Synthesis, Characterisation and Antibacterial Activity of Some Indole Derivatives and their Inclusion Complexes with β-Cyclodextrin

Introduction

β-Cyclodextrin is an oligosaccharide containing seven D-glucose units forming a cyclic ring. The seven D-glucose units joined through  1,4-alpha linkages in such a way as to form a ring, a chain bracelet each link of which in pyranose hexagon.Mendez et al¹ studies the formation of amphiphilic click cluster of β-cyclodextrin with DNA and effect of structural modification of DNA complexes and their delivery properties. The diameter of  β-cyclodextrin ring is about twice the ring of 18-crown-6 and its hole (4.5Å across) is twice broad. The hole of cyclic ring of β-cyclodextrin is expected to entrap a small or linear molecule to form a inclusion complexes.  The interaction products of B-cyclodextrin with bioactive molecules may be physiological interest and we have motivated us to synthesised some of the new products with these Schiff bases. Isatin (indole-2,3 dione) derivatives that possess wide spectrum of medicinal activities like antibacterial², antiviral³, antihelmintic⁴, herbicidal⁵, fungicidal⁶ and antitubercostatic⁷ properties. Isatin and indole were formerly extracted from natural products and these were reported to possess immense medicinal activities^8-14. The Schiff bases of isatin (indole-2,3 dione) with semicarbazone, thiosemicarbazone and thiocarbohydrazide are expected to be get interrupted into β-cyclodextrin hole to form inclusion complexes and these may possess physiological activity. The hydrazide and hydrazine derivatives of isatin and substituted isatin possess pronounced anticonvulsant activities^15-21. A number of Schiff bases of isatin derivatives have also been used as neurotoxic agent¹⁷. The epilepsy is a well known  neurological disorder characterized by unprovoked  seizure of convulsion and thus efforts have directed for its drug development. The isatinsemicarbazones have also been used as an excellent anticonvulsant drug^18-19. The compound 3-(3′,4′-dihydro-2-methymarcapto-4-oxoquinozoline-3′-yl) iminoisatin has been proved to be most active anticonvulsant molecule possessing  neurotoxicity²⁰. A number of new N-methyl/N-acetyl-5-substituted isatin-3-semicarbazones have been reported to possess anticonvulsant activity  by Pandeya and Raja²¹. Since last decades a number of isatin derivatives have been shown  to possess potential hypnotic²², antibacterial^23-24, monoamine oxidaze (MAO) inhibitory²⁵  and antioxidant²⁶ properties. However much attention has been focused on chemistry of isatin derivatives due to its biological potentiality like anticancer, anti-protozoa, cytotoxic, their presence in hippocampus cerebellum brain and DNA cleavage¹⁶.

Isatin derivatives have been found in some mammalian tissues where their function is as modulator of biochemical processes²⁶. Isatin has been reported as “Tribulin” and identified as a selective inhibitor of monoamine oxidaze (MAO)²⁵. A new series of 5-hydroxyisatin derivatives has been synthesized by hydroxylation of aromatic ring in isatin and these has been shown to possess anti-anxiety effect⁵. These immense medicinal activities of isatin derivatives motivated us to study the inclusion complexes of Schiff bases with β-cyclodextrin(β-cyclod), Bisisatinyl thiocarbohydrazide (BisttcabH), isatinylthiosemicarbazone (IsttscabH) and isatinylsemicarbazone (IstscabH). Therefore, in the present paper, we report the synthesis and characterisation of inclusion complexes of IstscabH, IsttscabH and BisttcabH with  β-cyclodextrin and investigated bio activity of some bacteria namely Bacillus subtilis,  Staphylococcus and P. mirabilis.

Experimental

Methods and Material

β-Cyclodextrin and isatin were obtained from Sigma Aldrich and semicarbazide, thiosemicarbazide, thiocarbohydrazide used were either BDH or E Merck and was extra pure reagents. Solvents used were obtained from BDH. The result of  C, H, N, S were obtained from IIT Patna using vario MICRO  V-3 Elemental analyser. The IR spectra were recorded on Shimadzu IR Spectrophotometer in KBr disc in the range of 400 to 600 cm^-1. All  UV visible spectra were recorded in ethanol or dimethylformamide (DMF) in the range of 200-800 nm using a Shimadzu Spectrophotometer No. 2000 at IIT Patna. Similarly, ¹H NMR and ¹³C NMR were recorded in DMSO on Bruker 400 MHz NMR spectrophotometer at IIT Patna. The molecular weight of inclusion product  determined by Rast method in Camphor²⁷. Bacterial screen were performed in the Department of Biochemistry, Patna University, Patna.

