Synthesis and Anti-inflammatory Study of Novel N-substituted Hydro-acridine-1,8-diones and Bis-hexahydroacridine-1,8-dione Derivatives

Large number of natural and synthetic acridine scaffold compounds exhibit broad spectrum of biological and physical properties [1-5]. Although many researchers have devoted their studies on synthesis of acridine compounds and their pharmaceutical applications as anti-tumor [6-9], anti-bacterial [10], anti-malarial [11] and anti-inflammatory agents [12]. Acridinediones, in particular, have been identified as anti-malarial and anti-tumor agents [13-15]. Hexahydroacridine-1,8-dione derivatives are also reported to possess important properties such as high fluorescence efficiency [16]. As a consequence, the interest of organic chemists in the synthesis or structure modifications of hydroacridinone derivatives remains high. It has also been discovered that the introduction of a substituted group to the nitrogen atom of hexahydroacridine-1,8-diones leads to enhance the fluorescence activity [17,18]. Recently, Hubschwerlen et al. found that the introduction of a cyclopropyl group to the nitrogen atom of the pyridine ring results in a wide spectrum of anti-bacterial activities [19]. However, the introduction of a 2-hydroxypropyl or tosylated propyl groups to the nitrogen atom has not been reported yet.


Introduction
Large number of natural and synthetic acridine scaffold compounds exhibit broad spectrum of biological and physical properties [1][2][3][4][5]. Although many researchers have devoted their studies on synthesis of acridine compounds and their pharmaceutical applications as anti-tumor [6][7][8][9], anti-bacterial [10], anti-malarial [11] and anti-inflammatory agents [12]. Acridinediones, in particular, have been identified as anti-malarial and anti-tumor agents [13][14][15]. Hexahydroacridine-1,8-dione derivatives are also reported to possess important properties such as high fluorescence efficiency [16]. As a consequence, the interest of organic chemists in the synthesis or structure modifications of hydroacridinone derivatives remains high. It has also been discovered that the introduction of a substituted group to the nitrogen atom of hexahydroacridine-1,8-diones leads to enhance the fluorescence activity [17,18]. Recently, Hubschwerlen et al. found that the introduction of a cyclopropyl group to the nitrogen atom of the pyridine ring results in a wide spectrum of anti-bacterial activities [19]. However, the introduction of a 2-hydroxypropyl or tosylated propyl groups to the nitrogen atom has not been reported yet.
On other hand, many studies showed that dimerization of acridine compounds enhance their biological activities. They were first developed as tumorostatic agents compared to those of the respective mono acridines [20][21][22]. Synthesis of such dimeric ligands, have been employed to improve the local concentration of the bioactive species and to avoid the cellular efflux mechanisms associated with multi drug resistance to the respective monomeric counter parts [23,24]. Bis-acridines have also demonstrated bioactivity in mice infected with Plasmodium berghei [22] and in vitro against Plasmodium falciparum and try panosomatid parasites [25]. Such aforementioned facts inspired us to synthesis and investigate the anti-inflammatory potency of some new N-substituted hydroacridine-1,8-diones and their dimer derivatives.

Experimental
All melting points are uncorrected and were determined by Kofeler melting point apparatus. IR (cm -1 ) spectra were recorded (KBr disc) on a Shimadzu DR-8001 spectrophotometer. 1 H-NMR and 13 C-NMR (DMSO-d 6 or CDCl 3 ) spectra were recorded at 400 MHz on a Varian Mercury-300 BB at Sohag University, the chemical shift is expressed in δ value (ppm) using TMS as an internal reference Evaluation of Anti-

Abstract
We report in this study the synthesis of some new N-substituted hexahydro-acridine-1,8-dione compounds including some new N-2-hydroxypropylhexahydro-acridine-1,8-diones and their tosylatedoctahydroacridine-1,8-dione derivatives in addition to some bis-hexahydroacridine-1,8-diones via a one pot reaction technique. Moreover, in vivo anti-inflammatory evaluation for some newly synthesized compounds has been investigated. The highly alkylated bishydroacridine-1,8-dione 5f showed a higher anti-inflammatory potency more than the non-alkylated and the standard employed indomethacin. The structure of all new products has been characterized by IR, 1H-NMR and 13 C-NMR.
inflammatory effect was carried out by Faculty of Medicine, Assuite University.

Synthesis of tosylatedhexahydroacridinediones (4a-d)
General procedure: A solution of (1 mmol) of the hexahydroacridine-1,8-dione derivatives 3a-d, (1 mmol) of tosyl chloride and a catalytic amount of triethylamine in (20 mL) ethanol was refluxed and monitored by TLC till completion after 3 hrs. The reaction mixture was allowed to cool down to room temperature and the obtained solid product was collected by filtration, dried under vacuum and crystallized from the appropriate solvent to afford the corresponding tosylatedacridinediones4a-d respectively.
The molecular structure of the products 3a-f was elucidated from their IR, 1 H-NMR and 13 C-NMR spectra. The IR spectrum of these products in KBr showed broad bands at 3397-3421Cm -1 assigned for both of phenolic and alcoholic OH groups, and strong bands ranged from 1630-1648Cm -1 related to carbonyl groups. Broad bands characteristic of stretching the aliphatic CH 3  Green Chemistry been also observed at 2874-2965 Cm -1 . The 1 H-NMR spectrum of 3a-f showed clearly the presence of phenolic OH at δ=10-10.9 ppm and alcoholic OH at δ=3.2-3.5 ppm. Singlet sharp peaks at δ=1.05, 0.99, 0.89 ppm were attributed to the alcoholic CH 3 and the four methyl groups of dimedone scaffold respectively in 3d. The other two clear singlet peaks were observed at δ=3.6 and 5.06 ppm assigned for the CH of the hydro pyridine ring and the CH of the isopropanol group respectively. The 13 C-NMR spectrum of 3a-f supported the presence of carbonyl at the range δ=199-196 ppm, and aliphatic carbon signals appeared in the regular region (see experimental). Moreover, compound 3a has been further confirmed by refluxing the corresponding xanthenone 7 [26] with amino-iso-propanol (Scheme 2). The spectral data of the authentic product was identical with 3a.

