Engaging thieno[2,3-b]indole-2,3-dione for the efficient synthesis of spiro[indoline-3,4′-thiopyrano[2,3-b]indole] by reaction with N-substituted isatilidenes

Article abstract of DOI:10.1002/jhet.4147

A simple and efficient method, proceeding through a new mechanistic pathway, for the synthesis of spiro[indoline-3,4-thiopyrano[2.3-b]indole derivatives have been developed by exploiting the reaction of thieno[2,3-b]indole-2,3-dione with N-substituted isatilidenes. The compounds synthesized have been screened for antibacterial activity. The generality of the reaction and mechanistic rationale are presented.

Full text of DOI:10.1002/jhet.4147

Engaging thieno[2,3-b]indole-2,3-dione for the efficient synthesis of spiro[indoline-
,4’-thiopyrano[2,3-b]indole] by reaction with N-substituted isatilidenes
3
a
a
*a
a
b
Noble V Thomas, Vidya Sathi, Ani Deepthi, Sruthi S. L. and Sidharth Chopra
*
a
Ani Deepthi, Asst. Professor, Department of Chemistry, University of Kerala, Kariavattom,
Thiruvananthapuram 695581, Kerala State, India
b
Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow 226031, Uttar
Pradesh, India
Email: anideepthi@gmail.com
Abstract
A simple and efficient method, proceeding through a new mechanistic pathway, for the synthesis
of spiro[indoline-3,4-thiopyrano[2.3-b]indole derivatives have been developed by exploiting the
reaction of thieno[2,3-b]indole-2,3-dione with N-substituted isatilidenes. The compounds
synthesized have been screened for antibacterial activity. The generality of the reaction and
mechanistic rationale are presented.
Keywords: Thieno[2,3-b]indole-2,3-dione, isatilidene, thiopyran, Michael addition, elimination
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Introduction
[
1]
The presence of thieno[2,3-b]indole moiety 1 in thienodolin A, (Figure 1) an alkaloid with
plant growth regulating properties, and the biological activities associated with 1 has triggered
[2,3]
scientific interest in this molecule over the years.
exhibit various biological properties such as anti-tubercular, anti-inflammatory, acetylcholine
Thienoindole derivatives are known to
[4-6]
esterase inhibitory and CNS disorder inhibition.
In addition, very recent studies have shown
that thienoindoles can be used to prepare antibody-drug conjugates (ADC) which can act as
anticancer agents by specifically recognizing ALK protein which appears on the surface of
[
7]
nueroblastoma cells.
Principally, indole derivatives are the most widely distributed
heterocycles in nature having numerous applications in areas of pharmaceuticals and
[
8-10]
agrochemicals.
Apart from the biological interests associated with indoles and thienoindoles,
studies have also proved that the latter are suitable for the construction of organic photovoltaic
devices as well as they represent an electron-rich heteroaromatic moiety which can be used for
[
11]
the construction of D-π-A type systems (Figure 2).
O
NH2
S
S
N
N
Cl
H
H
A
1
Figure 1. Thienodolin and thieno[2,3-b]indole
O
CN
NC
S
NC
X
S
n
S
S
N
N
Alkyl
Alkyl
Alkyl = Ethyl, 2-ethyl hexyl
X = CN, CO R, CO H
2
2
n = 0, 1, 2, 3
Figure 2. Representative examples for D-π-A systems using thieno[2,3-b]indole
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Bergman and Berg have together reported the synthesis of thieno[2,3-b]indole-2,3-dione 2 and
[
12,13]
found that it undergoes ring expansion on treatment with diazomethane.
The susceptibility
of 2 towards ring opening was also evident when it yielded esterthione 3 on reaction with sodium
methoxide in methanol and compound 4 on reaction with o-phenylene diamine. In the light of
[
14]
this report and due to our continued interest in the synthesis of spirofused compounds,
we
decided to explore the reactivity of thieno[2,3-b]indole-2,3-dione towards electrophilic systems
like acetylenic esters and isatilidenes. The results of our investigations with isatilidenes is the
subject matter of this paper. Coincidentally, the product that we obtained from our reaction was
same as that obtained by Majumdar et.al. through the one-pot reaction of indole-2-thione, isatin
[
15,16]
and malononitrile (Scheme 1).
Although the product was reported, what enthralled us was
the unexpected pathway of the reaction resulting in the thiopyran ring formation by loss of two
carbon atoms, probably by involvement of the solvent.
