Synthesis of some methoxy- and hydroxy-phenazine-1-carboxylic acids

摘要

J.C.S. Perkin ISynthesis of Some Methoxy- and Hydroxy-phenazine-I -carboxylic Acids tBy Philip K. Brooke, S. Richard Challand, Michael E. Flood, Richard B. Herbert, Frederick G. Holliman,'The synthesis of the naturally occurring 6- and 9-hydroxyphenazine-1 -carboxylic acids, (3) and (4). is described.Methyl 6-methoxyphenazine-1 -carboxylate (1 2) is identified as a metabolite from Streptomyces luteoreticuli, anda metabolite of Pseudomonas aureofaciens is identified as 2-hydroxyphenazine-I -carboxylic acid (2) by com-parison with synthetic material.and P. Nicholas Ibberson, Department of Organic Chemistry, The University, Leeds LS2 9JTTHE elaboration of phenazine metabolites by micro-organisms often involves hydroxylation of one or moreof the aromatic rings. This reaction may involve simplehydroxylation as in the transformation of phenazine-l-carboxylic acid (1) into Z-hydroxyphenazine-l-carb-oxylic acid (2) in Pseudowzonas aureofaciens cu1tures.lAlternatively a more complex process may be involved,as in the conversion of (1) into pyocyanin (7) by Ps.aeruginosa or of 6-hydroxyphenazine-1-carboxylic acid(3) into iodinin (8) by Brevibacterium iodinum; in thesecases the carboxy-function is replaced by a hydroxy-group.In the course of our studies on phenazine bio-synthesis we have synthesised both the hydroxy-acids(2) and (3) as well as 9-hydroxyphenazine-l-carboxylicacid (4) which is also a microbial metab~lite.~We recently developed a useful method for the syn-thesis of substituted phenazines which is particularlywell suited to the preparation of l-carboxyphenazinederivative^.^ The method involves the reductive cyclis-ation of appropriately substituted 2-nitrodiphenylamineswith sodium borohydride in ethanolic sodium ethoxide,and was applied t o the synthesis of B-methoxyphenazine-1 -carboxylic acid (5) and 9-methoxyphenazine- 1 -carb-oxylic acid (6) from compounds (9) and (lo), respect-ively.As expected the cyclisation of (9) gave 8-methoxy-phenazine-l-carboxylic acid (1 1) along with the required6-methoxy-isomer ( 5 ) , but the facility with which (9)could be prepared from 2-bromo-3-nitrobenzoic acid andnz-anisidine outweighed the advantage of an unambigu-ous route to (5) which would have required a much lessaccessible 2-nitrodiphenylamine. The two isomericphenazines were readily separated and were distinguishedby their n.m.r.spectra.In addition to the methoxy-acids small amounts oft Preliminary communication, R. B. Herbert, F. G. Holliman,M. E. Flood, R. B. Herbert, and F. G. Holliman, J.C.S.R. B. Herbert, F. G. Holliman, and P. N. Ibberson, J.C.S.N. N. Gerber, J . Heterocj~clic Chem., 1969, 6, 297.S. R. Challand, R. B. Herbert, and F. G. Holliman, Chem.P. J. Culhane, Org. Synth.., Coll. Vol. I, 1944, p. 125.P. K. Brooke, Ph.D. Thesis, University of Leeds, 1973.and