The hitherto unknown 6-amino-4-(2-chloro-5-nitrophenyl)-3-methyl-1,4-dihydropyrano[2,3-c] pyrazole-5-carbonitrile 1a was synthesized. Both 1a and its 2,4 dichlorophenyl derivative 1b were utilized as building blocks for the preparation of novel class of pyrazolopyrano-[oxazines 2a–d and pyrimidinones 3a–d]. Synthesis of these compounds was achieved by two alternative acylation steps followed by ammonolysis. The structures of the synthesized compounds were elucidated by spectral data and elemental analysis. Screening and evaluation of these products as antimicrobial agents showed that the derivatives 1b, 2s, 3b, and 3d possess a potent activity.
1. Introduction
2-Amino-3-cyano-4-(H)pyran derivatives [1, 2] represent a group of heterocycles of special interest due to their biological activities as antimicrobial, antioxidant, antifungal, and antimycobacterial [3–5]. Meanwhile, they have been utilized as building block for the synthesis of important heterocycles including pyrazopyranopyrimidines [6], chromenooxazine [7], and pyrrolopyranopyrazole [8]. Heterocycles containing the oxazine nucleus were found to possess a wide range of valuable biological properties like analgesic, anti-inflammatory antileukemic, antimalarial [9–11], antipyretic, anticonvulsant, and antimicrobial activities [12–16]. Benzo-1,3-oxazines are also known to be biologically active, demonstrating antirheumatic, antianginal, antihypertensive effects, cytotoxic [17, 18], and antiosteoclastic bone resorption activities [19]. Efavirenz, a trifluoromethyl-1,3-oxazine-2-one, is a nonnucleoside reverse transcriptase inhibitor which displays significant activity against HIV-1 mutant strains [20]. 1,3-Oxazine derivatives are also known to function as progesterone receptor agonists [21]. Naphthoxazines are found to possess psychostimulating and antidepressant activity and are used in the treatment of Parkinson’s disease [22, 23]. Only few reports are available regarding the antimicrobial activity of pyrazolopyranooxazinones [24]. Based on these reports, in this paper we devoted our efforts to construct new pyrazolopyranooxazinones and pyrimidinone heterocycles, as well as screening and evaluation of their antimicrobial activity [25, 26].
2. Experimental
All melting points were determined on an electrothermal apparatus and are uncorrected. The infrared spectra were recorded in potassium bromide disks on Pye Unicam SP-3-300 and Shimdazu FTIR 8101 PC Infrared spectrophotometers. The 1H-NMR was recorded on a Varian Mercury VX-300 NMR spectrometer. 1H-NMR spectra were run at 300 MHz and on a Varian Gemini 200 MHz, Bruker AC-200 MHz using TMS as internal standard in deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6). Chemical shifts are quoted in δ and were related to that of the solvents. The mass spectra were recorded on a Shimadzu GC-MS QP1000 EX 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 cheeked by TLC.
2.1. Chemistry2.1.1. General Procedure for Synthesis of Compounds 1a and 1b
A mixture of 2-chloro-5-nitrobenzaldehyde or 2,4-dichlorobenzaldehyde (5 mmol), malononitrile (5 mmol), hydrazine monohydrate (5 mmol), and ethyl acetoacetate (5 mmol) in n-butanol (15 ml) containing few drops of piperidine was heated under reflux for 5 h. The separated solid was filtered off, dried, and crystallized from proper solvent to give compounds 1a and 1b, respectively.
2.1.2. General Procedure for Synthesis of Compounds 2a and 2b
A solution 1a or 1b (5 mmol) in freshly distilled acetic anhydride (20 ml) was refluxed on a hot plate for 24 h, and excess of acetic anhydride was removed using rotary evaporator. The solid remains after evaporation were crystallized from the proper solvent to give compounds 2a and 2b, respectively.
