Synthesis, Characterization and Evaluation of Pharmacological Potential of inclusion Complexes of 2(([1,3,4] Thiadiazino [6,5-b] indol-3-ylimino) methyl) Phenol and its Derivatives with β -Cyclodextrin

Rabinarayana Sahu¹, Pramoda Kumar Das², Bamakanta Garnaik¹*

¹P. G. Department of Chemistry, Berhampur University, Bhanja Bihar- 760007 Odisha, India

²Kendrapara Autonomous College, Kendrapara, Odisha

*Corresponding Author E-mail: bama_61@rediffmail.com

ABSTRACT:

Extensive biochemical and pharmacological studies of indole derivatives have confirmed their highly effectiveness against various strains of micro organisms as well as have various therapeutic effects. Inspired by these observations, in this research work, some substituted indoles i.e. 2(([1,3,4] Thiadiazino [6,5- b] indol-3-ylimino) methyl)substituted phenols are synthesized and their inclusion complexes have been prepared with β -Cyclodextrin to enhance their stability, solubility, bio-accessibility and hence pharmacological activities. The formation of both synthesized pharmacophores and their inclusion complexes are established on the basis of elemental, thermal and spectral (UV, FT-IR, ¹HNMR etc.)studies. The comparative biological screening of the synthesized compounds and their inclusion complexes are made for different bacterial strains, fungi and antioxidant character. The results of the research authenticate the fact that the inclusion complexes of the synthesized compounds exhibit profound antibacterial, antifungal and radical scavenging property with respect to their bared compounds.

KEYWORDS: Substituted Indole, β –Cyclodextrin, Inclusion complex, antibacterial, antifungal, antioxidant

INTRODUCTION:

Indole is found to be a versatile pharamacophore and serves as precursor in many pharmaceuticals. The incorporation of indole nucleus in certain organic compounds has made it multifaceted heterocyclic possessing significant biological activities like antifungal [1], antiviral [2], anti-cancer [3], anti-tubercular [4], anti- malarial [5], antimicrobial [6], anti-tumor [7], anti-inflammatory [8], antidepressant [9], antioxidant [10], and many others.

Indole myriad derivatives are receiving considerable attention of researchers over the years. Hence with an objective of discovering potent pharmacological activities, four different newly Substituted indoles were synthesized by condensing substituted Salicylaldehydes with a key compound, 2-amino-1,3,4-Thiadiazino [6,5 b] indole. The key compound is synthesized by methanolic refluxation of indole-2,3-dione with Thiosemicarbazide [11].

The structure of the synthesized compounds have been analysed spectroscopically (UV, IR, NMR), which provide very valuable information about their structural features. But the solubility of these compounds in aqueous medium is found to be poor, which is a measure stumbling block in showing their bio accessibility and hence their pharmacological activities. The main difficulty of using the compound as a therapeutic agent is its poor bioavailability and metabolic instability. To enlist the improvement in their solubility and stability, the complexation of the synthesized compounds have been done with a suitable host molecule i.e. β – cyclodextrin, a useful molecular encapsulant [12]. Also the synthesized Schiff’s bases obtained from salicylaldehydes have strong ability to form inclusion complexes with β – cyclodextrin. The complex forming tendency of β – cyclodextrin is mainly due to its special architecture i.e. hydrophilic outer and a lipophillic inner cavity. The lipophillic cavity of β – cyclodextrin facilitates a microenvironment into which apolar moieties of suitable dimension can enter to form inclusion complex. The formation and stability of inclusion complexes are verified from measurement of thermodynamic stability constant and change in free-energy etc.

The potential of the synthesized compounds before and after the inclusion complex formation are examined for antibacterial, antifungal and antioxidant activities in order to know their drug efficiency. In comparision, it is found that the inclusion complexes possess promising antibacterial, considerable antifungal and remarkable antioxidant activities as compared to their respective compounds.

