King abdul aziz uneversity, colleg of Scinece, chemistry departmaent, jeddah, Saudi Arabia 2

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1 7 Modern Organic Chemistry Research, Vol., o. 4, ovember 07 ynthesis of ome ew Fluorinated Fused eteropolycyclic itrogen ystems Containing pyrazolo[3,4-d]pyrimidines Moiety and Their Effects on Cellobiase Activity Produced by Aspergillus nidulans Fungi Dina A. Bakhotmah *, Reda M. Abdel-Rahman, Montha fakhrji, Abrar kamal King abdul aziz uneversity, colleg of cinece, chemistry departmaent, jeddah, audi abia King abdul aziz uneversity hospital, Jeddah, audi abia * Abstract. ew Fluorinated fused heteropolycyclic nitrogen systems containing pyrazolo[3,4-d] pyrimidines moieties have been synthesized from the ring closure reaction of the corresponding hydrazine derivatives with bifunctional compounds. tructure of the new fluorinated systems obtained has been established from elemental and spectral analysis. The effect of these compounds was evaluated on the activity of cellobiase produced by Aspergillus nidulans fungi. Keywords: Pyrimidines, fluoropyrazol, cellobiase activity, hydrazine derivatives. Introduction Recently, ydrazine group bearing,,4-triazines are used to synthesize an important heterocyclic nitrogen system with various functional group [], in addition both the pyrazoles [] and pyrimidines [3] exhibit medicinal, pharmacological and biological activities. The introduction of fluorine C-F and CF 3 group to heterocyclic systems often improves and enhances their properties. Thus, this work tends to synthesize some more new fluorinated fused heteropolycyclic nitrogen systems containing pyrazolo[3,4- d]pyrimidines moieties starting from hydrazine-pyrazolopyrimidines, in view of their effects on the cellobiase activity produced by Aspergillus nidulans fungi.. Chemistry The 6-hydrazino-4-(4 -fluorophenyl)-3-methyl--phenylpyrazolo[3,4-d]pyrimidines () were obtained from hydrazinolysis of compound [4]. Pyrimidine is used as a starting material for building a number of fused polyheterocyclic nitrogen systems. Thus, ring closure reactions of compound with triethyl orthoformat (TOF) in reflux tetrahydrofuran (TF) produced the pyrazolo[4,3 :5,6]pyrimido[,3- d][,,4]triazole 3, while the reaction with diethylcarbonate or carbon disulfide in reflux DMF furnished pyrazolo[4,3 :5,6]pyrimido[,3-c][,,4]triazole-6-one 4 and pyrazolo[4,3 :5,6]pyrimido[,3-c][,,4]triazole -6-thione 5 (cheme ). In addition, fused heteropolycyclic nitrogen systems 6 and 7 were obtained from the reaction of with benzoic acid hydrazide or isonicotinic acid hydrazine in DMF under reflux (scheme ). The nucleophilic attack of of acid hydrazide was followed by ring closure reaction by elimination of O molecule (figure ).

2 Modern Organic Chemistry Research, Vol., o. 4, ovember O EtO,reflux,6h TOF DMF, reflux, h O EtO OEt TF, reflux, 4h - EtO 3 4 O = F C DMF, reflux, 3h - 5 cheme. ynthesis of triazole-6-one 4 and triazole-6-thione 5. CO DMF, reflux,h 6 COCl DMF heat h 5 CO EtO, reflux,5h 7 = F cheme. ynthesis of fused heteropolycyclic nitrogen systems 6 and 7. O C DMF - O O - O, reflux 6 ph Figure. uggested formation of compound 6. The nucleophilic attack of - dithioic formic acid hydrazide on mercapto group of compound in DMF under reflux for h gave -substituted-thiohydrazide 8. [5] The further eterocyclization of compound 8 furnished compound 5 (scheme 3).

