Synthesis, characterization and in vitro antitubercular evaluation of some schiff bases of substituted indoles and their inclusion complexes with β-cyclodextrin

Article abstract of DOI:10.14233/ajchem.2018.20986

Indole derivatives are best known for their biological importance as bactericides and fungicides which encouraged us to synthesize some Schiff bases of substituted indoles i.e. 2(([1, 3, 4]thiadiazino[6, 5-b]indol-3-ylimino)methyl)substituted phenols. The

Full text of DOI:10.14233/ajchem.2018.20986

Asian Journal of Chemistry; Vol. 30, No. 3 (2018), 556-560
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2018.20986
Synthesis, Characterization and in vitro Antitubercular Evaluation of Some Schiff
Bases of Substituted Indoles and their Inclusion Complexes with β-Cyclodextrin
1
1
2
2
1,*
PRAMODA KUMAR DAS , RABINARAYANA SAHU , PRAKASINI SATAPATHY , DASARATHI DASH and BAMAKANTA GARNAIK
1P.G. Department of Chemistry, Berhampur University, Bhanja Bihar-760 007, India
2ICMR-Tuberculosis Laboratory, Regional Medical Research Center, Bhubaneswar-751 023, India
*Corresponding author: E-mail: bama_61@rediffmail.com
Received: 9 August 2017;
Accepted: 31 October 2017;
Published online: 31 January 2018;
AJC-18743
Indole derivatives are best known for their biological importance as bactericides and fungicides which encouraged us to synthesize some
Schiff bases of substituted indoles i.e. 2(([1,3,4]thiadiazino[6,5-b]indol-3-ylimino)methyl)substituted phenols. The synthesized compounds
are found to exhibit antitubercular activities. With a view to enhance solubility induced potential anti tubercular activities, these synthesized
compounds are encapsulated with β-cyclodextrin. Through elemental and spectral (UV, IR, 1H NMR) characterization, the architecture of
the compounds and their inclusion complexes are confirmed. They are also screened for in vitro antitubercular activities by using rifampicin
as the reference standard. The synthesized compounds exhibited good antitubercular activity but their inclusion complexes exhibited
profound antitubercular activities as compared to their respective compounds.
Keywords: Schiff bases, Inclusion complex, Antitubercular activity, β-Cyclodextrin.
Therefore to enlist the improvement in their solubility and
pharmacological activity, the inclusion complex of the com-
pounds are prepared with β-cyclodextrin, a useful molecular
encapsulant [10] (a host compound). The complex forming
tendency of β-cyclodextrin is mainly due to its special struc-
tural design i.e. hydrophilic outer and a hydrophobic interior
[11]. The host guest stoichiometry of the complex has been
ascertained from the aqueous phase solubility studies of the
compound. The change in free energy and stability constant
of the inclusion complexes have also been determined, which
reveals that the formation of inclusion complex is thermodyna-
mically permissible [12]. The antitubercular activity of this
synthesized compounds and their respective inclusion comp-
lexes are studied by absolute concentration method and from
the findings of the study it is revealed that the inclusion comp-
lexes of the compounds exhibit profound antitubercular
properties in comparison to their respective compounds.
INTRODUCTION
Indole nucleus is continuously drawing interest for the
development of newer drug moiety due to its wide range of
pharmacological activities such as antibacterial [1], antifungal
[2], antioxidant [3], antiviral [4], antitubercular [5], anticancer
[6], antitumor [7], anti-inflammatory [8], etc. Since this nucleus
has shown quite good response as an antitubercular agent, it
has become an interest in the field of research. Further, since
there are fewer antitubercular drugs available and there are
ever increasing fear of drug resistance, it is becoming more
and more essential to synthesize some more new drugs with
higher potency and low toxicity. So with an objective of explo-
ring the potent antitubercular activities, four different Schiff
bases of indole moiety and multiple functional groups are
synthesized by condensing salicylaldehyde and 5-substituted
salicylaldehydes with a key compound, 2-amino-1,3,4-
thiadiazino[6,5-b]indole. The key compound is synthesized
by methanolic refluxing of indole-2,3,dione with thiosemi-
carbazide [9]. The structure of the sysnthsized compounds have
been analyzed spectroscopically [UV, IR and PMR], which
provide a valuable information about their structural features.
