One-Pot Synthesis of Isoxazole-Fused Tricyclic Quinazoline Alkaloid Derivatives via Intramolecular Cycloaddition of Propargyl-Substituted Methyl Azaarenes under Metal-Free Conditions

A practical method was developed for the convenient synthesis of isoxazole-fused tricyclic quinazoline alkaloids. This procedure accesses diverse isoxazole-fused tricyclic quinazoline alkaloids and their derivatives via intramolecular cycloaddition of methyl azaarenes with tert-butyl nitrite (TBN). In this method, TBN acts as the radical initiator and the source of N–O. Moreover, this protocol forms new C–N, C–C, and C–O bonds via sequence nitration and annulation in a one-pot process with broad substrate scope and functionalization of natural products.


Substrate Scope
After the optimum conditions were established, the substrate scope of 2-methyl-3-(prop-2-yn-1-yl)quinazolin-4(3H)-one for the formation of 4H,6H-isoxazolo [3 ,4 :3,4]pyrrolo[2,1b]quinazolin-6-one 2 was evaluated. As shown in Figure 2, a series of electron-donating groups on the phenyl ring of 1, such as the methyl groups at the C-4, C-4,6, C-5,6 position (1b-1d) and the methoxyl group at the C-5 position (1e), could participate in this reaction to afford the corresponding products in 73-83% yields (2b-2e). To our delight, 1 with a phenyl group in the position of C-5 (1g-1h) gave the corresponding products smoothly in 75% and 50% yields, respectively. However, 1f was not tolerated in the reaction under the optimized conditions, giving the desired product in a very low yield. Meanwhile, electron-drawing groups on the phenyl ring of 1 proceeded well via our protocol, such as the fluoro group at the position C-5, C-4,5 (1i-1j), the chloro and nitro group at the C-4 position (1k-1l), leading to the desired products in good yields (60-76%). To our delight, heteroatom-contained substrate 1m was also suitable for this reaction to afford the desired product 2m in 46% yield. In addition, the substrates of the substituted alkynyl group were also tested; those results showed that alkynyl substituted substrate (1n) was unsuitable for this reaction.