J. D. Kynnersley, Tetrahedron Letters, 1968, 1907,Perkin I , 1972, 622.Chem. Cowma., 1972, 355.Comm., 1970, 1423.the corresponding ethoxy-compounds were also isolated,presumably as a result of direct nucleophilic displacementof the methoxy-group in the phenazine by ethoxide ion.If the cyclisation of (9) was carried out in t-butylalcohol containing sodium t-butoxide the only phen-azines isolated were (5) and (11).Cyclisation of (10) inethanolic sodium ethoxide additionally gave smallamounts of phenazine-l-carboxylic acid (l), formed as aresult of cyclisation on to the carbon atom bearing themethoxy-group in (10).In the n.m.r. spectrum of the 9-methoxy-acid (6) thesignal at highest field (6 7.80) can be assigned to theproton at C-8 on the basis of substituent effects; theabsence of this signal in the n.m.r. spectrum of 6,s-dideuterio-9-methoxyphenazine-1-carboxylic acid con-firmed this assignment. However this signal does notshow the more usual splitting into a doublet or doubledoublet cf.the spectrum of (5), where the high field(6 7.72) doublet is apparent with J 7.5 Hz. Insteadthe spectrum is ' deceptively simple ' in that this signal(X) appears as a triplet with a coupling constant ( J 4.5Hz) which is expected7 of an ABX system where, ashere, 6 a ~ _t 0; the coupling conforms to the expectedvalue of (JAX + J s x ) / ~ . The corresponding signal in thespectrum of the methyl ester of (6) is more complex inshowing four strong transitions in which the outer twolines are separated by the expected value of JAx + JBx.This is also expected of an ABX system in which 6AB issmall. 8Attempted demethylation of (6) with boron tribro-mide and boron trichloride lo proved unsatisfactorybut success was achieved with aluminium chloride.ll9-Hydroxyphenazine-l-carboxylic acid (4) was obtained,R.J. Abraham and H. J. Bernstein, Canad. J . Chem., 1961,39, 216.L. M. Jackman and S. Sternhell, 'Applications of NuclearMagnetic Resonance Spectroscopy in Organic Chemistry,'Pergamon, Oxford, 2nd edn., 1969. * J. fiv. F. McOmie arid M. L. Watts, Chem. am2 Ind., 1963,1658.lo F. M. Dean, J. Goodchild, L. E. Houghton, J. A. Martin,R. B. Morton, B. Parton, A. W. Price, and S. Somvchien, Tetya-hedron Lettevs, 1966, 4163; IV. Gerrard and hl. F. Lappert, Claem.Rev., 1955, 58, 1081.Cf. 1;. E. King, N. G. Clark, and P. M. H. Davis, J . Chejn.Soc., 1949, 30121976 2249with properties similar to those reported for materidisolated from microbial culture and also synthe-~ i s e d , ~ J ~ by a less convenient route.CO,H H R2 DNQR ' NO?bsol;0-Me(71-0OH 1OH0-( 8 IOMe(121 R:C02Me(13)R: OMeRluminium chloride was also successfully employed forthe demethylation of 6-methoxyphenazine-l-carboxylicacid (5) to (3).The hydroxy-acid (3) was subsequentlyshown to be an important intermediate in the biosyn-thesis of iodinin (8).2913Several methoxy-phenazine derivatives have recentlybeen isolated from Stre@towyces Zuteoreticuli c~1tures.l~One of these was identified l5 as the 6-methoxy-deriva-tive (12). The quoted physical data l4 for this compoundclosely correspond to those obtained for the methoxy-l2 K. I'oshida, J . Plzarnz.SOC. Japan, 1964, 84, 703.l3 R. B. Herbert, F. G. Holliman, and I?. N. Ibberson, Tetra-hedrogz Letters, 1974, 151.l4 S. Yamagishi, Y. Koyama, Y. Fukusa, N. Koyomura, J.