and CH 2 groups have
The alcoholic group in 3a-d has been tosylated by tosyl chloride in TEA to afford the corresponding tosylatedoctahydro-acridine-1,8-diones 4a-d via elimination of HCl. IR spectra showed a clear characteristic peak at 1177 (in 4c), 1178 (in 4d), 1180(in 4b) and 1183Cm -1 (in 4a) for (O=S=O) group. The 1 H-NMR supported the presence of a multiplet peaks at δ=5-5.06 ppm assigned for the CH-O tosylate and a singlet peak for the CH 3 tolyl has been observed at the range δ=2.5-2.9 ppm. Furthermore 13 C-NMR confirmed the existence carbonyl at average δ=197 ppm. Two clear peaks have been also observed at δ=19 and 22 ppm attributed to the CH 3 tolyl and CH 3 propyl groups respectively.
Gratifyingly, the symmetry function in the structures of the new products 5a,b,e,f (Scheme 1) has been unambiguously confirmed by both 1 H-NMR and 13 C-NMR spectra. The 13 C resonance of the two carbon atoms of the ethyl linkage have been observed in all compounds 5a,b,e,f at the average δ=50-56 ppm and the 1 H-NMR confirmed the four proton resonance of the ethyl linkage as a symmetrical triplet peak between δ=2.0-2.5 ppm. Moreover 13 C-NMR confirmed the existence of the peaks of four carbonyl groups between δ=204-196 ppm for all compounds 5a,b,e,f.

Carrageenan-induced paw oedema standard method in rats:
Anti-inflammatory activity screening for the chosen compounds 3c, 5a, 5b and 5f was determined in vivo by the acute carrageenan-induced paw oedema standard method in rats [27]. Adult albino rats of either sex (pregnant female animals were excluded) weighing 160-190 g were divided into 6 groups of 6 animals each. To reduce the variability of oedema response, rats were fasted overnight, then on the next day (day of experiment), animals were uniformly hydrated by giving 3 ml of water per rat orally. Indomethacin (reference standard) and the tested compounds (20 mg/kg body weight) were suspended in saline solution by the aid of few drops of Tween 80 (to improve wettebility of particles) and given orally one hour before induction of inflammation. The control group was given saline solution containing few drops of Tween 80.
Carrageenan paw oedema was induced according to a modified method of Winter et al. [27,28] by subcutaneous injection of 1% solution of carrageenan in saline (0.1 ml/rat) into the subplanter region of the right hind paw of rats. The thickness of rat paw was measured by mercury digital micrometer at different time intervals, at zero time and after one, two three, four and five hours of carrageenan injection. The oedema was determined from the difference between the thickness of injected and non-injected paws.
Data were collected, checked, revised and analyzed. Quantitative variables from normal distribution were expressed as means ± SE "standard error". The significant difference between groups was tested by using one-way ANOVA followed by post hoc test [29] at p<0.05 and p<0.01.
The results of the anti-inflammatory activity were expressed as percentage inhibition of oedema thickness in treated animals in comparison with the control group according to the following equation (Table 1, Figure 1).
Where V R represents the mean right paw thickness, V L represents the mean left paw thickness, (V R -V L ) control represents the mean increase in paw thickness in the control group of rats and (V R -V L ) treated represents the mean increase in paw thickness in rats treated with the tested compounds [30,31].

Discussion
The anti-inflammatory activity of four representative synthesized compounds (5a,b,f and 3c) was determined by the carrageenan induced paw oedema standard method in rats [27,28]. Generally, it has been observed from the obtained results, (Table 1, Figure 1), that all the tested compounds show considerable anti-inflammatory activity. In addition, compounds 5f exhibit better anti-inflammatory properties (57.53 % inhibition of oedema) than that of the used reference standard indomethacin (54.9 % inhibition of oedema).
The effect of substituents on the anti-inflammatory potency has been considered by comparing the activity of the bis-hydroacridine derivatives5a,b and 5f. The compound 5bshowed higher antiinflammatory potency (39.72) compared to 5.47 for 5a. This might attributed to the existence of the halogen atom. On other hand, we found the bis-hydroacridinedione5f bearing 8 alkyl groups exhibits even better anti-inflammatory potency than 5b. The N-substituted Green Chemistry alcohol of the mono-hydroacridinedione3c showed more effectiveness as an anti-inflammatory agent compare to the bis-hydroacridine-dione5a.
In general, we concluded that bis-hydroaccridinediones bearing more electron donating alkyl groups showed high anti-inflammatory effect than the standard indomethacin. This means that the electron donating alkyl groups in hydroacridines could enhance their anti-inflammatory activity.