Synthesis of Novel Benzodifuranyl; 1,3,5-Triazines; 1,3,5-Oxadiazepines; and Thiazolopyrimidines Derived from Visnaginone and Khellinone as Anti-Inflammatory and Analgesic Agents

Novel (4-methoxy or 4,8-dimethoxy)-3-methyl-N-(6-oxo-2-thioxo-1,2,3, 6-tetrahydro- pyrimidin-4-yl) benzo [1,2-b: 5, 4-b’] difuran-2-carboxamide (5a–b) has been synthesized by the reaction of visnagenone–ethylacetate (2a) or khellinone–ethylacetate (2b) with 6-aminothiouracil in dimethylformamide or refluxing of benzofuran-oxy-N-(2-thioxopyrimidine) acetamide (4a–b) in sodium ethoxide to give the same products (5a,b) in good yields. Thus, compounds 5a–b are used as an initiative to prepare many new heterocyclic compounds such as 2-(4-(3-methylbenzodifuran- 2-carbox-amido) pyrimidine) acetic acid (6a–b), N-(thiazolo[3, 2-a]pyrimidine)-3-methylbenzo- difuran-2-carboxamide (7a–b), N-(2-thioxopyrimidine)-methylbenzodifuran-2-carbimidoylchloride (8a–b), N-(2-(methyl-thio) pyrimidine)-3-methylbenzodifuran-2-carbimidoylchloride (9a–b), N-(2, 6 -di(piperazine or morpholine)pyrimidine)-1-(3-methylbenzodifuran)-1-(piperazine or morpholine) methanimine(10a–d), 8-(methylbenzodifuran)-thiazolopyrimido[1,6-a][1,3,5]triazine-3,5-dione (11a –b), 8-(3-methyl benzodifuran)-thiazolopyrimido[6,1-d][1,3,5]oxadiazepine-trione (12a–b), and 2,10 -di(sub-benzylidene)-8-(3-methylbenzodifuran)-thiazolopyrimido[6,1-d][1,3,5]oxadiazepine-3,5,11- trione (13a–f). All new chemical structures were illustrated on the basis of elemental and spectral analysis (IR, NMR, and MS). The new compounds were screened as cyclooxygenase-1/ cyclooxygenase-2 (COX-1/COX-2) inhibitors and had analgesic and anti-inflammatory activities. The compounds 10a–d and 13a–f had the highest inhibitory activity on COX-2 selectivity, with indices of 99–90, analgesic activity of 51–42% protection, and anti-inflammatory activity of 68%–59%. The inhibition of edema for the same compounds, 10a–d and 13a–f, was compared with sodium diclofenac as a standard drug.

