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

Authors: Naglaa F. H. Mahmoud; Eman A. El-Bordany; Galal A. Elsayed
Journal: Journal of Chemistry
 (2017)

---

## Abstract

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]oxazin-5-one2 has been prepared and utilized as a starting material for novel pyrazolopyranopyrimidinones3,5,6, and7a–c and pyrazolopyranopyrimidines4,9,10, and11 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.


---

## Body

## 1. Introduction

Heterocyclic compounds are one of the most important molecules in the synthesis of pharmacologically active compounds [1–4]. Enaminonitriles have been utilized as starting materials for synthesis of huge number of heterocyclic compounds containing pyranopyrimidine [5–9] and pyridopyrimidine [10–13] moieties. Oxazines [14] and quinazolines [15] are natural products which exhibit a widespread spectrum of biological activity including antitumor [16–18], anti-inflammatory [19], and antiplatelet [20] properties. In view of these reports and as a continuation of our previous work on heterocyclic chemistry [21–27], we aimed to incorporate the pyrano moiety into the 1,5-position of oxazinone and pyrimidinone ring systems to obtain new heterocyclic system which is anticipated to possess notable chemical and pharmacological activities. So we utilized enaminonitrile in the synthesis of pyrano[2,3-d][1,3]oxazin-5-one derivative and pyrano[2,3-d]pyrimidine and pyrano[2,3-d]pyrimidine-5-one derivatives in addition to studying their antioxidant activities.


## 2. Results and Discussion

### 2.1. Chemistry

Our present investigation aimed to synthesize of 6-amino-4-(4-methoxyphenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile1 [28], which was used as a precursor for the synthesis of previously unreported 4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one2 as new heterocyclic compound containing oxazine and pyranopyrazole moieties. Compound1 has been prepared by four-component one-pot reaction [29] and three-component one-pot reaction [30] (Scheme 1).Scheme 1Treatment of1 by freshly distilled acetic anhydride afforded2 as a sole product. The structure of2 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; 1H-NMR spectrum showed a singlet at δ 12.52 ppm of NH, a doublet at δ 7.162–7.136 ppm and δ 6.84–6.81 ppm of aromatic protons, a singlet at δ 4.7 ppm of benzylic proton, and a singlet at δ 3.68 ppm of three protons for OCH3 group. Ammonolysis of2 by fusion with ammonium acetate and/or formamide afforded 4-(4-methoxyphenyl)-3,7-dimethyl-4,6-dihydro-pyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine-5(1H)-one3. The structure of3 was confirmed by the IR spectrum which exhibited strong absorption bands at 3200, 3281, 1664, and 1610 cm−1 corresponding to νNH, νC=O, and νC=N, respectively and lacked a band corresponding to lactonic carbonyl group and also the presence of acidic hydrogen in the 1H-NMR spectrum at δ 12.38. The electrophilicity of the lactonic carbonyl group of compound2 was studied by nucleophilic reaction with 1,2-diaminobenzene and thiosemicarbazide, as 1,4-dinucleophile, to afford 4-(4-methoxyphenyl)-3,7-dimethyl-4-hydrobenzimidazolo[1,2,c]pyrazolo[4′3′:5,5]pyrano[2,3-d]pyrimidine4 and N-[4-(4-methoxyphenyl)-3,7-dimethyl-5-oxo-1,4-dihydro-pyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-6(5H)-yl]thiourea5, as sole products. The pyrazolopyranopyrimidine derivative4 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 compound4 showed strong absorption bands at 3180 and 1609 cm−1 corresponding to νNH and νC=N. The IR spectrum of compound5 showed well defined absorption bands at 3251, 3158, 3121, 1665, 1619, and 1254 cm−1 corresponding to νNH2, νNH, νC=O, νC=N, and νC=S. Compound2 was allowed to react with primary amines, namely, hydrazine hydrate, 4-methylaniline, 4-methoxyaniline, and/or 4-aminoaniline, to afford the pyrazolopyranopyrimidine derivatives6 and7a–c, respectively (Scheme 2).Scheme 24-(4-Methoxyphenyl)-3,7dimethy-l-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-thione8 was synthesized by sulfurization of3 using Lawson’s reagent or by reaction of thiourea with 5-chloro-4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine9. Compound9 was prepared by chlorination of3 by POCl3 and PCl5 mixture. Hydrazinolysis of9 afforded 5-hydrazino-4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine10.IR spectrum of compound10 showed strong absorption bands at 3215, 3156, 3161, and 1612 cm−1 corresponding to νNH2, νNH, and νC=N. Also the structure was confirmed by 1H-NMR spectrum that showed a singlet at δ 12.01 ppm of NH, multiplet at δ 7.83 ppm of aromatic protons, a singlet at δ 4.8 ppm of benzylic proton, a singlet at δ 3.7 ppm of three protons for OCH3 group, and a singlet at δ 5.354 of NH2.Acetylation of10 by refluxing with acetic anhydride gave the diacetyl derivative11 and not the expected triazolo derivative12. The structure of11 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 δ 2.678 ppm for 2CH3 in 1H-NMR spectrum (Scheme 3).Scheme 3


