a library of twenty three carbohydrate-fused pyrano[3,2-c]quinolone hybrid
compounds were prepared. The carbohydrate part or free NH in these hybrid molecules can also
be further modified and diversified to enhance their biological activities during the lead
optimization process. We have shown that N-methyl carbohydrate-fused pyrano[3,2-c]quinolone
hybrid can be obtained using this protocol. Subsequently, we demonstrated that the selected
library members displayed low micromolar (3.53-9.68 µM) and selective antiproliferative
activity.
Experimental section
General Experimental Methods.
All experiments were performed in an oven-dried apparatus and in anhydrous solvents using
CEM microwave synthesizer. High resolution mass spectra were obtained from a qudrapole/TOF
mass spectrometer with an ESI source. Solvents were distilled by standard distillation procedure
1
13
and stored in 4Å and 3Å molecular sieves. H (400 MHz) and C (100 MHz) NMR spectra were
1
13
recorded with a Bruker AMX-400 MHz instrument. H and C chemical shifts are referenced to
1
13
the solvents residual signals (CDCl
3
: δ 7.26 for H NMR and δ 77.16 for C NMR) and reported
in parts per million (ppm) at 25 °C, the peak at δ 1.56 corresponds to water (O-H) in CDCl
3
.
Coupling constants are expressed in hertz (Hz). Reactions were monitored by thin-layer
chromatography carried out on 0.25 mm E. Merck silica gel plates (60F-254), Spots were
visualized by phosphomolybdic acid and 10% H
uncorrected and recorded with an electrothermal melting point apparatus. 4-hydroxyquinolones
1-11 and 34), 2-C-formyl galactal 1a and 2-C-formyl glucal 1b were freshly prepared in
2
SO
4
in ethanol. Melting points (m.p.).were
(
laboratory as per reported procedures. Organocatalysts (L-prolin, pyrrolidine and piperidine) and
different anilines used for preparing 4-hydroxyquinolones were purchased from Sigma-Aldrich.
General Experimental Procedure for reaction optimization: To a 10 mL microwave vial
3
,4,6-tri-O-benzyl-2-C-formyl D-galactal 1a (100 mg, 0.225 mmol) and 4-hydroxyquinolone 1
(43.51 mg, 0.270 mmol) were added in different combinations of organocatalysts (Pyrrolidine,
Pipridine and L-proline, 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 15 min under microwave
condition. The completion of reaction was monitored by TLC (R
Hexane, v/v). After completion, the reaction was quenched by adding 5 mL of NaHCO
and the reaction mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic
layer was dried over anhydrous Na SO and concentrated in vacuum to get the crude product.
f
= 0.56, 3:7 = Ethylacetate:
3
solution
2
4
The crude product was purified by flash column chromatography, which furnished the designed
product 12 in good yield (71%) as an amorphous solid. Different organocatalysts and solvent
combinations furnished the same product 12 in different yield percentages (Table 1).
1
2

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General experimental procedure for synthesis of compounds 12-22, 35 and 23a/b-33a/b.
A mixture of 3,4,6-tri-O-benzyl-2-C-formyl D-galactal 1a (200 mg, 0.450 mmol), various 4-
hydroxyquinolones 1-11 (0.540 mmol) and pyrrolidine (8 mg, 0.225 mmol) was heated in a
microwave vial using 2 mL of EtOAc:AcOH (1:0.01) at 80 °C (100W) for 15 min under
microwave condition. The progress of reaction was monitored by TLC (3:7 = Ethylacetate:
Hexane, v/v). After completion, the reaction mixture was quenched by adding 5 mL of NaHCO
solution and the reaction mixture was extracted with ethyl acetate (3 × 5 mL). The combined
organic layer was dried over anhydrous Na SO and concentrated in vacuum to get the crude
3
2
4
product. The crude product was purified by flash column chromatography to obtain the pure
products 12-22 in good to very good yield (Scheme 1). A similar reaction protocol was adopted
for the synthesis of compounds 23a/b-33a/b starting from 3,4,6-tri-O-benzyl-2-C-formyl D-
glucal 1b and respective 4-hydroxyquinolone 1-11 (Scheme 2). The reaction time taken in the
case of compounds 22 and 33a/b was 20 min. Compounds 23a/b-33a/b were isolated as an
1
inseparable mixture (1:1) of two epimers. Therefore in H NMR spectra of these compounds,
protons were integrated and reported as a doubled number.
(
8R,9R,10R)8,9-bis(benzyloxy)10((benzyloxy)methyl)8,9,10,11a-tetrahydropyrano[3′,2′,5,6]
pyrano[3,2-c]quinolin-6(5H)–one (12). Purple amorphous solid, m.p.: 190 °C; yield 71%, R =
f
1
0
.56 (3:7 = Ethylacetate: Hexane, v/v), H NMR (CDCl
3
, 400 MHz) δ 10.58 (brs, 1H, NH), 7.95
(
d, J = 8.0 Hz, 1H, H-1), 7.47-7.19 (m, 18H, ArH), 7.17 (s, 1H, H-7), 6.10 (s, 1H, H-11a), 4.98
d, J = 11.6 Hz, 1H, CH Ph), 4.88 (d, J = 12.4 Hz, 1H, CH Ph), 4.71 (d, J = 12.0 Hz, 1H,
Ph), 4.65 (d, J = 12.4 Hz, 1H, CH Ph), 4.47 (d, J = 12.0 Hz, 1H, CH Ph), 4.40 (d, J = 11.2
Hz, 1H, CH Ph), 4.17 (brs, 1H, H-8), 4.00 (brs, 1H, H-9), 3.87 (t, J = 6.8 Hz, 1H, H-10), 3.62
dd, J = 9.2 Hz and J = 6.0 Hz, 1H, H-13a), 3.52 (dd, J = 9.6 Hz and J = 6.4 Hz, 1H, 13b), C
NMR (CDCl3, 100 MHz) δ 162.3, 154.8, 138.3, 137.8, 137.7, 137.4, 130.9, 128.6, 128.5, 128.4,
(
2
2
CH
2
2
2
2
1
3
(
1
1
28.1, 127.9, 127.8, 127.6, 127.4, 125.8, 122.5, 122.3, 115.8, 114.1, 112.9 (C-7), 104.3, 97.0 (C-
1a), 79.7 (C-8), 76.4 (C-9), 75.0 (C-10), 74.2, 73.5, 71.7 (3 × CH Ph), 68.8 (C-13),
2
+
HRMS(ESI), calcd, m/z C37
H33NO
6
, [M+H] 588.2381; Found: 588.2405.
(
8R,9R,10R)-8,9-bis(benzyloxy)–10-((benzyloxy)methyl)–4-fluoro-8,9,10,11a-tetrahydro-
pyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (13) Yellow amorphous solid, m.p.: 140 °C;
1
yield 68%, R
f
= 0.64 (3:7 = Ethylacetate: Hexane, v/v), H NMR (CDCl
3
, 400 MHz) δ 8.79 (s,
1
H), 7.72 (d, J = 8.4 Hz, 1H, H-1), 7.39-7.09 (m, 17H, ArH), 7.19 (s, 1H, H-7), 6.09 (s, 1H, H-
1a), 4.95 (d, J = 12.0 Hz, 1H, CH Ph,), 4.85 (d, J = 12.4 Hz, 1H, CH Ph), 4.69 (d, J = 12.0 Hz,
1
2
2
1
3