Preparation of Isatin Derivatives

Isatinderivatives, isatinylsemicarbazone, (IstscabH), isatinylthiosemicarbazone (IsttscabH) and bisisatinylthiocarbohydrazone (BisttcabH) were prepared by condensing isatin in stoichiometric proportion with semicarbazide, thiosemicarbazide and thiocarbohydrazide in aqueous ethanol containing a few drops of acetic acid at reflux temperature.

Procedure

0.01 Millimole of isatin in 30ml ethanol and 0.01millimole of semicarbazide (or thiosemicarbazide hydrochloride) dissolved in 40-50 ml hot aqueous ethanol containing 1 ml of acetic acid were mixed together and refluxed on a steam bath  for 40-50 minutes when desired product separated as bright yellow or orange yellow products. The products were cooled and collected on buckner funnel, washed with cold aqueous ethanol and dried over  in a desiccator. The dried products were crystallised from hot ethanol and analysed. The analytical results and melting point of products are recorded in Table 1. The bisisatinylthiocarbohydrazide were prepared as above taking  millimole of isatin and 0.01  millimole of thiocarbohydrazide in hot aqueous ethanol 50 ml  containing 1 ml acetic acid. The product was separated as orange yellow compound and recrystallized with ethanol-dioxane mixture.

Preparation of Inclusion Complexes of Isatin Derivatives with  β-Cyclodextrin

0.001 millimole of β-cyclodextrin was dissolved in 100 ml aqueous ethanol (1:1) and treated with 0.012 millimole of appropriate isatin derivatives (IstscabH, IsttscabH or BisttcabH) dissolved in 100 ml hot aqueous ethanol (75%). The mixed solution were refluxed for two hours at steam bath and cooled at room temperature when some unreacted ligand separated which was removed by filtration and filtrates were left in air for slow evaporation at room temperature. After 3 to 4 days cream yellow product separated when the volume of mixed solution was about  20-25 ml (1/8 of original volume). The products were collected on a buckner funnel and washed with cold aqueous ethanol and dried in a desiccator over CaCl₂. The molecular weight of ligands and inclusion products were determined in Camphor by Rast method²⁷. The analytical results C, H, N, S  percentage, molecular weight and melting point of products are given in Table 1. The IR, electronic absorption spectra and ¹H NMR as well as ¹³C NMR spectrum of isatin derivatives and their inclusion complexes were recorded and results are shown in Table-2, 3, 4.

Table 1: Analytical Results and Physical Data of Isatin Derivatives and Their Inclusion Complexes with β-Cyclodextrin

Sr. No.	Compound &        Formula	Elemental Analysis, Found (Cal) (%)				Moleculalar weight Found (cal)  in (g)	Decomposition    Temperature       in (°C)
		C	H	N	S
1.	IstscabH (C9H8N4O2)	52.71 (52.93)	4.12 (3.95)	27.28 (27.44)		204.31	278
2.	IsttscabH (C9H8N4SO)	48.73 (49.06)	3.81 (3.66)	25.19 (25.44)	14.31  (14.55)	220.31	237
3.	BisttcabH (C17H12N6O2S)	55.81 (56.04)	3.41 (3.28)	22.92 (23.08)	8.91   (8.79)	364.57	302
4.	[β-cydxIstscabH] (C51H78N4O37)	45.41 (45.74)	5.71 (5.83)	4.38 4.18		1312.21 (1339.29)	287-290
5.	[β-cydxIsttscabH] (C51H78N4SO36)	45.01 (45.20)	5.93 (5.76)	4.01 (4.14)	2.18   2.36	1332.31 (1355.35)	252 – 273
6.	[β-cydxBisttcabH] (C59H82N6O37S)	47.01 (47.26)	5.68 (5.47)	5.43 (5.61)	2.01  (2.14)	1461.41 (1499.55)	295-297