OMe
HN
O
HO
O
O
o-phenylene
diamine
NH
S
O
S
NaOMe
S
Bergmann, 2017
N
MeOH
N
H
H
N
H
4
2
3
H
N
O
O
CN
CN
EtOH
80 °C
S
+
+
CN Majumdar, 2012
NH2
O
N
N
H
N
H
S
H
6
NC
O
CN
O
methanol
1h, reflux
CO Me
2
S
O
6 +
This work
+
N
CO Me
2
N
H
2
5
H
Scheme 1. Comparison of reported work and present work
Results and Discussion
As an initial experiment, 2 was treated with N-methyl isatilidene 5b in methanol at room
temperature. The progress of the reaction was monitored using TLC and the reaction was found
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to be complete in 12 hours. Addition of a few drops of water to the reaction mixture resulted in
the precipitation of a compound 6b. The reaction conditions were then optimized and it was
observed that in solvents like THF, acetonitrile, dichloromethane and toluene, the reaction did
not yield any product at all while very good yields of the product was obtained in
methanol/ethanol under reflux conditions in 1 hour. It was observed that one pot reaction of
thieno[2,3-b]indole-2,3-dione, malononitrile and N-methyl isatin at 80 °C resulted in lower yield
for the product, again indicating that the reaction proceeded via another pathway in our case,
yielding the same product. The generality of the reaction was then tested using various N-alkyl
isatins and cyanoacrylates (Table 1). N-methyl-thieno[2,3-b]indole-2,3-dione was also found to
react similarly yielding 6j-l.
Figure 3. ORTEP diagram of 6g
-1
In the IR spectrum of 6b, the ketone carbonyl stretching was seen at 1637 cm while nitrile
-1
-1
stretch was observed at 2186 cm and peak at 3170 cm corresponded to the NH stretch. In the
1
H NMR spectrum, the singlets at δ 11.6 and δ 3.27 ppm were attributed to the NH and NCH3
1
3
protons respectively. In the C NMR spectrum, signals at δ 176.7 and δ 71.7 ppm corresponded
to the amide carbonyl and carbon atom bonded to the nitrile group respectively. The quarternary
carbon atom was seen as a peak at 51.5 ppm. In the high resolution mass spectrum (ESIMS) the
[
M+1] peak was observed at m/z 359.0977. The structure was finally confirmed based on single
crystal X-ray analysis (CCDC 2020515, Figure 3).
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Table 1. Generality of the reaction
R2
O
E
CN
O
N
O
O
MeOH
CO Me
2
S +
E
1
h, reflux
+
CO2Me
N
N
N
R1
S
NH2
R1
R2
2
5a-l
6a-l
R = H, Me
1
a
b
Entry
Product
R1
H
R2
E
Yield (%)
1
2
3
4
5
6
7
8
9
6a
6b
6c
6d
6e
6f
H
CN
CN
CN
CN
CN
CN
CN
93
91
90
92
93
89
91
89
92
91
93
92
H
Me
Et
H
H
Pr
H
Bu
Allyl
H
6g
6h
6i
H
CH Ph
2
H
H
COOEt
COOEt
CN
H
CH Ph
2
1
1
0
1
2
6j
Me
Me
Me
Pr
H
6k
6l
CN
1
CH Ph
CN
2
a
All reactions were done by refluxing 2 (0.15 mmol) and 5 (0.15 mmol)
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b
in MeOH (4 ml) for 1 h. Isolated yield.
The unusual mechanism of the reaction can be explained along the following lines. Under reflux
condition (or at room temperature for longer time) thieno[2,3-b]indole-2,3-dione 2 ring opens to
[
12]
form the enol intermediate 3. A blank experiment done by stirring 2 in methanol under the
1
13
same conditions also yielded 3 and its structure was confirmed by FTIR, H and C NMR
analysis (supporting information). The enol 3 can co-exist with its thiol tautomer 3’ which
undergoes Michael addition with isatilidene producing thione I. The latter probably cyclizes to
form the imine II which tautomerizes to the enamine 6 via elimination of a molecule of dimethyl
oxalate in presence of methanol. In a separate experiment, the reaction of enol 3 with isatilidene
under the same conditions also furnished the product 6 indicating that the reaction occurs
through 3 (Scheme 2). The formation of the by-product dimethyl oxalate (or diethyl oxalate in
case the reaction is done with ethanol) was confirmed by titrating the mother liquor, obtained
after filtering the product 6 with aqueous KMnO which indicated stoichiometric formation of
4
dimethyl oxalate (mother liquor was hydrolysed prior to the titration). Further from the LCMS
analysis of the crude reaction mixture, a peak at m/z 118.250 indicated the presence of the anion
[17]
formed from dimethyl oxalate. The reaction proceeded with slightly lower yields in ethanol
probably because the ring opening of 2 in ethanol is not as feasible as it is in methanol.
O
O
HO
R
CO Me
O
CO Me
2
2
O
N
MeOH
S
S
SH
NC
N
N
N
H
H
H
C
2
3
3'
N
5
MeOH
MeO COC
R
2
MeO COC
N
2
6
O
N R
O
CN
CO Me
CN
-
2
CO Me
2
NH2
N
H
N
S
S C
I
II
NH
Scheme 2. Mechanistic rationale
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Contrastingly, in the one pot synthesis of spirooxindole annulated thiopyran derivatives reported
by Majumdar et al., the enol form of indole-2-thione adds in a Michael fashion to the isatilidene
formed by the Knoevenagel condensation of N-substituted isatin and ethyl
cyanoacetate/malononitrile leading to the intermediate (similar to the proposed thione
intermediate in our mechanism) which then undergo intramolecular S-acylation to give the
[
15]
product.
Whereas the current reaction is initiated through ring opening by the attack of
methanol (a weak nucleophile under reflux conditions) and the product is formed by elimination
of dimethyl oxalate.
