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Laccase-mediated chemoselective C-4 arylation of 5-aminopyrazoles
Laccase-mediated chemoselective C-4 arylation of 5-aminopyrazoles
Shahedi Mansour Investigation Methodology Writing – original draft 1
Shahani Rojina Conceptualization Data curation Software 1
Omidi Niloofar Validation Writing – original draft 1
https://orcid.org/0000-0002-0407-1469
Habibi Zohreh Supervision 1 *
Yousefi Maryam Investigation Project administration Writing – review & editing 2
Mohammadi Mehdi Project administration Supervision Writing – review & editing 3 *
1 Department of Organic Chemistry, Shahid Beheshti University, Tehran, Iran
2 Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
3 Bioprocess Engineering Department, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Shaik Afzal Basha Editor
Vignan Pharmacy College, INDIA
Competing Interests: NO authors have competing interests.

* E-mail: z_habibi@sbu.ac.ir (ZH); m.mohammadi@nigeb.ac.ir (MM)
18 9 2024
2024
19 9 e030803626 3 2024
17 7 2024
© 2024 Shahedi et al
2024
Shahedi et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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.

The author(s) received no specific funding for this work. Data AvailabilityAll relevant data are within the manuscript and its Supporting information files.
Data Availability

All relevant data are within the manuscript and its Supporting information files.
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pmcIntroduction

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 [5–8]. 5-Amino pyrazoles are a subclass of pyrazoles that contain an amino group (-NH2) linked to the pyrazole ring [9, 10]. 5-Amino pyrazoles have shown ant-inflammatory [11], anti-cancer [12] and anti-microbial [13, 14] properties.

5-Aminopyrazoles functionalization is of great interest in pharmaceutical and material chemistry, where the goal is to develop novel molecules with improved properties [15–19]. Acylation [20], alkylation [21–23, sulfonylation [24], cyclization [25, 26], and arylation [27, 28] are some popular techniques for functionalization of 5-aminopyrazoles. These reactions can produce amides, alkylated amines, sulfonyl amines, pyrazolo[1,5-a] pyrimidines, and arylated pyrazoles, respectively. Recently, the C-4 functionalized 5-aminopyrazoles have been proved to present anti-inflammatory properties [29], inhibitory effect on cyclin-dependent kinases [30], and anti-proliferative activity against MCF7 [31].

Because of their various characteristic and uses, aryl-substituted 5-aminopyrazoles at C-4 position have recently garnered a lot of interest [32–35]. However, the C-4 arylation of 5-aminopyrazoles can be challenging because of the presence of NH2 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].

10.1371/journal.pone.0308036.g001 Fig 1 Competitive arylation of 5-aminopyrazoles.

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).

10.1371/journal.pone.0308036.g002 Fig 2 Previous works and this work.

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.

Materials and methods

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 F254. All organic synthesis products were purified by preparative thin-layer chromatography (TLC), (CAMAG® instrument, in-house prepared 20 × 20 cm silica plates) and characterized by NMR spectroscopy. 1H and 13C NMR spectra were recorded at 300 (75) MHz on a Bruker Avance spectrometer using DMSO-d6 and CDCl3 as solvents. The chemical shifts were referenced to the solvent signals at δH/C 2.49/39.50 ppm (DMSO-d6) and δH/C 7.26/77 ppm (CDCl3) relative to TMS. Melting points were determined with a Thermo Scientific 9100 melting point apparatus and are uncorrected. Mass spectra were recorded with an Agilent Technologies (HP) 5973 mass spectrometer.

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 1H NMR spectroscopy to confirm their structure and purity.

10.1371/journal.pone.0308036.g003 Fig 3 Synthesis of 5-aminopyrazoles 1a-1h.

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.

Results

3-(4-bromophenyl)-1-phenyl-1H-pyrazol-5-amine (1a) cream solid, isolated yield = 85%, 1H NMR (300 MHz, DMSO-d6) δ 7.5–7.9 (m, 8H), 7.37 (m, 1H), 6.0 (s, 1H), 5.5 (s, 2H).

1,3-diphenyl-1H-pyrazol-5-amine (1b) brown solid, isolated, yield = 97% 1H NMR (300 MHz, Chloroform-d) δ 7.8 (d, J = 7.5 Hz, 2H), 7.6 (d, J = 7.8 Hz, 2H), 7.5 (t, J = 7.9 Hz, 2H), 7.4 (m, 4H), 6.0 (d, J = 2.3 Hz, 1H), 4.1 (s, 2H).