### 2.2. 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 Table1. Compounds1,4,7a, and10 exhibit a similar antioxidant activity to ascorbic acid, used as standard. Compounds8 and11 exhibited moderate antioxidant activity, while2,3,7b,5, and9 showed lower activity.Table 1
The antioxidant activities of the synthesized compounds.
Compound number
Antioxidant activity
Bleomycin dependent DNA damage
Absorbance
Inhibition %
1
0.111
78.6
0.098
2
0.427
17.9
0.127
3
0.419
19.4
0.135
4
0.062
88.1
0.083
5
0.393
24.4
0.138
7a
0.083
84.0
0.091
7b
0.428
17.7
0.151
8
0.274
47.3
0.089
9
0.446
14.2
0.142
10
0.060
88.5
0.066
11
0.355
31.7
0.112
Ascorbic acid
0.065
89.2
0.076
ABTS
0.520
—
—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–Fe3+ 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 Fe3+ to Fe2+. The synthesized compounds were selected for bleomycin dependent DNA damage testing (Table 1).Results in Table1 showed that compounds1,4,7a,8, and10 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 compounds1,4,7a, and10 which were found to very strong cytotoxic compounds. Compound8 was also considered strongly cytotoxic. Finally, compounds3,5, and11 were moderate in their cytotoxic activity and compounds2,7b, and9 were weakly cytotoxic (Table 2).Table 2
The cytotoxic activity of some compounds against human tumor cells.
Compounds
In vitro cytotoxicity IC50 (µg/ml)•
HePG2
HCT-116
PC3
MCF-7
5-FU
7.9
±
0.12
5.3
±
0.14
8.3
±
0.25
5.4
±
0.21
1
9.4
±
0.27
13.5
±
1.65
16.9
±
1.43
26.8
±
1.52
2
82.1
±
5.34
95.9
±
6.34
>100
>100
3
45.6
±
3.21
51.1
±
3.50
61.1
±
4.96
37.2
±
2.07
4
8.4
±
0.30
9.0
±
0.41
16.0
±
1.30
14.6
±
1.36
5
36.2
±
3.22
52.2
±
3.67
41.3
±
2.65
40.1
±
3.22
7a
8.7
±
0.26
13.61
±
.
48
17.9
±
1.57
19.5
±
1.81
7b
63.5
±
3.98
>100
85.2
±
6.31
85.5
±
5.62
8
16.4
±
1.04
31.4
±
2.64
19.9
±
1.68
26.9
±
1.91
9
52.0
±
2.83
69.2
±
4.05
47.2
±
2.54
58.5
±
3.54
10
8.3
±
0.41
11.2
±
0.86
8.9
±
0.50
9.1
±
0.65
11
22.1
±
1.56
37.8
±
1.93
26.4
±
1.79
30.0
±
2.80
•IC50 (µg/ml): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), and above 100 (noncytotoxic); 5-FU = 5-fluorouracil.


### 2.3. 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.The1H-NMR and 13C-NMR were recorded at a Varian Mercury VX-300 MHz and 75 MHz, respectively, using TMS as internal standard in deuterated chloroform (CDCl3) 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 F254.


### 2.4. Synthesis of 6-Amino-4-(4-methoxyphenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(1)

Method 1.
An equimolar mixture of anisaldehyde (10 mmol, 1.36 mL), ethyl acetoacetate (10 mmol, 1.3 mL), hydrazine hydrate (0.5 mL), and malononitrile (10 mmol, 0.66 g) was stirred for 30 min at room temperature in the presence of triethyl amine and water (5 ml). The precipitate was filtered off and recrystallized from ethanol, yield (81%).Method 2.
A mixture of 3-methyl-5-pyrazolone (10 mmol, 0.98 g) and arylidine (10 mmol, 1.1 g) was refluxed in (30 mL) ethanol and 5 drops of piperidine for 2 h. The precipitate was filtrated off and recrystallized from ethanol to give solid 1, yield (87%).Method 3.
A mixture of 3-methyl-5-pyrazolone (10 mmol, 0.98 g), anisaldehyde (10 mmol, 1.36 mL), and malononitrile (10 mmol, 0.66 g) was refluxed in (30 mL) ethanol in presence of 5 drops of piperidine for 2 h. The precipitate was filtrated off and recrystallized from ethanol to give solid 1, yield (80%)


### 2.5. Synthesis of 4-(4-Methoxyphenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one(2)

A mixture of1 (10 mmol, 2.82 g) and freshly distilled acetic anhydride (20 mL) was heated under reflux for 3 h. The solid that deposited after distilling the excess solvent was collected and recrystallized from ethanol to give(2).m.p. over 300°C. Anal. Calcd. for C17H15N3O4 (325.32): C, 62.76; H, 4.65; N, 12.92. Found: C, 63.07; H, 4.29; N, 13.10. FTIR (KBr) (cm−1): 3157 (NH), 1732 (C=O), 1624 (C=N), 1606 (C=C). 1H-NMR (DMSO-d6): δH (ppm) 12.52 (s, 1H, NH, D2O exchangeable), 7.162–7.136 (d, J=7.8, 2H, Ar-H), 6.84–6.81 (d, J=9, H, Ar-H), 4.7 (s, 1H, benzylic proton), 3.68 (s, 3H, OCH3), 2, 16 (s, 3H, CH3) and 1.831 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6):δ 173.7, 165, 163.4, 161.9, 157.2, 138.6, 134.2, 130.3, 130.3, 114.4, 114.5, 112.9, 99.8, 55.5, 34.4, 15.7, 13.4. MS,m/z (%): 325 (M•+, 6), 281 (27), 241 (13), 218 (100), 107 (28).


### 2.6. Synthesis of 4-(4-Methoxyphenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine-5-(1H)-one(3)

A mixture of2 (10 mmol, 3.25 g) and formamide (10 mL) or excess ammonium acetate was refluxed for 4 h. After cooling white solid was collected, washed with water, dried, and recrystallized from benzene: ethanol mixture (1 : 1) to give(3).m.p. over 300°C Anal. Calcd. for C17H16N4O3 (324.33): C, 62.95; H, 4.97; N, 17.27. Found: C, 62.07; H, 4.92; N, 17.10. FTIR (KBr) (cm−1): 3200, 3281 (NH), 1664 (C=O), 1610 (C=N) 1H-NMR (DMSO-d6): δH (ppm) 12.381 (s, 1H, NH, D2O exchangeable), 12.01 (s, 1H, NH, D2O exchangeable), 7.359–7.334 (d, J=7.5, 2H, Ar-H), 6.790–6.762 (d, J=8.4, 2H, Ar-H), 4.899 (s, 1H, benzylic proton), 3.668 (s, 3H, OCH3), 2.176 (s, 3H, CH3) and 1.791 (s, 3H, CH3). MS,m/z (%): 324 (M•+, 16), 281 (19), 240 (24), 217 (100), 107 (51).