-I.Ohishi, S. Hamamichi, and T. Arai, J . Phnmz. Soc. Japan, 1971,91, 351.l 6 S. l'amanaka, Chiba Igakkai Zasshi, 1972, 48, 63 (Chem..4bs., 1972, 77, 162, 986).ester (12) prepared from the corresponding acid (5), andthe structure for the metabolite is accordingly confirmed.I,6-Dimethoxyphenazine (13) is, like (12), a metaboliteof S. ZuteoreticuZi and, by analogy with the biosynthesisof iodinin (8),2 may reasonably be thought of as arisingfrom either 6-methoxy- or 6-hydroxy-phenazine-l-carboxylic acid.Cultures of Ps. aureofaciem produce an abundance ofphenazine-l-carboxylic acid (1) and two minor meta-bolites.16 The structure (2), assigned to one of thesemainly on the basis of spectroscopic evidence,17J8 wassubstantiated by showing inter alia that a degradationproduct was identical with 2-methoxy-l-methylphen-azine,l8 and was confirmed by X-ray analysis of themetabolite it~e1f.l~ We have independently confirmedthe correctness of the structural conclusion in a synthesisof (2) by application of the earlier and more general, butless convenient route to substituted phenazines, namelythe oxidative cyclisation of 2-aminodiphenylamines inboiling nitrobenzene.In this case the diphenylaminewas (la), readily obtained by reduction of (15), itselfprepared by condensation of methyl 2-amino-6-methoxy-benzoate and o-iodonitrobenzene.Cyclisation of (14)gave methyl 2-methoxyphenazine-l-carboxylate (16),identical with a sample of the dimethyl derivative ofnaturally occurring (2).Demethylation of synthetic (16) with tin(I1) chlorideand hydrochloric acid l8 gave, in our experience, lowyields of methyl 2-hydroxyphenazine-l-carboxylate (17).Moreover treatment of this ester with aqueous alkaliresulted in decarboxylation as well as hydrolysis (cf. ref.18). On the other hand demethylation with borontrichloride lo afforded 2-methoxyphenazine-l-carboxylicacid (18). No further reaction occurred even in thepresence of a large excess of boron trichloride, butisolation of this product and further treatment withboron trichloride gave, in good yield, 2-hydroxyphen-azine-l-carboxylic acid (2), identical with the naturalproduct. Two steps are apparently necessary for thecomplete demethylation of (16) ; the complex formedon ester demethylation must be broken before cleavageat the aryl methoxy-group can be effected.EXPERIMENTALN.m.r.spectra were recorded at 90 MHz. M.p.s weredetermined with a Kofler hot-stage apparatus. Chromato-graphy refers to column chromatography on Kieselgel G(nach Stahl) .2O Where preparative t.1.c. was used instead,this is stated.N-(3-fWethoxyphenyl)-3-nitroanthuanilic A c i d (9) .