Green Synthesis of Substituted Anilines and Quinazolines from Isatoic Anhydride-8-amide

Synthetic methods used to generate substituted anilines and quinazolines, both privileged pharmacological structures, are cumbersome, hazardous or, in some cases, unavailable. We developed a straightforward method for making isatoic anhydride-8-amide from isatin-7-carboxylic acid as a tool to easily produce a range of quinazoline and substituted aniline derivatives using adaptable pH-sensitive cyclization chemistry. The approaches are inexpensive, simple, fast, efficient at room temperature and scalable, enabling the synthesis of both established and new quinazolines and also highly substituted anilines including cyano derivatives.

www.nature.com/scientificreports www.nature.com/scientificreports/ at position 4, which we expected to compete for interactions with the amide intermediate in a counterclockwise (leftward) cyclization reaction, leading to the formation of phthalimide compounds (Fig. S3b). Consistent with this model, isatin-4-acid 1b, methyl ester 1c, primary amide 1d, C3-hydrazide 1e, and alkyl substitution at position 1 1f, 1g produced 3-amino-phthalimide 2b or 3-methylamino-phthalimide 2f (Table 2a, entries 1-6). As expected, moving the acid to the fifth position 1h prevents cyclization (Table 2a, entry 7). In addition to supporting the proposed IAA mechanism, we also showed for the first time how phthalimides, used in the synthesis of quinazoline 5-acid derivatives from an isatin derivative, are formed 16 . These sulfuric acid-based methods are easier to perform than established procedures for generating phthalimides involving urea 17 , ammonium carbonate at elevated temperature, or reduction using palladium 18 . This method also provides synthetic pathways to a wide range of N-substituted phthalimides, whereas prior methods are limited to 3-methylamino-phthalimide 17,19 .
While it is theoretically possible that the clockwise (rightward) cyclization required to form IAA may occur with either an acid, ester, or amide, the acid at position 7 is preferred (Table 2b, entries 8-9). Cyclization also depends on the availability of the secondary amine at position 1, as shown by failure to produce N-alkyl IAA when an alkyl group is introduced at position 1 (Table 2b, entry 10,11). Surprisingly, alkyl substitution at first position yielded a cyano aniline for both acid and esters 2k, 2l (Table 2b, entries 10, 11), suggesting that the combined effects of electron withdrawal by the acid and N-alkyl groups contribute to forming nitrile from the primary amide. Of note, known methods for the synthesis of n-alkylated nitrile-acid nitrile-ester involve using corrosive chemicals, high temperatures, and purification composed of multistep reactions, which are eliminated with the current method 20 . To assess whether incorporating a secondary amide at position 8 is possible, we started with enamines at position 3 1m, 1n, although they yielded IAA instead of a secondary amide (Table 2b, entries 12, 13). This finding suggested that enamines are unstable under acidic conditions. Substitution on the phenyl ring with bromine at position 5 1o was also tolerated, giving 2o in excellent yield (Table 2b, entry 14). To exclude the possibility that cyclization may occur using an n-alkyl acid at position 1 we tested reactions with isatin-N-propanoic acid and obtained an open chain product 2p (Table 2b, entry 15), instead of oxazine, suggesting that oxazine is less stable than benzoxazine.