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1
1
8
H, CH
1.6 Hz, 1H, CH
.8 Hz and J = 6.4 Hz, ,1H, H-13a), 3.52 (dd, J = 9.6 Hz and J = 6.4Hz, 1H, C-13b), C NMR
2
Ph), 4.62 (d, J = 12.4 Hz, 1H, CH
2
Ph), 4.47 (d, J = 11.6 Hz, 1H, CH
2
Ph), 4.42 (d, J =
2
Ph), 4.16 (brs, 1H, H-8), 4.00 (brs, 1H, H-9), 3.85 (m, 1H, H-10), 3.62(dd, J =
1
3
(CDCl3, 100 MHz) δ 160.7, 146.5, 140.9, 140.0, 138.2, 137.6, 128.6, 128.6, 128.5, 128.4, 128.2,
1
28.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 126.9, 126.7, 126.7, 122.0, 120.9, 118.3, 115.6,
14.7, 112.6 (C-7), 105.4, 96.9 (C-11a), 79.5 (C-8), 76.4 (C-9), 75.1 (C-10), 74.3, 73.6, 71.7 (3 ×
1
CH
2
Ph), 68.8 (C-13). HRMS(ESI), calcd, m/z C37
H
32FNO
, [M+H]+ 606.2286; Found:
6
6
06.2277.
(
8R,9R,10R)-8,9-bis(benzyloxy)–10-((benzyloxy)methyl)–3–phenoxy-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (14). Off white solid, m.p.: 172
1
°
C; yield 69%, R
f
= 0.64 (3:7 = Ethylacetate: Hexane, v/v), H NMR (CDCl3, 400 MHz) δ 10.18
(
s, 1H, NH), 7.88 (d, J = 8.4 Hz, 1H, H-1), 7.39-7.05 (m, 20H, ArH), 6.86-6.76 (m, 3H, ArH),
.07 (s, 1H, H-11a), 4.96 (d, J = 12.4 Hz, 1H, CH Ph), 4.86 (d, J = 12.0 Hz, 1H, CH Ph), 4.70
d, J = 11.2 Hz, 1H, CH Ph), 4.63 (d, J = 12.4 Hz, 1H, CH Ph), 4.46 (d, J = 12.0 Hz, 1H,
Ph), 4.39 (d, J = 11.2 Hz, 1H, CH Ph), 4.15 (brs, 1H, H-8), 3.98 (brs, 1H, H-9), 3.84 (t, J =
6
2
2
(
2
2
CH
2
2
4
6
1
1
7
6
.8 Hz,1H, H-10), 3.61 (dd, J = 7.8 Hz and J = 6.0 Hz, 1H, H-13a), 3.51 (dd, J = 8.4 Hz and J =
13
.4 Hz, 1H, H-13b); C NMR (CDCl3, 100 MHz,) δ 162.3, 160.1, 154.8, 139.0, 138.3, 137.8,
29.9, 128.6, 128.4, 128.1, 127.9, 127.8, 127.6, 127.4, 125.3, 125.0, 124.8, 124.3, 119.9, 119.1,
18.0, 113.4, 112.9 (C-7), 109.6, 103.8, 102.9, 97.0 (C-11a), 79.6 (C-8), 75.9 (C-9), 75.0 (C-10),
4.2, 73.5, 71.7 (3× CH
2
Ph), 68.8 (C-13), HRMS(ESI), calcd, m/z C43
H
37NO
, [M+H]+
7
80.2643; Found: 680.2634.
(
8R,9R,10R)-8,9-bis(benzyloxy–1-((benzyloxy)methyl)–4–nitro-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (15) Orange amorphous solid,
1
m.p.: 180 °C; yield 65%, R
f
= 0.58 (3:7 = Ethylacetate: Hexane, v/v), H NMR (CDCl3, 400
MHz) δ 11.12 (brs, 1H, NH), 8.47 (d, J = 8.4 Hz, 1H, H-3), 8.32 (d, J = 7.6 Hz, 1H, H-1), 7.40-
7
4
.27 (m, 16H, ArH), 7.17 (s, 1H, H-7), 6.11 (s, 1H, H-11a), 4.93 (d, J = 12.0 Hz, 1H, CH
.85 (d, J = 12.0 Hz, 1H, CH Ph), 4.68 (d, J = 11.2 Hz, 1H, CH Ph), 4.63 (d, J = 12.4 Hz, 1H,
Ph), 4.47 (d, J = 12.0 Hz ,1H, CH Ph), 4.40 (d, J = 12.0 Hz, 1H, CH Ph), 4.18 (brs, 1H, C-
), 4.00 (d, J = 2.0 Hz, 1H, H-9), 3.87 (m, 1H, H-10), 3.61 (dd, J = 9.6 Hz and J = 6.0 Hz, 1H,
2
Ph),
2
2
CH
2
2
2
8
13
H-13a), 3.49 (dd, J = 9.6 Hz and J = 6.4 Hz, 1H, H-13b), C NMR (CDCl3, 100 MHz) δ 160.1,
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4

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1
1
9
53.0, 140.0, 138.0, 137.6, 137.5, 132.9, 130.5, 128.6, 128.5, 128.4, 127.2, 128.1, 128.0, 127.8,
27.7, 127.6, 127.4, 126.9, 121.0, 116.9, 112.0 (C-7), 105.7, 96.9 (C-11a), 79.5 (C-8), 76.58 (C-
), 75.24 (C-10), 74.4, 73.6, 71.8 (3 × CH Ph), 68.7 (C-13), HRMS(ESI), calcd, m/z
2
+
C
37
H
32
N
2
O
8
, [M+H] 633.2231; Found: 633.2224.
(
8R,9R,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)–4-triflouromethoxy-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)–one (16) Yellow amorphous solid,
1
m.p.: 195 °C; yield 63%, R
f
= 0.71 (3:7 = Ethylacetate: Hexane, v/v), H NMR (CDCl3, 400
MHz) δ 8.92 (s, 1H), 7.88 (d, J = 8.4 Hz, 1H, H-1), 7.39-7.26 (m, 16H, ArH), 7.18 (s, 1H, H-7),
.09 (s, 1H, H-11a), 4.95 (d, J = 12.0 Hz, 1H, CH Ph), 4.85 (d, J = 12.4 Hz, 1H, CH Ph), 4.68
d, J = 11.6 Hz, 1H, CH Ph), 4.62 (d, J = 12.4 Hz, 1H, CH Ph), 4.46 (d, J =11.6 Hz, 1H,
Ph), 4.40 (d, J = 12.0Hz 1H, CH Ph), 4.16 (brs, 1H, H-8), 3.99 (brs, 1H, H-9), 3.86 (t, J =
6
2
2
(
2
2
CH
2
2
6
6
1
1
7
6
.4 Hz, 1H, H-10), 3.61 (dd, J = 9.2 Hz and J = 6.4 Hz, 1H, H-13a), 3.50 (dd, J = 9.6 Hz and J =
.4 Hz, 1H, H-13b), 13C NMR (CDCl3, 100 MHz) δ 161.0 (C-6), 153.8, 138.2, 137.6, 136.1,
29.9, 128.3, 128.5, 128.4, 128.2, 127.9, 127.8, 127.8, 127.6, 127.3, 126.9, 121.7, 121.6, 121.1,
19.3, 116.1, 112.6 (C-7), 106.5, 96.9 (C-11a), 79.6 (C-8), 76.5 (C-9), 75.1 (C-10), 74.3, 73.6,
+
1.7 (3 × CH
72.2204.
2
Ph), 68.8 (C-13), HRMS(ESI), calcd, m/z C38
H
32
F
3
NO
7
, [M+H] 672.2204;Found:
(
8R,9R,10R)-8,9-bis(benzyloxy)–10-((benzyloxy)methyl)-3-triflouromethyl-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)–one (17). Yellow amorphous solid,
1
m.p.: 139 °C; yield 66%, R
f
= 0.41 (3:7 = Ethylacetate: Hexane, v/v), H NMR (CDCl
3
,
4
1
1
00MHz), δ 10.95 (s, 1H, NH), 8.04 (d, J = 7.6 Hz, 1H, H-1), 7.50-7.29 (m, 18H, ArH), 6.13 (s,
H, H-11a), 4.97 (d, J = 10.4 Hz, 1H, CH Ph), 4.87 (d, J = 12.4 Hz, 1H, CH Ph), 4.71 (d, J =
2.0 Hz, 1H, CH Ph), 4.65 (d, J = 12.4 Hz, 1H, CH Ph), 4.47 (d, J = 11.6 Hz, 1H, CH Ph), 4.41
Ph), 4.19 (brs, 1H, H-8), 4.02 (brs, 1H, H-9), 3.88 (m, 1H, H-10), 3.64
2
2
2
2
2
(
(
d, J = 11.2 Hz, 1H, CH
dd, J = 9.6 Hz and J = 5.2 Hz, 1H, H-13a), 3.52 (dd, J = 9.0 Hz and J =5.2 Hz, 1H, H-13b), C
, 100 MHz) δ 162.4, 158.4, 153.8, 138.1, 138.0,137.6, 137.4, 136.8, 128.6, 128.4,
28.2, 128.0, 127.7, 127.4, 126.1, 124.9, 123.5, 118.7, 112.4 (C-7), 106.0, 96.9 (C-11a), 89.6,
2
1
3
NMR (CDCl
3
1
7
9.5 (C-8), 76.4 (C-9), 75.1 (C-10), 74.3, 73.6, 71.8 (3 × CH Ph), 68.7 (C-13), HRMS(ESI),
2
+
calcd, m/z C38
H
32
F
3
NO
6
, [M+H] 656.2254; Found: 656.2276.
1
5

