Novel Ring Systems: Spiro[Cycloalkane] Derivatives of Triazolo- and Tetrazolo-Pyridazines

In orderto synthesize new pyridazine derivatives anellated with different nitrogen heterocyclic moieties, spiro[cycloalkane]pyridazinones were transformed into the corresponding thioxo derivatives via a reaction with phosphorus pentasulfide. The reaction of the formed 2,3-diazaspiro[5.5]undec-3-ene-1-thiones with hydrazine provided the corresponding 1-hydrazono-2,3-diazaspiro[5.5]undec-3-ene, whose diazotization led to the desired spiro[cyclohexane-1,8′-tetrazolo[1,5-b]pyridazines. The reaction of dihydropyridazinethiones with benzhydrazide afforded the corresponding 7H-spiro[[1,2,4]triazolo[4,3-b]pyridazin-8,1′-cyclohexanes]. As a result of our work, seven new pyridazinethione intermediates were prepared, which served as starting materials for the synthesis of two kinds of new ring systems: tetrazolo-pyridazines and triazolo-pyridazines. The six new annulated derivatives were characterized by physicochemical parameters. The new N-heterocycles are valuable members of the large family of pyridazines.

The spirocycles have recently gained more attention, as they usually have a high Fsp 3 character, and the three-dimensionality is better in the case of flat aromatic rings. The use of spiro building blocks better optimizes physicochemical parameters, structural novelty, and higher patentability. There are well-known examples among the spiro compounds, e.g., the anxiolytic agent buspirone, the angiotensin receptor blocker irbesartan, the antifungal natural product griseofulvin, or the spironolactone with sterane skeleton. The кopioid The spirocycles have recently gained more attention, as they usually have a high Fsp 3 character, and the three-dimensionality is better in the case of flat aromatic rings. The use of spiro building blocks better optimizes physicochemical parameters, structural novelty, and higher patentability. There are well-known examples among the spiro compounds, e.g., the anxiolytic agent buspirone, the angiotensin receptor blocker irbesartan, the antifungal natural product griseofulvin, or the spironolactone with sterane skeleton. The к opioid agonist enadoline seemed to be the most potent к selective analgesic. Most of the recently published spiro compounds contain five and six membered rings, but there are examples for 4-membered or 7-membered spirocyclic systems too [19][20][21][22][23].
Realizing that the use of the nitrogen containing heterocycles and the spiro motif may be advantageous, a few spiro[cycloalkane]pyridazinone derivatives were synthesized by us (Scheme 1) [24,25]. Depending on the substituents, the starting anhydrides (2-oxaspiro [4.4]nonane-1,3-dione (1a) and 2-oxaspiro[4.5]-1,3-dione (1b)) were converted to the desired keto-carboxylic acids (2 and 3) either by the Friedel-Crafts reaction or the Grignard reaction. This was followed by the ring closure with hydrazine hydrate in refluxing ethanol to give the corresponding pyridazinone derivatives (4a-d, 4e and 5a,b) in high yields [24,25]. Realizing that the use of the nitrogen containing heterocycles and the spiro motif may be advantageous, a few spiro[cycloalkane]pyridazinone derivatives were synthesized by us (Scheme 1) [24,25]. Depending on the substituents, the starting anhydrides (2-oxaspiro[4.4]nonane-1,3-dione (1a) and 2-oxaspiro[4.5]-1,3-dione (1b)) were converted to the desired keto-carboxylic acids (2 and 3) either by the Friedel-Crafts reaction or the Grignard reaction. This was followed by the ring closure with hydrazine hydrate in refluxing ethanol to give the corresponding pyridazinone derivatives (4a-d, 4e and 5a,b) in high yields [24,25]. The spirocycles have recently gained more attention, as they usually have a high Fsp 3 character, and the three-dimensionality is better in the case of flat aromatic rings. The use of spiro building blocks better optimizes physicochemical parameters, structural novelty, and higher patentability. There are well-known examples among the spiro compounds, e.g., the anxiolytic agent buspirone, the angiotensin receptor blocker irbesartan, the antifungal natural product griseofulvin, or the spironolactone with sterane skeleton. The к opioid agonist enadoline seemed to be the most potent к selective analgesic. Most of the recently published spiro compounds contain five and six membered rings, but there are examples for 4-membered or 7-membered spirocyclic systems too [19][20][21][22][23].
In this work, we described the use of spiro[cycloalkane]pyridazinone derivatives (4ad, 5a,b) in the synthesis of new triazolo-pyridazine and tetrazolo-pyridazine derivatives via the corresponding thioxo intermediates.

