Synthesis of Diversified Pyrazolo[3,4-b]pyridine Frameworks from 5-Aminopyrazoles and Alkynyl Aldehydes via Switchable C≡C Bond Activation Approaches

A cascade 6-endo-dig cyclization reaction was developed for the switchable synthesis of halogen and non-halogen-functionalized pyrazolo[3,4-b]pyridines from 5-aminopyrazoles and alkynyl aldehydes via C≡C bond activation with silver, iodine, or NBS. In addition to its wide substrate scope, the reaction showed good functional group tolerance as well as excellent regional selectivity. This new protocol manipulated three natural products, and the arylation, alkynylation, alkenylation, and selenization of iodine-functionalized products. These reactions demonstrated the potential applications of this new method.


Introduction
A series of natural products and biologically active molecules contain pyrazolo [3,4b]pyridine as a key structural motif [1,2]. Several of these compounds are effective antienterovirals, antimalarials, anticancer agents, and kinase inhibitors ( Figure 1) [3][4][5]. This has inspired the development of efficient methods to construct these compounds and has become a hot topic in modern organic synthesis. Recently, catalytic carbon-carbon bond activation has emerged as a useful tool to build complex molecules rapidly and efficiently [6][7][8][9]. There are versatile intermediates involved in these reactions, which could be trapped in situ by a second molecule that triggers subsequent tandem reactions [10][11][12][13][14][15]. The nucleophilic/electrophilic addition reactions of alkynes are well-known and provide a convenient way to synthesize functionalized molecules [16][17][18][19][20]. The high reactivity, good selectivity, excellent functional-group tolerance, and mild reaction conditions of these reactions have inspired significant research over the past few decades. Generally, this process forms highly active intermediates using transition metals, such as Ag, Au, Rh, Cu, and Co [21][22][23][24][25][26][27], or electrophiles like I 2 , NXS (X = I, Br, Cl), Se, S, and P (Scheme 1a) [28][29][30][31][32][33][34]. Each reagent type sees significant use in the development of synthetic methodologies and applications to prepare bioactive compounds or complex naturally occurring skeletons. However, to our knowledge, among those strategies, a direct and efficient protocol for the selective synthesis of polysubstituted and functionalized fused heterocycles, such as halogen-functionalized pyrazolo [3,4-b]pyridine frameworks, by C≡C bond activation has seldom been described. Thus, developing convenient and sustainable synthetic methods to build these high-value compounds merits attention. As a kind of synthetic block with bifunctional groups (C≡C and carbonyl), alkynyl aldehydes are essential synthons with rich and unexpected chemical properties [35][36][37][38]. Tandem cyclization reactions using alkynyl aldehydes as synthons yields a variety of heterocycles. Generally, tandem cyclization occurs in one of two ways, 5-exo-dig or 6endo-dig cyclizations. For example, some efficient synthesis strategies have been reported for the synthesis of multi-substituted thiazoles, imidazo[1,2-a]pyridines, and imidazoles by using alkynyl aldehydes as synthons via 5-exo-dig cyclization [39][40][41]. The 6-endo-dig cyclization of alkynyl aldehydes is an alternative method to construct complex fused ring systems (Scheme 1b) [42][43][44]. These protocols typically use simple starting materials, with good functional tolerance and high yields. Inspired by these achievements, we sought to selectively activate the C≡C bond by changing the reaction conditions to obtain a series of compounds with a pyrazolo [3,4-b]pyridine structure core.
Next, various 5-aminopyrazoles and alkynyl aldehydes were tested to determine the scope of iodine-functionalized products (Scheme 4). These reactions produced the corresponding products 5a-5j 58-68% yields. Meanwhile, 3-methylisoxazol-5-amine tolerated the reaction conditions and reacted with 3-phenylpropiolaldehyde (2a) to generate 5k in moderate yield. When iodine was replaced by NBS, the expected compounds 5l-5r were obtained in moderated yields (53-66%). However, after many trials, the Cl-functionalized product 5s was not obtained. To demonstrate the applicability of this method, we modified natural products (Scheme 5a). For example, estrone, formononetin, and eudistomin Y 1 are all biologically active natural products, and these compounds have phenolic hydroxyl groups that undergo conversion into trifluoromethane sulfonates. Those sulfonates undergo Sonogashira coupling and deketalization to afford alkynaldehyde intermediates (6, 8, and 10). By using these alkynaldehyde intermediates as substrates for this protocol, we successfully obtained three natural product functionalized pyrazolo [3,4-b]pyridines in moderate to good yields (7, 9, and 11). Because heteroaryl iodides are highly useful functional structures in synthetic organic chemistry, additional applications of iodine-functionalized products were conducted (Scheme 5b) [45]. A series of coupling reactions were examined to form iodine-functionalized products, including Suzuki, Sonogashira, and Heck couplings that yielded the expected products 13-15 in good yields. Furthermore, selenization of iodinefunctionalized products afforded 16 in very good yield (78%). The reaction of 1a and 2a was scaled up to 5 mmol to illustrate the potential applications of this method; 3a and 5a formed 71% and 53% yields, respectively (Scheme 6). This promising result lays a good foundation for large-scale syntheses. Relevant control experiments were conducted to probe the reaction mechanism for the formation of pyrazolo [3,4-b]pyridine frameworks. When the reaction of 1a with 2a was conducted without acid for 2 h, it afforded 3a and 3a in 40% and 46% yields, respectively, (Scheme 7a). Intermediate 3a was confirmed by TLC-MS(APCI), LC-HRMS, and NMR (the details can be seen in Supplementary Materials). In addition, intermediate 3a transform to 3a in 88% yield under standard conditions (Scheme 7b). These results suggested that 3a may serve as the intermediate in this reaction. To illustrate regioselectivity, we chose cinnamaldehyde (2a ) as a substrate to react with 1a under standard conditions. Compared with a standard simple, 3-methyl-1,4-diphenyl-1H-pyrazolo [3,4-b]pyridine (17) was obtained in 45% yield, no product 3a was observed (Scheme 7c). These results confirmed the regioselectivity of this method, as it only afforded the C6 substituted pyrazolo [3,4b]pyridine for alkynyl aldehydes substrates. Furthermore, when 1 eq. of iodine was added, non-iodinated and iodized products 3a and 5a were detected in 46% and 30% yields, respectively (Scheme 7d). Considering the aforementioned control experiments and earlier works [46][47][48], a reaction mechanism is shown in Scheme 8 using 1a with 2a as a typical reaction. Initially, 3-methyl-1phenyl-1H-pyrazol-5-amine (1a) undergoes condensation with 3-phenylpropiolaldehyde (2a) to form intermediate 3a . Next, the silver salt coordinates to the alkyne of 3a to form intermediate A; this undergoes 6-endo-dig cyclization to form B. Finally, B undergoes demetallation to afford product 3a (Scheme 8, pathway A). Similarly, I 2 or NBS adds to a triple bond that leads to intermediate A', which undergoes 6-endo-dig cyclization to form B', followed by acid loss from B' to obtain 5a or 5l (Scheme 8, pathway B).

