Synthesis of 3-N-Sugar-substituted-2, 4(1H, 3H)-quinazolinediones as Anti-Angiogenesis Agents

A series of novel 3-N-sugar-substituted quinazolinediones were synthesized through the cyclization of the intermediate 2-aminobenzamides using triphosgene as the condensing reagent. Their anti-angiogenesis activities were investigated. The compound 3-(2'-aminoglucosyl)-2,4-(1H,3H)-quinazolinedione, (5d) showed good anti-angiogenesis activity.


Introduction
The development of an effective anti-cancer drug is still a major challenge in the field of drug discovery. It was reported that aminopeptidase N (APN) plays a crucial role in the degradation and invasion of extracellular matrices by fibrosarcoma cells [1] . It is also important in the proliferation and the activation of pathogenic T-cells [2]. Several APN inhibitors were prepared to treat inflammatory disease, autoimmune disease, allogenic rejection reactions and allergies. In addition, APN antagonists were found to specifically inhibit angiogenesis in chorioallantoic membranes and in the retina, thus suppressing tumor growth. Therefore, APN was believed to be involved in angiogenesis and can serve as a target for the development of anti-cancer drugs [3][4][5].

OPEN ACCESS
The quinazolinediones have inhibitory activities towards some amino peptidases, such as puromycin-sensitive aminopeptidase (PSA) [5] and aminopeptidase N [1]. One representative compound, PAQ-22 (Figure 1), showed potent and specific PSA inhibiting activity with an IC 50 of 0.09 μg/mL [5]. The inhibitory mechanism of these compounds was through non-competition, as revealed by the Lineweaver-Burk plot analysis. Structure-activity relationship studies indicated that tautomerism of the imidobenzoylketone group of the cyclic imide moiety of these kinds of inhibitors was important for the inhibitory activity [5]. It is reported that glucosamine, a type of amino-sugar, possesses immunosuppressive activity and could be beneficial as an immunosuppressive agent [6][7]. Water-soluble conjugates of glucosamine and glucosamine 6-sulfate were reported showing immunomodulatory and anti-angiogenesis properties, These derivatives of glucosamine could function synergistically to prevent scar tissue formation [8][9][10].
To prepare the intermediate 2-nitro-(N-sugar-substituted) benzamides from aminosugars, condensations of protected amino-sugars with 2-nitrobenzoyl chloride or with 2-nitrobenzoic acid activated by DCC (or EDC/HOBt) were attempted, but all these efforts failed. It was reported that the unprotected amino-sugars could be used directly to synthesize related amides [20][21], and following this method, the key N-sugar o-nitrobenzamide intermediates were obtained with isolated yields of 40-47%.

Instruments and apparatus
1 H-and 13 C-NMR spectra were recorded on a Varian VXR 300 MHz spectrometer with Me 4 Si as the internal standard and CDCl 3 or Me 2 SO-d 6 as solvent. Optical rotations were measured at 25 ºC with an AA-10R polarimeter. The progress of reactions was monitored by silica-gel GF 254 TLC plates. Detection was performed by examination under UV light and by 15% H 2 SO 4 in EtOH. Preparative TLC was performed on silica-gel GF 254 plates and column chromatography was on silica-gel H. The inhibitory activity of anti-angiogenesic was assayed under chick chorioallantoic membrances (CAM) model, monitored by biological dissection microscope of DM-1 and recorded with a NIKON S610 digital camera.

Synthesis and Spectral Data of 2a-c
Aminosugar 1a-c (2.9 g, 15 mmol), o-nitrobenzoic acid (3.2 g, 16.5 mmol) and 1-hydroxy benzotriazole (HOBt) (4.9 g, 36.3 mmol) were dissolved in DMF (80 mL). The mixture was cooled to 0 ºC and stirred for 30 min. Then a solution of dicyclohexylcarbodiimide (DCC, 3.8 g, 18.2 mmol) in DMF (15 mL) was added dropwise. The mixture was stirred for 18 h at room temperature and filtered. The filtrate was evaporated to dryness under reduced pressure, and the residue was purified by column chromatography on silica gel to give 2a-c. Glucosamine hydrochloride (7.8 g, 36 mmol) and sodium methoxide (2.25 g, 41.7 mmol) were added to methanol (100 mL). The mixture was stirred for 20 min and then evaporated to dryness under vacuum. The residue was dissolved in DMF (200 mL), followed by the addition of o-nitrobenzoic acid (5.1 g, 30 mmol) and 1-hydroxybenzotriazole (HOBt, 9.5 g, 72 mmol). The mixture was cooled to 0 ºC and stirred for 30 min. Then the solution of dicyclohexylcarbodiimide (DCC, 6.9 g, 36 mmol) in DMF (25 mL) was added dropwise. The mixture was stirred for 20 h at room temperature and filtered. The filtrate was evaporated to dryness under reduced pressure, and the residue was purified through column chromatography on silica gel to yield 5.

Synthesis and Spectral Data of 3a-d
The appropriate 2-nitro-(N-sugar-substituted) benzamide 2a-d (2.0 g) was dissolved in pyridine (50 mL), followed by the addition of acetic anhydride (25 mL). The solution was stirred at room temperature overnight and evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate and washed sequentially with saturated sodium hydrogen carbonate solution, saturated brine and water. The organic layer was dried over anhydrous Na 2 SO 4 and evaporated under reduced pressure to give 3a-d as yellow solids.

Synthesis and Spectral Data of 4a-c
The appropriate compound 3a-c (2.0 g, 7.4 mmol) was dissolved in THF (50 mL) and acetic acid (5 mL). Under stirring, zinc power (1.3 g, 20 mmol) was added slowly. The mixture was then refluxed for 2 h, cooled to room temperature, and filtered through a short column of silica gel. The eluent was evaporated to dryness under vacuum. The residue was dissolved in ethyl acetate and washed sequentially with saturated sodium hydrogen carbonate solution, saturated brine and water. The organic layer was dried over anhydrous Na 2 SO 4 and evaporated under reduced pressure to afford the compounds 4a-c as yellow solids.

Synthesis and Spectral Data of 5a-d
Compounds 4a-d (300 mg) were dissolved in ClCH 2 CH 2 Cl (50 Ll), then triphosgene (140 mg, 0.54 mmol) was added. The mixture was refluxed for 6h and cooled to room temperature. CH 2 Cl 2 (50 mL) was added and the organic layer was washed with saturated sodium hydrogen carbonate solution, saturated brine and water. The organic layer was dried over anhydrous Na 2 SO 4, evaporated under reduced pressure to dryness, and purified with column chromatography on silica gel to yield white solids of 5a-d.

Anti-angiogenesic Inhibitory Activity of the Target Compounds
The eggs were cut and chicken embryos were incubated under 37.5 °C for 7 days. When the CAM's diameter had grown to 1-3cm, solutions of the compounds was added to each chicken embryo with PBS as control. The results were recorded by camera under a dissection microscope [22].

Conclusions
In summary, several novel 3-N-sugar-substituted quinazolinediones were synthesized and their antiangiogenesis activities were tested. An efficient method, using triphosgene as the carbonylation condensation reagent, was developed for the synthesis of N-sugar-substituted quinazolinediones. This method might be useful in the future for the preparation of similar derivatives.