Efficient protocol to quinazolino[3,2-d ][1,4]benzodiazepine-6,9-dione via Staudinger-aza-Wittig cyclization: application to synthesis of Asperlicin D

Tandem Staudinger and intramolecular aza-Wittig reactions followed by cyclodehydration of the linear N -[ N -(2-azidobenzoyl)-2-aminobenzoyl]glycine ethyl ester furnished the tetracyclic quinazolino[3,2-d ][1,4]benzodiazepine-6,9-dione ring system found in some biologically active natural alkaloids. This method was successfully implemented to synthesize asperlicin D from a linear peptide containing ester and azido terminal groups.


Results and Discussion
Retrosynthetically, we envisioned that these alkaloids could be derived from two consecutive intramolecular reactions from linear peptide 6 containing two termini capable of reacting with each other (Scheme 1).Therefore, azido and ester groups were selected to study the feasibility of intramolecular aza-Wittig cyclization [15][16][17][18]  As shown in Scheme 2, our study commenced with the coupling of isatoic anhydride (10) with ethyl glycinate in dry acetonitrile followed by acylation with freshly prepared 2azidobenzoyl chloride.This reaction furnished N-[N-(2-azidobenzoyl)-2-aminobenzoy]glycine ethyl ester 11, as a model substrate, in good yield (80%).
6][17][18][19] The formation of these intermediates was confirmed by their acidic hydrolysis (PhSO 3 H, H 2 O, THF) at ambient temperature.Iminophosphoranes 12 and 13 gave the known amine 16, 13,14 whereas 14 and 15 gave the corresponding amidophosphates 17 and 18, respectively.Similar results were obtained when iminophosphorane intermediates 12, 13, 14 and 15 were passed through a short column of silica gel.The amidophosphates (17 and 18) were isolated in pure form by column chromatography and their structures were confirmed by spectral data and elemental analysis.Next, we turned to explore the viability of tandem cyclization reactions of Staudinger intermediates 12-15 shown in Scheme 2. Suitable conditions for the intramolecular aza-Wittig reaction were optimized employing 12. Initial attempts to promote cyclization of this intermediate at reflux temperature in toluene or xylene were unsuccessful even after an extended reaction time (48 h).However, when cyclization was conducted in boiling mesitylene for 40 h it furnished a significant amount of the expected imino ether, 7-ethoxy-quinazolino [3,2d] [1,4]benzodiazepine (19) along with its hydrolyzed product quinazolino [3,2d] [1,4]benzodiazepine (20)  20 in 20% and 15% yield, respectively, after separation by column chromatography.The two products were isolated in variable proportions depending on the reaction time and temperature.The methylene protons of the seven-membered ring in 19 were observed at δ 5.87 (d, 13.2 Hz) and 3.83 (d, 13.2 Hz), indicating that they are non-equivalent on the NMR time scale due to the significant barrier to flipping of the ring.Furthermore the methylene protons of the ethoxy group were displayed at δ 4.47 and 4.24 as two broad peaks.Since the reaction conditions were anhydrous, it seemed likely that the hydrolysis of 19 to 20 occurred on the silica gel during purification.Moreover, the imino ether 19 was cleanly hydrolyzed to 20 in wet THF containing a catalytic amount of PhSO 3 H.To simplify the separation of the desired product 20 from the reaction mixture after conducting the reaction in boiling mesitylene, the crude product was hydrolyzed after concentration to give 20 in 30-40% yield.The structure of the final product 20 was assigned based on the reported NMR spectral data and also by elemental analysis.The spectral data of 20 were identical to those reported previously. 20he yield of the final product 20 was significantly improved by switching to the more reactive iminophosphorane 13.Stirring 11 and (Bu) 3 P in mesitylene at room temperature for 2 h then at reflux for 40 h, followed by hydrolysis, gave 20 in 55% yield.Lower yields of 20 (<30%) were obtained by performing the reaction in boiling xylene.The results of these experiments are summarized in Table 1.The cyclization of 14 failed in boiling xylene, affording only the hydrolysis product, amidophosphate 17.However, 20 was obtained in 20% yield along with the amidophosphate 17 by conducting the reaction in mesitylene at reflux temperature.Similarly, 15 furnished 20 along with the corresponding amidophosphate 17. a In method A: the reactions were conducted in mesitylene at reflux temperature for 40 h.b In method B: the reactions were conducted in sealed tube at 200 °C for 5 h.
An alternative procedure for the cyclization of the intermediates 12-15 was also explored.Thus, the cyclization process was performed in sealed tube.Table 1 details the efficiency of the cyclization of intermediates 12-15 at 200 °C in sealed tube followed by hydrolysis.In general, the yield of the desired product was enhanced.
Furthermore, implementation of Wipf methodology (Ph 3 P/I 2 /Et 3 N) 21 with 16 afforded an inseparable mixture of products.Fortunately, we observed that the starting material 16 was completely consumed and a new product 21 was isolated (10% yield) in impure form when the reaction was conducted with excess reagent (Ph 3 P/I 2 /Et 3 N).We believe that the product 21 is formed from 20 after it has been formed in the reaction mixture.Thus, to confirm this proposal, compound 20 was allowed to react with excess Wipf reagent (Ph 3 P/I 2 /Et 3 N).This reaction furnished 21 in moderate yield (50%).Moreover, derivative 22 was isolated when 20 stirred with a mixture of Ph 3 P/I 2 /Bu 3 N. Utilizing the experimental conditions established for the unsubstituted quinazolino [3,2-d] [1,4]benzodiazepine (20), we next explored the application of these conditions to prepare asperlicin D (4) starting from tryptophan azido derivative 23.This starting material was prepared in a one-pot reaction in good yield (>70%) by condensation of isatoic anhydride (10) with L-tryptophan methyl ester followed by acylation with freshly prepared 2-azidobenzoyl chloride (Scheme 3).Staudinger iminophosphorane intermediate 24 was generated in situ by stirring 23 with (Ph) 3 P at room temperature until the evolution of nitrogen gas ceased (2 h).Initial attempts to promote cyclization of 24 at reflux in benzene or xylene were unsuccessful even after an extended reaction time.However, the TLC indicated the consumption of 24 when the reaction was conducted in boiling mesitylene for 40 h.This reaction afforded two products (by TLC).Fortunately, after hydrolysis (H 2 O, THF, PhSO 3 H) the crude reaction mixture furnished the natural product 4 in 30-40% yield together with amine 25. 14 The two products were formed in variable proportions depending on the reaction time and temperature.The yield of asperlicin D was improved using (Bu) 3 P in mesitylene at reflux temperature.The isolated asperlicin D showed spectral properties identical to those previously described for the natural product. 4,13,14