Substrate Scope
After the optimum conditions were established, the substrate scope of 2-methyl-3-(prop-2-yn-1-yl)quinazolin-4(3H)-one for the formation of 4H,6H-isoxazolo[3′,4′:3,4]pyrrolo[2,1-b]quinazolin-6-one 2 was evaluated. As shown in Figure 2, a series of electrondonating groups on the phenyl ring of 1, such as the methyl groups at the C-4, C-4,6, C-5,6 position (1b-1d) and the methoxyl group at the C-5 position (1e), could participate in this reaction to afford the corresponding products in 73-83% yields (2b-2e). To our delight, 1 with a phenyl group in the position of C-5 (1g-1h) gave the corresponding products smoothly in 75% and 50% yields, respectively. However, 1f was not tolerated in the reaction under the optimized conditions, giving the desired product in a very low yield. Meanwhile, electron-drawing groups on the phenyl ring of 1 proceeded well via our protocol, such as the fluoro group at the position C-5, C-4,5 (1i-1j), the chloro and nitro group at the C-4 position (1k-1l), leading to the desired products in good yields (60-76%). To our delight, heteroatom-contained substrate 1m was also suitable for this reaction to afford the desired product 2m in 46% yield. In addition, the substrates of the substituted alkynyl group were also tested; those results showed that alkynyl substituted substrate (1n) was unsuitable for this reaction.  To further investigate the universality of our method, a variety of substituted 2-methyl-3-(prop-2-yn-1-yloxy)quinolines were surveyed. As shown in Figure 3, 2-methyl-3-(prop-2-yn-1-yloxy)quinoline (3a) was smoothly transformed into the corresponding product with a yield of 80%. Substrates of substituted groups on the phenyl ring 3b and 3c were applied to this reaction, which yielded products 4b and 4c in 77% and 24% yields,  To further investigate the universality of our method, a variety of substituted 2-methyl-3-(prop-2-yn-1-yloxy)quinolines were surveyed. As shown in Figure 3, 2-methyl-3-(prop-2yn-1-yloxy)quinoline (3a) was smoothly transformed into the corresponding product with a yield of 80%. Substrates of substituted groups on the phenyl ring 3b and 3c were applied to this reaction, which yielded products 4b and 4c in 77% and 24% yields, respectively. Then, ester-substituted substrates in the C-4 position were also screened, and all substrates were transformed smoothly to obtain the desired products in moderate to good yields (4d-4h, 56-79%). Moreover, different amide-substituted substrates on the C-4 position (3i-3j) were tested, processing the corresponding products 4i and 4j in 43% and 51% yields, respectively. Furthermore, to evaluate the application of the present reaction. Several natural products were modified via our protocol. As shown in Figure 4, 2-methyl-3-(prop-2-yn-1yloxy)quinoline-4-carboxylic acid reacted with natural alcohols to generate ester derivates 3. The L-menthol and sugar methyl 2,3-O-isopropylideneisopropylidene-β-D-ribofuranoside could be involved in this protocol, giving the desired products (4k-4l) in 22-35% yields. The 3m phytol derivate was smoothly transformed into the corresponding product with a yield of 67%. Two natural steroids, cholesterol and stigmasterol, were also screened, affording the corresponding products (4n-4o) in 29-33% yields. Furthermore, to evaluate the application of the present reaction. Several natural products were modified via our protocol. As shown in Figure 4, 2-methyl-3-(prop-2-yn-1yloxy)quinoline-4-carboxylic acid reacted with natural alcohols to generate ester derivates 3. The L-menthol and sugar methyl 2,3-O-isopropylideneisopropylidene-β-D-ribofuranoside could be involved in this protocol, giving the desired products (4k-4l) in 22-35% yields. The 3m phytol derivate was smoothly transformed into the corresponding product with a yield of 67%. Two natural steroids, cholesterol and stigmasterol, were also screened, affording the corresponding products (4n-4o) in 29-33% yields.

Mechanism Experiments
Several control experiments were carried out to explore the reaction mechanism (Scheme 2). The reaction was halted completely with only trace amounts of 2a, when 3.5 equivalent of radical scavenger 2,2,6,6-tetramethyl-1-piperidinyl (TEMPO) was added to the standard reaction. These results indicated that the reaction was conducted possibly through a radical pathway. Then, 2-methyl-3-(prop-2-yn-1-yl)quinazolinone (1a) and TBN were reacted under standard conditions for 20 min to address the possible intermediates.
Only 2a 4H,6H-isoxazolo-pyrrolo[2,1-b]quinazolin-6-one was detected by MS (APCI), because the intermediate nitrile oxide F (Scheme 3) shares the same relative molecular mass as 2a. Then, we tried other ways to prove them by conducting substrates 5a 2-methyl-3-phenyl quinazoline-4(3H)-one under standard conditions for 20 min to detect 5ac nitrile oxides via MS (APCI) (see Supplementary Materials). A group of intermolecular reactions was used to explore the reaction mechanism further by using 5a, 5ab, and phenylacetylene. Under optimal conditions, the desired product 6a was afforded in yields of 56% and 65%, respectively. These results disclosed that nitrile oxide was the potential intermediate for this protocol.