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(
8R,9S,10R)-8,9-bis(benzyloxy)–10-((benzyloxy)methyl)–4–isopropyl-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3, 2-c]quinolin-6(5H)-one (18). Amorphous solid, m.p.:
1
1
8
1
75 °C; yield 65%, R
f
= 0.64 (3:7 = Ethylacetate: Hexane, v/v), H NMR (CDCl
3
, 400 MHz,) δ
.81 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H, H-1), 7.39-7.19 (m, 16H, ArH), 7.18 (s, 1H, H-7), 6.09 (s,
H, H-11a), 4.96 (d, J =11.6 Hz, 1H, CH Ph), 4.86 (d, J = 12.4 Hz, 1H, CH Ph), 4.69 (d, J =11.6
Ph), 4.63 (d, J = 12.4 Hz , 1H, CH Ph), 4.46 (d, J = 12.0 Hz, 1H, CH Ph), 4.40 (d, J
11.6 Hz, 1H, CH Ph), 4.16 (brs, 1H, H-8), 3.99 (brs, 1H, H-9), 3.85 (t, J = 6.0 Hz, 1H, H-10),
.62 (dd, J = 8.9 Hz and J = 6.4 Hz, 1H, H-13a), 3.52 (dd, J = 8.9 Hz and J = 6.4 Hz, 1H, H-
2
2
Hz, 1H, CH
2
2
2
=
2
3
1
1
1
7
1
3
3b), 3.16 (m, 1H, CH of isopropyl), 1.33 (d, 6H, Me ), C NMR (CDCl3, 100 MHz,) δ 161.5,
2
55.1, 138.3, 137.8, 134.5, 133.3, 128.5, 128.4, 128.1, 127.9, 127.8, 127.6, 127.3, 127.2, 125.8,
22.2, 120.5, 114.3, 112.8 (C-7), 104.0, 97.0 (C-11a), 79.7 (C-8), 76.4 (C-9), 75.0 (C-10), 74.2,
3.5, 71.7 (3×CH
2
Ph), 68.8 (C-4), 26.8 (CH of-CHMe ), 22.8 (Me), 22.7 (Me), HRMS(ESI),
2
+
calcd, m/z C40
H39NO
6
, [M+K] 668.2409;Found: 668.2422.
(
8R,9R,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-3-chloro-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (19). Yellow amorphous solid,
1
m.p.: 185 °C; yield 64%, R
f
= 0.60 (3:7 = Ethylacetate: Hexane, v/v), H NMR (CDCl
3
, 400
MHz) δ 10.02 (brs, 1H, NH), 7.94 (d, J = 8 Hz,1H, H-1), 7.41-7.16 (m, 18H, ArH), 6.10 (s, 1H,
H-11a), 4.97 (d, J = 12.0 Hz, 1H, CH Ph), 4.87 (d, J = 12.4 Hz, 1H, CH Ph), 4.71 (d, J = 12.0
Hz, 1H, CH Ph), 4.64 (d, J = 12.4 Hz, 1H, CH Ph), 4.46 (d, J = 11.6 Hz, 1H, CH Ph, 4.40 (d, J =
1.6 Hz, 1H, CH Ph), 4.17 (brs, 1H, H-8), 4.00 (brs, 1H, H-9), 3.87-3.84 (m, 1H, H-10), 3.62
dd, J = 9.6 Hz and J = 5.6 Hz, 1H, H-13a), 3.52 (dd, J = 9.6 Hz and J = 6.4 Hz, 1H, H-13b), C
NMR (CDCl , 100 MHz) δ 162.2, 154.8, 138.3, 137.8, 137.7, 137.3, 130.9, 128.6, 128.4, 128.2,
28.0, 129.9, 127.8, 127.7, 127.6, 127.4, 125.8, 122.6, 122.4, 115.8, 114.1, 112.9 (C-7), 104.3,
2
2
2
2
2
1
2
1
3
(
3
1
9
7.0 (C-11a), 79.6 (C-8), 76.4 (C-9), 75.0 (C-10), 74.2, 73.5, 71.7, 68.8 (C-13), HRMS(ESI),
+
calcd, m/z C37
H
32ClNO
6
, [M+Na] 644.187; Found: 644.1916.
(
8R,9R,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-3-bromo-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (20). Off white amorphous solid,
1
m.p.: 150 °C; yield 63%, R
f
= 0.45 (3:7 = Ethylacetate: Hexane, v/v), H NMR (CDCl3,400
MHz) δ 9.71 (brs, 1H), 7.95 (d, J = 7.6 Hz, 1H, H-1), 7.45-7.18 (m, 17H, ArH), 7.17 (s, 1H, H-
), 6.10 (s, 1H, H-11a), 4.96 (d, J = 12.0 Hz, 1H, CH Ph), 4.87 (d, J = 12.4 Hz, 1H, CH Ph),
7
2
2
1
6