The synthesized compounds are found to exhibit good anti-
tubercular activities. But the sparingly soluble property of the
synthesized compounds in aqueous medium may reduce their
bio-accessibility and hence their pharmacological activity.
EXPERIMENTAL
All the chemicals used in the present investigation are of
analytical reagent grade (AR) and procured from SigmaAldrich.
Aqueous solution of the compounds are prepared by using
doubly distilled water. All the melting points are determined
in open glass capillaries with the help of thermonic melting
point apparatus and are uncorrected. Samples are routinely
purified by crystallization and checked by TLC.

Vol. 30, No. 3 (2018)
Synthesis and in vitro Antitubercular Evaluation of Some Schiff Bases of Substituted Indoles 557
Absorption spectra are recorded on Shimadzu-1800 UV-
visible spectrophotometer. IR spectra of the compounds and
inclusion complexes are recorded as KBr pellets on a Shimadzu
8400 FTIR spectrophotometer. PMR spectra (CDCl3) are
measured on NMR spectrometer (300 MHz) using TMS as an
internal standard (chemical shift in δ, ppm).
Synthesis of 2([1,3,4]thiadiazino[6,5b]indol-3-
ylimino)methyl-4-substituted phenols: The synthesis of the
Schiff bases are carried out mainly according to the route in
Scheme-I [13].
Step-2: Synthesis of 2-amino-1,3,4-thiadiazino[6,5b]-
indole: About 0.013 mol of 3-thiosemicarbazido indol-2-one
is taken in a beaker. 1 mL of cold concentrated H2SO4 is added
to it. The mixture is then set aside overnight. The ice-cold
water is added to the beaker containing mixture followed by
few drops of liquid ammonia till neutralization. The solid mass
is found which is filtered, washed with distilled water and dried.
The dried residue is recrystallized from ethanol to yield 2-amino-
1,3,4-thiadiazino[6,5b]indole.
Step-3: Synthesis of 2(([1,3,4]thiadiazino[6,5b]indol-
3-ylimino)methyl)phenol): To salicylaldehyde (reagent grade,
0.01 mol) dissolved in DMF (50 mL), 2-amino-1,3,4-thiadia-
zino[6,5b]indole (0.01 mol) is added. The mixture is refluxed
for 6 h in the presence of 0.6 mL glacial acetic acid. The end
point of the reaction is checked by TLC and excess of the
solvent is distilled out. It is then poured over crushed ice,
which gives a precipitate. The precipitate is filtered through
Whatmann 42 filter paper, washed with distilled water and
then dried in open air to give a crude product which is then
recrystallized form ethanol to give pure compound of
2(([1,3,4]thiadiazino[6,5 b]indol-3-ylimino)methyl)phenol)
(compound – P)
O
S
NH2HNCNH2
N
O
H
CH3OH
N
NH
C
NH
2
N
H
O
S
By following the same procedure other 03 compounds
(Q, R, S) are also synthesized.
Phase solubility measurements: As per Higuchi-Connor
method the extent of solubility of the compounds in aqueous
medium with different concentrations of β-cyclodextrin (0-7
mML) is studied [14].
N
N
C
NH
2
N
OH
HS
Synthesis of inclusion complexes: Among different
methods, co-precipitation method [15] is convenient for the
preparation of inclusion complexes of the compounds (P,Q,
R, S).