Table 2 MIC (µg/mL) against ESKAPE pathogen panel
MIC (µg/mL)
S.No
Compound
E. coli
S. aureus
ATCC
K.
A.
P.
ATCC
pneumoniae
BAA 1705
baumannii aeruginosa
2
5922
29213
BAA 1605
ATCC
7853
2
1
2
6a
6b
6c
6d
6e
6f
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
32
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
1
3
4
8
5
32
8
6
7
6g
6h
6i
8
8
32
16
0.125
9
1
0
Levo floxacin 0.0156
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The antibacterial activity of the synthesized compounds 6a-i were screened using microbroth
dilution assay at CSIR-Central Drug Research Institute, Lucknow, India and the results are
summarized in Table 2. It can be seen that compounds 6c, 6d, 6e and 6f showed increased
activity against S. aureus ATCC 29213 with an MIC value of 8 µg/mL. To determine the
selectivity Index, 6c, 6d, 6e and 6f were tested to determine their cytotoxicity against Vero cells.
Upon analysis, it was determined that the compounds were cytotoxic at MIC levels, thus
precluding further experiments.
Conclusions
In conclusion, we have described a new route for the catalyst-free synthesis of spiro[indoline-
3
,4’-thiopyrano[2,3-b]indole] in methanol/ethanol and have evaluated their antibacterial
property. It was seen that the compounds 6c, 6d, 6e and 6f showed increased activity against S.
aureus ATCC 29213 with lower MIC values but were found to be cytotoxic. It has been
observed that thieno[2,3-b]indole-2,3-dione is a promising molecule for discovering new
reactions and its reactivity towards other electrophilic systems like dimethyl acetylene
dicarboxylate and transformations of the products thus obtained are underway in our laboratory.
Experimental Section
NMR spectra were recorded on a 400 MHz Bruker Avance FTNMR spectrometer. Chemical
shifts are reported relative to TMS as the internal standard. IR spectra were recorded on an
Agilent Cary 630 FTIR spectrometer. The oxindole, isatin, malononitrile and Lawesson’s reagent
were purchased from Spectrochem Pvt. Ltd and were used as such without further purification.
[
12]
Thieno[2,3-b]indole-2,3-dione was synthesized following the literature.
Commercial grade
solvents were used. Analytical thin layer chromatography was performed on silica gel coated on
aluminium sheets which was visualized using UV light of 254 nm.
General procedure
Procedure for the synthesis of thieno[2,3-b]indole-2,3-dione: Lawesson’s reagent (1.74 g, 10
mmol) was taken in a 250 ml RB flask and dissolved in 75 ml dry THF. Oxindole (1.5 g, 11.2
mmol) was added to it and stirred for 12 h at room temperature. The reaction mixture was then
dried on rotary evaporator, dissolved in minimum amount of acetonitrile and kept for stirring.
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Dropwise addition of oxalyl chloride (1.27 g, 10 mmol) followed by 5 minutes stirring and
[
12]
filtration yields the required thieno[2,3-b]indole-2,3-dione.
Compound 3 (E)-methyl 2-hydroxy-2-(2-thioxoindolin-3-ylidene)acetate:
Synthetic procedure: In a 50 ml dried RB flask, thieno[2,3–b]indole-2,3-dione 50 mg (0.25
mmol) was stirred with 4 ml methanol under room temperature for 4 hours. After the reaction
was complete, (monitored by TLC) water was added to the reaction mixture. The yellow
precipitate formed was filtered dried and weighed (58 mg, 98%) and was subjected to spectral
characterization.
-1
Characterization: Mp 190-191°C: FTIR (cm ): 3360, 2917, 2186, 1732, 1635, 1624, 1464,
1
1
426. H NMR (400 MHz, DMSO-d ), δ, ppm (J, Hz): 3.90 (s, 3H, -OMe), 7.26-7.33 (m, 2H,
6
1
3
ArH), 7.52 (m, 1H, ArH), 7.77 (m, 1H, ArH), 12.80 (s, 1H, -NH). C NMR (DMSO-d , 100
6
MHz): , ppm 53.2, 112.7, 113.1, 119.7, 123.7, 124.6, 126.3, 137.7, 143.2, 166.0, 180.0. Anal.
Calculated (%) for C H NO S: C, 56.16; H, 3.89; N, 5.95; S, 13.63 Found: C, 56.13; H, 3.84; N,
1
1
9
3
5
.97. S, 13.65
Representative procedure for the synthesis of spiro[indoline-3,4'-thiopyrano[2,3-b]indole
Synthesis of 6b: In a 50 ml RB flask, thieno[2,3–b]indole-2,3-dione 30 mg (0.15 mmol) was
refluxed with N-methyl isatilidene 32 mg (0.15 mmol) in methanol (10 ml) for 1 h. After
completion of the reaction as indicated by TLC, solvent was removed and the residue was
collected and washed with minimum amount of methanol-water (1:1) mixture and was filtered.
The precipitate thus collected was thoroughly dried and weighed (48 mg) and was subjected to
spectral characterization. The mother liquor containing dimethyl oxalate was hydrolysed by
adding H SO and refluxing for 2h. The concentration of oxalic acid thus obtained was estimated
2
4
by permanganometric titration with 0.01N KMNO solution. Estimated normality of the oxalic
4
acid from the titration was 0.019 N and the weight of dimethyl oxalate in whole of the filtrate
was calculated to be 16 mg. Thus, it was found that for every molecule of 6b formed, I molecule
of dimethyl oxalate was generated.