### 2.7. Synthesis of 4-(4-Methoxyphenyl)-3,7-dimethyl-4-hydrobenzoimidazolo[1,2-c]pyrazolo[4′,3′:5,5]pyrano[3,2-d]pyrimidine(4)

A mixture of 2 (10 mmol, 3.25 g) and 2-amino aniline (10 mmol, 1.08 g) in n-butanol (20 mL) was heated under reflux for 4 h. The solid that separated after concentration and cooling was filtered off and recrystallized from petroleum ether at 60–80°C.m.p. 102–104°C Anal. Calcd. For C23H19N5O2 (397): C, 69.51; H, 4.82; N, 17.62. Found: C, 69.26; H, 4.78; N, 17.23. FTIR (KBr) (cm−1): 3180 (NH), 1609 (C=N) 1H-NMR (DMSO-d6): δH (ppm) 11.252 (s, 1H, NH, D2O exchangeable), 8.023–7.215 (m, 8H, Ar-H), 5.099 (s, 1H, benzylic proton), 3.212 (s, 3H, OCH3), 2.801 (s, 3H, CH3) and 2,176 (s, 3H, CH3). MS,m/z (%): 395 (M•+, 7), 317 (9), 290 (12), 267 (10), 181 (25), 80 (100).


### 2.8. Synthesis of N-[4-(4-Methoxyphenyl)-3,7-dimethyl-5-oxo-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine-6(5H)-yl]thiourea(5)

A mixture of2 (10 mmol, 3.25 g) and thiosemicarbazide (10 mmol, 0.91 g) in dimethylformamide (20 mL) was heated under reflux for 6 h. The solid that was separated after concentration and cooling was filtered off, washed with water, and recrystallized from ethanol to give(5).m.p. 265–267°C Anal. Calcd. for C18H18N6O3S (398.44): C, 54.26; H, 4.55; N, 21.09; S, 8.05. Found: C, 53.70; H, 4.19; N, 20.83. FTIR (KBr) (cm−1): 3251, 3158, 3121 (NH2, NH), 1665 (CO), 1619 (C=N) and 1254 (C=S). 1H-NMR (DMSO-d6): δH (ppm) 11.912 (s, 1H, NH, D2O exchangeable), 7.923–7.751 (m, 4H, Ar-H), 8.213 (s, 1H, NH, D2O exchangeable), 6.754 (s, 2H, NH2, D2O exchangeable), 5.013 (s, 1H, benzylic proton), 3.934 (s, 3H, OCH3), 2.178 (s, 3H, CH3) and 1.987 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6):δ182.7, 163.2, 161.9, 160.3, 158.5, 157.1, 139.8, 135.2, 130.3, 130.3, 114.6, 114.6, 113.8, 102.9, 55.9, 35.7, 22.3, 13.3,. MS,m/z (%): 398 (M•+, 23), 323 (20), 291 (18), 107 (100), 75 (21).


### 2.9. Synthesis of 6-Amino-4-(4-methoxyphenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one(6)

To a solution of2 (10 mmol, 3.25 g) in 20 mL of ethanol, hydrazine hydrate (0.5 mL) was added. The reaction mixture was refluxed for 4 h. The separated solid was filtered, dried, and recrystallized from ethanol to give(6).m.p. 268–290°C Anal. Calcd. for C17H17N5O3 (339.35): C, 60.17; H, 5.05; N, 20.64. Found: C, 60.07; H, 4.94; N, 20.53. FTIR (KBr) (cm−1): 3325, 3210, 3154 (NH2, NH), 1669 (CO), 1609 (C=N). 1H-NMR (DMSO-d6): δH (ppm) 12.012 (s, 1H, NH, D2O exchangeable), 7.632–7.115 (m, 4H, Ar-H), 6.845 (s, 2H, NH2, D2O exchangeable), 5.013 (s, 1H, benzylic proton), 3.798 (s, 3H, OCH3), 2.867 (s, 3H, CH3) and 1.872 (s, 3H, CH3). MS,m/z (%): 322 (M•+ -NH3, 23), 282 (19), 240 (10), 80 (100).


### 2.10. General Procedure for the Synthesis of Pyrazolopyranopyrimidine(7a–c)

A solution of2 (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(7a–c).


### 2.11. 4-(4-Methoxyphenyl)-3,7-dimethyl-6-(p-tolyl)-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one(7a)

Solvent: ethanol m.p. 278–280°C. Anal. Calcd. for C24H22N4O3 (414.46): C, 69.55; H, 5.35; N, 13.52. Found: C, 68.67; H, 4.89; N, 12.98. FTIR (KBr) (cm−1): 3191 (NH), 1682 (CO), 1608 (C=N). 1H-NMR (DMSO-d6): δH (ppm) 10.972 (s, 1H, NH, D2O exchangeable), 7.963–7.785 (m, 8H, Ar-H), 4.876 (s, 1H, benzylic proton), 3.912 (s, 3H, OCH3), 2.911 (s, 3H, CH3) and 2.176 (s, 3H, CH3) and 1.987 (s, 3H, CH3). MS,m/z (%): 414 (M•+, 17), 386 (8), 307 (19), 107 (100).