3 74 Modern Organic Chemistry Research, Vol., o. 4, ovember 07 - DMF, reflux,h - = F 8 C DMF reflux 4 h - 8 C 5 cheme 3. ynthesis of -substituted-thiohydrazide 8. The hydrazo and azo compounds exhibit an important attention due to its application in the industry and agriculture field [6,7]. Thus, interaction between compounds and in isopropyl alcohol under reflux afforded the hydrazo-derivative 9, which upon simple oxidation gave the azo compound 0. (cheme 4). 3. Result and Discussion The Ultraviolet (UV) spectrum for synthesis compounds, 3 and 4 showed λmax at 430 (.79), 437 (0.35) and 540 (0.85) nm. The fused heteropolycyclic nitrogen system bears both a chromophor and oxochrom probes. In addition, the hetero-conjugation systems formed enhance the λ max of compound 4 in comparison with compound. The IR spectroscopy spectrum of new synthesized systems showed lakes of functional group of,,4-triazin at 3300 cm -, compound 4 showed an interesting type of enolketo form systems exhibited at Ѵ cm-. On the other hand, compounds 5 and 8 recorded two functional groups and C= at Ѵ and 80 respectively. + isopropyl alcohol reflux, 7h - The hydrazo-compound 9 oxidation heat 4h /dry C 6 6 = F The azo-compound 0 cheme 4. ynthesis of azo compound 0 via hydrazo-derivative 9 oxidation.

4 Modern Organic Chemistry Research, Vol., o. 4, ovember The MRspectrum of both compounds 4, 5 and 0 showed two broad singlet for proton at δ 9 and 3.5 ppm, while compound 8 exhibited two different singlet for and protons at δ 8.5 and 4.8 ppm respectively. Compounds 3, 6, 7 and 0 showed a lack of peaks. Additionally, 3 CMR spectrum showed as expected a various aliphatic and aromatic carbons at 4, 9-5 and C-F, C=, C- and C-C carbons at δ 44, 4, 0 ppm respectively. Only compounds 4 and 5 showed carbonyl C=O and thionyl carbon C= carbons at δ 5 and 6 ppm. The mass fragmentation pattern of the fluorinated heterocyclic systems exhibited a base peak at m/e 95(00%), attribute to 4-fluorophenyl radicals. 4. Experiments lting points determined with an electrochermal Bibly turat cientific melting Point sample (UK). A Perkin Elmer Model RXI-FT IR system 5559 was used for Recording IR spectra of the prepared compounds (cm-). A Bruker advance DPX 400 MZ model using TM as internal standard was used for recording the and 3 CMR spectra of the compounds on DMO-d 6 (ppm). A GC-M-GP 000 Ex model was used for recording the mass spectra of the compounds (Mz). Electronic spectra recorded in ethanol on himadzu UV and visible 30 IPC pectrophotometer (nm). Elemental analysis was performed in micro analytical Center of Cairo University, Cairo, Egypt. 6-ydrazino-4-(4ˋ-fluorophenyl)--phenyl-3-methyl-pyrazolo[3,4d]pyrimidines () A mixture of 5 (g, 6.0 mmol) and hydrazine hydrate (00%, 0.56g, 6.0 mmol) in EtO (40 ml) refluxed for 8 h, cooled. The resulting solid collected by filtration and crystallized from EtO to give compound as orang crystals. Yield 93%, m.p ºC. Analytical data; Found: C,64.5;, 4.30;,4.88 %. Calculated for C F (334); C,64.67;, 4.49;, 5.0 %. UV (nm) ʎ max : 430 ( ε.79). IR (cm-) Ѵ: 3300, 300 (,), 99 (aliph. C), 60 (deformation ), 60 (C=C), 59 (C=), 497 (deformation of C 3 ), 7(C-F), 903, 787(ph). MR (ppm) δ: 9.53 (s,,), (m,4, ar), (s,,ar), (d,,ar),,4 (s,3, C3). 