One-Pot Synthesis of Triazolobenzodiazepines Through Decarboxylative [3 + 2] Cycloaddition of Nonstabilized Azomethine Ylides and Cu-Free Click Reactions

A one-pot synthesis of triazolobenzodiazepine-containing polycyclic compounds is introduced. The reaction process involves a decarboxylative three-component [3 + 2] cycloaddition of nonstabilized azomethine ylides, N-propargylation, and intramolecular click reactions.


Results and Discussions
Reaction conditions for the synthesis of proline 4a through one-pot [3 + 2] cycloaddition were developed using 1:1.2:1 of 2-azidebenzaldehyde 1a, 2-aminoisobutyric acid 2a, and N-ethylmaleimide 3a in the presence of 0.3 equiv. of AcOH for decarboxylation [43] (Table 1). After screening Molecules 2019, 24, 601 3 of 7 solvents including 2-methyltetrahydrofuran, toluene, EtOH and CH 3 CN as well as reaction time and temperature, it was found that a reaction using CH 3 CN as a solvent at 110 • C for 6 h afforded 4a in 93% LC (liquid chromatography) yield with a dr (diastereomer) of 6:1 (Table 1, entry 6). The stereochemistry of 4a was determined according to the literature report [38].

Results and Discussions
Reaction conditions for the synthesis of proline 4a through one-pot [3 + 2] cycloaddition were developed using 1:1.2:1 of 2-azidebenzaldehyde 1a, 2-aminoisobutyric acid 2a, and N-ethylmaleimide 3a in the presence of 0.3 equiv. of AcOH for decarboxylation [43] (Table 1). After screening solvents including 2-methyltetrahydrofuran, toluene, EtOH and CH3CN as well as reaction time and temperature, it was found that a reaction using CH3CN as a solvent at 110 °C for 6 h afforded 4a in 93% LC (liquid chromatography) yield with a dr (diastereomer) of 6:1 (Table 1, entry  6). The stereochemistry of 4a was determined according to the literature report [38]. Decarboxylative [3 + 2] cycloaddition product 4a was then used for the development of conditions for the N-propargylation and sequential click reaction for the synthesis of triazolobenzodiazepine 6a. In the presence of K2CO3, 4a reacted with propargyl bromide in CH3CN at 80 °C for 2 h to give 5a in 94% LC yield ( Table 2, entries 2-5). Without separation, the reaction mixture was used for intramolecular click reaction at 100 °C under the catalysis of Cu salts ( Table 2, entries 2-4). The CuI-catalyzed click reaction gave 6a in 89% LC yield, which is better than the reactions catalyzed with CuCl or CuBr. In our previous work, the intramolecular click reaction was accomplished under microwave heating and Cu-free conditions [32]. In this work, N-propargylation compound 5a generated under the microwave heating was continuously heated at 150 °C for 1 h to give 6a in 88% LC yield without CuI catalyst ( Decarboxylative [3 + 2] cycloaddition product 4a was then used for the development of conditions for the N-propargylation and sequential click reaction for the synthesis of triazolobenzodiazepine 6a. In the presence of K 2 CO 3 , 4a reacted with propargyl bromide in CH 3 CN at 80 • C for 2 h to give 5a in 94% LC yield ( Table 2, entries 2-5). Without separation, the reaction mixture was used for intramolecular click reaction at 100 • C under the catalysis of Cu salts ( Table 2, entries 2-4). The CuI-catalyzed click reaction gave 6a in 89% LC yield, which is better than the reactions catalyzed with CuCl or CuBr. In our previous work, the intramolecular click reaction was accomplished under microwave heating and Cu-free conditions [32]. In this work, N-propargylation compound 5a generated under the microwave heating was continuously heated at 150 • C for 1 h to give 6a in 88% LC yield without CuI catalyst (Table 2, entry 6). A Cu-free control reaction of 5a under conventional heating at 100 • C for 3 h only gave 5% of 6a (Table 2, entry 5).  After establishing the three-component [3 + 2] cycloaddition, N-propargylation, and sequential click reactions for 6a shown in Tables 1 and 2, we then aimed to combine these three reactions in one pot. After modification of the conditions shown in Tables 1 and 2, the best conditions for the one-pot synthesis was to conduct the decarboxylative [ After establishing the three-component [3 + 2] cycloaddition, N-propargylation, and sequential click reactions for 6a shown in Tables 1 and 2, we then aimed to combine these three reactions in one pot. After modification of the conditions shown in Tables 1 and 2, the best conditions for the one-pot synthesis was to conduct the decarboxylative [3 + 2] cycloaddition in MeCN under conventional heating at 110 • C for 6 h, then to perform the N-propargylation and spontaneous Cu-free click reaction under microwave heating at 150 • C for 1 h to give 6a in 76% LC yield (Table 3, entry 3). A control reaction using CuI as a catalyst for the click reaction didn't give a better yield (Table 3, entry 4). Table 3. Conditions for the one-pot synthesis of 6a a .

