Fischer indolisation of N-(α-ketoacyl)anthranilic acids into 2-(indol-2-carboxamido)benzoic acids and 2- indolyl-3,1-benzoxazin-4-ones and their NMR study

Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. Accepted Manuscript Organic & Biomolecular Chemistry


Introduction
Molecules containing indole 1 and anthranilic acid 2 scaffolds are ubiquitous in many natural products with diverse biological activities. Thus, it is not surprising that the compounds featuring both the indole and the anthranilic acid fragments are encountered in nature. Alkaloids cephalandole C and cephalinone B, isolated from the native orchid plant Cephalanceropsis gracilis, 3 and the alkaloids secofascaplysins, isolated from the sponge Fascaplysinopsis reticulate, 4 consist of the 2-(indol-2-carboxamido)benzoic acid 1 substructures, for example ( Figure 1). Derivatives of 2-(indol-2carboxamido)benzoic acid 1 are receiving attention because of their antibacterial activities. 5 In material sciences, the 2-(indol-2carboxamido)benzoic acid derivatives are used as UV absorbers. 6 Compounds with indol-2-carboxamide scaffold are potent inhibitors of HIV-1 replication (Delavirdine, Rescriptor®) 7 and the androgen receptor binding function 3 (BF3), 8 and were identified as hydrogenbonding organocatalysts for the ring-opening polymerization of cyclic esters. 9 3,1-Benzoxazin-4-ones serve as valuable precursors for the preparation of fused heterocycles including an important pharmacophore, quinazolin-4-one. 10 It is also noteworthy that 3,1benzoxazin-4-ones are potent inhibitors of the human leukocyte elastase, 11 human chymase, 12 chymotrypsin 13 and proteases of herpes simplex type 1, 14 human cathepsin G 15 and serine, 16 for example. Avenalumin I, phytoalexin of the 3,1-benzoxazin-4-one structure that is inhibitory to the growth of rust fungi, was isolated from oat leaves ( Figure 1). 17 Surprisingly, despite the biological relevance and synthetic potential of 2-(indol-2-carboxamido)benzoic acids 1, to our knowledge, the only published approach to access these compounds makes use of N-acylation of anthranilic acids with 1H-indol-2carboxylic acid derivatives. 18 As an alternative to this, herein we report the protocol that is based on the Fischer indolisation of N-(αketoacyl)anthranilic acids 2 (Scheme 1). Optimization of the reaction conditions and the scope as well as multinuclear NMR spectral analysis of the products is reported. The Fischer indolisation is known to proceed through an intermediately formed hydrazone, which then undergoes several consecutive transformations. It isomerises to an enamine, which after protonation rearranges to an imine and then a cyclic aminal. Acid catalysed elimination of ammonia from the latter finally gives rise to the indole ring. The above mentioned hydrazone can in principle be pre-assembled by condensation of aryl hydrazine with an appropriate carbonyl compound. Herein, we decided to compare the stepwise protocol that proceeds through the isolated phenylhydrazone 3 (Route A, Scheme 3) with the direct-one in which N-(αketoacyl)anthranilic acids 2 is treated with phenylhydrazine directly into the 2-(indol-2-carboxamido)benzoic acid 1 (Route B).
To examine the stepwise protocol, Route A, phenylhydrazones 3a,c-e were initially prepared by the condensation of N-(αketoacyl)anthranilic acids 2a,c-e with phenylhydrazine. The isolated phenylhydrazones were in the subsequent step subjected to the thermally induced rearrangement into 2-(indol-2-carboxamido)benzoic acids 1a,c-e. The results are collected in Table  1. Although the preparation of the phenylhydrazones 3a,c-e proceeded in high 78-88% yields, the subsequent indolisation step into the target 2-(indol-2-carboxamido)benzoic acids 1a,c-e returned low 17-33% yields.
