Selective Synthesis and Photoluminescence Study of Pyrazolopyridopyridazine Diones and N-Aminopyrazolopyrrolopyridine Diones

The newly designed luminol structures of pyrazolopyridopyridazine diones and N-aminopyrazolopyrrolopyridine diones were synthesized from versatile 1,3-diaryfuropyrazolopyridine-6,8-diones, 1,3-diarylpyrazolopyrrolopyridine-6,8-diones, or 1,3-diaryl-7-methylpyrazolopyrrolopyridine-6,8-diones with hydrazine monohydrate. Photoluminescent and solvatofluorism properties containing UV–Vis absorption, emission spectra, and quantum yield (Φf) study of pyrazolopyridopyridazine diones and N-aminopyrazolopyrrolopyridine diones were also studied. Generally, most of pyrazolopyrrolopyridine-6,8-diones 6 exhibited the significant fluorescence intensity and the substituent effect when compared with N-aminopyrazolopyrrolopyridine diones, particularly for 6c and 6j with a m-chloro group. Additionally, the fluorescence intensity of 6j was significantly promoted due to the suitable conjugation conformation. Based on the quantum yield (Φf) study, the value of compound 6j (0.140) with planar structural skeletal was similar to that of standard luminol (1, 0.175).


Introduction
Sleep-disorders are one of the largest public health concerns in the whole world [1]. New functionalized pyrazolo [3,4-b]pyrrolo [3,4-d]pyridine derivatives were enthusiastically investigated to develop the increased potency and reduced side effects of novel sedative/hypnotic drug compounds for treatment of sleep-disorders [2,3]. On the other hand, pyrazolopyridopyridazine diones are well-known as the versatile precursors for synthesis of pyrazolopyridopyridazine phosphodiesterase type 5 (PDE5) inhibitors [4,5]. In recent years, chemiluminescent luminol derivatives have been an attractive detection technique in analytical applications such as presumptive test agents for latent blood detection [6][7][8][9], high-performance liquid chromatography (HPLC) [10,11], DNA, immunoassay, and cancer screening detection [12,13]. Since now, many newly designed luminol structures have Furthermore, N-aminophthalimides were considered as phthalazine 1,4-dione tautomeric pairs [20,21]. N-Amino maleimides with pyridine heterocycle series also presented as a very important privileged substructure in organic synthesis for preparing diverse biologically active molecules [22]. Typically, the most important pharmacological effects that have been reported are potential antimicrobial [22] and anticancer activities [23]. Herein, we judiciously explore the insertion of pyridazinedione and N-Amino maleimide units into the pyrazolopyridine core ring for construction of the new designed luminol structures 6a-j and 7a-i from versatile 1,3diarylpyrazolopyrrolopyridine-6,8-diones 11. Observably, we found that the series of pyridazinediones 6a-j would not only provide conjugation systems but also allow to modify the fluorescence intensity and biological activity ( Figure 2).

Results and Discussion
Initially, dimethyl 1,3-diphenyl-1H-pyrazolo [3,4-b]pyridine-4,5-dicarboxylate 8 and diethyl 1,3diphenyl-1H-pyrazolo [3,4-b]pyridine-4,5-dicarboxylate 9 were prepared by following our previously reported literature [24] from N,N-diisopropylamidinyl pyrazolylimine and chosen as the model substrate for this investigation on the construction of pyrazolopyridopyridazine diones 6a (Scheme 1). Compounds 8 and 9 were reacted with hydrazine hydrate at reflux in methanol or ethanol solution under the basic condition for 24-36 h [25,26]. However, all the efforts for the predominant formation of 6a were unsuccessful. We also attempted to perform the hydrolysis of ester groups of compounds 8 and 9 under basic conditions to obtain 1,3-diphenyl-1H-pyrazolopyridine-4,5-dicarboxylic acid 10 [27,28]. Subsequently, pyrazolopyridine-4,5-dicarboxylic acid 10 was refluxed with hydrazine in acetic acid to carry out the cyclization for 8 h, but without success (Scheme 1). Furthermore, N-aminophthalimides were considered as phthalazine 1,4-dione tautomeric pairs [20,21]. N-Amino maleimides with pyridine heterocycle series also presented as a very important privileged substructure in organic synthesis for preparing diverse biologically active molecules [22]. Typically, the most important pharmacological effects that have been reported are potential antimicrobial [22] and anticancer activities [23]. Herein, we judiciously explore the insertion of pyridazinedione and N-Amino maleimide units into the pyrazolopyridine core ring for construction of the new designed luminol structures 6a-j and 7a-i from versatile 1,3-diarylpyrazolopyrrolopyridine-6,8-diones 11. Observably, we found that the series of pyridazinediones 6a-j would not only provide conjugation systems but also allow to modify the fluorescence intensity and biological activity ( Figure 2).