General Information
Aminopyrazoles and NBS were purchased from Shanghai Shaoyuan Co. Ltd. (Shanghai, China) 3-substituted propiolaldehyde and Ag(CF 3 CO 2 ) were purchased from Leyan. Unless stated otherwise, all solvents and commercially available reagents were obtained from commercial suppliers and used without further purification. In addition, petroleum ether (b.p. 60-90 o C) was distilled prior to use for Column chromatography. Non-commercial starting materials were prepared as described below or according to literature procedures. TLC analysis was performed using pre-coated glass plates. Column chromatography was performed using silica gel (200-300 mesh). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Advance 400 MHz spectrometer at ambient temperature using the non or partly deuterated solvent as internal standard ( 1 H: δ 7.26 ppm and 13 C{ 1 H}: δ 77.0 ppm for CDCl 3 ; 1 H: δ 2.50 ppm and 13 C{ 1 H}: δ 40.0 ppm for DMSO-d 6 ). Chemical shifts (δ) are reported in ppm, relative to the internal standard of tetramethylsilane (TMS). The coupling constants (J) are quoted in hertz (Hz). Resonances are described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad) or combinations thereof. High resolution mass-spectrometric (HRMS) were obtained on an Apex-Ultra MS equipped with an electrospray source. Melting points were determined using SGW X-4 apparatus and not corrected. The X-ray diffraction data for the crystallized compound were collected on a Bruker Smart APEX CCD area detector diffractometer (graphite monochromator, Mo Kα radiation, λ = 0.71073 Å) at 296(2) K. All the heating procedures were conducted with an oil bath.