3 CMR (ppm) δ: 44 (C-F), 9.9 (C=), 9.07, 8.94, 8.89, 7.68, 6.66, 6.03(ar carbons, (C-), 4.4(C-C3). m/s = 35(M+ O, %), 95(00%). -enyl-3-methyl-4-(4`-fluorophenyl)pyrazolo[4`,3`:5,6]pyrimido[,3-c][,,4]triazole (3) A mixture of 4 (0.4g,.0 mmol) and TOF (0.48g,.0 mmol) in DMF (7 ml) refluxed for h and cooled. The resulting solid collected by filtration and crystallized from DMF to give compound 3. Yield 9%, m.p.9-94 ºC. Analytical data; Found: C,66.00;, 3.5;,4. %. Calculated for C F (334); C,66.7;, 3.77;, 4.4%. UV (nm) ʎ max : 430 ( ε.79). IR (cm-) Ѵ: 90, 850 (aliph.c), 59, 58 (C=), 49(deformation of C 3 ), 0 (C-F), 903,788 (ph). 4-yl-3-methyl--phenyl--pyrazolo[4`,3`:5,6]pyrimido[,3-c][,,4]triazole-6-(7)one (4) A mixture of 4 (0.6g, 0.0 mol) and diethyl carbonate (0.36g, 0.00 mol) with tetrahydrofuran TF (30 ml) refluxed for 4h, cooled. The resulting solid collected by filtration and crystallized from TF to give compound 4 as pale yellow crystal. Yield 9%, m.p. 0- ºC. Analytical data; Found: C,6. 98;, 3.55;,3.0%. Calculated for C FO (36); C,63.5;, 3.87;, 3.6 %. IR (cm-) γ : 3090 (), 99, 849 (aliph. C), 650 (C=O) 60, 59,58 (C=), 458 (deformation C3), 9 (C-F), 903,83 (ph). IR (cm-) Ѵ: (b, ), 90 (aliph. C), 59,58 (C=), 498 (deformation C3), 366 (C), (C-F), 90,83 (ph). MR (ppm) δ: , (each d,, ar), (m,5, ar), 7.30, , (each s,, ar), 4.4 (s,,o),,5 (s, 3, C 3 ). 4-yl-3-methyl--phenyl--pyrazolo[4`,3`:5,6]pyrimido[,3-c][,,4]triazole-6-(7)thion (5) A mixture of 4 (0.g, 0. 6 mmol) and C (0.4g, 0. 6 mmol) in DMF (0 ml) refluxed for 3h, cooled. The resulting solid collected by filtration and crystallized from DMF to give compound 5 as yellow crystals. Yield 88%, m.p ºC. Analytical data; Found: C,60.3;, 3.55;,.00;, 8. %. Calculated for C F (377); C,60.47;, 3.7;,.8;, 8.48%. IR (cm-) γ : 3080 (), 850,90 (aliph. C), 59,608 (C=), 450,498 (deformation of C 3 ), 365 (C), 60 (C-F), 68(C=), 788,903 (ph). MR (ppm) δ: 9.53 (s,,), , (each d,, ar), , (each s,,aromatic), (m,4,ar), , (each d,, ar),.4 (s,3,c 3 ). 3 CMR (ppm) δ: 6.8 (C=),38 (C-F),37 (C-F), 9.9 (C=),.7, 6.64, 8.93 (ar-c), 09 (C-C),.93 (C-C3). m/s (Int.y.): 379 (M+, 3.), 37 (5.), 09 (3), 83 (), 57 (8), 95 (00).

5 76 Modern Organic Chemistry Research, Vol., o. 4, ovember 07,6-Diphenyl-8-(4`-fluorophenyl)-9-methyl-pyrazolo[4`,3`:5,6]pyrimido[,3-c][,,4]triazole (6) A mixture of 4 (0.4g,.0 mmol) and benzoic acid hydrazide (0.85g,.0 mmol) in DMF (5 ml) refluxed for h, cooled. The resulting solid collected by filtration and crystallized from DMF to give compound 6 as pale brown powder. Yield 94%, m.p ºC. Analytical data; Found: C,7.30;, 3.85;,9.79 %. Calculated for C F (40); C,7.4;, 4.04;, 0.00 %. IR (cm - ) γ : 840, 97 (aliph C), 584,67 (C=), 489 (deformation of C 3 ), 03(C-F), 763,89 (ph). MR (ppm) δ: , 7, (each d,, ar), , (each d,, ar), , , (each m,, ar),.5 (s,3,c 3 ). 