Synthesis and Characterization of Novel Methyl (3)5-(N-Boc-piperidinyl)-1H-pyrazole-4-carboxylates

Series of methyl 3- and 5-(N-Boc-piperidinyl)-1H-pyrazole-4-carboxylates were developed and regioselectively synthesized as novel heterocyclic amino acids in their N-Boc protected ester form for achiral and chiral building blocks. In the first stage of the synthesis, piperidine-4-carboxylic and (R)- and (S)-piperidine-3-carboxylic acids were converted to the corresponding β-keto esters, which were then treated with N,N-dimethylformamide dimethyl acetal. The subsequent reaction of β-enamine diketones with various N-mono-substituted hydrazines afforded the target 5-(N-Boc-piperidinyl)-1H-pyrazole-4-carboxylates as major products, and tautomeric NH-pyrazoles prepared from hydrazine hydrate were further N-alkylated with alkyl halides to give 3-(N-Boc-piperidinyl)-1H-pyrazole-4-carboxylates. The structures of the novel heterocyclic compounds were confirmed by 1H-, 13C-, and 15N-NMR spectroscopy and HRMS investigation.

The heterocyclic tripeptide Gly-Pro-Glu I, containing an L-proline residue, is a neuroprotective compound for the control of neurodegenerative processes such as Parkinson's Heterocyclic amino acids have been applied widely as building blocks for the preparation of DNA-encoded chemical libraries, including heterocyclic hybrid and peptide compounds [35][36][37][38][39]. In general, a DNA-encoded library of target component molecules should have a high degree of structural and functional diversity, taking into account diversity-oriented synthesis (DOS) [40]. For example, a highly specific and potent p38α kinase tripeptide-type inhibitor (VPC00628) V containing the residue of 3-amino-1-phenyl-1H-pyrazole-4-carboxylic acid has been identified directly from a multimillion-membered DNA-encoded molecule library that was prepared using highfidelity yoctoReactor (yR) technology [41].

Results and Discussion
Numerous methods for forming pyrazole ring systems have been developed. The most common synthetic method for the production of pyrazoles is the condensation of the corresponding hydrazine derivative, which acts as a double nitrogen nucleophile, with three carbon units containing compounds such as 1,3-dicarbonyl and 2,3-unsaturated carbonyl, or enamine [45][46][47]. Rosa et al. [48] developed a simple and efficient method for preparing both regioisomers of 4,5-substituted N-phenylpyrazoles from β-enamino diketones and phenylhydrazine, and the regiochemistry of the reaction was protic or aprotic solvent depen-dent. A patent [49] was obtained for the synthesis of 4-(piperidin-4-yl)-N-phenylpyrazole derivatives from β-enamino diketones with 4-fluoro-and 4-methoxyphenylhydrazines.
Our strategy for the synthesis of methyl 3(5)-(N-Boc-piperidinyl)-1H-pyrazole-4carboxylates according to the enamine method is described in Schemes 1 and 2, and Figure 2. The synthetic sequence started with preparing β-keto esters 2a-c by treating N-Boc protected piperidine acids 1a-c with 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum's acid) in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 4-dimethylaminopyridine (DMAP), and further methanolysis of Meldrum's acid adduct [50,51]. Compounds 2a-c were treated with N,N-dimethylformamide dimethyl acetal (DMF·DMA) to obtain β-enamino diketones 3a-c [49]. In the next step, we investigated the formation of 3(5)-substituted-1H-pyrazoles 5 and 6 via the key intermediates 4 and 4 (Scheme 2). Optimization of the coupling reaction conditions was undertaken, choosing 3a and phenylhydrazine as a model system (Table 1). An investigation of the reaction course and regioselectivity was carried out in various solvents, and the LC/MS and 1 H-NMR spectral data of the crude reaction mixture of intermediate compound 4a and products 5a, 6a were analyzed after 1 and 18 h (Table 1). EtOH was used as a polar protic (Table 1, entry 1), ACN as a polar aprotic (Table 1, entry 2), and CCl 4 as a nonpolar solvent (Table 1, entry 3). As a result, the reaction in EtOH provided high regioselectivity (99.5%) and good yield (78%) of 5a and just traces of its regioisomer 6a (Table 1, entry 1). Similarly, the reaction in ACN resulted in 5a as the main product (75%), and 6a was obtained with a 3% yield (Table 1, entry 2). The poorest yield and regioselectivity were observed when the reaction mixture was stirred in CCl 4 . In this case, 5a formed as a major product with 54% yield, and regioisomer 6a was obtained with 9% yield (Table 1,  entry 3). During optimization of the reaction conditions in different solvents, 1 H-NMR analysis of the crude reaction mixture after 1 h also showed the formation of intermediate compound 4a, which was successfully isolated for structure elucidation. The regioisomer 6a formed as a minor isomer via intermediate 4 a which resulted from the nucleophilic attack of a secondary amino group of phenylhydrazine on β-enamino diketone 3a.  In the case of intermediate compound 4a, the key information for structure elucidation was obtained from the 15 N-NMR data. In the 1 H-15 N HMBC spectrum of 4a, the 15 N shift of δ −241.2 ppm was assigned to nitrogen N a , due to the correlation with the neighboring protons 2 (6 )-H (δ 6.81 ppm) from the phenyl moiety ( Figure 3). The 1 H-15 N HSQC experiment indicated that proton N a -H (δ 6.24 ppm) had one-bond connectivity with the aforementioned nitrogen N a at δ −241.2 ppm, while proton N b -H (δ 11.72 ppm) generated a cross peak with nitrogen N b at δ −275.1 ppm. The formation of compound 4a was also confirmed by a NOESY experiment, which exhibited NOEs between the 2 (6 )-H protons at δ 6.81 ppm and the enamine proton at δ 8.28 ppm. However, the configuration of the (2E or 2Z)-isomer of compound 4a is not yet known.