-A solu-tion of 2-bromo-3-nitrobenzoic acid (2.47 g), m-anisidine(1.48 g), and anhydrous sodium carbonate (1.5 g) in peiityll6 A. J. Kluyver, J . Bacteriol., 1956, 72, 406; W. C. Haynes.F. H.Stodola, J. XI. Locke, T. G. Pridham, 13. F. Conway, V. E.Sohns, and R. W. Jackson, ibid., p. 412; RI. E. Levitch and P.Rietz, Biochemistry, 1966, 5, 689.17 J. I. Toohey, C . D. Nelson, and G. Krotkov, Canad. J .Botany, 1965, 43, 1055.18 E. S. Olsenand J. H. Richards, J . Org. Chew., 1967,32, 2887.l9 N. Jones, R. Marsh, and J. H. Richards, unpublished resultsquoted in ref. 18.20 B. J. Hunt and W. Rigby, Clzeun. and Ind., 1967, 18682250 J.C.S. Perkin Ialcohol (50 ml) was refluxed for 4 11. Steam distillation leftan oil which was taken into dichloromethane. The solutionwas washed with dilute mineral acid and dried. Evapor-ation, and recrystallisation of the residue from ethylacetate-hexane gave yellow prisms of the anthranilic acid(9) (1.6 g, 56), m.p.189-191" (Found: C, 57.9; H, 4.15;N, 9.8. C,,H,,N,O, requires C, 58.4; H, 4.15; N, 9.7y0),6-Methoxyphenazine- l-carboxylic A cid (5) .-A solution ofthe anthranilic acid (9) (0.60 g), sodium borohydride (0.15g), and sodium ethoxide ( 2 ~ ) in ethanol (100 ml) wasrefluxed for 20 h. The precipitate was collected and dis-solved in water. The solution was filtered, acidified, andextracted with chloroform. The extract was dried andevaporated. The yellow solid thus obtained was chromato-graphed (benzene containing 40 ethyl acetate). Fourcompounds were eluted in the following order : 8-ethoxy-phenazine-l-carboxylic acid (0.028 g, 5), m.p. 237.5-239"(from aq. EtOH), vmaX (KC1) 1735 cm-l; Lax. (EtOH)265 (log E 4.49), 368 (3.74), and 415 nm (3.66); m/e 268( M c ) , 224 (M' - 44), and 196; 6 (CF,*CO,H) 9.20 (1 H,dd, J 1.5 and 7.5 Hz, C-2 H), 9.00 (1 H, dd, J 1.5 and 8.5J 8.5 and 7.5 Hz, C-3 H), 7.97 (1 H, dd, J 2.5 and 10.5J 7.0 Hz), and 1.70 (3 H, t, J 5.0 Hz) (Found: C, 66.9;H, 4.35; N, 10.25.C15Hl,N20, requires C, 67.1; H, 4.5;w, 10.45) ; 8-nzethoxyplienazine-l-cnrboxylic acid (1 1)(0.143 g, 27y0), m.p. 292-293" (from benzene-chloroform) ;vnlztx. (Nujol) 1730 cni-l; hmx. (EtOH) 258 (log E 4.57), 365(3.49), and 400 nm (3.38) ; nz/e 256, 254 (M'), 210 (M+ - 44),and 167; 8 (CF,-C0,D) 9.27 (1 H, d, J 7.5 Hz, C-2 H),H), 8.33 (1 H, dd, J 8.5 and 7.5 Hz, C-3 H), 8.1-7.9 (2 H,m, C-7 and -9 H), and 4.37 (3 H, s) (Found: C, 66.2; euro;3,4.05; N, 10.75. C1,H,,N,O, requires C, 66.2; H, 3.95;N, 11.0) ; 6-ethoxyphenazine-l-carboxylic acid (0.035 g,6), m.p.238.5-239.5" (from aq. EtOH); vwx. (KCl)1730 cm-l; hmx. (EtOH) 264 (log E 4.54), 370 (3.94), and425 nni (3.48); m/e 270, 268 (M'), and 224 (Mf - 44); 6(CF,.CO,H) 9.31 (1 H, dd, J 1.5 and 7.5 Hz, C-2 H), 9.02(1 H, dd, J 1.5and 9.0Hz, C-4H), 