A facile one step synthesis of N -2 substituted 3-phenyliminoisoindolinones from N -(2-carboxybenzoyl)-anthranilic acid and the design of reverse-turn mimetics

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Introduction
Generally 3-iminoisoindolinone derivatives are pigments with excellent color strength and suitable for pigmenting organic materials of high molecular weight, such as ethyl cellulose, acetyl cellulose, nitrocellulose, polyamide, polyester, natural resins and synthetic resins. 1 Further iminoisoindolinones are used to prepare nanosized organic pigments which are used effectively as additives for crystal growth during pigment synthesis. 23-Iminoisoindolinone itself is found to form inclusion complexes with βand γ-cyclodextrins. 3Interestingly it has been observed that 3iminoisoindolinone can bind to an enzyme possessing N-iminylamidase activity isolated from pig liver. 4Although 3-iminoisoindolinone and its derivatives are important for diverse kinds of applications, there has been only a limited number of methods available in the literature for their synthesis.As for example iminoisoindolinone pigments are obtained by condensation of suitably substituted isoindolinones with diamines. 1,2The synthesis of 3-phenyliminoisoindolinone is achieved by refluxing 1-amino-3-phenyliminoisoindoline with phthalonitrile. 5In this report we wish to present a general and efficient method for the synthesis of N-2 substituted 3phenyliminoisoindolinones from easily prepared N-(2-carboxybenzoyl)anthranilic acid.
It is well established that reverse-turns play important roles in stabilizing tertiary structures, initiating folding and facilitating intermolecular recognition. 6There have been increasing efforts to rationally design and synthesize biologically active non-peptidic analogues of peptide reverse turns. 7Since enzymes like mammalian imidase recognizes 3-iminoisoindolinone as substrate for hydrolysis, 4 it will be quite pertinent to design reverse turns on a 3-iminoisoindolinone scaffold.They will provide an opportunity to explore the substrate specificity, chemoselectivity and the mechanism of enzymatic hydrolysis of compounds bearing the N-iminylamide functional group.

Results and Discussion
Initially we thought that N-(2-carboxybenzoyl)anthranilic acid 1, the alkaline hydrolysis product of 2-phthalimidobenzoic acid, 8 could be a reverse-turn inducing scaffold.Therefore, we were trying to develop reverse-turn mimetics through DCC mediated coupling of 1 with various anilines, amines and amino acids.Surprisingly we observed that stirring of 1 with p-toluidine in the presence of DCC in DMF produces an unusual product N-2 substituted 3phenyliminoisoindolinone 2a in very good yield (Scheme 1, Table 1, Entry 1).Under similar conditions, 1 also produces 2b by reaction with benzylamine (Entry 2).We have proposed a mechanism for this reaction.The intermediate 3 which is formed through the DCC mediated coupling of 1 with p-toluidine and benzylamine, undergoes intramolecular nucleophilic attack of the amide NH to the phthalamide CO to produce the isoindolinone based intermediate 4.
Subsequently dehydration produces the final compounds 2a and 2b.Previous reports show that 3-phenyliminoisoindolinone can be prepared by refluxing 1-amino-3-phenyliminoisoindoline with phthalonitrile. 5Synthesis of compounds 2a and 2b by this method is not straightforward.Some synthetic optimization is necessary to achieve the target.The present method represents a novel one step reaction to derive N-2 substituted 3-phenyliminoisoindolinones such as 2a and 2b starting from 1.
The formation 2a and 2b in the above method indicates that the natural L-α-amino acids would be good candidates for reaction with 1 in a similar fashion to provide a useful route to achieve reverse-turn mimetics on the basis of a 3-phenyliminoisoindolinone scaffold.In fact, the DCC mediated coupling of 1 with methyl esters of various amino acids such as Leu, Phe, Ile and Val produces 3-phenyliminoisoindolinones 2c-f in very good yields (Scheme 1, Table 1, Entry 3-6).The structures of compounds 2a-f were confirmed by their IR, 1 H NMR, and 13 C NMR spectra.The X-ray crystal structures of 2c and 2d further confirm the product formation. 9,10The formation of 2a-f through the coupling of 1 with p-toluidine, benzylamine and various amino acids demonstrates the synthetic potential and generality of the present method.
The DCC mediated reaction of 1 with anilines containing electron withdrawing groups such as methyl m-aminobenzoate and p-nitroaniline produces different products 5a and 5b (Scheme 2, Table 1, Entry 7 and 8).Since the coupling of methyl m-aminobenzoate and p-nitroaniline with 1 is slow, the intermediate 2-phthalimidobenzoic acid 6 is formed in a higher rate than intermediate 3. Finally coupling of 6 with anilines produces 5a and 5b (Scheme 2).