Discrimination between regioisomeric compounds 5a and 6a was based on data from 1 H-13 C HMBC, 1 H-15 N HMBC, and 1 H-1 H NOESY experiments ( Figure 3). The 1 H-15 N HMBC experiment of the major regioisomer 5a revealed three-bond correlations between the piperidine 4 -H proton at δ 3.10 ppm and the phenyl group 2 (6 )-H protons at δ 7.34 ppm, with the pyrazole N-1 "pyrrole-like" nitrogen at δ -160.3 ppm [52,53]. The 1 H-1 H NOESY spectrum of 5a exhibited NOEs between the phenyl group 2 (6 )-H protons and the 4 -H proton from the piperidine moiety. The second regioisomer 6a was easily identified by utilizing a similar approach. The minor regioisomer 6a exhibited a strong three-bond connectivity between the piperidine proton 4 -H (δ 3.43 ppm) and the pyrazole N-2 "pyridine-like" nitrogen at δ -81.5 ppm, while the phenyl group protons 2 (6 )-H (δ 7.68 ppm) showed three-bond connectivity with the pyrazole N-1 "pyrrole-like" nitrogen at δ -165.7 ppm. Moreover, the pyrazole 5-H proton in the 1 H-13 C HMBC spectrum showed a three-bond connectivity with the phenyl group C-1 carbon at δ 139.3 ppm. Finally, confirmation of these regiochemical assignments was obtained from the 1 H-1 H NOESY 6a spectrum, showing only the NOEs between the phenyl group 2 (6 )-H protons and the pyrazole 5-H proton (δ 8.34 ppm).
The optimal conditions for the regioselective synthesis of methyl 5-(N-Boc-piperidinyl)-1H-pyrazole-4-carboxylate 5a were applied to the synthesis of other pyrazoles to evaluate the scope of the methodology (Figure 2). β-Enamino diketone 3a was coupled with different phenylhydrazines to give corresponding products 5b-h with fair to good yields. No obvious effect of the phenylhydrazine substituent on the reaction yield was observed. A reaction of β-enamino diketone 3a with methylhydrazine provided a corresponding tert-butyl 4-[4-(methoxycarbonyl)-1-methyl-1H-pyrazol-5-yl]piperidine-1-carboxylate 5i with a 51% yield. To our delight, the reactions of chiral β-enamino diketones 3b,c with phenyl-, (4-methylphenyl)-or [3-(trifluoromethyl)phenyl]hydrazines formed products 5j-o, also with good yields. While analyzing the LC/MS and 1 H-NMR spectral data of crude cyclization reaction mixtures, the formation of the regioisomeric 6b-o was observed at trace amounts. The structure of compounds 5b-o was determined by analogous NMR spectroscopy experiments as described above.
Next, having β-enamino diketone 3a, we also performed a cyclocondensation reaction with hydrazine hydrate under the conditions described above, and the formation of tautomeric 3(5)-substituted NH-pyrazole 7 was established by NMR analysis (Scheme 3, Figure 4). Scheme 3. Two tautomers, 7a and 7b, of 3(5)-substituted NH-pyrazole (7) and regioisomers 5i, 6i, and compound 8. The prototropic tautomerism of NH-pyrazoles is well documented in many scientific studies, including with the use of multinuclear dynamic NMR spectroscopy [54][55][56]. In general, the annular tautomerism of 3(5)-1H-pyrazoles in solution under normal conditions is a very rapid process on the NMR time scale, and the determination of tautomeric ratios can usually be achieved only at low temperatures [57]. We carried out NMR studies of compound 7 at 25 • C in a diluted CDCl 3 solution ( Figure 4). The 1 H-NMR spectrum of compound 7 revealed a narrow singlet of the pyrazole ring proton resonating at δ 7.96 [3(5)-H] and two singlets for methyl ester and Boc moiety protons in the area of δ 3.83 (OCH 3 ) and 1.47 [C(CH 3 ) 3 ] ppm, respectively. The 13 C-NMR spectrum provided important information; as expected, the characteristic signal of the pyrazole C-4 carbon at δ 110.1 ppm remained sharp, while the other two signals of pyrazole ring carbons 3(5)-C resonated at δ 138.7 and 153.6 ppm and appeared broadened. It is known that the broadening of NMR spectral lines very often reflects dynamic structural transformations of molecules in solution [58]. Therefore, the observed broadness of relevant C-3 and C-5 pyrazole carbon signals is due to the coalescence of individual signals to average signals, indicating tautomeric equilibrium of 7 (7a and 7b). In addition, the pyrazole NH proton (δ 11.52 ppm) exhibited NOEs not only with the pyrazole ring proton at 7.96 ppm but also with the 3 -H piperidine protons at 1.70 ppm, which is only possible in the case of annular tautomerism 7.