Synthetic Procedures
Typical Procedure (TP 1) for the Synthesis of 3 and 4 Taking 3a as an Example. A 25 mL pressure vial was charged with 1a (34.6 mg, 0.20 mmol, 1.0 equiv.), 2a (26 mg, 24.5 uL, 0.20 mmol, 1.0 equiv.), Ag(CF 3 CO 2 ) (4.4 mg, 0.02 mmol, 10 mol%), TfOH (9 mg, 5.3 uL, 0.06 mmol, 30 mol%) and DMAc (1.5 mL).The vial was sealed and the reaction mixture was stirred at 100 • C for 2 h under air atmosphere (monitored by TLC). After the reaction was completed, and added 50 mL water to the mixture, then extracted with EtOAc 3 times (3 × 50 mL). The solution was dried over anhydrous Na 2 SO 4 , concentrated under reduced pressure, and dried under vacuum. The residue was purified by flash column chromatography by using ethyl acetate/petroleum ether mixture to obtain the corresponding product 3a.
Typical Procedure (TP 2) for the Synthesis of 5 Taking 5a as an Example. A 25 mL pressure vial was charged with 1a (34.6 mg, 0.20 mmol, 1.0 equiv.), 2a (26 mg, 24.5 uL, 0.20 mmol, 1.0 equiv.), I 2 (101.5 mg, 0.40 mmol, 2.0 equiv.), TfOH (30 mg, 17.6 uL, 0.20 mmol, 1.0 equiv.) and DMSO (2.0 mL). The vial was sealed, and the reaction mixture was stirred at 100 • C for 6 h under air atmosphere (monitored by TLC). After the reaction was completed, and added 50 mL water to the mixture, then extracted with EtOAc 3 times (3 × 50 mL). The extract was washed with 10% Na 2 S 2 O 3 solution, dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by flash column chromatography by using ethyl acetate/petroleum ether mixture to obtain the corresponding product 5a.
General Procedure for Synthesis of 13. K 2 CO 3 (0.4 mmol, 2.0 equiv.), phenylboronic acid (0.26 mmol, 1.3 equiv.) and PdCl 2 (PPh 3 ) 2 (5 mol%) were added to a solution of 5a (0.2 mmol, 1.0 equiv.) in a 5:1 solvent mixture of dioxane and water. The reaction mixture was heated to 90 • C and stirred at this temperature until complete consumption of 5a was observed (monitored by TLC). After cooling to room temperature, the mixture was diluted with a mixture of EA and water and the aqueous layer was extracted with EtOAc (3 × 50 mL). dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by flash column chromatography by using ethyl acetate/petroleum ether mixture to obtain the desired product 13 in 88% yield.
General Procedure for Synthesis of 14. 5a (0.2 mmol, 1 equiv.), PdCl 2 (PPh 3 ) 2 (5 mol%), CuI (10 mol%) and phenylacetylene (0.3 mmol, 1.5 equiv.) were added to a 25 mL Schlenk flask with a stir bar under an Ar atmosphere. Then DMF (2 mL) and TEA (1 mL) were added sequentially. The reaction mixture was then stirred at 90 • C. Afterwards 15 mL of water were added, and the reaction mixture was extracted with EtOAc (3 × 50 mL). The combined organic fractions were washed with brine and dried over Na 2 SO 4 . After filtration, the solvent was removed under reduced pressure. The residue was purified by flash column chromatography by using ethyl acetate/petroleum ether mixture to obtain the desired product 14 in 85% yield.
The combined organic fractions were washed with brine and dried over Na 2 SO 4 . After filtration, the solvent was removed under reduced pressure. The residue was purified by flash column chromatography by using ethyl acetate/petroleum ether mixture to obtain the desired product 15 in 75% yield.
General Procedure for Synthesis of 16. Adapting a literature procedure [49], A 25 mL Schlenk flask with a stir bar was charged with 5a (0.2 mmol, 1.0 equiv.), diphenyl diselenide (0.14 mmol, 0.7 equiv.) CuI (0.02 mmol, 10 mol%) and Cs 2 CO 3 (0.4 mmol, 2.0 equiv.) in MeCN (2.0 mL). The vial was sealed and the resulting mixture was stirred at 80 • C for 24 h under an Ar atmosphere. After the reaction completed, and added 50 mL water to the mixture, then extracted with EtOAc 3 times (3 × 50 mL). The extract was washed with brine, dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by flash column chromatography by using ethyl acetate/petroleum ether mixture to obtain the desired product 16 in 78% yield.

Conclusions
In summary, a cascade 6-endo-dig cyclization reaction was developed for the switchable synthesis of halogen and non-halogen-functionalized pyrazolo [3,4-b]pyridines from 5aminopyrazoles and alkynyl aldehydes. This method afforded diversified pyrazolo [3,4b]pyridine frameworks via C≡C bond activation with silver, iodine, or NBS. The protocol was characterized by a wide substrate scope, good functional group tolerance, and excellent regional selectivity. The structural modification of estrone, formononetin, and eudistomin Y 1 provided new ideas for syntheses of drug molecules. Iodine functionalization allowed several additional transformations, including arylation, alkenylation, alkynylation, and selenization to fabricate useful molecules.

Patents
A patent (Yantai University, CN 112300157, and 2021 A) has been derived from this manuscript. The patent is entitled Novel pyrazolopyridine compound with antitumor activity and preparation method thereof.