The IR spectra of 10a and 10c revealed strong absorption broad bands at 3385-3390 cm -1 characteristic of the (br, NH) groups. The 1 H-NMR spectrum of 10a indicated three singlet broad signals at δ 10.10, 10.30, and 10.50 ppm conforming to the three protons of the three NH groups, which were D 2 O exchangeable. All new compounds were confirmed by their correct compatible spectrum data and elemental analyses values (see the Experimental section, Scheme 3).  [1,3,5]oxadiazepine-3,5,11(2H,10H)-trione (12a,b) respectively, in good yields. In addition, the second method involved the refluxing of 7a or 7b with formamide or chloroacetyl-chloride in acetic acid, in the presence of zinc dust, to afford the conforming the same products (11a-b and 12a-b), respectively. The formation of 11a-b and 12a-b from the corresponding 7a-b may proceed through an initial reduction of compounds 7a-b, followed by the cyclocondensation of the intermediates produced with the ketones followed by a necessary final reduction step to produce 11a, b and 12a, b. The IR spectrum of 11a showed absorption bands at 1684 and 1680 cm -1 of two carbonyl amide groups and 1632 cm -1 to the C=N group. The 1 H-NMR spectrum of 11a revealed six singlet signals at δ 2.32, 3.94, 5.85, 7.20, 7.35, and 8.10 ppm, agreeing with the three protons of methyl, three protons of methoxy, one proton of thiazole, one proton of phenyl, one proton of triazine, and one proton of the pyrimidine ring, respectively and exhibited two doublet signals at δ 4.10 and 4.17 ppm approving the two protons of CH2, thiazole ring. Hence, CH2 protons are diastereotopic pairs to give the germinal coupling (J = 6.90 Hz). The 13 C-NMR spectrum of 11a exhibited signals at δ 166.7 and 169.8 ppm corresponding to two carbon atoms of the two carbonyl groups. Likewise, the IR spectrum of 12a displayed absorption bands at 1688, 1684, and 1679 cm -1 corresponding to three carbonyl amide groups, respectively. Besides, the 13 C-NMR spectrum of 12a showed absorption signals at δ 68.8 ppm, corresponding to one carbon atom of the thiazole, 87.3 ppm, corresponding to one carbon atom of pyrimidine, 102.6 ppm, corresponding to one carbon atom of the phenyl, and 164.8, 167.9, and 172.9 ppm, corresponding to the three carbon atoms of the carbonyl groups. Furthermore, the refluxing of 12a or 12b with a suitable aromatic aldehyde, namely, benzaldehyde, 4-chlorobenzaldehyde, or 4-methoxybenzaldehyde respectively, in dioxane solution having a catalyst amount of piperidine for an extended time gave the products 2,10-di(substituted-benzylidene)-8-((4-methoxy or 4,8-dimethoxy)-3-methylbenzo [1,2- [1,3,5]oxadiazepine-3,5,11(2H,10H)-trione (13a-f) in high yields. In another way, Similarly, in the first method, compound 7a or 7b reacted with formamide or α-halo-ketones as chloroacetyl-chloride in DMF, in the presence of anhydrous potassium carbonate to afford the corresponding 8-(4-methoxy or 4, [1,3,5]triazine-3,5-dione (11a,b), and 8-(4-methoxy or 3,5]oxadiazepine-3,5,11(2H,10H)-trione (12a,b) respectively, in good yields. In addition, the second method involved the refluxing of 7a or 7b with formamide or chloroacetyl-chloride in acetic acid, in the presence of zinc dust, to afford the conforming the same products (11a-b and 12a-b), respectively. The formation of 11a-b and 12a-b from the corresponding 7a-b may proceed through an initial reduction of compounds 7a-b, followed by the cyclocondensation of the intermediates produced with the ketones followed by a necessary final reduction step to produce 11a, b and 12a, b. The IR spectrum of 11a showed absorption bands at 1684 and 1680 cm-1 of two carbonyl amide groups and 1632 cm-1 to the C=N group. The 1H-NMR spectrum of 11a revealed six singlet signals at δ 2.32, 3.94, 5.85, 7.20, 7.35, and 8.10 ppm, agreeing with the three protons of methyl, three protons of methoxy, one proton of thiazole, one proton of phenyl, one proton of triazine, and one proton of the pyrimidine ring, respectively and exhibited two doublet signals at δ 4.10 and 4.17 ppm approving the two protons of CH2, thiazole ring. Hence, CH2 protons are diastereotopic pairs to give the germinal coupling (J = 6.90 Hz). The 13 C-NMR spectrum of 11a exhibited signals at δ 166.7 and 169.8 ppm corresponding to two carbon atoms of the two carbonyl groups. Likewise, the IR spectrum of 12a displayed absorption bands at 1688, 1684, and 1679 cm-1 corresponding to three carbonyl amide groups, respectively. Besides, the 13C-NMR spectrum of 12a showed absorption signals at δ 68.8 ppm, corresponding to one carbon atom of the thiazole, 87.3 ppm, corresponding to one carbon atom of pyrimidine, 102.6 ppm, corresponding to one carbon atom of the phenyl, and 164.8, 167.9, and 172.9 ppm, corresponding to the three carbon atoms of the carbonyl groups. Furthermore, the refluxing of 12a or 12b with a suitable aromatic aldehyde, namely, benzaldehyde, 4-chloro-benzaldehyde, or 4-methoxybenzaldehyde respectively, in dioxane solution having a catalyst amount of piperidine for an extended time gave the products 2,10-di(substituted-benzylidene)-8-((4-methoxy or 4, [1,3,5]oxadiazepine-3,5,11(2H,10H)-trione (13a-f) in high yields. In another way, we obtained the same products (13a-f) via reacting 12a or 12b with a proper aromatic aldehyde and anhydrous sodium acetate in glacial acetic acid with acetic anhydride for 8-10 h. The 1 H NMR spectrum of 13a displayed two singlets at 8.04 and 8.10 ppm conforming to the two methine protons (2CH). The mass spectra of 13a, 13b, 13c, 13d, 13e, and 13f exposed molecular ion peaks at m/z 629 (M + , 90%), 698 (M + , 92%), 689 (M + , 88%), 659 (M + , 88%), 728 (M + , 95%), and 719 (M + , 94%), respectively, All spectral analyses of new compounds are described in the Experimental section (Scheme 4).