Mechanism Experiments
Several control experiments were carried out to explore the reaction mechanism (Scheme 2). The reaction was halted completely with only trace amounts of 2a, when 3.5 equivalent of radical scavenger 2,2,6,6-tetramethyl-1-piperidinyl (TEMPO) was added to the standard reaction. These results indicated that the reaction was conducted possibly through a radical pathway. Then, 2-methyl-3-(prop-2-yn-1-yl)quinazolinone (1a) and TBN were reacted under standard conditions for 20 min to address the possible intermediates. Only 2a 4H,6H-isoxazolo-pyrrolo[2,1-b]quinazolin-6-one was detected by MS (APCI), because the intermediate nitrile oxide F (Scheme 3) shares the same relative molecular mass as 2a. Then, we tried other ways to prove them by conducting substrates 5a 2-methyl-3phenyl quinazoline-4(3H)-one under standard conditions for 20 min to detect 5ac nitrile oxides via MS (APCI) (see Supplementary Materials). A group of intermolecular reactions was used to explore the reaction mechanism further by using 5a, 5ab, and phenylacetylene. Under optimal conditions, the desired product 6a was afforded in yields of 56% and 65%, respectively. These results disclosed that nitrile oxide was the potential intermediate for this protocol. Based on the evidence presented above and the related literature [75][76][77], a plausible reaction pathway was proposed (Scheme 3). Firstly, TBN was transformed to NO and t-BuO radicals via thermal homolysis.

General Information
Analytical thin layer chromatography (TLC) was performed using pre-coated silica gel HF254 glass plates. Column chromatography was performed using silica gel (200-300 mesh). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Advance Based on the evidence presented above and the related literature [75][76][77], a plausible reaction pathway was proposed (Scheme 3). Firstly, TBN was transformed to NO and t-BuO radicals via thermal homolysis.

General Information
Analytical thin layer chromatography (TLC) was performed using pre-coated silica gel HF254 glass plates. Column chromatography was performed using silica gel (200-300 mesh). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Advance 500 MHz spectrometer at ambient temperature using DMSO-d 6 or CDCl 3 as the solvent with tetramethylsilane (TMS) as the internal standard at room temperature ( 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 39.5 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 spectra were obtained on Thermo Scientific Q-Exactive (ESI mode). Melting points were determined using SGW X-4 apparatus and were not corrected.

