Inclusion Complexes of Acridone and Its Semicarbazone Derivative With β- Cyclodextrin: - A Thermodynamic, Spectral and Antimicrobial Study

¹Sunakar Panda and ²Swapna Sankar Nayak

¹PG Department of Chemistry, Berhampur University, Bhanja Vihar 760007, India.

²S.B.R. Govt. Women’s (Auto) College, Berhampur 760001, India

*Corresponding Author E-mail: swapna_nayak7@yahoo.com

ABSTRACT

Both acridone and its semicarbazone derivative being insoluble in polar medium, may have poor pharmacological activity. To enhance bio-accessibility of these drugs, the inclusion complexes of acridone and its derivative have been prepared with β- cyclodextrin. The spectral characteristics (UV-Vis, FTIR) of these compounds have been studied with and without inclusion complex formation. The phase solubility studies reveal 1:1 stoichiometry between guest and host. The determination of thermodynamic stability constants indicates weak intermolecular forces between the compounds and β- cyclodextrin. The thermodynamic parameters DG, DH, DS of the complexes have been calculated, the values of which suggest that complex formation is spontaneous and exothermic in nature. The antimicrobial activity of acridone and its semicarbazone derivative along with their inclusion complexes against the microbes like Escherichia coli an, Pseudomonas aeruginosa have been studied.

KEYWORDS: Acridone semicarbazone, Inclusion complex, Thermodynamic stability, Antimicrobial study.

INTRODUCTION:

Acridone and its derivatives are important anthracene analogue heterocyclic compounds in which a carbon in the middle ring is replaced by a ‘N’ atom. Their DNA affinity and intercalative properties make it an important pharmacophore for the designing of several chemotherapeutic agents (anti cancer, anti bacterial, anti protozoal).^1.2,3 These are pharmacologically acceptable, efficacious in preventing and treating diseases such as asthma, allergic rhinitis, atopic dermatitis, uticaria, gastrointestinal allergies etc.⁴

Since bio-accessibility of a drug depends upon its solubility, one of the factors limiting the pharmacological activities of acridone and its derivatives is their insoluble nature in aqueous solutions. The solubility of these compounds can be enhanced by forming inclusion (host-guest) complexes with cyclodextrins (CDs) which in turn increases their drug efficiency .^5,6 Among the natural CDs, only the more available β form has certain prospects in applications because the other forms(α, γ) are expensive. β-CD is capable of forming complexes with other compounds in both solid and liquid state.

Although a series of 10-N-Susbstituted acridones, bearing alkyl side chains with tertiary amine groups at the terminal position have been reported, there are few report regarding the synthesis of acridone derivatives involving the keto group.^6,7 In this paper an attempt has been made to synthesize acridone and its semicarbazone derivative in their purest forms. Respective inclusion complexes of these compounds with β-CD have been synthesized. Finally, the spectral and thermodynamic properties of the compounds and their inclusion complexes have been studied. As these compounds contain quinolone group, they are expected as potential drugs against some bacteria and accordingly antimicrobial activity studies have been made.

Table – 1       Analytical Data

Sl No	Compound	m.p. 0 c	Colour	Elemental Analysis Found (calculated) %				lmax nm	IR (KBr)l cm ^-1
				C	H	N	O
1	Acridone	350	Greenish yellow	80 (80.2)	4.8 (4.6)	8.4 (8.2)	7.0 (7.18)	403,385	1674 (C=O) 3274( N-H) 1161 (C-N) 1633 (C=C) 1572 (ring)
2	Acridone/ ß-CD complex	359	Yellow	--	--	--	---	401,383	1662 (C=O) 3270 (N-H) 1155 (C-N)
3	Acridone- semicarbazone	292	Bright yellow	66.62 (66.66)	4.8 (4.76)	22.25 (22.22)	6.32 (6.35)	409,381	1560 (C=N) 1635 (C=O) 1159 (C-N) 1535 (N-H)
4	Acridone- semicarbazone/ ß-CD complex	306	Yellow	-	-	-	-	406,379	1556 (C=N) 1633 (C=O) 1156 (C-N) 1532 (N-H)

EXPERIMENTAL:

MATERIAL AND METHODS:

Apparatus and materials:

All chemicals are procured form the local market and are of suitable Anal R grade. Double distilled water is used as the solvent for dilution. Other solvents employed are redistilled before use. The elemental analysis has been performed in a CHN analyzer. Electronic  spectra are recorded on Shimadzu UV-1700 spectrophotometer while IR spectra are recorded in KBr pellets in the 400- 4000 cm ^-1 region in a Shimadzu 8400 S FT- IR spectrophotometer. Melting points are recorded by open capillary method. Antimicrobial screening by Kirby-Bauer method has been done by employing Muller Hinton agar plates in normal saline medium and sterilised cotton swabs.