The Inhibition Test of COX Enzymes in vitro
In Table 1, the IC50 results of COX inhibition and the selectivity index (SI) are shown. The evaluation results evidenced that all compounds displayed efficient inhibitory activities on COX-2 enzymes, with IC50 values that were lower than those of COX-1, in the range of 0.04-0.46 μM,

The Inhibition Test of COX Enzymes in vitro
In Table 1, the IC 50 results of COX inhibition and the selectivity index (SI) are shown. The evaluation results evidenced that all compounds displayed efficient inhibitory activities on COX-2 enzymes, with IC 50 values that were lower than those of COX-1, in the range of 0.04-0.46 µM, compared to COX-2. Moreover, the inhibitory activity against COX-1 ranged from 4.15 to 18.80 µM. The compounds 10a-d, 13a-f, 12a-b, and 11a-b had the highest inhibitory activities with IC 50 values of 4.15-4.60, 5.10-5.79, 6.10-6.40, and 6.60-6.85 µM to COX-1, respectively, and 0.042-0.048, 0.053-0.064, 0.070-0.075, and 0.080-0.085 µM to COX-2, respectively, indicating the highest selectivity index of 99-81 respectively. In addition, 10a-d, 13a-f, 12a-b, 11a-b, and 7a-b compounds exhibited the highest COX-2 selectivity index.   Table 2 shows the results of the analgesic activity of the new compounds in vivo via applying the writhing test [4,[30][31][32][33][34][35], with results evaluated by comparing them to the same dose (10 mg/kg) of the standard drug sodium diclofenac. The writhing response was calculated, and protection (%) was calculated. Compounds 10a-d, 13a-f, and 12a-b evidenced the highest analgesic activities among the synthesized compounds, 48-51%, 42-47%, and 40-41% respectively, which were nearly the same activities of the drug sodium diclofenac (51%). In addition, the analgesic activities of the aforementioned compounds agreed with their anti-inflammatory activity. On the other hand, compounds 4a-b, 5a-b, and 6a-b showed the lowest activities, which may be due to several pharmacokinetic parameters that affected the absorption and degradation of these compounds. Values are expressed as mean ± SEM (n = 5). Data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test. A p-value was considered significant if it was less than 0.05 compared to control; (a) Significantly different from the control group; (b) Significantly different from the diclofenac group.

Anti-Inflammatory Activity
From the past results, the determined compounds 10a-d, 13a-f, 12a-b, 11a-b, and 7a-b possessed the highest anti-inflammatory activities. A carrageenan-induced paw edema in rats was used to evaluate the anti-inflammatory activity of these compounds [4,[30][31][32][33][34]36]. Most of the compounds evidenced significant (p < 0.05) anti-inflammatory activity, via reducing paw height; hence, rat paw edema was reduced after three hours in comparison with the control group, as shown in Table 3. The compounds 10a-d, 13a-f, 12a-b, and 11a-b, among all the synthesized compounds, displayed the highest anti-inflammatory activity, which appeared from the first hour of inflammation with edema inhibition percentages of 86-65%, 64-59%, 58-57%, and 56-55%, respectively. This was superior to the drug sodium diclofenac, with a rapid onset of action after one hour, and the time continued until the third hour after compound administration. Values are expressed as mean ± SEM (n = 5). Data were analyzed using one-way ANOVA followed by a Bonferroni post hoc test. A p-value was considered significant if it was less than 0.05 compared to control; a Significantly different from the control group; b Significantly different from the diclofenac group.