4-(2-Chloro-5-nitrophenyl)-3,7-dimethyl-1,4-dihydro-5H-pyrazolo[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2a). Pale brown crystals, m.p. > 300°C (DMF), yield 65%. Anal. Calcd. for C16H11N4O5Cl (374.74): C, 51.28; H, 2.96; Cl, 9.46; N, 14.95; O, 21.35. Found: C, 51.26; H, 2.94; Cl, 9.47; N, 14.95; O, 21.33. IR (KBr, υ cm−1): 3170 (NH), 1735 (C=O), 1626 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.58 (s, 1H, NH, pyrazole, exch. with D2O), 7.66–8.09 (m, 3H, Har), 5.58 (s, 1H, benzylic), 3.72 (s, 3H, CH3), 2.21 (s, 3H, CH3, pyrazole). MS m/z (%): 374 (M∙+; 9.87), 375 (4.28), 322 (100), 338 (63.35), 217 (59.76), 176 (29.75).
2.1.3. General Procedure for Synthesis of Compounds 2c and 2d
A solution of 1a or 1b (5 mmol) and benzoyl chloride (20 ml) was refluxed on a hot plate for 24 h, and excess of benzoyl chloride was removed using rotary evaporator. The solid remains after evaporation were crystallized to give compounds 2c and 2d, respectively.
1-Benzoyl-4-(2-chloro-5-nitrophenyl)-3,methy-7,phenyll-4-hydro-5H-pyrazolo-[4′,3′:5,6]pyrano[2,3-d][1,3]oxazin-5-one (2c). Pale brown crystals, m.p. > 300°C (toluene), yield 60%. Anal. Calcd. for C28H17N4O6-Cl (540.91): C, 62.17; H, 3.17; Cl, 6.55; N, 10.36; O, 17.75. Found: C, 62.16; H, 3.15; Cl, 6.57; N, 10.36; O, 17.76. FTIR (KBr, υ cm−1): 1758 (C=O), 1715 (C=O), 1613 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 8.33–7.17 (m, 13H, Har), 6.15 (s, 1H, benzylic), 1.23 (s, 3H, CH3). MS m/z (%): 540 (M∙+;1.53), 445 (29.38), 327 (22.16), 230 (44.69), 175 (100).
2.1.4. General Procedure for Synthesis of Compounds 3c and 3d
A mixture of 2a and/or 2b (5 mmol) and ammonium acetate (15 mmol) was refluxed on a hot plate for 20 h, and the reaction mixture was cooled and then poured into cold water. The separated solid was filtered off, dried, and crystallized from the proper solvent to give products 3a and 3d, respectively.
2.1.5. General Procedure for Synthesis of Compounds 3c and 3d
A mixture of 2c and/or 2d (5 mmol) and ammonium acetate (30 mmol) was refluxed on a hot plate for 17 h, the reaction mixture was cooled and then poured into cold water, and the separated solid was filtered off, dried, and crystallized from proper solvent to give compounds 3c and 3d, respectively.
4-(2-Chloro-5-nitrophenyl)-3-methyl-7-phenyl-4,6-dihydropyrazolo[4′,3′:5,6]-pyrano[2,3-d]pyrimidin-5(1H)-one (3c). Pale yellow crystals, m.p. > 300°C (ethanol), yield 63%. Anal. Calcd. for C21H14N5O4-Cl (435.82): C, 57.87; H, 3.24; Cl, 8.13; N, 16.07; O, 14.68. Found: C, 57.86; H, 3.23; Cl, 8.11; N, 16.07; O, 14.67. FTIR (KBr, υ cm−1): 3311 (NH), 1662 (C=O), 1605 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.75 (s, 1H, NH, pyrazole, exch. with D2O), 12.24 (s, 1H, NH, exch. with D2O), 8.11–7.51 (m, 8H, Har), 5.31 (s, 1H, benzylic), 3.28(s, 3H, CH3). MS m/z (%): 436 (M∙+; 9.01), 400.03 (36.80), 297 (73.08), 279 (100).
