Structural Identification between Phthalazine-1,4-Diones and N-Aminophthalimides via Vilsmeier Reaction: Nitrogen Cyclization and Tautomerization Study

N-Aminophthalimides and phthalazine 1,4-diones were synthesized from isobenzofuran-1,3-dione, isoindoline-1,3-dione, furo [3,4-b] pyrazine-5,7-dione, or 1H-pyrrolo [3,4-c] pyridine-1,3-dione with monohydrate hydrazine to carry out the 5-exo or 6-endo nitrogen cyclization under the different reaction conditions. Based on the control experimental results, 6-endo thermodynamic hydrohydrazination and kinetical 5-exo cyclization reactions were individually selective formation. Subsequently, Vilsmeier amidination derivatization was successfully developed to probe the structural divergence between N-aminophthalimide 2 and phthalazine 1,4-dione 3. On the other hand, the best tautomerization of N-aminophthalimide to diazinone was also determined under acetic acid mediated solution.

Heterocycles containing phthalazine 1,4-dione moiety have been reported to possess different pharmacological properties including anti-inflammatory, cardiotonic vasorelaxant, anticonvulsant [13], antihypertensive [14], antibacterial [15], anti-cancer [16], and carbonic anhydrase enzyme activity [17]. On the other hand, phthalimide group was conceived as a nitrogen source [18], for the direct introduction of masked amino function via the classical Gabriel protocol [19,20] as well as for the protection of amino groups [21][22][23]. N-aminophthalimides can be considered as phthalazine 1,4-dione tautomeric pairs. The structural arrangement of hydrazine derivatives is the mainly associated with the interconversion of imine−enamine [24,25]. Herein, we selectively synthesize N-aminophthalimide and phthalazine 1,4-dione derivatives in via the thermodynamic-kinetic control conditions. They will provide as the precursors for constructing the pharmacological heterocyclic compounds (PDE5 inhibitors) [26,27] or the chemiluminescent luminol derivatives [28].                    The reaction condition at -20 The reaction condition at 0 • C within 4h. c Compound 2f and 3f were provided and prepared from our previous work [28]. The reaction condition at -20 °C within 4h. b The reaction condition at 0 °C within 4h. c Compound 2f and 3f were provided nd prepared from our previous work [28].  Vilsmeier amidination methodology was essentially examined for the applicable protected utilization of primary amines. The usual method was directly treating primary amines with dimethylformamide (DMF) and coupling agents including POCl3, P2O5, PCl5, he reaction condition at -20 °C within 4h. b The reaction condition at 0 °C within 4h. c Compound 2f and 3f were provided nd prepared from our previous work [28].  Vilsmeier amidination methodology was essentially examined for the applicable protected utilization of primary amines. The usual method was directly treating primary amines with dimethylformamide (DMF) and coupling agents including POCl3, P2O5, PCl5, Vilsmeier amidination methodology was essentially examined for the applicable protected utilization of primary amines. The usual method was directly treating primary amines with dimethylformamide (DMF) and coupling agents including POCl 3 , P 2 O 5 , PCl 5 , (COCl) 2 , PyBOP, SOCl 2 , acyl chlorides, trifluoroacetic anhydride (TFAA), or sulfonyl chloride to give the corresponding amidine products [37][38][39]. To further probe the structural divergence, pyrazolopyridopyridazine diones 2f and N-aminopyrazolopyrrolopyridine-6,8diones 3f were selected as model cases for the further control experiments [28]. At first, we employed Vilsmeier reagent (halomethyleniminium salt) [29][30][31][32][33] to compounds 2f and 3f (Scheme 1). The reactions were individually monitored by TLC method. When compound 2f was completely consumed for 4 h at 65 • C, the corresponding acquired amidination product 4 was formed and obtained in 89% yield without producing chlorinated compound 5. The structure of compound 4 was fully characterized by spectroscopic methods and single-crystal X-ray diffraction study. Based on 1 H NMR spectroscopic characterization, compound 4 possesses singlet signal of pyridine ring proton H a around 9.03 ppm, and significant amidinyl moiety signals of iminium proton H b around 7.70 ppm