MATERIALS AND METHODS:

All the chemicals used in the present investigation are of analytical reagent grade (AR) and procured from sigma Aldrich. 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.

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 spectro photometer. PMR spectra (CDCl₃) are measured on NMR spectrometer (300 mz) using TMS as an internal standard (chemical shift in δ , ppm ).

Synthesis of 2([1,3,4] Thiadiazino [6,5 b]indole -3-ylimino) methyl -4- substituted phenols:

The synthesis of the above titled compounds are carried out mainly according to the route in scheme – 1 which involves the following steps.[13]

Step -1 synthesis of 3-thiosemicarbazido-indol-2-one

A mixture of methanolic solution of isatin (0.0136 mole) and thiosemicarbazide (0.013 mole) is refluxed for 1 hour in a 500 ml R.B 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 recrystallised by using methanol. The percentage of yield is 80% and the melting point is 235⁰C.

Step – 2 Synthesis of 2- amino-1,3,4-thiadiazino[6,5 b] indole:

About 0.013 mole of 3-thiosemicarbazido indol-2-one is taken in a beaker. 1ml 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 recrystallised from ethanol to yield 2- amino-1,3,4-thiadiazino[6,5 b] indole.

Step -3 Synthesis of 2(([1,3,4]thiadiazino[6,5 b]indol-3-ylimino) methyl) phenol):

To salicylaldehyde (reagent grade,0.01mole) dissolved in DMF (50ml), 2-amino-1,3,4-thiadiazino[6, 5 b] indole (0.01 mole) is added. The mixture is refluxed for 6 hours 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 recrystallised form ethanol to give pure compound of 2(([1,3,4]thiadiazino[6,5 b]indol-3-ylimino) methyl) phenol) (compound – C) By following the same procedure other 03 compounds (D,E,F) are 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 (0-7mMl) is studied [14].

Synthesis of inclusion complexes:

Among different methods, co-precipitation method [15] is convenient for the preparation of inclusion complexes of the compounds (C,D,E,F).

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 calculated using Benesi-Hilderband equation [16].

1/∆A=1/∆ε+ 1/ K_T [Guest]_o∆ε.[ β-CD]

Where ∆A is change in absorbance, ∆ε is change in absorption coefficient, K_T is stability constant, [Guest] 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

Stability Constant (K_T) = Intercept/Slope

The value of ΔG at 298 K is calculated by using the equation:

ΔG = -RT ln K_T , where K_T is the stability constant.

Antibacterial study:

The in vitro biological screening of the newly synthesized compounds and their inclusion complexes are undertaken for few bacterial strains such as Escherichia coli, P. Vulgaris and Staphylococcus aureus by cup plate method using nutrient agar medium [17-18].

Evaluation of Antifungal activity:

The in vitro activity of the newly synthesized compounds and their respective inclusion complexes are undertaken against 24 hours cultures of two fungi namely Aspergillus niser and Candida albicans by cup plate technique [19]. The compounds are tested at a concentration of 0.002 mg/ml in DMF. With the help of sterile cork borer (8 mm) cups are cut and into each of these cups 0.1 ml of the test solution, standard drug and the control (DMF) are placed separately under aseptic condition. The zone of inhibition is compared with the standard drug (Micanazole) after 72 hours of incubation at 37⁰C. The results are recorded in Table VI .

Evaluation of Antioxidant activity:

The free radical entrapping capacity of the synthesized compounds and their inclusion complexes are evaluated by DPPH scavenging assay method, adopted by Tagashira and Ohtake [20]. 0.1mM solution of DPPH in ethanol is prepared and 1ml of this solution is mixed with 3ml of the sample solution in water at various concentrations. The mixture is incubated for 30 minutes at 300K. Then the absorbance of the samples are measured spectrophotometrically at 517nm against a blank. The percentage of inhibition is calculated by the following equation.

% of Inhibition = A₀ – A /A₀ ×100 .

Where, A₀ is the absorbance of the control (without sample) and A is the absorbance of the test sample.