1-phenyl-3-(p-tolyl)-1H-pyrazol-5-amine (1c) cream solid, isolated yield = 93% 1H NMR (300 MHz, Chloroform-d) δ 7.7 (d, J = 7.6 Hz, 2H), 7.6 (d, J = 7.8 Hz, 2H), 7.5 (t, J = 7.6 Hz, 2H), 7.3–7.4 (m, 1H), 7.2 (d, J = 7.7 Hz, 2H), 6.0 (s, 1H), 4.4 (s, 2H), 2.4 (s, 3H).

3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-5-amine (1d) cream solid, isolated yield = 83% 1H NMR (300 MHz, DMSO-d6) δ 7.7 (m, 5H), 7.3–7.6 (m, 4H), 5.9 (s, 1H), 5.5 (s, 2H).

4-(5-amino-3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)benzene-1,2-diol (3a) brown solid, melting point: 170–172°C, isolated yield = 62%, 1H NMR (300 MHz, DMSO-d6) δ 9.0 (s, 2H), 7.7 (d, J = 7.9 Hz, 2H), 7.5 (m, 4H), 7.3–7.4 (m, 3H), 6.8 (d, J = 8.0 Hz, 1H), 6.6 (d, J = 2.1 Hz, 1H), 6.5 (dd, J = 8.0, 2.1 Hz, 1H), 4.9 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 147.2, 145.9, 145.0, 144.6, 139.5, 133.5, 131.5, 129.64, 124.0, 123.6, 121.2, 120.9, 117.6, 116.6, 104.1. MS: (EI, 70 eV): m/z = 423 [M+]. Anal. Calcd. for C21H16BrN3O2: C, 59.73; H, 3.82; N, 9.95. Found: C, 59.66; H, 3.81; N, 9.87.

4-(5-amino-1,3-diphenyl-1H-pyrazol-4-yl)benzene-1,2-diol (3b) brown solid, melting point: 145–147°C, isolated yield = 75%, 1H NMR (300 MHz, DMSO-d6) δ 8.9 (d, 2H), 7.7 (d, J = 7.8 Hz, 2H), 7.5 (t, J = 7.7 Hz, 2H), 7.4 (d, J = 7.0 Hz, 2H), 7.3–7.4 (m, 1H), 7.3 (d, J = 7.2 Hz, 3H), 6.7 (d, J = 7.6 Hz, 1H), 6.6 (s, 1H), 6.5 (d, J = 8.2 Hz, 1H), 4.8 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 148.4, 145.8, 144.8, 144.47, 139.6, 134.3, 129.6, 128.4, 127.7, 126.9, 123.4, 121.3, 117.7, 116.5, 104.2. MS: (EI, 70 eV): m/z = 343 [M+]. Anal. Calcd. for C21H17N3O2: C, 73.45; H, 4.99; N, 12.24. Found: C, 73.51; H, 4.31; N, 12.17.

4-(5-amino-1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)benzene-1,2-diol (3c) white solid, melting point: 188–190°C, isolated yield = 83%, 1H NMR (300 MHz, DMSO-d6) δ 8.7–9.0 (m, 2H), 7.7–7.8 (m, 2H), 7.5–7.6 (m, 2H), 7.3–7.4 (m, 3H), 7.0–7.2 (m, 2H), 6.7–6.9 (m, 1H), 6.6–6.7 (m, 1H), 6.5 (d, J = 9.2 Hz, 1H), 4.8 (s, 2H), 2.3 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 148.5, 145.8, 144.7, 144.4, 139.7, 136.9, 131.5, 129.6, 129.0, 127.6, 126.8, 124.5, 123.4, 121.3, 117.7, 116.5, 104.1, 21.2. MS: (EI, 70 eV): m/z = 357 [M+]. Anal. Calcd. for C22H19N3O2: C, 73.93; H, 5.36; N, 11.76. Found: C, 73.96; H, 5.40; N, 11.71.

