SECTION C Organic Chemistry a-Aminoacyl Derivatives of Aminobenzoic Acid and of Amino-oxy-acids by Reaction of their Hydrochlorides with Amino-acid N-Carboxy- an h yd rides By Y. Knobler," S. Bittner, D. Virov, and Max Frankel, Department of Organic Chemistry, The Hebrew Uni- versity of Jerusalem, Jerusalem, Israel The reaction of amino-acid N-carboxyanhydrides with hydrochlorides of aminobenzoic acids and of amino- oxy-acids gave amino-acid amides in a one-step synthesis. The coupling of L-alanine N-carboxyanhydride with anthranilic and with p-aminobenzoic acid hydrochloride gave peptide-like compounds of high optical activity. indicating the presence of little or no racemate. Glycyl and DL-phenylalanyl amides with aminobenzoic acid were obtained in this way, and trimers were formed by reaction of the N-carboxyanhydrides with p-aminobenzoyl- L-glutamic acid hydrochloride.Parallel procedures led to the u-, p-, and r-amido-oxy-peptides of amino-oxy-acetic, -propionic, and -butyric acid, respectively. THE reaction of amino-acid N-carboxyanhydrides with amine salts was outlined previously as a useful synthesis of aminoacyl derivatives of weakly basic amines.l The applicability of the reactions has been extended to give peptide-like derivatives of weakly basic amino- acids, such as aminobenzoic acids and amino-oxy-acids. These coupling reactions could be carried out with hydrochlorides of unprotected amino-acids, by virtue of the pronounced difference in ability to form salts between the starting material and the product. HCI NH *C,H,.CO,H RCH-CO -+ \o HC1,NH,=CHR-CO*NH*C6H4C02H + CO, HC1,NH,*CHRCO-NH*O-[CH2-jn*C0,H + CO, - NH-CO HCI,NH,*O*[CH&*CO~H n = I, 2, or 3 R = H, Me, CH,*Ph, or CH,*CH,*CO,*CH,-Ph Amino-acid N-carboxyanhydrides were coupled with hydrochlorides of anthranilic acid, @-aminobenzoic acid, and their esters by dissolving both reagents in dimethyl- formamide.The reaction proceeded at room tempera- ture with slow evolution of carbon dioxide. The products were obtained as precipitates from solution or after removal of the solvent (see Table 1). As carbon dioxide is the predominant by-product, the peptide-like derivatives prepared by this method could be obtained in a high state of purity. The facts that the reaction takes place in one step and that only weakly acidic conditions are required during reaction and 1 Y .Knobler, S. Bittner, andM. Frankel, J . Chem. SOL, 1964, a F. E. King, J. W. Clark-Lewis, D. A. A. Kidd, and G. R. 3941. Smith, J . Chem. SOL, 1954, 1039. recrystallisation made possible the production of amides with high specific rotations. This method thus compares well with others r e p ~ r t e d . ~ - ~ Optically active L-alan ylan t hranilic acid hydrochloride and L-alanyl-@-aminobenzoic acid hydrochloride were prepared ; their optical purity was proved by hydrolysis, which regenerated L-alanine with the correct rotation. The degree of racemisation during acylation of benzo- caine hydrochloride with L-alanine N-carboxyanhydride has been compared with that occurring during coupling by the dicyclohexylcarbodi-imide rneth~d.