and two peaks of NMe 2 around 2.97 and 3.02 ppm ( Figure 3). These results showed the free primary amine group of compound 2f was successfully converted into the amidinyl substituent. On the other hand, chlorination of compound 3f was accomplished without amidination product 4 formation by Vilsmeier reagent at reflux for 4 h, affording the corresponding product 5 with down-field proton signal H c of pyridine ring around 9.68 ppm in good yield (80%, Scheme 1 and Figure 3) [27]. Based on the above derivatization study, Vilsmeier reaction was conceived as the significant derivatization agent to identify isomers between 2f and 3f. (COCl)2, PyBOP, SOCl2, acyl chlorides, trifluoroacetic anhydride (TFAA), or sulfonyl chloride to give the corresponding amidine products [37][38][39]. To further probe the structural divergence, pyrazolopyridopyridazine diones 2f and N-aminopyrazolopyrrolopyridine-6,8-diones 3f were selected as model cases for the further control experiments [28]. At first, we employed Vilsmeier reagent (halomethyleniminium salt) [29][30][31][32][33] to compounds 2f and 3f (Scheme 1). The reactions were individually monitored by TLC method. When compound 2f was completely consumed for 4 h at 65 °C, the corresponding acquired amidination product 4 was formed and obtained in 89% yield without producing chlorinated compound 5. The structure of compound 4 was fully characterized by spectroscopic methods and single-crystal X-ray diffraction study. Based on 1 H NMR spectroscopic characterization, compound 4 possesses singlet signal of pyridine ring proton Ha around 9.03 ppm, and significant amidinyl moiety signals of iminium proton Hb around 7.70 ppm and two peaks of NMe2 around 2.97 and 3.02 ppm ( Figure 3). These results showed the free primary amine group of compound 2f was successfully converted into the amidinyl substituent. On the other hand, chlorination of compound 3f was accomplished without amidination product 4 formation by Vilsmeier reagent at reflux for 4 h, affording the corresponding product 5 with down-field proton signal Hc of pyridine ring around 9.68 ppm in good yield (80%, Scheme 1 and Figure 3) [27]. Based on the above derivatization study, Vilsmeier reaction was conceived as the significant derivatization agent to identify isomers between 2f and 3f. (COCl)2, PyBOP, SOCl2, acyl chlorides, trifluoroacetic anhydride (TFAA), or sulfonyl chloride to give the corresponding amidine products [37][38][39]. To further probe the structural divergence, pyrazolopyridopyridazine diones 2f and N-aminopyrazolopyrrolopyridine-6,8-diones 3f were selected as model cases for the further control experiments [28]. At first, we employed Vilsmeier reagent (halomethyleniminium salt) [29][30][31][32][33] to compounds 2f and 3f (Scheme 1). The reactions were individually monitored by TLC method. When compound 2f was completely consumed for 4 h at 65 °C, the corresponding acquired amidination product 4 was formed and obtained in 89% yield without producing chlorinated compound 5. The structure of compound 4 was fully characterized by spectroscopic methods and single-crystal X-ray diffraction study. Based on 1 H NMR spectroscopic characterization, compound 4 possesses singlet signal of pyridine ring proton Ha around 9.03 ppm, and significant amidinyl moiety signals of iminium proton Hb around 7.70 ppm and two peaks of NMe2 around 2.97 and 3.02 ppm ( Figure 3). These results showed the free primary amine group of compound 2f was successfully converted into the amidinyl substituent. On the other hand, chlorination of compound 3f was accomplished without amidination product 4 formation by Vilsmeier reagent at reflux for 4 h, affording the corresponding product 5 with down-field proton signal Hc of pyridine ring around 9.68 ppm in good yield (80%, Scheme 1 and Figure 3) [27]. Based on the above derivatization study, Vilsmeier reaction was conceived as the significant derivatization agent to identify isomers between 2f and 3f.  