Our synthetic method simplifies isatin transformations considerably compared to prior methods. Synthesis of isatoic anhydride from isatin previously required using reagents such as peroxides 21,22 , phenyliodide 23 , and NBS 24 ; making benzamide derivatives from isatin required using chromic acid 25 , peroxide/phosphate buffer systems 26 , or

Scope of Quinazolines originating from iAA
We next focused on the conversion of IAA to quinazoline derivatives, (Table 4). IAA readily transformed to quinazoline-8-carboxylic acid 14a by heating or exposure to basic conditions (Table 4, entry 1). Interestingly, while attempting to make tertiary butyl esters from IAA by using potassium tertiary butoxide, we instead obtained either 14a at room temperature or 9a at 50 °C, indicating the tunability and versatility of the IAA reactions. The corresponding quinazoline-8-amide 15 was achieved by adding amine and coupling reagent to in situ generated quinazoline-8-acid at 100 °C (Table 4, entry 2). To show that N3-substituted quinazoline can be obtained, we treated IAA with an amine followed by PhNCS, yielding an N3-substituted quinazoline 16 ( www.nature.com/scientificreports www.nature.com/scientificreports/ Alternatively, N3-substituted quinazoline 17 was obtained by treating with 1 equivalent of an amine source in acetic acid, suggesting that the secondary amide is preferred over primary amide during cyclization (Table 4, entry 4). An attempt to convert p-amide to nitrile using TFAA in pyridine unexpectedly gave a 2-substituted quinazoline 18, showing that amide is preferred over acid during cyclization and also indicating the possibility of generating a series of 2-substituted quinazolines. (Table 4, entry 5).
We noted that a number of quinazoline-based inhibitors, including inhibitors of BRAF and p38, utilize C2 and N3 substitutions 31 . We speculated that these substitutions could be obtained by reactions with amidine-containing compounds, if they react at C4. Accordingly, the combination of benzimidine with IAA resulted in 2-substituted quinazolines-8-amide 19 in excellent yields (Table 4, entry 6). N-substituted benzamidine also leads to isolating 19, indicating selectivity of primary amide formation for secondary amide during cyclization. Surprisingly, 2-amino benzamidine gave a similar product 20, showing selectivity of benzimidine-NH 2 over phenyl-NH 2 ( Table 4, entry 7). The corresponding quinazoline-8-cyano derivatives 21 can be synthesized by treating in situ-generated nitrile-TEA salt with amines and coupling reagents ( Table 4, entry 8).
Developing IAA and its derivatives opens new options for generating highly substituted anilines and functionalized quinazolines. The versatility is multiplied by the availability of many anthranilic acid derivatives, allowing to easily generate IAA derivatives that contain functional group positions 4, 5, and 6, which may lead to generating quinazolines functionalized in the 5, 6, and 7 positions (Fig. S5); a challenging and perhaps impossible transformation at the quinazoline stage. Likewise, a few examples of anilines with 3 ortho groups, either as intermediates or end products, are found in the literature. This raises the prospect of generating novel highly substituted anilines with functional groups that were not previously considered in medicinal and industrial chemistry. www.nature.com/scientificreports www.nature.com/scientificreports/ Materials and Methods chemicals. Starting materials, reagents, and solvents were purchased from commercial sources and used as received, unless stated otherwise. Isatin derivatives were purchased from Enamine, a Sigma-Aldrich partner. Melting points were determined using μTherm°Cal 10 (Analab scientific Pvt. Ltd.) melting point apparatus and are not corrected. Reaction progress was monitored by thin layer chromatography on Merk's silica plates. 1 H and 13 C NMR spectra were recorded on Varian 400 MHz instruments using TMS as internal standard. Mass spectrometry data were recorded on Shimadzu LCMS 2010 mass spectrometer. IR spectrometry data were recorded on a FTIR Perkin Elmer Spectrum 100 spectrometer as KBr pellets with absorption in cm −1 .
Single crystal x-ray diffraction of IAA. Crystals grew as clusters of colorless thin needles by slow evaporation from acetone and dioxane using a starting concentration of 5 mg/ml. Diffraction data were collected from a crystal with approximate dimensions of 0.38 × 0.06 × 0.04 mm on an Agilent Technologies SuperNova Dual Source diffractometer using a μ-focus Cu K α radiation source (λ = 1.5418 Å) with collimating mirror monochromators, and at 100 K using an Oxford 700 Cryostream low temperature device. Details of crystal data, data collection, and structure refinement are listed in Tables S1-7. Data collection, unit cell refinement, and data reduction were performed using Agilent Technologies CrysAlisPro V 1.171.39.46. The structure was identified by direct methods and refined by full-matrix least-squares on F 2 with anisotropic displacement parameters for the non-H atoms using SHELXL-2016/6 32,33 . Structure analysis was aided by using PLATON 34 and WinGX 1.64. The hydrogen atoms on carbon were calculated in ideal positions with isotropic displacement parameters set to 1.2 × U eq of the attached atoms. The hydrogen atoms bound to the nitrogen atoms were located in a ΔF map and refined with isotropic displacement parameters.