Results and Discussion

The elemental analysis C, H, N, S of inclusion products of isatin derivatives (IstscabH, IsttscabH and BisttcabH) with β-cyclodextrin (C₄₂H₇₀O₃₅), (β-Cydx) correspond to compositions β-cydx-Istscabh (C₅₁H₇₈N₄O₃₇), β-cydx-Isttscabh (C₅₁H₇₈N₄OS) and β-cydx-BisttcabH (C₅₉H₈₂N₆O₃₆S). These products are insoluble in cold water but dissolve slightly in boiling water. These products are soluble in DMF and DMSO without decomposition. The molecular weight of inclusion complexes determined by Rast method in Camphor and these approximately correspond to calculated molecular weight which supports the formation of inclusion product. The melting point of isatin derivatives and their inclusion products with β-cyclodextrin show wide variation in their physical properties from each other. β-Cyclodextrin in general on heating started decomposing with melting above 200°C but inclusion complexes are stable below 240°C. The inclusion complexes of β-cydx-IstscabH and β-cydx-IsttscabH decompose slowly at 285°C and 290°C respectively. The electrical conductance values of isatin derivatives and their inclusion complexes were measured qualitatively at room temperature in DMF solution. All Schiff bases and their inclusion products show electrical conductivity in the range 70-72 ohm^-1 mol^-1 cm² indicating that in these Schiff bases as well as inclusion products. The Schiff bases NH protons get ionised in DMF solution. The ionic behaviour of NH or tautomeric SH (Thione-thiol) proton has also been supported by ¹H NMR spectra of isatin derivatives²⁸.

Infrared Spectra

The IR spectrum (Figure-IR₁) of IstscabH shows ν(NH₂) + ν(NH) vibrations^31-32 as broad band between 3286-3156 cm^-1. The  ν(CO) carbomide band of isatin ring and carbohydrazone ν(CO) are observed as very strong  band at 1715 and 1693 cm^-1 respectively³². The hydrazine diazene ν(C=N) and benzene ring ν(C=C) bands are also located as very strong band at 1603 cm^-1. The  ν(NH₂ ) and ν(NH ) could be assigned to a strong bands located at 1471 cm^-1 and 1451 cm^–1. The ν(C-N), ν(N-N) and benzene ring skeletal bands³³ can be assigned to IR bands at 1391, 1350, 1197, 1163 and 1109 cm^-1. The isatin ring NH, C-H and semicarbanzone part NH as well as NH₂ deformation vibration of IstscabH can be assigned to IR bands at 1052, 951, 787, 749 and 664.6 cm^-1. The  IR spectrum of its inclusion complex (Figure-IR₂) with  β-cyclodextrin retains most of its IR vibrations but ν(NH),  ν(NH₂) and ν(CO) of IstscabH observed as at wave number 3350-3151 cm^-1 and 1691 cm^-1 respectively indicating that hydrogen bonds of NH₂ of free ligand is cleaved in the inclusion complexes³¹. The ν(CO) to shifted to lower wave number due to hydrogen bonding of hydroxy (OH) of β-cyclodextrin. The IR spectrum of Isatinylthiosemicarbazone (IsttscabH) (Figure-IR₃) shows ν(C=O) of isatin part at 1727 cm^-1 and diazine ν(C=N) band at 1615 cm^-1. The ring skeletal vibrations of IsttscabH are observed at 1593 cm^–1. The ν(C=S) is observed at 1460, 1331, 1322 and 1201 cm^-1 as strong and sharp band. The IR spectrum of IsttscabH in inclusion complex (Figure-IR₄) showed some changed in its vibrations but much of IR vibration at 1026 cm^–1 are retained without major change. The inclusion products of β-cyclod-IsttscabH shows as number of new bonds due to inclusion of β-cyclod. The ν(CO) is shifted to lower vibration and observed at 672 cm^–1 due to hydrogen bonding but ν(C=S) vibration is shifted to higher wave number and observed at 1128 cm^–1  as very strong band. These changes in IR vibration supported inclusion of IsttscabH with β-cyclodextrin. The IR spectrum of bis(isatinyl)thio-carbohydrazone (BisttcabH) (Figure-IR₅) shows strong ν(C=O) vibration of (NH-CO) group of isatin ring at 1687 cm^-1 indicating strong hydrogen bonding but in its inclusion complex of  β-cyclodextrin, it is shifted to 1737 cm^–1 and partial at 1687cm^-1 (Figure-IR₆). The ν(C=N) of BisttcabH is located at 1617 cm^-1 is observed at 1618 cm^-1 in inclusion complex. The ν(NH) of BisttcabH and its inclusion product observed at 1492 cm^-1. A number of IR vibrations of BisttcabH are retained in its inclusions complexes. The extent of hydrogen bonding of free BisttcabH is cleaved and observed with reduced intensity in its inclusion complexes. The shift of bands in IR spectrum of isatin derivatives in its inclusion complexes support the inclusion between interacting molecules without chemical reaction.