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4
.70 (d, J = 12.0 Hz, 1H, CH
CH
H-10), 3.62 (dd, J = 9.6 Hz and J = 5.6 Hz, 1H, H-13a), 3.51 (dd, J = 9.6 Hz and J = 6.7 Hz, 1H,
2
Ph), 4.64 (d, J = 12.4 Hz,1H, CH
2
Ph), 4.46 (d, J = 12.0 Hz, 1H,
2
Ph), 4.40 (d, J = 11.6 Hz, 1H, CH Ph), 4.17 (brs, 1H, H-8), 4.00 (brs, 1H, H-9), 3.86 (m, 1H,
2
1
3
H-13b), C NMR (CDCl
3
, 100 MHz) δ 162.2, 154.8, 138.3, 137.8, 137.7, 137.3, 130.9, 128.6,
1
28.4, 128.2, 128.0, 129.9, 127.8, 127.7, 127.6, 127.4, 125.8, 122.6, 122.4, 115.8, 114.1, 112.9
(
C-7), 104.3, 97.0 (C-11a), 79.6 (C-8), 76.4 (C-9), 75.0 (C-10), 74.2, 73.5, 71.7 (3 × CH Ph),
2
+
6
8.8 (C-13), HRMS(ESI), calcd, m/z C37
H
32BrNO
6
, [M+H] 666.1486; Found: 666.1500.
(
8R,9R,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-4-triflouromethyl-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (21). Brown amorphous solid,
1
m.p.: 130 °C; yield 67%, R
f
= 0.79 (3:7 = Ethylacetate: Hexane, v/v), H NMR (CDCl3, 400
MHz) δ 8.17 (d, J = 8.0 Hz, 1H, H-3), 7.75 (d, J = 7.6 Hz, 1H, H-1), 7.39-7.27 (m, 17 H, ArH),
7
1
1
3
.17 (s, 1H, H-7), 6.10 (s, 1H, H-11a), 4.95 (d, J = 11.6 Hz, 1H, CH
H, CH Ph), 4.68 (d, J = 12.0 Hz, 1H, CH Ph), 4.63 (d, J = 12.4 Hz, 1H, CH
2.0 Hz, 1H, CH Ph), 4.40 (d, J = 12.0 Hz, 1H, CH Ph), 4.16 (brs, 1H, H-8), 3.99 (brs, 1H, H-9),
.86 (t, J = 6.4 Hz, 1H, H-10), 3.61 (dd, J = 9.2 Hz and J = 6.0 Hz, 1H, H-13a), 3.50 (dd, J = 9.2
2
Ph), 4.85 (d, J = 12.4 Hz,
2
2
2
Ph), 4.47 (d, J =
2
2
1
3
Hz and J = 6.4 Hz, 1H, H-13b), C NMR, (CDCl3,100 MHz) δ 160.3 (C-6), 153.7, 128.6, 128.5,
1
28.5, 128.4, 128.3, 128.3, 128.2, 127.9, 127.9, 127.8, 127.6, 127.4, 127.2, 126.9, 121.4, 115.6,
12.3 (C-7), 106.2, 102.7, 96.9 (C-11a), 79.5 (C-8), 76.5 (C-9), 75.1 (C-10), 74.3, 73.6, 71.7 (3 ×
1
CH
2
Ph), 68.7 (C-13). HRMS(ESI), calcd, m/z C38
H
32
F
3
NO
, [M+H]+ 656.2254; Found:
6
6
56.2276.
(
8R,9R,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2-chloro-3-triflouromethyl
-
8
,9,10,11a-tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (22). Off white
amorphous solid, m.p.: 182 °C; m.p.: 190 °C; yield 68%, R = 0.56 (3:7 = Ethylacetate: Hexane,
f
1
v/v), H NMR (CDCl
3
, 400 MHz) δ 11.32 (brs, 1H, NH), 8.00 (s, 1H, H-4), 7.61 (s, 1H, H-1),
7
.42-7.24 (m, 15H, ArH), 7.20 (s, 1H, H-7), 6.12 (s, 1H, H-11a), 4.99 (d, J = 11.6 Hz, 1H,
CH
2
Ph), 4.86 (d, J = 12.4 Hz, 1H, CH
Ph), 4.48 (d, J = 12.0 Hz, 1H, CH
H, H-8), 4.04 (brs, 1H, H-9), 3.89 (t, J = 5.6 Hz, 1H, H-10), 3.64 (dd, J = 9.2 Hz and J = 6.0
2
Ph), 4.72 (d, J = 11.6 Hz, 1H, CH
2
Ph), 4.65 (d, J = 12.4
Hz, 1H, CH
2
2
Ph), 4.41 (d, J = 12.0 Hz, 1H, CH
2
Ph), 4.19 (brs,
1
1
3
Hz, 1H, H-13a), 3.53 (dd, J = 9.6 Hz and J = 6.4 Hz, 1H, H-13b), C NMR (CDCl
3
, 100 MHz) δ
161.6, 152.5, 138.0, 137.5, 128.6, 128.4, 128.2, 128.0, 127.9, 127.8, 127.4, 125.0, 112.3 (C-7),
1
7

Organic & Biomolecular Chemistry
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1
1
06.9, 96.9 (C-11a), 79.4 (C-8), 76.4 (C-9), 75.1 (C-10), 74.4, 73.6, 71.8 (3 × CH Ph), 68.5 (C-
2
+
3), HRMS(ESI), calcd, m/z C38
H31ClF
3
NO
6
, [M+K] 728.1424; Found: 728.1420.
(
[
8R,9S,10R)8,9bis(benzyloxy)10((benzyloxy)methyl)8,9,10,11a-tetrahydropyrano
3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (23a/b). Yellow amorphous solid, m.p.: 180 °C;
1
yield 70%, R
f
= 0.41 (4:6 = Ethylacetate: Hexane, v/v), H NMR (CDCl
3
, 400 MHz) δ 11.82 (s,
1
H, NH), 8.00-7.95 (m, 2H), 7.49-7.18 (m, 37H, Ar H), 7.18 (s, 1H, H-7 of 23a), 7.07 (s, 1H, H-
of 23b), 6.61 (s, 1H, H-11a of 23a), 6.01 (s, 1H, H-11a of 23b), 4.99 (d, J = 12.0 Hz, 1H,
Ph), 4.95 (d, J = 10.8 Hz, 1H, CH Ph), 4.75 (d, J = 12 Hz, 1H, CH Ph,), 4.68 (d, J = 12.8
Hz, 1H, CH Ph), 4.62 (d, J = 12.8 Hz, 1H, CH Ph), 4.57 (d, J = 11.2 Hz, 1H, CH Ph), 4.54 -
.49 (m, 3H, CH Ph), 4.50 (d, J = 11.2 Hz, 1H, CH Ph), 4.44 (d, J = 12.0 Hz, 1H, CH Ph), 4.37
d, J = 11.6 Hz, 1H, CH , 100
Ph), 4.26 -4.23 (m, 2H), 3.86-3.67 (m, 8H), 13C NMR (CDCl
MHz) δ 162.3, 157.8, 154.6, 138.1, 137.9, 137.8, 137.8, 137.7, 137.5, 128.5, 128.5, 128.4, 128.3,
7
CH
2
2
2
2
2
2
4
2
2
2
(
2
3
1
1
7
28.0, 127.9, 127.9, 127.6, 127.6, 122.5, 122.4, 127.3, 126.2, 123.3, 123.2, 122.6, 122.5, 122.4,
20.9, 116.0, 115.9, 114.2, 114.0, 111.5, 109.7, 106.3, 104.3, 102.0, 98.5, 96.2, 82.3, 81.1, 79.0,
8.8, 76.4, 75.1, 73.5, 73.3, 73.3, 73.0, 72.9, 71.5, 70.1, 69.0, 68.7, HRMS(ESI), calcd, m/z
+
C
37
H33NO
6
, [M+H] 588.2381; Found: 588.2405.
(
8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-4-flouro-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (24a/b). Brown amorphous
1
solid, m.p.: 120 °C; yield 65%, R
CDCl
f
= 0.60 (1:1 = Ethylacetate: Hexane, v/v), H NMR (400 MHz,
3
) δ 9.62 (brs, 2H, NH), 7.77 (s, 2H), 7.42-7.15 (m, 34H, ArH), 7.10 (s, 1H, H-7 of 24a),
.99 (s, 1H, H-7 of 24b), 6.59 (s, 1H, H-11a of 24a), 5.99 (s, 1H, H-11a of 24b), 5.01-4.89 (m,
H, CH Ph), 4.71-4.62 (m, 4H, CH Ph), 4.59 (d, J = 12.0 Hz, 2H, CH Ph), 4.52 (d, J = 11.2 Hz,
H, CH Ph), 4.44 (d, J = 12.4 Hz, 1H, CH Ph), 4.40 (d, J = 10.4 Hz, 1H, CH Ph), 4.33 (d, J =
1.2 Hz, 1H, CH , 100
Ph), 4.20 (s, 2H), 3.99 (s, 1H), 3.82-3.69 (m, 7H), 13C NMR (CDCl
6
2
1
1
2
2
2
2
2
2
2
3
MHz) δ 160.9, 160.8, 156.8, 138.3, 137.8, 137.6, 137.4, 137.2, 128.5, 128.4, 128.3, 128.1, 128.0,
1
7
27.9, 127.8, 127.7, 127.6, 127.1, 126.9, 122.1, 116.0, 111.2, 104.9, 98.7, 96.1, 82.1, 81.0, 78.7,
5.1, 73.5, 73.3, 72.9, 72.4, 71.5, 70.6, 70.1, 68.3, 67.7; HRMS(ESI), calcd, m/z C37
H
32FNO ,
6
+
[
M+H] 606.2286; Found: 606.2277.
(
8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-3-phenoxy--8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one
(25a/b).
Light
yellow
1
8