### 2.12. 4,6-Bis(4-methoxyphenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one(7b)

Solvent: ethanol. m.p. over 300°C Anal. C; alcd. for C24H22N4O4 (430.46): C, 66.97; H, 5.15; N, 13.02. Found: C, 66.17; H, 4.98; N, 13.41 FTIR (KBr) (cm−1): 3154 (NH), 1685 (CO), 1611 (C=N). 1H-NMR (DMSO-d6): δH (ppm) 11.241 (s, 1H, NH, D2O exchangeable), 8.054–7.815 (m, 8H, Ar-H), 5.045 (s, 1H, benzylic proton), 3.912 (s, 6H, 2 OCH3), 2.832 (s, 3H, CH3), 2.092 (s, 3H, CH3) and 1.898 (s, 3H, CH3). MS,m/z (%): 430 (M•+, 8), 402 (7), 295 (19), 80 (100).


### 2.13. 6-(4-Aminophenyl)-4-(methoxyphenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one(7c)

Solvent: dioxane. m.p. over 300°C Anal. Calcd. for C23H21N5O3 (415.44): C, 66.49; H, 5.09; N, 16.86. Found: C, 66.16; H, 4.92; N, 16.16. FTIR (KBr) (cm−1): 3312, 3215, 3145 (NH2, NH), 1679 (CO), 1611 (C=N). 1H-NMR (DMSO-d6): δH (ppm) 9.471 (s, 1H, NH, D2O exchangeable), 7.439–7.005 (m, 8H, Ar-H), 6.450 (s, 2H, NH2, D2O exchangeable), 4.713 (s, 1H, benzylic proton), 3.803 (s, 3H, OCH3), 2.367 (s, 3H, CH3) and 1.912 (s, 3H, CH3). MS,m/z (%): 415 (M•+, 10), 398 (9), 372 (20), 77 (100).


### 2.14. Synthesis of 4-(4-Methoxyphenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine-5(1H)-thione(8)

A mixture of9 (10 mmol, 3.4 g) and thiourea (10 mmol, 0.76 g) in ethanol (20 mL) was heated under reflux for 6 h. The separated solid was filtered, dried, and recrystallized from ethanol to give(8).m.p. 218–220°C. Anal. Calcd. for C17H16N4O2S (340): C, 59.98; H, 4.74; N, 16.46. Found: C, 60.03; H, 4.91; N, 16.10. FTIR (KBr) (cm−1): 3158, 3121 (NH), and 1225 (C=S). 1H-NMR (DMSO-d6): δH (ppm) 11.912 (s, 1H, NH, D2O exchangeable), 10.112 (s, 1H, NH, D2O exchangeable), 7.923–7.751 (m, 4H, Ar-H), 4.731 (s, 1H, benzylic proton), 3.876 (s, 3H, OCH3), 2.112 (s, 3H, CH3) and 1.831 (s, 3H, CH3). MS,m/z (%): 340 (M•+, 16), 281 (19), 241 (23), 233 (100), 107 (18).


### 2.15. Synthesis of 5-Chloro-4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine(9)

A mixture of3 (10 mmol, 3.24 g), phosphorus oxychloride (15 mL), and phosphorus pentachloride (150 mmol, 3.13 g) was heated in water bath at 80°C for 8 h. The reaction mixture after cooling was poured onto crushed ice and the solid obtained was filtered off and recrystallized from ethanol to give pale brown solid(9).m.p. 198–200°C. Anal. Calcd. for C17H15ClN4O2 (342): C, 59.57; H, 4.41; N, 10.34. Found: C, 58.97; H, 3.95; N, 10.10. FTIR (KBr) (cm−1): 3123 (NH), 1620 (C=N) 1H-NMR (DMSO-d6): δH (ppm) 12.012 (s, 1H, NH, D2O exchangeable), 7.832–7.615 (m, 4H, Ar-H), 4.832 (s, 1H, benzylic proton), 3.843 (s, 3H, OCH3), 2.546 (s, 3H, CH3) and 1.687 (s, 3H, CH3). MS,m/z (%): 342 (M•+, 10), 344 (25), 235 (12), 237 (30), 214 (28), 201 (100), 107 (30).


### 2.16. Synthesis of 5-Hydrazino-4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine(10)

A mixture of9 (10 mmol, 3.4 g) and hydrazine hydrate (0.5 mL) in (30 mL) ethanol was heated under reflux for 5 h. The solid separated after concentration and cooling was filtered off and recrystallized from ethanol to give(10).m.p. 243–245°C Anal. Calcd. for C17H18N6O2 (338.36): C, 60.34; H, 5.36; N, 24.84. Found: C, 61.07; H, 5.23; N, 24.73. FTIR (KBr) (cm−1): 3215, 3156, 3161 (NH2, NH), 1612 (C=N) and. 1H-NMR (DMSO-d6): δH (ppm) 12.012 (s, 1H, NH, D2O exchangeable), 8.123–7.836 (m, 4H, Ar-H), 6.013 (s, 1H, NH, D2O exchangeable), 5.354 (s, 2H, NH2, D2O exchangeable), 4.803 (s, 1H, benzylic proton), 3.734 (s, 3H, OCH3), 2,576 (s, 3H, CH3) and 2.087 (s, 3H, CH3). MS,m/z (%): 338 (M•+, 12), 307 (21), 244 (11), 201 (100), 107 (18).


### 2.17. Synthesis of 5-(2N,N-diacetylhydrazino)-4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine(11)

A mixture of10 (10 mmol, 3.3 g) was heated under reflux with 30 mL of fresh distilled acetic anhydride for 8 h. The excess acetic anhydride was removed by distillation and the separated product was filtered off and recrystallized from ethanol to give(11).m.p. 278–280°C Anal. Calcd. for C21H22N6O4 (422.44): C, 59.71; H, 5.25; N, 19.81. Found: C, 60.17; H, 4.96; N, 19.23. FTIR (KBr) (cm−1): 3211, 3198 (NH), 1732, 1695 (CO), and 1619 (C=N). 1H-NMR (DMSO-d6): δH (ppm) 12.901 (s, 1H, NH, D2O exchangeable), 7.902–7.714 (m, 4H, Ar-H), 6.213 (s, 1H, NH, D2O exchangeable), 5.630 (s, 1H, benzylic proton), 3.919 (s, 3H, OCH3), 2.013 (s, 3H, CH3), 2.678 (s, 6H, 2CH3) and 1.987 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6): δ 176.6, 174.6, 173.8, 171.8, 163.5, 160.2, 157.2, 138.7, 130.7, 130.0, 130.0, 114.6, 114.6, 113.1, 98.2, 56.0, 39.3, 25.2, 21.4, 21.4, 12.8. MS,m/z (%): 422 (M•+, 13), 322 (17), 307 (100), 201 (23), 107 (28), 101 (16).