Scheme 2
Single crystal X-ray diffraction studies show that both 2c and 2d adopt turn structures around the central 3-phenyliminoisoindolinone scaffold (Figures 1 and 2).Although a previous report showed that 3-phenyliminoisoindolinone prefers the anti conformation in the solid state, 5 in 2c and 2d the centrally placed 3-phenyliminoisoindolinone moiety adopts the syn conformation.This helps 2c and 2d to attain a turn structure around the imino C=N bond.The torsion angle C(41)-N(51)-C(52)-N( 10) is -5.2(2) o at iminoisoindolinone moiety and N(10)-C9-C4-C3 = 7.1(2) o at the anthranilic acid moiety are responsible for inducing a turn structure in 2c (Table 2).The corresponding torsion angles values 0.9(4) o and 3.0(4) o in 2d ensure a turn conformation.A β-turn is defined as a tetrapeptide sequence in which the α-carbon distance between first and fourth residue is less than 7 Å. 11Crystal structure analysis reveals that the distance between the α-carbon atoms (between C(41) and C(1)) in 2c and 2d are 6.32 and 4.95 Å respectively, well within the limit for satisfying the criteria of β-turn mimetics (Figures 1, 2).The only major difference in the conformation of the backbone of 2c and 2d can be seen in the torsion angle C( 9   In the 1 H NMR solvent titration it has been observed that by increasing the percentage of (CD 3 ) 2 SO in CDCl 3 from 0 to 8.1 % (v/v) the net changes in the chemical shift (∆δ) values for Leu-NH in 2c and Phe-NH in 2d are negligible, 0.00, and 0.02 ppm respectively (Figure 3).The result indicates that in the solution phase Leu-NH of 2c and Phe-NH of 2d are strongly hydrogen bonded preferably with the C=O of the methyl ester group of the remaining amino acid within the same molecule to stabilize the 11-membered turn structures (Figure 4).Further the ability of 2c and 2d to adopt a turn structure in solution was also evaluated by CD spectroscopy (Figure 5).The spectra were measured in methanol (1.5 mM) and showed a similar behaviour: two negative minima, one at 207-210 nm and a second one at 230-235 nm and a positive maximum at 119-223 nm were displayed by both the pseudopeptides 2c and 2d.The CD pattern is found to be quite similar to that of reported β-hairpin mimetics containing diketopiperazine as turn inducing scaffold. 15Therefore, the results of solvent dependent NMR titrations and CD spectroscopy favor the conclusion that both 2c and 2d are folded into turn structures in the solution phase.Generally 3-iminoisoindolinone derivatives are color pigments. 1They are also useful for preparing nanosized organic pigments. 2In the present study 2a and 2b are found to be white.Interestingly all the compounds 2c-f derived from the amino acids are found to be yellow in color.The photograph of yellow crystals of 2d is presented in Figure 6.

Conclusions and out look
In summary, we have found a general and efficient method for the preparation of N-2 substituted 3-phenyliminoisoindolinones by DCC mediated coupling of N-(2-carboxybenzoyl)anthranilic acid with activated anilines, amines and amino acids.The present method is a simple one-step reaction involving easily prepared starting materials.Single crystal X-ray diffraction studies and solution phase NMR and CD studies on two pseudopeptides reveal that 3phenyliminoisoindolinone is a turn-inducing scaffold.Apart from developing reverse-turns this scaffold could be utilized in engineering the turn regions of β-hairpins and multiple antiparallel β-strands.The exploration of the substrate specificity, chemoselectivity and catalytic mechanism of hydrolysis of enzyme mammalian imidase 4 with respect to these turns and hairpins may provide more insights.