Phase solubility measurements:

The aqueous phase solubility of Acridone and its semicarbazone at various concentrating of β-CD has been studied by Higuchi-Connors method.⁸ Accurately weighed sample of these compounds in quantities exceeding their aqueous solubility are shaken in a rotary flash shaker at room temperature with aqueous solution of β-CD in increasing concentration (0-10 mM/L) in a series of stoppered conical flask for a period of 48 hours till equilibrium is established. The solutions are filtered through Whatman No1 paper and are analyzed in a UV-Vis spectrophotometer at 380-420 nm range. The various values of OD at l _max have been plotted against different concentration of β -CD.

Syntheses of Acridone and its semicarbazone derivative:⁹.

0.2 mole of N-phenylanthranilic acid (I) in 100 ml of conc. H₂SO₄ is refluxed in a 500 ml flask on a boiling water bath for four hours and then poured into a 1L flask containing hot water slowly and carefully. The yellow precipitate formed is filtered after boiling for few minutes and then the moist solid is again boiled for five minutes with a solution

of 0.28 mole Na₂CO₃ in 400ml of distilled water. The precipitate is collected with suction and washed well with water. After drying, the crude acridone (II) obtained is then recrystalised form a mixture of aniline and acetic acid.

1gm of semicarbazide hydrochloride and 1.5 gm of crystallized sodium acetate are dissolved in 10 ml water to which 0.5 gm of acridone is added and shaken. Alcohol is added till turbidity disappeared to give a clear solution. Then the solution is refluxed for 2 hours on a water bath with condenser. The resulting solution is poured carefully into ice-cold water where the crystals of acridone-semicarbazone (III) are obtained. These are recrystallised from alcohol and water mixture and finally dried.

The synthesis of acridone and its semicarbazone derivative is shown in scheme – 1

Scheme - 1

Table – 2   Thermo dynamical Data at 298 0 K

Sl No.	Compound	K(M^-1)	DG kJ / mol	DH kJ / mol	DS kJ / mol
1	Acridone / ß-CD complex	104	-11.5	-43.7	-0.11
2	Acridone- semicarbazone/ ß-CD complex	108.3	-11.6	-46.8	-0.12

Syntheses of inclusion complexes:

These have been done by Co-precipitation method. ^10,11 The solution of the synthesized compounds are prepared in required concentrations (0.03M) and were added drop wise to previously stirred β-CD solution. The mixtures are stirred at room temperature for 48 hours, filtered. Then the content is and cooled for another 48 hours in refrigerator. Finally the precipitate obtained is filtered through G-4 crucible, washed with distilled water and dried in air for 24 hours.

Study of thermodynamic properties:

The thermodynamic stability constant  (K_T) at room temperature of the complexes are  calculated using  Benesi-Hilderbrand relation.¹² The stability constant K (during de-encapsulation) of each complex has been calculated with increasing temperature.   The slope of the linear plot of ln K  vs.  I/T  gives rise to the calculation  of DH (change in free enthalpy) and then DS (change in entropy) was calculated using  the integrated from of the van’t Hoff equation.

The value of DG was calculated from the value of K_T  at 298 ⁰ k using the equation:

Study of Antimicrobial activity:

The disk diffusion method for antimicrobial susceptibility test is the Kirby-Bauer method^13,14 . Muller-Hinton agar plates with normal saline medium have been used for this test. The bacterial inoculums are prepared by making a direct saline suspension of colonies of same morphological type that are selected from an 18-24 hour agar plate. The turbidity with sterile saline is adjusted. Within 15 minutes after adjusting the turbidity, a sterile non-toxic swab is dipped on an applicator into the adjusted suspension. A maximum of 5 disks on a100 mm plate are placed on the surface of the agar plate. The plates are inverted and are

placed in an aerobic incubator at 35⁰ C. After 16-18 hours of incubation, the diameters of zones of complete inhibition are measured. The zone sizes are interpreted by referring to standard antibiotics (manufacturer provided standard table) and the report on the organism to be susceptible (S), intermediate (I) or resistant (R) are made.

RESULTS AND DISCUSSION:

Syntheses of acridone and acridone semicarbazone:

The syntheses of acridone and acridone semicarbazone are confirmed from elemental analysis and IR- data as shown in (Table-1). The elemental composition nearly matches with theoretical data. Infrared data of C=0 _str at 1674 cm^-1, N-H _str at 3274 cm^-1, C-N _str at 1161 cm^-1 etc. suggest formation of acridone. Similarly, C=N _str at 1560 cm^-1, C=O _str at 1635 cm^-1, C-N _str at 1159 cm^-1 N-H _deformation  at 1533 cm^-1 etc suggest the formation of  acridone semicarbazone. In addition, both acridone and its derivatives differ significantly in their melting points (Table-1)

Syntheses of inclusion complex:

The syntheses of inclusion complexes of acridone and acridone semicarbazone are confirmed from melting point data, colour and spectral characteristics. (UV-Vis and IR) (Table-1). The melting point of acridone is 350 ⁰C while that of inclusion complex with β-CD is 359 ⁰C. A higher melting point of inclusion complex than acridone itself is due to the fact that extra amount of thermal energy is required for the later to bring it out of β-CD cavity.  Similarly melting point of acridone semicarbazone is 292 ⁰C, but that of its inclusion complex is 306 ⁰C.