## 2.1. Chemistry

Our present investigation aimed to synthesize of 6-amino-4-(4-methoxyphenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile1 [28], which was used as a precursor for the synthesis of previously unreported 4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one2 as new heterocyclic compound containing oxazine and pyranopyrazole moieties. Compound1 has been prepared by four-component one-pot reaction [29] and three-component one-pot reaction [30] (Scheme 1).Scheme 1Treatment of1 by freshly distilled acetic anhydride afforded2 as a sole product. The structure of2 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; 1H-NMR spectrum showed a singlet at δ 12.52 ppm of NH, a doublet at δ 7.162–7.136 ppm and δ 6.84–6.81 ppm of aromatic protons, a singlet at δ 4.7 ppm of benzylic proton, and a singlet at δ 3.68 ppm of three protons for OCH3 group. Ammonolysis of2 by fusion with ammonium acetate and/or formamide afforded 4-(4-methoxyphenyl)-3,7-dimethyl-4,6-dihydro-pyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine-5(1H)-one3. The structure of3 was confirmed by the IR spectrum which exhibited strong absorption bands at 3200, 3281, 1664, and 1610 cm−1 corresponding to νNH, νC=O, and νC=N, respectively and lacked a band corresponding to lactonic carbonyl group and also the presence of acidic hydrogen in the 1H-NMR spectrum at δ 12.38. The electrophilicity of the lactonic carbonyl group of compound2 was studied by nucleophilic reaction with 1,2-diaminobenzene and thiosemicarbazide, as 1,4-dinucleophile, to afford 4-(4-methoxyphenyl)-3,7-dimethyl-4-hydrobenzimidazolo[1,2,c]pyrazolo[4′3′:5,5]pyrano[2,3-d]pyrimidine4 and N-[4-(4-methoxyphenyl)-3,7-dimethyl-5-oxo-1,4-dihydro-pyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-6(5H)-yl]thiourea5, as sole products. The pyrazolopyranopyrimidine derivative4 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 compound4 showed strong absorption bands at 3180 and 1609 cm−1 corresponding to νNH and νC=N. The IR spectrum of compound5 showed well defined absorption bands at 3251, 3158, 3121, 1665, 1619, and 1254 cm−1 corresponding to νNH2, νNH, νC=O, νC=N, and νC=S. Compound2 was allowed to react with primary amines, namely, hydrazine hydrate, 4-methylaniline, 4-methoxyaniline, and/or 4-aminoaniline, to afford the pyrazolopyranopyrimidine derivatives6 and7a–c, respectively (Scheme 2).Scheme 24-(4-Methoxyphenyl)-3,7dimethy-l-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-thione8 was synthesized by sulfurization of3 using Lawson’s reagent or by reaction of thiourea with 5-chloro-4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine9. Compound9 was prepared by chlorination of3 by POCl3 and PCl5 mixture. Hydrazinolysis of9 afforded 5-hydrazino-4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine10.IR spectrum of compound10 showed strong absorption bands at 3215, 3156, 3161, and 1612 cm−1 corresponding to νNH2, νNH, and νC=N. Also the structure was confirmed by 1H-NMR spectrum that showed a singlet at δ 12.01 ppm of NH, multiplet at δ 7.83 ppm of aromatic protons, a singlet at δ 4.8 ppm of benzylic proton, a singlet at δ 3.7 ppm of three protons for OCH3 group, and a singlet at δ 5.354 of NH2.Acetylation of10 by refluxing with acetic anhydride gave the diacetyl derivative11 and not the expected triazolo derivative12. The structure of11 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 δ 2.678 ppm for 2CH3 in 1H-NMR spectrum (Scheme 3).Scheme 3


## 2.2. 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 Table1. Compounds1,4,7a, and10 exhibit a similar antioxidant activity to ascorbic acid, used as standard. Compounds8 and11 exhibited moderate antioxidant activity, while2,3,7b,5, and9 showed lower activity.Table 1
The antioxidant activities of the synthesized compounds.
Compound number
Antioxidant activity
Bleomycin dependent DNA damage
Absorbance
Inhibition %
1
0.111
78.6
0.098
2
0.427
17.9
0.127
3
0.419
19.4
0.135
4
0.062
88.1
0.083
5
0.393
24.4
0.138
7a
0.083
84.0
0.091
7b
0.428
17.7
0.151
8
0.274
47.3
0.089
9
0.446
14.2
0.142
10
0.060
88.5
0.066
11
0.355
31.7
0.112
Ascorbic acid
0.065
89.2
0.076
ABTS
0.520
—
—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–Fe3+ 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 Fe3+ to Fe2+. The synthesized compounds were selected for bleomycin dependent DNA damage testing (Table 1).Results in Table1 showed that compounds1,4,7a,8, and10 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 compounds1,4,7a, and10 which were found to very strong cytotoxic compounds. Compound8 was also considered strongly cytotoxic. Finally, compounds3,5, and11 were moderate in their cytotoxic activity and compounds2,7b, and9 were weakly cytotoxic (Table 2).Table 2
The cytotoxic activity of some compounds against human tumor cells.
Compounds
In vitro cytotoxicity IC50 (µg/ml)•
HePG2
HCT-116
PC3
MCF-7
5-FU
7.9
±
0.12
5.3
±
0.14
8.3
±
0.25
5.4
±
0.21
1
9.4
±
0.27
13.5
±
1.65
16.9
±
1.43
26.8
±
1.52
2
82.1
±
5.34
95.9
±
6.34
>100
>100
3
45.6
±
3.21
51.1
±
3.50
61.1
±
4.96
37.2
±
2.07
4
8.4
±
0.30
9.0
±
0.41
16.0
±
1.30
14.6
±
1.36
5
36.2
±
3.22
52.2
±
3.67
41.3
±
2.65
40.1
±
3.22
7a
8.7
±
0.26
13.61
±
.
48
17.9
±
1.57
19.5
±
1.81
7b
63.5
±
3.98
>100
85.2
±
6.31
85.5
±
5.62
8
16.4
±
1.04
31.4
±
2.64
19.9
±
1.68
26.9
±
1.91
9
52.0
±
2.83
69.2
±
4.05
47.2
±
2.54
58.5
±
3.54
10
8.3
±
0.41
11.2
±
0.86
8.9
±
0.50
9.1
±
0.65
11
22.1
±
1.56
37.8
±
1.93
26.4
±
1.79
30.0
±
2.80
•IC50 (µg/ml): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), and above 100 (noncytotoxic); 5-FU = 5-fluorouracil.


