Flexible synthesis of isomeric pyranoindolones and evaluation of cytotoxicity towards HeLa cells

Article abstract of DOI:10.1007/s12039-016-1070-8

A hybrid pharmacophore approach for the synthesis of isomeric pyranoindolones was achieved by employing gold(III) chloride-catalyzed cycloisomerization of alkyne-tethered indole carboxylic acids in good to excellent yield. All the synthesized compounds were evaluated for their tumor cell growth inhibitory activity against human cervix adenocarcinoma (HeLa) which revealed that three compounds exhibited activity comparable with the standard cis-platin (IC₅₀ = 0.08 μM). Molecular docking of all the compounds in Vaccinia H1-Related (VHR) Phosphatase receptor also supported that compound 7d as the most active with a free energy of binding as ?8.27 kcal/mol. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s12039-016-1070-8

c
J. Chem. Sci. ꢀ Indian Academy of Sciences.
DOI 10.1007/s12039-016-1070-8
Flexible synthesis of isomeric pyranoindolones and evaluation of
cytotoxicity towards HeLa cells
J C JEYAVEERANa, CHANDRASEKAR PRAVEENb,∗, Y ARUNc, A A M PRINCEa
and P T PERUMALc
aDepartment of Chemistry, Ramakrishna Mission Vivekananda College, Mylapore, Chennai 600 004, India
bFunctional Materials Division, Central Electrochemical Research Institute (CSIR laboratory),
Karaikudi 630 006, India
cOrganic Chemistry Division, Central Leather Research Institute (CSIR laboratory), Adyar,
Chennai 600 020, India
e-mail: chandrasekar.praveen@gmail.com
MS received 17 November 2015; revised 10 January 2016; accepted 6 March 2016
Abstract. A hybrid pharmacophore approach for the synthesis of isomeric pyranoindolones was achieved
by employing gold(III) chloride-catalyzed cycloisomerization of alkyne-tethered indole carboxylic acids in
good to excellent yield. All the synthesized compounds were evaluated for their tumor cell growth inhibitory
activity against human cervix adenocarcinoma (HeLa) which revealed that three compounds exhibited activity
comparable with the standard cis-platin (IC50 = 0.08 μM). Molecular docking of all the compounds in Vaccinia
H1-Related (VHR) Phosphatase receptor also supported that compound 7d as the most active with a free energy
of binding as −8.27 kcal/mol.
Keywords. Pyranoindolones; positional isomers; gold catalysis; chemical synthesis; cytotoxicity;
molecular docking.
1. Introduction
include [(Cp*RhCl2)2]/Cu(OAc)2 catalyzed oxidative
coupling of indole-3-carboxylic acid with internal
alkynes,4 Pd(OAc)2/AgCO3 catalyzed annulation of
allenes with indole-2-carboxylic acids,5 cycloisomer-
ization of indole acetylenic acids by π-Lewis acid.3d,6
Given the significance of these methodologies, a cat-
alytic protocol that accomplishes the general and flex-
ible synthesis of isomeric pyranoindolones remains to
be developed. In this context, we herein report the syn-
thesis and cytotoxic evaluation of pyrano[4,3-b]indol-
1(5H)-ones towards HeLa cells. The results of chem-
ical synthesis as well as comparison of the biological
data with our previously reported pyrano[3,4-b]indol-
1(9H)-one scaffolds are disclosed in this paper.
In recent years, homogeneous gold catalysis was fre-
quently applied in medicinal chemistry for the synthesis
of bioactive natural products and clinical drugs.1 Ever
since its first use in homogeneous gold catalysis, the
catalytic application of AuCl3 has been used in numer-
ous organic transformations.2 Our group was engaged
in the development and applications of gold cat-
alyzed methodologies for the synthesis of hetero- and
carbocycles.3a−j Towards this end, we have previously
communicated the AuCl3 catalyzed cycloisomeriza-
tion of 3-alkynyl-indole-2-carboxylic acid leading to
pyrano[3,4-b]indol-1(9H)-ones, which exhibited mod-
erate cytotoxicity towards HeLa cells.3d As a logical
extension, we envisaged the synthesis of correspond-
ing positional isomer, pyrano[4,3-b]indol-1(5H)-ones
by utilising 2-alkynyl-indole-3-carboxylic acid instead
of 3-alkynyl-indole-2-carboxylic acids (figure 1). At
the outset, the success of such a deceptively simple
variant could not be taken for granted, since these
compounds may show improved levels of cytotoxicity.
Surprisingly, the synthesis of these types of compounds
has scarcely been investigated. Some rare examples
2. Experimental
2.1 Materials and instruments
All commercially available solvents and reagents were
used without further purification. Solutions in organic
solvents were dried with anhydrous sodium sulphate.
Solvents were evaporated under reduced pressure.
Melting points were obtained using open capillaries and
are uncorrected. Infrared (IR) spectra were recorded on
∗For correspondence

J C Jeyaveeran et al.
was on the order of Real = 1 h 13 m 54.67 s, CPU = 1 h
08 m54.57 s, System=11.75 s on the Pentium machine.
2.2 Methods and Characterization
2.2a General Procedure for the esterification of
indole-2-carboxylic acids 2a and 2b: To an ice-cold
solution of indole-2-carboxylic acid (5.0 g) in methanol
(60 mL) was added concentrated sulphuric acid (5 mL).
The reaction mixture was refluxed for 24 h and concen-
trated under reduced pressure. To this reaction mixture
was added ice-cold water (50 mL) and extracted with
EtOAc (3 × 50 mL). The organic layer was dried over
anhydrous Na2SO4, filtered and concentrated to afford
analytically pure product (98% yield) which was used
for the next step without further purification.
Figure 1. Structures of isomeric pyranoindolones.
a Perkin-Elmer FTIR spectrophotometer as KBr pel-
lets for solid compounds and neat sample for liquid
1
compounds. H and 13C NMR spectra were obtained
in DMSO-d6 and CDCl3 on a JEOL spectrometer at
500 and 125 MHz, respectively. Proton chemical shifts
(δ) are relative to tetramethylsilane (TMS, δ = 0.00)
as internal standard and expressed in parts per mil-
lion. The number of protons (n) for a given resonance
was indicated as nH. Spin multiplicities are given as
s (singlet), d (doublet), t (triplet), dd (doublet of dou-
blet), dt (doublet of triplet), ddd (doublet of doublet of
doublet) and m (multiplet). Coupling constants (J) are
given in hertz. Elemental analyses were recorded using
a Thermo Finnigan FLASH EA 1112CHN analyzer.All
the compounds gave C, H and N analysis within 0.5%
of the theoretical values. Column chromatography was
performed using a mixture of petroleum ether and ethyl
acetate on silica gel (100–200 mesh, SRL, India). Ana-
lytical TLC was performed on precoated plastic sheets
of silica gel G/UV-254 of 0.2 mm thickness (Macherey-
Nagel, Germany) using analytical grade solvents and
visualized with iodine spray (10% w/w I2 in silica gel),
UV light (λ = 254 and 365 nm) and alkaline KMnO4
solution. Human cervical cancer cell line (HeLa) was
obtained from National Centre for Cell Science (Pune,
India). Cells were cultured in Dulbecco’s modified
Eagle medium (DMEM) (Sigma Chemical Co.) con-
taining glucose (5 mM) and supplemented with 10%
fetal bovine serum and maintained at 37◦C in a humid-
ified incubator with an atmosphere of 5% CO2. The
in vitro cytotoxicity of the compounds was evaluated
2.2b General procedure for the iodination of indole-
2-carboxylic acid methyl ester 3a and 3b: To a solu-
tion of indole-2-carboxylic acid methyl ester (5.0 g) in
15 mL of DMF was added powdered KOH (3.0 equiv)
and stirred for 15 min. To this mixture was added a
solution of iodine (1.1 equiv) in DMF (15 mL) and
stirred for 3 h. After completion of the reaction as indi-
cated by TLC, the reaction mixture was quenched with
10% NaHSO3 and extracted with EtOAc (3 × 30 mL).
The EtOAc extract was repeatedly washed with 10%
Na2S2O3 and aqueous ammonia. The combined organic
extract was dried over anhydrous Na2SO4 and concen-
trated under reduced pressure to afford the pure product
(90% yield) which was used for the next step without
further purification.
2.2c General procedure for the N-alkylation of 3-
iodo-indole-2-carboxylic acid methyl ester 4a, 4b, 4d
and 4e: To a mixture of NaH (2.0 equiv) in DMF
(3 mL) kept at 0◦C at N2 atmosphere was added a solu-
tion of 3-iodo-indole-2-carboxylate (1.0 equiv) in DMF
(2 mL) and stirred for 15 min. To this mixture was
added the corresponding alkyl halide (1.2 equiv) and
stirred for 3 h at 0◦C to room temperature. After com-
pletion of the reaction as indicated by TLC, the reac-
tion mixture was quenched with water and extracted
with EtOAc (3 × 10 mL). The organic layer was dried
over anhydrous Na2SO4, filtered, concentrated and puri-
fied by column chromatography on silica gel using
petroleum ether/EtOAc to afford the pure product.
using MTT colourimetric assay. Absorbance (at λmax
)
was read on Tenac 200 plate reader at 570 nm. The IC50
values were calculated using the PrismPad computer
program (GraphPad Software, Inc., San Diego, CA)
and were defined as concentration of drug causing 50%
inhibition in absorbance compared with control (vehi-
cle) cells. All computations for molecular docking stud-
ies were carried out on a Dell Desktop D510 personal 2.2c1 1-Methyl-3-iodo-1H-indole-2-carboxylic acid
computer (2.4 GHz Pentium 4 processor, Intel, Santa methyl ester (4a): 91% yield; Yellow solid; M.p. 64–
Clara, CA) running Red Hat Enterprise Linux Client 65◦C; IR (KBr): 2925, 2360, 1704, 1455, 1247, 1100,
release 5.5. The time required for each simulation run 741 cm−1. 1H NMR (500 MHz, CDCl3): δH 3.97 (s, 3H);