4-(5-amino-1,3-diphenyl-1H-pyrazol-4-yl)-5-methylbenzene-1,2-diol (3d) brown solid, melting point: 145–147°C, isolated yield = 91%, 1H NMR (300 MHz, DMSO-d6) δ 8.7 (d, 2H), 7.7 (d, J = 7.8 Hz, 2H), 7.5 (t, J = 7.6 Hz, 3H), 7.4 (m, 3H), 7.2–7.3 (m, 2H), 6.7 (s, 1H), 6.5 (s, 1H), 4.7 (s, 2H), 1.9 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 148.3, 145.1, 145.0, 143.6, 139.8, 134.7, 129.6, 128.6, 128.5, 127.6, 126.6, 123.5, 123.1, 123.0, 118.9, 118.0, 103.4, 19.5. MS: (EI, 70 eV): m/z = 357 [M+]. Anal. Calcd. for C22H19N3O2: C, 73.93; H, 5.36; N, 11.76. Found: C, 73.91; H, 5.31; N, 11.67.

4-(5-amino-1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-5-methylbenzene-1,2-diol (3e) brown solid, melting point: 198–200°C, isolated yield = 93%, 1HNMR (300 MHz, DMSO-d6) δ 8.7 (d, 2H), 7.7 (d, J = 7.7 Hz, 2H), 7.5(t, J = 8.0 Hz, 3H), 7.3 (t, J = 7.0 Hz, 2H), 7.0 (d, J = 7.7 Hz, 2H), 6.7 (s, 1H), 6.5 (s, 1H), 4.7 (s, 2H), 2.2 (s, 3H), 1.9 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 148.3, 145.0, 144.9, 143.6, 136.8, 131.88, 129.6, 129.12, 126.6, 123.1, 118.0, 103.3, 21.2, 19.5. MS: (EI, 70 eV): m/z = 371 [M+]. Anal. Calcd. for C23H21N3O2: C, 74.37; H, 5.70; N, 11.31. Found: C, 74.31; H, 5.72; N, 11.37.

4-(5-amino-3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)benzene-1,2-diol (3f) brown solid, melting point: 143–145°C, isolated yield = 54%, 1H NMR (300 MHz, DMSO-d6) δ 7.72 (d, J = 7.9 Hz, 2H), 7.53 (t, J = 7.6 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 7.9 Hz, 3H), 6.78 (d, J = 8.0 Hz, 1H), 6.61 (s, 1H), 6.50 (d, J = 8.1 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ 147.2, 146.0, 145, 144.7, 139.5, 133.2, 132.3, 129.6, 129.3, 128.6, 127.0, 123.9, 123.5, 121.2, 117.7, 116.7, 104.1. MS: (EI, 70 eV): m/z = 377 [M+]. Anal. Calcd. for C21H16ClN3O2: C, 66.76; H, 4.27; N, 11.12. Found: C, 66.71; H, 4.31; N, 11.17.

4-(5-amino-1-(4-methoxyphenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)benzene-1,2-diol (3g) pale brown solid, melting point: 145–147°C, isolated yield = 94%, 1H NMR (300 MHz, DMSO-d6) δ 8.70 (d, J = 137.2 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 7.9 Hz, 2H), 7.06 (d, J = 8.3 Hz, 4H), 6.75 (d, J = 8.1 Hz, 1H), 6.66–6.53 (m, 1H), 6.53–6.34 (m, 1H), 4.71 (s, 2H), 3.80 (s, 3H), 2.25 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 158.2, 147.9, 145.8, 144.5, 144.4, 136.7, 132.6, 131.6, 129.0, 127.6, 125.4, 124.6, 121.2, 117.7, 116.5, 114.7, 103.6, 55.8, 21.2. MS: (EI, 70 eV): m/z = 387 [M+]. Anal. Calcd. for C23H21N3O3: C, 71.30; H, 5.46; N, 10.85. Found: C, 71.21; H, 5.31; N, 10.77.

4-(5-amino-1,3-di-p-tolyl-1H-pyrazol-4-yl)benzene-1,2-diol (3h) pale brown solid, melting point: 165–167°C, isolated yield = 91%, 1H NMR (300 MHz, DMSO-d6) δ 8.99 (s, 2H), 7.60 (d, J = 8.0 Hz, 2H), 7.44–7.23 (m, 4H), 7.09 (d, J = 7.9 Hz, 2H), 6.77 (d, J = 8.0 Hz, 1H), 6.68–6.56 (m, 1H), 6.56–6.43 (m, 1H), 4.79 (s, 2H), 2.38 (s, 3H), 2.28 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 148.2, 145.8, 144.5, 144.4, 137.2, 136.8, 136.2, 131.6, 130.03, 129.0, 127.6, 124.6, 123.5, 121.2, 117.7, 116.5, 103.9, 21.3, 21.1. MS: (EI, 70 eV): m/z = 371 [M+]. Anal. Calcd. for C23H21N3O2: C, 74.37; H, 5.70; N, 11.31. Found: C, 74.41; H, 5.61; N, 11.47.