## 2.3. 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.The1H-NMR and 13C-NMR were recorded at a Varian Mercury VX-300 MHz and 75 MHz, respectively, using TMS as internal standard in deuterated chloroform (CDCl3) 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 F254.


## 2.4. Synthesis of 6-Amino-4-(4-methoxyphenyl)-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(1)

Method 1.
An equimolar mixture of anisaldehyde (10 mmol, 1.36 mL), ethyl acetoacetate (10 mmol, 1.3 mL), hydrazine hydrate (0.5 mL), and malononitrile (10 mmol, 0.66 g) was stirred for 30 min at room temperature in the presence of triethyl amine and water (5 ml). The precipitate was filtered off and recrystallized from ethanol, yield (81%).Method 2.
A mixture of 3-methyl-5-pyrazolone (10 mmol, 0.98 g) and arylidine (10 mmol, 1.1 g) was refluxed in (30 mL) ethanol and 5 drops of piperidine for 2 h. The precipitate was filtrated off and recrystallized from ethanol to give solid 1, yield (87%).Method 3.
A mixture of 3-methyl-5-pyrazolone (10 mmol, 0.98 g), anisaldehyde (10 mmol, 1.36 mL), and malononitrile (10 mmol, 0.66 g) was refluxed in (30 mL) ethanol in presence of 5 drops of piperidine for 2 h. The precipitate was filtrated off and recrystallized from ethanol to give solid 1, yield (80%)


## 2.5. Synthesis of 4-(4-Methoxyphenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one(2)

A mixture of1 (10 mmol, 2.82 g) and freshly distilled acetic anhydride (20 mL) was heated under reflux for 3 h. The solid that deposited after distilling the excess solvent was collected and recrystallized from ethanol to give(2).m.p. over 300°C. Anal. Calcd. for C17H15N3O4 (325.32): C, 62.76; H, 4.65; N, 12.92. Found: C, 63.07; H, 4.29; N, 13.10. FTIR (KBr) (cm−1): 3157 (NH), 1732 (C=O), 1624 (C=N), 1606 (C=C). 1H-NMR (DMSO-d6): δH (ppm) 12.52 (s, 1H, NH, D2O exchangeable), 7.162–7.136 (d, J=7.8, 2H, Ar-H), 6.84–6.81 (d, J=9, H, Ar-H), 4.7 (s, 1H, benzylic proton), 3.68 (s, 3H, OCH3), 2, 16 (s, 3H, CH3) and 1.831 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6):δ 173.7, 165, 163.4, 161.9, 157.2, 138.6, 134.2, 130.3, 130.3, 114.4, 114.5, 112.9, 99.8, 55.5, 34.4, 15.7, 13.4. MS,m/z (%): 325 (M•+, 6), 281 (27), 241 (13), 218 (100), 107 (28).


## 2.6. Synthesis of 4-(4-Methoxyphenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine-5-(1H)-one(3)

A mixture of2 (10 mmol, 3.25 g) and formamide (10 mL) or excess ammonium acetate was refluxed for 4 h. After cooling white solid was collected, washed with water, dried, and recrystallized from benzene: ethanol mixture (1 : 1) to give(3).m.p. over 300°C Anal. Calcd. for C17H16N4O3 (324.33): C, 62.95; H, 4.97; N, 17.27. Found: C, 62.07; H, 4.92; N, 17.10. FTIR (KBr) (cm−1): 3200, 3281 (NH), 1664 (C=O), 1610 (C=N) 1H-NMR (DMSO-d6): δH (ppm) 12.381 (s, 1H, NH, D2O exchangeable), 12.01 (s, 1H, NH, D2O exchangeable), 7.359–7.334 (d, J=7.5, 2H, Ar-H), 6.790–6.762 (d, J=8.4, 2H, Ar-H), 4.899 (s, 1H, benzylic proton), 3.668 (s, 3H, OCH3), 2.176 (s, 3H, CH3) and 1.791 (s, 3H, CH3). MS,m/z (%): 324 (M•+, 16), 281 (19), 240 (24), 217 (100), 107 (51).