8-(4`-Fluorophenyl)-6-(pyridine-4`-yl)--phenyl-9-methylpyrazolo[4`,3`:5,6]pyrimido[,3- c][,,4] triazole (7) A mixture of compound 5 (0.6g,.0 mmol) and isonicotinic acid (0.g,.0 mmol) in EtO (5 ml) refluxed for 5h and cooled. The resulting solid was collected by filtration and crystallized from EtO to give compound 7 as yellow crystals. Yield 70%, m.p ºC. Analytical data; Found: C,7.30;, 3.85;,9.79 %. Calculated for C F (4); C,68.40;, 3.80;, 3. %. IR (cm-) γ : 849, (aliph C), 584,59, 66 (C=),488(deformation of C3), 03 (C-F), (phenyl). MR (ppm) δ: , 7, (each d,,ar), , (each d,, ar), , , (each m,, ar),.5 (s,3,c 3 ). -(Pyrazolo[4,3-d]pyrimidin-3-yl)dithioicformic acid hydrazide (8) A mixture of 5 (0.6g,.0 mmol) and dithioic formic acid hydrazide 0 (0.6g,.0 mmol) in EtO (30 ml) refluxed for 4h, cooled. The resulting solid was collected by filtration and crystallized from EtO to give compound 8 as yellow crystals. Yield 94%, m.p ºC. Analytical data; Found: C,55.40;, 3.55;,0.33;,5.40 %. Calculated for C F (40); C,55.60;, 3.65;, 0.48;,5.60 %. IR (cm - ) γ : (b, ), 99 (aliph C), 584,65 (C=), 489 (deformation of C3), 03(C- F), 80 (C-), 80,933 (ph). MR (ppm) δ: 8.5, 9.0 (each s,, ), (each d,,ar), , (each m,4, ar), , , (each s,3, ar), 4.8 (s,,),.5(s,3,c 3 ). 3 CMR (ppm) δ: 6 (C=), 5 (C-), 38.3 (C-F), (C=),.6, 6.6, 8.9 (ar carbons), 09 (C-C), 4.4 (C-C 3 ). Pyrazolo[4`,3`:5,6]pyrimido[,3-c][,,4]triazole-6-thione (5) Compound 8 (0.5g,.0mmol) and dry DMF (5 ml) were refluxed for 3h, cooled. The resulting solid was collected by filtration and crystallized from DMF to give compound 5. Yield 94%, m.p ºC. ynthesis of hydrazo-compound (bis-compound),-di-(-phenyl-3-methyl-4-arylpyrazolopyrimidin-6-yl)hydrazine (9) A mixture of compound (0.6g,.0 mmol) and compound (0.6g,.0 mmol) in isopropyl alcohol (60 ml) refluxed for 6h, cooled and concentrated. The resulting solid collected by filtration and crystallized from ethanol to give compound 9 as yellow crystal. Yield 89%, m.p ºC. Analytical data; Found: C,67.64;, 3.85;,.89 %. Calculated for C F (636) ; C,67.9;, 4.08;,.0%. UV(nm) ʎ max : 434 (Ѵ.60). IR (cm-) γ : 350 (), 980 (aliph C), 35 (aza - group), 58,590 (C=), 450,498 (deformation of C3), 347 (C), 30 (C-F), 80,908 (ph). MR (ppm) δ: (d,3,aromatic), 7.53 (s,,ar), (d, d,, ar), 7.33, 7.34, 7.35 (each s,3,ar), (, ),.5 (s,3,c3).,-diheteroaryl azo compound (0) A mixture of compound 9 (0.6g, 0.9 mmol) and sulphur powder (0.08g, 0.9 mmol) in dry benzen (35 ml) was refluxed for 4h, cooled. The resulting solid collected by filtration and crystallized from benzene to give compound 0 as yellow powder. Yield 9%, m.p ºC. Analytical data; Found: C,67.90;, 3.55;,.80 %. Calculated for C F (634); C,68.3;, 3.78;,.08 %. UV(nm) ʎ max : 540 (Ѵ, 0.90). IR (cm - ) γ : 850, 95 (aliph. C), 867 (=), 58,59 (C=), 48,496 (deformation C3), 335 (C), 5 (C-F), 803,906 (ph). MR (ppm) δ: , (each d,,ar), (m,0, ar), (d,, ar), (m, 4, ar),.5 (s,,c 3 ). 3 CMR (ppm) δ: 4 (C-F), (C=), 37 (C-),.3,.6, 8.6, 8.65, 8.9, 9.9,9.5 (ar. carbons), (C-C), 4.5 (C-C3). m/s (Int.y.): 636 (M+, 0.5%), 303 (3), 08 (8), 56 (5.8), 3 (8), 95 (00), 5(5).