Experimental Section
General procedure for products 2a and 2b (2.0 g, 7.02 mmol) of compound 1 was dissolved in dimethylformamide (DMF, 8 mL).Appropriate anilines and amines such as p-toluidine and benzylamine(42.12mmol) were added to the former solution followed by addition of DCC (4.34 g, 21.06 mmol) in an ice-cold condition.The reaction mixture was stirred at room temperature for 1 day.The precipitated dicyclohexylurea (DCU) was filtered off.The filtrate was diluted with ethylacetate.The organic layer was washed with 1N HCl (3 x 30 mL), brine, 1M Na 2 CO 3 solution (3 x 30 mL) and then again with brine.The solvent was dried over anhydrous Na 2 SO 4 and evaporated in vacuo, giving a light yellow gum.The products were purified by column chromatography over silica gel (ethyl acetate-petroleum ether).The final compounds were fully characterized by 300 MHz 1 H NMR spectroscopy, 75MHz 13  General procedure for products 2c-f (2.0 g, 7.02 mmol) of compound 1 was dissolved in dimethylformamide (DMF, 8mL).Methyl esters of various amino acids such as Leu, Phe, Ile and Val obtained from their hydrochloride (42.12 mmol) were added to the former solution followed by addition of DCC (4.34 g, 21.06 mmol) and HOBT (1.90 g, 14.04 mmol) in an ice-cold (solvent) solution. 16The reaction mixture was stirred at room temperature for 1 day.The precipitated dicyclohexylurea (DCU) was filtered off.The filtrate was diluted with ethyl acetate.The organic layer was washed with 1N HCl (3x30mL), brine, 1M Na 2 CO 3 solution (3x30mL) and then again with brine.The solvent was dried over anhydrous Na 2 SO 4 and evaporated in vacuo, giving a light yellow gum.The products were purified by column chromatography over silica gel (ethylacetate-petroleum ether).The final compounds were fully characterized by 300 MHz 1 H NMR spectroscopy, 75MHz 13 C NMR spectroscopy, IR spectroscopy.Single crystals were grown from a mixture of ethyl acetatepetroleum ether (for 2c) and chloroform-petroleum ether (for 2d) by slow evaporation and were stable at room temp.General procedure for products 5a and 5b 5a and 5b were produced following the similar procedure as that of 2a and 2b described previously.Here 1 was coupled with methyl m-aminobenzoate and p-nitroaniline in presence of DCC to produce 5a and 5b respectively (

NMR experiments
All 1 H NMR and 13 C NMR study was recorded on a Bruker Avance 300 model spectrometer operating at 300, 75 MHz respectively.The peptide concentration was in the range 1-10 mM in CDCl 3 and d 6 -DMSO for 1 H NMR and 30 -40 mM in CDCl 3 and d 6 -DMSO for 13 C NMR. Solvent titration experiments were carried out at a concentration of 10mM in CDCl 3 with gradual addition of d 6 -DMSO from 0-8.1 % v/v approximately.

Circular Dichroism Spectroscopy
A methanolic solution of peptide 2c and 2d (1.5 mM as final concentration) was used for obtaining the spectra.Far-UV CD measurement was recorded at 25 o C with a 0.5sec averaging time, a scan speed of 50 nm/min, using a JASCO spectropolarimeter (J 720 model) equipped with a 0.1 cm pathlength cuvette.The measurement was taken at 0.2nm wavelength interval, 2.0 nm spectral bandwidth and five sequential scans were recorded for the sample.6) o , U = 1427.36(13)Ǻ 3, dcalc = 1.330 gcm -3 ; 7820 and 5862 independent data for 2c and 2d respectively were collected with MoKα radiation at 150K using the Oxford Diffraction X-Calibur CCD System.The crystals were positioned at 50 mm from the CCD.321 frames were measured with a counting time of 10s.Data analyses were carried out with the CrysAlis program. 9The structures were solved using direct methods with the Shelxs97 program. 10The non-hydrogen atoms were refined with anisotropic thermal parameters.The hydrogen atoms bonded to carbon were included in geometric positions and given thermal parameters equivalent to 1.2 times those of the atom to which they were attached.The structures were refined on F 2 using Shelxl97 10 to respectively R1 0.0440 and 0.0548; wR2 0.0987 and 0.1403 for 5103 and 4656 reflections with I>2σ(I).Crystallographic data have been deposited at the Cambridge Crystallographic data Centre with reference number CCDC 699562 and 699563 for peptides 2c and 2d respectively.

13 Figure 1 .
Figure 1.The ORTEP diagram of 2c including atom numbering scheme.Thermal ellipsoids are shown at the level of 50% probability.

Figure 2 .
Figure 2. The ORTEP diagram of 2d including atom numbering scheme.Thermal ellipsoids are shown at the level of 50% probability.

3 Figure 3 .
Figure 3. Plot of solvent dependence of NH chemical shifts (δ in ppm) of pseudopeptides 2c and 2d at varying concentration of DMSO-d 6 in CDCl 3.

Figure 4 .
Figure 4. Schematic diagram of intramolecular hydrogen bond of 2c and 2d in the solution phase.

Figure 6 .
Figure 6.Photograph of the crystals of compound 2d.