Furthermore, we applied this reliable procedure to reactants 11d-j bearing p-Cl-Ph, p-Br-Ph, p-Me-Ph, p-OMe-Ph, p-CN-Ph, p-NO2-Ph, and m-Cl-Ph at the N-1 position and phenyl and H at C-3 position of pyrazolic ring. Various substituted reactants 11d-j were demonstrated to proceed smoothly. Both electron-donating and electron-withdrawing substituents were all well-tolerated in good yields (69-84%, Entries 10-16, Table 1). All of 1,3-diarylpyrazolopyrrolopyridine-6,8-diones 6aj were fully characterized by spectroscopic methods. For example, compound 6a presented one singlet at δ 9.41 ppm for pyrazolopyridine ring N=CH-C=C in 1 H-NMR and two peaks at δ 153.1 and 155.7 ppm for pyridazine dione carbon O=C-NH in 13 C-NMR spectrum. Its IR absorptions showed peaks at 3161 cm −1 for stretching of the -NH group and at 1014 cm -1 for stretching of the N-N group.
For the further controlled experiment for photoluminescence study, we also tried to prepare a series of N-aminopyrazolopyrrolopyridine diones 7a-i as the comparison cases (Scheme 2). Treatment of pyrazolopyrrolopyridine-6,8-diones 11a-i with 5.0 equivalents of hydrazine hydrate in EtOH/H2O co-solution was performed in an ice-bath to room temperature for 48 h. The corresponding N-aminopyrazolopyrrolopyridine diones 7a-i were obtained in 71-87% yields and characterized by spectroscopic methods. For example, compound 7a presented one singlet peak at δ 8.83 ppm for pyrazolopyridine ring N=CH-C=C in 1 H-NMR and two peaks at δ 164.1 and 164.4 ppm for phthalimide moiety carbon O=C-NH in 13 C-NMR spectrum. Its IR absorptions showed peaks at 3172 and 3276 cm −1 for stretching of the -NH2 group and at 1014 cm −1 for stretching of the N-N group. Due to the structural skeletons being very similar between 6 and 7, the identify method should be our next future evaluation.
Furthermore, we applied this reliable procedure to reactants 11d-j bearing p-Cl-Ph, p-Br-Ph, p-Me-Ph, p-OMe-Ph, p-CN-Ph, p-NO 2 -Ph, and m-Cl-Ph at the N-1 position and phenyl and H at C-3 position of pyrazolic ring. Various substituted reactants 11d-j were demonstrated to proceed smoothly. Both electron-donating and electron-withdrawing substituents were all well-tolerated in good yields (69-84%, Entries 10-16, Table 1). All of 1,3-diarylpyrazolopyrrolopyridine-6,8-diones 6a-j were fully characterized by spectroscopic methods. For example, compound 6a presented one singlet at δ 9.41 ppm for pyrazolopyridine ring N=CH-C=C in 1 H-NMR and two peaks at δ 153.1 and 155.7 ppm for pyridazine dione carbon O=C-NH in 13 C-NMR spectrum. Its IR absorptions showed peaks at 3161 cm −1 for stretching of the -NH group and at 1014 cm −1 for stretching of the N-N group.