The IAA (C 9 H 6 N 2 O 4 ) crystal demonstrated a monoclinic P21/C space group with 4 crystallographically unique molecules in the asymmetric unit. The absolute configuration was the same for all molecules in the asymmetric unit. The function, Σw(|F o | 2 − |F c | 2 ) 2 , was minimized, where w = 1/[(σ(F o )) 2 + (0.085 * P) 2 + (0.0727 * P)] and P = (|F o | 2 + 2|F c | 2 )/3. R w (F 2 ) refined to 0.127, with R(F) equal to 0.0444 and a goodness of fit, S = 1.06. Definitions used for calculating R(F), R w (F 2 ) and the goodness of fit, S, are given below. The data were checked for secondary extinction effects but no correction was needed. Neutral atom scattering factors and values used to calculate the linear absorption coefficient are from the International Tables for X-ray Crystallography 35 . All figures www.nature.com/scientificreports www.nature.com/scientificreports/ were generated using SHELXTL/PC V5.03 (Siemens Analytical X-ray Instruments, Inc., Madison, Wisconsin, USA). (E/Z)-3-hydrazineylidene-2-oxoindoline-4-carboxylic acid (1e): 1.0 mmol of (190 mg) Isatin-7-acid was cooled to 0 °C in a 50 ml round bottom flask. Five milliliters of concentrated sulfuric acid were added to the solution, followed by 1.0 mmol (186 mg) of tosyl hydrazine. The mixture was stirred at room temperature for 3 h. Cold water was poured on the solution to allow it neutralization. After extraction with DCM (50 ml × 2), the combined organic layer was washed with water, dried over MgSO 4, and filtered. The filtrate was concentrated to obtain 163 mg of product in 81% yield. 1  1-methyl-2,3-dioxoindoline-4-carboxylic acid (1f): 150 mg of Methyl 1-methyl-2,3-dioxoindoline-7-carboxylate (0.68 mmol) was dissolved in water: THF 1:1 (10 mL: 10 mL), to which 3.0 eq of LiOH (6.9 mmol, 60 mg) was added and stirred at ambient temperature overnight. The solution was concentrated using a rotary evaporator and the residue was dissolved in alkaline water (pH 11). Unreacted starting material was then removed by extraction using diethyl ether. The aqueous layer was then acidified with HCl and extracted twice with diethyl ether. The ether extract was washed with water and dried over MgSO 4 . A filtrate was concentrated and showed to be pure by NMR in 80% yield (113 mg). 1 H NMR (400 MHz, DMSO-d 6 ): δ 7.71 (t, J = 7.9 Hz, 1H), 7.27 (d, J = 7.9 Hz, 2H), 3.13 (s, 3H) 37 . www.nature.com/scientificreports www.nature.com/scientificreports/ Methyl 1-methyl-2,3-dioxoindoline-4-carboxylate (1g): Isatin-7-acid (285 mg, 1.5 mmol) was dissolved in 20 mL of DMF, to which 2.2 eq of potassium carbonate (455 mg) was added, followed by 4.0 eq of methyl iodide. This mixture was stirred overnight at room temperature, concentrated using a rotary evaporator, and then dissolved in water. The product was extracted with dichloromethane twice, washed with water, dried over MgSO 4 , and filtered. The solution was further concentrated with a rotary evaporator to obtain an essentially pure compound in 81% yield (280 mg), 1 H NMR (400 MHz, Chloroform-d): δ 7.66 (t, J = 7.9 Hz, 1H), 7.46 (dd, J = 7.9, 0.8 Hz, 1H), 7.05 (dd, J = 7.9, 0.9 Hz, 1H), 3.97 (s, 3H), 3.27 (s, 3H). 