~ The L- alanyl-@-aminobenzoic acid ethyl ester hydrochloride obtained was treated with benzyloxycarbonyl chloride.The derivative produced (N-benzyloxycarbonyl-L-alanyl- p-aminobenzoic acid ethyl ester) was also prepared by condensing N-benzyloxycarbonyl-L-alanine with benzo- caine by use of dicyclohexylcarbodi-imide. No signifi- cant difference between the specific rotations of the compounds was observed. Compounds related structurally to folic acid were synthesised by treating p-aminobenzoyl-L-glutamic acid hydrochloride with DL-phenylalanine, glycine, or y- benzyl-L-glutamic acid N-carboxyanhydride. Amino-acid N-carboxyanhydrides were coupled with amino-oxy-acid hydrochlorides in ethanol-water at room temperature. The pronounced nucleophilic activity of the amino-oxy-group and the high degree of dissociation of the amino-oxy-acid hydrochlorides in water allow interaction in aqueous ethanol.Data for W. Langebeck and D. Weisbrod, J . prakt. Chern., 1965, 28, R. F. Lloyd, C. G. Skinner, W. Shive, and R. J. Stedman, J. C. Sheehan and G. P. Hess, J . Amer. Chem. SOG., 1955, 78. J . Medicin. Chem., 1965, 8, 398. 77, 1067.1822 J. Chem. SOC. (C), 1969 glycyl, DL-phenylalanyl, and y-benzyl-L-glutamyl amido- oxypeptides of amino-oxyacetic, P-amino-oxypropionic, and y-amino-oxybutyric acid are given in Table 2. EXPERIMENTAL L-Alanine N-carboxyanhydride was prepared by the method of Bailey.s N-Benzyloxycarbonyl-L-alanine was obtained as described by Bergman and Zervas.' Anthran- ilic acid ethyl ester hydrochloride and fi-aminobenzoic acid ethyl ester (benzocaine) hydrochloride were prepared (1) R1 a PhCH, b PhCH, c H d H e L-P~CH,*CO,-[CH~], f PhCH, g PhCH, h H i H (11) n a 1 H b 1 PhCH, methylformamide (5 ml.) by shaking for some minutes.The solution, protected from moisture (CaCl,), was left at room temperature for 40 hr., during which time carbon dioxide was evolved. After filtration and evaporation (35"/2 mm.) the residue was dissolved in ethanol (charcoal). The hydrochloride (2.05 g., 85%) crystallised on addition of ether and light petroleum. Washed with ether and dried (P20,) it melted at 220-228", [ol]D23 -40". Recrystall- isation from propan-2-ol-ether or ethanol-ether with addi- tion of light petroleum gave crystals (80% recovery), m.p. 228-230" [Found: C, 49.0; H, 5.4; Cl, 14-7; N, 11.3; TABLE 1 a-Aminoacylamidobenzoic acids NH2*CHR1*CO*NH*C6H,*CO*R2 (I) M.p.111" 190 255-260 t 238 172 145 142 250-252 241-243 Yield 70 72 90 * 75 * 45 38 44 75 * 85 * (%) c (%I - I Formula Calc. Found Cl,H,,N,03 69.2 69.0 Cl,H16N,03 67.6 67.3 CllHl,N203C1 51.1 50.9 CgHllN203C1 46.7 46.5 ClgH,,N20S 64.0 63.7 C18H,,N,03 69.2 68.9 Cl,Hl,N20, 67-6 66.9 Cl1Hl,N,O3C1 51.1 51.0 CgHllNz03CI 46.7 46.7 R * Isolated as hydrochloride. t Lit.,, 244-246" (decornp.). TABLE 2 Amido-oxy-peptides NH,*CHR*CO*NH*O*[CH,],*CO,H (11) H (%I -- Calc. Found 6.4 6*4 5.7 5-8 5.8 5.7 4.8 5.0 5.7 5.6 6.4 6-3 5.7 5.7 5-8 5.9 4-8 4.9 N (70) 7 - 7 Calc. Found 9.0 8-9 9.8 9.7 10.8 10.8 12.1 12.3 7.9 8.0 9.0 8.8 9.8 9.7 10.8 10.6 12.1 12.1 c (%) H (%) N (%) Yield 7- r b M.p.(%) Formula Calc. Found Calc. Found Calc. Found 170" 74 C4H8N,0, 32.4 32.4 5.4 5.6 18.9 18.5 177 60 CllH14N204 55.5 55.2 5.9 5.8 11.8 11.6 C * 1 L-P~CH,*CO,.[CH~], 147 58 C14H18N,0, 54.2 54.2 5.8 6.8 9.0 9.1 d 2 H 161 58 C6HloN204 37.0 37.1 6.2 6.1 17.3 17.1 e 2 PhCH, 133 60 Cl,Hl,N,04 57.1 56.8 6.4 6.3 11.1 10.9 f 3 H 159 72 C6H,,N204 40-9 40.7 6.9 6.7 15.9 16.0 g 3 PhCH, 181 59 C13HI,N20, 58.6 58.3 6.8 6.9 10.5 10.3 * [ c t ] ~ * ~ +21.6" (c 12.5 in N-HC1). by esterification of the acids in ethanol saturated with hydrogen chloride. 