Laccase-mediated chemoselective C-4 arylation of 5-aminopyrazoles

Chemoselective arylation of 5-aminopyrazoles was performed through oxidative formation of orthoquinones from catechols catalyzed by Myceliophthora thermophila laccase (Novozym 51003), and subsequently nucleophilic attack of 5-aminopyrazole to the catechol intermediates. The C-4 arylated products were obtained under extremely mild conditions without the need for amine protection or halogenation of the substrates. From this method, 10 derivatives with moderate to good efficiency (42–94%) were prepared.


Introduction
The synthesis of heterocyclic compounds or their derivatives is of great interest in organic chemistry due to their diverse properties [1,2].Heterocycles that contain nitrogen are very important due to their presence in various medicinal and natural compounds, and finding new routes for expanding the library of these compounds has been the subject of many studies [3,4].Pyrazoles are five-membered aromatic heterocyclic rings made up of three carbon atoms and two neighboring nitrogen atoms [5][6][7][8].5-Amino pyrazoles are a subclass of pyrazoles that contain an amino group (-NH 2 ) linked to the pyrazole ring [9,10].5-Amino pyrazoles have shown ant-inflammatory [11], anti-cancer [12] and anti-microbial [13,14] properties.
Because of their various characteristic and uses, aryl-substituted 5-aminopyrazoles at C-4 position have recently garnered a lot of interest [32][33][34][35].However, the C-4 arylation of 5-aminopyrazoles can be challenging because of the presence of NH 2 group that facilitates the competitive N-6 arylation (Fig 1).This can lead to undesired side reaction thus lowering the yield of the desired product [32].Therefore, controlling the selectivity of the reaction in favor of C-4 arylation is crucial.Furthermore, the presence of bulky groups on either the arylating reagent or 5-aminopyrazole may result in lower reactivity of C-4 position in the reaction [36].
To overcome these challenges, halogenation of the C-4 position followed by performing the Suzuki-Miura cross-coupling reaction has been proposed [32] (Fig 2a).As an alternative for cross-coupling, the simultaneous protection of the amine group and halogenation of the target carbon has been adopted [32] (Fig 2b).Direct arylation has also been used to introduce an aryl group onto the C-4 position, bypassing the amine protection of 5-aminopyrazoles or pre-functionalization of the arylating reagent.However, performing the reaction in high temperature and necessity of using toxic solvents such as dioxane are some drawbacks of this approach [36] (Fig 2c).We here report a novel laccase-catalyzed strategy for the chemoselective arylation of 5-aminopyrazoles at C-4 position under extremely mild condition without the need for pre-activation of the target carbon or protecting the amine group (Fig 2d).
Laccases are multi-copper oxidases whose active site consists of four copper centers and are classified into three groups: type 1 (one copper, T1), type 2 (one copper, T2), and type 3 (two copper, T3) [37].Laccases use aerial oxygen as an oxidant and produce water as the only byproduct, which is important in terms of green chemistry.These enzymes have various applications such as bioremediation [38], biosensors [39], textiles [40], food [41], and synthesis of organic compounds [42].Laccases catalyzed the synthesis of various compounds through the oxidation of, for example, phenols (catechols) to their active intermediates (orthoquinons) which generally have a redox potential in the range of laccases [37].The laccase-catalyzed synthesis of benzofurans [43], benzothia-zoles [44], and functionalization of C-H bonds [45,46] have been previously well-documented.Following the recent studies conducted by our group to implement laccases as green catalysts in the synthesis of organic compounds [47,48], enzymatic arylation of 5-aminopyrazoles is presented here for the first time.

General remarks
All reagents are commercially available and used without further purification.Solvents used for extraction and purification were distilled before use.Myceliophthora thermophila laccase (Novozym 51003) was a generous gift from Novozymes (Copenhagen, Denmark).Reactions were monitored by thin-layer chromatography (TLC) using silica gel 60 F 254 .All organic synthesis products were purified by preparative thin-layer chromatography (TLC), (CAMAG 1 instrument, in-house prepared 20 × 20 cm silica plates) and characterized by NMR spectroscopy. 1 H and 13 C NMR spectra were recorded at 300 (75) MHz on a Bruker Avance spectrometer using DMSO-d6 and CDCl 3 as solvents.The chemical shifts were referenced to the solvent signals at δH/C 2.49/39.50ppm (DMSO-d6) and δH/C 7.26/77 ppm (CDCl 3 ) relative to TMS.Melting points were determined with a Thermo Scientific 9100 melting point apparatus and

Synthesis of α-bromoketones
15 mmol of the corresponding methyl ketone was dissolved in 10 mL of glacial acetic acid and 18 mmol of bromine solution was added dropwise to the reaction medium at room temperature.After the consumption of starting materials, the reaction mixture was poured into ice and the precipitate was filtered, washed with water, and dried at room temperature.

Synthesis of α-cyanoketones
α-cyanoketones were prepared according to literature [49]: 10 mmol of the prepared α-bromoketone was dissolved in a mixture of water and ethanol with a ratio of 1:5 and stirred in an ice water bath.Then 30 mmol of sodium cyanide was added to the reaction mixture and stirred for 16 h at room temperature.After the completion of the reaction monitored by thin layer chromatography, 5 mL of water was added to the reaction mixture and filtered.Then, 8 mL of concentrated hydrochloric acid was added to the filtrate to remove excess sodium cyanide (this is done due to the release of hydrogen cyanide gas under fume hood).After the complete removal of hydrogen cyanide gas, the resulting mixture was extracted three times with ethyl acetate.The organic phase was dried under reduced pressure and obtained precipitate dried at room temperature.