Serum Level of Interleukin-1 Beta (IL-1 β)
At the end of the paw edema test, serum was synthesized for ELISA to determine the interleukin-1 beta (IL-1 β) concentration in the most important groups. The determined compounds were 10a-d, 13a-f, 12a-b, and 11a-b (shown in Table 4). Compounds 10a-d showed the most significant reduction in IL-1 β concentration, which established the in vitro inhibition of COX activity and so inhibited the inflammatory intermediator in this pathway. Table 4. Serum level of interleukin-1 beta (IL-1 β) after paw edema in rats. Values are expressed as mean ± SEM (n = 5). Data were analyzed using one-way ANOVA followed by the Bonferroni test as a post hoc test. A p-value was considered as significant if it was less than 0.05 compared to control; a Significant from normal animals; b Significant from paw edema-bearing animals.

General Information
All melting points were taken on an Electrothermal IA 9100 series digital melting point apparatus (Shimadzu, Tokyo, Japan). Elemental analyses were performed on a Vario EL (Elementar, Langenselbold, Germany). Microanalytical data were processed in the microanalytical center, Faculty of Science, Cairo University and National Research Centre. The IR spectra (KBr disc) were recorded using a Perkin-Elmer 1650 spectrometer (Waltham, MA, USA). NMR spectra were determined using JEOL 270 MHz and JEOL JMS-AX 500 MHz (JEOL, Tokyo, Japan) spectrometers with Me4Si as an internal standard. Mass spectra were recorded on an EI Ms-QP 1000 EX instrument (Shimadzu, Japan) at 70 eV.  The final product precipitated was filtered off and washed with (100 mL) water, dried, and crystallized from the proper solvent to give 5a and 5b. Method B: A solution of compound 4a (3.89 g, 0.01 mol) or 4b (4.19g, 0.01 mol) in sodium ethoxide solution (prepared by dissolving 0.23 g of sodium metal in 40 mL ethanol) was heated under reflux with stirring for 5-7 h under control (TLC). The reaction mixture was allowed to cool and poured into cold water (100 mL) and neutralized via hydrochloric acid. The solid product was precipitated, which was filtered off and crystallized from the proper solvent to give 5a and 5b, respectively. A mix of 5a (3.71 g, 0.01 mol) or 5b (4.01 g, 0.01 mol), chloroacetic acid (0.94 g, 0.01 mol), and (0.02 mol) anhydrous sodium acetate was stirred under reflux in 45 mL of glacial acetic acid and 25 mL of acetic anhydride for 4-6 h. The reaction solution was cooled and poured into cold water (100 mL). The deposited precipitate was filtered off and crystallized from suitable solvent to yield 6a and 6b, respectively.