Conc.H2SO4
HO
N
N
R
Study of thermodynamic properties: From plots of inverse
of change in absorbance versus inverse concentration of β-
cyclodextrin the stability constants of the complexes are calcu-
lated using Benesi-Hilderband equation [16]:
C
NH
2
N
S
C
O
H
R = H (Compound P)
Glacial CH3COOH
R = 5-Cl (Compound Q)
R = 5-Br (Compound R)
R = 5-NO2 (Compound S)
1/∆A = 1/∆ε + 1/KT [Guest]o ∆ε·[β-CD]
HO
N
where ∆A is change in absorbance, ∆ε is change in absorption
coefficient, KT is stability constant, [Guest]o is the concentration
of compound and [β-CD] is the molar concentration of β-
cyclodextrin. The values of stability constants for all the
complexes are calculated using the relation
N
C
R
N
C
N
S
H
2(([1,3,4]Thiadiazino[6,5-b]indol-3-ylimino)methyl)phenol (P)
2(([1,3,4]Thiadiazino[6,5-b]indol-3-ylimino)methyl)-4-chlorophenol (Q)
2(([1,3,4]Thiadiazino[6,5-b]indol-3-ylimino)methyl)-4-bromophenol (R)
Stability constant (KT) = Intercept/Slope
2(([1,3,4]Thiadiazino[6,5-b]indol-3-ylimino)methyl)-4-nitrophenol (S)
The value of ∆G at 298 K is calculated by using the
equation:
Scheme-I
Step-1: Synthesis of 3-thiosemicarbazido-indol-2-one:
A mixture of methanolic solution of indole-2,3-dione (0.0136
mol) and thiosemicarbazide (0.013 mol) is refluxed for 1 h in
a 500 mL round bottom flask. The end point of the reaction is
checked by thin layer chromatography. Excess of methanol is
distilled out and the content is cooled. Yellow precipitate is
appeared after the addition of refluxed mixture into ice cold
water.
In order to obtain 3-thiosemicarbazido-indol-2-one in pure
form, the residue is washed with distilled water, dried and
recrystallized by using methanol. The percentage of yield is
80 % and the melting point is 235 °C.
∆G = -RT ln KT
where KT is the stability constant.
Antitubercular activity: Antitubercular activity was carried
out by absolute concentration method [17]. M. tuberculosis
H37RV, a laboratory strain culture suspension was prepared
and matched with McFarland 1.0 standard. The suspension
was diluted 1:100; 0.02 mL of the dilution was used for inocu-
lation onto LJ (Lowensen-Jensen) medium in the control tube
(without drug) as well as tubes containing graded concentration
of the compounds and inclusion complexes to be tested. Six
concentrations (1, 2, 4, 10, 20 and 40 µg/mL) of each com-
pounds and inclusion complexes were tested and rifampicin

558 Das et al.
Asian J. Chem.
(40 µg/mL) and isoniazid (0.2 µg/mL) was taken as reference
standard. Resistance was expressed in terms of the lowest
concentration of the drug that inhibits the growth i.e. MIC.
Viability of the organism and inoculums size greatly affects
the method.
The melting point of inclusion complexes of respective
compounds are always marked with increased value which
may be assumed through the fact that an additional thermal
energy is required for de-encapsulating the compound from
the β-cyclodextrin cavity (Table-2). The IR frequency data 746
(C-Sstr.), 1211(C-N str.), 1338 (C-O str.), 1471, 1541, 1616
(Ar., C=C str.), 1714 (C=N str.), 3041(C-H str.) confirms the
presence of these groups in the compound. There is a noticeable
change in the IR data in all compounds after encapsulation
(absorption frequencies shift towards higher energy side),
which is featured to the fact that there are some weak inter-
actions within the hydrophobic cage of β-cyclodextrin (Table-
3). The host-guest complexation is further supported by NMR
data (Table-3). When the NMR data of the compounds are
compared with inclusion complexes, signals of different protons
reveal that all the protons undergo smaller shifts (towards
upfield in case of all the compounds) after encapsulation. These
shifts can be explained on the basis of shielding of protons
from the applied magnetic field in the cavity of β-cyclodextrin.
Different graphs are drawn with a definite concentration
of the synthesized compounds vs. different conc. (0-10 mM)
of β-cyclodextrin. From the graphs it is clear that solubility of
the compounds in aqueous medium steadily increase as a
function of the concentration of β-cyclodextrin up to 5th point
followed by a smooth decline (Fig. 1). This indicates that the
concentration at 5th point is the most appropriate one for getting
the higher yield of inclusion complex. Better correlation coeffi-
cients are obtained which have values close to unity, which
assumes the stoichiometry of these complexes may be 1:1 [18].
