Synthesis and Pharmacological Activities of Pyrano [ 2 , 3-d ] pyrimidine and Pyrano [ 2 , 3-d ] pyrimidine-5-one Derivatives as New Fused Heterocyclic Systems

Various fused oxazine such as 4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo [4,3:5,6]pyrano[2,3-d][1,3]oxazin5-one 2 has been prepared and utilized as a starting material for novel pyrazolopyranopyrimidinones 3, 5, 6, and 7a–c and pyrazolopyranopyrimidines 4, 9, 10, and 11 which are expected to possess considerable chemical and pharmacological activities. The structures of the new compounds have been elucidated by spectroscopic data and elemental analysis. The antioxidant and anticancer activities of synthesized products have been evaluated.

Treatment of by freshly distilled acetic anhydride afforded as a sole product.The structure of was elucidated by spectral analysis.The IR spectrum showed a strong absorption band at 1732 cm −1 corresponding to lactonic carbonyl group and the disappearance of the cyano group.Also the structure was confirmed by elemental analysis; 1 H-NMR spectrum showed a singlet at  12.52 ppm of NH, a doublet at  7.162-7.136ppm and  6.84-6.81ppm of aromatic protons, a singlet at  4.7 ppm of benzylic proton, and a singlet at  3.68 ppm of three protons for OCH 3 group.[2,3-d]pyrimidin-6(5H)-yl]thiourea , as sole products.The pyrazolopyranopyrimidine derivative was formed via ring opening of the oxazine ring by an amino group followed by ring closer and then dehydration with the other amino group.The IR spectrum of compound showed strong absorption bands at 3180 and 1609 cm −1 corresponding to ] NH and ] C=N .The IR spectrum of compound showed well defined absorption bands at 3251, 3158, 3121, 1665, 1619, and 1254 cm −1 corresponding to ] NH2 , ] NH , ] C=O , ] C=N , and ] C=S .Compound was allowed to react with primary amines, namely, hydrazine hydrate, 4-methylaniline, 4-methoxyaniline, and/ or 4-aminoaniline, to afford the pyrazolopyranopyrimidine derivatives and a-c, respectively (Scheme 2).IR spectrum of compound showed strong absorption bands at 3215, 3156, 3161, and 1612 cm −1 corresponding to ] NH2 , ] NH , and ] C=N .Also the structure was confirmed by 1 H-NMR spectrum that showed a singlet at  12.01 ppm of NH, multiplet at  7.83 ppm of aromatic protons, a singlet at  4.8 ppm of benzylic proton, a singlet at  3.7 ppm of three protons for OCH 3 group, and a singlet at  5.354 of NH 2 .
Acetylation of by refluxing with acetic anhydride gave the diacetyl derivative and not the expected triazolo derivative .The structure of was confirmed by the spectral data such as the IR which showed the absorbance for the carbonyl group at 1732 and 1695 cm −1 and the two methyl groups signals at  2.678 ppm for 2CH 3 in 1 H-NMR spectrum (Scheme 3).

Pharmacological Activity.
Antioxidant activity using 2,2  -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) inhibition.The prepared compounds were tested for antioxidant activity as reflected in their ability to inhibit oxidation in rat brain and kidney homogenates, from the result in Table 1.Compounds , , a, and exhibit a similar antioxidant activity to ascorbic acid, used as standard.Compounds and exhibited moderate antioxidant activity, while , , b, , and showed lower activity.
Bleomycin Dependent DNA Damage.The bleomycins are a family of glycopeptides antibiotics [31] that are routinely used as antitumor agents.The bleomycin assay has been adopted for assessing the prooxidant activity of food antioxidants.The antitumor antibiotic bleomycin binds iron ions and DNA.If the samples are able to reduce bleomycin-Fe 3+ to bleomycin-Fe, DNA degradation in the system will be stimulated, resulting in a positive test for prooxidant activity.DNA degradation is accompanied by the formation of a product similar to malondialdehyde.
L-Ascorbic acid was used as the reducing agent to reduce Fe 3+ to Fe 2+ .The synthesized compounds were selected for bleomycin dependent DNA damage testing (Table 1).
Results in Table 1 showed that compounds , , a, , and have the ability to protect DNA from the damage induced by bleomycin.On the other hand, the rest of the compounds exhibited weak activities.

Cytotoxic Activity of Some Compounds against Human Tumor
Cells.The prepared compounds were tested for cytotoxic activity against four human tumor cells.Best results were observed for compounds , , a, and which were found to very strong cytotoxic compounds.Compound was also considered strongly cytotoxic.Finally, compounds , , and were moderate in their cytotoxic activity and compounds , b, and were weakly cytotoxic (Table 2).

Experimental.
All melting points were measured on a Gallenkamp electric melting point apparatus and are uncorrected.The infrared spectra were recorded in potassium bromide disks on a pyeUnicam SP-3-300 and Shimdazu FTIR 8101 PC infrared spectrophotometers.
The 1 H-NMR and 13 C-NMR were recorded at a Varian Mercury VX-300 MHz and MHz, respectively, using TMS as internal standard in deuterated chloroform (CDCl 3 ) or deuterated dimethyl sulfoxide (DMSO-d6).Chemical shifts are measured in ppm.The mass spectra were recorded on a Shimadzu GCMS-QP-1000EX mass spectrometer at 70 eV.Elemental analyses were carried out at the Microanalytical Center of Cairo University.All the reactions and the purity of the new compounds were followed and checked by TLC using TLC aluminum sheets silica gel F 254 .

General Procedure for the Synthesis of Pyrazolopyranopyrimidine ( a-c).
A solution of (10 mmol, 3.25 g) in dimethylformamide (20 mL) and (10 mmol) of 4-methyl aniline, 4-methoxyaniline, and/or 4-amino aniline was heated under reflux for 5 h.The solid that was separated out after concentration and cooling was recrystallized from the proper solvent to give ( a-c).[2,3-d] [2,3-d]

Table 1 :
The antioxidant activities of the synthesized compounds.

Table 2 :
The cytotoxic activity of some compounds against human tumor cells.