4-(2,4-Dichlorophenyl)-3-methyl-7-phenyl-4,6-dihydropyrazolo[4′,3′:5,6]-pyrano[2,3-d]pyrimidin-5(1H)-one (3d). Pale brown crystals, m.p. > 300°C (ethanol), yield 60%. Anal. Calcd. for C21H14N4O2Cl2 (425.27): C, 59.31; H, 3.32; Cl, 16.67; N, 13.17; O, 7.52. Found: C, 59.30; H, 3.30; Cl, 16.65; N, 13.15; O, 7.51. FTIR (KBr, υ cm−1): 3392 (NH), 1673 (C=O), 1559 (C=N). 1H-NMR (DMSO- d6) δ (ppm): 12.57 (s, 1H, NH, pyrazole, exch. with D2O), 12.15 (s, 1H, NH, exch. with D2O), 8.21–7.22 (m, 8H, Har), 5.42 (s, 1H, benzylic), 3.26 (s, 3H, CH3). MS m/z (%): 425 (M∙+; 22.7), 424.05 (100), 426 (63.9).
2.2. Antimicrobial Assay by Agar Cup Plate Method
The sample was prepared by dissolving 0.005 g in 2 ml of DMSO and 100 µl (containing 250 µg) was used in this test. The antimicrobial activity of different samples was investigated by the agar cup plate method. Four different test microbes, namely, Staphylococcus aureus (G+ve), Pseudomonas aeruginosa (G−ve), Candida albicans (yeast), and Aspergillus niger (fungus), were used. Nutrient agar plates were heavily seeded uniformly with 1 ml of 105–106 cells/ml in case of bacteria and yeast. A potato dextrose agar plate seeded by the fungus was used to evaluate the antifungal activities. Then a hole was made in media by gel cutter (cork borer number 4) in sterile condition. Then one drop of melted agar was poured into hole and allowed to solidify to make a base layer. After that specific amount of culture filtrate (0.1 ml) was poured into the hole. Then plates were kept at low temperature (4°C) for 2–4 hours to allow maximum diffusion. The plates were then incubated at 37°C for 24 hours for bacteria and at 30°C for 48 hours in upright position to allow maximum growth of the organisms. The antimicrobial activity of the test agent was determined by measuring the diameter of zone of inhibition expressed in millimeter. The experiment was carried out more than once and mean of reading was recorded [27, 28].
3. Results and Discussion3.1. Chemistry
In order to study their utility as precursors to annulated heterocycles, 6-amino-4-aryl-3-methyl pyranopyrazole-5-carbonitrile derivatives 1a,b were synthesized employing the previously reported [29] multicomponent reaction (Scheme 2).
Examination of IR spectrum of 1a showed absorption frequencies at υ 3393, 3305, and 3141 cm−1 due to NH and NH2 groups, respectively, in addition to a strong absorption band which appeared at 2192 cm−1 referring to the presence of the cyano (C≡N) group. A compelling evidence for this observation was provided by 13C-NMR spectrum that showed a singlet at δ 95.58 ppm. In addition, 1H-NMR spectrum of the assigned compound displayed signals at δ 12.21 ppm and 7.10 ppm due to absorption of the former NH and NH2 group protons, respectively (which disappeared upon deuteration). Further, the mass spectrum showed the EI-fragment at m/z=331 due to the molecular ion peak. Meanwhile, the structure of the previously reported derivative 1b was confirmed by identity of melting point and IR spectrum data with the literature [29].
With the aim of constructing new annulated heterocycles containing pyranooxazine moiety, these prepared compounds were subjected to reaction with acetic anhydride at the reflux temperature. Successfully, this reaction went readily to afford 4-(2-chloro-5-nitrophenyl)- and 4-(2,4-dichlorophenyl)-3,7-dimethyl-1,4-dihydro-5H pyrazolo [4′,3′:5,6] pyrano [2,3-d][1,3]oxazin-5-one 2a and 2b, respectively (Scheme 3). The suggested mechanism for the formation of the latter compounds could be visualized as shown in Scheme 4.