RESULTS AND DISCUSSION:

All the four compounds (C, D, E, F) are synthesized in their crystalline solid forms with maximum purity. The maximum 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 (C,D,E,F) and their inclusion complexes have been elucidated from physical properties (Table 2), elemental composition and spectral data such as UV, IR and ¹H NMR (Table III). The composition of elements present in the compounds derived through CHN analyser resembles with theoretical data (Table-1).

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 interactions 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.

Table 1: Elemental Composition of the compounds

Compound	Elemental Analysis
Compound	C	H	N	S	O	Cl	Br
C	50.00(49.42)	31.25(31.16)	12.50(12.35)	3.12(3.01)	3.12(3.02)
D	50.00(49.42)	28.12(31.16)	12.50(12.35)	3.12(3.01)	3.12(3.02)	3.12(3.01)
E	50.00(49.42)	28.12(31.16)	12.50(12.35)	3.12(3.01)	3.12(3.02)		3.12(3.01)
F	47.05(49.42)	26.47(26.16)	14.70(14.55)	2.94(2.87)	8.82(8.50)

Table 2: Some physical properties of the synthesized compounds and complexes

Sl No.	Compound/Complex	Molecular formula	Molecular weight	Colour	M.P. (⁰C)	Yield(%)
1	Compound- C	C₁₆H₁₀N₄OS	306	Light yellow	232-237	78
1	I.C._C			Pale yellow	260-265	73
2	Compound- D	C₁₆H₉N₄OSCl	340.5	Light brown	87-92	70
2	I.C._D			Dull white	255-260	75
3	Compound- E	C₁₆H₉N₄OSBr	385	Light brown	110-115	78
3	I.C._E			white	250-255	73
4	Compound- F	C₁₆H₉N₅O₃S	351	yellow	215-220	70
4	I.C._F			Pale yellow	270-275	75

Table 3: Spectral data of synthesized compounds and complexes

Sl No.	Compound/ Inclusion	UV λ_Max(nm)	IR (KBr) cm^-1	NMR
1	Compound C	354	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., Ar-H)	¹H NMR (CDCl₃) : d 6.99-7.9(d,4H, Ar-H), 7.02-7.60(m,4H, Ar-H),5.32(s,1H, OH),8.35 (s, 1H, CH)
2	Inclusion C	356	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-CD)	¹H NMR (CDCl₃) : d 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
3	Compound D	342	748 (C-Sstr.), 1251(C-N str.), 1338 (C-O str.) 1485(Ar., C=C str.), 1714 (C=N str.), 3120(C-H str., Ar-H)	¹H NMR (CDCl₃) : d 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)
4	Inclusion D	345	754(C-Sstr.), 1361 (C-N str.), 1541 (Ar., C=C str.), 1728 (C=N str.), 3307(H-bonding with β-CD)	¹H NMR (CDCl₃) d 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)
5	Compound E	349	746 (C-Sstr.), 1487 (Ar., C=C str.), 1714 (C=N str.), 3041(C-H str., Ar-H)	¹H NMR (CDCl₃) : d 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)
6	Inclusion E	351	756(C-Sstr.), 1541(Ar., C=C str.), 1716 (C=N str.), 3253(H-bonding with β-CD)	¹H NMR (CDCl₃) : d 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)
7	Compound F	345	740(C-Sstr.), 1253(C-N str.), 1581, 1622 (Ar., C=C str.), 1699 (C=N str.),3054(C-H str., Ar-H)	¹H NMR (CDCl₃) : d 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)
8	Inclusion F	348	754(C-Sstr.), 1256 (C-N str.), 1585, 1631 (Ar., C=C str.), 1725 (C=N str.), 3244(H-bonding with β-CD)	¹H NMR (CDCl₃) : d 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)

Table V : Antioxidant activity of the synthesized compounds and their inclusion complexes

Compound and complexes

Conc.(μg/ml)