Same procedure was followed for the synthesis of all other derivatives.
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Compound
6a
2'-amino-2-oxo-9'H-spiro[indoline-3,4'-thiopyrano[2,3-b]indole]-3'-
carbonitrile: White powder (47 mg, 93%); mp 266-267 °C (reported melting point of this
1
5
-1
compound in literature is > 230 °C) ; FTIR (cm ): 3405, 3303, 3167, 2187, 1692, 1620, 1564,
1
1
471, 1438. H NMR (400 MHz, DMSO-d ), δ, ppm (J, Hz): 6.37 (d, 1H, J 8.0 Hz, ArH), 6.75 (t,
6
1
H, J 7.6 Hz, ArH), 6,92-701 (m, 4H, ArH), 7.05 (s, 2H, -NH ), 7.28 (t, 2H, J 8.0 Hz, ArH),
2
1
3
1
0.71 (s, 1H, -NH), 11.63 (s, 1H, -NH). C NMR (DMSO-d6, 100 MHz): , ppm 51.9, 72.1,
03.7, 110.1, 111.4, 117.0, 118.1, 120.0, 121.7, 122.9, 123.0, 124.7, 125.3, 129.4, 134.2, 136.8,
42.0, 153.5, 178.3. Anal. Calculated (%) for C H N OS, C, 66.26; H, 3.51; N, 16.27; S, 9.31;
1
1
1
9
12
4
Found: C, 66.22; H, 3.55; N, 16.28, S, 9.34
Compound 6b 2'-amino-1-methyl-2-oxo-9'H-spiro[indoline-3,4'-thiopyrano[2,3-b]indole]-
-1
3
'-carbonitrile: White powder (48 mg, 91%); mp 229-231 °C; FTIR (cm ): 3408, 3322, 3170,
1
2
186, 1689, 1637, 1562, 1470, 1432. H NMR (400 MHz, DMSO-d ), δ, ppm (J, Hz): 3.27 (3H,
6
s, N-CH ), 6.20 (d, 1H, J 8.0 Hz ArH), 6.72 (t, 1H, J 8.0 Hz, ArH), 6.95-7.07 (m, 3H, ArH), 7.10
3
(
s, 2H, -NH ), 7.23 (d, 1H, J 8.0 Hz, ArH), 7.28 (d, 1H, J 8.0 Hz, ArH), 7.36-7.40 (m, 1H, ArH),
2
1
3
1
1.6 (s, N-H). C NMR (DMSO-d6, 100 MHz): , ppm 51.5, 71.7, 103.5, 109.2, 119.2, 120.5,
1
20.8, 121.8, 122.7, 123.1, 123.6, 124.4, 124.5, 125.0, 129.5, 133.4, 136.6, 136.8, 153.7, 176.7.
HRMS: MS Calculated: m/z value for [M+1]: 359.0977, observed value for [M+1]: 359.0977.
Anal. Calculated (%) for C H N OS: C, 67.02; H, 3.94; N, 15.63; S, 8.95. Found: C, 67.03; H,
2
0
14
4
3
.91; N, 15.59; S, 8.99
Compound 6c 2'-amino-1-ethyl-2-oxo-9'H-spiro[indoline-3,4'-thiopyrano[2,3-b]indole]-3'-
-1
carbonitrile: White powder (48 mg, 89%); mp 239-241 °C; FTIR (cm ): 3389, 3279, 3170,
1
2
186, 1687, 1637, 1624, 1564, 1471, 1436. H NMR (400 MHz, DMSO-d ), δ, ppm (J, Hz): 1.23
6
(
t, 3H, J 7.2 Hz, -CH CH ), 3.83 (q, 2H, J 7.2 Hz, N-CH CH ), 6.26 (d, 1H, J 8.0 Hz, Ar-H), 6.72
2 3 2 3
(
t, 1H, J 8.0 Hz, ArH), 6.95-7.06 (m, 3H, ArH), 7.08 (s, 2H, -NH ), 7.24 (d, 1H, J 8.0 Hz, ArH),
2
1
3
7
.29 (d, 1H, J 8.0 Hz, ArH), 7.35-7.39 (m, 1H, ArH), 11.6 (s, 1H, -NH). C NMR (DMSO-d6,
00 MHz): , ppm 12.9, 35.0, 51.4, 71.9, 103.4, 109.2, 111.5, 116.9, 117.8, 120.0, 121.8, 123.1,
23.4, 124.6, 125.2, 129.5, 133.6, 136.8, 142.3, 153.5, 176.2. Anal. Calculated for C H N OS:
1
1
2
1
16
4
C, 67.72; H, 4.33; N, 15.04; S, 8.61 Found: C, 67.70; H, 4.29; N, 15.07; S, 8.63