Study of spectral characteristics:

The drug recipient interaction are better identified by employing IR spectrophotometry  as an useful tool. The absorption maxima are shown to undergo a distinct blue shift after their inclusion complex formation with β-CD (Table-1). This observation clearly demonstrates transference of the compound from a more protic environment (aqueous media) to a less protic environment (cavity of β-CD). The compound and β-CD interaction leading to inclusion complex formation is further supported by IR data (Table-1). It is seen that the IR- stretching frequencies due to different bonds     (C=O, N-H, C-N etc. in case of acridone and C=N, C=O, C-N etc. in case of acridone semicarbazone) undergo a downward shift towards lower energy and the peaks become broader, weaker and smoother. Such changes in IR- spectral characteristics due to inclusion complex formation may be attributed to development of weak interaction like H-bonding, vander-Waal forces and hydrophobic interactions between host and guest molecules.¹⁵

Phase solubility studies:

The phase solubility plots of acridone and acridone semicarbazone with and without inclusion complex formation with β-CD are shown in Fig. 1. In both the cases it is seen that there is a linear increase in solubility of these compounds with increasing concentration of β-CD. At a higher concentration of β-CD, a small negative deviation is observed. Since the slopes of both plots are less than unity, the stoichiometry of the inclusion complexes is 1:1.¹⁶

The thermodynamic stability constants ( K_T) of inclusion complexes are determined by following Benesi-Hilderbrand relation.

1/DA=1/DÎ+1/K[guest]₀ DÎ . 1/[b-CD]₀

Good linear correlations (Fig. 2) are obtained for a plot of 1/∆A verses 1/[β-CD]₀ for  acridone and acridone semicarbazone. The values of K_T for both the complexes are calculated using the relation:

The K_T values for these inclusion complexes i.e. acridone- β-CD and acridone semicarbazone- β-CD are found to be 104 M^-1and 108.3M^-1 respectively. The data obtained are within 100 to 1000M^-1 (ideal values) indicating appreciable stabilities for the inclusion complexes. ¹⁶

Table- 3   Antimicrobial Susceptibility Test          

Sample   (1mM) Organism	A	AA	D	DD
E. coli	S (10)	S(15)	S(12)	S (17)
P. aeruginosa	S(21)	S (27)	S (24)	S (33)

A= Acridone, AA=Acridone/ ß-CD;  D=Acridonesemicarbazone, DD= Acridonesemicarbazone/ ß-CD

Thermodynamic Properties:

The thermodynamic parameters associated with binding of acridone and its semicarbazone with β-CD for 1:1 stoichiometry have also been calculated by determining the K values at different temperatures. The K values are found to decrease with increasing temperature (de encapsulation) as expected for an exothermic process.¹⁷ The plot of ln K as a function of inverse absolute temperature produced linear plots (Fig.3). In each case, the slope corresponds to (-DH / R).¹⁸  From this value and value of   K_T  at 298 K,  DG, DS and DH have been calculated (Table 2).

As can be seen from the table, DG values are negative for both the complexes. These data clearly demonstrate the spontaneous formation of inclusion complexes. Secondly, the DH and DS values are negative at 298 K which suggests that the complex formation is an exothermic and enthalpy controlled process. The negative enthalpy change is due to stabilization of the compound within the cavity of β-CD by weak intermolecular forces as suggested earlier. The small negative entropy change is due to steric barrier caused by less free movement of guest molecules. The study further suggests that change in entropy (DS) in destabilizing inclusion complexes is compensated by change in enthalpy (DH).¹⁹

Antimicrobial Screening:

The results obtained in the antimicrobial susceptibility test by Kirby-Bauer method (Table-3) show that acridone and its semicarbazone derivative are more susceptible to Gram negative bacteria Pseudomonas aeruginosa than Gram positive bacteria E. coli. Further their sensitivity to these microbes increases significantly from non-complexed state to complexed state. This is well explained by considering the enhanced solubility and in the later state. Thus these drugs become more bio-available and bio-accessible to the tissues leading to increased antimicrobial activity.

CONCLUSION

From the above results and discussions it is clear that the solubility of acridone and its derivatives can be improved by inclusion complex formation with β-CD which is a very good analytical tool for enhancing the bio-availability of drugs. The study of these complexes furnishes information about non-covalent intermolecular forces binding the “host – guest” molecules. The negative DG, DH, DS values support the formation of such complexes. Cyclodextrins are now widely used for the stabilization of many drugs^20.  . Acridone and its derivatives show antibacterial activity which can further be enhanced by forming their inclusion complexes.

ACKNOWLEDGEMENT:

The authors acknowledge the Principal, Institute of Pharmacy and Technology, Salipur  (India) for IR spectra investigation in their laboratory and DR. P.M. Panda and DR. S. Padhy of  Department of Microbiology,M.K.C.G. Medical College and Hospitals, Berhampur (India) for providing the facilities to study antimicrobial activity.

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Received on 21.08.2009        Modified on 09.10.2009

Accepted on 30.10.2009        © AJRC All right reserved

Asian J. Research Chem. 2(4):Oct.-Dec. 2009 page 539-543