The disappointing overall results of the stepwise procedure prompted us to test the direct approach, outlined as Route B in Scheme 3. In screening for the optimal reaction conditions, a mixture of selected N-(α-ketoacyl)anthranilic acid 2 and phenylhydrazinium chloride was heated under reflux in different solvents including acetonitrile, acetic acid, methanol and water. Since Lewis 22 and Brønsted acids 23 are well known to promote the Fischer indolisation, we selected to test hydrochloric acid, sulphuric acid, acetic acid, zinc(II) chloride and bismuth(III) nitrate pentahydrate (Bi(NO 3 ) 3 5H 2 O) 24 as acid catalysts (Table 2). Good results in terms of the reaction time and the product yields were obtained for the reactions in boiling acetonitrile either in the presence of  3,4,[9][10][11][12][13] or acetic acid (Entry 8). However, the highest yields of the products 1 were seen by conducting the reaction in boiling acetic acid in the absence of the additives (Entry 1). As expected, the heating of N-(αketoacyl)anthranilic acid 2a in aqueous hydrochloric or sulphuric acid resulted in an amide bond hydrolysis to produce anthranilic acid (Entries 6 and 7). Having identified the optimal reaction conditions we turned to examine the scope of the reaction. Several N-(α-ketoacyl)anthranilic acids (2a-l) were allowed to react with phenylhydrazinium chloride in boiling acetic acid, which afforded the target 2-(indol-2carboxamido)benzoic acids (1a-l) in good to excellent yields of the isolated products (Table 3). In few instances, the reactions were accompanied by the formation of small amounts of by-products, which were isolated and identified as 3,1-benzoxazin-4-ones 4 and phenylhydrazides 5. The only exception to this was compound 2m, which afforded naphthoxazinone 4m as the sole product, with the anthranilic acid 1m being undetected in the reaction mixture (Entry 13).
In principle, the formation of 2-indolyl-3,1-benzoxazin-4-ones 4 could be realised by two different pathways as shown in Scheme 4. An initial formation of 2-acyl-3,1-benzoxazin-4-one 4' through the Path a could be followed by the Fischer indolisation with phenylhydrazinium chloride. This pathway was, however, ruled out on the basis of the 3,1-benzoxazin-4-ones reactivity considerations.
In the presence of nucleophiles these compounds are prone to undergo rapid ring opening into the anthranilic acid derivatives (vide infra) suggesting that somehow higher amounts of the phenylhydrazides 5 should have been formed in the reactions shown in Table 3. The Path a was also ruled out experimentally by heating compound 2a in neat acetic acid under the reflux conditions in the absence of phenylhydrazinium chloride, which resulted in no detectable formation of 2-acyl-3,1-benzoxazin-4-one 4'. This left the Path b, i.e. the initial Fischer indolisation of the N-(αketoacyl)anthranilic acid 2 with phenylhydrazinium chloride into 2-(indol-2-carboxamido)benzoic acid 1 and subsequent cyclodehydration into 2-indolyl-3,1-benzoxazin-4-one 4, as the most plausible.
anthranilic acid is N-acylated and the carboxylic group is transformed into a mixed anhydride intermediate. This intermediate then undergoes an intramolecular nucleophilic displacement of the carboxylate ion from the anhydride moiety by the amide in its iminol form. 26 In turn, heating the 2-(indol-2-carboxamido)benzoic acid 1 in acetic acid is unlikely to produce mixed anhydride. Since an intramolecular nucleophilic attack of the carboxylic group to the amide is also unlikely because of the low electrophilicity of the amide, the formation of 3,1-benzoxazin-4-ones 4 could best be rationalized through the intramolecular nucleophilic attack of the iminol 1' to the protonated carboxylic group as shown in Scheme 4. The formation of naphthoxazinone 4m as the sole product from 2m ( Table 3, Entry 13) could be accounted for by an enhanced resonance stabilisation of iminol 1m', the result of an extended conjugation through the naphthalene ring.