Compound 6d 2'-amino-1-propyl-2-oxo-9'H-spiro[indoline-3,4'-thiopyrano[2,3-b]indole]-
-1
3
'-carbonitrile: White powder (53 mg, 93%); mp 249-251 °C; FTIR (cm ): 3412, 3328, 3155,
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1
2
3
184, 1688, 1620, 1563, 1475, 1431. H NMR (400 MHz, DMSO-d ), δ, ppm (J, Hz): 0.93 (t,
6
H, J = 7.2 Hz, –CH CH CH ), 1.66-1.72 (m, 2H, –CH CH CH ), 3.71 -3.79 (m, 2H, -
2
2
3
2
2
3
CH CH CH ), 6.22 (d, 1H, J 8.2 Hz, ArH), 6.72 (t, 1H, J 7.2 Hz, ArH), 6.95–7.09 (m, 3H, ArH),
2
2
3
7
.11 (s, 2H, NH ), 7.23 (d, 1H, J 8.0 Hz, ArH), 7.30 (d, 1H, J 8.4 Hz, ArH), 7.37 (t, 1H, J 7.6 Hz,
2
1
3
ArH), 11.68 (s, 1H, -NH). C NMR (DMSO-d6, 100 MHz): , ppm 13.7, 20.9, 51.5, 72.0,
1
1
8
03.5, 109.4, 111.5, 116.9, 117.9, 120.0, 121.8, 123.2, 123.4, 124.6, 125.2, 129.5, 133.4, 136.9,
43.0, 143.5, 153.7, 176.7. Anal. Calculated (%) for C H N OS: C, 68.37; H, 4.69; N, 14.50; S,
2
2
18
4
.30; Found: C, 68.33; H, 4.67; N, 14.52; S, 8.31
Compound 6e 2'-amino-1-butyl-2-oxo-9'H-spiro[indoline-3,4'-thiopyrano[2,3-b]indole]-3'-
-1
carbonitrile: White powder (55 mg, 93%); mp 256-258 °C; FTIR (cm ): 3402, 3289, 3165,
1
2
186, 1689, 1567, 1473, 1428. H NMR (400 MHz, DMSO-d ), δ, ppm (J, Hz): 0.88 (t, 3H, J 7.2
6
Hz, –CH CH CH CH ), 1.33-1.38 (m, 2H, –CH CH CH CH ), 1.61-1.67 (m, 2H, –
2
2
2
3
2
2
2
3
CH CH CH CH ), 3.75-3.80 (m, 2H, -CH CH CH CH ), 6.19 (d, 1H, J 8.4 Hz, ArH), 6.70 (t,
2
2
2
3
2
2
2
3
1
H, J 7.6 Hz, ArH), 6.95-7.05 (m, 2H, ArH), 7.07 (s, 2H, NH ), 7.15–7.41 (m, 4H, ArH), 11.66
2
1
3
(
s, 1H, NH). C NMR (DMSO-d6, 100 MHz): , ppm 14.1, 20.0, 29.6, 42.9, 51.4, 71.9, 103.4,
1
09.3, 111.5, 116.9, 119.9, 120.5, 120.8, 121.8, 122.7, 123.2, 124.6, 125.2, 129.5, 133.4, 136.6,
53.6, 176.6. Anal. Calculated (%) for C H N OS, C, 68.98; H, 5.03; N, 13.99; S, 8.01; Found:
1
2
3
20
4
C, 68.95; H, 5.07; N, 13.97; S, 8.05
Compound 6f 2'-amino-1-allyl-2-oxo-9'H-spiro[indoline-3,4'-thiopyrano[2,3-b]indole]-3'-
-1
carbonitrile: White powder (52 mg, 93%); mp. 257-259 °C; FTIR (cm ): 3419, 3308, 3164,
1
2
183, 1693, 1627, 1558, 1464, 1424. H NMR (400 MHz, DMSO-d ), δ, ppm (J, Hz): 4.34-4.39
6
(
m, 2H, -CH -CH=CH ), 5.19-5.36 (m, 2H, CH -CH=CH ), 5.82-5.92 (m, 1H, CH -CH=CH ),
2 2 2 2 2 2
6
.17 (d, 1H, J 8.0 Hz, ArH), 6.69 (t, 1H, J 7.6 Hz, ArH),. 6.94-7.11 (m, 4H, ArH), 7.13 (s, 2H, -
1
3
NH ), 7.29-7.38 (m, 2H, ArH), 12.10 (s, 1H, -NH). C NMR (DMSO-d6, 100 MHz): , ppm
2
4
1
6
9.0, 51.5, 71.7, 103.3, 109.8, 111.6, 116.9, 117.5, 118.1, 119.9, 120.8, 121.7, 123.4, 123.6,
24.6, 125.2, 129.4, 131.9, 133.3, 137.0, 154.1, 176.5. Anal. Calculated (%) for C H N OS: C,
2
2
16
4