4-(5-amino-1-(4-bromophenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)benzene-1,2-diol (3i) pale brown solid, melting point: 142–144°C, isolated yield = 42%, 1H NMR (300 MHz, DMSO-d6) δ 8.57 (s, 2H), 7.70 (s, 4H), 7.32 (d, J = 7.9 Hz, 2H), 7.09 (d, J = 7.8 Hz, 2H), 6.75 (d, J = 8.1 Hz, 1H), 6.58 (s, 1H), 6.47 (d, J = 8.0 Hz, 1H), 4.94 (s, 2H), 2.27 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 148.9, 145.8, 144.9, 144.5, 139.0, 137.1, 132.4, 131.2, 129.1, 127.7, 125.2, 124.3, 121.3, 119.2, 117.7, 116.5, 104.5, 21.3. MS: (EI, 70 eV): m/z = 437 [M+]. Anal. Calcd. for C22H18BrN3O2: C, 60.56; H, 4.16; N, 9.63. Found: C, 60.40; H, 4.11; N, 9.67.

4-(5-amino-3-(4-chlorophenyl)-1-(p-tolyl)-1H-pyrazol-4-yl)benzene-1,2-diol (3j) pale brown solid, melting point: 162–164°C, isolated yield = 59%, 1H NMR (300 MHz, DMSO-d6) δ 8.67 (s, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.50–7.39 (m, 2H), 7.34 (dd, J = 8.3, 5.5 Hz, 4H), 6.78 (d, J = 7.9 Hz, 1H), 6.61 (s, 1H), 6.49 (d, J = 8.0 Hz, 1H), 4.85 (s, 2H), 2.38 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 146.9, 145.9, 144.9, 144.6, 137.0, 136.4, 133.3, 132.23, 130.1, 129.3, 128.5, 124.1, 123.6, 121.2, 117.6, 116.6, 103.9, 21.1. MS: (EI, 70 eV): m/z = 391 [M+]. Anal. Calcd. for C22H18ClN3O2: C, 67.43; H, 4.63; N, 10.72. Found: C, 67.40; H, 4.61; N, 10.69.

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®). 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).

10.1371/journal.pone.0308036.g004 Fig 4 The model reaction.

10.1371/journal.pone.0308036.t001 Table 1 Optimization study.

Entrya	Enzyme (U)	Solvent system	Time (h)	Isolated yield (%)	
1	1000b	MeCN/ phosphate buffer pH 8, 10 mM (1:2)	24	0	
2	1000	EtOAc/ phosphate buffer pH 8, 10 mM (1:2)	4	55	
3	1000	EtOAc/citrate buffer pH 4.5, 10 mM (1:2)	4	89	
4	1000	EtOH/citrate buffer pH 4.5, 10 mM (1:2)	24	N.R.	
5	1000	MeCN/citrate buffer pH 4.5, 10 mM (1:2)	24	N.R.	
6	500	EtOAc/citrate buffer pH 4.5, 10 mM (1:2)	12	48	
7	0	EtOAc/citrate buffer pH 4.5, 10 mM (1:2)	24	N.R.	
8	1000	EtOAc /phosphate buffer pH 7, 10 mM (1:2)	8	21	
9	1000	MeCN/phosphate buffer pH 7, 10 mM (1:2)	24	N.R.	
aReaction conditions: 1c (0.1 mmol), 2a (0.15 mmol). bThe laccase activity as given by the supplier. N.R.: no reaction

1HNMR, 13CNMR and Mass spectrometry was used to prove the chemical structure of the products. The 1HNMR 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 13CNMR 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.

10.1371/journal.pone.0308036.g005 Fig 5 Chemical shifts of 3c in 1HNMR.

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).

10.1371/journal.pone.0308036.g006 Fig 6 Scope of reaction (Reaction conditions: Laccase (1000 U), 1 (0.1 mmol), 2 (0.15 mmol)).

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 imine-enamine tautomerization to afford the corresponding product 3b.

10.1371/journal.pone.0308036.g007 Fig 7 Proposed mechanism.

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.

Supporting information

S1 File 1H, 13C NMR and mass spectrum of all synthesized derivatives.

(DOCX)

The authors would like to acknowledge Novozymes for kindly providing enzymes for this research.
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