If the mechanism shown in Scheme 4 is operating, it can be expected that a prolonged reaction time and/or an elevated reaction temperature would work beneficially to the formation of 3,1benzoxazin-4-one 4. Indeed, by prolonging the heating of compound 1j with phenylhydrazinium chloride in acetic acid (bp AcOH = 118 C) from 12 h to 40 h increased the yield of the expected product 4j from 15% to 65% (compare entries 1 and 2 from Table 4). The use of the higher boiling propanoic acid (bp = 141 C) in place of acetic acid afforded the 3,1-benzoxazin-4-one 4j in 72% yield already within 17 h (Entry 3). It is noteworthy that these reaction conditions can potentially be utilized as a convenient one-pot protocol for the preparation of 3,1-benzoxazin-4-ones 4 from N-(αketoacyl)anthranilic acids 2 and aryl hydrazines. The synthetic methodologies towards 3,1-benzoxazin-4-ones, other than those utilizing anthranilic acids, have been reviewed. 27 Table 4 The influence of the reaction time and temperature on transformation of 2-(indol-2-carboxamido)benzoic acid 1j into 3,1-benzoxazin-4-one 4j. Minute amounts of the hydrazides 5 also accompanied the formation of 2-(indol-2-carboxamido)benzoic acids 1 ( Table 3). As it is less likely that under the applied reaction conditions phenylhydrazinium chloride reacts with the carboxyl group of either the starting N-(α-ketoacyl)anthranilic acids 2 or the product 2-(indol-2-carboxamido)benzoic acids 1, it is reasonable to assume that the formation of hydrazides 5 proceeds through the ring-opening at the 3,1-benzoxazin-4-ones 4. Smooth reactivity of the 3,1-benzoxazin-4ones towards different nucleophiles is well documented. 26,28 In our case the reactivity of compound 4f towards phenylhydrazine to form 5f was independently confirmed in boiling toluene as the reaction solvent (Table 5). Analogously, treatment with n-butylamine gave the appropriate amide 6f.

Entry
Aqueous sodium hydroxide in DMSO mediated complete hydrolysis of 3,1-benzoxazin-4-ones 4f,j,m to the corresponding 2-(indol-2-carboxamido)benzoic acids 1f,j,m (Table 5). High tendency of related 3,1-benzoxazin-4-ones for hydrolysis into the corresponding anthranilic acids has been documented. 29,30 With these results in mind it can be assumed that higher quantities of the 3,1benzoxazin-4-one products 4 are actually formed during the Fischer indolization of N-(α-ketoacyl)anthranilic acids 2 shown in Table 3 as they were actually isolated. During the isolation workup the latter most probably partly hydrolyse back into the 2-(indol-2carboxamido)benzoic acids 1. The elemental composition of all the compounds under investigation was confirmed by combustion analysis and highresolution mass spectrometry with electrospray ionization. In addition, low resolution mass spectra with electron impact ionization and the infrared spectra were provided. In the latter, the characteristic absorption bands belonging to the indole N-H, C=O and C=N bonds were identified, where appropriate.