Figure 1: IR1 (IstscabH), (C9H8N4O2) Click here to View figure

Figure 2: IR2 (IstscabH) (C9H8N4O2) Click here to View figure

Figure 3: IR3 (IsttscabH), (C9H8N4OS) Click here to View figure

Figure 4: IR4 (IsttscabH) (C9H8N4OS) Click here to View figure

Figure 5: IR5 (BisttcabH) (C17H12N6O2S) Click here to View figure

Figure 6: IR6 (IsttscabH) (C9H9N5OS) Click here to View figure

Table 2: Diagonostic IR Bands (In Cm^–1) of Schiff Bases and their Inclusion Products with their Assignment

	Compound  & Formula	ν(NH2)+ν(NH) +ν(OH)	ν(C=O)	ν(CºN) azine	d(NH)	ν(C=S)+ ν(C–O)	d(C-H) and ring deformation band
1.	IstscabH (C9H8N4O2)	3286 m, 3156 m	1715 vs, 1693 vs	1603 st	1471 st	1052 st	787 m, 741 m
2.	[(β-cyclod) (IstscabH)] (C51H78N4O37)	3350 m, 3151 m	1710 st, 1691 vs	1599 st	1452 st	1044 st	780 m, 750 m
3.	IsttscabH (C9H8N4SO)	3354 m, 3216 m	1727 st	1615 st	1460 st	1026 m	768 m, 734 m
4.	[(β-cyclod) (IsttscabH)] (C51H78N4SO36)	3518 m, 3335 m 3216 m, 3164 m	1672 st	1622 st 1593 m	1463 st	1128 st	794 m, 759 m 741 m
5.	BisIsttcabH (C17H12N6O2S)	3292 m, 3264 m 2971 m	1687 st	1617	1492	1155 vs	788, 765, 670
6.	[(β-cyclod) (BisIsttscabH)] (C59H82N6O37S)	3294, 3246, 2972, 2803	1737	1618	1492, 1460	1202, 1013	825, 788, 753, 660

Electronic Spectra Studies

The electronic absorption spectrum of isatinylsemicarbazone (IstscabH) (Figure-UV₁) in ethanol observed at 204, 206 and 209 nm assignable to σ®σ* transition. The strong band at 239, 246  and 315 nm are assigned to π®π* transitions for isatin ring and amide (NH-CO) group. The broad and weak absorption band at 330 nm is assigned as n®π* transition²⁹. The inclusion complex displays electronic transition located at 205, 207, 210, 238, 245 and  349 nm. The n®π* transition of IstscabH is shifted to higher wave number and observed at 349 nm in inclusion complexes. The shift in the electronic spectrum of isatinylthiosemicarbazone(Isttscabh) (Figure-UV₂) in ethanol shows σ®σ* transition at 202, 206 and 210 nm and π®π* band at 240, 268 nm. The strong band at 364 nm is assigned to charge transfer transition of (C=S) group³⁰. The medium band at 268 nm is attributed to n®p* transition. The inclusion complex of IsttscabH with β-cyclodextrin shows σ®σ* transition at 202,204, 206, and 212 nm and π®π* transition at 236 and 254 nm. The medium band at 286 nm is attributed to n®π* transition. The charge transfer band near 370-380 nm in inclusion complex is very broad. The change pattern of electronic absorption band supported the inclusion of IsttscabH in β-cyclodextrin. The electronic absorption band of BisttcabH (Figure-UV₃) shows strong absorption band at 206, 210 nm assigned to σ®σ* transition  and 245 nm band as π®π* transition. The weak absorption band at 275 nm in BisttcabH is tentatively attributed to n®π* transition. The strong and broad absorption at 360 nm is assigned to charge transfer transition involving  thioamide group (HNC=S). The inclusion product of BisttcabH displays a number of σ®σ* transition at 202, 212 and 218 nm.  The charge transfer transition of BisttcabH was observed as very broad band between 350-380 nm indicating association and formation of inclusion complex.