Synthetic Procedures
Compounds 1a-1l were prepared according to the referenced literature (Scheme 4) [78]. To corresponding 2-aminobenzoic acid (1 mmol) was added acetic anhydride (5 mmol), and the mixture was warmed to 120 • C for 3 h with stirring. The mixture was then concentrated in vacuo (50 • C) to remove excess acetic anhydride (bp 138 • C) to give a dry solid. Ammonium hydroxide (28% NH 3 , 100 mL) was added, and the mixture was heated to 95 • C for 4 h. The mixture was cooled, vacuum filtered and the resultant solid washed with water, saturated with NaHCO 3 solution and more water. The 3-bromopropyne (1.5 equiv.) was dropped into the solution of the obtained solid, t-BuOK (1.3 equiv.) and DMF under Argon atmosphere at 0 • C to r.t overnight. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the corresponding products [79]. concentrated in vacuo (50 °C) to remove excess acetic anhydride (bp 138 °C) to give a dry solid. Ammonium hydroxide (28% NH3, 100 mL) was added, and the mixture was heated to 95 °C for 4 hrs. The mixture was cooled, vacuum filtered and the resultant solid washed with water, saturated with NaHCO3 solution and more water. The 3-bromopropyne (1.5 equiv.) was dropped into the solution of the obtained solid, t-BuOK (1.3 equiv.) and DMF under Argon atmosphere at 0 °C to r.t overnight. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the corresponding products [79]. Compound 1m was prepared according to the referenced literature with some modification (Scheme 5) [80]. A Schlenk flask was charged with a magnetic stirrer, evacuated and backfilled with argon. 2-Chloronicotinic acid (0.5 mmol) and acetamidine hydrochloride (0.75 mmol) in EtOH (3 mL) were added under Argon atmosphere. After 10 min of stirring, Cs2CO3 (1 mmol) was added to the flask. Then, 15 min later, CuI (0.1 mmol) was added to the flask. The mixture was stirred at 80 °C for 12 h. After completion of the reaction, the mixture was filtered, and the solvent of the filtrate was removed with the aid of a rotary evaporator. The residue was purified by column chromatography on silica gel to provide the desired quinazolinone. The 3-Bromopropyne (1.5 equiv.) was dropped into the solution of the obtained solid, t-BuOK (1.3 equiv.), and DMF under argon atmosphere at 0 °C to overnight. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the desired products. Compound 1m was prepared according to the referenced literature with some modification (Scheme 5) [80]. A Schlenk flask was charged with a magnetic stirrer, evacuated and backfilled with argon. 2-Chloronicotinic acid (0.5 mmol) and acetamidine hydrochloride (0.75 mmol) in EtOH (3 mL) were added under Argon atmosphere. After 10 min of stirring, Cs 2 CO 3 (1 mmol) was added to the flask. Then, 15 min later, CuI (0.1 mmol) was added to the flask. The mixture was stirred at 80 • C for 12 h. After completion of the reaction, the mixture was filtered, and the solvent of the filtrate was removed with the aid of a rotary evaporator. The residue was purified by column chromatography on silica gel to provide the desired quinazolinone. The 3-Bromopropyne (1.5 equiv.) was dropped into the solution of the obtained solid, t-BuOK (1.3 equiv.), and DMF under argon atmosphere at 0 • C to overnight. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the desired products. 7-Chloro-2-methylquinazolin-4(3H)-one was prepared from the above procedure. A Schlenk flask was charged with a 7-chloro-2-methylquinazolin-4(3H)-one (0.3 mmol), corresponding to phenylboronic acid (0.45 mmol), Pd(OAc)2 (0.05 mmol), Sphos (0.03 mmol) and K3PO4 (2.4 mmol), and was heated in toluene (2 mL) at 80 °C for 24 h. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the corresponding products [81]. The 3bromopropyne (1.5 equiv.) was dropped into the solution of the obtained solid, t-BuOK (1.3 equiv.), and DMF under argon atmosphere at 0 °C to r.t overnight. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the desired products (Scheme 6).  7-Chloro-2-methylquinazolin-4(3H)-one was prepared from the above procedure. A Schlenk flask was charged with a 7-chloro-2-methylquinazolin-4(3H)-one (0.3 mmol), corresponding to phenylboronic acid (0.45 mmol), Pd(OAc) 2 (0.05 mmol), Sphos (0.03 mmol) and K 3 PO 4 (2.4 mmol), and was heated in toluene (2 mL) at 80 • C for 24 h. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the corresponding products [81]. The 3-bromopropyne (1.5 equiv.) was dropped into the solution of the obtained solid, t-BuOK (1.3 equiv.), and DMF under argon atmosphere at 0 • C to r.t overnight. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the desired products (Scheme 6). ethyl acetate/petroleum ether mixture to obtain the corresponding products [81]. The 3bromopropyne (1.5 equiv.) was dropped into the solution of the obtained solid, t-BuOK (1.3 equiv.), and DMF under argon atmosphere at 0 °C to r.t overnight. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the desired products (Scheme 6).  1 mmol), NCS (0.1 mmol) and AcOH (0.1mmol) were heated in acetonitrile (2 mL) at 100 °C for 10 h under argon atmosphere. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the corresponding products 2a.
