Synthesis of New Visnagen and Khellin Furochromone Pyrimidine Derivatives and Their Anti-Inflammatory and Analgesic Activity

6-[(4-Methoxy/4,9-dimethoxy)-7-methylfurochromen-5-ylideneamino]-2-thioxo-2,3-dihydropyrimidin-4-ones 1a,b were prepared by reaction of 6-amino-2-thiouracil with visnagen or khellin, respectively. Reaction of 1a,b with methyl iodide afforded furochromenylideneaminomethylsulfanylpyrimidin-4-ones 2a,b. Compounds 2a,b were reacted with secondary aliphatic amines to give the corresponding furochromen-ylideneamino-2-substituted pyrimidin-4-ones 3a-d. Reaction of 3a-d with phosphorus oxychloride yielded 6-chlorofurochromenylidenepyrimidinamines 4a-d, which were reacted with secondary amines to afford furochromenylideneamino-2,6-disubstituted pyrimidin-4-ones 5a-d. In addition, reaction of 5a-d with 3-chloropentane-2,4-dione gave 3-chloro-furochromenylpyrimidopyrimidines 6a-d. The latter were reacted with piperazine and morpholine to give 1-(furochromenyl)-pyrimidopyrimidine-3,6,8-triylpiperazines or-3,6,8-triylmorpholines 7a-d. The chemical structures of the newly synthesized compound ware characterized by IR, 1H-NMR, 13C-NMR and mass spectral analysis. These compounds were also screened for their analgesic and anti-inflammatory activities. Some of them, particularly 3-7, exhibited promising activities.


Introduction
Khellin and visnagen, 4,9-dimethoxy-or 4-methoxy-7-methyl-furo[3,2-g]chromen-5-one, respectively ( Figure 1) are obtained from fruits and seeds of the plant Ammi visnaga. The khellin and visnagen extract has been widely employed as a herbal medicine in the treatment of angina [1]. Khellin is used as a spasmolytic agent and for kidney stone treatment [2]. Khellin and visnagen extract significantly prolongs the induction time of nucleation of calcium oxalate [3]. Khellin has been used for photochemotherapeutic treatment of vitiligo and psoriasis [4] and the photodynamic properties of khellin and visnagen in their photoreaction with DNA have been studied [5]. Recently, khellin was proved to have phototherapeutic properties that are similar to those of the psoralens, but with substantially lower phototoxic effects and DNA mutation effects. Its penetration into the hair follicles is enhanced by encapsulating it into liposomes. Subsequent activation of khellin with UV light stimulates the melanocytes in the hair follicles [6]. The fact that furochromone derivatives are known to have anti-inflammatory properties [7] and analgesic properties [8], prompted us to synthesize and investigate such properties in khellin and visnagen derivatives as typical furochromones. From a photobiological point of view, furochromones show valuable phototoxicity toward various kinds of microorganisms and also valuable genotoxic activity on various biological substrates [9,10]. In the present work, we planned the synthesis of various furochromenylidenylpyrimidine derivatives representing new heterocyclic compounds. These compounds were also screened for their anti-inflammatory and analgesic activities.
The steps of the suggested mechanism are shown in Scheme 2. The 1 H-NMR spectrum of 6a, for example, showed a doublet at 1.15 ppm, a multiplet at 3.14 and a triplet at 3.92, which support the proposed reduced structure 6. Finally, the 3-chlorofurochromenepyrimido/pyrimidines reacted with either piperazine or morpholine in boiling methanol to give the corresponding 3,6,8-tripiperazin-1-yl or 3,6,8-trimorpholin-1-yl derivatives 7a-d (Scheme 3). Compatible analytical and spectral data were obtained for compounds 7a-d (see Experimental).

Anti-Inflammatory Activity
The anti-inflammatory activity was evaluated in rats by the carrageenan-induced paw edema test. The data (Table 1) indicated that all the tested compounds protected rats from carrageenan-induced inflammation, and that some of the tested compounds (6a-d and 7a-d) are more potent than our previously reported ones [15,16]. Compounds 2-5 showed similar and higher anti-inflammatory activity than diclofenac sodium.

Analgesic Activity
The analgesic activity was determined by the hot-plate test (central analgesic activity) and acetic acid induced writhing assay. The results (Tables 2 and 3) revealed that all tested compounds exhibited significant activity. Most of the tested compounds have nearly the same activity as the reference drug and the remaining tested compound have good activities in central analgesic activity. Also compound 7c exhibited activities higher than the reference.