13   1-methyl-2,3-dioxoindoline-7-carboxylic acid (1k): 500 mg of Methyl 1-methyl-2,3-dioxoindoline-7-carboxylate (2.3 mmol) were dissolved in water: THF 1:1 (25 mL: 25 mL), to which 3.0 eq of LiOH (6.9 mmol, 165 mg) was added and stirred at ambient temperature overnight. The solution was concentrated using a rotary evaporator and the residue was dissolved in alkaline water (pH 11). Unreacted starting material was then removed by extraction using diethyl ether. The aqueous layer was acidified with HCl and extracted twice with diethyl ether. The ether extract was washed with water, and then dried over Methyl 1-methyl-2,3-dioxoindoline-7-carboxylate (1l): Isatin-7-acid (570 mg, 3.0 mmol) was dissolved in 20 mL of DMF, to which 2.2 eq of potassium carbonate (910 mg) was added, followed by 4.0 eq of methyl iodide. This mixture was stirred overnight at room temperature. The solution was concentrated using a rotary evaporator and dissolved in water. The product was extracted with dichloromethane twice, washed with water, dried over MgSO 4 , filtered, and concentrated on rotary evaporator to obtain an essentially pure compound in 91% yield (595 mg). 1  www.nature.com/scientificreports www.nature.com/scientificreports/ (t, J = 7.6 Hz, 1H), 3.88 (s, 3H), 3.09 (s, 3H). 13  (E,Z)-3-(isopropylimino)-2-oxoindoline-7-carboxylic acid (1m): Isatin-7-acid and isopropyl amine were combined in ethanol at equimolar ratio. A catalytic amount of acetic acid was added to the solution and heated to reflux for 3 h. The solution was concentrated using a rotary evaporator and then poured into water which allowed a precipitate to form. The filtrate was washed with water and dried to obtain the essentially pure E/Z mixture of product in quantitative yield. 1 13 (1n): 1 mmol of (190 mg) Isatin-7-acid in 50 ml round bottom flask was cooled to 0 °C. Sulfuric Acid was added to this 5 ml concentrate, followed by 1 mmol (186 mg) of tosyl hydrazine. A dark brown color developed immediately. The mixture was stirred at room temperature for 3 h. Upon addition to cold water, a red-colored solid precipitate developed and was recovered by filtration. The filtrate was washed with water and dried to give the product in quantitative yield (180 mg). 1 (1o): 382 mg of Isatin-7-Acid was dissolved in 2.0 ml of concentrated sulfuric acid at room temperature. Liquid bromine at 1.2 equivalents (195 mg) was added at room temperature. The solution was stirred while heating at 100 °C, until bromine completely dissolved in sulfuric acid (10-15 min). This was further cooled to room temperature and poured into cold water, to form an orange to yellow precipitate. This was filtered and washed with water. We obtained 520 mg of product. ( www.nature.com/scientificreports www.nature.com/scientificreports/ Method 1-2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine-8-carboxamide (2a): 1.0 g (5.5 mmol, 1.0 eq) of 2,3-dioxoindoline-7-carboxylic acid (1a) was added to a 50 ml round bottom flask equipped with a gas bubbler. Later 100 ml of 1 N NaOH were added dropwise. The solution was cooled to 0 °C and spiked with 10 ml of concentrated sulfuric acid. After 10 min, 410 mg (6.3 mmol, 1.2 eq) of sodium azide were added gradually over a period of 10 min. The solution was stirred at 0 °C-RT for 1 h and left at room temperature for 1 h. The reaction was then combined with 100 ml of cold water to form a precipitate. The precipitate was recovered using a Buckner flask and dried at room temperature to obtain 860 mg of product (80%). MP, 237 °C. 1 H NMR (400 MHz, DMSO-d 6    2-amino-3-carbamoylbenzoic acid, free base (3a): 1.0 g (5.5 mmol, 1.0 eq) of 2,3-dioxoindoline-7-carboxylic acid (1a) was added dropwise to a 50 ml round bottom flask equipped with gas bubbler and containing 100 ml of 1 N NaOH. The solution was cooled to 0 °C and spiked with 10 ml of concentrated sulfuric acid. After 10 min, 410 mg (6.3 mmol, 1.2 eq) of sodium azide were added gradually over a period of 10 min. The solution was stirred at 0 °C-RT for 1 h and left at room temperature for 1 h. A precipitate formed after adding the reaction to 100 ml of cold water. NaOH solution was used to adjust to pH 8. The product was recovered by filtration, washed with water and dried at room temperature to get 760 mg pale yellow solid in 80%. 