By using Benesi-Hilderband relation, thermodynamic stability
constants (KT) of host-guest complexes were determined.
Good linear correlations were obtained for a plot of 1/∆A
versus 1/[β-CD]o for compounds as shown compounds in
Fig. 2.
RESULTS AND DISCUSSION
All the four compounds (P, Q, R, S) are synthesized in
their crystalline solid forms with maximum purity. The maxi-
mum inclusion conc. of β-cyclodextrin has been determined
from aqueous phase solubility study (Fig. 1). The inclusion
complexes of the synthesized bioactive compounds having
indole moiety are prepared with β-cyclodextrin. The structures
of the compounds (P, Q, R, S) and their inclusion complexes
have been elucidated from physical properties (Table-2), ele-
mental composition and spectral data such as UV, IR and 1H
NMR (Table-3). The composition of elements present in the
compounds derived through CHN analyzer resembles with
theoretical data (Table-1).
1.0
0.9
0.8
0.7
0.6
0.5
0.4
Compound P
Compound Q
0.3
Compound R
Compound S
0.2
The values of KT for all the complexes were calculated
using the relation. KT = Intercept/Slope. The KT values of the
inclusion complexes of compounds with β-cyclodextrin were
found to be 735.71, 660.10, 860.86, 291.25 M-1 respectively
(Table-4). The data obtained are within a standard range (100
0
0.001
0.002
0.003
0.004
0.005
0.006
Different concentration of
β-cyclodextrin
Fig. 1. Plot of absorbance vs. β-cyclodextrin conc. of the compounds with
β-cyclodextrin
TABLE-1
ELEMENTAL ANALYSIS RESULTS OF THE COMPOUNDS
Elemental analysis (%): Found (calcd.)
Compound
C
H
N
S
O
Cl
Br
P
Q
R
S
50.00 (49.42)
50.00 (49.42)
50.00 (49.42)
47.05 (49.42)
31.25 (31.16)
28.12 (31.16)
28.12 (31.16)
26.47 (26.16)
12.50 (12.35)
12.50 (12.35)
12.50 (12.35)
14.70 (14.55)
3.12 (3.01)
3.12 (3.01)
3.12 (3.01)
2.94 (2.87)
3.12 (3.02)
3.12 (3.02)
3.12 (3.02)
8.82 (8.50)
–
–
3.12 (3.01)
–
3.12 (3.01)
–
–
–
TABLE-2
SOME PHYSICAL PROPERTIES OF THE SYNTHESIZED COMPOUNDS AND COMPLEXES
Compound/Complex
Compound-P
I.C.P
m.f.
C16H10N4OS
m.w.
306.0
Colour
Light yellow
Pale yellow
Light brown
Dull white
Light brown
White
m.p. (°C)
232-237
260-265
87-92
255-260
110-115
250-255
215-220
270-275
Yield (%)
78
73
70
75
78
73
70
75
Compound-Q
I.C.Q
Compound-R
I.C.R
Compound-S
I.C.S
C16H9N4OSCl
C16H9N4OSBr
C16H9N5O3S
340.5
385.0
351.0
Yellow
Pale yellow

Vol. 30, No. 3 (2018)
Synthesis and in vitro Antitubercular Evaluation of Some Schiff Bases of Substituted Indoles 559
TABLE-3
SPECTRAL DATA OF SYNTHESIZED COMPOUNDS AND COMPLEXES
Compound/
Inclusion
UV λmax
(nm)
IR (KBr, νmax, cm-1)
NMR
1H NMR (CDCl3): δ 6.99-7.9 (d, 4H, Ar-H), 7.02-7.60
675 (C-Sstr.), 1211 (C-N str.), 1338 (C-O str.), 1474, 1543,
1618 (Ar., C=C str.), 1691 (C=N str.), 3041 (C-H str.)