For further investigation into the reactivity of Vilsmeier amidination derivatization, Vilsmeier reaction was carried out using different substrates including N-aminophthalimides 2a-e at 50 • C for 0.5 h. Various substituted reactants 2a-e were demonstrated to perform the reactions smoothly, regardless of whether electron-donating or electron-withdrawing substituents, and the corresponding amidination products 6−10 were afforded in 74−88% yields ( Table 2). All products 6-10 were fully characterized by spectroscopic methods, and they actually presented singlet peak for the significant amidinyl moiety signals of iminium proton H and two peaks of NMe 2 in 1 H-NMR. Subsequently, a series of phthalazine 1,4diones 3a-e were treated with Vilsmeier reagent (POCl 3 /DMF) at 65 • C or 80 • C for 2-4 h. The chlorination happened smoothly to afford the desired products 11-15 in high yields (82−90%, Table 2), except for 3d (31%). Owing to the electron-rich property of nitrogen atoms on the aromatic motif of compound 3d, the complicated aromatic substitution and polylization were proceeded. All chlorinated products 11-15 were also fully characterized by spectroscopic methods, and two peaks for the significant dione moieties were converted into -N = 13 C-Cl singlet signal at δ 153-157 ppm in 13 C-NMR spectrum. Therefore, Vilsmeier reagent (POCl 3 /DMF) was used as the derivatization reagent for the different reactive phenomenon to distinguish N-aminophthalimides 2 and phthalazine-1,4-diones 3.  For further investigation into the reactivity of Vilsmeier amidination derivatization, Vilsmeier reaction was carried out using different substrates including N-aminophthalimides 2a-e at 50 °C for 0.5 h. Various substituted reactants 2a-e were demonstrated to perform the reactions smoothly, regardless of whether electron-donating or electronwithdrawing substituents, and the corresponding amidination products 6−10 were afforded in 74−88% yields ( Table 2). All products 6-10 were fully characterized by spectroscopic methods, and they actually presented singlet peak for the significant amidinyl moiety signals of iminium proton H and two peaks of NMe2 in 1 H-NMR. Subsequently, a series of phthalazine 1,4-diones 3a-e were treated with Vilsmeier reagent (POCl3/DMF) at 65 °C or 80 °C for 2-4 h. The chlorination happened smoothly to afford the desired products 11-15 in high yields (82−90%, Table 2), except for 3d (31%). Owing to the electronrich property of nitrogen atoms on the aromatic motif of compound 3d, the complicated aromatic substitution and polylization were proceeded. All chlorinated products 11-15 were also fully characterized by spectroscopic methods, and two peaks for the significant dione moieties were converted into -N = 13 C-Cl singlet signal at δ 153-157 ppm in 13 C-NMR spectrum. Therefore, Vilsmeier reagent (POCl3/DMF) was used as the derivatization reagent for the different reactive phenomenon to distinguish N-aminophthalimides 2 and phthalazine-1,4-diones 3. Table 2. Derivatization results of N-aminophthalimides 2a-e and phthalazine 1,4-diones 3a-e with Vilsmeier reagent. For further investigation into the reactivity of Vilsmeier amidination derivatization, Vilsmeier reaction was carried out using different substrates including N-aminophthalimides 2a-e at 50 °C for 0.5 h. Various substituted reactants 2a-e were demonstrated to perform the reactions smoothly, regardless of whether electron-donating or electronwithdrawing substituents, and the corresponding amidination products 6−10 were afforded in 74−88% yields ( Table 2). All products 6-10 were fully characterized by spectroscopic methods, and they actually presented singlet peak for the significant amidinyl moiety signals of iminium proton H and two peaks of NMe2 in 1 H-NMR. Subsequently, a series of phthalazine 1,4-diones 3a-e were treated with Vilsmeier reagent (POCl3/DMF) at 65 °C or 80 °C for 2-4 h. The chlorination happened smoothly to afford the desired products 11-15 in high yields (82−90%, Table 2), except for 3d (31%). Owing to the electronrich property of nitrogen atoms on the aromatic motif of compound 3d, the complicated aromatic substitution and polylization were proceeded. All chlorinated products 11-15 were also fully characterized by spectroscopic methods, and two peaks for the significant dione moieties were converted into -N = 13 C-Cl singlet signal at δ 153-157 ppm in 13 C-NMR spectrum. Therefore, Vilsmeier reagent (POCl3/DMF) was used as the derivatization reagent for the different reactive phenomenon to distinguish N-aminophthalimides 2 and phthalazine-1,4-diones 3. Table 2. Derivatization