Page 19 of 61
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DOI: 10.1039/C7OB03186F
1
amorphous solid, m.p.: 160 °C; yield 64%, R
f
= 0.66 (1:1 = Ethylacetate: Hexane, v/v), H NMR
(CDCl3, 400 MHz) δ 10.81 (brs, 1H, NH), 10.20 (brs, 1H, NH), 7.94-7.89 (m, 2H), 7.43-6.99 (m,
4
4H, ArH), 6.96 (s, 1H, H-7 of 25a) 6.75 (s, 1H, H-7 of 25b), 6.56 (s, 1H, H-11a of 25a), 5.97 (s,
H, H-11a of 25b), 4.96 (d, J = 12.8 Hz, 1H, CH Ph), 4.90 (d, J = 8.8 Hz, 1H, CH Ph), 4.70 (d, J
12.0 Hz, 1H, CH Ph), 4.65 (d, J = 12.0 Hz, 1H, CH Ph), 4.60-4.33 (m, 8H, CH Ph), 4.21-4.19
m, 1H), 3.85-3.49 (m, 9H), C NMR (CDCl , 100 MHz) δ 162.4, 160.8, 160.2, 157.8, 156.7,
56.6, 154.6, 140.0, 139.1,138.1, 137.9, 137.7, 137.3, 130.0, 129.6, 129.4, 128.5, 128.4, 128.3,
1
2
2
=
2
2
2
1
3
(
3
1
1
1
7
6
28.0, 127.9, 127.8, 127.7, 127.6, 125.4, 124.5, 124.4, 123.4, 122.1, 120.1, 120.0, 119.9, 118.2,
16.7, 113.5, 111.6, 109.5, 104.0, 98.6, 96.1, 82.2, 81.0, 79.0, 78.7, 76.3, 75.1, 73.5, 73.3, 73.2,
+
2.7, 71.5, 70.0, 69.0, 68.6, HRMS(ESI), calcd, m/z C43
80.2634.
H37NO
7
, [M+H] 680.2643; Found:
(
8R,9S,10R)-8,9-bis(benzyloxy)–10-((benzyloxy)methyl)–4-nitro-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (26a/b). Yellow amorphous
1
solid, m.p.: 179 °C; m.p.: 190 °C; yield 62%, R
f
= 0.72 (1:1 = Ethylacetate: Hexane, v/v), H
NMR (CDCl
3
, 400 MHz) δ 11.11 (brs, 1H), 11.08 (brs, 1H), 8.48-8.44 (m, 2H), 8.33-8.31 (m,
H), 7.40-7.08 (m, 30H, ArH), 7.05 (d, J = 1.6 Hz, 1H), 6.94 (d, J = 1.6 Hz, 1H), 6.80 (s, 1H, H-
of 26a), 6.58 (s, 1H, H-7 of 26b), 6.36 (s, 1H, H-11a of 26a), 5.97 (s, 1H, H-11a of 26b), 4.91
Ph), 4.88 (d, J = 10.8 Hz, 1H, CH Ph), 4.70 (d, J = 10.2 Hz, 1H,
Ph), 4.67 (d, J = 10.2 Hz, 1H, CH Ph), 4.61 (d, J = 12.0 Hz, 1H, CH Ph), 4.56 (d, J = 9.6
Ph), 4.53 (d, J = 9.6 Hz, 1H, CH Ph), 4.51 (d, J = 9.8 Hz, 1H, CH Ph), 4.48 (d, J =
.8 Hz, 1H, CH Ph), 4.42 (d, J = 9.8 Hz, 1H,CH Ph), 4.38 (d, J = 9.8 Hz, 1H, CH Ph), 4.29 (d, J
11.2 Hz, 1H, CH Ph), 4.20-4.18 (m, 2H), 3.79-3.57 (m, 8H), CNMR (CDCl3, 100 MHz) δ
2
7
(
d, J = 11.6 Hz, 1H, CH
2
2
CH
2
2
2
Hz, 1H, CH
2
2
2
9
2
2
2
1
3
=
2
1
1
7
60.0, 156.2, 152.9, 137.6, 137.5, 137.3, 137.1, 128.6, 128.4, 128.3, 128.2, 128.1, 128.0 127.9,
27.8, 127.7, 121.2, 121.1, 110.6, 96.2, 95.4, 82.1, 81.0, 78.6, 78.5, 75.5, 75.2, 73.6, 73.5, 73.3,
+
3.0, 71.6, 71.2, 70.3, 68.9, 68.5; HRMS(ESI), calcd, m/z C37
H
32
N
2
O
8
, [M+H] 633.2231;
Found: 633.2224.
8R,9S,10R)-8,9-bis(benzyloxy–10-((benzyloxy)methyl)–4-triflouromethoxy-8,9,10,11a-
(
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)–one (27a/b). Light yellow sticky
1
solid, m.p.: 130 °C; m.p.: yield 63%, R
f
= 0.80 (1:1 = Ethylacetate: Hexane, v/v), H NMR
(CDCl3, 400 MHz) δ 10.98 (brs, 1H, NH), 10.95 (brs, 1H, NH), 7.96-7.91 (m, 2H, ArH) 7.46-
1
9