## 2.7. Synthesis of 4-(4-Methoxyphenyl)-3,7-dimethyl-4-hydrobenzoimidazolo[1,2-c]pyrazolo[4′,3′:5,5]pyrano[3,2-d]pyrimidine(4)

A mixture of 2 (10 mmol, 3.25 g) and 2-amino aniline (10 mmol, 1.08 g) in n-butanol (20 mL) was heated under reflux for 4 h. The solid that separated after concentration and cooling was filtered off and recrystallized from petroleum ether at 60–80°C.m.p. 102–104°C Anal. Calcd. For C23H19N5O2 (397): C, 69.51; H, 4.82; N, 17.62. Found: C, 69.26; H, 4.78; N, 17.23. FTIR (KBr) (cm−1): 3180 (NH), 1609 (C=N) 1H-NMR (DMSO-d6): δH (ppm) 11.252 (s, 1H, NH, D2O exchangeable), 8.023–7.215 (m, 8H, Ar-H), 5.099 (s, 1H, benzylic proton), 3.212 (s, 3H, OCH3), 2.801 (s, 3H, CH3) and 2,176 (s, 3H, CH3). MS,m/z (%): 395 (M•+, 7), 317 (9), 290 (12), 267 (10), 181 (25), 80 (100).


## 2.8. Synthesis of N-[4-(4-Methoxyphenyl)-3,7-dimethyl-5-oxo-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine-6(5H)-yl]thiourea(5)

A mixture of2 (10 mmol, 3.25 g) and thiosemicarbazide (10 mmol, 0.91 g) in dimethylformamide (20 mL) was heated under reflux for 6 h. The solid that was separated after concentration and cooling was filtered off, washed with water, and recrystallized from ethanol to give(5).m.p. 265–267°C Anal. Calcd. for C18H18N6O3S (398.44): C, 54.26; H, 4.55; N, 21.09; S, 8.05. Found: C, 53.70; H, 4.19; N, 20.83. FTIR (KBr) (cm−1): 3251, 3158, 3121 (NH2, NH), 1665 (CO), 1619 (C=N) and 1254 (C=S). 1H-NMR (DMSO-d6): δH (ppm) 11.912 (s, 1H, NH, D2O exchangeable), 7.923–7.751 (m, 4H, Ar-H), 8.213 (s, 1H, NH, D2O exchangeable), 6.754 (s, 2H, NH2, D2O exchangeable), 5.013 (s, 1H, benzylic proton), 3.934 (s, 3H, OCH3), 2.178 (s, 3H, CH3) and 1.987 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6):δ182.7, 163.2, 161.9, 160.3, 158.5, 157.1, 139.8, 135.2, 130.3, 130.3, 114.6, 114.6, 113.8, 102.9, 55.9, 35.7, 22.3, 13.3,. MS,m/z (%): 398 (M•+, 23), 323 (20), 291 (18), 107 (100), 75 (21).


## 2.9. Synthesis of 6-Amino-4-(4-methoxyphenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one(6)

To a solution of2 (10 mmol, 3.25 g) in 20 mL of ethanol, hydrazine hydrate (0.5 mL) was added. The reaction mixture was refluxed for 4 h. The separated solid was filtered, dried, and recrystallized from ethanol to give(6).m.p. 268–290°C Anal. Calcd. for C17H17N5O3 (339.35): C, 60.17; H, 5.05; N, 20.64. Found: C, 60.07; H, 4.94; N, 20.53. FTIR (KBr) (cm−1): 3325, 3210, 3154 (NH2, NH), 1669 (CO), 1609 (C=N). 1H-NMR (DMSO-d6): δH (ppm) 12.012 (s, 1H, NH, D2O exchangeable), 7.632–7.115 (m, 4H, Ar-H), 6.845 (s, 2H, NH2, D2O exchangeable), 5.013 (s, 1H, benzylic proton), 3.798 (s, 3H, OCH3), 2.867 (s, 3H, CH3) and 1.872 (s, 3H, CH3). MS,m/z (%): 322 (M•+ -NH3, 23), 282 (19), 240 (10), 80 (100).


## 2.10. General Procedure for the Synthesis of Pyrazolopyranopyrimidine(7a–c)

A solution of2 (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(7a–c).


## 2.11. 4-(4-Methoxyphenyl)-3,7-dimethyl-6-(p-tolyl)-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one(7a)

Solvent: ethanol m.p. 278–280°C. Anal. Calcd. for C24H22N4O3 (414.46): C, 69.55; H, 5.35; N, 13.52. Found: C, 68.67; H, 4.89; N, 12.98. FTIR (KBr) (cm−1): 3191 (NH), 1682 (CO), 1608 (C=N). 1H-NMR (DMSO-d6): δH (ppm) 10.972 (s, 1H, NH, D2O exchangeable), 7.963–7.785 (m, 8H, Ar-H), 4.876 (s, 1H, benzylic proton), 3.912 (s, 3H, OCH3), 2.911 (s, 3H, CH3) and 2.176 (s, 3H, CH3) and 1.987 (s, 3H, CH3). MS,m/z (%): 414 (M•+, 17), 386 (8), 307 (19), 107 (100).


## 2.12. 4,6-Bis(4-methoxyphenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one(7b)

Solvent: ethanol. m.p. over 300°C Anal. C; alcd. for C24H22N4O4 (430.46): C, 66.97; H, 5.15; N, 13.02. Found: C, 66.17; H, 4.98; N, 13.41 FTIR (KBr) (cm−1): 3154 (NH), 1685 (CO), 1611 (C=N). 1H-NMR (DMSO-d6): δH (ppm) 11.241 (s, 1H, NH, D2O exchangeable), 8.054–7.815 (m, 8H, Ar-H), 5.045 (s, 1H, benzylic proton), 3.912 (s, 6H, 2 OCH3), 2.832 (s, 3H, CH3), 2.092 (s, 3H, CH3) and 1.898 (s, 3H, CH3). MS,m/z (%): 430 (M•+, 8), 402 (7), 295 (19), 80 (100).