General Procedure for Synthesis of N-(6-chloro-2-thioxo-2, 3-dihydropyrimidin-4-yl)-(4-methoxy or 4, 8-dimethoxy)-3-methyl benzo [1,2-b: 5,4-b'] difuran-2-carbimidoyl chloride (8a, b)
A solution of 5a (3.71 g, 0.01 mol) or 5b (4.01 g, 0.01 mol) in dry dioxane (30 mL) was treated with 20 mL of phosphorus oxy-chloride, and the mixture was stirred under reflux for 4-6 h. The reaction mixture was allowed to cool to room temperature. Then, it was poured into cold water (100 mL), whereby a solid was separated, filtered off, and crystallized from a suitable solvent to give 8a and 8b, respectively. To a warmed ethanolic potassium hydroxide solution (prepared by dissolving 10 mmol of KOH in 50 mL ethanol), 8a (4.08 g, 0.01 mol) or 8b (4.38 g, 0.01 mol) was added, and the heating was continued for 35 min. Then, the mixture was allowed to cool to room temperature, and methyl iodide (0.62 mL, 0.01 mol) was added. The mixture was stirred under reflux for 6-8 h; then, it was cooled to room temperature and poured into cold water (100 mL). The final solid precipitated was filtered off, washed with water, and the product was dried and crystallized from a suitable solvent to yield 9a and 9b, respectively. The compound was obtained from the reaction of 8a with methyl iodide as brownish crystals, which were crystallized from DMF in 80% yield, m.p. In a warm solution of 9a (4.22 g, 0.01 mol) or 9b (4.52 g, 0.01 mol) in glacial acetic acid (40 mL) or absolute methanol (40 mL), freshly distilled secondary (2 • ) amines, piperazine (0. 86 g, 0.01 mol), or morpholine (0.87 mL, 0.01 mol) were added. The reaction mixture was stirred under reflux for 5-7 h with TLC; then, it was allowed to cool to 0 • C for 4 h. The solid obtained was filtered, washed with water (100 mL), dried, and recrystallized from an appropriate solvent to produce 10a-d.  Method A: A mixture of compound 7a (4.11 g, 0.01 mol) or 7b (4.41 g, 0.01 mol) and formamide (10 mL) or chloro-acetyl-chloride (1.13 g, 0.01 mol) was stirred under reflux in dimethylformamide (40 mL) in the presence of anhydrous potassium carbonate (0.015 mol) for 18-20 h under control (TLC). The reaction mixture was allowed to cool to room temperature, poured into water (100 mL), and neutralized. The solid formed was filtered off, dried, and crystallized from appropriate solvent to produce 11a, b and 12a, b respectively. Method B: The stirring of mixture of compound 7a (4.11 g, 0.01 mol) or 7b (4.41 g, 0.01 mol) and formamide (10 mL) or chloro-acetyl-chloride (1.13 g, 0.01 mol) in glacial acetic acid (45 mL), activated zinc dust (1.3 g, 0.02 mol) was added portionwise at room temperature over a period of 1 h. Stirring was continued for an additional 3-5 h. The reaction mixture was heated on a water bath (85-95 • C) for 4-6 h under control (TLC). After allowing the reaction mixture to cool to room temperature, it was poured into cold water (100 mL). The precipitate was filtered, washed with water, dried, and crystallized to produce 11a, b and 12a, b respectively. The compound was obtained from the reaction of 7a with formamide in DMF as pale yellow crystals, which were crystallized from benzene in 91% yield, m.p. The compound was obtained from the reaction of 7b with formamide in DMF as brownish crystals, which were crystallized from toluene in 90% yield, m.p. >350 • C. IR (ν, cm -1 ) KBr: 3062 (CH-aryl), 2944 (CH-aliph), 1686 and 1682 (2CO, amide), and 1630 (C=N). 1 13  The compound was obtained from the reaction of 7b with chloro-acetyl-chloride in DMF as yellowish crystals, which were crystallized from Pet. ether in 70% yield, m.p. >350 • C. IR (ν, cm -1 ) KBr: 3045 (CH-aryl), 2935 (CH-aliph), 1686, 1682, and 1677 (3CO, amide), and 1631 (C=N). 1   was stirred and refluxed for 12-15 h (TLC under control). The reaction solution was cooled, and the formed precipitate was filtered off, dried, and recrystallized from the proper solvent to give 13a-f. Method B: A mixture from one of the compounds 12a (4.53 g, 0.01 mol) or 12b (4.83 g, 0.01 mol), the appropriate aromatic aldehyde (0.02 mol), and 0.02 mol of anhydrous sodium acetate was stirred under reflux in 40 mL of glacial acetic acid and 20 mL of acetic anhydride for 8-10 h. The reaction mixture was allowed to cool to room temperature and poured into cold water (100 mL). The deposited precipitate was filtered off and crystallized from an appropriate solvent to produce 13a-f. The compound was obtained from the reaction of 12a with benzaldehyde (2.12 g, 0.02 mol) in dioxane as yellowish crystals, which were crystallized from ethanol in 76% yield, m.p. >350 • C. IR (ν, cm -1 ) KBr: 3070 (CH-aryl), 2960 (CH-aliph), 1690, 1687, and 1684 (3CO, amide), and 1638 (C=N). 1  All the procedures adopted were approved and agreed upon by Animal Ethical committee of Mansoura University, Faculty of Pharmacy (Department of Pharmacognosy), 35516, Egypt.

Human and Animal Rights
No humans were used in the study. The research was conducted in accordance with the ethical standards. All care and use guidelines for laboratory animals were followed.

Chemicals and Drugs
Carrageenan and glacial acetic acid were purchased from Sigma-Aldrich (St. Louis, Mo, USA). Celecoxib, indomethacin, and diclofenac sodium were purchased from Pfizer (New York, NY, USA). The interleukin 1 beta (IL-1 b) ELISA Kit was purchased from Reddot Biotech. Inc. (Kelowna, BC, Canada).