8 p-Aminobenzoyl-L-glutamic acid was obtained as described by King, Acheson, and Spensley.9 The N-carbox9anhydrides of glycine and DL-phenylalanine were prepared by the procedure of Farthing; lo y-benzyl- N-carboxy-L-glutamate anhydride was made by the method of Blout and Karlson. l1 @-Amino-oxypropionic acid hydrochloride was prepared by condensation of 8- bromopropionic acid with aqueous sodium benzohydroxam- ate and hydrolysis of p-benzamido-oxypropionic acid (formed along with phenylcarbamoyl benzohydroxamate) with 6% hydrochloric acid.12 L-Alanylanthranilic Acid Hydrochloride.-L-Alanine N- carboxyanhydride (1.15 g., 0.01 mole) and anthranilic acid hydrochloride (1.73 g., 0.01 mole) were dissolved in di- J.L. Bailey, J. Chem. Soc., 1950, 3461. ' M. Bergman and L. Zervas, Ber., 1932, 65, 1192. I? D. VorlBnder and F. Meyer, Annulen, 1902, 320, 135; H. @ F. E. King, R. M. Acheson, and P. C. Spensley, J . Chem. Salkowski, Bey., 1895, 28, 1921. SOL, 1949, 1401. N(Van Slyke), 6-0. C1,H13C1N203 requires C, 49.1; H, 5.4; C1, 14.5; N, 11.5; N(Van Slyke), 5.8y0], [olIDz3 -40.0" (c 1 in N-HCl), A,, (Nujol) 3.8, 4.1, and 4.9 (NH,+), 5.9 (GO, un-ionised carboxy-group), 6.0 (amide I), 6-2 (NH,+, C=C), 6.3 (ortho-substituted phenyl), 6-55 (amide 11), and 13.3 (ortho-substituted phenyl) p.Optical purity. L-Alanylanthranilic acid hydrochloride (0.275 g.) was heated under reflux in 5~-hydrochloric acid (20 ml.); the rotation of the solution gradually changed to the value for L-alanine (freed by hydrolysis). After 29 hr. under reflux the solution was treated with charcoal and filtered; 3-7.2" (calc. as L-alanine, c 0-5 in ~N-HC~). For comparison a solution (in ~N-HC~) of L- alanine (0.1 g., 1 mol.) and anthranilic acid (0.154 g., 1 mol.) was heated under reflux under the same conditions; it then lo A.C. Farthing, J . Chem. Soc., 1950, 3213. l1 E. R. Blout and R. H. Karlson, J . Amer. Chew Soc., 1956, l2 N. Frydman, M.Sc. Thesis, Hebrew University of Jerusalem, 78, 941. 1965.Org. 1823 showed [a],,,, +7-2" (c 0.5 in ~N-HC~); the same optical activity (f0-1") was recorded if the solution was not heated. The same specific rotations were obtained for the hydrolys- ates of the amino-acid amide hydrochloride from the first precipitation. This shows that the products obtained in the reaction are optically pure and do not arise from preferential removal of stereoisomers. ~-Alanyl-p-aminobenzoic Acid Hydrochloride.-A solution of p-aminobenzoic acid (1.73 g . , 0.01 mole) and L-alanine N-carboxyanhydride (1.15 g., 0.01 mole) in dimethylf orm- amide was kept at room temperature for 40 hr.and then worked up as before. Recrystallised from ethanol-ether on addition of light petroleum, the hydrochloride (2 g., Slyo) melted at 262-265' (decomp.) [Found: C, 49.2; H, 5.3; C1, 14.5; N, 11.2; N(Van Slyke), 5.7%], [a(IDz3 +28-3O (c 1 in N-HCl), A,, (Nujol) 3.7, 3.9, and 5.0 (NH,'), 5-9 (CO,H), 6.0 (amide I), 6-25 (NH,+, C=C), 6.5 (amide II), and 12.95 (para-substituted phenyl) p. Optical purity. L-Alanyl-p-aminobenzoic acid hydro- chloride (0.275 g.) was heated under reflux in 5~-hydro- chloric acid for 24 hr. The optical activity of the hydrolys- ate, purified with charcoal, was the same as that of a solu- tion of L-alanine (0.1 g.) and of p-aminobenzoic acid (0.154 g.) in 5~-hydrochloric acid (20 ml.) : [a] t 7 - 2 " ( k 0 .1 ) . A hydrolysate of twice-recrystallised dipeptide hydrochloride had the same specific rotation. ~-Alanyl-p-aminobenzoic Acid Ethyl Ester Hydrochloride.