The IR spectra of 2a and 2b displayed the band characteristic to the 6-membered oxazinone carbonyl group at υ 1738–1735 cm−1, however, they lack the bands corresponding to both the amino and nitrile functionalities. The 1H-NMR spectra showed the disappearance of the former group protons signal. This means that these groups have been involved in acylation and ring closure processes (Scheme 4). Further, the mass spectra showed the EI-fragment at m/z=374 and 364 due to the molecular ion peaks of both 2a and 2b, respectively.
On the other hand, in treatment of 1a and 1b with benzoyl chloride as a coreactant and a cosolvent, the products 1-benzoylpyrazolopyranooxazinones 2c and 2d were obtained. The 1H-NMR spectra showed the absence of a signal characteristic to NH proton indicating that the reaction might involve benzoylation of the latter group. This has been confirmed from the molecular ion peak shown at m/z=540 and 530 of 2c and 2d, respectively (cf. Section 2).
Ammonolysis of 2a and 2b was carried out either by fusion with amm. acetate or by boiling with formamide. Both reactions yielded the corresponding 3,7-dimethyl-pyrazolopyranopyrimidinones 3a and 3b, respectively. However, the 3-methyl-7-phenyl derivatives 3c and 3d were produced via ammonolysis using ammonium acetate or formamide of the lactonic carbonyl and the N-benzoyl functionalities of derivatives 2c and 2d, respectively (cf. Scheme 5). The IR spectra of the assigned products 3a–d showed the disappearance of the lactone carbonyl absorption and instead the lactam carbonyl frequency was exhibited at the range of υ 1662–1646 cm−1, in addition to the band displayed at υ 3420–3311 cm−1 due to the cyclic lactam NH functionality. The 1H-NMR spectra of the latter NH group proton appeared at δ 12.2 ppm (cf. Section 2). The suggested mechanism for the formation of 3c, d could be visualized as shown in Scheme 5.
3.2. Antimicrobial Study
Antibiotic resistance is a growing problem; some of this is due to the overuse of antibiotics in human, but some of it is probably due to the use of antibiotics as growth promoters in food of animals, so there is a growing demand for new antibiotics. The synthesized new pyrazolopyranopyrimidines and pyrazolopyranooxazinones were evaluated for their in vitro antimicrobial efficacy against four strains, namely, Staphylococcus aureus (G+ve), Pseudomonas aeruginosa (G−ve), Candida albicans (yeast), and Aspergillus niger (fungus). Neomycin was used as standard drug. Based on the results of zone of inhibition, data in Table 1 revealed that compounds 1b, 3b, 2d, and 3d exhibit strong activities and compounds 2a, 2b, and 1a exhibit moderate activities, whereas 3a, 2c, and 2b exhibit week antimicrobial activities compared with neomycin as standard drug.
The antimicrobial activities of the synthesized compounds as inhibition zone in mm diameter per mg sample.
Sample code number
Clear zone (ϕmm)
Staphylococcus aureus
Pseudomonas aeruginosa
Candida albicans
Aspergillus niger
Control: DMSO
0.0
0.0
0.0
0.0
Neomycin
22
28
30
0.0
3d
17
16
17
15
2d
18
20
18
15
2c
14
15
0
12
2a
16
12
0
0
1a
16
13
0
0
3a
13
14
17
0
3c
16
13
14
0
3b
18
17
16
10
1b
25
25
32
15
2b
13
14
13
0
Slandered drug (neomycin 100 micrograms per 100 microliters).
Competing Interests
The authors declare that they have no competing interests.