Organic & Biomolecular Chemistry
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7
.15 (m, 34H, Ar H), 7.16 (s, 1H, H-7 of 27a), 7.06 (s, 1H, H-7 of 27b), 6.63 (s, 1H, H-11a of
7a), 6.01 (s, 1H, H-11a of 27b), 5.01 (d, J = 12.0 Hz, 1H, CH Ph), 4.95 (d, J = 10.8 Hz, 1H,
Ph), 4.69 (d, J = 12.4 Hz, 1H, CH Ph), 4.54-4.63 (m, 5H,
Ph), 4.43 (d, J = 12.0 Hz, 1H, CH Ph), 4.35 (d, J = 11.6
Ph), 4.25-4.24 (m, 2H), 4.07-4.04 (m, 1H), 3.87-3.64 (m, 7H), C NMR (CDCl3,
00 MHz) δ 162.4, 162.1, 157.0, 153.5, 153.3, 138.0, 137.8, 137.5, 137.4, 137.2, 136.9, 130.1,
2
2
CH
2
2
Ph), 4.73 (d, J = 12.0 Hz, 1H, CH
2
2
CH
Ph), 4.51 (d, J = 11.2 Hz, 1H, CH
2
2
1
3
Hz, 1H, CH
2
1
1
1
28.5, 128.4, 128.3, 128.0 127.9, 127.90, 127.7, 124.3, 122.45, 123.5, 118.7, 113.0, 111.4, 111.0,
06.9, 98.8, 96.1, 82.2, 81.0, 78.7, 76.4, 75.2, 73.5, 73.3, 73.0, 72.3, 71.5, 70.2, 68.9, 68.4,
+
HRMS(ESI), calcd, m/z C38
H
32
F
3
NO
7
, [M+H] 672.2204; Found: 672.2231.
(
8R,9S,10R)-8,9-bis(benzyloxy)–10-((benzyloxy)methyl)–3-triflouromethyl-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (28a/b). Off white amorphous
1
solid, m.p.: 140 °C; yield 67%, R
f
= 0.66 (1:1 = Ethylacetate: Hexane, v/v), H NMR (CDCl3,
4
7
2
00 MHz) δ 12.04 (s, 1H, NH), 11.87 (s, 1H, NH), 8.15-8.10 (m, 1H), 7.95 (m, 1H), 7.94 (s, 1H),
.74 (m, 1H), 7.57 (s, 1H), 7.53 (s, 1H), 7.50 (s, 1H), 7.43 (d, 3H, J = 6.8 Hz), 7.15-7.38 (m,
6H, ArH), 6.63 (s, 1H, H-11a of 28a), 6.03 (s, 1H, H-11a of 28b), 4.94 (m, 2H, CH Ph), 4.70
Ph), 4.67(d, J = 12.0 Hz, 1H, CH Ph), 4.64 (brs, 1H, CH Ph), 4.58 (d, J
Ph), 4.44-4.53 (m, 4H, CH Ph), 4.39 (d, J = 12.8, Hz, 1H, CH Ph), 4.34 (d, J
Ph), 4.20-4.24 (m, 2H), 3.63-3.99 (m, 8H), C NMR (CDCl3, 100 MHz) δ
2
(
d, J = 11.6 Hz, 1H, CH
2
2
2
=
=
10.8 Hz, 1H, CH
2
2
2
1
3
11.6 Hz, 1H, CH
2
1
1
8
65.2, 162.1, 157.6, 157.0, 153.6, 143.5, 138.0, 137.8, 137.5,137.4, 137.2, 136.8, 130.1, 128.5,
28.4, 128.3, 128.0, 127.9, 127.7, 127.94, 124.3, 123.5, 118.7, 113.0, 106.9, 98.8, 96.1, 82.2,
1.0, 78.7, 76.4,75.2, 73.5, 73.3, 73.0, 72.3, 71.5, 70.2, 68.9, 68.4, 68.3, HRMS(ESI), calcd, m/z
+
C
38
H
32
F
3
NO
6
, [M+H] 656.2254; Found: 656.2276.
(
8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)–4-isopropy-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (29a/b). Amorphous solid, m.p.:
1
1
1
10 °C; yield 66%, R
f
= 0.66 (1:1 = Ethylacetate: Hexane, v/v), H NMR (CDCl3, 400 MHz) δ
0.22 (s, 1H, NH), 9.94 (s, 1H, NH), 8.00-7.85 (m, 2H), 7.44-7.12 (m, 33H, ArH), 7.11 (s, 1H,
H-7 of 29a), 7.00 (s, 1H, H-7 of 29b), 6.56 (s, 1H, H-11a of 29a), 5.97 (s, 1H, H-11a of 29b),
.96 (d, J = 12.0 Hz, 1H, CH Ph), 4.91 (d, J = 10.8 Hz, 1H, CH Ph), 4.71 (d, J = 11.6 Hz, 1H,
Ph), 4.64 (d, J = 12.0 Hz, 1H, CH Ph), 4.59 (d, J = 11.6 Hz, 1H, CH Ph), 4.54 - 4.45 (m, 5H,
Ph), 4.40 (d, J = 12.0 Hz, 1H, CH Ph), 4.33 (d, J = 11.6 Hz, 1H, CH Ph), 4.21 (s, 2H), 4.00-
4
2
2
CH
2
2
2
2
CH
2
2
2
0

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DOI: 10.1039/C7OB03186F
3
.99 (m, 1H), 3.83-3.60 (m, 7H), 3.47-3.37 (2H, CH of CH(Me)
2
), 1.31 (d, J = 4.8 Hz, 12H,
13
CH(Me) ), C NMR (CDCl3, 100 MHz) δ 164.3, 161.7, 161.5, 158.1, 155.0, 138.2, 137.9, 137.8,
2
1
1
9
2
37.7, 137.4, 137.2, 135.4, 134.7, 133.7, 128.5, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7, 127.6,
27.4, 126.9, 126.3, 123.3, 122.4, 122.3, 121.2, 120.9, 120.6, 114.4, 114.3, 111.4, 105.1, 104.1,
8.6, 96.3, 82.3, 81.2, 79.3, 78.8, 76.5, 75.1, 73.5, 73.3, 72.8, 71.5, 70.0, 69.0, 68.8, 68.4, 26.8,
+
6.8, 22.9, 22.8. HRMS(ESI), calcd, m/z C40
H39NO
6
, [M+K] 668.2409; Found: 668.2422.
(
8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-3-chloro-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (30a/b). Off white amorphous
1
solid, m.p.: 150 °C; yield 68%, R
f
= 0.57 (4:6 = Ethylacetate: Hexane, v/v), H NMR (CDCl3,
4
00 MHz) δ 11.67 (brs, 2H, NH), 7.92-7.84 (m, 3H), 7.44-7.11 (m, 33H, Ar H), 7.12 (s, 1H, H-7
of 30a), 7.03 (s, 1H, H-7 of 30b), 6.60 (s, 1H, H-11a of 30a), 5.98 (s, 1H, H-11a of 30b), 4.98-
4
4
4
1
1
9
.92 (m, 1H, CH
2
Ph), 4.73(d, J = 10.2 Hz, 1H, CH
2
Ph), 4.67 (d, J = 12.0 Hz, 1H, CH
Ph), 4.36 (d, J = 12 Hz, 1H, CH
2
Ph), 4.60-
.51 (m, 7H, CH
2
Ph), 4.44 (d, J = 11.6 Hz, 1H, CH
2
2
Ph), 4.26-
1
3
.22 (m, 2H), 4.01-4.00 (m, 1H), 3.85-3.64 (m, 7H), C NMR (CDCl3,100 MHz) δ 160.1, 160.0,
55.8, 152.6, 137.0, 136.7, 136.6, 136.4, 136.2, 129.8, 129.1, 127.5, 127.4, 127.3, 126.9, 126.8,
26.6, 126.5, 126.2, 121.9, 121.3, 120.8, 120.7, 120.2, 115.0, 114.9, 110.1, 105.5, 104.5, 97.7,
5.1, 81.1, 80.0, 77.6, 77.5, 75.4, 74.1, 72.4, 72.3, 72.2, 71.7, 70.4, 69.0, 67.9, 67.6,
+
HRMS(ESI), calcd, m/z C37
H
32ClNO
6
, [M+Na] 644.187; Found: 644.1916.
(
8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-3-bromo-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (31a/b). Off white amorphous
1
solid, m.p.: 185 °C; yield 66%, R
f
= 0.62 (4:6 = Ethylacetate: Hexane, v/v), H NMR (CDCl3,
4
3
4
00 MHz) δ 12.17 (brs, 1H NH), 7.63 (m, 1H), 7.47-7.05 (m, 37H, ArH), 7.05 (s, 1H, H-7 of
1b), 6.66 (s, 1H, H-11a of 31a), 6.03 (s, 1H, H-11a of 31b), 4.96 (d, J = 10.4 Hz, 2H, CH Ph),
.94 (m, 1H, CH Ph), 4.70 (d, J = 10.4 Hz, 2H, CH Ph), 4.65 (d, J = 9.6 Hz, 1H, CH Ph), 4.60
Ph), 4.55 (d, J = 10.4 Hz, 1H, CH Ph), 4.51 (d, J = 9.4 Hz, 1H, CH Ph),
.46 (d, J = 12.0 Hz, 1H, CH Ph), 4.40 (d, J = 11.6 Hz, 1H, CH Ph), 4.23-4.29 (m, 2H), 4.10-
.13 (m, 1H), 3.64-3.84 (m, 7H), 13 C NMR (CDCl
, 100 MHz) δ 160.5, 153.3, 139.2, 138.4,
2
2
2
2
(
d, J = 9.8 Hz, 2H, CH
2
2
2
4
4
1
1
2
2
3
37.2, 136.9, 136.7, 136.6, 136.3, 129.0, 127.5, 127.3, 127.2, 127.0, 126.9, 126.8, 126.7, 126.6,
26.4, 125.6, 120.3, 116.5, 112.1, 97.6, 94.6, 81.2, 80.1, 77.6, 76.1, 74.1, 72.4, 72.3, 72.1, 71.8,
2
1