- A solution of p-aminobenzoic acid ethyl ester hydro- chloride (2-02 g., 0.01 mole) and L-alanine N-carboxy- anhydride (1.15 g., 0.01 mole) in dimethylformamide was kept at room temperature for 20 hr. The solvent was removed (35"/2 mm.) and the residue was dissolved in ethanol (charcoal). Addition of ether and an excess of light petroleum precipitated the crude ester hydrochloride (2.3 g.), m.p. 245-248", $15.8" (c 1 in 80% EtOH) and +25-9" (c 1 in N-HCl). Recrystallised from ethanol- ether and light petroleum the product (2.05 g., 75%) melted at 250-252" [Found: C, 52.7; H, 6.1; Cl, 13.2; N, 10.3; N(Van Slyke), 5-2; OEt, 16.8.Cl,H,7C1N,0, requires C, 52-8; H, 6.3; C1, 13.0; N, 10-3; N(Van Slyke), 5.2; OEt, 16.5%], [a]D23 +17-5" (G 1 in 80% EtOH), +27.5" (G 1 in N-HCl), A,, (Nujol) 3-85, 3.5-4.1, and 5.2 (NH3+), 5.88sh-5.92 (ester and amide I), 6.25 (NH,', C=C), 6-45 (amide 11), and 12.95 (para-substituted phenyl) p. N-Benzyloxycarbonyl-L-alanyl-p-arninobenzoic Acid Ethyl Ester.-(a) To a stirred solution of ethyl L-alanyl-p-amino- benzoic acid ethyl ester hydrochloride (0.546 g., 0.002 mole) and benzyloltycarbonyl chloride (0.375 g., 0-0022 mole) in chloroform (10 ml.), triethylamine in chloroform (5 ml.) was added (during & hr.), with the temperature kept be- tween -5 and 0". The mixture was stirred for a further 30 min.at room temperature. Chloroform was added (10 ml.) and the mixture was washed with 1% hydrochloric acid (15 ml.) with stirring and cooling (0"). Washing with 1 % hydrochloric acid was repeated and the chloroform solution was shaken with water (2 x 15 ml.). The chloro- form solution was dried (MgSO,) and evaporated under reduced pressure, and the solid residue was recrystallised from ethyl acetate and light petroleum. The benzyloxy- carbonyl derivative (0-6 g., 81%) melted at 155" (Found: C, 64.9; H, 6.2; N, 7-5; OEt, 11.9. C,,H,,N,O, requires C, 64-9; H, 6.0; N, 7-6; OEt, 12.1y0), @IDz3 -43.0" (G 1 in EtOH), [aID2, -27.7" (G 1 in AcOH), lmL (Nujol) 3-05 (NH), 5-8 (urethane CO), 5-93 (conjugated ester CO), 6.03 (amide I), and 6.55 (amide 11) ; overall yield based on L-alanine (b) N-Benzyloxycarbonyl-L-alanine, m.p.88", [aIDz3 -14.5" (c 1 in AcOH) (2.23 g., 0.01 mole) was dissolved in chloroform (50 ml.) and dicyclohexylcarbodi-imide (2.06 g., 0.01 mole) was added to the cooled (0") and stirred solution. p-Aminobenzoic acid ethyl ester hydroehloride (2.02 g., 0.01 mole) and triethylamine (1.0 g., 0.001 mole) were added and the mixture was stirred for 30 min. with cooling, then for 3 hr. without cooling. It was left overnight a t room temperature and dicyclohexylurea was filtered off. An excess of light petroleum precipitated the product mixed with triethylamine hydrochloride. The filtrate was con- centrated and more crude product was obtained on addition of light petroleum. Washing with 2-3% hydrochloric acid and recrystallisation from ethanol-water gave the benzyloxycarbonyl ester (1.6 g., 59%), m.p.146-148". Recrystallised from ethyl acetate and light petroleum it melted at 152-153" and had an i.r. spectrum identical with that of the product obtained by method (a) (Found: C, 65.1; H, 5.9; N, 7.8; OEt, 12*2%), [aIDz3 -41.2" (c 1 in EtOH), [=IDz3 -27.2" (c 1 AcOH); overall yield based on L-alanine 40-42%. ~~-Phenylalanyl-p-arninobenzoyl-L-glutamic Acid.