Percentage of inhibition

Mean percentage of inhibition ± SD

Trial - 1

Trial - 2

Trial -3

Compound - C

100

500

38.6

57.8

38.9

58.2

39.1

57.6

38.86 ± 0.19

57.86 ± 0.24

IC. C

100

500

47.8

68.2

48.2

67.8

47.6

68.4

47.86 ± 0.24

68.13 ± 0.24

Compound - D

100

500

37.8

52.4

38.2

52.6

37.9

52.4

37.96 ± 0.17

52.46 ± 0.09

IC. D

100

500

40.2

61.3

40.7

61.6

40.8

60.9

40.56 ± 0.26

61.26 ± 0.28

Compound – E

100

500

40.4

54.6

40.6

54.5

41.0

54.0

40.66 ± 0.24

54.36 ± 0.26

IC. E

100

500

50.6

62.5

50.8

62.8

51.2

62.3

50.86 ± 0.24

62.53 ± 0.20

Compound – F

100

500

48.2

58.8

47.6

58.3

48.2

58.5

48.0 ± 0.28

58.53 ± 0.20

IC. F

100

500

58.6

65.5

59.1

65.7

59.3

66.1

59.00 ± 0.29

65.76 ± 0.35

STANDARD (ASCORBIC ACID)

100

500

74.4

89.9

74.5

90.0

74.0

90.1

74.30 ± 0.21

90.00 ± 0.08

This increase of bactericidal activity of the inclusion complexes may be due to the enhanced solubility of the synthesized medicinally active molecules which makes them more bioactive to specific infected tissues leading to increased drug activity [23]. The free radical entrapping activity of the compound increases significantly after caging in the core of β-CD as shown in Table V. This can be correlated to the higher solubility of the compounds due to inclusion complex formation there by increasing the bio-accessibility [24]. The standard deviation of all the three experimental data shows a very good result.

Higher bio-accessibility of compounds helps to increase their entrapping nature towards reactive oxygen species (free radicals), for which the antioxidant character of the compounds is increased. All the compounds (C, D, E, F) show a good activity towards the three fungal organisms. The antifungal behavior of the compounds noticeably enhanced after entering in the cavity of β- cyclodextrin which is ascertained from the zone of inhibition (Table-VI).

Table VI: Antifungal activity of the synthesized compounds and their inclusion complexes

Compound/complex	Diameter of zone of inhibition in mm
Compound/complex	*A, Niger*	*C. Albicans*
Compound C	08	09
Inclusion Complex Of C	12	13
Compound D	07	08
Inclusion complex of D	13	12
Compound E	09	07
Inclusion complex of E	14	13
Compound F	09	10
Inclusion complex of F	15	15
Control	0	0
Standard	20	22

ACKNOWLEDGEMENT:

The authors are thankful to Dr. J. Panda, Asst. Professor of Roland Institute of Pharmaceutical science, Berhampur, Odisha, India for providing necessary facilities to examine antibacterial, antifungal, and antioxidant behaviour of the compounds.

CONCLUSION:

The present research work successfully synthesized inclusion complexes of Indole derivatives with β- cyclodextrin. The results of research concludes that the encapsulation of the synthesized compounds in the cavity of β- cyclodextrin is the most suitable mechanism to increase the stability, solubility and the pharmacological activity in comparision to their respective compounds.

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Received on 16.08.2017 Modified on 20.09.2017

Asian J. Research Chem. 2017; 10(6):757-764.

DOI: 10.5958/0974-4150.2017.00128.6

Sl No.	Inclusion complex of Compound	Equilibrium Constant K_T in M^-1	ΔG=-2.303RTlog KΔG (kJ/mol)	Correlation coefficient(r)
1	I.C._C	735.71	-16. 466	0.9987
2	I.C._D	660.10	-16.195	0.9756
3	I.C._E	860.86	-16.858	0.9899
4	I.C._F	291.25	-14.155	0.9887