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DOI: 10.1039/C7OB03186F
+
7
6
0.5, 69.0, 68.1, 67.4, HRMS(ESI), calcd, m/z C37
66.1599.
H
32BrNO , [M+H] 666.1486; Found:
6
(
8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-4-triflouromethyl-8,9,10,11a-
tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (32a/b). Yellow amorphous
1
solid, amorphous solid, m.p.: 155 °C; yield 63%, R
f
= 0.50 (3:7 = Ethylacetate: Hexane, v/v), H
NMR (CDCl
3
, 400 MHz) δ 12.52 (s, 1H, NH), 12.18 (s, 1H, NH), 8.75 (d, J = 15.6 Hz, 1H), 8.21
d, J = 7.6 Hz, 1H), 7.78 (m, 2H, ArH), 7.43-7.15 (m, 32H, ArH), 7.07 (s, 1H, H-7 of 32a), 6.97
s, 1H, H-7 of 32b), 6.59 (s, 1H, H-11a of 32a), 5.99 (s, 1H, H-11b of 32b), 4.95 (d, J = 12.8 Hz,
(
(
2
H, CH
2.8 Hz, 1H, CH
Ph), 4.33 (d, J = 11.6 Hz, 1H, CH
2
Ph), 4.92 (d, J = 12.8 Hz, 1H, CH
Ph), 4.55 (m, 3H, CH Ph), 4.47 (d, J = 12.0 Hz, 1H, CH
Ph), 4.20 (d, 2H, H-10), 3.98 (s, 1H), 3.82-3.59
, 100 MHz) δ 165.8, 164.0, 162.4, 161.2, 159.2, 159.1, 155.8, 152.6,
37.0, 136.7, 136.5, 136.4, 136.1, 134.9, 132.8, 132.2, 129.0, 127.5, 127.4 ,127.3, 127.0, 126.9,
2
Ph), 4.64 (d, J = 12.4 Hz, 2H, CH
2
Ph), 4.60 (d, J =
1
2
2
2
Ph), 4.39 (d, J = 12.4
Hz, 1H, CH
2
2
1
3
(
m, 7H), C NMR (CDCl
3
1
1
1
6
27.7, 127.6, 126.3, 125.5, 125.4, 117.7, 117.2, 114.6, 114.5, 114.1, 110.8, 109.8, 109.2, 105.1,
04.2, 97.8, 95.1, 81.1, 80.0, 77.6, 77.5, 75.5, 74.1, 72.5, 74.1, 72.5, 72.4, 72.3, 71.9, 70.5, 69.1,
+
7.9, 67.6, HRMS(ESI), calcd, m/z C38
H
32
F
3
NO
6
[M+H] 656.2254; Found: 656.2276.
(
8R,9S,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-2-chloro-3-triflouromethyl
-
8
,9,10,11a-tetrahydropyrano[3′,2′,5,6]pyrano[3,2-c]quinolin-6(5H)-one (33a/b). Yellowish
1
amorphous solid, m.p.: 186 °C; yield 61%, R = 0.67 (3:7 = Ethylacetate: Hexane, v/v), H NMR
f
(
CDCl3, 400 MHz) δ 12.35 (s, 1H, NH), 11.64 (s, 1H, NH), 8.09 (s, 1H), 7.70 (d, J = 8.4 Hz,
H), 7.50-7.17 (m, 32H, ArH), 7.11 (s, 1H, H-7 of 33a), 7.01 (s, 1H, H-7 of 33b), 6.66 (s, 1H, H-
1a of 33b), 5.95 (s, 1H, H-11a of 33a), 5.00-4.97 (m, 2H,CH Ph), 4.71-4.35 (m, 10H, CH Ph),
.26-4.21 (m, 2H), 4.03-4.00 (m, 1H), 3.90-3.72 (m, 7H), C NMR (CDCl , 100 MHz) δ 162.2,
1
1
4
1
1
7
2
2
1
3
3
61.0, 159.2, 155.8, 151.7, 150.9, 137.8, 137.5, 137.0, 134.8, 128.5, 128.4, 128.3, 128.2, 128.1,
28.0, 127.9, 127.8, 127.7, 124.4, 123.2, 123.2, 116.4, 115.5, 111.0, 106.9, 95.9, 82.1, 80.7, 77.2,
6.1, 75.3, 73.6, 73.5, 73.4, 73.3 72.8, 71.6, 70.3, 68.8, 68.0, HRMS(ESI), calcd., m/z
+
C
38
H31ClF
3
NO
6
, [M+K] 728.1424; Found: 728.1420.
(
8R,9R,10R)-8,9-bis(benzyloxy)-10-((benzyloxy)methyl)-5-methyl-8,9,10,11a-tetrahydro
pyrano[3',2':5,6]pyrano[3,2-c]quinolin-6(5H)-one (35). White amorphous solid, m.p.: 180 °C;
1
yield 71%, Rf = 0.56 (2 : 8 = Ethylacetate: Hexane, v/v), H NMR (CDCl
3
, 400 MHz) δ 8.01 (d,
2
2