For the further controlled experiment for photoluminescence study, we also tried to prepare a series of N-aminopyrazolopyrrolopyridine diones 7a-i as the comparison cases (Scheme 2). Treatment of pyrazolopyrrolopyridine-6,8-diones 11a-i with 5.0 equivalents of hydrazine hydrate in EtOH/H 2 O co-solution was performed in an ice-bath to room temperature for 48 h. The corresponding N-aminopyrazolopyrrolopyridine diones 7a-i were obtained in 71-87% yields and characterized by spectroscopic methods. For example, compound 7a presented one singlet peak at δ 8.83 ppm for pyrazolopyridine ring N=CH-C=C in 1 H-NMR and two peaks at δ 164.1 and 164.4 ppm for phthalimide moiety carbon O=C-NH in 13 C-NMR spectrum. Its IR absorptions showed peaks at 3172 and 3276 cm −1 for stretching of the -NH 2 group and at 1014 cm −1 for stretching of the N-N group. Due to the structural skeletons being very similar between 6 and 7, the identify method should be our next future evaluation. characterized by spectroscopic methods. For example, compound 7a presented one singlet peak at δ 8.83 ppm for pyrazolopyridine ring N=CH-C=C in 1 H-NMR and two peaks at δ 164.1 and 164.4 ppm for phthalimide moiety carbon O=C-NH in 13 C-NMR spectrum. Its IR absorptions showed peaks at 3172 and 3276 cm −1 for stretching of the -NH2 group and at 1014 cm −1 for stretching of the N-N group. Due to the structural skeletons being very similar between 6 and 7, the identify method should be our next future evaluation.  Table 2. The pyrazolopyridopyridazine dione 6a has better solubility in polar organic solvents, such as DMSO > THF > acetone, but N-aminopyrazolopyrrolopyridine dione 7a has the solubility only in highly polar solvents like DMSO. Luminol (1) was also measured and used as the standard sample. The UV-Vis absorption spectra of the compounds 6a and 7a in all the studied solvents were almost nearly the same; their absorption property is independent of the solvent polarity ( Figure 3 and Table 2). All these compounds exhibit two highly intense absorption maxima peaks. Among these two, the first one was a high energy absorption between 253 nm and 286 nm for 6a and 7a probably due to the π−π* transition of the aryl core [35] while the low energy band between 329 nm and 366 nm is attributed to the intramolecular charge transfer transition (ICT). However, the rigidity in the structure of compounds 6a and 7a exhibited the stronger blue-shifted absorption (~15 nm) than luminol (1) in DMSO solution, as shown in Figure 3 and Table 2. In comparison with 6a and 7a, they demonstrated a similar absorption intensity, and compound 6a has obvious red-shift~20 nm with respect to 7a. Table 2. UV-Vis absorption maximum and fluorescence emission peak wavelength of luminol (1), pyrazolopyridopyridazine dione 6a and N-aminopyrazolopyrrolopyridine dione 7a in the different solvents.