Results and Discussion
Our plan was to synthesize derivatives, where the pyridazine ring was fused with another N-heterocyclic moiety (9 and 10).
In this work, we described the use of spiro[cycloalkane]pyridazinone derivatives (4a-d, 5a,b) in the synthesis of new triazolo-pyridazine and tetrazolo-pyridazine derivatives via the corresponding thioxo intermediates.

Results and Discussion
Our plan was to synthesize derivatives, where the pyridazine ring was fused with another N-heterocyclic moiety (9 and 10).

Synthesis of Pyridazinethiones
Phosphorus pentasulfide or the Lawesson reagent may be used for the thionation of an amide function [26]. The earlier described spiro[cycloalkane]pyridazinones (4a-d and 5a,b) reacted with phosphorus pentasulfide in the refluxing toluene. According to thin layer chromatography (TLC), the starting material was consumed after 6-8 h. After the workup comprising extraction, concentration, and preparative TLC purification, the products (6a-d and 7a,b) were isolated in yields of 40-89% (Scheme 2). The new compounds (6a-d and 7a,b) were characterized by 1 H and 13 C nuclear magnetic resonance spectroscopy (NMR), as well as high resolution mass spectrometry (HRMS). During the preparation of the p-methoxyphenyl[spirocyclohexane]pyridazinone (4a), the isomeric pyridazinone derivative (4e) was isolated as a byproduct. The formation of the two isomers was explained earlier [24]. The thionation reaction with phosphorus pentasulfide led to the corresponding pyridazinethione (6e) in a yield of 14% (Scheme 3). During the preparation of the p-methoxyphenyl[spirocyclohexane]pyridazinone (4a), the isomeric pyridazinone derivative (4e) was isolated as a byproduct. The formation of the two isomers was explained earlier [24]. The thionation reaction with phosphorus pentasulfide led to the corresponding pyridazinethione (6e) in a yield of 14% (Scheme 3).

Preparing the Tetrazole Derivatives through Hydrazones
After reacting the thioxo derivatives 6a-c with hydrazine hydrate, the corresponding hydrazones 8a-c were isolated in 48-61% yields. The 4-(phenyl)-2,3-diazaspiro [5.5]undec-3-ene-1-thiones (6a-c) and hydrazine hydrate in THF had to be stirred at reflux for 5 h. It was observed that compounds (8a-c) were unstable when standing at room temperature. After a week, they began decomposing. However, after preparation, intermediates 8a-c were immediately reacted further in a diazotization reaction. The tetrazolo-pyridazinones (9a-c) were obtained in 45-77% yields (Scheme 4) after a preparative TLC purification. Compounds 8a-c and 9a-c were new and were characterized by 1 H and 13 C NMR, as well as HRMS. A similar method was applied in the sphere of benzodiazepines, where the extended conjugation helped the transformation [27]. In the case of spiro compounds 8a-c, the reaction was new.  The physicochemical parameters were calculated using ChemDraw, but only Molinspiration software was suitable for calculating logP values *.   (Table 2)).  The physicochemical parameters were calculated using ChemDraw, a drugs under development.

Preparing the Triazole Derivatives from Thioxo Compounds with Acid Hydrazides
Our next purpose was to synthesize triazole derivatives starting from the thioxo compounds (6a-c). The thioxo compounds (6a-c) were refluxed with benzoic acid hydrazide (11) in n-butanol. After evaporation and chromatographic purification, the triazolepyridazinone derivatives (10a-c) were isolated in yields of 14-42% (Scheme 5). This annulation technique was also applied in the sphere of benzodiazepines [33], where the extended aromatic conjugation may be advantageous for the reaction. Similarly to the preparation of tetrazoles, this reaction is new in case of spiro derivatives 10a-c. After the preparative TLC chromatographic separation, the three new products (10a-c) were characterized by 1 H and 13 C NMR, as well as HRMS. Table 3 summarizes the physicochemical parameters of the triazolo-pyridazinones (10a-c). The introduction of an additional aromatic ring increased the log P and clogP values, and decreased the Fsp 3 character.