Compound P
354
356
(m, 4H, Ar-H), 5.32 (s, 1H, OH), 8.35 (s, 1H, CH)
754 (C-S str.), 1215 (C-N str.), 1338 (C-O str.), 1471,
1539, 1651 (Ar., C=C str.), 1732 (C=N str.) 3371 (H-
bonding with β-cyclodextrin)
748 (C-Sstr.), 1251 (C-N str.), 1338 (C-O str.), 1485 (Ar.,
C=C str.), 1714 (C=N str.), 3120 (C-H str)
1H NMR (CDCl3): δ 6.25-6.99 (d, 4H, Ar-H), 6.6-7.1
(m, 4H, Ar-H), 4.98 (s, 1H, OH), 7.85 (s, 1H, CH
Inclusion P
1H NMR (CDCl3): δ 6.7-7.9 (d, 5H, Ar-H), 7.20-7.60
(m, 2H, Ar-H), 5.30 (s, 1H, OH), 8.40 (s, 1H, CH)
1H NMR (CDCl3) δ 6.10-7.10 (d, 5H, Ar-H), 6.8-7.2
(m, 2H, Ar-H), 4.75 (s, 1H, OH), 7.85 (s, 1H, CH)
1H NMR (CDCl3): δ 6.81-7.70 (d, 5H, Ar-H), 7.25-7.55
(m, 2H, Ar-H), 5.28 (s, 1H, OH), 8.50 (s, 1H, CH)
1H NMR (CDCl3): δ 6.20-7.10 (d, 5H, Ar-H), 6.50-7.10
(m, 2H, Ar-H), 4.65 (s, 1H, OH), 7.90 (s, 1H, CH)
1H NMR (CDCl3): δ 7.2-8.5 (d, 5H, Ar-H), 7.3-7.70 (m,
2H, Ar-H), 5.25 (s, 1H, OH), 8.37 (s, 1H, CH)
1H NMR (CDCl3): δ 6.5-7.9 (d, 5H, Ar-H), 6.8-7.2 (m,
2H, Ar-H), 4.35 (s, 1H, OH), 7.70 (s, 1H, CH)
Compound Q
Inclusion Q
Compound R
Inclusion R
Compound S
Inclusion S
342
345
349
351
345
348
754 (C-Sstr.), 1361 (C-N str.), 1541 (Ar., C=C str.), 1728
(C=N str.), 3307 (H-bonding with β-cyclodextrin)
746 (C-Sstr.), 1487 (Ar., C=C str.), 1714 (C=N str.), 3041
(C-H str.)
756 (C-Sstr.), 1541 (Ar., C=C str.), 1716 (C=N str.), 3253
(H-bonding with β-cyclodextrin)
740 (C-Sstr.), 1253 (C-N str.), 1581, 1622 (Ar., C=C str.),
1699 (C=N str.)
754 (C-Sstr.), 1256 (C-N str.), 1585, 1631 (Ar., C=C str.),
1725 (C=N str.), 3244 (H-bonding with β-cyclodextrin)
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
(Table-4) for the inclusion complexes of compounds P, Q, R
and S, respectively. The negative value of free energy change
indicates that the inclusion complex formation is a thermo-
dynamically allowed process.
Compound P
Compound Q
Compound R
Compound S
TABLE-4
THERMODYNAMIC STABILITY CONSTANT AND
FREE ENERGY CHANGE OF INCLUSION COMPLEXES
Inclusion
complex of
compound
Equilibrium
constant
∆G = -2.303
RT log K
∆G (kJ/mol)
Correlation
coefficient (r)
(KT, M-1)
I.C.P
I.C.Q
I.C.R
I.C.S
735.71
660.10
860.86
291.25
-16. 466
-16.195
-16.858
-14.155
0.9987
0.9756
0.9899
0.9887
1.0
0
200
400
600
800
1000
1/β-Cyclodextrin concentration
The data obtained from the antitubercular studies (Table-5)
concludes that inclusion complexes of the respective comp-
ounds (P, Q, R, S) show good result. The result showed mini-
mum inhibitory concentration for inclusion complexes are
lower (2 µg/mL) than the compounds (4 µg/mL). This may be
explained on the basis of solubility induced bio-accessibility
after encapsulation within host cavity [20].
Fig. 2. Plot of 1/absorbance vs. 1/β-cyclodextrin concentration
to 1000 M-1). This explains the appreciable stabilities of the
inclusion complexes through host-guest interaction like van
der Waal’s force, hydrophobic interaction etc. [19].