Conclusions
The present work demonstrates the viability of aminophosphorane intermediates having a secondary amide proton to provide a one-step entry to quinazolino[1,4-d]benzodiazepine ring system via tandem intramolecular aza-Wittig reaction followed by cyclodehydration performed on a linear peptide.We have devised a general procedure with simple reagents for accomplishing successive cylization reactions via Staudinger intermediates.The power of this approach has resulted in the synthesis of asperlicin D.

Experimental Section
General Procedures.Melting points (mp) were determined on an electrothermal digital melting point apparatus and are uncorrected.Infrared (IR) spectra were recorded using a Nicolet-Impact 410 FT-IR spectrophotometer.Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded on Bruker, Avance DPX-300 (300 MHz), Bruker 250 spectrometers.Tetramethylsilane (TMS) was used as an internal reference.The spectral data are reported in delta (δ) units relative to TMS reference line.Carbon-13 nuclear magnetic resonance ( 13 C NMR) spectra were taken using a Bruker Avance DPX-300 (75.5 MHz) spectrometer and signals are reported in delta (δ) units relative to TMS reference using the solvent peaks (CDCl 3 ) as internal standard.Mass spectra were recorded with a Mariner Biospectrometry Workstation 4.0 by Applied Biosystems.

Ethyl N-{2-[(2-azidobenzoyl)amino]benzoyl} glycinate (11)
Isatoic anhydride (0.327 g, 2 mmol) was added to ethyl glycinate hydrochloride (0.279 g, 2 mmol) dissolved by heating in acetonitrile (7 mL) containing triethylamine (0.212 g, 2.1 mmol).The mixture was heated and allowed to reflux for 3 h.The reaction mixture was cooled to 0 °C and then triethylamine (0.404 g, 4 mmol) was added.Then a solution of freshly prepared 2-azidobenzoyl chloride (0.417 g, 2.3 mmol) in acetonitrile (2 mL) was added to the mixture.The reaction mixture was stirred at 0 °C for 0.5 h and at room temperature for 24 h.The mixture was concentrated and extracted with ethyl acetate (2 x 40 mL) and water (20 mL).The combined organic layers were washed with brine, dried with MgSO 4 and concentrated.The residue was purified by column chromatography on silica gel (30% ethyl acetate in hexane) to afford 11 (0.559 g, 77%

General procedure for synthesis of 20 using sealed tube reactions
A tube containing a pulverized mixture of azide 11 (0.367 g, 1 mmol) and phosphorus(III) reagent (1.1 mmol) and mesitylene (5 mL) was sealed under reduced pressure and kept in oven at 190-200 °C for 5 h.The tube was cooled, wrapped with towel and crushed.The crude reaction mixture was stirred in wet-THF containing catalytic amount of PhSO