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DOI: 10.1039/C7OB03186F
J = 6.4 Hz, 1H, H-1), 7.52 (m, 1H, 2H), 7.39-7.28 (m, 16H, ArH), 6.08 (s, 1H, H-11a), 4.95 (d, J
11.2 Hz, 1H, CH Ph), 4.86 (d, J = 12.0 Hz, 1H, CH Ph), 4.69 (d, J = 11.6 Hz, 1H, CH Ph),
.63 (d, J = 12.0 Hz, 1H, CH Ph), 4.47 (d, J = 11.6 Hz, 1H, CH Ph), 4.40 (d, J = 10.8 Hz, 1H,
Ph), 4.16 (brs, 1H, H-9), 3.99 (brs, 1H, H-8), 3.85(brs, 1H, H-10), 3.62 (brs, 1H, H-13a),
=
2
2
2
4
2
2
CH
2
1
3
3
1
1
7
6
.70 (s, 1H, N-CH
3
), 3.54 (brs, 1H, H-13b); C NMR (CDCl , 100 MHz) δ 160.7, 152.9, 138.9,
3
38.3, 137.8, 128.6, 128.5, 128.4, 128.1, 127.9, 127.8, 127.6, 127.5, 127.4, 127.3, 125.9, 123.1,
21.8, 114.9, 113.9 (C-7), 113.7, 104.3, 96.9 (C-11a), 79.7 (C-10), 76.5(C-9), 75.0 (C-8), 74.2,
3.5, 71.7 (3 × CH
2
Ph), 68.8 (C-13), 29.4 (CH
3
); HRMS(ESI), calcd, m/z C38
H
35NO , [M+H]+
6
02.25; Found: 602.26.
Cell Culture. The human breast cancer cell line MCF-7 and liver cancer cell line HepG2 were
cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, St. Louis, USA)
supplemented with 10% heat inactivated FBS (Fetal Bovine Serum, Gibco, USA) and 1%
penicillin-streptomycin (Gibco Canada) in humidified CO incubator at 37 ºC. The cells were
2
maintained as a monolayer in 100 mm culture plate. The cells used for each experiment were of
less than 8 passage number. Subculturing of cells was performed every third day, followed by
trypsin EDTA treatment.
Cell Viability Assay. Cell viability assay was performed as per standard protocols. Briefly,
MCF-7 and HepG2 cells were trypsinized (Trypsin-EDTA, Invitrogen) and seeded at a density of
5
0,000 cells per well in complete media in a 12-well flat-bottomed plate, followed by incubation
at 37 °C and 5% CO for 12-14 hours. The cells were then treated with all synthesized
2
carbohydrate-fused pyrano[3,2-c]quinolones (n = 22) for 48 hours at five different
concentrations, 1 µM, 5 µM, 10 µM, 20 µM and 25 µM respectively. DMSO was used as vehicle
control and Doxorubicin, (a DNA damaging compound) is taken as positive control. After
treatment, cells were observed under microscope for 48 hours. After 48 hours, medium from the
plates was discarded and cells were washed with Phosphate Buffer Saline (PBS) followed by
gentle shaking for removal of dead cells. Cells were then trypsinized and resuspended in 1 mL
DMEM. 10 µl of cell suspension was added to hemocytometer and viable cells were counted
from each four quadrant followed by calculating average cell viability for each compound.
Percent viability was calculated as number of cells in the treated well divided by number of cells
in the untreated well (DMSO treated) × 100. IC50 values were determined by plotting values of
percent cell viability against different concentrations of each of those most active compounds.
Conflict of interest
There are no conflicts of interest to declare.
2
3

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DOI: 10.1039/C7OB03186F
Acknowledgements
Authors are thankful to the Shiv Nadar University (SNU) 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). AG is a recipient of the
IYBA Award from Department of Biotechnology (DBT). SD acknowledges Shiv Nadar
University for providing JRF assistantship.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://.
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1
448.
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4

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55–458.
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2
6

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Design and efficient synthesis of new carbohydrate fused pyrano[3,2-c]quinolone derivatives and
their submicromolar anticancer activity.

Organic & Biomolecular Chemistry
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DOI: 10.1039/C7OB03186F
Supplementary material
Stereoselective synthesis of natural product inspired carbohydrate
fused pyrano[3,2-c]quinolones as antiproliferative agents
a
a
b
b
a,c
Priti Kumari, Chintam Narayana, Shraddha Dubey, Ashish Gupta and Ram Sagar
a
Department of Chemistry, School of Natural Sciences, Shiv Nadar University (SNU), NH91, Tehsil-Dadri, Gautam
Buddha Nagar, Uttar Pradesh, 201314, India.
Department of Life Sciences, School of Natural Sciences, Shiv Nadar University (SNU), NH91, Tehsil-Dadri,
b
Gautam Buddha Nagar, Uttar Pradesh, 201314, India.
c
Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, 221005, India.
*
E-mail (Corresponding author): ram.sagar@snu.edu.in; ashish.gupta@snu.edu.in,
S. No.
Contents
Page No.
S2
1
2
3
4
5
6
Synthesis of Compound 1a and 1b
Synthesis and NMR data of 4-hydroxyquinolones1-11
S2-S6
1
13
Copies of H NMR and C NMR of pyrano[3,2-c]quinolones12-33a/b
S7-S28
S29-S30
1
1
1
13
NOE, H- H COSY and H- C HSQC spectra of compound 12
1
13
Copies of H & C NMR of 34 and N-methyl pyrano[3,2-c]quinolones 35 S31-S32
Cell viability data for all compounds against MCF-7 and HepG2 cell lines S33-S34
S1

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General experimental procedures
Synthesis of 2-C-formyl galactal 1a and 2-C-formyl glucal 1b: In a 100 ml round bottom flask
0 ml anhydrous DMF was added followed by dropwise addition of POCl (0.6 mL, 21.6 mmol)
3
3
at 0 °C and resulting mixture was stirred for 30 min under Argon atmosphere. A solution
dissolved in anhydrous DMF) of 3,4,6-tri-O-benzyl-D-galactal (2.99 g, 7.20 mmol) was added
(
to this dropwise within 30 min. The resulting reaction mixture was stirred at 0 °C to room
temperature for 5 h which shows disappearance of starting material (TLC). Reaction mixture was
quenched by slow addition of (within 30 min) of chilled NaHCO (30 mL) solution at 0 °C.
3
Mixture was extracted with ethyl acetate (3 × 30mL).and combined organic layer washed with
brine solution (3 × 30mL).then dried over anhydrous Na SO . The combined organic layer was
2
4
evaporated under vaccume to get crude residue which was purified though flash column
chromatography and pure 2-C-formyl galactal 1a was isolated in 94 % isolated yield (3.0 g).
Similarly using 3,4,6-tri-O-benzyl-D-galucal and adopting above protocol 2-C-formyl glucal 1b
1
was obtained in 54 % (1.7g) isolated yield.
Synthesis of 4-hydroxyquinolones 1-11: In an oven dried 25 ml round bottom flask Aniline (1
mL, 1.024 gm 10.99 mmol) and Meldrum’s Acid (1.583 gm,10.99 mmol ) were taken and heated
at 80 °C for 2 h. After consumption of starting material (TLC), added ethylacetate and aquous
solution of sodium bicarbonate. Resulting suspension partitioned in separating funnel and
aqueous layer washed with ethyl acetate (3 × 50 mL). The aqueous layer acidified with conc.HCl
until pH 1-2 then extracted with dichloromethane (3 × 35 mL). The combined organic layer dried
over anhydrous Na SO filtered and evaporated on rotary evaporator to get solid residue which
2
4
was forwarded next step without further purification. The solid residue (1.9 g) and Eaton’s
reagent (9.5 g, 5 equiv/w) was heated at 80 °C for 1 h or till disappearance of starting material
(TLC). The reaction mixture cooled down and added ice cold water and keep stirring for 30 min
then solid precipitate appeared which was filtered using Buchner funnel under vaccum till
2
dryness to get almost pure 4-hydroxyquinolone 1 in 80 % yield (1.41gm). Similar reaction
protocol was followed for preparation of various other 4-hydroxyquinolone (2-11 and 34) using
corresponding anilines (Table S1).
References
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. N.G. Ramesh, K.K. Balasubramanian, Tetrahedron Lett. 1991, 32 , 3875-3878.
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S2