results of N-aminophthalimides 2a-e and phthalazine 1,4-diones 3a-e with Vilsmeier reagent. For further investigation into the reactivity of Vilsmeier amidination derivatization, Vilsmeier reaction was carried out using different substrates including N-aminophthalimides 2a-e at 50 °C for 0.5 h. Various substituted reactants 2a-e were demonstrated to perform the reactions smoothly, regardless of whether electron-donating or electronwithdrawing substituents, and the corresponding amidination products 6−10 were afforded in 74−88% yields ( Table 2). All products 6-10 were fully characterized by spectroscopic methods, and they actually presented singlet peak for the significant amidinyl moiety signals of iminium proton H and two peaks of NMe2 in 1 H-NMR. Subsequently, a series of phthalazine 1,4-diones 3a-e were treated with Vilsmeier reagent (POCl3/DMF) at 65 °C or 80 °C for 2-4 h. The chlorination happened smoothly to afford the desired products 11-15 in high yields (82−90%, Table 2), except for 3d (31%). Owing to the electronrich property of nitrogen atoms on the aromatic motif of compound 3d, the complicated aromatic substitution and polylization were proceeded. All chlorinated products 11-15 were also fully characterized by spectroscopic methods, and two peaks for the significant dione moieties were converted into -N = 13 C-Cl singlet signal at δ 153-157 ppm in 13 C-NMR spectrum. Therefore, Vilsmeier reagent (POCl3/DMF) was used as the derivatization reagent for the different reactive phenomenon to distinguish N-aminophthalimides 2 and phthalazine-1,4-diones 3. To explore the interconversion reactivity of the tautomerization, the solvent scope was first examined by using 7-aminopyrazolopyrrolopyridine-6,8-dione 2f. Compound 2f was screened and refluxed in the various solvents including CH2Cl2, THF, EtOH, MeCN, toluene, dioxane, and DMSO for 24 h. However, the reactions in CH2Cl2, EtOH, toluene recovered the starting material 2f without conversion happening (Table 3 entries 1-3). The To explore the interconversion reactivity of the tautomerization, the solvent scope was first examined by using 7-aminopyrazolopyrrolopyridine-6,8-dione 2f. Compound 2f was screened and refluxed in the various solvents including CH 2 Cl 2 , THF, EtOH, MeCN, toluene, dioxane, and DMSO for 24 h. However, the reactions in CH 2 Cl 2 , EtOH, toluene recovered the starting material 2f without conversion happening (Table 3 entries 1-3). The use of polar THF, MeCN, dioxane, and DMSO led to lower interconversion ratios of 2f/3f from 93/7 to 88/12 (Table 3, entries 4-7). Subsequently, Brønsted-Lowry acids including acetic acid (AcOH), methanesulfonic acid (TsOH), methanesulfonic chloride (TsCl), and trifluoroacetic acid (TFA) were studied for the interconversion reaction at reflux for 4 h (Entries 8-11, Table 3). Several experimental observations are worthy to discuss: we firstly found that the conversion ratios were improved under acidic condition (Entries 8-11, Table 3). Secondly, under the strong acid such as trifluoroacetic acid (pKa = 0.30), p-toluenesulfonic acid (TsOH, pKa = −1.9), and methanesulfonic chloride (TsCl), the low conversion ratio and decomposed products were observed (Entries 8-10, Table 3). To explore the interconversion reactivity of the tautomerization, the solvent scope was first examined by using 7-aminopyrazolopyrrolopyridine-6,8-dione 2f. Compound 2f was screened and refluxed in the various solvents including CH2Cl2, THF, EtOH, MeCN, toluene, dioxane, and DMSO for 24 h. However, the reactions in CH2Cl2, EtOH, toluene recovered the starting material 2f without conversion happening (Table 3 entries 1-3). The use of polar THF, MeCN, dioxane, and DMSO led to lower interconversion ratios of 2f/3f from 93/7 to 88/12 (Table 3, entries 4-7). Subsequently, Brønsted-Lowry acids including acetic acid (AcOH), methanesulfonic acid (TsOH), methanesulfonic chloride (TsCl), and trifluoroacetic acid (TFA) were studied for the interconversion reaction at reflux for 4 h (Entries 8-11, Table 3). Several experimental observations are worthy to discuss: we firstly found that the conversion ratios were improved under acidic condition (Entries 8-11, Table 3). Secondly, under the strong acid such as trifluoroacetic acid (pKa = 0.30), p-toluenesulfonic