Pyranoindolones and Cytotoxicity
4.05 (s, 3H); 7.22 (t, 1H, J = 6.8 Hz); 7.32 (d, 1H, over anhydrous Na2SO4, filtered, concentrated and puri-
J = 8.4 Hz); 7.39 (t, 1H, J = 6.8 Hz); 7.56 (d, 1H, fied by column chromatography on silica gel using
J = 7.6 Hz). 13C NMR (125 MHz, CDCl3): δC 33.1, petroleum ether/EtOAc to afford the pure product.
51.7, 110.5, 121.5, 124.0, 126.2, 128.8, 130.3, 130.4,
139.0, 161.9. GC-MS: m/z = 315 [M+]. Anal. Calcd
2.2d1 1-Butyl-3-iodo-1H-indole-2-carboxylic acid me-
thyl ester (4c): 74% yield; Brown oil; IR (neat): 2955,
for C11H10INO2: C, 41.93; H, 3.20; N, 4.45%. Found:
C, 42.11; H, 3.16; N, 4.39%.
1
1710, 1463, 1351, 1245, 1199, 1115, 740 cm−1. H
NMR (500 MHz, CDCl3): δH0.93 (t, 3H, J = 7.6 Hz);
2.2c2 1-Ethyl-3-iodo-1H-indole-2-carboxylic acid me- 1.33–1.37 (m, 2H); 1.73–1.76 (m, 2H); 3.98 (s, 3H);
thyl ester (4b): 85% yield; Brown oil; IR (neat): 2928, 4.53 (t, 2H, J = 7.6 Hz); 7.22 (t, 1H, J = 6.9 Hz);
1706, 1465, 1218, 1110, 745 cm−1. 1H NMR (500 MHz, 7.33–7.37 (m, 2H); 7.56 (d, 1H, J = 7.6 Hz).13C
CDCl3): δH 1.49 (t, 3H, J = 6.9 Hz); 3.97 (s, 3H); 4.45 NMR (125 MHz, CDCl3): δC13.9, 20.2, 32.8, 45.9,
(q, 2H, J = 6.8 Hz); 7.21 (t, 1H, J = 6.8 Hz); 7.34–7.37 51.7, 110.7, 121.5, 124.126.1, 128.2, 130.3, 138.5,
(m, 2H); 7.56 (d, 1H, J = 7.6 Hz). 13C NMR (125 MHz, 161.8. GC-MS: m/z = 357 [M+]. Anal. Calcd for
CDCl3): 15.8, 41.0, 51.7, 110.5, 121.5, 121.6, 124.1, C14H16INO2: C, 47.08; H, 4.52; N, 3.92%. Found: C,
126.0, 126.1, 130.5, 138.0, 161.3. GC-MS: m/z = 329 47.21; H, 4.49; N, 3.85%.
[M+]. Anal. Calcd for C12H12INO2: C, 43.79; H, 3.67;
2.2d2 3-Iodo-5-methoxy-1-butyl-1H-indole-2-carbox-
ylic acid methyl ester (4f): 72% yield; Colourless
N, 4.26%. Found: C, 43.89; H, 3.65; N, 4.24%.
2.2c3 3-Iodo-5-methoxy-1-methyl-1H-indole-2-car- solid; M.p. 96–98◦C; IR (KBr): 2952, 1706, 1500,
1
boxylic acid methyl ester (4d): 92% yield; Yellow 1254, 1204 cm−1. H NMR (500 MHz, CDCl3): δH
solid; M.p. 107–108◦C; IR (KBr): 2900, 2360, 1692, 0.92 (t, 3H, J = 7.6 Hz); 1.29–1.36 (m, 2H); 1.69–1.75
1446, 1218 cm−1. H NMR (500 MHz, CDCl3): δH (m, 2H); 3.89 (s, 3H); 3.96 (s, 3H); 4.50 (t, 2H, J =
1
3.89 (s, 3H); 3.96 (s, 3H); 4.01 (s, 3H); 6.91 (s, 1H); 6.9 Hz); 6.92 (d, 1H, J = 2.2 Hz); 7.03 (dd, 1H, J1 =
7.02 (d, 1H, J = 9.1 Hz); 7.22 (d, 1H, J = 9.1 Hz).13C 2.3 Hz, J2 = 9.1 Hz); 7.24 (d, 1H, J = 9.1 Hz). 13C
NMR (125 MHz, CDCl3): δC 32.2, 51.6, 55.8, 103.5, NMR (125 MHz, CDCl3): δC 13.9, 20.2, 33.0, 46.0,
111.7, 118.1, 128.6, 130.6, 134.4, 155.5, 161.8. GC- 51.6, 55.8, 103.6, 111.9, 118.0, 128.1, 130.6, 133.8,
MS: m/z = 345 [M+]. Anal. Calcd for C12H12INO3: C, 155.5, 161.7. GC-MS: m/z = 387 [M+]. Anal. Calcd
41.76; H, 3.50; N, 4.06%. Found: C, 41.91; H, 3.46; N, for C15H18INO3: C, 46.53; H, 4.69; N, 3.62%. Found:
4.02%.
C, 46.81; H, 4.65; N, 3.55%.
2.2c4 3-Iodo-5-methoxy-1-ethyl-1H-indole-2-carbox- 2.2d3 Methyl-1-benzyl-3-iodo-5-methoxy-1H-indole-
ylic acid methyl ester (4e): 83% yield; Brown oil; IR 2-carboxylate (4g): 78% yield; Colourless solid;
1
(neat): 2919, 1706, 1455, 1204, 830 cm−1. H NMR M.p. 98–100◦C; IR (KBr): 2938, 2360, 1706, 1500,
(500 MHz, CDCl3): δH 1.47 (t, 3H, J = 6.8 Hz); 3.86 (s, 1255, 1206 cm−1. 1H NMR (500 MHz, CDCl3): δH 3.89
3H); 3.94 (s, 3H); 4.42 (q, 2H, J = 7.6 Hz); 6.90 (d, 1H, (s, 3H); 3.90 (s, 3H); 5.78 (s, 2H); 6.96 (d, 1H, J =
J = 2.3 Hz); 6.99 (d, 1H, J = 8.4 Hz); 7.19 (d, 1H, J = 2.3 Hz); 6.99–7.02 (m, 3H); 7.20–7.26 (m, 4H). 13C
9.1 Hz). 13C NMR (125 MHz, CDCl3): δC 14.3, 41.1, NMR (125 MHz, CDCl3): δC 49.3, 51.7, 55.8, 103.7,
55.7, 61.2, 103.6, 111.6, 117.9, 118.0, 127.6, 130.7, 112.2, 118.4, 126.1, 127.4, 128.4, 128.7, 128.9, 130.9,
133.2, 155.5, 161.1. GC-MS: m/z = 359 [M+]. Anal. 134.2, 137.9, 155.8, 161.6. GC-MS: m/z = 421 [M+].
Calcd for C13H14INO3: C, 43.47; H, 3.93; N, 3.90%. Anal. Calcd for C18H16INO3: C, 51.32; H, 3.83; N,
Found: C, 43.21; H, 3.97; N, 3.96%.
3.33%. Found: C, 51.50; H, 3.79; N, 3.25%.
2.2d General procedure for the N-alkylation of 3- 2.2e General procedure for the synthesis of 3-ethynyl-
iodo-indole-2-carboxylic acid methyl ester 4c, 4f and indole-2-carboxylic acid methyl ester 5a–j: To a
4g: To a mixture of KOH (3.0 equiv) in DMSO (3 mL) mixture of 3-iodo-indole-2-carboxylic acid methyl
kept at 0◦C was added, 3-iodo-indole-2-carboxylate ester (1.0 mmol), Pd(Ph3P)2Cl2 (5.0 mol%) and CuI
(1.0 equiv) and stirred for 30 min. To this mixture was (5.0 mol%) under N2 atmosphere was added triethy-
added the corresponding alkyl halide (5.0 equiv) and lamine (5 mL) and stirred for 15 min. To this reac-
stirred for 6 h at 0◦C to room temperature. After com- tion mixture was added the appropriate terminal alkyne
pletion of the reaction as indicated by TLC, the reac- (1.1 mmol) and stirred at room temperature for 6 h.
tion mixture was quenched with water and extracted After completion of the reaction as indicated by TLC,
with EtOAc (3 × 15 mL). The organic layer was dried the reaction mixture was quenched with water (50 mL)

J C Jeyaveeran et al.
3H); 7.60 (d, 2H, J = 6.9 Hz). 13C NMR (125 MHz,
CDCl3): δC 32.5, 51.8, 55.8, 83.2, 95.5, 101.5, 104.2,
111.5, 117.7, 124.2, 128.0, 128.4, 129.0, 131.5, 133.9,
155.5, 162.1. MS (EI): m/z = 319 [M+]. Anal. Calcd
for C20H17NO3: C, 75.22; H, 5.37; N, 4.39%. Found:
C, 75.01; H, 5.33; N, 3.33%.
and extracted with EtOAc (3 × 15 mL). The organic
layer was dried over anhydrous Na2SO4, filtered, con-
centrated and purified by column chromatography on
silica gel using petroleum ether/EtOAc to afford the
pure product.
2.2e1 1-Methyl-3-phenylethynyl-1H-indole-2-carboxylic
acid methyl ester (5a): 88% yield; Brown paste; IR
2.2e5 5-Methoxy-1-ethyl-3-phenylethynyl-1H-indole-
2-carboxylic acid methyl ester (5e): 84% yield;
Brown paste; IR (neat): 2340, 1704, 1438, 1209 cm−1.
1H NMR (500 MHz, CDCl3): δH 1.39 (t, 3H, J =
6.9 Hz); 3.90 (s, 3H); 3.91 (s, 3H); 4.01 (s, 3H); 4.59 (q,
2H, J = 6.9 Hz); 7.05–7.07 (m, 1H); 7.24 (s, 1H); 7.30–
7.39 (m, 4H); 7.58–7.60 (d, 2H, J = 8.4 Hz). 13C NMR
(125 MHz, CDCl3): δC 15.7, 404, 51.8, 55.8, 83.2,
95.4, 101.6, 111.5, 117.7, 124.2, 128.0, 128.2, 128.6,
131.4, 131.5, 132.9, 155.5, 161.9. MS (EI): m/z = 333
[M+]. Anal. Calcd for C21H19NO3: C, 75.66; H, 5.74;
N, 4.20%. Found: C, 75.51; H, 5.70; N, 4.15%.
1
(neat): 2379, 1707, 1472, 1268, 1119, 745 cm−1. H
NMR (500 MHz, CDCl3): δH 4.02 (s, 3H); 4.03 (s, 3H);
7.27 (t, 1H, J = 7.6 Hz); 7.32–7.41 (m, 5H); 7.64 (d,
2H, J = 7.6 Hz); 7.91 (d, 1H, J = 8.4 Hz). 13C NMR
(125 MHz, CDCl3): δC 32.3, 51.9, 83.2, 95.7, 105.0,
110.5, 121.4, 121.7, 124.2, 126.0, 127.9, 128.1, 128.5,
128.9, 131.6, 138.4, 162.1. MS (EI): m/z = 289 [M+].
Anal. Calcd for C19H15NO2: C, 78.87; H, 5.23; N,
4.84%. Found: C, 79.10; H, 5.15; N, 4.76%.
2.2e2 1-Ethyl-3-phenylethynyl-1H-indole-2-carboxylic
acid methyl ester (5b): 86% yield; Brown oil; IR
1
(neat): 2976, 1701, 1469, 1247, 1124 cm−1. H NMR
2.2e6 5-Methoxy-1-butyl-3-phenylethynyl-1H-indole-
2-carboxylic acid methyl ester (5f): 72% yield;
Brown paste; IR (neat): 2938, 2360, 1706, 1446, 1203,
745 cm−1. 1H NMR (500 MHz, CDCl3): δH 0.93 (t, 3H,
J = 7.6 Hz); 1.32–1.37 (m, 2H); 1.74–1.77 (m, 2H);
3.91 (s, 3H); 4.00 (s, 3H); 4.54 (t, 2H, J = 7.6 Hz);
7.05 (dd, 1H, J1 = 2.3 Hz, J2 = 9.2 Hz); 7.22 (d, 1H,
J = 3.05 Hz); 7.29–7.37 (m, 4H); 7.59 (d, 2H, J =
6.8 Hz). 13C NMR (125 MHz, CDCl3): δC 13.9, 20.2,
32.8, 45.3, 51.9, 55.8, 83.3, 95.5, 101.5, 104.3, 111.8,
117.7, 124.2, 128.0, 128.4, 128.5, 128.6, 131.5, 133.3,
155.4, 161.9. MS (EI): m/z = 361 [M+]. Anal. Calcd
for C23H23NO3: C, 76.43; H, 6.41; N, 3.88%. Found:
C, 76.61; H, 6.36; N, 3.81%.
(500 MHz, CDCl3): δH1.52 (t, 3H, J = 6.8 Hz); 4.05
(s, 3H); 4.51 (q, 2H, J = 6.9 Hz); 7.27–7.31 (m, 1H);
7.36 (d, 1H, J = 7.6 Hz); 7.39–7.44 (m, 4H); 7.65 (d,
2H, J = 7.6 Hz); 7.95 (d, 1H, J = 8.4 Hz). 13C NMR
(125 MHz, CDCl3): δC 15.7, 40.3, 51.9, 83.3, 95.5,
110.5, 121.4, 121.9, 124.3, 126.0, 126.1, 128.0, 128.1,
128.5, 131.5, 131.6, 137.5, 161.5. MS (EI): m/z = 303
[M+]. Anal. Calcd for C20H17NO2: C, 79.19; H, 5.65;
N, 4.62%. Found: C, 79.31; H, 5.62; N, 4.57%.
2.2e3 1-Butyl-3-phenylethynyl-1H-indole-2-carboxylic
acid methyl ester (5c): 79% yield; Yellow liquid;
IR (neat): 2926, 1708, 1463, 1359, 1244, 1200, 1124,
745 cm−1. 1H NMR (500 MHz, CDCl3): δH 0.93 (t, 3H,
J = 6.9 Hz); 1.33–1.40 (m, 2H); 1.75–1.79 (m, 2H);
4.02 (s, 3H); 4.55 (t, 2H, J = 7.6 Hz); 7.28–7.33 (m,
1H); 7.38 (d, 1H, J = 7.6 Hz); 7.41–7.50 (m, 4H);
7.69 (d, 2H, J = 7.6 Hz); 8.00 (d, 1H, J = 8.4 Hz). 13C
NMR (125 MHz, CDCl3): δC 13.6, 20.1, 32.6, 45.6,
52.0, 83.7, 95.6, 110.7, 121.6, 124.5, 126.1, 126.3,
127.9, 128.2, 128.6, 131.6, 131.8, 137.7, 161.7. MS
(EI): m/z = 331 [M+]. Anal. Calcd for C22H21NO2: C,
79.73; H, 6.39; N, 4.23%. Found: C, 79.51; H, 6.44; N,
4.30%.
2.2e7 5-Methoxy-1-benzyl-3-phenylethynyl-1H-indole-
2-carboxylic acid methyl ester (5g): 85% yield;
Brown solid; M.p. 100–102◦C; IR (KBr): 2945, 1705,
1
1450, 1239, 1201, 692 cm−1. H NMR (500 MHz,
CDCl3): δH 3.91 (s, 3H); 3.96 (s, 3H); 5.82 (s, 2H);
7.01–7.04 (m, 2H); 7.19–7.28 (m, 6H); 7.32–7.40 (m,
4H; 7.61 (d, 1H, J = 6.8 Hz). 13C NMR (125 MHz,
CDCl3): δC 48.6, 51.9, 55.8, 83.1, 95.8, 101.7, 105.1,
112.1, 118.0, 124.1, 126.2, 127.4, 128.1, 128.5, 128.7,
128.9, 129.1, 131.6, 133.7, 137.8, 155.7, 161.7. MS
(EI): m/z = 395 [M+]. Anal. Calcd for C26H21NO3: C,
78.97; H, 5.35; N, 3.54%. Found: C, 79.11; H, 5.27; N,
3.44%.
2.2e4 5-Methoxy-1-methyl-3-phenylethynyl-1H-indole-
2-carboxylic acid methyl ester (5d): 90% yield;
Orange solid; M.p. 182–184 ◦C; IR (KBr): 2919, 1692,
1
1498, 1450, 1190, 878 cm−1. H NMR (500 MHz, 2.2e8 Methyl-3-(hex-1-ynyl)-1H-indole-2-carboxylate
◦
CDCl3): δH 3.91 (s, 3H); 4.00 (s, 3H); 4.04 (s, 3H); (5h): 67% yield; Yellow solid; M.p. 98–99 C; IR
7.06 (dd, 1H, J1 = 2.3 Hz, J2 = 9.1 Hz); 7.33–7.39 (m, (KBr): 3317, 1692, 1450, 1217, 736 cm−1. H NMR
1