Figure 7: UV1 (IstscabH) (C9H8N4O2) Click here to View figure

Figure 8: UV2 (IsttscabH) (C9H8N4OS) Click here to View figure

Figure 9: UV3 (BisIsttscabH) (C17H12N6O2S) Click here to View figure

Table 3: Electronic Absorption Bands (In Nm) of Istscabh/Isttscabh/Bisttcabh and their Inclusion Complexes with Β-Cyclodextrin with Assignment

Sl. No.	Compound &            & Formula	Transition in nm
		σ®σ*	p®p*	n®p*	CT
1.	IstscabH (C9H8N4O2)	204, 206 209	239 m, 246 m 315 m	330 m
2.	IsttscabH (C9H8N4SO)	202, 206, 210	240 m, 268 m	360 sh	364 st
3.	BisttcabH (C17H12N6O2S)	206, 210	245 m	275 w	360 st
4.	[(β-cyclod)(IstscabH)] (C51H78N4O37)	205, 207, 210	238 m, 245 m	349 m
5.	[(β -cyclod)(IsttscabH)] (C51H78N4SO36)	202, 204 206	236 m, 254 m	286 w	370-380 st
6.	[(β -cyclod)(BisttcabH)] (C59H82N6O37S)	202, 212, 218	245 m	280 w	350-380 st

CT = Charge Transfer, m = medium, st = strong, w = weak, sh = shoulder

NMR Spectra

The proton (H) and ¹³C NMR spectra of some representative products are shown in (Figure- ¹H NMR₁, ¹³C NMR₁, ¹H NMR₂, ¹³C NMR₂). The NMR spectrum of isatinylsemicarbozone (IstscabH) shows NH₂ proton signals at d=3.44 ppm and phenyl ring proton signals as multiplete between =6.89 and 7.61 ppm (Figure-¹H NMR₁). The isatinyl (NH) and semicarbozone (–NH–) proton signals  are observed at 10.19, 10.72, 11.1 and 11.75 ppm suggested acidic nature of NH. The acidic nature may be due to keto-enol tautomerism of (HNC= O) group. The inclusion product with β-cyclodextrin shows cyclodextrin CH protons signal at multiplete at =3.31-3.47 ppm. The phenyl ring (C-H) protons of isatin ring signals were observed as multiplete between =6.89 – 7.89 ppm²⁹. A broad signal located at d=5.12-5.73 ppm is assigned as hydroxyl (OH) proton of β-cyclodextrin. The NH proton signal of indole ring and semicarbazone group amide (CONH) in inclusion product is located at 11.18 and 12.48 ppm suggested that NMR signals isatinylsemicarbazone is affected in inclusion complex. The ¹³C NMR signals of β-cyclodextrin carbons are observed  as multiplete between d=38.22-40.57 ppm. The (Figure-¹³C NMR₁) signals of IstscabH phenyl ring carbon signals are observed as multiplete between d=112.57-142.37 ppm. The ketoamide (C=O) carbon signals were located at d=155.52, 156.45 ppm and d=163.17, 165.30 ppm in spectrum of free IstscabH ¹³C NMR. These signals are raised to higher ppm values in inclusion complexes (Table-4). The proton NMR spectrum of isatinylthiosemicarbazone (Figure-¹H NMR₂) shows NH₂ proton signals at d=3.32-4.84 ppm and 5.68-5.73 ppm indicating two different isomeric structure of IsttscabH. The benzene ring proton signals are located between d=6.91-8.66 ppm as multiplete. The thioamide (N-H) proton signals are observed at d=11.18 and 12.48 ppm. In ¹³C NMR spectrum (Figure- ¹³C NMR₂) ¹³C signals of β-cyclod carbons are observed as multiplete between d = 39.31-40.56 ppm. The phenyl ring carbon ¹³C NMR of IsttscabH were observed between d=102.40-142.80 ppm and carbamide ¹³C NMR signals at d=163.09, 179.17 and 181.63 ppm. The proton and ¹³C NMR spectrum of its inclusion complex shows relatively broad proton NMR signals. The β-cyclodextrin CH proton signals are affected due to its association with isatinylthiosemicarbazone on inclusion and observed at d=2.51 and 2.56 ppm. The ν(OH) signal was observed at =3.68-3.72 ppm as board band. The ¹³C NMR signal of β-cyclodextrin was observed between d= 39.30-68.54 ppm which is higher than free β-cyclodextrin signals. In inclusion complexes, the phenyl ring ¹³C NMR signals were located between =110.36-143.37 ppm these are higher than free IsttscabH suggested the association of inclusion complexes. The ¹H NMR spectrum of bis(isatinyl)thiocarbohydrazone(BisttcabH) shows phenyl ring CH proton signals between d=6.88-7.55 ppm²⁹. The isatin ring NH and thioamide   HNCS proton signals were observed at d=11.02 and 13.34 ppm indicating their acidic nature. The ¹³C NMR  signals of methyl carbon in solvent DMSO were observed at d=39.27-40.52 ppm. The phenyl ring carbon ¹³C NMR signals were located between d=112.57 and d=149.57 ppm. The ¹³C NMR signals located  at 155.85, 159.82 and 183.85 ppm in spectrum were assigned to (C=S), (C=N) and (CO) carbon atoms respectively. In inclusion complexes these ¹³C NMR signals of (C=S),(C=N) and (CO) carbon were observed at higher ppm values than free isatinyl derivatives  (=158.25, 161.32 and 188.63 ppm).