8.53 (1 H, dd, J 7.5and9.0 Hz, C-3 H), 8.41 (1 H, dd, J 7.5 and 8.5 Hz, C-8 H),8.19 (1 H, dd, J 1.0and 8.5Hz, C-9H), 7.64 (1 H, dd, J 1.0and 7.5 Hz, C-7 H), 4.64 (2 H, q, J 7.0 Hz), and 1.72 (3 H, t ,J 7.0 Hz) (Found: C, 67.3; H, 4.65; N, 10.2) ; 6-methoxy-flhenazine-l-carboxylic acid (5) (0.182 g, 34y0), m.p. 292.5-293" (from aq. EtOH); v,, (KC1) 1720 cm-l; La,(EtOH) 264 (log E 4.67), 365 (3.94), and 428 nm (3.40);m/e 254 (M+), 210 (Mf - 44, nz* 174), and 181 (M+-C-2 H), 9.11 (1 H, d, J 9.0 Hz, C-4 H), 8.7-8.35 (2 H, m,C-3 and -8 H), 8.28 (1 H, d, J 9.0 Hz, C-9 H), 7.72 (1 H, d,J 7.5 Hz, C-7 H), and 4.43 (3 H, s ) (Found: C, 65.85; H,3.85; N, 10.65).A mixture of 6-methoxy- (12) and 8-niethoxy-phen-azine-l-carboxylic acid (13) was obtained by reductivecyclisation of (9) (0.60 g) with sodium borohydride (0.15 g)and sodium t-butoxide ( 2 ~ ) in refluxing t-butyl alcohol for18 h (experiment with T.ETHERINGTON) .Metlzyl 8-Methoxyphenazine-l-carboxylate.-The acid (1 1)was esterified with an excess of ethereal diazornethane inmethanol-dimethylforamide. Preparative t .l.c. (276MeOH in CHCl,) gave methyl 8-methoxyphenazine- 1-carboxylate (66), m.p.100-101" (from aq. EtOH) ;vmx. (Nujol) 1730 cm-l; A,,, (EtOH) 254 (log E 4.84),Hz, C-4 H), 8.73 (1 H, d, J 10.5 Hz, C-6 H), 8.28 (1 H, dd,Hz, C-7 H), 7.92 (1 H, d, J 2.5 Hz, C-9 H), 4.62 (2 H, 9,9.03 (1 H, d, J 8.5 Hz, C-4 H), 8.63 (1 H, d, J 10.5 Hz, C-644) -29, WZ* 1591; 6 (CF,CO,D) 9.39 (1 H, d, J 7.5 Hz,356 (3.95), and 397 nm (3.90); m/e 268 ( M f ) ; 8 (CDCI,)8.34 (1 H, dd, J 1.5 and 8.5 Hz, C-2 H), 8.22 (1 H, dd, J1.5 and 7.0 Hz, C-4 H), 8.08 (1 H, d, J 10.0 Hz, C-6 H),7.71 (1 H, dd, J 7.0 and 8.5 Hz, C-3 H), 7.55 (1 H, dd, J2.5 and 10.0 Hz, C-7 H), 7.47 (1 H, d, J 2.5 Hz, C-9 H),4.07 (3 H, s), and 4.01 (3 H, s) (Found: C, 66.9; H, 4.7;N, 10.15. C,,H1,N,O, requires C, 67.1; H, 4.5; N,10.45).Methyl 6-Methoxyfihelzazine- 1-carboxylate (1 2) .-Esteramp;fication of (5) with diazomethane as above gave methyl 6-methoxyphenazine- 1-carboxylate (69 yo), m.p.139- 14 1 " (fromaq. EtOH); vmx. (KCl) 1 725 cm-l; Lx (EtOH) 264 (log E4.69), 348 (3.74), 366 (3.93), and 415 nm (3.42); m/e 270239, 223, 210, 209, 199, 184, and 156; 6 (CDCI,) 8.55 (1 H,dd, J 1.5 and 8.5 Hz, C-2 H), 8.25 (1 H, dd, J 1.5 and 7.0Hz, C-4 H), 8.0-7.4 (3 H, m, C-3, -8, and -9 H), 7.09 (1 H,dd, J 1.5 and 8.0 Hz, C-7 H), 4.17 (3 H, s ) , and 4.09 (3 H, s)(Found: C , 66.85; H, 4.55; N, 10.5).6-Hydroxyphenazine- 1-carboxyh Acid (3) .-A mixture of6-niethoxyphenazine- l-carboxylic acid (0.048 g) , anhydrousaluminium chloride (0.20 g), and dry benzene (10 ml) wasrefluxed for 7 h. The residue obtained after removal of thebenzene was treated with ice-water, aqueous sodiumhydroxide ( 2 ~ ; 5 ml) was added, and the mixture washeated at 100 "C for 1 h.The solution was acidified andextracted with chloroform. This extract was dried.Evaporation gave 6-hydroxjq5henazine- 1-carboxylic acid(0.029 g, 64y0), m.p. 250-251" (from aq. EtOH); vmx. (KC1)1 695 cm-l; Amax.