Pyranoindolones and Cytotoxicity
(500 MHz, CDCl3): δH 0.99 (t, 3H, J = 6.9 Hz); 1.56– 2.2g General procedure for the synthesis of pyrano
1.61 (m, 2H); 1.65–1.70 (m, 2H); 2.58 (t, 2H, J = [3,4-b]-indol-1(9H)-ones 7a-j: To a mixture of 3-
6.9 Hz); 4.00 (s, 3H); 7.20 (t, 1H, J = 6.9 Hz); 7.34 ethynyl-indole-2-carboxylic acid (1.0 mmol) in acetoni-
(t, 1H, J = 8.4 Hz); 7.38 (d, 1H, J = 7.6 Hz); 7.78 (d, trile (1 mL) under N2 atmosphere was added AuCl3
1H, J = 7.6 Hz); 9.25 (s, 1H). 13C NMR (125 MHz, (5 mol%) and refluxed for the specified time (table 1).
CDCl3): δC 13.8, 19.7, 22.0, 31.0, 52.2, 72.8, 97.5, After completion of the reaction as indicated by TLC,
105.2, 112.0, 121.2, 121.8, 126.2, 127.4, 129.6, 135.6, the reaction mixture was concentrated under reduced
162.2. MS (EI): m/z = 255 [M+]. Anal. Calcd for pressure and poured into water, and extracted with ethyl
C16H17NO2: C, 75.27; H, 6.71; N, 5.49%. Found: C, acetate (3 × 10 mL). The organic layer was dried over
75.51; H, 6.66; N, 5.41%.
anhydrous Na2SO4, filtered, concentrated, and puri-
fied by column chromatography on silica gel using
petroleum ether/ethyl acetate to afford the pure product.
2.2e9 Methyl-3-(2-phenylethynyl)-1H-indole-2-carbox-
ylate (5i): 85% yield; Brown solid; M.p. 188–189 ◦C;
IR (KBR): 3298, 1673, 1455, 1252, 740 cm−1. 1H NMR
(500 MHz, CDCl3): δH 4.05 (s, 3H); 7.25 (t, 1H, J =
7.6 Hz); 7.34–7.40 (m, 4H); 7.43 (d, 1H, J = 7.6 Hz);
7.61 (d, 2H, J = 7.6 Hz); 7.89 (d, 1H, J = 7.6 Hz). 13C
NMR (125 MHz, CDCl3): δC 52.4, 82.1, 96.1, 104.3,
112.1, 121.6, 121.8, 123.8, 126.5, 128.0, 128.3, 128.4,
129.2, 131.7, 135.6, 161.9%. MS (EI): m/z = 275
[M+]. Anal. Calcd for C18H13NO2: C, 78.53; H, 4.76;
N, 5.09%. Found: C, 78.85; H, 4.72; N, 5.02%.
2.2g1 9-Methyl-3-phenylpyrano[3,4-b]indol-1(9H)-one
(7a): 84% yield; Yellow solid; M.p. 168–170◦C; IR
1
(KBr): 1711, 1471, 1222, 1063, 742 cm−1. H NMR
(500 MHz, DMSO-d6, variable temperature): δH 4.02
(s, 3H); 7.22–7.23 (m, 1H); 7.36–7.56 (m, 5H); 7.81–
7.85 (m, 3H); 8.02–8.04 (m, 1H). 13C NMR (125 MHz,
DMSO-d6, variable temperature): δC 31.6, 98.3, 111.6,
120.9, 121.2, 121.4, 122.3, 124.8, 125.6, 128.3, 129.4,
129.5, 132.6, 141.5, 151.6, 156.1. MS (EI): m/z = 275
[M+]. Anal. Calcd for C18H13NO2: C, 78.53; H, 4.76;
N, 5.09%. Found: C, 78.69; H, 4.72; N, 5.03%.
2.2e10 5-Methoxy-3-phenylethynyl-1H-indole-2-car-
boxylic acid methyl ester (5j): 87% yield; Brown
solid; M.p. 202–203◦C; IR (KBr): 3289, 1687, 1469,
1209, 1020 cm−1. 1H NMR (500 MHz, CDCl3+
DMSO-d6): δH 3.79 (s, 3H); 3.90 (s, 3H); 6.89 (d, 1H,
J = 9.1 Hz); 7.09 (s, 1H); 7.23–7.30 (m, 4H); 7.50 (d,
2H, J = 6.9 Hz); 10.85 (s, 1H). 13C NMR (125 MHz,
CDCl3+ DMSO-d6): δC 47.2, 50.9, 78.1, 90.6, 96.3,
97.9, 109.0, 112.8, 119.2, 123.2, 123.6, 123.7, 124.6,
126.7, 126.8, 150.4, 157.0. MS (EI): m/z = 305 [M+].
Anal. Calcd for C19H15NO3: C, 74.74; H, 4.95; N,
4.59%. Found: C, 74.51; H, 4.91; N, 4.51%.
2.2g2 9-Ethyl-3-phenylpyrano[3,4-b]indol-1(9H)-one
(7b): 85% yield; Orange solid; M.p. 70–75◦C; IR
(KBr): 1712, 1469, 1220, 1061, 739 cm−1.1H NMR
(500 MHz, CDCl3): δH 1.44 (t, 3H, J = 7.6 Hz); 4.66
(q, 2H, J = 7.6 Hz); 7.24 (t, 1H, J = 6.9 Hz); 7.29
(s, 1H); 7.35 (d, 1H, J = 6.9 Hz); 7.41–7.43 (m, 3H);
7.49 (t, 1H, J = 6.9 Hz); 7.85–7.89 (m, 3H). 13C NMR
(125 MHz, CDCl3): δC 15.9, 39.6, 97.1, 110.7, 120.4,
120.9, 121.4, 121.7, 124.9, 125.9, 127.9, 128.8, 129.1,
132.6, 140.4, 152.4, 156.5. MS (EI): m/z = 289 [M+].
2.2f General procedure for the alkaline hydrolysis of
3-ethynyl-indole-2-carboxylic acid methyl ester 6a-j:
To a solution of methanol (3 mL), ethanol (1 mL)
and water (2 mL) was added 3-ethynyl-indole-2-
carboxylate (500 mg) and stirred at room temperature
for 5 min. To this reaction mixture was added NaOH
(1.5 equiv) and stirred at 75◦C for 1 h. After comple-
tion of the reaction as indicated by TLC, the reaction
mixture was concentrated under reduced pressure. The
residue was quenched with water (10 mL) and extracted
with Et2O (3 × 15 mL) to remove the organic impuri-
ties. The aqueous extract was acidified (pH = 1 to 2)
with 10% HCl and extracted with EtOAc (3 × 15 mL).
The combined organic extract was dried over anhy-
drous Na2SO4 and concentrated under reduced pressure
to afford pure product (6a–j), which was used for the
next step.
Table 1. AuCl3 catalyzed synthesis of pyrano[3,4-b]
indol-1(9H)-ones.
Entry Substrate Producta Time (min) Yield (%)b
1
2
3
4
5
6
7
8
9
10
6a
6b
6c
6d
6e
6f
6g
6h
6i
7a
7b
7c
7d
7e
7f
7g
7h
7i
20
20
30
05
05
15
15
20
45
40
84
85
83
92
91
88
87
79
75
77
6j
7j
1
aAll the Products were characterized by IR, H NMR, 13C
NMR and MS
bIsolated yield after column chromatography