1 (3b): Method B. 206 mg of IAA (2b) was dissolved in 1.0 ml of concentrated sulfuric acid at room temperature. A 1.1 equivalent (180 mg) of liquid bromine was added to the solution at room temperature. This was stirred while heating at 100 °C until the bromine was completely dissolved (10-15 min). After cooling to room temperature and adding to cold water, an orange yellow precipitate formed and was recovered by filtration and washed with additional water. We obtained 240 mg of product. (93% yield). 4 series, Method A: A 0.1 mmol IAA in polar solvent was stirred at reflux for 2 h, cooled to room temperature, and concentrated. The residue was titrated with chloroform-hexane to generate the pale white solid product in 73-83% yield. 4 series, Method B: A 0.1 mmol IAA in 1.0 ml of solvent was combined with 0.2 mmol (28 mg) K 2 CO 3 and stirred at room temperature (12 h). After complete disappearance of the IAA peak by GC, the reaction mixture was added to water and extracted with dichloromethane. This was further washed with water, dried over MgSO 4 , and recovered by filtration. 73% yield 40 .  192.52, 170.95, 149.78, 135.49, 133.91, 118.63, 117.01, 113.97 (6): Method B. A suspension of 0.1 mmol of IAA, ammonium acetate (0.4 mmol), and acetic acid (5 mL) was heated at reflux for 2 h. The reaction mixture was cooled to room temperature and evaporated under reduced pressure. Five milliliters of water were added to the residue to form a precipitate. This was recovered by filtration, washed with water, and dried at room temperature; 16 mg of product, 95% yield. (6): Method C. A suspension of 0.1 mmol of IAA and ammonium carbonate (0.4 mmol) in DMSO was heated at 50 °C for 6 h. Water was added to form a precipitate. The precipitate was recovered by filtration, washed with water, and dried at room temperature; 16 mg of product in 95% yield.

2-aminoisophthalamide
2-aminoisophthalamide (6): Method D. A suspension of 0.1 mmol of IAA and ammonium acetate (0.4 mmol) in DMSO was heated at 50 °C for 6 h. Water was added to the residue to form a precipitate. The precipitate was recovered by filtration, washed with water, and dried at room temperature; 16 mg of product, 95% yield.
General procedure for Compound-7 derivatives: To a 0.1 mmol of IAA in 1.0 mL of DMSO and 0.1 mmol of amine was added and stirred at 50 °C for 6 h. The reaction was followed by GC. Upon completion, this was added to 5 ml of water and acidified to pH 2, filtered and dried. Product yield was 85-95%.   171.89, 156.32, 152.17, 152.09, 135.85, 132.97, 130.06, 124.53, 120.08, 115.98, 112.98, 110.45, 110.16, 97.64, 56.04, 28.86. MS (m/z): 340.1 (M + 1).   N-(1-(2-methoxyethyl) www.nature.com/scientificreports www.nature.com/scientificreports/ 2-amino-3-cyanobenzoic acid (9a): Method C. 0.1 mmol (14 mg) of K 2 CO 3 were added to 0.1 mmol of IAA in 1.0 ml of DMSO and heated at 50 °C for 3 h. The reaction was added to water and acidified to pH 2. A solid product was recovered by filtration and dried to obtain 16 mg of pale-yellow solid in 98% yield. General procedure compound 10a-e cyano-ester Series: Method A. 0.1 mmol (14 mg) of K 2 CO 3 were added to 0.1 mmol of IAA in 1.0 ml of DMSO and heated at 50 °C for 3 h. After the complete disappearance of the IAA peak by GC, alkyl halide was added and stirred at room temperature for another 12 h. The reaction was extracted with dichloromethane to remove the unreacted alkyl halide and then added to water. The solution was acidified to pH 3, extracted with dichloromethane, and washed with water. The product was dried over MgSO 4 and recovered by filtration. The filtrate was concentrated to obtain product in 80-95% yield.