Compound
Solvent λmax/nm of UV-Vis λmax/nm of PL  Consequently, we investigated the photoluminescence properties of the compounds 6a and 7a with luminol (1). For the fluorescence spectra, as shown in Figure 3 and Table 2, both the fluorescence intensity and the maximal position slightly varied depending on the solvent. Compound 6a displayed a characteristic emission band of the excitation wavelengths between 400 and 600 nm, and the λmaxs of PL was ~480 nm with the intense greenish-blue fluorescence in Figures 3 and 4. For compound 7a, it's emission spectrum was between 350 and 550 nm, and the λmaxs of PL was ~450 nm with the intense bluish-green fluorescence in Figures 3 and 4. Compounds 6a and 7a exhibited a red-shift ~80 nm or ~60 nm as compared to luminol (1). Therefore, new luminol analogues 6a and 7a were efficiently conjugate and connect two chromophores (pyrazole and pyridine) to lead to an increase of aromaticity and provide the greenish-blue or bluish-green fluorescent materials (Table 2 and Figure  4) [36]. Particularly, the best positive solvatofluorism phenomenon was presented in CH2Cl2 solution. It was also beneficial for the visibility of the naked eye due to the bathochromic (red-shift) phenomenon from blue color to green (Figure 4). Consequently, we investigated the photoluminescence properties of the compounds 6a and 7a with luminol (1). For the fluorescence spectra, as shown in Figure 3 and Table 2, both the fluorescence intensity and the maximal position slightly varied depending on the solvent. Compound 6a displayed a characteristic emission band of the excitation wavelengths between 400 and 600 nm, and the λ max s of PL was~480 nm with the intense greenish-blue fluorescence in Figures 3 and 4. For compound 7a, it's emission spectrum was between 350 and 550 nm, and the λ max s of PL was~450 nm with the intense bluish-green fluorescence in Figures 3 and 4. Compounds 6a and 7a exhibited a red-shift 80 nm or~60 nm as compared to luminol (1). Therefore, new luminol analogues 6a and 7a were efficiently conjugate and connect two chromophores (pyrazole and pyridine) to lead to an increase of aromaticity and provide the greenish-blue or bluish-green fluorescent materials (Table 2 and Figure 4) [36]. Particularly, the best positive solvatofluorism phenomenon was presented in CH 2 Cl 2 solution. It was also beneficial for the visibility of the naked eye due to the bathochromic (red-shift) phenomenon from blue color to green (Figure 4). Moreover, the maximum of fluorescence wavelength and intensity, as shown in Figure 3, significantly vary with the diluted solvent. Further, we surprisingly observed the more significant solvent effect on compound 6a when compared with compound 7a. As shown in Figure 3, similar fluorescence spectra but a significant difference in intensity (∼6 times) were observed in varying solvents. Of note, it was interesting that toluene, THF, EA, and CH2Cl2 had differences in their polarity (toluene: 0.099, THF: 0.207, EA: 0.228, CH2Cl2: 0.309, with respect to the reference polarity of DMSO: 0.444) [37,38]. However, for the above solvents, we observed a strong intensity, in comparison to that for protic or/and polar solvent (DMSO). The intensities of fluorescence bands were reversed in protic or/and polar solvents. Therefore, the solvent polarity modulation of fluorescence was quite interesting. It was well studied that amide tautomer of pyrazolopyridopyridazine dione 6a was efficiently produced in toluene, THF, EA, and CH2Cl2 solvents [39][40][41][42][43][44]. In alcoholic (protic) and DMSO solvent, there exists competition between intermolecular bonding of the nearest hydrogen with the hydroxyimine tautomer of 6-hydroxypyrazolopyridopyridazin-9-one 6a. Therefore, different intensities of behavior were observed in different polarity solutions. On the other hand, the different fluorescence intensity between structural isomers 6a and 7a was also observed [45]. The aromaticity of compound 6a possessed the bathochromic shift of fluorescence maximum λmax by 12 nm and ∼4 times significant intensity in CH2Cl2 solution when compared with compound 7a (Table  2 and Figure 3) [46]. However, the intramolecular and intermolecular hydrogen bondings between the amino and carbonyl groups of N-aminopyrazolopyrrolopyridine dione 7a were formed to lead to the poor intensity in solution.