J C Jeyaveeran et al.
Anal. Calcd for C19H15NO2: C, 78.87; H, 5.23; N, 20.1, 33.0, 44.7, 55.9, 97.2, 101.6, 112.0, 119.4, 121.1,
4.84%. Found: C, 78.00; H, 4.28; N, 5.41%.
121.5, 124.8, 125.1, 128.8, 128.9, 132.7, 136.3, 151.8,
154.8, 156.6. MS (EI): m/z = 347 [M+]. Anal. Calcd
for C22H21NO3: C, 76.06; H, 6.09; N, 4.03%. Found:
C, 75.89; H, 6.14; N, 4.13%.
2.2g3 9-Butyl-3-phenylpyrano[3,4-b]indol-1(9H)-one
(7c): 83% yield; Colourless liquid; IR (neat): 2949,
1
1714, 1059, 743 cm−1. H NMR (500 MHz, CDCl3):
δH 0.94 (t, 3H, J = 7.6 Hz); 1.36–1.42 (m, 2H); 1.84–
1.85 (m, 2H); 4.68 (t, 2H, J = 7.6 Hz); 7.27 (t, 1H,
J = 7.6 Hz); 7.36–7.39 (m, 2H); 7.43–7.53 (m, 4H);
7.89–7.94 (m, 3H).13C NMR (125 MHz, CDCl3): δC
14.2, 22.7, 31.7, 44.6, 97.1, 111.0, 120.9, 121.4, 121.7,
124.9, 125.9, 127.8, 128.8, 129.1, 132.7, 140.9, 152.5,
156.6. MS (EI): m/z = 317 [M+]. Anal. Calcd for
C21H19NO2: C, 79.47; H, 6.03; N, 4.41%. Found: C,
79.69; H, 6.20; N, 4.35%.
2.2g7 9-Benzyl-6-methoxy-3-phenylpyrano[3,4-b]indol-
1(9H)-one (7g): 87% yield; Yellow solid; M.p. 186–
188◦C; IR (KBr): 3446, 1694, 1233, 1069, 764,
1
693 cm−1. H NMR (500 MHz, CDCl3): δH 3.89 (s,
3H); 5.89 (s, 2H); 7.13 (d, 1H, J = 7.6 Hz); 7.21–
7.26 (m, 5H); 7.30 (s, 1H); 7.33–7.39 (m, 3H); 7.45
(t, 2H, J = 7.6 Hz); 7.89 (d, 2H, J = 7.6 Hz). 13C
NMR (125 MHz, CDCl3): δC 48.2, 55.9, 97.2, 101.8,
112.5, 119.6, 121.9, 124.9, 125.7, 127.1, 127.6, 128.8,
128.9, 129.1, 132.8, 136.4, 137.5, 152.2, 155.1, 156.8,
163.6. MS (EI): m/z = 381 [M+]. Anal. Calcd for
C25H19NO3: C, 78.72; H, 5.02; N, 3.67%. Found: C,
78.89; H, 4.98; N, 3.60%.
2.2g4 6-Methoxy-9-methyl-3-phenylpyrano[3,4-b]indol-
1(9H)-one (7d): 92% yield; Brown solid; M.p. 138–
140◦C; IR (KBr): 3254, 2550, 1666, 1569, 1365, 1057,
1
658 cm−1. H NMR (500 MHz, Acetone-d6, variable
temperature): δH 3.86 (s, 3H); 4.14 (s, 3H); 7.18 (s, 1H);
7.38–7.55 (m, 5H); 7.74 (s, 1H); 7.89–7.91 (m, 2H).
13C NMR (125 MHz, Acetone-d6, variable tempera-
ture): δC 30.7, 55.1, 97.5, 101.7, 110.9, 111.9, 119.2,
121.2, 121.6, 124.4, 124.7, 128.7, 128.8, 128.9, 136.9,
151.3, 155.2. MS (EI): m/z = 305 [M+]. Anal. Calcd
for C19H15NO3: C, 74.74; H, 4.95; N, 4.59%. Found:
C, 74.99; H, 4.90; N, 4.51%.
2.2g8 3-Butylpyrano[3,4-b]indol-1(9H)-one (7h): 79%
yield; Colourless solid; M.p. 158–160◦C; IR (KBr):
3261, 1688, 1621, 748 cm−1. 1H NMR (500 MHz,
CDCl3+ DMSO-d6): δH 0.86 (t, 3H, J = 6.9 Hz);
1.31–1.33 (m, 2H); 1.60–1.63 (m, 2H); 2.54 (t, 2H,
J = 6.9 Hz); 6.62 (s, 1H); 7.11 (t, 1H, J = 7.6 Hz); 7.34
(t, 1H, J = 7.6 Hz); 7.50 (d, 1H, J = 8.4 Hz); 7.73 (d,
1H, J = 8.4 Hz); 11.17 (s, 1H). 13C NMR (125 MHz,
CDCl3+ DMSO-d6): δC 13.8, 22.1, 29.6, 33.3, 98.5,
113.2, 120.5, 120.6, 121.3, 121.5, 126.0, 127.6, 140.3,
156.7, 158.3. MS (EI): m/z = 241 [M+]. Anal. Calcd
for C15H15NO2: C, 74.67; H, 6.27; N, 5.81%. Found: C,
74.89; H, 6.24; N, 5.75%.
2.2g5 9-Ethyl-6-methoxy-3-phenylpyrano[3,4-b]indol-
1(9H)-one (7e): 91% yield; Brown solid; M.p. 118–
120◦C; IR (KBr): 3447, 1711, 1460, 1237, 1056,
1
759 cm−1. H NMR (500 MHz, Acetone-d6): δH 1.37
(t, 3H, J = 6.9 Hz); 3.86 (s, 3H); 4.70 (q, 2H, J =
7.6 Hz); 7.17 (dd, 1H, J1 = 2.3 Hz, J2 = 6.9 Hz); 7.37
(t, 1H, J = 7.6 Hz); 7.46 (t, 2H, J = 7.6 Hz); 7.56–7.58
(m, 2H); 7.76 (s, 1H); 7.89 (d, 2H, J = 6.9 Hz). 13C
NMR (125 MHz, Acetone-d6): δC 15.4, 39.4, 55.1,
97.5, 101.9, 111.9, 119.3, 120.4, 121.9, 124.5, 125.1,
128.8, 128.9, 133.0, 135.8, 151.4, 155.1, 155.6. MS
(EI): m/z = 319 [M+]. Anal. Calcd for C20H17NO3: C,
75.22; H, 5.37; N, 4.39%. Found: C, 75.39; H, 5.33; N,
4.32%.
2.2g9 3-Phenylpyrano[3,4-b]indol-1(9H)-one (7i): 75%
yield; Yellow solid; M.p. 171–173◦C; IR (KBr): 3401,
1
2921, 1686, 742 cm−1. H NMR (500 MHz, CDCl3 +
DMSO-d6): δH 7.13 (t, 1H, J = 6.8 Hz); 7.17–7.19 (m,
1H); 7.27 (d, 1H, J = 6.8 Hz); 7.34–7.37 (m, 3H);
7.39 (s, 1H); 7.45–7.50 (m, 1H); 7.79 (d, 1H, J =
7.6 Hz); 7.84 (d, 1H, J = 7.6 Hz); 11.94 (s, 1H). 13C
NMR (125 MHz, CDCl3+ DMSO-d6): δC 97.7, 113.4,
120.8, 121.4, 121.9, 124.3, 124.8, 125.6, 127.7, 128.9,
129.0, 132.8, 140.6, 151.9, 157.0. MS (EI): m/z = 261
[M+]. Anal. Calcd for C17H11NO2: C, 78.15; H, 4.24;
N, 5.36%. Found: C, 78.39; H, 4.19; N, 5.26%.
2.2g6 9-Butyl-6-methoxy-3-phenylpyrano[3,4-b]indol-1
(9H)-one (7f): 88% yield; Yellow liquid; IR (neat):
1
2928, 1711, 1501, 1239, 1060, 762 cm−1. H NMR
(500 MHz, CDCl3): δH 0.92 (t, 3H, J = 7.6 Hz); 1.34–
1.38 (m, 2H); 1.81–1.84 (m, 2H); 3.91 (s, 3H); 4.64 2.2g10 6-Methoxy-3-phenylpyrano[3,4-b]indol-1(9H)-
(t, 2H, J = 6.8 Hz); 7.17 (dd, 1H, J1 = 11.45; J2 = one (7j): 77% yield; Brown solid; M.p. 232–234◦C;
1
2.3 Hz); 7.29 (d, 1H, J = 2.3 Hz); 7.33 (s, 1H); 7.36– IR (KBr): 3223, 1707, 1480, 1219, 1080 cm−1. H
7.39 (m, 2H); 7.44 (t, 2H, J = 7.6 Hz); 7.88 (d, 2H, NMR (500 MHz, CDCl3+ DMSO-d6): δH 3.78 (s, 3H);
J = 7.6 Hz). 13C NMR (125 MHz, CDCl3): δC 13.9, 6.98 (d, 1H, J = 6.9 Hz); 7.23–7.27 (m, 2H); 7.32–7.38

Pyranoindolones and Cytotoxicity
(m, 4H); 7.77 (d, 2H, J = 7.6 Hz); 11.86 (s, 1H). 13C
NMR (125 MHz, CDCl3+ DMSO-d6): δC 55.7, 97.8,
101.4, 114.3, 119.2, 121.7, 122.2, 124.7, 125.0, 128.9
(2 carbon), 132.9, 135.9, 151.3, 154.7, 156.9. MS (EI):
m/z = 291 [M+]. Anal. Calcd for C18H13NO3: C,
74.22; H, 4.50; N, 4.81%. Found: C, 74.49; H, 4.44; N,
4.73%.
layer was dried over anhydrous Na2SO4, filtered, con-
centrated and purified by column chromatography on
silica gel using petroleum ether/EtOAc to afford the
pure product.
2.2j1 Methyl 2-bromo-1-methyl-1H-indole-3-carbox-
ylate (11a): 92% yield; colourless liquid; IR (neat):
1
2919, 2355, 1709, 1457, 1245, 1108, 751 cm−1. H
NMR (500 MHz, CDCl34): δH 3.75 (s, 3H); 4.01 (s,
3H); 7.42 (d, 1H, J = 7.6 Hz); 7.55 (t, 1H, J = 8.4 Hz);
2.2h Experimental procedure for the synthesis of 2-
bromo-indole-3-carbaldehyde 9a: To a solution of
anhydrous DMF (27.5 g) in anhydrous 1,2-dichloro-
ethane (100 mL) was added POBr3 (26.8 g, 0.0937 mol)
dropwise at 0◦C under argon atmosphere. The resulting
mixture was allowed to stir for 30 min followed by addi-
tion with a solution of 2-oxindole (5.0 g, 0.0375 mol)
in dichloroethane (100 mL). The resulting mixture was
stirred at room temperature for 24 h and quenched with
ice-cold solution of excess NaOAc.3H2O. The prod-
uct was extracted with dichloroethane (3 × 100 mL),
dried over anhydrous sodium sulphate and evaporated
under reduced pressure. The crude product was further
purified by recrystallization using 95% EtOH to afford
5.9 g (82% yield) of analytically pure compound 9a as
a beige solid; M.p. 220 to 222◦C. 1H NMR (500 MHz,
DMSO-d6): δH 7.00–7.05 (m, 2 H), 7.21 (d, 1H, J =
7.6 Hz), 8.02 (d, 1H, J = 7.6 Hz), 9.80 (s, 1H), 12.21 (s,
1H).
7.67 (t, 1H, J = 8.4 Hz); 8.21 (d, 1H, J = 7.6 Hz). 13
C
NMR (125 MHz, CDCl3): δC 33.4, 51.9, 109.9, 110.3,
111.1, 120.5, 121.8, 122.2, 128.9, 138.8, 164.1. MS
(EI): m/z = 267 [M+], 269 [M+2]
2.2j2 Methyl 2-bromo-1-ethyl-1H-indole-3-carboxyl-
ate (11b): 89% yield; colourless solid; M.p. 73–75◦C;
IR (KBr): 2925, 1705, 1459, 1218, 1009, 741 cm−1. 1H
NMR (500 MHz, CDCl3): δH 1.42 (t, 3H, J = 7.6 Hz);
3.95 (s, 3H); 4.32 (q, 2H, J = 7.6 Hz); 7.45 (d, 1H, J =
7.6 Hz); 7.95 (t, 1H, J = 8.4 Hz); 8.03 (t, 1H, J =
8.4 Hz); 8.44 (d, 1H, J = 7.66 Hz). 13C NMR (125 MHz,
CDCl3): δC 14.6, 43.0, 52.6, 110.6, 111.2, 112.1, 120.0,
122.8, 123.1, 132.1, 138.6, 166.7. MS(EI): m/z = 281
[M+], 283 [M+2]
2.2k General procedure for the N-alkylation of 3-
iodo-indole-2-carboxylic acid methyl ester 11c and
11d: To a mixture of KOH (3.0 equiv) in DMSO
(3 mL) kept at 0◦C was added, 2-bromo-indole-2-
carboxylic acid methyl ester 10a (1.0 equiv) and stirred
for 30 min. To this mixture was added the correspond-
ing alkyl halide (5.0 equiv) and stirred for 6 h at 0◦C to
room temperature. After completion of the reaction as
indicated by TLC, the reaction mixture was quenched
with water and extracted with EtOAc (3 × 15 mL). The
organic layer was dried over anhydrous Na2SO4, fil-
tered, concentrated and purified by column chromatog-
raphy on silica gel using petroleum ether/EtOAc to
afford the pure product.
2.2i Experimental procedure for the oxidative esterifi-
cation of 9a to 10a: To a solution of 2-bromo-indole-
3-carbaldehyde (5.0 g, 0.0223 mol) in dry MeOH
R
(100 mL) was added oxoneꢀ (3.56 g, 0.0234 mol) por-
tion wise and stirred at room temperature. After 3 h,
the reaction mixture was quenched with water (100 mL)
and extracted with dichloromethane (3 × 100 mL). The
organic layer was dried over anhydrous sodium sul-
phate and concentrated under reduced pressure to afford
the ester which was used for the next step without
further purification.
2.2j General procedure for the N-alkylation of 2- 2.2k1 Methyl 2-bromo-1-butyl-1H-indole-3-carbox-
bromo-indole-3-carboxylic acid methyl ester 11a and ylate (11c): 75% yield; Colourless solid; M.p. 113–
11b: To a mixture of NaH (2.0 equiv) in DMF (3 mL) 115◦C; IR (KBr): 2959, 1715, 1460, 1358, 1256, 1202,
kept at 0◦C at N2 atmosphere was added a solution 1144, 746 cm−1. 1H NMR (500 MHz, CDCl3): δH 0.88
of 2-bromo-indole-3-carboxylic acid methyl ester 10a (t, 3H, J = 7.6 Hz); 1.29–1.35 (m, 2H); 1.65–1.71 (m,
(1.0 equiv) in DMF (2 mL) and stirred for 15 min. To 2H); 3.97 (s, 3H); 4.21 (t, 2H); 7.34 (d, 1H, J = 8.1 Hz);
this mixture was added the corresponding alkyl halide 7.75 (t, 1H, J = 8.4 Hz); 8.01 (t, 1H, J = 8.4 Hz); 8.38
(1.2 equiv) and stirred for 3 h at 0◦C to room temper- (d, 1H, J = 8.1 Hz). 13C NMR (125 MHz, CDCl3): δC
ature. After completion of the reaction as indicated by 13.5, 20.3, 33.3, 52.8, 53.7, 110.1, 110.9, 111.2, 119.7,
TLC, the reaction mixture was quenched with water 121.9, 122.6, 129.6, 138.6, 166.3. MS(EI): m/z = 309
and extracted with EtOAc (3 × 10 mL). The organic [M+], 311 [M+2]