2-amino-5-bromo-3-cyanobenzoic acid
General procedure for 10a-e cyano-ester Series: Method B.0.3 mmol of trimethylamine were added to a mixture of 0.15 mmol IAA in DMSO and heated at 50 °C for 3 h. After the complete disappearance of the IAA peak by GC, alkyl halide was added and stirred at room temperature overnight. The reaction was extracted with dichloromethane to remove the unreacted alkyl halide and then added to water. The solution was acidified to pH 3, extracted with dichloromethane, and washed with water. The product was dried over MgSO 4 and recovered by filtration. The filtrate was concentrated to obtain product in 80-95% yield.
General procedure compound 10a-e, cyano-ester: Method C. 0.3 mmol of triethylamine were added to 0.15 mmol of IAA in 1.0 ml of DMSO and heated at 50 °C for 3 h. After the complete disappearance of the IAA peak by GC, 0.12 mmol of BOP or HBTU and 0.1 mmol of Amines/alcohols/thiols were added and stirred at room temperature overnight. The reaction was added to water and acidified. The product was filtered and washed with water, then dried over vacuum to obtain the product in 85-96% yield. General procedure compound 12a-g, cyano-amide: 0.3 mmol of triethylamine were added to 0.15 mmol of IAA in 1.0 ml of DMSO and heated at 50 °C for 3 h. After the complete disappearance of the IAA peak by GC, 0.12 mmol of BOP or HBTU and 0.1 mmol of amines were added and stirred at room temperature overnight. The reaction was added to water and acidified. The product was filtered and washed with water, then dried over vacuum to obtain the product in 85-96% yield.  -N-(1-(tert-butyl)-1H-benzo[d]imidazol-2-yl)-3-cyanobenzamide (12f): Method C. 45 mg, pale white solid, 93% yield. 1 H NMR (400 MHz, DMSO-d 6 ): δ 8.40 (dd, J = 7.9, 1.7 Hz, 1H), 7.82 (dd, J = 7.6, 1.6 Hz, 1H), 7.59 (ddd, J = 10.7, 7.5, 1.8 Hz, 2H), 7.24-7.10 (m, 2H), 6.70 (t, J = 7.7 Hz, 1H), 1.93 (s, 9H). 13 N-(1-(2-methoxyethyl) (13): 206 mg of IAA (2a) were dissolved in 1.0 ml of concentrated sulfuric acid at room temperature. A 2.2 equivalent (350 mg) of liquid bromine was added to this solution at room temperature. This was stirred at 100 °C, until the bromine completely dissolved in sulfuric acid (10-15 min). The mixture was cooled to room temperature and added to cold water until a pale yellow precipitate formed. The precipitate was recovered by filtration, washed with additional water, and dried to give 280 mg of product (95% yield). 1  2,4-dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxylic acid (14a): Method B. 0.1 mmol of IAA in DMSO was combined with 0.11 mmol (1.2 eq) of potassium tert-butoxide and stirred at room temperature overnight. The reaction mixture was poured into water and acidified to pH 2 to form a precipitate. The precipitate was recovered by filtration using a Buckner flask and dried at room temperature to obtain 20 mg of product (90%).

Data Availability
Crystallographic model data is available through the CCDC under identifier 1896630. Requests for materials should be addressed to K.D.W. (kenneth.westover@utsouthwestern.edu).