For further investigation of substituent efficiency of compounds 6 and 7 in photoluminescence properties, we synthesized a series of pyrazolopyridopyridazine diones 6a-j and Naminopyrazolopyrrolopyridine diones 7a-i bearing various substituents including o-, m-and p-Cl, p-Br, p-Me, p-OMe, p-CN, and p-NO2 groups in N1-phenyl ring of pyrazole moiety. Generally, most of the substituents such as o-, m-and p-Cl, p-Br, p-Me, and p-CN in N1-phenyl of pyrazolic ring of compounds 6 possessed the blue-shift phenomenon range ~10 to 30 nm with significant fluorescence intensity when compared with compound 6a, particularly for 6c with meta-chloro group ( Figure 5). For compounds 6g and 6i with the strong electron-donating (p-OMe) or electron-withdrawing groups (p-NO2), they exhibited negative photoluminescence properties ( Figure 5). While we modified the skeletal structure of pyrazolopyridopyridazine dione 6j, in which Ph-group was replaced to H atom Moreover, the maximum of fluorescence wavelength and intensity, as shown in Figure 3, significantly vary with the diluted solvent. Further, we surprisingly observed the more significant solvent effect on compound 6a when compared with compound 7a. As shown in Figure 3, similar fluorescence spectra but a significant difference in intensity (∼6 times) were observed in varying solvents. Of note, it was interesting that toluene, THF, EA, and CH 2 Cl 2 had differences in their polarity (toluene: 0.099, THF: 0.207, EA: 0.228, CH 2 Cl 2 : 0.309, with respect to the reference polarity of DMSO: 0.444) [37,38]. However, for the above solvents, we observed a strong intensity, in comparison to that for protic or/and polar solvent (DMSO). The intensities of fluorescence bands were reversed in protic or/and polar solvents. Therefore, the solvent polarity modulation of fluorescence was quite interesting. It was well studied that amide tautomer of pyrazolopyridopyridazine dione 6a was efficiently produced in toluene, THF, EA, and CH 2 Cl 2 solvents [39][40][41][42][43][44]. In alcoholic (protic) and DMSO solvent, there exists competition between intermolecular bonding of the nearest hydrogen with the hydroxyimine tautomer of 6-hydroxypyrazolopyridopyridazin-9-one 6a. Therefore, different intensities of behavior were observed in different polarity solutions. On the other hand, the different fluorescence intensity between structural isomers 6a and 7a was also observed [45]. The aromaticity of compound 6a possessed the bathochromic shift of fluorescence maximum λ max by 12 nm and ∼4 times significant intensity in CH 2 Cl 2 solution when compared with compound 7a (Table 2 and Figure 3) [46]. However, the intramolecular and intermolecular hydrogen bondings between the amino and carbonyl groups of N-aminopyrazolopyrrolopyridine dione 7a were formed to lead to the poor intensity in solution.
For further investigation of substituent efficiency of compounds 6 and 7 in photoluminescence properties, we synthesized a series of pyrazolopyridopyridazine diones 6a-j and N-aminopyrazolo pyrrolopyridine diones 7a-i bearing various substituents including o-, mand p-Cl, p-Br, p-Me, p-OMe, p-CN, and p-NO 2 groups in N1-phenyl ring of pyrazole moiety. Generally, most of the substituents such as o-, mand p-Cl, p-Br, p-Me, and p-CN in N1-phenyl of pyrazolic ring of compounds 6 possessed the blue-shift phenomenon range~10 to 30 nm with significant fluorescence intensity when compared with compound 6a, particularly for 6c with meta-chloro group ( Figure 5). For compounds 6g and 6i with the strong electron-donating (p-OMe) or electron-withdrawing groups (p-NO 2 ), they exhibited negative photoluminescence properties ( Figure 5). While we modified the skeletal structure of pyrazolopyridopyridazine dione 6j, in which Ph-group was replaced to H atom on C-3 position of pyrazolic ring, the blue-shift phenomenon was remarkably observed in photoluminescence spectra. Additionally, the fluorescence intensity of 6j was significantly promoted about 2.3 times in comparison with compound 6a (Figure 5). Based on the result of the substituent study, we conceived that compound 6j was an effective substrate that possessed suitable conjugation conformation without the torsion effect to facilitate the photoluminescence properties [26]. For compounds 7a-i bearing the above various substituents, they provided the weak fluorescence intensity [45] and possessed the blue-shift phenomenon when compared with 7a, except for 7c with m-chloro group and 7h with p-CN group ( Figure 6). Generally, compounds 7a-i were the inappropriate photoluminescent substrates [45].