J C Jeyaveeran et al.
2.2k2 Methyl 1-benzyl-2-bromo-1H-indole-3-carbox- CDCl3): δH 0.91 (t, 3H, J = 7.6 Hz); 1.32–1.37 (m,
ylate (11d): 80% yield; Colourless solid; M.p. 153– 2H); 1.69–1.75 (m, 2H); 3.95 (s, 3H); 4.19 (t, 2H, J =
155◦C; IR (KBr): 2949, 1716, 1455, 1255, 1209, 6.8 Hz); 7.35–7.45 (m, 4H); 7.55 (d, 2H, J = 6.9 Hz);
767 cm−1. 1H NMR (500 MHz, CDCl3): δH 3.88 (s, 3H); 7.77 (t, 1H, J = 7.8 Hz); 7.92 (t, 1H, J = 7.8 Hz); 8.03
5.67 (s, 2H); 7.27–7.34 (m, 2H); 7.39–7.42 (m, 2H); (d, 1H, J = 7.1 Hz). 13C NMR (500 MHz, CDCl3): δC
7.77 (d, 1H, J = 8.2 Hz); 7.86–7.94 (m, 2H); 8.19 (d, 13.5, 20.4, 32.4, 52.1, 52.3, 86.6, 90.1, 109.4, 110.5,
1H, J = 8.2 Hz). 13C NMR (125 MHz, CDCl3): δC 51.3, 111.2, 119.5, 121.3, 121.9, 122.4, 127.1, 128.3, 128.5,
55.9, 110.1, 110.9, 111.4, 120.1, 121.8, 122.9, 126.2, 133.3, 138.1, 165.4. MS(EI): m/z = 331 [M+]
127.2, 129.0, 134.3, 137.9, 138.2, 166.8. MS(EI):
m/z = 343 [M+], 345 [M+2]
2.2l4 Methyl 1-benzyl-2-(phenylethynyl)-1H-indole-
3-carboxylate (12d): 91% yield; Yellow solid; M.p.
168–170◦C; IR (KBr): 3356, 2948, 1701, 1245, 1211,
2.2l General procedure for the synthesis of 2-ethynyl-
indole-3-carboxylic acid methyl ester 12a-j: To a mix-
713 cm−1. 1H NMR (500 MHz, CDCl3): δH 3.99 (s, 3H);
5.71 (s, 2H); 7.21–7.34 (m, 5H); 7.43–7.51 (m, 3H);
7.58–7.73 (m, 5H); 7.98 (d, 1H, J = 6.9 Hz). 13C NMR
(500 MHz, CDCl3): δC 51.9, 56.0, 87.2, 90.1, 109.5,
110.1, 110.4, 120.0, 121.4, 122.1, 122.5, 125.5, 126.5,
127.4, 128.3, 128.5, 128.7, 132.6, 136.9, 137.2, 166.6.
MS(EI): m/z = 365 [M+]
ture of 2-bromo-indole-3-carboxylic acid methyl ester
(1.0 mmoL), Pd(Ph3P)4 (5.0 mol%) and CuI (5.0 mol%)
under N2 atmosphere was added triethylamine (5 mL)
and stirred for 30 min. To this reaction mixture was
added the appropriate terminal alkyne (1.1 mmol) and
stirred at room temperature for 10 h. After completion
of the reaction as indicated by TLC, the reaction mix-
ture was quenched with water (50 mL) and extracted
with EtOAc (3 × 15 mL). The organic layer was dried
over anhydrous Na2SO4, filtered, concentrated and puri-
fied by column chromatography on silica gel using
petroleum ether/EtOAc to afford the pure product.
2.2l5 Methyl 2-(hex-1-yn-1-yl)-1-methyl-1H-indole-
3-carboxylate (12e): 74% yield; Colourless solid;
M.p. 103–105◦C; IR (KBr): 3315, 1695, 1455, 1211,
746 cm−1. 1H NMR (500 MHz, CDCl3): δH 0.88 (t, 3H,
J = 7.6 Hz); 1.31–1.35 (m, 2H); 1.41–1.44 (m, 2H);
3.72 (s, 3H); 4.08 (s, 3H); 7.28 (d, 1H, J = 8.4 Hz);
7.57 (t, 1H, J = 7.2 Hz); 7.71 (t, 1H, J = 8.4 Hz); 7.89
(d, 1H, J = 7.1 Hz). 13C NMR (500 MHz, CDCl3):
δC 13.0, 21.4, 22.1, 30.5, 36.3, 51.4, 76.5, 77.2, 109.8,
110.7, 112.3, 119.5, 120.4, 122.1, 127.9, 136.7, 167.1.
MS(EI): m/z = 269 [M+]
2.2l1 Methyl 1-methyl-2-(phenylethynyl)-1H-indole-
3-carboxylate (12a): 85% yield; Yellow solid; M.p.
89–91◦C; IR (KBr): 3381, 2382, 1711, 1475, 1265,
1101, 752 cm−1. 1H NMR (500 MHz, CDCl3): δH 3.87
(s, 3H); 4.01 (s, 3H); 7.36–7.59 (m, 6H); 7.67 (t, 1H,
J = 7.6 Hz); 7.82 (t, 1H, J = 7.6 Hz); 8.14 (d, 1H,
J = 8.1 Hz). 13C NMR (500 MHz, CDCl3): δC 37.0,
52.1, 88.1, 90.2, 110.1, 111.9, 122.4, 122.9, 124.1,
125.3, 126.6, 126.8, 129.5, 130.3, 132.1, 136.8. 165.5.
MS(EI): m/z = 289 [M+]
2.2l6 Methyl 1-ethyl-2-(hex-1-yn-1-yl)-1H-indole-3-
carboxylate (12f): 77% yield; Colourless solid; M.p.
109–111◦C; IR (KBr): 3325, 1702, 1457, 1218, 739
1
cm−1. H NMR (500 MHz, CDCl3): δH 0.91 (t, 3H,
J = 7.1 Hz); 1.31 (t, 3H, J = 7.6 Hz); 1.37–1.41 (m,
2H); 1.44–1.47 (m, 2H); 2.42 (t, 2H, J = 6.9 Hz); 4.01
(s, 3H); 4.25 (q, 2H, J = 7.6 Hz); 7.34 (d, 1H, J =
8.1 Hz); 7.78–7.85 (m, 2H); 7.91 (d, 1H, J = 8.2 Hz).
13C NMR (500 MHz, CDCl3): δC 13.3, 14.3, 21.4, 21.9,
30.5, 42.1, 51.8, 76.8, 77.6, 109.8, 110.2, 111.1, 119.5,
121.5, 122.6, 127.6, 138.0, 166.1. MS(EI): m/z = 283
[M+]
2.2l2 Methyl 1-ethyl-2-(phenylethynyl)-1H-indole-3-
carboxylate (12b): 87% yield; Yellow solid; M.p.
103–105◦C; IR (KBr): 3398, 2975, 1709, 1467, 1250,
1126, 745 cm−1. 1H NMR (500 MHz, CDCl3): δH 1.27
(t, 3H, J = 7.7 Hz); 3.92 (s, 3H); 4.19 (q, 2H, J =
7.7 Hz); 7.38–7.48 (m, 4H); 7.59 (d, 2H, J = 6.9 Hz);
7.71 (t, 1H, J = 7.2 Hz); 7.86 (t, 1H, J = 7.2 Hz); 8.04
(d, 1H, J = 7.6 Hz). 13C NMR (500 MHz, CDCl3): δC
14.3, 42.5, 51.6, 86.8, 90.4, 109.7, 110.4, 110.9, 119.7,
121.8, 122.4, 126.9, 137.1, 165.7. MS(EI): m/z = 303
[M+]
2.2l7 Methyl 1-benzyl-2-(hex-1-yn-1-yl)-1H-indole-
3-carboxylate (12g): 72% yield; Pale yellow solid;
M.p. 134–136◦C; IR (KBr): 3346, 1699, 1461, 1213,
1
749 cm−1. H NMR (500 MHz, CDCl3): δH 0.90 (t,
2.2l3 Methyl 1-butyl-2-(phenylethynyl)-1H-indole-3- 3H, J = 7.6 Hz); 1.35–1.44 (m, 4H); 2.55 (t, 2H, J =
carboxylate (12c): 81% yield; Pale yellow solid; M.p. 6.9 Hz); 4.05 (s, 3H); 7.22–7.35 (m, 5H); 7.53–7.78 (m,
114–116◦C; IR (KBr): 3367, 2925, 1710, 1465, 1365, 3H); 7.91 (d, 1H, J = 7.6 Hz). 13C NMR (500 MHz,
1
1240, 1210, 1129, 763 cm−1. H NMR (500 MHz, CDCl3): δC 13.1, 21.3, 21.9, 31.0, 52.3, 56.2, 76.5,

Pyranoindolones and Cytotoxicity
77.4, 109.6, 110.2, 111.3, 119.4, 120.1, 122.4, 125.5, and concentrated under reduced pressure to afford pure
126.6, 127.8, 128.0, 135.4, 136.8, 165.4. MS(EI): product (13a-j), which is used for the next step without
m/z = 345 [M+]
purification.
2.2l8 Methyl 1-benzyl-2-(hept-1-yn-1-yl)-1H-indole-
3-carboxylate (12h): 67% yield; Yellow solid; M.p.
151–153◦C; IR (KBr): 3317, 1703, 1455, 1218, 749
cm−1. 1H NMR (500 MHz, CDCl3): δH 0.89 (t, 3H, J =
7.6 Hz); 1.27–1.51 (m, 6H); 2.49 (t, 2H, J = 6.9 Hz);
3.88 (s, 3H); 5.55 (s, 2H); 7.26–7.7.58 (m, 6H); 7.69–
7.74 (m, 2H); 7.95 (d, 1H, J = 8.1 Hz). 13C NMR
(500 MHz, CDCl3): δC 14.3, 21.5, 28.3, 30.5, 30.8,
51.9, 55.5, 78.7, 79.9, 109.5, 111.1, 111.7, 120.1, 121.2,
122.4, 125.4, 126.3, 126.9, 127.5, 135.3, 137.3, 167.5.
MS(EI): m/z = 359 [M+]
2.2n General procedure for the synthesis of pyrano
[4,3-b]-indol-1(5H)-ones 14a-j: To a mixture of 2-
ethynyl-indole-3-carboxylic acid 13a-j (1.0 mmoL) in
acetonitrile (1 mL) under N2 atmosphere was added
AuCl3 (5 mol%) and refluxed for the specified time
(table 2). After completion of the reaction as indicated
by TLC, the reaction mixture was concentrated under
reduced pressure and poured into water, and extracted
with ethyl acetate (3 × 10 mL). The organic layer was
dried over anhydrous Na2SO4, filtered, concentrated,
and purified by column chromatography on silica gel
using petroleum ether/ethyl acetate to afford the pure
product.
2.2l9 Methyl 1-benzyl-2-(3-hydroxy-3-methylbut-1-
yn-1-yl)-1H-indole-3-carboxylate (12i): 65% yield;
Colourless paste; IR (CH2Cl2): 3489, 3401, 1702,
1449, 1219, 768 cm−1 1H NMR (500 MHz, CDCl3):
δH 1.55 (s, 6H); 2.64 (brs, 1H); 3.77 (s, 3H); 5.46 (s,
2H); 7.19–7.37 (m, 6H); 7.49–7.65 (m, 2H); 7.77–7.84
(m, 1H). 13C NMR (500 MHz, CDCl3): δC 33.1, 50.9,
55.8, 77.7, 80.8, 110.9, 111.9, 112.8, 120.3, 121.5,
122.3, 125.4, 126.5, 128.4, 128.7, 136.8, 138.4, 166.0.
MS(EI): m/z = 348 [M++1]
2.2n1 5-Methyl-3-phenylpyrano[4,3-b]indol-1(5H)-
one (14a): 80% yield; Colourless solid; M.p. 77–
79◦C; IR (KBr): 3398, 1708, 1476, 11198, 1166, 758
1
cm−1. H NMR (500 MHz, CDCl3): δH 4.00 (s, 3H);
6.38 (s, 1H); 7.30–7.45 (m, 5H); 7.62–7.76 (m, 3H);
8.01 (d, 1H, J = 6.9 Hz). 13C NMR (500 MHz, CDCl3):
δC 33.9, 98.3, 108.5, 109.9, 120.0, 121.9, 122.1,
125.0, 125.8, 127.4, 127.7, 128.1, 130.4, 136.7, 160.1,
165.4. MS(EI): m/z = 275 [M-H]−; Anal. Calcd for
C18H13NO2: C, 78.53; H, 4.76; N, 5.09%. Found: C,
79.01.49; H, 4.69; N, 4.98%.
2.2l10 Methyl 1-benzyl-2-(5-hydroxypent-1-yn-1-yl)-
1H-indole-3-carboxylate (12j): 60% yield; Colour-
less paste; IR (CH2Cl2): 3456, 3389, 1688, 1458, 1212,
745 cm−1 1H NMR (500 MHz, CDCl3): δH 1.63–1.68
(m, 2H); 2.52 (brs, 1H); 2.55 (t, 2H, J = 6.9 Hz);
3.55–3.63 (m, 2H); 3.93 (s, 3H); 5.45 (s, 2H); 7.28–
7.53 (m, 6H); 7.62–7.68 (m, 2H); 7.72–7.76 (m, 1H).
13C NMR (500 MHz, CDCl3): δC 14.8, 31.5, 52.4, 54.9,
62.1, 79.9, 86.7, 109.2, 109.8, 110.8, 119.1, 120.3,
121.7, 125.7, 126.5, 127.2, 128.5, 138.5, 139.1, 161.1.
MS(EI): m/z = 348 [M++1]
2.2n2 5-Ethyl-3-phenylpyrano[4,3-b]indol-1(5H)-
one (14b): 78% yield; Colourless solid; M.p. 83–
85◦C; IR (KBr): 3388, 1715, 1475, 1230, 1055, 775
cm−1.1H NMR (500 MHz, CDCl3): δH1.28 (t, 3H, J
= 7.6 Hz); 4.58 (q, 2H, J = 7.6 Hz); 6.40 (s, 1H);
Table 2. AuCl3 catalyzed synthesis of pyrano[4,3-b]
indol-1(5H)-ones.
2.2m General procedure for the alkaline hydroly-
sis of 2-ethynyl-indole-3-carboxylic acid methyl ester
13a-j: To a solution of methanol (3 mL), ethanol (1 mL)
and water (2 mL) was added 2-ethynyl-indole-3-car-
boxylate (500 mg) and stirred at room temperature for
5 min. To this reaction mixture was added NaOH (1.5
equiv) and stirred at 75◦C for 1 h. After completion of
the reaction as indicated by TLC, the reaction mixture
was concentrated under reduced pressure. The residue
was quenched with water (10 mL) and extracted with
Et2O (3 × 15 mL) to remove the organic impurities. The
aqueous extract was acidified (pH = 1 to 2) with 10%
HCl and extracted with EtOAc (3 × 15 mL). The com-
bined organic extract was dried over anhydrous Na2SO4
Entry Substrate Producta Time (min) Yield (%)b
1
2
3
4
5
6
7
8
9
10
13a
13b
13c
13d
13e
13f
13g
13h
13i
14a
14b
14c
14d
14e
14f
14g
14h
14i
50
60
90
40
90
90
90
90
90
90
80
78
75
81
74
73
67
65
47
42
13j
14j
1
aAll the Products were characterized by IR, H NMR, 13C
NMR and MS
bIsolated yield after column chromatography