Compound 4a-4c were prepared according to the referenced literature (Scheme 7) [82]. A solution of MeONa (1.5 mmol) in MeOH was added to a stirred solution of the corresponding 2-nitrobenzaldehyde (1.5 mmol) and a chloracetone (1.5 mmol) in MeOH (3.5 mL) at room temperature overnight. After the reaction was completed, the resulting precipitate was filtered off, the mixture was quenched carefully with water (3 × 50 mL) and with saturated NH4Cl (1 × 10 mL), and the product was isolated as a corresponding (2-(2-nitrophenyl)oxiran-1-yl)(aryl)methanone. A solution of Na2S2 O4 (5 mmol) in H2O (65 mL) was added to a solution of (2-(2-nitrophenyl)oxiran-1-yl)(aryl)methanone (1 mmol) in dioxane (65 mL). The reaction mixture was allowed to cool down to room temperature after reflux for 3 h and was poured into water (500 mL). The resulting precipitate was filtered off, washed with water (2 × 50 mL) and dried in air to give the corresponding 2-arylquinolin-3-ols. The 3-bromopropyne (3 equiv.) was dropped into the solution of the obtained solid, K2CO3 (3 equiv.), and acetonitrile under argon atmosphere at 0 °C to r.t  1 mmol), NCS (0.1 mmol) and AcOH (0.1mmol) were heated in acetonitrile (2 mL) at 100 • C for 10 h under argon atmosphere. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the corresponding products 2a.
Compound 4a-4c were prepared according to the referenced literature (Scheme 7) [82]. A solution of MeONa (1.5 mmol) in MeOH was added to a stirred solution of the corresponding 2-nitrobenzaldehyde (1.5 mmol) and a chloracetone (1.5 mmol) in MeOH (3.5 mL) at room temperature overnight. After the reaction was completed, the resulting precipitate was filtered off, the mixture was quenched carefully with water (3 × 50 mL) and with saturated NH 4 Cl (1 × 10 mL), and the product was isolated as a corresponding (2-(2-nitrophenyl)oxiran-1-yl)(aryl)methanone. A solution of Na 2 S 2 O 4 (5 mmol) in H 2 O (65 mL) was added to a solution of (2-(2-nitrophenyl)oxiran-1-yl)(aryl)methanone (1 mmol) in dioxane (65 mL). The reaction mixture was allowed to cool down to room temperature after reflux for 3 h and was poured into water (500 mL). The resulting precipitate was filtered off, washed with water (2 × 50 mL) and dried in air to give the corresponding 2-arylquinolin-3-ols. The 3-bromopropyne (3 equiv.) was dropped into the solution of the obtained solid, K 2 CO 3 (3 equiv.), and acetonitrile under argon atmosphere at 0 • C to r.t overnight. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the desired products [83,84]. overnight. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the desired products [83,84]. To a solution of 3-hydroxy-2-methyl-4-quinolinecarboxylic acid (1.1 equiv.), ROH or secondary amine (1.0 equiv.), DCC (1.1 equiv.) and DMAP (0.1 equiv.) were added in CH2Cl2 overnight to obtain 3-hydroxy-2-methylquinoline-4-carboxylate [85]. The 3-bromopropyne (3 equiv.) was dropped into the solution of the obtained solid, K2CO3 (3 equiv.), and acetonitrile under argon atmosphere at 0 °C to r.t overnight. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the desired products (Scheme 8). To a solution of 3-hydroxy-2-methyl-4-quinolinecarboxylic acid (1.1 equiv.), ROH or secondary amine (1.0 equiv.), DCC (1.1 equiv.) and DMAP (0.1 equiv.) were added in CH 2 Cl 2 overnight to obtain 3-hydroxy-2-methylquinoline-4-carboxylate [85]. The 3-bromopropyne (3 equiv.) was dropped into the solution of the obtained solid, K 2 CO 3 (3 equiv.), and acetonitrile under argon atmosphere at 0 • C to r.t overnight. After the reaction was completed, 50 mL water was added to the mixture and then extracted with EtOAc 3 times (3 × 50 mL). The extract was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude residues were purified by column chromatography using ethyl acetate/petroleum ether mixture to obtain the desired products (Scheme 8).

Conclusions
In summary, a facile and practical 1,3-dipolar cycloaddition reaction that accessed a wide variety of isoxazole-fused tricyclic quinazoline alkaloids and their derivatives has been developed under metal-free conditions. In this system, methyl azaarenes were transformed into nitrile oxides in situ by using TBN as the radical initiator and source of N-O without transition metal. This strategy has broad substrate applicability and good functional group tolerance with facile manipulation of readily available starting materials. Natural product modifications confirmed the practical utility of this synthetic method.