Molecules 2020, 25, x 8 of 16 on C-3 position of pyrazolic ring, the blue-shift phenomenon was remarkably observed in photoluminescence spectra. Additionally, the fluorescence intensity of 6j was significantly promoted about 2.3 times in comparison with compound 6a ( Figure 5). Based on the result of the substituent study, we conceived that compound 6j was an effective substrate that possessed suitable conjugation conformation without the torsion effect to facilitate the photoluminescence properties [26]. For compounds 7a-i bearing the above various substituents, they provided the weak fluorescence intensity [45] and possessed the blue-shift phenomenon when compared with 7a, except for 7c with m-chloro group and 7h with p-CN group ( Figure 6). Generally, compounds 7a-i were the inappropriate photoluminescent substrates [45].  The quantum yields (Φf) of luminol (1) and pyrazolopyridopyridazine diones 6a, 6c, and 6j were measured in the CH2Cl2 solution using quinine sulfate in 0.05M H2SO4 (Φf = 0.60) as the standard (excitation wavelength 350 nm) [47,48]. The quantum yields (Φf) values of luminol (1) and pyrazolopyridopyridazine diones 6a, 6c, and 6j were estimated as 0.175, 0.056, 0.067, and 0.140 in CH2Cl2 solution, respectively, indicating that the Φf value of 6j was similar to that of luminol (1, Table  3). Moreover, we also investigated the quantum yields of 6j in various solvents by using the same condition. The estimated values order trendy was as 0.218 (THF) > 0.209 (Toluene) > 0.140 (CH2Cl2) > 0.083 (acetone) > 0.049 (EA), indicating THF provided the largest Φf value among them (Table 3). On the other hand, most of the quantum yields (Φf) pyrazolopyridopyridazine diones 6a-i in CH2Cl2 solution were predicted to be an almost identical value (ca. 0.05-0.06). Interestingly, the high Φf value on C-3 position of pyrazolic ring, the blue-shift phenomenon was remarkably observed in photoluminescence spectra. Additionally, the fluorescence intensity of 6j was significantly promoted about 2.3 times in comparison with compound 6a ( Figure 5). Based on the result of the substituent study, we conceived that compound 6j was an effective substrate that possessed suitable conjugation conformation without the torsion effect to facilitate the photoluminescence properties [26]. For compounds 7a-i bearing the above various substituents, they provided the weak fluorescence intensity [45] and possessed the blue-shift phenomenon when compared with 7a, except for 7c with m-chloro group and 7h with p-CN group ( Figure 6). Generally, compounds 7a-i were the inappropriate photoluminescent substrates [45].  The quantum yields (Φf) of luminol (1) and pyrazolopyridopyridazine diones 6a, 6c, and 6j were measured in the CH2Cl2 solution using quinine sulfate in 0.05M H2SO4 (Φf = 0.60) as the standard (excitation wavelength 350 nm) [47,48]. The quantum yields (Φf) values of luminol (1) and pyrazolopyridopyridazine diones 6a, 6c, and 6j were estimated as 0.175, 0.056, 0.067, and 0.140 in CH2Cl2 solution, respectively, indicating that the Φf value of 6j was similar to that of luminol (1, Table  3). Moreover, we also investigated the quantum yields of 6j in various solvents by using the same condition. The estimated values order trendy was as 0.218 (THF) > 0.209 (Toluene) > 0.140 (CH2Cl2) > 0.083 (acetone) > 0.049 (EA), indicating THF provided the largest Φf value among them (Table 3). On the other hand, most of the quantum yields (Φf) pyrazolopyridopyridazine diones 6a-i in CH2Cl2 solution were predicted to be an almost identical value (ca. 0.05-0.06). Interestingly, the high Φf value The quantum yields (Φ f ) of luminol (1) and pyrazolopyridopyridazine diones 6a, 6c, and 6j were measured in the CH 2 Cl 2 solution using quinine sulfate in 0.05M H 2 SO 4 (Φ f = 0.60) as the standard (excitation wavelength 350 nm) [47,48]. The quantum yields (Φ f ) values of luminol (1) and pyrazolopyridopyridazine diones 6a, 6c, and 6j were estimated as 0.175, 0.056, 0.067, and 0.140 in CH 2 Cl 2 solution, respectively, indicating that the Φ f value of 6j was similar to that of luminol (1, Table 3). Moreover, we also investigated the quantum yields of 6j in various solvents by using the same condition. The estimated values order trendy was as 0.218 (THF) > 0.209 (Toluene) > 0.140 (CH 2 Cl 2 ) > 0.083 (acetone) > 0.049 (EA), indicating THF provided the largest Φ f value among them (Table 3). On the other hand, most of the quantum yields (Φ f ) pyrazolopyridopyridazine diones 6a-i in CH 2 Cl 2 solution were predicted to be an almost identical value (ca. 0.05-0.06). Interestingly, the high Φ f value of 6j was obtained and possibly caused by a particular improvement in the planar skeletal conformation (Table 3 and Figure 7). Molecules 2020, 25, x 9 of 16 of 6j was obtained and possibly caused by a particular improvement in the planar skeletal conformation (Table 3 and Figure 7).