8.73; H, 4.19; N, 14.57; S, 8.34. Found: C, 68.70; H, 4.15; N, 14.59; S, 8.38
Compound 6g 2'-amino-1-benzyl-2-oxo-9'H-spiro[indoline-3,4'-thiopyrano[2,3-b]indole]-3'-
-1
carbonitrile: White powder (60 mg, 93%); mp 231-233 °C; FTIR (cm ): 3410, 3311, 3181,
1
2
182, 1691, 1622, 1562, 1466, 1435. H NMR (400 MHz, DMSO-d ), δ, ppm (J, Hz): 5.04 (q,
6
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2
H, J 9.6 Hz -NCH ), 6.09 (t, 1H, J 5.6 Hz, ArH), 6.61 (t, 1H, J 5.6 Hz, ArH), 6.95-7.03 (m, 2H,
2
ArH), 7.07–7.11 (m, 2H, ArH), 7.16 (s, 2H, NH ), 7.28-7.31 (m, 5H, ArH), 7.44 (m, 2H, ArH)
2
1
3
1
1
1
4
1.70 (s, 1H, NH), C NMR (DMSO-d6, 100 MHz): , ppm 43.7, 51.6, 71.7, 79.1, 79.7, 103.5,
09.9, 111.4, 117.1, 118.2, 119.9, 121.8, 123.3, 123.7, 124.5, 125.3, 128.0, 128.7, 128.9, 129.4,
33.3, 136.4, 136.8, 142.5, 154.0, 177.0. Anal. Calculated (%) for C H N OS, C, 71.87; H,
2
6
18
4
.18; N, 12.89; S, 7.38; Found: C, 71.83; H, 4.22; N, 12.93; S, 7.35
Compound 6h ethyl 2'-amino-2-oxo-9'H-spiro[indoline-3,4'-thiopyrano[2,3-b]indole]-3'-
-1
carboxylate: White powder (52 mg, 90%); mp 237-238 °C; FTIR (cm ): 3408, 3304, 3259,
1
2
3
108, 1717, 1685, 1643, 1575, 1453. H NMR (400 MHz, DMSO-d ), δ, ppm (J, Hz): 0.81 (t,
6
H, J 9.6 Hz -OCH CH ), 4.09 (q, 2H, J 5.6 Hz, -OCH CH ), 6.73-6.79 (m, 2H, ArH) 6.84-6.85
2
3
2
3
(d, 1H, J 7.2 Hz, ArH) 6.88-6.96 (m, 3H, ArH), ), 7.07-7.13 (m, 1H, ArH) 7.22 (d, 1H, J 8.0 Hz
1
3
ArH), 8.18 (s, 2H, NH ), 10.49 (s, 1H, NH), 11.48 (s, 1H, NH). C NMR (DMSO-d6, 100
2
MHz): , ppm 13.5, 52.2, 59.4, 90.2, 105.7, 109.1, 117.8, 117.8, 119.7, 121.4, 121.4, 122.0,
1
23.3, 124.7, 127.8, 136.8, 137.9, 142.4, 154.6, 167.9, 180.6. Anal. Calculated (%) for
C H N O S, C, 64.43; H, 4.38; N, 10.73; S, 8.19; Found: C, 64.46; H, 4.35; N, 10.77; S, 8.17.
21
17
3
3
Compound 6i Ethyl 2'-amino-1-benzyl-2-oxo-9'H-spiro[indoline-3,4'-thiopyrano[2,3-
-1
b]indole]-3'-carboxylate: White powder (65 mg, 92%); mp 229-230 °C; FTIR (cm ): 3417,
1
3
313, 3163, 2188, 1713, 1692, 1623, 1554, 1476, 1432. H NMR (400 MHz, DMSO-d ), δ, ppm
6
(
J, Hz): 1.04 (t, 3H, J 8.0 Hz, OCH CH ), 4.70 (q, 2H, J 8.0 Hz, -OCH CH ), 5.01(m, 2H, -
2 3 2 3
NCH Ph), 6.34 (d, 2H, J 8.0 Hz, ArH), 6.96 -7.00 (m, 2H, ArH) 7.11-7.16 (m, 2H, ArH), 7.25-
2
1
3
7
.39 (m, 7H, ArH) 7.30 (s, 2H, -NH ), 12.43 (s, 1H, -NH). C NMR (DMSO-d6, 100 MHz): ,
2
ppm 13.1, 43.3, 52.6, 56.0, 89.6, 109.8, 111.8, 112.3, 118.1, 119.6, 120.8, 122.5, 125.3, 126.4,
27.8, 127.9, 128.0, 128.1, 129.0, 132.9, 133.0, 135.9, 136.6, 150.8, 176.9, 183.5 Anal.
Calculated for C H N O S, C, 69.83; H, 4.81; N, 8.73; S, 6.66; Found C, 69.87; H, 4.83; N,
1
2
8
23
3
3
8
.70; S, 6.62.