acid (TsOH, pKa = −1.9), and methanesulfonic chloride (TsCl), the low conversion ratio and decomposed products were observed (Entries 8-10, Table 3). For further investigations, the timed programming of the thermodynamic conversion of compound 2f was carried out under acetic acid (AcOH) solution and shown in Figure  4. The reaction mixture was sampled at 1.5, 2.3, 3.5, and 5 h and detected by the 1 H-NMR spectroscopic method. This result showed that compound 2f was gradually converted to For further investigations, the timed programming of the thermodynamic conversion of compound 2f was carried out under acetic acid (AcOH) solution and shown in Figure 4. The reaction mixture was sampled at 1.5, 2.3, 3.5, and 5 h and detected by the 1 H-NMR spectroscopic method. This result showed that compound 2f was gradually converted to the thermodynamic stable product 3f (Figure 4). Finally, transformation reaction was equilibrated at reflux for more than 5 h, and the conversion ratio was obtained proximately 6/94 (2f/3f, Entry 11 of Table 4 and Figure 4). Based on the above experimental result, acetic acid was conceived as the best acidic solvent with 6/94 conversion ratio. Fortunately, 2f can be successfully and smoothly transformed to more thermodynamically stable product 3f by refluxing in acidic medium [40,41]. To further demonstrate the reliable of conversion procedure, N-aminophthalimides 2a-e were also used as starting materials at reflux for 8-9 h. Fortunately, compounds 2a-e can be smoothly transformed to give the corresponding thermodynamically pyrazolopyridopyridazine diones 3a-e under acetic acid solvent, with the ratio of 2a-e/3a-e from 6/94 to 1/99 (Table 4). equilibrated at reflux for more than 5 h, and the conversion ratio was obtained proximately 6/94 (2f/3f, Entry 11 of Table 4 and Figure 4). Based on the above experimental result, acetic acid was conceived as the best acidic solvent with 6/94 conversion ratio. Fortunately, 2f can be successfully and smoothly transformed to more thermodynamically stable product 3f by refluxing in acidic medium [40,41]. To further demonstrate the reliable of conversion procedure, N-aminophthalimides 2a-e were also used as starting materials at reflux for 8-9 h. Fortunately, compounds 2a-e can be smoothly transformed to give the corresponding thermodynamically pyrazolopyridopyridazine diones 3a-e under acetic acid solvent, with the ratio of 2a-e/3a-e from 6/94 to 1/99 (Table 4).    ble of conversion procedure, N-aminophthalimides 2a-e were also used as starting materials at reflux for 8-9 h. Fortunately, compounds 2a-e can be smoothly transformed to give the corresponding thermodynamically pyrazolopyridopyridazine diones 3a-e under acetic acid solvent, with the ratio of 2a-e/3a-e from 6/94 to 1/99 (Table 4).  ble of conversion procedure, N-aminophthalimides 2a-e were also used as starting materials at reflux for 8-9 h. Fortunately, compounds 2a-e can be smoothly transformed to give the corresponding thermodynamically pyrazolopyridopyridazine diones 3a-e under acetic acid solvent, with the ratio of 2a-e/3a-e from 6/94 to 1/99 (Table 4).  ble of conversion procedure, N-aminophthalimides 2a-e were also used as starting materials at reflux for 8-9 h. Fortunately, compounds 2a-e can be smoothly transformed to give the corresponding thermodynamically pyrazolopyridopyridazine diones 3a-e under acetic acid solvent, with the ratio of 2a-e/3a-e from 6/94 to 1/99 (Table 4).  stable product 3f by refluxing in acidic medium [40,41]. To further demonstrate the relia-ble of conversion procedure, N-aminophthalimides 2a-e were also used as starting materials at reflux for 8-9 h. Fortunately, compounds 2a-e can be smoothly transformed to give the corresponding thermodynamically pyrazolopyridopyridazine diones 3a-e under acetic acid solvent, with the ratio of 2a-e/3a-e from 6/94 to 1/99 (Table 4).  stable product 3f by refluxing in acidic medium [40,41]. To further demonstrate the relia-ble of conversion procedure, N-aminophthalimides 2a-e were also used as starting materials at reflux for 8-9 h. Fortunately, compounds 2a-e can be smoothly transformed to give the corresponding thermodynamically pyrazolopyridopyridazine diones 3a-e under acetic acid solvent, with the ratio of 2a-e/3a-e from 6/94 to 1/99 (Table 4).