Figure 10: 1H NMR1 (IstscabH) (C9H8N4O2) Click here to View figure

Figure 11: 13C NMR2 (IstscabH) (C9H8N4O2) Click here to View figure

Figure 12: 1H NMR2 (IsttscabH) (C9H8N4OS) Click here to View figure

Figure 13: 13C NMR2 (IsttscabH) (C9H8N4OS) Click here to View figure

Table 4: ¹H NMR and ¹³C NMR Signals of Schiff Bases and their Inclusion Products

Compound	1H NMR signals in ppm	13C NMR Signals in ppm
Isatinylsemicarbazone (IstscabH)	d = 3.44 (NH2) proton, d = 6.89, 7.61 benzene (CH) proton, d = 10.79, 10.72, 11.10, 11.75 ring (NH) and (C=NH) proton	d = 112.57-142.37 phenyl (C-H) carbon, d = 155.52, 156.45 (CO) carbon, d = 163.17, 165.30 ring (CO),  (C=N) carbon
[(β-cyclod)(IstscabH)]	d = 3.31-3.47 (N-H) proton β-cyclod, d = 6.91-7.61 benzene (CH) proton, d = 5.12-5.23 (OH) proton of β-cyclod, d = 10.71, 11.11 and 12.12 ring (NH) and carbazone (NH) proton signal	d = 116.51-143.37 benzen ring 13C, d=38.22-40.57 multiplete β-cyclod carbon, d = 161.32, 167.15 carbazone (C=O) carbon d = 168.21-170.31, 172.21 (C=N) isatin ring (C=O) carbon
Isatinylthiosemicarbazone (IsttscabH)	d = 3.32, 4.84 (NH2) proton, d = 6.91-8.66 phenyl ring (CH) proton, d = 11.18, 12.48 thioamide and ring (NH) proton	d = 102.70-142.80 phenyl ring carbon, d=39.31-40.56 multiplete β-cyclod C, d = 163.09, 179.17 (C=O) and (C=S) carbon
[(β-cyclod)(IsttscabH)]	d = 3.28, 4.18 (NH2) proton, d = 6.92-8.58 ring (C-H) proton, d = 5.21-5.46 hydroxy (OH) proton of β-cyclod, d = 11.31-12.52 thioamide ring (NH) proton	d = 110.36-143.37 phenyl ring carbon, d = 164.13, 175.23, 189.13 (C=N), (CO), (C=S) carbon