J C Jeyaveeran et al.
7.33–7.49 (m, 5H); 7.60–7.77 (m, 3H); 7.98 (d, 1H, 2H, J = 7.1 Hz); 4.49 (q, 2H, J = 7.6 Hz); 5.86 (s, 1H);
J = 8.4 Hz). 13C NMR (500 MHz, CDCl3): δC 15.5, 7.44–7.56 (m, 3H); 7.67–7.74 (m, 1H). 13C NMR (500
45.3, 98.1, 108.3, 109.7, 120.2, 121.1, 122.0, 124.6, MHz, CDCl3): δC 13.9, 15.2, 22.5, 26.4, 33.6, 43.6,
125.5, 127.1, 127.6, 128.4, 130.0, 136.4, 160.3, 165.2. 98.7, 109.2, 110.8, 119.8, 122.9, 123.6, 127.2, 139.4,
MS(EI): m/z = 289 [M+NH4]+; Anal. Calcd for 161.5, 166.4. MS(EI): m/z = 269 [M+]. Anal. Calcd
C19H15NO2: C, 78.87; H, 5.23; N, 4.84%. Found: C, for C17H19NO2: C, 75.81; H, 7.11; N, 5.20%. Found:
79.21; H, 5.16; N, 4.75%.
C, 76.25; H, 7.19; N, 5.32%.
2.2n3 5-Butyl-3-phenylpyrano[4,3-b]indol-1(5H)-one
(14c): 75% yield; Colourless solid; M.p. 100–102◦C;
IR (KBr): 3401, 2952, 1721, 1065, 753 cm−1. 1H NMR
(500 MHz, CDCl3): δH 0.91 (t, 3H, J = 7.6 Hz); 1.42–
1.78 (m, 4H); 4.18 (t, 2H, J = 6.9 Hz); 6.31 (s, 1H);
7.32–7.49 (m, 5H); 7.61–781 (m, 3H); 8.02 (d, 1H, J
= 7.6 Hz). 13C NMR (500 MHz, CDCl3): δC 13.2, 20.6,
33.9, 54.7, 97.9, 108.9, 109.9, 120.8, 121.8, 122.5,
124.3, 125.8, 127.7, 127.9, 128.8, 130.7, 136.7, 1609,
165.9. MS(EI): m/z = 316 [M-H]−; Anal. Calcd for
C21H19NO2: C, 79.47; H, 6.03; N, 4.41%. Found: C,
78.99; H, 6.11; N, 4.55%.
2.2n7 5-Benzyl-3-butylpyrano[4,3-b]indol-1(5H)-one
(14g): 67% yield; Colourless solid; M.p. 103–105◦C;
1
IR (KBr): 3148, 3025, 1705, 1625, 832, 717 cm−1. H
NMR (500 MHz, CDCl3): δH (0.91 (t, 3H, J = 7.6 Hz);
1.29–1.38 (m, 4H); 2.00 (t, 2H, J = 6.9 Hz); 5.82 (s,
2H); 5.91 (s, 3H); 7.22–7.48 (m, 7H); 7.77 (d, 1H, J
= 8.1 Hz); 7.94 (d, 1H, J = 8.1 Hz). 13C NMR (500
MHz, CDCl3): δC 14.2, 22.5, 25.9, 33.3, 51.3, 100.1,
108.5, 110.1, 119.9, 122.4, 125.3, 125.7, 127.7, 129.2,
133.9, 139.9, 161.8, 167.6. MS(EI): m/z = 330 [M-
H]−; Anal. Calcd for C22H21NO2: C, 79.73; H, 6.39; N,
4.23%. Found: C, 80.09; H, 6.50; N, 4.37%.
2.2n4 5-Benzyl-3-phenylpyrano[4,3-b]indol-1(5H)-
one (14d): 81% yield; Pale yellow solid; M.p. 210–
212◦C; νmax(KBr)/cm−1 3364, 2960, 2921, 2850, 2358,
2.2n8 5-Benzyl-3-pentylpyrano[4,3-b]indol-1(5H)-one
(14h): 65% yield; Colourless solid; M.p. 120–122◦C;
IR (KBr): 3155, 3035–2865, 1715, 1625, 1610, 848
1
1
cm−1. H NMR (500 MHz, CDCl3): δH 0.89 (t, 3H,
2345, 2079, 1715, 1455, 1260, 799; H NMR (500
MHz, CDCl3): δH 5.51 (s, 2H); 6.94 (s, 1H); 7.11–7.17
(m, 2H); 7.30–7.40 (m, 6H); 7.42–7.50 (m, 3H); 7.85–
7.95 (m, 2H); 8.25–8.35 (m, 1H); 13C NMR (500 MHz,
CDCl3): δC 47.7, 91.4, 100.9, 110.3, 121.9, 123.3,
124.8, 125.3, 126.1, 126.6, 128.6, 129.3, 129.6, 130.8,
132.6, 135.9, 139.2, 146.7, 159.1, 159.6; MS(EI):
m/z = 352 [M+H]+; Anal. Calcd for C24H17NO2: C,
82.03; H, 4.88; N, 3.99%. Found: C, 81.79; H, 4.95; N,
4.09%.
J = 6.6 Hz); 1.25–1.33 (m, 4H); 1.66–1.80 (m, 2H);
2.55 (t, 2H, J = 8.1 Hz); 5.39 (s, 2H); 6.25 (s, 1H);
7.02–7.11 (m, 3H); 7.24–7.33 (m, 6H); 7.00–8.04 (m,
1H). 13C NMR (500 MHz, CDCl3): δC 14.2, 22.6,
27.0, 31.2, 34.5, 47.6, 92.8, 99.5, 110.0, 121.3, 122.9,
124.5, 126.2, 128.1, 129.2, 135.6, 138.3, 146.6, 160.1,
164.5. MS(EI): m/z = 346 [M+H]+; Anal. Calcd for
C23H23NO2: C, 79.97; H, 6.71; N, 4.05%. Found: C,
80.25; H, 6.77; N, 4.16%.
2.2n5 3-Butyl-5-methylpyrano[4,3-b]indol-1(5H)-one
(14e): 74% yield; Colourless solid; M.p. 87–89◦C; IR
2.2n9 5-Benzyl-3-(2-hydroxypropan-2-yl)pyrano[4,3-
b]indol-1(5H)-one (14i): 47% yield; Colourless
solid; M.p. 155–157◦C; IR (KBr): 3589, 3449, 3115–
1
(KBr): 3055, 3012, 17 01, 1622, 1611, 812 cm−1. H
1
2915, 1700, 1681, 834 cm−1. H NMR (500 MHz,
NMR (500 MHz, CDCl3): δH 0.91 (t, 3H, J = 7.6 Hz);
1.27–1.44 (m, 4H); 4.04 (s, 3H); 5.87 (s, 1H); 7.62
(d, 1H, J = 8.1 Hz); 7.69–7.75 (m, 2H); 7.96 (d, 1H,
J = 8.1 Hz). 13C NMR (500 MHz, CDCl3): δC 14.1,
22.5, 25.9, 33.2, 33.9, 99.9, 109.8, 110.2, 119.2, 122.3,
123.2, 127.8, 138.9, 161.4, 167.8. MS(EI): m/z = 255
[M+]. Anal. Calcd for C16H17NO2: C, 75.27; H, 6.71;
N, 5.49%. Found: C, 74.89; H, 6.79; N, 5.61%.
CDCl3): δH 1.65 (s, 6H); 3.02 (brs, 1H); 5.30 (s, 2H);
6.81 (s, 1H); 6.99–7.06 (m, 2H); 7.22–7.37 (m, 6H);
8.19–8.25 (m, 1H). 13C NMR (500 MHz, CDCl3): δC
28.6, 47.0, 71.9, 89.8, 99.5, 110.0, 121.2, 122.9, 124.1,
124.6, 126.2, 128.0, 129.0, 135.6, 138.3, 146.4, 159.5,
168.5. MS(EI): m/z = 332 [M-H]+; Anal. Calcd for
C21H19NO3: C, 75.66; H, 5.74; N, 4.20%. Found: C,
76.01; H, 5.65; N, 4.08%.
2.2n6 3-Butyl-5-ethylpyrano[4,3-b]indol-1(5H)-one
(14f): 73% yield; Colourless solid; M.p. 94–96◦C; IR 2.2n10 5-Benzyl-3-(3-hydroxypropan-2-yl)pyrano[4,
1
(KBr): 3148, 2978, 1712, 1625, 1605, 834 cm−1. H 3-b]indol-1(5H)-one (14j): 42% yield; Orange solid;
NMR (500 MHz, CDCl3): δH 0.88 (t, 3H, J = 6.9 Hz); M.p. 148–150◦C; IR (KBr): 3415, 2925, 2859, 1695,
1
1.28 (t, 3H, J = 7.6 Hz); 1.31–1.1.38 (m, 4H); 1.89 (t, 1559, 832, 789 cm−1. H NMR (500 MHz, CDCl3):