TabassumS.GovindarajuS.KhanR.-U.PashaM. A.Ultrasound mediated, iodine catalyzed green synthesis of novel 2-amino-3-cyano-4H-pyran derivatives2015241710.1016/j.ultsonch.2014.12.0062-s2.0-84922445199MansoorS. S.LogaiyaK.AswinK.SudhanP. N.An appropriate one-pot synthesis of 3,4-dihydropyrano[c]chromenes and 6-amino-5-cyano-4-aryl-2-methyl-4H-pyrans with thiourea dioxide as an efficient, reusable organic catalyst in aqueous medium20159221322610.1016/j.jtusci.2014.09.008SaundaneA. R.VijaykumarK.VaijinathA. V.Synthesis of novel 2-amino-4-(5′-substituted 2′-phenyl-1H- indol-3′-yl)-6-aryl-4H-pyran-3-carbonitrile derivatives as antimicrobial and antioxidant agents20132371978198410.1016/j.bmcl.2013.02.0362-s2.0-84875219741NakibT.BezjakV.RashidS.FullamB.MeeganM.The synthesis and antifungal activity of 2-amino-4-aryl-4H,5H-[1]benzothiopyrano[4,3-b]pyran-3-carbonitriles and 2-alkoxy-4-aryl-5H-[1]benzothiopyrano[4,3-b]pyridine-3-carbonitriles199126222123010.1016/0223-5234(91)90033-J2-s2.0-0025821353KumarR. R.PerumalS.SenthilkumarP.YogeeswariP.SriramD.An atom efficient, solvent-free, green synthesis and antimycobacterial evaluation of 2-amino-6-methyl-4-aryl-8-[(E)-arylmethylidene]-5,6,7,8-tetrahydro-4H-pyrano[3,2-c]pyridine-3-carbonitriles200717236459646210.1016/j.bmcl.2007.09.0952-s2.0-35648942163HeraviM. M.MousavizadehF.GhobadiN.TajbakhshM.A green and convenient protocol for the synthesis of novel pyrazolopyranopyrimidines via a one-pot, four-component reaction in water20145561226122810.1016/j.tetlet.2014.01.0042-s2.0-84894973233MadkourH. M. F.AfifyA. A. E.ElsayedG. A.SalemM. S.Use of enaminonitrile moiety in heterocyclic synthesis2008402147159ElziatyA. K.MostafaO. E. A.El-BordanyE. A.NabilM.MadkourH. M. F.Access to new pyranopyrazoles and related heterocycles201451727735TakimotoC. H.CalvoE.Principles of oncologic pharmacotherapy2008London, UKUBM Medica4258KalirajanR.SivakumarS. U.JubieS.GowrammaB.SureshB.Synthesis and biological evaluation of some heterocyclic derivatives of Chalcones20091127342-s2.0-77953275172VennerstromJ. L.MaklerM. T.AngerhoferC. K.WilliamsJ. A.Antimalarial dyes revisited: xanthenes, azines, oxazines, and thiazines19953912671267710.1128/aac.39.12.2671SinghC.ParwanaH. K.SinghG.Synthesis of 3,6-diaryl-2H, 3H, 4H, 5H, 6H-[1,3]-oxazine-2-thiones as potential anticonvulsants19955751982022-s2.0-0028845075LatifN.MishrikyN.AssadF. M.Carbonyl and thiocarbonyl compounds. XIX. Intramolecular cyclization of (2-nitroethenyl)aryl N-arylcarbamates: synthesis of newer series of 3,4-dihydro-2H-1,3-oxazin-2-ones and their antimicrobial activities19823551037104310.1071/ch98210372-s2.0-0019997677CassadyJ. M.ChanK. K.FlossH. G.LeistnerE.Recent developments in the maytansinoid antitumor agents200452112610.1248/cpb.52.12-s2.0-3042748791TurgutZ.PelitE.KöycüA.Synthesis of new 1,3-disubstituted-2,3-dihydro-1H-naphth-[1,2e][1,3] oxazines200712334535210.3390/120303452-s2.0-34147099443MohamedS. F.YoussefM. M.AmrA. E.