Immunofluorescence Analysis of the Rat Liver Following Intraperitoneal Injection of GdCl 3 or Zymosan
Immunofluorescence double staining showed increased numbers of myeloperoxidase positive (MPO + ) cells (recruited granulocytes) as early as 3 h (hours) after GdCl 3 administration ( Figure 1B) compared to untreated animals ( Figure 1A) while the number of MPO + cells decreased at 24 h after treatment ( Figure 1C). MPO + cells were mainly located near the portal vessel and in close vicinity to the liver macrophages. The same liver sections showed a progressive reduction of ED-1 positivity after GdCl 3 administration, mainly near the portal vessel (Figure 1) while ED-2 positivity remained unchanged (Figure 2). Double immunofluorescence staining showed few ED-1 + and MPO + , and ED-2 + and MPO + cells in close contact to each other near the portal area (Figures 1 and 2).
Double immunofluorescence staining of liver sections after Zymosan treatment with antibodies against ED-1, ED-2 and MPO showed an increased number of MPO + cells at 3 h ( Figure 3B,D), which were located near the portal vessels but also through the liver parenchyma, compared to controls ( Figure 3A). Double immunofluorescence staining showed few MPO + /ED-1 + as well as MPO + /ED-2 + cells near the portal area ( Figure 3A-D). Under the optimized conditions for the one-pot synthesis [44], 13 analogues of triazolobenzodiazepines 6a-m were synthesized using different sets of azidobenzaldehydes 1 (R 1 = H, CF 3 , Br, Cl, NO 2 ), amino acids 2 (R 2 = H, Me; R 3 = Me, Ph, i-Pr), and maleimides 3 (R 4 = Me, Et, Ph, Bn, 4-Br-Ph) ( Table 4). The reactions of five different maleimides with 2-aminoisobutyric acids and 2-azidebenzaldehyde gave 6a-e in 55-65% isolated yields. The substitution groups on the benzaldehydes had some influence on the product yield. For example, the azidobenzaldehydes bearing electron-withdrawing groups, such as Br and CF 3 , gave 6f and 6g in lower yields (59% and 35%), while the azidobenzaldehyde with the strong electron-withdrawing group NO 2 gave no product of 6m. The reactions of glycine and leucine with azidobenzaldehydes (R 1 = H, Br, Cl) and maleimides (R 4 = Me, Et) gave 6h-l in 44-55% yields. The stereochemistry of product 6 was established during the step of the decarboxylative [3 + 2] cycloaddition, which was determined according to the literature report [38]. Table 4. One-pot synthesis of triazolobenzodiazepines 6 a .
The proposed mechanism for the synthesis of product 6a is outlined in Scheme 2. The condensation of 2-azidebenzaldehyde 1a and 2-aminoisobutyric acid 2a give oxazolidin-5-one I. It then underwent decarboxylation to form the nonstabilized azomethine ylide II for [3 + 2] cycloaddition with 3a to form 4a. Formation of 5a through propargylation followed by continuous heating for intramolecular click reaction affords product 6a. There are several reports in literature which demonstrated that intramolecular click reactions in one-pot synthesis could be achieved a Reaction conditions, see [44]. b Isolated yield. The proposed mechanism for the synthesis of product 6a is outlined in Scheme 2. The condensation of 2-azidebenzaldehyde 1a and 2-aminoisobutyric acid 2a give oxazolidin-5-one I. It then underwent decarboxylation to form the nonstabilized azomethine ylide II for [3 + 2] cycloaddition with 3a to form 4a. Formation of 5a through propargylation followed by continuous heating for intramolecular click reaction affords product 6a. There are several reports in literature which demonstrated that intramolecular click reactions in one-pot synthesis could be achieved under Cu-free conditions [10,15,32,45,46]. a Reaction conditions, see [44]. b Isolated yield.
The proposed mechanism for the synthesis of product 6a is outlined in Scheme 2. The condensation of 2-azidebenzaldehyde 1a and 2-aminoisobutyric acid 2a give oxazolidin-5-one I. It then underwent decarboxylation to form the nonstabilized azomethine ylide II for [3 + 2] cycloaddition with 3a to form 4a. Formation of 5a through propargylation followed by continuous heating for intramolecular click reaction affords product 6a. There are several reports in literature which demonstrated that intramolecular click reactions in one-pot synthesis could be achieved under Cu-free conditions [10,15,32,45,46]. Scheme 2. Mechanism for one-pot synthesis of 6a.

Summary
A one-pot synthesis of fused-triazolobenzodiazepines was developed using readily available amino acids, maleimides, and 2-azidebenzaldehydes for decarboxylative [3 + 2] cycloaddition of nonstabilized azomethine ylides, followed by N-propargylation and a Cu-free intramolecular click Scheme 2. Mechanism for one-pot synthesis of 6a.

Summary
A one-pot synthesis of fused-triazolobenzodiazepines was developed using readily available amino acids, maleimides, and 2-azidebenzaldehydes for decarboxylative [3 + 2] cycloaddition of nonstabilized azomethine ylides, followed by N-propargylation and a Cu-free intramolecular click reaction. This is a highly efficient and operational simple reaction process for fused-triazolobenzodiazepines, and only CO 2 and H 2 O were generated as byproducts.
Supplementary Materials: The following are available online. 1 H-NMR, 13 C-NMR, and 19 F-NMR spectra of final products.
Author Contributions: X.M. and X.F. developed above reactions; W.Q., W.Z., and B.W. expanded the substrates scope; X.F. and W.Z. conceived the project; W.Z. supervised the project and revised the manuscript.