Pyranoindolones and Cytotoxicity
δH 1.69 (brs, 1H); 1.91–2.05 (m, 2H), 2.75 (t, 2H, J = side chains were replaced using the ADT. Gasteiger
7.6 Hz), 3.70 (t, 2H, J = 6.4 Hz), 5.38 (s, 2H), 6.35 charges were added to each atom and merged the non-
(s, 1H), 7.03–7.12 (m, 2H); 7.22–7.38 (m, 6H); 8.15– polar hydrogen atoms to the protein structure. The
8.25 (m, 1H). 13C NMR (500 MHz, CDCl3): δC 30.3, hydrogen bond distance between donor and acceptor
31.1, 47.2, 61.9, 93.0, 99.6, 110.0, 121.5, 122.9, 124.6, atoms was defined as 1.9 Å with a tolerance of 0.5 Å,
124.5, 126.3, 128.1, 129.3, 135.7, 138.5, 146.7, 160.1, and the acceptor– hydrogen–donor angle was not less
163.5. MS(EI): m/z = 351 [M+NH4]+; Anal. Calcd than 120◦. Then, the structures were saved in PDBQT
for C21H19NO3: C, 75.66; H, 5.74; N, 4.20%. Found: file format for further studies in ADT. ChemDraw Ultra
C, 75.35; H, 5.79; N, 4.28%.
7.0 (ChemOffice 2002) was used to draw the Ligand 2D
structures. Then, Chem3D Ultra 7.0 was used to convert
2D structure into 3D, and semi-empirical AM1 method
was used to minimize the energy. Minimum RMS gra-
dient of 0.100 was set in each iteration to minimize the
energy. All structures were saved as .pdb file format
for input to ADT. All the ligand structures were then
saved in PDBQT file format, to carry out docking in
ADT. A grid box with dimension of 40 × 40 × 40 Å3
with 0.375 Å spacing centred on −0.908, 0.681, −6.463
was created around the binding site of co-crystallised
ligand on 3F81 protein using ADT. The centre of the
box was set at co-crystallised ligand centre and grid
energy calculations were carried out. AutoDock dock-
ing calculation was done using default parameters and
50 docked conformations were generated for each com-
pound. Genetic algorithms were used to calculate the
energy of the binding interactions. The outputs were
exported to PyMOL for visual inspection of the binding
modes and interactions of the compounds with amino
acid residues in the active sites.
2.3 Experimental procedure for the evaluation of
cytotoxicty
The HeLa cells were seeded into 96-well plates in
100 μL of medium containing 5% fetal bovine serum
(FBS), at plating density of 10,000 cells/well and incu-
bated at 37◦C, 5% CO2, 95% air and 100% rela-
tive humidity for 24 h prior to addition of samples.
The samples were solubilized in DMSO and diluted
in serum free medium. After 24 h, 100 μL of the
medium containing the samples at various concentra-
tions were added and incubated at 37◦C, 5% CO2,
95% air and 100% relative humidity for 48 h. Tripli-
cate was maintained and the medium containing with-
out samples were served as control. After 48 h, 15 μL
of MTT (5 mg/mL) in Dulbecco’s phosphate buffered
saline (DPBS) was added to each well and incubated at
37◦C for 4 h. The medium with MTT was then flicked
off and the formed formazan crystals were solubilized
in 100 μL of DMSO and then the absorbance was mea-
sured at 570 nm using micro plate reader. The per-
centage growth of inhibition of was calculated by com-
paring the results of the test with those of the control
using the formula: % inhibition = (AC−AT)/AC × 100,
where AC is the absorbance of control and AT is the
absorbance of test. From the results nonlinear regres-
sion graph was plotted between % cell growth inhibi-
tion and Log10 concentration (μM). The half maximal
inhibitory concentration (IC50 value) was determined
using GraphPad Prism software.
3. Results and Discussion
3.1 Chemistry
3.1a Chemical synthesis of pyrano[3,4-b]indol-1(9H)-
ones: The synthetic pathway of pyrano[3,4-b]indol-
1(9H)-ones is outlined in scheme 1. Indole-2-carbox-
ylic acid 1a-b was esterified with methanol using cat-
alytic sulphuric acid to afford the corresponding methyl
ester 2a-b.7 The esters were then subjected to C3-
iodination using molecular iodine to afford 3-iodo-
indole-2-carboxylate 3a-b.8 N-alkyl-3-iodo-indole-2-
carboxylate 4a-g was obtained by N-alkylation reaction
2.4 Molecular docking
Molecular docking simulation was performed using the of 3a-b using respective alkyl halide and base.9 3-
AutoDock Tools (ADT) version 1.5.6 and AutoDock (Ethynyl)-indole-2-carboxylate 5a-j was obtained by
version 4.2.5.1 docking program. Three dimensional coupling terminal alkynes with 3-iodo indoles 3a-b and
structure of VHR receptor was obtained from the Pro- 4a-j under standard Sonagashira condition.10 Hydroly-
tein Data Bank (PDB ID: 3F81). The co-crystallized sis of 5a-j was achieved using aqueous sodium hydrox-
ligand in the 3F81 structure was removed. Then, the ide in an ethanol-methanol solution to give 3-(ethynyl)-
water molecules present with the crystal were deleted, indole-2-carboxylic acid 6a-j.11 Having synthesized the
the polar hydrogen atoms were added, lower occupancy starting materials in satisfactory yields, we then focused
residue structures were deleted, and any incomplete our attention on the cyclization of substrates 6a-j.

J C Jeyaveeran et al.
Scheme 1. (a) H2SO4 (catalytic), MeOH, reflux, 24 h (b) I2 (1.2 equiv.), KOH (3.0 equiv.), DMF, rt, 6 h (c) (for 4a, 4b,
4d and 4e) NaH (2.0 equiv), DMF, 30 min at 0◦C then R2X (3.0 equiv), 0◦C to rt, 6 h (for 4c, 4f and 4g) KOH (3.0 equiv),
DMSO, 30 min at 0◦C then R2X (5.0 equiv), 0◦C to rt, 6 h (d) PdCl2(Ph3P)2 (5 mol%), CuI (5 mol%), alkyne, (1.1 equiv),
Et3N, 6 h, rt (e) NaOH (1.5 equiv), MeOH/EtOH / H2O (3:1:2), 75◦C, 1 h (f) AuCl3 (5 mol%), MeCN, reflux.
Cycloisomerization of substrates 6a-j in the presence of the presence of C4-hydrogen of the indolone ring.
5 mol% of AuCl3 in refluxing acetonitrile gave the pyra- In 13C NMR spectrum the lactone carbonyl and C4-
noindolones 7a-j with good to excellent yields as single carbon resonated at δC = 155.2–163.6 and 97.1–
regioisomer. The results were shown in table 1, which
indicate that the reaction proceeded at a short reaction
time in N-alkyl indoles than unsubstituted indoles. Sig-
nificant increase in yield of the product was observed in
N-alkyl indoles 7a-g, whereas a slight decrease in the
product yield was observed in N-unsubstituted indoles
7h-j. These observations suggest that the presence of
electron releasing N-alkyl group enhances the electron
density of the triple bond, thus coordinating with the
Lewis acidic AuCl3 with ease. The presence of elec-
tron releasing substituent (MeO-) in the aryl ring also
enhances the reaction as evidenced by the short reac-
tion time and excellent yield 7d-g. The structures of all
the synthesized compounds were confirmed by spectral
98.5 ppm respectively. All these findings confirmed the
formation of products.
3.1b Chemical synthesis of pyrano[4,3-b]indol-1(5H)-
ones: With an efficient protocol for the synthesis of
pyrano[3,4-b]indol-1(9H)-ones in hand, we next set out
to investigate the scope and limitations of our gold cat-
alytic system towards the synthesis of its positional
isomer, pyrano[4,3-b]indol-1(5H)-ones. The required
2-(Ethynyl)-indole-2-carboxylic acids can be easily
prepared according to standard literature procedures
as shown in scheme 2. Vilsmeir bromoformylation
of oxindole 8a affords 2-bromo-indole-3-carbaldehyde
9a in excellent yield.12 Oxidative esterification of 9a
with oxone in methanol gave the ester 10a.13 N-
alkylation of 10a was performed with appropriate
alkyl halide and base which results in the formation
of N-alkyl derivatives 11a-d.9 By subjecting 11a-d
and terminal acetylenes under standard Sonogashira
1
data (FTIR, H NMR, 13C NMR and EI-MS) and ele-
mental analyses. Pyrano[3,4-b]indol-1(9H)-one deriva-
tives 7a-j showed C=O stretching bands in the region
of 1666–1714 cm−1 in their IR spectrum, indicating the
1
presence of a δ- lactone ring. In H NMR spectrum, a
prominent singlet at δH = 6.62–7.76 ppm ascertained

Pyranoindolones and Cytotoxicity
◦
R
Scheme 2. (a) POBr3 (2.5 equiv.), DMF (10 equiv.), (CH2)2Cl2, 0 C to rt, 24 h (b) Oxoneꢀ (1.05 equiv.), MeOH, rt 3 h
(c) (for 11a and 11b) NaH (2.0 equiv), DMF, 30 min at 0◦C then R2X (3.0 equiv), 0◦C to rt, 6 h (for, 11c and 11d) KOH (3.0
equiv), DMSO, 30 min at 0◦C then R2X (5.0 equiv), 0◦C to rt, 6 h (d) Pd(Ph3P)4 (5 mol%), CuI (5 mol%), terminal alkyne,
(1.1 equiv), Et3N, 6 h, rt (e) NaOH (1.5 equiv), MeOH / EtOH / H2O (3:1:2), 75◦C, 1 h (f) AuCl3 (5 mol%), MeCN, reflux.
cross-coupling reaction yields the acetylenic derivatives 3.2 Biology
12a-j.10 Alkaline hydrolysis of 12a-j affords the req-
uisite 2-(ethynyl)-indole-3-carboxylic acid 13a-j.11 In In pursuance of our on-going project on bio-active
the light of our previous study,3d we subjected the heterocycles,3k−t an attempt was made to evaluate the
acetylenic precursors 13a-j with 5 mol% of AuCl3 cytotoxicity of compounds (7a-j and 14a-j) against
under refluxing condition. Gratifyingly, the reaction human adenocarcinoma cell lines. Towards this end,
proceeds and affords the corresponding products 14a- the MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-
j in moderate to good chemical yields (table 2). It is tetrazolium bromide] cell proliferation assay was used
noteworthy to mention that precursors 13aj led to rea- to evaluate the cytotoxic activity of the synthesized
sonable conversion with only an increase of the reac- compounds against HeLa cell lines.14 Results of the in
tion time as compared to its positional isomer 6a-j. This vitro anticancer activity were compared with reference
can be attributed to the reduced electrophilicity of car- drug cisplatin. Analysis of the screening data (table 3)
boxylic acid in C3-indole (as in 13a-j) compared to revealed that compounds 7d, 7e and 7j showed com-
that of C2-indole (as in 6a-j). The low yield observed parable anticancer potency with IC50 values of 0.22,
in compounds 14i and 14j can be rationalized by the 1.2 and 0.33 μM, respectively with respect to the refer-
decrease in Lewis acidity of the catalyst by interact- ence drug, cisplatin (IC50 = 0.08 μM). The results indi-
ing with the hydroxyl function and eventually the cat- cated that the presence of methoxy substituent on the
alytic efficiency of the overall process. Similar to our C-6 position was found to be essential for activity, since
previous results,3d we found that the cycloisomerization compounds without 6-MeO groups showed a signifi-
follows 6-endodig regiochemistry leading to δ-lactone cant loss of potency. A small substituent like methyl on
and no γ -lactone was observed as evidenced by crude the nitrogen atom 7d (IC50 = 0.22 μM) exhibited max-
NMR analysis. This can be ascribed to the energetic imum potency compared to other analogues. Replace-
inaccessibility or greater strain of 5-5 ring formation ment of the N-methyl group with an N-ethyl group 7e
over 5-6 ring formation. The formation of δ-lactone (IC50 = 1.20 μM) decreased the potency appreciably,
ring in all compounds was ascertained by stretching whereas N-butyl group 7f (IC50 = 5.00 μM) showed
band between 1695 and 1721 cm−1 in their IR spec- even much reduced inhibitory potency. The loss of
1
trum. In H NMR spectrum a singlet between δH 5.86 activity was observed due to the increase on group size
and 6.81 ppm were observed, characteristic of the C4- from methyl to ethyl to butyl for compounds 7d-f. How-
hydrogen of 14a-j. Moreover all compounds exhibited ever, similar behaviour was not observed in molecules
two characteristic peaks in their 13C NMR spectrum devoid of 6-MeO group such as 7a, 7b and 7c as evi-
at δC = 97.9–100.1 and 163.5–168.5 ppm, thus con- denced by the inconsistent IC50 values (1.90, 11.31
firming the presence of C4-carbon and lactone carbonyl and 3.20 μM, respectively). Although the reason was
carbon of 14a-j, respectively.
not clear, it was attributed to the favourable structural