NMR study
The compounds 1,3-6 were fully characterized by 1 H, 13 C and 15 N NMR spectroscopy. The corresponding resonances were assigned on the basis of gradient-selected 2D NMR experiments including 1 H-1 H gs-COSY, 1 H-13 C gs-HSQC, 1 H-13 C gs-HMBC and 1 H-15 N gs-HMBC. The spectra of compounds 1, 3, 5 and 6 were recorded in DMSO-d 6 . For 3,1-benzoxazin-4-ones 4, which proved to rapidly hydrolyse in DMSO-d 6 into the 2-(indol-2-carboxamido)benzoic acids 1, less polar CDCl 3 was identified as a suitable solvent. Acetone-d 6 was used as an alternative for dissolving compound 4k due to its sparing solubility in CDCl 3 . Some characteristic spectral features are discussed below. For the atom numbering scheme, see Figure 2.  There is a wealth of 1 H and 13 C data 31 as well as 15 N NMR data 32 on indoles reported in the literature. Relatively unsubstituted indole ring in the compounds 1 and 4 enabled us to unequivocally assign all proton, carbon and nitrogen resonances via long-range 1 H-13 C and 1 H-15 N heteronuclear coupling pathways, which we found in agreement to those discussed in the literature. 33 For the indole ring in 2-(indol-2-carboxamido)benzoic acids 1 the order of shielding in the 13 Table 6). By changing the indole C-2' substituent from the carbamoyl group in compounds 1 into the 3,1-benzoxazin-4-one ring in 4, the most dramatic changes in the chemical shift are seen for the carbon atoms of the fused 5-membered ring. In comparison to compounds 1, the C2' atoms of the indole ring in 4 are shielded by ca. 4 ppm, whereas the C3' and C3a' atoms are deshielded by ca. 6 ppm and ca. 1 ppm, respectively. By changing the R 2 = Me to the R 2 = n-Pr in either 1 or 4, the carbon atom C3' becomes more shielded for ca. 5-6 ppm (Tables 6 and 7).
The three-bond long-range couplings were observed in the 1 H-15 N gs-HMBC spectra from H7' to the indole N1' resonance in both 1 and 4 ( Figure 2). In addition, a one-bond direct NH doublet response corroborated the assignment of N1' (Figure 3). The chemical shifts of the N1' atoms in indoles 1 appear at  130.2-136.9 ppm and are in good agreement with the literature values reported for Delavirdine. 34 In compounds 4, the indole nitrogen atoms N1' resonate in the narrow range of  120.3-121.4 ppm (Tables 6 and 7).
Systematic NMR investigations of 3,1-benzoxazin-4-ones are more scarce. 29,35 As pointed out by Osborne and Goolamali some early NMR data should be taken with care because these compounds often show high tendency for hydrolysis into the corresponding anthranilic acids, especially when measured in polar solvents such as DMSO-d 6 . 29 High susceptibility towards hydrolysis has been illustrated by 2-methyl-3,1-benzoxazin-4-one that reacts with water into 2-acetylaminobenzoic acid already in the solid state. 30 Osborne and Goolamali reported an unequivocal differentiation between anthranilic acids and 3,1-benzoxazin-4-ones that was achieved through determination of characteristic J CH coupling interactions in the carbonyl region of the proton coupled 13 C NMR spectra. 29 Herein, the proton, carbon and nitrogen resonances belonging to the anthranilic moiety in 1 and 3,1-benzoxazin-4-one group in 4 were rapidly assigned by using the gradient-selected 2D NMR experiments. The results are collected in Tables 6 and 7. Characteristic for the downfield regions of the 13 C NMR spectra of the acylanthranilic acids 1 were two signals appearing at  160.2-161.6 ppm and  166.5-169.7 ppm for the amide carbonyl and for the carboxylic group, respectively. In 3,1-benzoxazin-4-one 4, the downfield region of the spectra were occupied with three signals for C8a ( 133.1-147.8 ppm), C2 ( 151.0-154.3 ppm) and C4 ( 156.2-159.7 ppm). Our results are in agreement with the literature data. 29 Chemical shifts for the 3,1-benzoxazin-4-one nitrogen atoms in compounds 4 were in the range of  212.0-222.8 ppm (Table 7).