Animals
Male albino rats and mice were used to evaluate anti-inflammatory and analgesic activities, respectively. Animals were purchased from the animal house at The Holding Company for biological products, vaccines, and drugs (VACSERA, Giza, Egypt), and they were acclimatized for two weeks before the experiments at the animal house of Mansoura University, Faculty of Pharmacy (Department of Pharmacognosy). The animals were divided into different groups, five animals in each group. Food and water were supplied ad libitum to the animals.

In Vitro COX Assay
All new compounds 4-13 and derivatives were screened for in vitro COX inhibitory activity (Ibrahim et al. [37]) using an enzyme immunoassay (EIA) kit (Cayman Chemical Company, USA). The IC50 values for the compounds were determined using celecoxib as the reference drug, which is a selective COX-2 inhibitor medicine. Furthermore, the selectivity index for COX-2 (COX-1 IC 50 / COX-2 IC 50 ) was calculated for the compounds. The ability of the compounds to inhibit ovine COX-1 and the human recombinant COX-2 form was determined and evaluated using a COX inhibitor Screening Assay kit (catalogue no. 560131, Cayman Chemical, Ann Arbor, MI, USA) in agreement with instructions recommended and mentioned by the supplier.

Screening of Analgesic Activity
The analgesic activity of the compounds was performed using the writhing test as described by Gawade et al. [35]. Mice weight ranged from 25 to 30 g. Then, mice were divided into 18 different groups, each containing five animals. The animals fasted for 8 h before the experiment. Administration of the compounds (10 mg/kg, p.o.) was performed orally. Equal doses of diclofenac as the reference drug and saline as a negative control were also administrated orally. Writhing was induced one hour after compound administration by using 0.1 mL of 1% glacial acetic acid at a volume of 0.1 mL/10 g body weight. The number of writhing responses (stretching of the abdomen, extension of hind limbs, twisting of the trunk, and elongation of the body) observed was counted for 20 min. The protection percentile against acetic acid-induced writhing was calculated according to the following formula: Analgesic activity (%) = n − n n × 100 * where n is the mean number of writhes of the control group, and n' is the mean number of writhes of the tested compound group.

Evaluation of the Anti-Inflammatory Activity
The method of Paw edema reported by Puttaswamy et al. [36] was used to evaluate and screen the activity of the compounds that exhibited analgesic activity against inflammation. Albino rats (150-180 g) were used. The rats were divided into 11 experimental groups of five rats each. One hour after oral administration of the compounds (10 mg/kg), 0.1 mL of 1% carrageenan solution was injected in the left hind paw of each animal. Rat paw volumes were measured with a digital caliber plethysmometer (UGO Basile, Varese, Italy) used to measure inflammation height at 0, 1, 2, and 3 h after carrageenan injection. The percentage increase in inflammation in the left hind paw, in comparison to the uninjected right hind paw, was determined, calculated, and expressed as the amount of inflammation. All the tested compounds were orally administered at equivalent doses of the reference drug diclofenac (10 mg/kg, p. o.). The anti-inflammatory activity of the selected analgesic compounds after 3 h was expressed as percentage inhibition of edema, which was calculated according to the following equation: where V cont is the edema volume in the control group, and V test is the edema volume in the group of the screened compound.

ELISA Determination of IL1
IL1 is one of the important inflammatory mediators and is used to confirm the anti-inflammatory activities of new anti-inflammatory compounds. At the end of the paw edema test, blood samples were collected from the orbital axis of the eye under light ether anesthesia. Sera were separated after centrifugation of blood at 5000 rpm for 15 min; the sera were stored at -80 o C until the ELISA assay was performed according to the manufacturer instructions.

Statistical Analysis
Data were expressed as mean ± SEM. One-way analysis of variance, (ANOVA, GraphPad Software, La Jolla California, USA) followed by Bonferroni's post hoc test was used to analyze the data using the Statistical Package for Social Sciences, version 19 (SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered to be statistically significant.

Conclusions
The new heterocyclic compounds were evidenced to be interesting for the study of anti-inflammatory and analgesic activities, which afforded the data of COX-1 and COX-2 inhibition as shown in Tables 1-4 compounds 10a-d, 13a-f, and 12a-b exhibited the highest anti-inflammatory and analgesic activities (COX-1/COX-2), because these compounds