Preparing the Triazole Derivatives from Thioxo Compounds with Acid Hydrazides
Our next purpose was to synthesize triazole derivatives starting from the thioxo compounds (6a-c). The thioxo compounds (6a-c) were refluxed with benzoic acid hydrazide (11) in n-butanol. After evaporation and chromatographic purification, the triazole-pyridazinone derivatives (10a-c) were isolated in yields of 14-42% (Scheme 5). This annulation technique was also applied in the sphere of benzodiazepines [33], where the extended aromatic conjugation may be advantageous for the reaction. Similarly to the preparation of tetrazoles, this reaction is new in case of spiro derivatives 10a-c. After the preparative TLC chromatographic separation, the three new products (10a-c) were characterized by 1 H and 13 C NMR, as well as HRMS. Table 3 summarizes the physicochemical parameters of the triazolo-pyridazinones (10a-c). The introduction of an additional aromatic ring increased the log P and clogP values, and decreased the Fsp 3 character.

LC-MS Analysis, TLC, and Preparative TLC
Liquid chromatography-mass spectrometry (LC-MS) spectra were recorded on an Agilent 1100/ZQMSD (Santa Clara, CA, USA) instrument equipped with UV (220 nm and   1 H and 13 C chemical shifts are given on the delta scale as parts per million (ppm) with tetramethylsilane (TMS) ( 1 H, 13 C) or dimethylsulfoxide-d 6 ( 13 C) as the internal standard (0.00 ppm and 39.4 ppm, respectively). 1 H-1 H, direct 1 H-13 C, and long-range 1 H-13 C scalar spin-spin connectivity were established from 2D. correlation spectroscopy, total correlation spectroscopy, heteronuclear single quantum coherence and heteronuclear multiple bond correlation (COSY, TOCSY, HSQC, and HMBC) experiments. 1 H-1 H spatial proximities were determined using two-dimensional nuclear Overhauser effect spectroscopy or rotating-frame Overhauser effect spectroscopy (NOESY or ROESY) experiments. 15 N Chemical shifts were referenced to nitromethane (0.0 ppm) and were obtained from 1 H-15 N HMBC measurements. All pulse sequences were applied using the standard spectrometer software package. All experiments were performed at 298 K. NMR spectra were processed using VnmrJ 2.

Mass Spectrometry
HRMS and MS-MS analyses were performed on a Thermo Velos Pro Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) system. The ionization method was ESI operated in a positive ion mode. The protonated molecular ion peaks were fragmented by collisison-induced dissociation (CID) at a normalized collision energy of 35%. For the CID experiment helium was used as the collision gas. The samples were dissolved in methanol. Data acquisition and analysis were accomplished with Xcalibur software version 2.0 (Thermo Fisher Scientific, Bremen, Germany).

Synthesis of the Starting Materials
The preparation of the starting pyridazinone derivatives (4a-e and 5a,b) was described earlier [24,25]. (6a-e and 7a,b) The pyridazinone derivatives (4a-e and 5a,b) (1.0 mmol) were dissolved in toluene (60 mL) and phosphorus pentasulfide (0.67 g, 3.0 mmol) was added. The reaction mixture was stirred at reflux for 6-8 h. After the completion of the reaction, the mixture was washed with 5% NaHCO 3 solution (80 mL), and then with distilled water (80 mL). The organic layer was dried over MgSO 4 and then concentrated. The crude product was purified by preparative thin layer chromatography (eluent: heptane:dichloromethane:methanol/5:5:1) to give the thioxo derivatives as a yellow solids (6a-e and 7a,b).

General Procedure for the Preparation of Hydrazones (8a-c)
A solution of hydrazine monohydrate (0.10 mL, 1.2 mmol) in THF (5 mL) was added to the corresponding pyridazinone derivatives (6a-c) (0.40 mmol) in THF (15 mL) dropwise. The reaction mixture was stirred at reflux for 5 h, then the solvent was evaporated. The residue was dissolved in dichloromethane (20 mL) and washed with distilled water (2 × 10 mL). The organic layer was dried over MgSO 4 , filtered and evaporated. The crude product was purified by preparative thin layer chromatography (eluent: heptane:dichloromethane:methanol/5:5:1) to give the hydrazones (8a-c).