General Procedure
All reagents were purchased commercially. All reactions were carried out under argon or nitrogen atmosphere and monitored by TLC. Flash column chromatography was carried out on silica gel (230-400 mesh). Analytical thin-layer chromatography (TLC) was performed using pre-coated plates (silica gel 60 F-254) purchased from Merck Inc. Flash

General Procedure
All reagents were purchased commercially. All reactions were carried out under argon or nitrogen atmosphere and monitored by TLC. Flash column chromatography was carried out on silica gel (230-400 mesh). Analytical thin-layer chromatography (TLC) was performed using pre-coated plates (silica gel 60 F-254) purchased from Merck Inc. Flash

General Procedure
All reagents were purchased commercially. All reactions were carried out under argon or nitrogen atmosphere and monitored by TLC. Flash column chromatography was carried out on silica gel (230-400 mesh). Analytical thin-layer chromatography (TLC) was performed using pre-coated plates (silica gel 60 F-254) purchased from Merck Inc. Flash

General Procedure
All reagents were purchased commercially. All reactions were carried out under argon or nitrogen atmosphere and monitored by TLC. Flash column chromatography was carried out on silica gel (230-400 mesh). Analytical thin-layer chromatography (TLC) was performed using pre-coated plates (silica gel 60 F-254) purchased from Merck Inc. Flash

General Procedure
All reagents were purchased commercially. All reactions were carried out under argon or nitrogen atmosphere and monitored by TLC. Flash column chromatography was carried out on silica gel (230-400 mesh). Analytical thin-layer chromatography (TLC) was

General Procedure
All reagents were purchased commercially. All reactions were carried out under argon or nitrogen atmosphere and monitored by TLC. Flash column chromatography was carried out on silica gel (230-400 mesh). Analytical thin-layer chromatography (TLC) was

General Procedure
All reagents were purchased commercially. All reactions were carried out under argon or nitrogen atmosphere and monitored by TLC. Flash column chromatography was carried out on silica gel (230-400 mesh). Analytical thin-layer chromatography (TLC) was performed using pre-coated plates (silica gel 60 F-254) purchased from Merck Inc. Flash column chromatography purification was carried out by gradient elution using n-hexane in ethyl acetate (EtOAc) unless otherwise stated. 1 H NMR spectra were recorded at 400 or 500 MHz and 13 C NMR spectra were recorded at 100 or 125 MHz, respectively, in CDCl 3 , DMSO-d 6 , or D 2 O solvent. The standard abbreviations s, d, t, q, and m refer to the singlet, doublet, triplet, quartet, and multiplet, respectively. Coupling constant (J), whenever discernible, has been reported in Hz. Infrared spectra (IR) were recorded in neat solutions or solids; and mass spectra were recorded using electron impact or electrospray ionization techniques. The reported wavenumbers are referenced to the polystyrene 1601 cm −1 absorption. ESI-MS analyses were performed on an Applied Biosystems API 300 mass spectrometer. High-resolution mass spectra were obtained from a JEOL JMS-HX110 mass spectrometer.