The value of free energy of activation has been calculated
and found to be -16.466, -16.195, -16.858 and -14.155 kJ/mol
TABLE-5
ANTITUBERCULAR ACTIVITY OF TEST COMPOUNDS/ INCLUSION COMPLEXES AND STANDARD DRUGS
Concentration (µg/mL)
Compound (C)/
Inclusion complex (IC)
1
2
4
10
20
40
C-P
IC-P
C-Q
IC-Q
C-R
IC-R
C-S
Growth
Growth
Growth
Growth
Growth
Growth
Growth
Growth
Growth
No growth
Growth
No growth
Growth
No growth
Growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
No growth
IC-S
No growth
Rifampicin (40 µg/mL)
Isoniazid (0.2 µg/mL)
No growth
No growth

560 Das et al.
Asian J. Chem.
7. E. Nassar, J. Am. Sci. Org., 6, 338 (2010).
ACKNOWLEDGEMENTS
8. G.P. Kalaskar, M. Girisha, M.G. Purohit, B.S. Thippeswamy and B.M.
Patil, Indian J. Heterocycl. Chem., 16, 325 (2007).
9. P.K. Das, R. Sahu and B.K. Garnaik, IOSR J. Appl. Chem., 9, 2265 (2016).
10. B. Yang, J. Lin, Y. Chen and Y. Liu, Bioorg. Med. Chem., 17, 6311
(2009);
The authors greatly acknowledge to The Director, Regional
Medical Research Centre (ICMR branch), Bhubaneswar, India
for providing the necessary facilities to examine antitubercular
activities of the compounds.
https://doi.org/10.1016/j.bmc.2009.07.060.
11. J. Szetjli, TIBTRCH, 7, 171 (1989).
12. S. Panda and J.K. Tripathy, Asian J. Chem., 23, 1631 (2011).
13. R. Sahu, P.K. Das and B. Garnaik, Pharmachem, 8, 176 (2016).
14. T. Higuchi and K.A. Connor, Adv. Anal. Chem. Instrum., 4, 117 (1965).
15. S.S. Nayak, S. Panda, P. Panda and M.S. Padhy, Bulg. Chem. Commun.,
42, 147 (2010).
REFERENCES
1. D.S. Mehta, K.H. Sikotra and H.V. Shah, Indian J. Chem., 44B, 2594
(2005).
2. H. Panwar, N. Chaudhary and S. Singh, Rasayan J. Chem., 4, 371 (2011).
3. N.V. Cinchana, P.S. Sujan Ganapathy and S.D. Shruthi, Res. J. Pharm.
Biol. Chem. Sci., 2, 353 (2011).
16. H.A. Benesi and J.H. Hildebrand, J. Am. Chem. Soc., 71, 2703 (1994);
https://doi.org/10.1021/ja01176a030.
4. M. Tichy, R. Pohl, H.Y. Xu, Y. Chen, F. Yokokawa, P.-Y. Shi and M.
Hocek, Bioorg. Med. Chem., 20, 6123 (2012);
17. C. Mamatha, M. Thangavel and V. Beaulah, Eur. J. Pharm. Med. Res.,
3, 382 (2016).
https://doi.org/10.1016/j.bmc.2012.08.021.
5. S. Lingala, R. Nerella, R. Cherukupally and A.K. Das, Int. J. Pharm.
Sci. Rev. Res., 6, 128 (2011).
6. B.C. Hong, Y. Jiang,Y. Chang and S. Lee, J. Chin. Chem. Soc. (Taipei),
53, 647 (2006);
18. S. Hazra and G.S. Kumar, RSC Advances, 5, 1873 (2015);
https://doi.org/10.1039/C4RA10204E.
19. J. Szetli, ed.:W.F. Smolen and L.A. Ball, Controlled Drug Bioavailability,
Wiley Interscience, New York, vol. 3, 365 (1985).
20. C. Nicolescu, C. Arama, A. Nedelcu and C.M. Monciu, Farmacia, 58,
78 (2010).
https://doi.org/10.1002/jccs.200600086.