Antibacterial Activity

Antibacterial activity of isatin Schiff bases IstscabH, IsttscabH, BisttcabH and their inclusion products with β-cyclodextrin have been screened  by growth inhibition technique using petri disc method for bacteria namely Bacillus Subtilis, P. mirabilis and Straphylococcus  aureus and the results of activities at concentration 100 and 200 ppm using Azithromycin and Ciprofloxacin as standard^34-35 and results are summerized in Table-5. The requisite amount of Schiff bases and inclusion products with β-cyclodextrin were dissolved in DMF to get 100 and 200 μgmL^–1 (ppm) solution. About 0.5 mL (containing 10⁷ microorganism per mL) of investigated mircroorganism was added to a sterile nutrient agar medium. Just before solidification and then poured on sterile perti discs and allowed to solidify using sterile Cork borer (6 mm in diameter) three holes are made in each disc and then 1 mL of tested inclusion product dissolved in DMF was poured in these holes. Finally the discs were in incubated at 35°C for 24 hours and zone of inhibition of growth were measured in mm against each bacteria. A blank containing DMF was measured and the inhibition was practically negligible. It was found that inclusion products showed greater antibacterial activity than Schiff bases. The higher activity of inclusion products with β-cyclodextrin compared to Schiff bases of isatin may be due to decrease in polarizability of inclusion product which  enhanced the lipophilicity of product than isatin Schiff bases leading to reduction of permeability of the cells resulting in interference with normal cell process. The results showed that in some cases the activities are comparable to Azithromycin and Ciprofloxacin.

Table 5: Antibacterial Screening of Isatin Schiff Bases Istscabh, Isttscabh, Bisttcabh and their Inclusion Products with β -Cyclodextrin

Compound	Antibacterial Activity zone of inhibition (mm)
	Staphylococcus		P. mirabilis		Bacillus Subtilis
Concentration ®	100 ppm	200 ppm	100 ppm	200 ppm	100 ppm	200 ppm
IstscabH IsttcabH BisttcabH [(β-cyclod) (IstscabH)] [(β -cyclod) (IsttscabH)] [(β -cyclod) (BisttcabH)] Ciprofloxacin Azithromycin	11 09 06 13 13 14 24 22	14 15 13 20 18 18 25 23	10 11 09 15 14 14 23 23	13 14 13 19 19 18 24 24	10 11 11 12 13 11 23 22	13 14 14 16 18 17 24 23

The comparison of IR, UV, ¹H NMR and ¹³C NMR spectral studies of Schiff bases and their inclusion complexes supported that these Schiff bases form stable  1:1 inclusion products and isolated in solid state.

One the basis of analytical results and spectral studies the following possible structure of expected inclusion complexes of β-cyclodextrin having 1:1 molar ratio  with isatinyl derivatives               is shown below.

Where,                                                                          is inclusion ligand (A). Isatinylsemicarbazone (istscabH), (B) Istalinylthiosemicarbazone (IsttscabH) and (C) Bisisatinyl-thiocarbohydrazone (BisttcabH). The structure is similar to supra molecule. 

Conclusions

From results of the element analysis, determination of molecular weight, spectral studies and comparison of  IR, UV and ¹H NMR as well as ¹³C NMR, it is inferred that isatin derivatives IstscabH, IsttscabH and BisttcabH form 1:1 inclusion complexes with β-cyclodextrin and these products are stable  and isolated in solid state. The antibacterial activity of inclusion products are enhanced than simple Schiff bases.

Acknowledgments

Authors are thankful to Principal, Jagdam College, Chapra, R.K. College Gridih for providing laboratory and library facilities available there,  Prof. D.N. Thakur, Head Chemistry, Patna University, Patna for providing advance laboratory facilities for some of experimental works and Authority of IIT Patna for recording IR, UV and NMR Spectra and for  C, H, N, S analysis. The cooperation of Authority  of Biochemistry Department of Patna University is thankfully acknowledged  for antibacterial screening.

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