6 Modern Organic Chemistry Research, Vol., o. 4, ovember Biological Activity The electron withdrawing/ donating nature of heterocyclic nitrogen systems in diamine influences the nucleophility of the amino group [8]. In addition, fused heteropolycyclic nitrogen systems exhibit marked biological and pharmacological effects obtained from ring closure reactions of asymmetric diamines [9-0]. Moreover, presence of fluorine atoms enhances the biological activity. Accordingly, this work is aimed to evaluate the effects of fluorinated heterocyclic systems on the cellobiase activity of some fungi. The effect of the newly synthesized fluorinated heteropolycyclic systems on the activity of cellobiase produced by Aspergillus idulans was studied according to the standard method. DMF was used as a solvent and a control, at p incubated at 50 C for hour. The released reducing sugar was estimated calorimetrically at 540 nm as an indicator for the enzyme activity. The results obtained were recorded in Table. Table. The effects of fluorinated hetero poly cyclic nitrogen systems on the cellobiase activity of A.. Fungi Concentration. itrogen Percent % Concentrations 0 µg/ml 00 µg/ml 000 µg/ml * Blank: 0.35 µg/ml (without substance or DMF). ** DMF: 0.04 µg/ml as solvent. Compound number From the results obtained in table we conclude that the overall activities of the tested compound are 0> 9> > 3> 4> 6, the highly activity of compounds 0 and 9 may be due to a higher nitrogen percent and the present of fluorine atoms. Thus the increasing of nitrogen percent in the active systems possibly enhances their activity. 6. Conclusion This investigation reported a novel synthetic application toward an interesting fluorine substituted fused heterocyclic nitrogen systems via ring closer reactions of hydrazinopyrazolopyrimidine with bifunctional compounds. These synthesis systems were evaluated as enzymatic effects towards cellobiase activity References. R. Abdel-Rahman, A. Asiri, A. Khan, WITDRAW: Reactivity of 3-hydrazono-,,4-triazines towards various strong electrophiles-ynthesis of heteropolycyclic nitrogen systems Targets a review. abian Journal of Chemistry, vol. 8, pp 800-8, 03.. D. Bakhotmah, R. Abdel-Rahman, A Review on the ynthesis and Chemistry of Bioactive Pyrazolines Bearing,,4-Triazine Moieties, Mini Rev. Org. Chem., vol. 3, no., pp. 6-77, M. Makki, D. Bakhotmah*, R. Abdel-Rahman, M. El-hahawy, Designing and ynthesis of ew Fluorine ubstituted Pyrimidine-Thion-5-Carbonitriles and the Related Derivatives as otochemical Probe Agents for Inhibition of Vitiligo Disease, International Journal of Organic Chemistry, vol., pp. 3-30, D. Bakhotmah, R. abdul-rahman, Montha fakhorji, ynthesis and chemical reactivity of some new fluorine substituted pyrazolopyrimidine derivatives and their effect on cellobiase activity produced by fungi, World Journal of Organic Chemistry, Vol. 5, no., R. Abdel-Rahman, M. Makki, A. Al-Romaizan, ynthesis of ovel Fluorine ubstituted Isolated and Fused eterobicyclic itrogen ystems Bearing 6-( -osphorylanilido)-,,4-triazin-5-one Moiety as Potential

7 78 Modern Organic Chemistry Research, Vol., o. 4, ovember 07 Inhibitor towards IV- Activity. International Journal of Organic Chemistry. vol. 4, 47, A. Abdelhamid, A. Elghandour, A. ussein, Y. Zaki, Reactions of ydrazonoyl alides 4:ynthesis of,,4- Triazoles,,3-Dihydro-,3,4-thiadiazoles, and Triazolo[4,3-a]pyrimidines. osphorus, ulfur, and ilicon and the Related Elements. Vol. 80, pp , P. Bhardwaj and. Gupta,,,4,5-Tetrazines as Platform Molecules for Energetic Materials and armaceuticals, Iranian J. Org. Chem., vol. 8, no. 3, pp , J. W. Fronabarger, R. D. Chapman, D. Gilardi, Facile entry into the 3,9-bis[,,4] triazolo-[,5-a:5,- d][,3,5]triazinium (5/6/5 tricyclic ) system, Tetrahedron Letters, vol. 47, Issue 44, pp , D. Bakhotmah, ynthesis of ovel α -Amino Acids Bearing,,4-Triazinone and teroidal Moieties as Enzymatic Affects (Cellobaiase Activity) Part II Current Organic ynthesis, vol., pp. -7, M. Ibrahim, R. Abdel-Rahman, A. Abdel-alim,. Ibrahim,. Allimony. ynthesis, Chemical Reactivity and Fungicidal Activity of Pyrido[,-b][,,4]triazine Derivatives. J. Braz. Chem. oc, vol. 0, no. 7, pp , 009.