It was not possible to obtain relevant information for the nitrogen atoms of the pyrazole ring N-1 and N-2 from the 15 N-NMR spectral data since 1 H-15 N HSQC and HMBC experiments showed no direct or long-range correlations with appropriate protons. Tautomeric compound 7 was alkylated with alkyl iodides (Scheme 3). It is known that N-alkylation of asymmetrically ring-substituted 1H-pyrazoles generally results in the formation of a mixture of regioisomeric N-substituted products [59]. Treatment of compound 7 with methyl iodide in the presence of KOH in DMF gave an inseparable mixture of regioisomers 5i and 6i in a ratio of about 1:5 and a total yield of 74%. However, alkylation of 1H-pyrazole-4-carboxylate 7 with ethyl iodide under analogous conditions afforded compound 8 as the sole product with a good 87% yield.
Discrimination of regioisomeric compounds 5i and 6i were based on 1 H-13 C HMBC, 1 H-15 N HMBC, and 1 H-1 H NOESY spectral data ( Figure 4). In the 1 H-15 N HMBC spectra of minor regioisomer 5i, a 15 N shift of δ −178.3 ppm was assigned to the "pyrrole-like" nitrogen N-1 due to the correlation of this signal with a piperidine ring proton 4 -H (δ 3.54 ppm). The 1 H-13 C HMBC experiment exhibited a three-bond correlation of the 1-CH 3 protons with a pyrazole quaternary carbon C-5 at δ 148.7 ppm. Moreover, the 1 H-1 H NOESY spectrum of 5i exhibited NOEs between the methyl group protons (1-CH 3 ) at 3.92 ppm and the piperidine proton 4 -H at δ 3.54 ppm. In the 1 H-15 N HMBC spectra of the major regioisomer 6i, an appropriate correlation between the piperidine ring proton 4 -H (δ 3.36 ppm) and the "pyridine-like" pyrazole N-2 nitrogen which resonated at δ −77.3 ppm could be observed. The 1 H-13 C HMBC spectral data of compound 6i provided a strong three-bond correlation of 1-CH 3 protons with pyrazole protonated carbon C-5 at δ 134.6 ppm. Finally, the regiochemistry of compound 6i was confirmed by a NOESY experiment, which exhibited NOEs between the 1-CH 3 protons and pyrazole proton 5-H (δ 7.78 ppm). The structure of compound 8 was determined by analogous NMR spectroscopy experiments as described above.
Pyrazole carboxylic acid amides, including anilides, have been known to play an important role in agrochemical research as fungicides [60,61]. Pyrazole-4-carboxylic acids 9a-c were used to obtain new anilide compounds (Scheme 4). First, 9a reacted with aniline in the presence of EDC·HCl, DMAP, and dichloromethane to give pyrazole anilide 10a. Moreover, chiral pyrazole anilide (R)-10b (100% ee) was obtained from carboxylic acid 9b, while the corresponding chiral anilide (S)-10c (96% ee) was synthesized from carboxylic acid 9c. The enantiomeric purity of prepared anilides 10b,c was evaluated by chiral HPLC analysis. As an example, HPLC analysis of enantiomeric samples of anilides 10b,c is shown in Figure 5.

Synthesis of tert-Butyl 3-and
To a solution of the corresponding l-(tert-butoxycarbonyl)piperidinecarboxylic acid (1a-c) (4 g, 17.4 mmol) in DCM (24 mL) cooled to 0 • C temperature Meldrum's acid (2.77 g, 19.2 mmol) was added followed by DMAP (4.26 g, 34.9 mmol). Then EDC·HCl (3.68 g, 19.2 mmol) was added in portions over 10 min. The reaction mixture was gradually warmed to r.t. and stirred for 16 h. The reaction solution was diluted with DCM (10 mL), washed with 1 M KHSO 4 (2 × 15 mL) and brine (20 mL). The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Then the residue was dissolved in MeOH (20 mL) and left under reflux for 5 h. The solvent was evaporated in vacuo. A solution of crude β-keto ester (2a-c) (4.7 g, 16.4 mmol) and N,Ndimethylformamide dimethyl acetal (4.4 mL, 32.8 mmol) in dioxane (24 mL) was stirred at 100 • C. After 5 h the solvent was removed under reduced pressure. Crude compounds 3a-c were carried forward without any further purification.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.