General Information
All reagents were used as obtained commercially. All reactions were carried out under argon or nitrogen atmosphere and monitored by thin-layer chromatography (TLC). Flash column chromatography was carried out on silica gel (230-400 mesh). Analytical thin-layer chromatography 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 was recorded at 400, 500, or 600 MHz and 13 C-NMR recorded at 100, 125, or 150 MHz, respectively, in DMSO-d6 as the 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, have been reported in Hz. Infrared spectra (IR) were recorded as neat solutions or solids; mass spectra were recorded using electron impact or electrospray ionization techniques. The wavenumbers reported 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 (HRMS) were recorded on a JEOL JMS-HX110 mass spectrometer with an electron ionization (EI) source The UV-visible absorption and emission spectra were performed on a Perkin-Elmer Lambda 265 and Perkin-Elmer LS50B, a fused quartz cuvette (10 mm × 10 mm) at room temperature, respectively. Quantum yields were obtained by using quinine sulfate (0.60 in 0.05 M Figure 7. Normalized fluorescence spectra of luminol and pyrazolopyridopyridazine diones 6a, 6c, and 6j in the CH 2 Cl 2 solution (excitation wavelength 350 nm).

General Information
All reagents were used as obtained commercially. All reactions were carried out under argon or nitrogen atmosphere and monitored by thin-layer chromatography (TLC). Flash column chromatography was carried out on silica gel (230-400 mesh). Analytical thin-layer chromatography 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 was recorded at 400, 500, or 600 MHz and 13 C-NMR recorded at 100, 125, or 150 MHz, respectively, in DMSO-d 6 as the 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, have been reported in Hz. Infrared spectra (IR) were recorded as neat solutions or solids; mass spectra were recorded using electron impact or electrospray ionization techniques. The wavenumbers reported 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 (HRMS) were recorded on a JEOL JMS-HX110 mass spectrometer with an electron ionization (EI) source The UV-visible absorption and emission spectra were performed on a Perkin-Elmer Lambda 265 and Perkin-Elmer LS50B, a fused quartz cuvette (10 mm × 10 mm) at room temperature, respectively. Quantum yields were obtained by using quinine sulfate (0.60 in 0.05 M H 2 SO 4 ) as a reference. Stock solutions (1 × 10 −3 M) of luminol (1), compounds of 6a-j and 7a-i were prepared in dimethyl sulfoxide (DMSO).

Determination of the Fluorescence Quantum Yield
The fluorescence quantum yield Φ x was determined through the comparative method. The quinine sulfate (Φ st = 0.60, λ ex = 350 nm) in H 2 SO 4 0.05 M was used as the standard, and it was calculated by following equation [48]: where st: standard; x: sample; Φ: quantum yield; A: absorbance at the excitation wavelength; D: area under the fluorescence spectra on an energy scale; n: the refractive index of the solution. In the process of detection, the absorbance should be controlled and lower than 0.1.