Compound 6j 2'-amino-9'-methyl-2-oxo-1-propyl-9'H-spiro[indoline-3,4'-thiopyrano[2,3-
-1
b]indole]-3'-carbonitrile: White powder (55.6 mg, 94%); mp 256-257 °C; FTIR (cm ): 3315,
1
3
251, 3185, 2179, 1698, 1635, 1561, 1531, 1464. H NMR (400 MHz, DMSO-d ), δ, ppm (J,
6
Hz): 0.841 (t, 3H, J 7.6 Hz, -NCH CH CH ), 1.57-1.64 (m, 2H, -NCH CH CH ), 3.26 (s, 3H,-
2
2
3
2
2
3
NCH ), 3.63-3.69 (m, 2H, -NCH CH2 CH ), 6.21 (d, 1H, J 8.0 Hz, ArH), 6.65-6.69 (m, 1H,
3
2
2
3
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ArH), 6.92-7.01 (m, 3H, ArH), 7.11 (s, 2H, -NH ), 7.17 (d, 1H, J 8.0 Hz, ArH), 7.28-7.37 (m,
2
1
3
2
H, ArH). C NMR (DMSO-d6, 100 MHz): , ppm 11.6, 20.8, 30.7, 41.7, 51.8, 72.2, 103.1,
09.4, 109.9, 117.0, 117.7, 120.2, 121.7, 123.4, 124.2, 125.3, 125.5, 129.6, 133.3, 137.8, 143.0,
53.9, 176.5. Anal. Calculated (%) for C H N OS, C, 68.98; H, 5.03; N, 13.99; S, 8.01; Found
1
1
2
3
20
4
C, 68.94; H, 5.00; N, 13.95; S, 8.03
Compound 6k 2'-amino-9'-methyl-2-oxo-9'H-spiro[indoline-3,4'-thiopyrano[2,3-b]indole]-
'-carbonitrile: White powder (50 mg, 94%); mp 274-275 °C (reported melting point of this
3
1
5
-1
compound in literature is > 250 °C) ; FTIR (cm ): 3431, 3259, 3155, 2186, 1691, 1613, 1568,
1
1
8
464, 1434. H NMR (400 MHz, DMSO-d ), δ, ppm (J, Hz): 3.70 (s, 3H, -NCH ) 6.37 (d, 1H, J
6
3
.0 Hz, ArH), 6.79 (q, 1H, J 7.6 Hz, ArH), 6,92-707 (m, 4H, ArH), 7.17 (s, 2H, -NH ), 7.26-7-44
2
1
3
(
m, 2H, ArH), 10.73(s, 1H, -NH). C NMR (DMSO-d6, 100 MHz): , ppm 30.7, 52.2, 72.4,
1
03.3, 109.9, 110.2, 117.1, 117.9, 120.2, 121.8, 122.9, 124.3, 125.2, 125.4, 129.5, 134.1, 137.8,
42.0, 152.7, 178.2. Anal. Calculated (%) for C H N OS, C, 67.02; H, 3.94; N, 15.63; S, 8.95;
1
2
0
14
4
Found: C, 67.01; H, 3.90; N, 15.67; S, 8.91.
Compound 6l 2'-amino-1-benzyl-9'-methyl-2-oxo-9'H-spiro[indoline-3,4'-thiopyrano[2,3-
-1
b]indole]-3'-carbonitrile: White powder (62 mg, 94%); mp 189-191 °C; FTIR (cm ): 3431,
1
3
3
6
271, 2183, 1691, 1605, 1572, 1464. H NMR (400 MHz, DMSO-d ), δ, ppm (J, Hz): 3.72 (s,
6
H, -NCH ), 5.02 (m, 2H, -NCH Ph), 6.08 (d, 1H, J 8.4 Hz, ArH), 6.65 (t, 1H, J 7.2 Hz, ArH),
3
2
.99-7.11 (m, 4H, ArH), 7.26-7.32 (m, 4H, ArH), 7.30 (s, 2H, -NH ), 7.41-7.44 (t, 3H, J 5.6 Hz,
2
1
3
ArH), C NMR (DMSO-d6, 100 MHz): , ppm 30.75, 52.0, 55.3, 72.0, 103.1, 109.9, 117.3,
1
18.0, 120.1, 121.8, 123.7, 124.2, 125.4, 125.5, 128.0, 128.9, 129.5, 133.2, 136.4, 137.9, 142.5,
53.2, 176.8. Anal. Calculated (%) for C H N OS, C, 72.30; H, 4.49; N, 12.49; S, 7.15; Found
1
2
7
20
4
C, 72.26; H, 4.53; N, 12.53; S, 7.11.
Antibacterial studies
Antibiotic susceptibility testing of DSF was conducted according to the CLSI guidelines using
the broth microdilution assay. 10 mg/mL stock solutions of test compounds were prepared in
DMSO. Bacterial cultures were inoculated in MHBII and optical density (OD) was measured at
6
00nm, followed by dilution to achieve ~106 CFU/mL. The compounds were tested from 64–0.5
mg/L in two-fold serial diluted fashion with 2.5 μL of each concentration added to well of a 96-
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well round bottom microtiter plate. Later, 97.5 μL of bacterial suspension was added to each well
containing either test compound or appropriate controls. The plates were incubated at 37°C for
1
8-24 h following which the MIC was determined. The MIC is defined as the lowest
concentration of the compound at which there is absence of visible growth. For each test
compound, MIC determinations were carried out independently three times using duplicate
samples.
Cell cytotoxicity of DSF
Cell toxicity was performed against Vero cells using the MTT assay. ~103 cells/well were
seeded in 96 well plate and incubated at 37°C in an5% CO2 atmosphere. After 24 h, compound
was added ranging from 100-12.5 µg/mL concentration and incubated for 72 h. After the
incubation was over, MTT was added in each well, incubated at 37°C for further 4 h, residual
medium was discarded, 0.1 mL of DMSO was added to solubilise the formazan crystals and OD
was taken at 540 nm for the calculation of CC50. CC50 is defined as the lowest concentration of
compound which leads to a 50% reduction in cell viability. Doxorubicin was used as positive
control and each experiment was repeated in triplicate.