-+- Aminobenzoyl-L-glutamic acid 9 (1.33 g., 0.005 mole) was dissolved in dry dimethylformamide (10 ml.). A 16% solution of hydrogen chloride in dimethylformamide (1.1 g., 0.005 mole) and DL-phenylalanine N-carboxyanhydride lo (0.96 g., 0.005 mole) were added and the solution was left at room temperature for 3 days. After removal of the solvent under vacuum, water (10 ml.) was added to the oily residue, and a small amount of undissolved material was filtered off.The clear solution was brought to pH 5.0 with triethylamine and left in the cold overnight. The product was obtained as an oil which was recrystallised from ethanol-water; m.p. 207-208" (Found: C, 60.8; H, 5.9; N, 10.3. C2,H,,N,0, requires C, 61.0; H, 5.6; N, 10.2%), [alpz5 +12-3' (G 6.2 in N-HC1). Glycyl-p-anzznobenzoyl-L-glutamic Acid.-Prepared as already described, from glycine N-carboxyanhydride lo (0.5 g., 0.005 mole), the product (1 g., 64%) melted at 255" (lit.,2 253") (Found: C, 51.8; H, 5.3; N, 12.8. Calc. for Cl4HI7N30: C, 52.0; H, 5.3; N, 13-0%), -4.2" (C 12.5 in N-HC1). y-Benzyl-~-glutanzyl-p-anzinobenzoyl-L-glutarnic Acid.- Prepared as before, from y-benzyl-N-carboxy-L-glutamate anhydride l1 (1.33 g., 0.005 mole), the product (1.40 g., 62%) melted at 168-169" [Found: C, 59.5; H, 5.6; N, 8.9; N(Van Slyke), 2.7. C,,H,7N,O, requires C, 59.4; H, 5.6; N, 8-7; N(Van Slyke), 2.9y0], [a]D25 +52" (c 12.5 in N-HC1).Glycylavvtinobenzoic Acids and Ethyl Ester Hydrochlorides. -Glycylaminobenzoic acids and esters were prepared by dissolving the hydrochloride of the aminobenzoic acid or of its ethyl ester with glycine N-carboxyanhydride (1 mol.) in dimethylformamide. The solution was kept at room temperature for 20 hr., the solvent was removed under vacuum, and the hydrochloride of the product was isolated and recrystallised as described for the L-alanyl peptides. Data for the following glycyl amides (Ic, d, h, and i) are recorded in Table 1.DL-Phenylalanylarninobenzoic Acids and Esters. General Procedure.-A solution of DL-phenylalanine N-carboxyan- hydride l o (0.01 mole) and the aminobenzoic acid hydro- chloride or its ester hydrochloride (0.01 mole) in dry di- methylformamide was kept at room temperature for 3 days, 52-54%.J. Chem. SOC. (C), 1969 protected from moisture (CaC1,). After filtration and re- moval of the solvent under vacuum, the oily residue was dissolved in water (25 ml.). Undissolved polymer and unchanged N-carboxyanhydride were filtered off, and to the clear solution triethylamine was added dropwise (to pH 6- 5-7.0). Cooling and scratching initiated precipitation within a few minutes, completed by storage overnight at 0".The amides and the esters were recrystallised from ethanol, propan-2-01, or ethanol-ether. The DL-phenylalanylaminobenzoic acid ethyl esters ob- tained were hydrolysed with 2~-sodium hydroxide. After filtration and acidification with conc. hydrochloric acid to pH 6.0, the precipitated DL-phenylalanyl amides were identified by analysis, mixed m.p., and i.r. spectra with those obtained from the acid hydrochlorides with DL-phenyl- alanine N-carboxyanhydride. Data for the amides (Ia, b, f, and g) are given in Table 1. A similar preparation, from y-benzyl-N-carboxy-L-glutam- ate anhydride 11 and p-aminobenzoic acid hydrochloride gave y-benzyl-L-glutamyl-p-awinobenzoic acid (Ie) (see Table 1). Amido-oxy-peptides of o-Amino-oxy-acids (11) .