## 2.13. 6-(4-Aminophenyl)-4-(methoxyphenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one(7c)

Solvent: dioxane. m.p. over 300°C Anal. Calcd. for C23H21N5O3 (415.44): C, 66.49; H, 5.09; N, 16.86. Found: C, 66.16; H, 4.92; N, 16.16. FTIR (KBr) (cm−1): 3312, 3215, 3145 (NH2, NH), 1679 (CO), 1611 (C=N). 1H-NMR (DMSO-d6): δH (ppm) 9.471 (s, 1H, NH, D2O exchangeable), 7.439–7.005 (m, 8H, Ar-H), 6.450 (s, 2H, NH2, D2O exchangeable), 4.713 (s, 1H, benzylic proton), 3.803 (s, 3H, OCH3), 2.367 (s, 3H, CH3) and 1.912 (s, 3H, CH3). MS,m/z (%): 415 (M•+, 10), 398 (9), 372 (20), 77 (100).


## 2.14. Synthesis of 4-(4-Methoxyphenyl)-3,7-dimethyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine-5(1H)-thione(8)

A mixture of9 (10 mmol, 3.4 g) and thiourea (10 mmol, 0.76 g) in ethanol (20 mL) was heated under reflux for 6 h. The separated solid was filtered, dried, and recrystallized from ethanol to give(8).m.p. 218–220°C. Anal. Calcd. for C17H16N4O2S (340): C, 59.98; H, 4.74; N, 16.46. Found: C, 60.03; H, 4.91; N, 16.10. FTIR (KBr) (cm−1): 3158, 3121 (NH), and 1225 (C=S). 1H-NMR (DMSO-d6): δH (ppm) 11.912 (s, 1H, NH, D2O exchangeable), 10.112 (s, 1H, NH, D2O exchangeable), 7.923–7.751 (m, 4H, Ar-H), 4.731 (s, 1H, benzylic proton), 3.876 (s, 3H, OCH3), 2.112 (s, 3H, CH3) and 1.831 (s, 3H, CH3). MS,m/z (%): 340 (M•+, 16), 281 (19), 241 (23), 233 (100), 107 (18).


## 2.15. Synthesis of 5-Chloro-4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine(9)

A mixture of3 (10 mmol, 3.24 g), phosphorus oxychloride (15 mL), and phosphorus pentachloride (150 mmol, 3.13 g) was heated in water bath at 80°C for 8 h. The reaction mixture after cooling was poured onto crushed ice and the solid obtained was filtered off and recrystallized from ethanol to give pale brown solid(9).m.p. 198–200°C. Anal. Calcd. for C17H15ClN4O2 (342): C, 59.57; H, 4.41; N, 10.34. Found: C, 58.97; H, 3.95; N, 10.10. FTIR (KBr) (cm−1): 3123 (NH), 1620 (C=N) 1H-NMR (DMSO-d6): δH (ppm) 12.012 (s, 1H, NH, D2O exchangeable), 7.832–7.615 (m, 4H, Ar-H), 4.832 (s, 1H, benzylic proton), 3.843 (s, 3H, OCH3), 2.546 (s, 3H, CH3) and 1.687 (s, 3H, CH3). MS,m/z (%): 342 (M•+, 10), 344 (25), 235 (12), 237 (30), 214 (28), 201 (100), 107 (30).


## 2.16. Synthesis of 5-Hydrazino-4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine(10)

A mixture of9 (10 mmol, 3.4 g) and hydrazine hydrate (0.5 mL) in (30 mL) ethanol was heated under reflux for 5 h. The solid separated after concentration and cooling was filtered off and recrystallized from ethanol to give(10).m.p. 243–245°C Anal. Calcd. for C17H18N6O2 (338.36): C, 60.34; H, 5.36; N, 24.84. Found: C, 61.07; H, 5.23; N, 24.73. FTIR (KBr) (cm−1): 3215, 3156, 3161 (NH2, NH), 1612 (C=N) and. 1H-NMR (DMSO-d6): δH (ppm) 12.012 (s, 1H, NH, D2O exchangeable), 8.123–7.836 (m, 4H, Ar-H), 6.013 (s, 1H, NH, D2O exchangeable), 5.354 (s, 2H, NH2, D2O exchangeable), 4.803 (s, 1H, benzylic proton), 3.734 (s, 3H, OCH3), 2,576 (s, 3H, CH3) and 2.087 (s, 3H, CH3). MS,m/z (%): 338 (M•+, 12), 307 (21), 244 (11), 201 (100), 107 (18).


## 2.17. Synthesis of 5-(2N,N-diacetylhydrazino)-4-(4-methoxyphenyl)-3,7-dimethyl-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine(11)

A mixture of10 (10 mmol, 3.3 g) was heated under reflux with 30 mL of fresh distilled acetic anhydride for 8 h. The excess acetic anhydride was removed by distillation and the separated product was filtered off and recrystallized from ethanol to give(11).m.p. 278–280°C Anal. Calcd. for C21H22N6O4 (422.44): C, 59.71; H, 5.25; N, 19.81. Found: C, 60.17; H, 4.96; N, 19.23. FTIR (KBr) (cm−1): 3211, 3198 (NH), 1732, 1695 (CO), and 1619 (C=N). 1H-NMR (DMSO-d6): δH (ppm) 12.901 (s, 1H, NH, D2O exchangeable), 7.902–7.714 (m, 4H, Ar-H), 6.213 (s, 1H, NH, D2O exchangeable), 5.630 (s, 1H, benzylic proton), 3.919 (s, 3H, OCH3), 2.013 (s, 3H, CH3), 2.678 (s, 6H, 2CH3) and 1.987 (s, 3H, CH3). 13C-NMR (75 MHz, DMSO-d6): δ 176.6, 174.6, 173.8, 171.8, 163.5, 160.2, 157.2, 138.7, 130.7, 130.0, 130.0, 114.6, 114.6, 113.1, 98.2, 56.0, 39.3, 25.2, 21.4, 21.4, 12.8. MS,m/z (%): 422 (M•+, 13), 322 (17), 307 (100), 201 (23), 107 (28), 101 (16).



---

*Source: 5373049-2017-07-17.xml*