Acknowledgements
N.V.T and V.S thank the UGC and KSCSTE for the financial support in terms of Junior research
fellowship. All authors thank CLIF, University of Kerala, Thiruvananthapuram for the spectral
and elemental analysis, CSIR-NIIST and IISER, Thiruvananthapuram for their valuable support.
Supplementary Material
1
13
The H NMR spectra, C NMR spectra, and CHN data of all new compounds can be found via
the ‘Supplementary content’ section of this article’s webpage
References
1
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. Olesen, P. H.; Hansen, J. B.; Engelstoft, M. J. Het. Chem. 1995, 32, 1641-1642 and
references cited therein
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4
. Egorov, D. L. Chem. Het. Comp. 2016, 52, 779-781.
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Chem. Lett. 2009, 19, 3006–3009.
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preparation and use. U.S. Patent 5, 783, 575, June 21, 1998.
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surface.html
8
9
1
1
. Zhang, Y. -C.; Jiang, F.; Shi, F. Acc. Chem. Res. 2020, 53, 425-446.
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1. Bakiev, A. N.; Irgashev, R. A.; Shklyaeva, E. V.; Vasyanin, A. N.; Abashev, G. G.;
Rusinov, G. L.; Charushin, V. N. Arkivoc 2018, (v), 11-19.
1
1
2. Berg, R.; Bergman, J. Tetrahedron Lett. 2017, 73, 5654-5658.
3. Satheesh, M.; Balachandran, A. L.; Devi, P. R.; Deepthi, A. Synth. Commun. 2018, 48,
5
82-587
1
4. Sathi, V.; Krishnan, P.; Jayasree, D. V.; Deepthi, A.; Biju, P. G. Synth. Commun. 2019,
9, 1592-1602.
4
1
1
1
5. Majumdar, K. C.; Ponra, S.; Nandi, R. K. Tetrahedron Lett. 2012, 53, 1732–1737.
6. Majumdar, K. C.; Ponra, S.; Ghosh, T. RSC Adv. 2012, 2, 1144–1152.
7. The LCMS data of the crude reaction mixture showing dimethyl oxalate peak at m/z
1
18.250 is included in the supporting information. Our attempts to find literature
precedence to dimethyl oxalate elimination type reactions however failed and so we
presume this is the first kind of such a report.
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O
NH2
S
S
N
N
Cl
H
H
A
1
Figure 1. Thienodolin and thieno[2,3-b]indole
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Figure 2. Representative examples for D-π-A systems using thieno[2,3-b]indole
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OMe
HN
O
HO
O
O
o-phenylene
diamine
NH
S
O
S
NaOMe
S
Bergmann, 2017
N
MeOH
N
H
H
N
H
4
2
3
H
N
O
O
CN
CN
EtOH
S
+
+
CN Majumdar, 2012
NH2
O
N
N
H
80 deg C
N
H
H
S
6
NC
O
CN
O
methanol
1h, reflux
CO Me
2
S
O
6 +
This work
+
N
CO Me
2
N
H
2
5
H
Scheme 1. Ring opening reactions of thieno[2,3-b]indole-2,3-dione
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Figure 3. ORTEP diagram of 6g
Table 1. Generality of the reaction
a
b
Entry
Product
6a
R1
H
H
H
H
H
H
H
H
H
R2
H
E
CN
Yield (%)
1
2
3
4
5
6
7
8
9
93
91
90
92
93
89
91
89
92
6b
Me
Et
CN
6c
CN
6d
Pr
CN
6e
Bu
Allyl
CN
6f
CN
6g
CH Ph
2
CN
6h
H
COOEt
COOEt
6i
CH Ph
2
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1
1
1
0
1
2
6j
6k
6l
Me
Me
Me
Pr
H
CN
CN
CN
91
93
92
CH Ph
2
a
All reactions were done by refluxing 2 (0.15 mmol) and 5 (0.15 mmol)
b
in MeOH (4 ml) for 1 h. Isolated yield.
O
O
HO
R
O
CO2Me
S
CO Me
2
O
N
MeOH
S
SH
NC
N
N
N
H
H
H
C
2
3
3'
N
5
MeOH
MeO COC
R
2
MeO COC
N
2
6
O
N
R
CO Me
CN
O
CN
-
2
CO Me
2
NH2
N
H
N
S
S
C
I
II
NH
Scheme 2. Mechanistic rationale
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Table 2 MIC (µg/mL) against ESKAPE pathogen panel
MIC (µg/mL)
S.No
Compound
E. coli
S. aureus
ATCC
K.
A.
P.
ATCC
pneumoniae
BAA 1705
baumannii aeruginosa
2
5922
29213
BAA 1605
ATCC
7853
2
1
2
3
4
5
6
7
8
9
6a
6b
6c
6d
6e
6f
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
32
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
1
8
32
8
6g
6h
6i
8
32
16
0.125
1
0
Levo floxacin 0.0156
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