Conclusions
The new ring systems prepared seem to be interesting for biological activity studies. Furthermore, the present investigation offers rapid and effective new procedures for the synthesis of the polycondensed new heterocyclic ring systems. Compounds 6a-d and 7a-d exhibited potent antiinflammatory and analgesic activities. It is worth mentioning that the incorporation of methoxy, dimethoxy, -furo[3,2-g]chromen, di-and tri-(piperazine or morpholine) and tetrahydropyrimido[1,6-a] pyrimidine moieties resulted in significant anti-inflammatory and analgesic activities. In conclusion, we report herein a simple and convenient route for the synthesis of some new heterocyclic compounds based on furochromene pyrimidine derivatives for anti-inflammatory and analgesic evaluation.

General
All melting points were taken on an Electrothermal IA 9100 series digital melting point apparatus (Shimadzu, Japan). Elemental analyses were performed at Vario EL (Elementar, Germany). Microanalytical data were processed in the microanalytical center, Faculty of Science, Cairo University and National Research Center. The IR spectra (KBr disc) were recorded using a Perkin-Elmer 1650 spectrometer (USA). 1 H-NMR spectra were determined using Jeol 270 MHz and Jeol JMS-AX 500 MHz (Jeol, Japan) spectrometers with Me 4 Si as an internal standard. Mass spectra were recorded on an EI Ms-QP 1000 EX instrument (Shimadzu) at 70 eV. Pharmacological evaluations were done by the Pharmacology unit, Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Egypt.

Animals
Female Sprague-Dawley rats (150-200 g) were used in the anti-inflammatory activity study. Swiss mice of both sexes weighing 25-30 g were used in analgesic activity tests. International principles and local regulations concerning the care and use of laboratory animals were taken into account. The animals had access to standard commercial diet and water at libitum and were kept in rooms maintained at 22 ± 1 °C with a 12 h light-dark cycle.

Anti-Inflammatory Activity (Carrageenan-Induced Rat Hind Paw Edema Model)
The method adopted essentially resembles that described in the literature [17]. Distilled water was selected as vehicle to suspend the standard drugs and the test compounds. Sprague-Dawley rats were starved for 18 h prior to the experiment. The animals were weighed, marked for identification and divided into 28 groups each containing six animals. Edema was induced in the left hind paw of all rats by subcutaneous injection of 0.1 mL of 1% (w/v) carrageenan in distilled water into their footpads. The 1st group was kept as control and was given the respective volume of the solvent (0.5 mL distilled water). The 2nd to 16th groups were orally administered aqueous suspension of the synthesized compounds in dose of 20 mg/kg 1 h before carrageenan injection. The last group (standard) was orally administered diclofenac sodium at a dose of 20 mg/kg as an aqueous suspension [18]. The paw volume of each rat was measured immediately by a mercury plethysmometer, before carrageenan injection and then hourly for 3 h post administration of aqueous suspension of the synthesized compounds. The edema rate and inhibition rate of each group were calculated as follows: Edema rate (E)% = Vt − Vo/Vo, Inhibition rate (I)% =Ec − Et/Ec where Vo is the volume before carrageenan injection (mL), Vt is the volume at t h after carrageenan injection (mL), Ec and Et are the edema rates of the control and treated groups, respectively.

Analgesic Activity Using Hot-Plate Test
The experiment was carried out as described in the literature [19], using a hot-plate apparatus, maintained at 53 ± 0.5 °C. The mice were divided into 28 groups of six animals each. The reaction time of the mice to the thermal stimulus was the time interval between placing the animal in the hot plate and when it licked its hind paw or jumped. The reaction time was measured prior to aqueous suspension of synthesized compounds and drug treatment (0 min). Group 1 was kept as normal control. The aqueous suspension of synthesized compounds was orally administered to mice of groups 2-16 at doses of 20 mg/kg. Mice of group 17 (reference) were orally treated with diclofenac sodium at a dose of 20 mg/kg body wt. The reaction time was again measured at 15 min and repeated at, 30, 60 and 90 min after treatment. To avoid tissue damage to the mice paws, cut-off time for the response to the thermal stimulus was set at 60 s. The reaction time was calculated for each synthesized compound and drug-treated group.

Analgesic Activity (Acetic Acid Induced Writhing Response Model)
The compounds were selected for investigating their analgesic activity in acetic acid induced writhing response in Swiss albino mice, following the method described in literature [20]. One hundred and two mice were divided into 28 groups (six in each group) starved for 16 h, pretreated as follows, the 1st group which served as control positive orally received distilled water in appropriate volumes. The 2nd to 16th groups received the aqueous suspension of synthesized compounds orally at a dose of 20 mg/kg. The last group orally received diclofenac sodium at a dose of 20 mg/kg. After 30 min, each mouse was administrated 0.7% of an aqueous solution of acetic acid (10 mL/kg) and the mice were then placed in transparent boxes for observation. The number of writhes was counted for 20 min after acetic acid injection. The number of writhes in each treated group was compared to that of a control group. The number of writhing was recorded and the percentage protection was calculated using the following ratio: % protection = (control mean − treated mean/control mean) × 100.