J C Jeyaveeran et al.
Table 3. Anticancer potency of compounds 7a-j and
14a-j in HeLa cell lines.
combination of 6-MeO and N-alkyl groups to exhibit
better anticancer activity. Interestingly, the absence of
alkyl substituent on the indole nitrogen 7j (IC50
=
Entry
Compound
IC50(μM)
0.33 μM) seems to be the second most active among
the tested compounds. In contrast, the related com-
pound 7i (IC50 = 19.1 μM) emerged as the least attrac-
tive compound in this series showing poor activity. A
moderate cytotoxity was observed for compound with
3-butyl group 7h (IC50 = 2.60 μM). Next, we turned
our attention in comparing the SAR of the pyrano[4,3-
b]indol-1(5H)-one series 14a-j (table 3). From the
analysis of the observed data, it was found that com-
pound 14d possessing C3-phenyl and N-benzyl sub-
stituent showed highest activity (IC50 = 0.69 μM).
Compound 14c which contains C3-butyl and N-butyl
groups emerged as the second most active in the series
(IC50 = 1.11 μM). However, replacement of N-butyl
by a small group like methyl (14a) marginally reduced
the inhibitory potency to 1.24 μM. By increasing the
chain length to ethyl at the nitrogen atom (14b), a slight
decrease in activity was observed (IC50 = 1.41 μM).
Switching from phenyl to aliphatic chains such as butyl
and pentyl as a C3-substituent (14e-h) exhibited only
moderate potency with the IC50 values ranging between
1.63 and 1.92 μM. Compounds possessing tertiary and
primary hydroxyl (14i and 14j) emerged as less active
among the tested series (IC5 = 2.02 and 2.25 μM,
respectively) suggesting the intolerance of polar sub-
stituent at the C3-site. These qualitative assessments
revealed that the presence of C3-phenyl substituent (as
in 14a-d) is essential for exhibiting good inhibitory
potency. An overall comparison of the results suggested
that pyrano[4,3-b]indol-1(5H)-ones 14a-j exert consis-
tent IC50 values (0.69-2-89 μM against the analogous
positional isomers 7a-j (0.22–19.11 μM).
1
2
3
4
5
6
7
8
7a
7b
7c
7d
7e
7f
7g
7h
7i
1.90
11.31
3.20
0.22
1.20
5.00
6.70
2.60
19.11
0.33
1.24
1.41
1.11
0.69
1.92
1.89
1.75
1.63
2.02
2.25
0.08
9
10
11
12
13
14
15
16
17
18
19
20
21
7j
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
cis-platin
3F81) crystal structures available in the Protein Data
Bank. A link between VHR and cervical cancer was
well established. Compared to normal keratinocytes,
VHR protein levels are found to be up regulated in sev-
eral cervix cancer cell lines including human papilloma
virus (CaSki, HeLa, SiHa, HT3 and C33). Biopsies
of the primary cervix cancer, squamous intra-epithelial
lesions and squamous cell carcinomas of the uterine
cervix revealed the higher expression levels of VHR
protein level. This clearly suggests that in the treatment
of cervical cancer, VHR might be a promising drug
target and VHR inhibiting small-molecules could be a
potential drug to cure the cervical cancer.17
To start with, the reproducibility of docking calcula-
tions was verified by extracting the co-crystallized lig-
3.3 Molecular docking
In pharmaceutical drug design, the discovery of small and from the complexes and submitted for one-ligand
molecules that predominantly undergo non-covalent run calculation. This reproduced top scoring conforma-
interactions such as hydrogen bonding, van der Waals, tion falling within root-mean-square deviation (RMSD)
π-π and π-cationic interactions in protein-ligand bind- value of 1.04 Å with bound X-ray conformation for
ing is an important concept.15 This in silico approach 3F81, suggesting this method is valid enough to be
involves the docking of synthesized compounds into used for docking studies of other compounds (figure S1
the active site of the 3D structure of the target, fol- in Supplementary Information). Docking simulation of
lowed by the calculation of free energy of binding all the synthesized compounds was performed in the
(FEB) of the protein-ligand complex. The binding same active site using the same protocol used for the
mode of the potent inhibitors was investigated using validation study (figure S2 in SI). For each of the
AutoDock Tools (ADT) version 1.5.6 and AutoDock test molecules, dockings were performed by taken into
version 4.2.5.1 docking program to rationalize the phar- 2.5 million energy evaluations. The conformations of
macological results.16 To gain insights of the observed docked ligand with VHR receptor were analyzed in
activity, we docked all the compounds to Vaccinia terms of energy, hydrogen bonding, hydrophobic and
H1-Related (VHR) Phosphatase receptor (PDB ID: π-π interaction. The final coordinates of the ligand and

Pyranoindolones and Cytotoxicity
receptor were saved after the clear analysis of ligand- noted that compound 7d with highest binding energy
receptor interactions. To investigate the interactions of (−8.27 kcal/mol) did exhibit significant IC50 value
ligand and receptor simulated conformations output (0.22 μM), thus establishing a correlation with the bio-
was exported to PyMOL software. The free energy of logical results.
binding (FEB) of all compounds were calculated from
the docking scores (table 4).
The results revealed that all the docked compounds
bind with the receptor and exhibits free energy of bind-
ing value between −5.00 and −8.27 kcal/mol. All the
compounds binds in the active site and the simulated
conformation exhibits various interactions with 16 ami-
no acids namely LEU-25, PRO-26, PHE-68, MET-69,
ASP-92, CYS-124, ARG-125, GLU-126, GLY-127,
TYR-128, SER-129, ARG-130, ARG-158, GLY-161,
PRO-162 and ASN-163 with non-covalent interactions
such as hydrophobic, hydrophilic, π-π interaction and
hydrogen bonding. Among all compounds docked, 7d
exhibits very high binding with 3F81 receptor and
forms two hydrogen bonds with TYR-128 and SER-129
amino acids which resulted in the binding energy of
−6.27 kcal/mol (figure S6 in SI). In compound 7d, oxy-
gen of O-CH3 interacts with the hydrogen of NH of
TYR-128 and forms a hydrogen bond with the bond
length of 2.7 Å. Similarly, oxygen of O-CH3 interacts
with the hydrogen of NH of SER-129 and forms a
hydrogen bond with the bond length of 2.2 Å. In addi-
tion, benzyl moiety exhibits hydrophobic interaction
with GLY-161 and PRO-162 in addition to π-π inter-
action with the phenyl ring of TYR-128. It is to be
4. Conclusions
In continuation to our previous work on the synthesis
of pyrano[3,4-b]indol-1(9H)-ones, we further extended
our studies towards the synthesis of the corresponding
pyrano[4,3-b]indol-1(5H)-ones. The synthetic poten-
tial of gold(III) chloride in the flexible synthesis of
isomeric pyranoindolones in excellent regioselectivity
and good chemical yields was also demonstrated. All
compounds were evaluated for their cytotoxic potency
towards HeLa cells and compared with the reference
drug cis-platin. The results of which indicated that
three candidates 7d, 7j and 14d exhibited signifi-
cant inhibitory potency with IC50 values of 0.22, 0.33
and 0.69 μM, respectively. Molecular docking with
VHR phosphatase receptor also supported the biologi-
cal results by showing compound 7d as the most active
with a free energy of binding −8.27 kal/mol. Further
investigations to delineate the scope and limitations of
the current protocol to biologically active natural prod-
ucts are actively underway in our laboratory and the
results will be disclosed in the due course.
Supplementary information (SI)
Table 4. FEB values of compounds on VHR phosphatase
receptor.
1
Copies of molecular docking pictures, H NMR, 13C
NMR and mass spectra of selected compounds are
provided in the Supplementary Information, which is
available at www.ias.ac.in/chemsci.
Entry
Compound
FEB (kcal/mol)a
1
2
3
4
5
6
7
8
7a
7b
7c
7d
7e
7f
7g
7h
7i
−6.39
−6.26
−6.67
−8.27
−6.13
−6.45
−6.43
−5.67
−6.25
−6.07
−5.89
−5.86
−6.10
−7.27
−5.17
−5.00
−6.31
−6.47
−6.77
−6.51
Acknowledgements
C.P. gratefully acknowledges the Science and Engineer-
ing Research Board (SERB), India for providing Fast
Track award under Young Scientist Scheme (SERB/
F/5666/2015-16). C.P. extends his acknowledgement to
Dr. Vijayamohanan K. Pillai, Director, CSIR-CECRI
and Dr. D. Jeyakumar, Head, Functional Materials
Division, CSIR-CECRI for providing infrastructure
facilities.
9
10
11
12
13
14
15
16
17
18
19
20
7j
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
References
1. (a) Fürstner A 2014 Acc. Chem. Res. 47 925; (b) Hashmi
A S K and Rudolph M 2008 Chem. Soc. Rev. 37 1766;
(c) Zhang Y, Luo T and Yang Z 2014 Nat. Prod. Rev. 31
489
aFree energy of binding (PDB ID: 3F81)

J C Jeyaveeran et al.
2. (a) Hashmi A S K, Schwarz L, Choi J-H and Frost T M 5. Suresh R R and Swamy K C K 2012 J. Org. Chem. 77
2000 Angew. Chem. Int. Ed. 39 2285; (b) Hashmi A S
K, Frost T M and J W Bats 2000 J. Am. Chem. Soc. 122
11553
6959
6. Ngi S I, Guilloteau V, Abarbri M and Thibonnet J 2011
J. Org. Chem. 76 8347
7. Agnusdei M, Bandini M, Melloni A and Umani-Ronchi
A 2003 J. Org. Chem. 68 7126
8. (a) Choshi T, Sada T, Fujimoto H, Nagayama C,
Sugino E and Hibino S 1997 J. Org. Chem. 62 2535;
(b) Sakamoto T, Nagano T, Kondo Y and Yamanaka H
1988 Chem. Pharm. Bull. 36 2248
9. (a) Sechi M, Derudas M, Dallocchio R, Dessí A,
Bacchi A, Sannia L, Carta F, Palomba M, Ragab O,
Chan C, Shoemaker R, Sei S, Dayam R and Neamati N
2004 J. Med. Chem. 47 5298; (b) Tsotinis A, Afroudakis
P A, Davidson K, Prashar A and Sugden D 2007 J. Med.
Chem. 50 6436
3. (a) Praveen C, Karthikeyan K and Perumal P T 2009
Tetrahedron 65 9244; (b) Praveen C, Kalyanasundaram
A and Perumal P T 2010 Synlett 777; (c) Praveen C and
Perumal P T 2011 Synlett 521; (d) Praveen C, Ayyanar
A and Perumal P T 2011 Bioorg. Med. Chem. Lett. 21
4170; (e) Praveen C and Perumal P T 2016 Chin. J.
Catal. 37 288; (f) Jeyaveeran J C, Praveen C, Arun Y,
Prince A A M and Perumal P T 2016 J. Chem. Sci.
128 73; (g) Praveen C, Sagayaraj Y W and Perumal
P T 2009 Tetrahedron Lett. 50 644; (h) Praveen C,
Kiruthiga P and Perumal P T 2009 Synlett 1990; (i)
Praveen C, Jegatheesan S and Perumal P T 2009 Syn-
lett 2795; (j) Praveen C and Perumal P T 2016 Synthe- 10. Sonogashira K, Tohda Y and Hagihara N 1975 Tetrahe-
sis 48 855; (k) Praveen C, Dheenkumar P, Muralidharan
dron Lett. 16 4467
D and Perumal P T 2010 Bioorg. Med. Chem. Lett. 11. Marchal E, Uriac P, Legouin B, Toupet L and van de
20 7292; (l) Praveen C, Ayyanar A and Perumal P T
Weghe P 2007 Tetrahedron 63 9979
2011 Bioorg. Med. Chem. Lett. 21 4072; (m) Praveen 12. Somei M, Sayama S, Naka K, Shinmoto K and Yamada
C, Iyyappan C, Girija K, Kumar K S and Perumal P T
F 2007 Heterocycles 73 537
2012 J. Chem. Sci. 124 451; (n) Praveen C, Nandakumar 13. Travis B R, Sivakumar M, Hollist G O and Borhan B
A, Dheenkumar P, Muralidharan D and Perumal P T
2003 Org. Lett. 5 1031
2012 J. Chem. Sci. 124 609; (o) Praveen C, Narendiran 14. (a) Alley M C, Scudiero D A, Monks A, Hursey M L,
S, Dheenkumar P and Perumal P T 2013 J. Chem. Sci.
125 1543; (p) Praveen C, Dheenkumar P and Perumal
P T 2013 J. Chem. Sci. 125 71; (q) Parthasarathy K,
Czerwinski M J, Fine D L, Abbott J G M, Shoemaker
R H and Boyd M R 1988 Cancer Res. 48 589; (b)
Mossman T 1983 J. Immunol. Methods 65 55
Praveen C, Balachandran C, Kumar P S, Ignacimuthu 15. Williams D H, Stephens E, O’Brien D P and Zhou M
S and Perumal P T 2013 Bioorg. Med. Chem. Lett.
2004 Angew. Chem. Int. Ed. 43 6596
23 2708; (r) Parthasarathy K, Praveen C, Kumar P S, 16. (a) Morris G M, Huey R, Lindstrom W, Sanner M F,
Balachandran C and Perumal P T 2015 RSC Adv. 5
15818; (s) Praveen C, Iyyappan C, Perumal P T and
Girija K 2012 Ind. J. Chem. 51B 498; (t) Praveen C,
Belew R K, Goodsell D S and Olson A J 2009 J. Com-
put. Chem. 30 2785; (b) Sanner M F 1999 J. Mol.
Graphics Mod. 17 57
Parthasarathy K, Kumar, P S and Perumal P T 2015 Ind. 17. Wu S, Vossius S, Rahmouni S, Miletic A V, Vang T,
J. Chem. 54B 373
4. Shimizu M, Hirano K, Satoh T and Miura M 2009 J.
Org. Chem. 74 3478
Vazquez-Rodriguez J, Cerignoli F, Arimura Y, Williams
S, Hayes T, Moutschen M, Vasile S, Pellecchia M,
Mustelin T and Tautz L 2009 J. Med. Chem. 52 6716