-G. E.KotbE. R.Antimicrobial activities of some synthesized pyridines, oxazines and thiazoles from 3-aryl-1-(2-naphthyl)prop-2-en-1-ones200876227930310.3797/scipharm.0804-092-s2.0-46249133609VermaV.SinghK.KumarD.KlapötkeT. M.StierstorferJ.NarasimhanB.QaziA. K.HamidA.JaglanS.Synthesis, antimicrobial and cytotoxicity study of 1,3-disubstituted-1H- naphtho[1,2-e][1,3]oxazines20125619520210.1016/j.ejmech.2012.08.0182-s2.0-84865998116BenameurL.BouazizZ.NeboisP.BartoliM.-H.BoitardM.FillionH.Synthesis of furonaphth[1,3]oxazine and furo[1,3]oxazinoquinoline derivatives as precursors for an o-quinonemethide structure and potential antitumor agents199644360560810.1248/cpb.44.6052-s2.0-0029883149TabuchiY.AndoY.KanemuraH.KawasakiI.OhishiT.KoidaM.FukuyamaR.NakamutaH.OhtaS.NishideK.OhishiY.Preparation of novel (Z)-4-ylidenebenzo[b]furo[3,2-d][1,3]oxazines and their biological activity200917113959396710.1016/j.bmc.2009.04.0172-s2.0-65549143033PierceM. E.ParsonsR. L.Jr.RadescaL. A.LoY. S.SilvermanS.MooreJ. R.IslamQ.ChoudhuryA.FortunakJ. M. D.NguyenD.LuoC.MorganS. J.DavisW. P.ConfaloneP. N.ChenC.-Y.TillyerR. D.FreyL.TanL.XuF.ZhaoD.ThompsonA. S.CorleyE. G.GrabowskiE. J. J.ReamerR.ReiderP. J.Practical asymmetric synthesis of Efavirenz (DMP 266), an HIV-1 reverse transcriptase inhibitor199863238536854310.1021/jo981170l2-s2.0-15644370082ZhangP.TerefenkoE. A.FensomeA.WrobelJ.WinnekerR.ZhangZ.Novel 6-aryl-1, 4-dihydrobenzo[d][and oxazine-2thiones as potent, selective, and orally active nonsteroidal progesterone receptor agonists20031371313131610.1016/S0960-894X(03)00128-8M JMillan.Di CaraB.HillM.JacksonM.JoyceJ. N.BrotchieJ.Mc GuireS.Crossman LA.JennerP.A.Gobert, J.L.Peglion and M.Brocco, Novel naphtoxazine agonist at Dopamine D3/D2 receptors: I. cellular, electrophysiological, and neurochemical profile in comparison with Ropinirole2004309921935JoyceJ. N.PresgravesS.RenishL.BorwegeS.OsredkarT.HagnerD.ReplogleM.PazSoldanM.MillanM. J.Neuroprotective effects of the novel D3/D2 receptor agonist and antiparkinson agent, S32504, in vitro against 1-methyl-4- phenylpyridinium (MPP+) and in vivo against 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP): a comparison to ropinirole2003184139340710.1016/s0014-4886(03)00353-42-s2.0-0344616886SunilD.Upadhya SadhanaH.SavithaRamaM.Synthesis, characterization and QSAR studies of some new 1, 3-oxazines as potent antimicrobial agents2013221519Abou-ElmagdW. S. I.El-ZiatyA. K.AbdalhaA. A.Ring transformation and antimicrobial activity of indolyl-substituted 2(3H)-furanones201521317918510.1515/hc-2015-00082-s2.0-84935885402El-SayedN. S.ShiraziA. N.El-MeligyM. G.El-ZiatyA. K.RowleyD.SunJ.NagibZ. A.ParangK.Synthesis of 4-aryl-6-indolylpyridine-3-carbonitriles and evaluation of their antiproliferative activity20145561154115810.1016/j.tetlet.2013.12.0812-s2.0-8489502342119764thLondon, UKELBSAbdel-AzizM. S.ShaheenM. S.El-NekeetyA. A.Abdel-WahhabM. A.Antioxidant and antibacterial activity of silver nanoparticles biosynthesized using Chenopodium murale leaf extract201418435636310.1016/j.jscs.2013.09.0112-s2.0-84905051932KiyaniH.SamimiH. A.GhorbaniF.EsmaieliS.One-pot, four-component synthesis of pyrano[2,3-c]pyrazoles catalyzed by sodium benzoate in aqueous medium20132419720610.5267/j.ccl.2013.07.002