Due to the lack of the 15 N NMR data no comparison with the literature could be done. In C8-unsubstituted 3,1-benzoxazin-4-ones 4b,c,f,h (R 6 = H), three-bond long-range couplings were observed in the 1 H-15 N gs-HMBC spectra from H8 to the N1 resonance as illustrated in Figure 2. The strongly electron donating C8-methoxy substituent in derivatives 4d,k (R 6 = OMe) enabled a four-bond long-range couplings from H7 to the N1 whereas no such correlation could be observed in the C8-methyl substituent analogues 4g,i,j (R 6 = Me). The amide nitrogen atoms (CONH) in acylanthranilic acids 1 resonate in the range of  113.9-129.5 ppm (Table 6). In analogy with the above, the cross-peaks between H3 and CONH resonance were observed in 1 H-15 N gs-HMBC spectra of 1a-c,h,l. In the 4methoxy substituted acylanthranilic acid 1c there was also a fourbond long-range coupling from H6 to the CONH. With the exception of 1l and 1m, the 1 H-15 N gs-HMBC spectra also featured the onebond direct NH doublet response. Interestingly, in 3-methylindoles 1a,b,g,h, five-bond long-range couplings were observed in the 1 H-15 N gs-HMBC spectra from the C3'CH 3 to the amide nitrogen (CONH) resonance. With the 2-(indol-2-carboxamido)benzoic acid 1b as a representative example, the above mentioned 1 H-15 N gs-HMBC spectral features are illustrated in Figure 3. Direct responses for the N1' and CONH resonances were observed as doublets at 132.7 ppm and 125.1 ppm, respectively. The spectrum also features three-bond long-range response to N1' and CONH from H7' and H3, respectively. Five-bond long-range correlation to CONH from C3'CH3 is also observed.

Conclusions
We have demonstrated that Fischer indolisation of N-(αketoacyl)anthranilic acids can serve as a mild and highly efficient alternative for the preparation of 2-(indol-2-carboxamido)benzoic acids. By simple changes in the reaction conditions, this protocol offers a potential to access 2-indolyl-3,1-benzoxazin-4-ones. The anthranilic acid, indol-2-carboxamide and 3,1-benzoxazin-4-one structural motifs discussed herein were fully characterized by 1 H, 13 C and 15 N NMR spectroscopy. The data presented will help in unequivocal identification of these classes of compounds.

General
The reagents and solvents were used as obtained from the commercial sources. Compounds 2a-k,m were prepared according to the literature procedure. 21 Column chromatography was carried out on Fluka Silica gel 60 (particle size 0.063-0.

Solvent and catalyst screening for the Fischer indolisation of 2 into 1 (Table 2)
A stirred mixture of N-(α-ketoacyl)anthranilic acid 2 (2.00 mmol), phenylhydrazinium chloride (310 mg, 2.15 mmol) and the catalyst in the solvent (Entry 1: 7.5 mL; Entries 2-13: 13 mL) was heated under reflux until TLC analysis indicated complete consumption of the starting material. The progress of the reaction was accompanied by the colour change of the reaction mixture from pale yellow to green and finally to dark red. The products were isolated as follows. Entries 1 and 5: The cooled reaction mixture was poured into ice water (35-60 mL). The solid was collected by filtration, washed with water (20-60 mL) and recrystallized from ethanol affording pure compound 1a. [9][10][11][12][13] The cooled reaction mixture was poured into ice water (35 mL). If oily organic phase was formed (Entry 13), the resulting mixture was stirred overnight. The resulting solid was collected by filtration, washed with 10% HCl (16 mL) and water (25 mL), and recrystallized from ethanol affording the appropriate pure compound 1. Entries 6 and 7: The cooled reaction mixture was filtered. The filter cake was washed with water (10 mL) and recrystallized from ethanol affording pure compound 1a. The filtrate was evaporated to dryness and residue was dissolved in a minimal amount of water (ca. 25 mL). The solution was made alkaline with 0.5 M NaOH, washed with benzene (3 × 5 mL) and carefully neutralised with 5% HCl. The precipitated anthranilic acid was filtered off as colourless crystals. Entry 8: The reaction mixture was concentrated in vacuo and the oily residue was triturated with water (10 mL) to give white precipitate of pure 1a, which was collected by filtration and washed with water (2 × 3 mL).