-The amido-oxy-peptides (IIa-g) were prepared by treatment of amino-oxy-acid hydrochlorides with N-carboxy- anhydrides in 3 : 1 ethanol-water.Data are summarised in Table 2. The preparation of glycylamino-oxyacetic acid (Ia) is characteristic. The preparation of y-amino-oxy- butyric acid hydrochloride, applied in the synthesis of (IIf and g), is also described. GlycyZamino-oxyacetic A cid (IIa) .-Amino-oxyacetic acid hemihydrochloride (0.55 g., 0.005 mole) and 36% hydro- chloric acid (0.21 ml., 0.0025 mole) were added to 3 : 1 ethanol-water (8 ml.), followed by glycine N-carboxy- anhydride (0.50 g., 0.005 mole). Evolution of carbon dioxide took place almost immediately and continued for about 20 min. The solution was then kept at room tempera- ture for 1 hr. and the solvent was removed under vacuum (40").The oily residue was dissolved in water (5 ml.) and cooled to 0", and impurities were filtered off. The clear solution was neutralised (to pH 7.0-7.5) with triethylamine, ethanol (8 ml.) was added, and the mixture was kept over- night at 0". The amido-oxy-peptide (0.55 g., 74%) precipit- ated as white crystals, m.p. 170" (decomp.). Addition of hydrochloric acid (0.5 equiv.) to the reaction mixture was necessary in the case of the hemihydrochloride of amino-oxyacetic acid. With the other amino-oxy-acid hydrochlorides no hydrogen chloride was necessary. y-Benzamido-oxybutyric Acid.-Benzohydroxamic acid (10.96 g.) in absolute ethanol (70 ml.) was added to a solu- tion of sodium ethoxide [from sodium (1.85 8.) in ethanol (70 ml.)]. To the mixture, which contained some precipit- ated sodium benzohydroxamate, ethyl y-bromoethyl- butyrate (7.80 g,) in absolute ethanol (70 ml.) was added. Stirring and heating to boiling caused dissolution. The solution was stirred at 55-60" for 48 hr., filtered, and evap- orated under vacuum to small bulk. The oily residue was suspended in %-sodium hydroxide (70 ml.) and the mixture was stirred for 12 hr. Impurities were filtered off and the solution was acidified with concentrated hydrochloric acid to pH 6.0; storage at 0" overnight caused precipitation of the acid (6-6 g., 74%), m.p. 108-109". Recrystallisation from water gave material, m.p. 112-113" (Found: C , 58.9; H, 6-0; N, 6.4. C11H,,N04 requires C, 59.2; H, 5.9; N, 6.3%). A second crop was obtained by acidifying the filtrate to pH 5.0 and storage in the cold for 24 hr. (total yield 82%). y-Amino-oxybutyric Acid HydrochZoyide.--y-Benzamido- oxybutyric acid (2.2 g., 0.01 mole) was heated under reflux in 4% hydrochloric acid (15 ml.) for 15 min. then cooled. Precipitated benzoic acid was filtered off and the solvent was removed under vacuum. The residue was dissolved in ethanol and ether was added. The hygroscopic acid hydrochloride (0-65 g., 42%) was washed with 4: 1 ether- ethanol; m.p. 129-130" (Found: C, 30.65; H, 6-6; N, 8.9. [9/545 Received, Mawh 28th, 19691 C,H,,ClNO, requires C, 30.85; H, 6.5; N, 9.0%).