Synthesis of 5-aminopyrazoles
5 mmol of cyanoaketone prepared in the previous step and 5.6 mmol of phenylhydrazine hydrochloride were dissolved in 15 mL of ethanol and refluxed for 12 h.After ensuring the completion of the starting materials, the solvent was minimized under reduced pressure, then the reaction mixture was poured into ice water and the precipitate was filtered and dried at room temperature (Fig 3).Selected 5-aminopyrazoles (1a, 1b, 1c, 1d) were characterized by 1 H NMR spectroscopy to confirm their structure and purity.

General procedure for synthesis 3a-j
A 100 mL round bottom flask with a magnetic stirrer bar was charged with a solution of 0.1 mmol of the corresponding 5-aminopyrazole, 0.15 mmol of catechol, 8 mL of 0.01 M citrate buffer pH 4.5, 4 mL of ethyl acetate and Myceliophthora thermophila laccase (1 mL) (1000 U) and the mixture was stirred under air.The reaction was monitored with TLC until it was completely consumed.Then the reaction mixture was diluted with EtOAc, the layers were separated and the aqueous phase was extracted with EtOAc (3 x 20 mL).The combined organic phases were dried with anhydrous sodium sulfate, and filtered, and the solvent was removed under reduced pressure.The reaction mixture was purified by preparative TLC (eluting with n-hexane/ ethyl acetate = 5/1 to 2/1), provided target compound 3.

Discussion
For the arylation of 5-aminopyrazole in C-4 position, the reaction between 5-amniopyrazole 1c and catechol 2a as a model reaction was firstly investigated (Fig 4).The first reaction was performed in phosphate buffer (pH 8, 10 mM) as a solvent and acetonitrile (2:1) as a co-solvent in the presence of laccase (Novozyme 51003 1 ).No detectable product was observed after 24 h of the reaction (Table 1 entry 1).By replacing the co-solvent with ethyl acetate (2:1), the desired product was obtained with a yield of 55% shortly after 4 h (Table 1 entry 2) probably due to the better solubility of the 5-aminopyrazole in ethyl acetate.To increase the reaction  efficiency, the solvent was changed to citrate buffer (pH 4.5, 10 mM).The results showed that lowering the pH caused to improve the yield to 89% within 4 h (Table 1 entry 3).As reported in previous studies, the reason for this is attributed to higher fungal laccase activity in acidic pH compared to basic pH.By further changing the co-solvent to ethanol and acetonitrile in the presence of citrate buffer as a solvent, no product was formed (Table 1 entries 4 and 5).Also by altering the amount of enzyme to 500 U, the reaction efficiency was effectively decreased to 48% (Table 1 entry 6) probably due to the lower concentration of ortho-quinone produced in the presence of a lower amount of enzyme.Furthermore, running a control reaction in the absence of the enzyme showed no product formation, clearly proving the catalytic function of laccase in the reaction (Table 1 entry 7).Performing the arylation of 5-aminopyrazole in citrate buffer (pH 4.5, 10 mM) as a solvent and ethyl acetate as a co-solvent within 4h was found to be the optimal condition of the reaction (Table 1 entry 3).
1 HNMR, 13 CNMR and Mass spectrometry was used to prove the chemical structure of the products.The 1 HNMR spectrum of 3c as a typical product of the enzymatic reaction showed a singlet with integration of 3 in 2.28 ppm, corresponding to the methyl group of the phenyl ring.The singlet peak at 4.85 ppm with integration of 2 corresponds to the amine group, clearly proving that the reaction is performed via nucleophilic attack of C4 to the ortho-quinone ring.Three peaks with the total integration of 3 at 6.49, 6.51 and 6.61 ppm correspond to catechol hydrogens.The rest of the peaks in aromatic area with integration of 9 can be attributed to the 2 remaining phenyl ringsin the structure.Two singlet peaks at 8.88 and 8.92 correspond to two hydroxyl groups.Compared to the spectrum of the corresponding starting material 1c, the singlet peak at 5.98 ppm was removed in the product, and the singlet peak at 4.38 ppm remained intact, which indicates the binding of catechol to the carbon of position 4 (Fig 5).In 13 CNMR spectrum of 3c, 18 peaks were observed while the desired compound has 22 carbon atoms.The increase in the height of some peaks in the aromatic region can be considered as an evidence for theoverlaping of some signals togatherof some carbons.The mass spectrum of the product 3c further proved its structure by showing the molecular ion (M + ) of 357, which belongs to the molecular mass of this product.The optimal conditions were then applied toexpand the scope of the reaction to the substrates with different substitutions (Fig 6).The results showed that when the electron-withdrawing substituent is placed on the 5-aminopyrazole ring, the reaction efficiency decreases (3a, 3f, 3i, and 3j), which can be attributed to the decrease in the nucleophilicity of 5-aminopyrazole.When nitrogen number 1 and carbon number 3 simultaneously had electron-donating substituents, the efficiency was associated with an increase (3g, 3h).In addition, the presence of methyl group on catechol ring at C-4 position facilitated the reaction toward higher efficiencies compared those performed with non-substituted catechol (3b compared to 3d and 3c compared to 3e).
The possible mechanism for the reaction was proposed based on the control experiments and the previous similar reports [37,39] on the mechanism of oxidative reactions catalyzed by laccases (Fig 7).The reaction goes through laccase-catalyzed oxidation of the catechol 2a to the ortho-quinone 4a.Then 5-aminopyrazole 1a attacks the quinone intermediate via imineenamine tautomerization to afford the corresponding product 3b.

Conclusions
In this research, for the chemoselective arylation of 5-aminopyrazoles in the C-4 position was introduced.This enzymatic route offered a simple and efficient method for the arylation reaction without prior protection of the amine group.The reaction was carried out in mild conditions without needing any toxic reagents which provides a safe approach in the synthesis of some heterocyclic medicinal compounds.