(EtOH) 269 (log ~4.47), 366 (3.76), 373(3.79),and 45Gnm (3.21); m/e 240 (M'), 196 (M' - 44), and 168(M+ - 44 - 28); 6 (CF,.CO,H), 9.34 (1 H, dd, J 1.0 and 7.0Hz, C-2 H), 9.00 (1 H, dd, J 1.0 and 8.5 Hz, C-4 H), 8.53(1 H, dd, J 8.0 and 9.0 Hz, C-8 H), 8.40 (1 H, dd, J 7.0and8.5Hz,C-3H),8.18(1H,dd, J9.0andl.OHz,C-9H),7.76 ( 1 H, dd, J 1.0 and 8.0 Hz, C-7 H) (Found: C , 65.0;H, 3.45; N, 11.45.C,,H,N,O, requires C, 65.0; H, 3.35;N, 11.7).N-(2-MethoxyphenyZ)-3-nitroanthranilic Acid ( 10) .-Amixture of 2-bromo-3-nitrobenzoic acid (3.04 g ) , o-anisidine (2.78 g), and sodium carbonate (2.2 g) was heatedin refluxing pentyl alcohol (60 ml) for 18 h. The aqueousresidue obtained after steam distillation was acidified. Theorange precipitate which formed was collected, washed withdichloromethane, and recrystallised (aq. EtOH) to give redprisms of the anthranilic acid (10) (1.48 g, 42y0), m.p. 229-231" (Found: C, 58.4; H, 4.25; N, 9.8. C,,H1,N,05requires C , 58.4; H, 4.15; N, 9.7).9-Methoxyphenazine- 1-carboxylic A cid (6) .-The anthra-nilic acid (10) (0.587 g) and sodium borohydride (0.087 g)were heated with sodium ethoxide (2M) in refluxing ethanol(60 ml) with stirring for 26 h.Water was added to dissolvethe precipitate which formed; the solution was filtered andthe ethanol removed under reduced pressure. Acidificationgave a precipitate which was collected. Chromatography(CHCl, containing 10 MeOH) gave 9-rnethoxyphenazi?ze-l-~arboxyli~ acid as the major product, m.p. 262.5-263'(from EtOH) (0.266 g, 52); vnlaX. (KCl) 1715 cm-l;Amax. (EtOH) 263 (log E 4.52), 366 (3.86), and 420sh nm(3.42) ; m/e 254 (M+), 253, 235 (M' - 1) - 18, m* 2191, 225(M+-l) -281, and 210 (Mf-44); 8 (CF,*CO,H) 9.29(1 H, dd, J 2.0 and 7.0 Hz, C-2 H), 9.00 (1 H, dd, J 2.0 and9.0 Hz, C-4 H), 8.40 (1 H, dd, J 7.0 and 9.0 Hz, C-3 H),8.32 (2 H, d, line separation 4.5 Hz, C-6 and -7 H), 7.80 (1 H,t, line separation 4.5 Hz, C-8 H), and 4.40 (3 H, s) (Found:(M' + 2), 268 (M'), 267 (M' - l ) , 254, 253 (Mf - 15)1976 2251C, 66.6; H, 4.1; N, 10.95.C,,H,,N,O, requires C, 66.2;H, 3.95; N, 11.0).Phenazine- 1-carboxylic acid (isolated as its methyl esterby chromatography after methylation of the crude cyclis-ation mixture) and 9-ethoxyphenazine- l-carboxylic acid(0.062 g, 11) were isolated as minor products. The latterhad m.p. 230-231.5"; v- (Nujol) 1730 cm-l; amp;(EtOH) 265 and 370 nm; m/e 268 (M+), 253 (M+ - 15, m*239), 235 (M+ - 15) - 18, m* 2191, and 224 (Mf - 44); 8(CF,.CO,H; 60 MHz) 9.29 (1 H, dd, J 1.0 and 7.0 Hz,C-2 H), 9.00 (1 H, dd, J 1.0 and 9.0 Hz, C-4 H), 8.8-8.2(3 H, ni, C-3, -6, and -7 H), 7.76 (1 H, t , line separation 4.5Hz, C-8 H), 4.7 (2 H, q, J 6.5 Hz), and 1.8 (3 H, t, J 6.5Hz) (Found: C, 67.25; H, 4.45; N, 10.5.C1,H,,N20,requires C, 67.1; H, 4.5; N, 10.45).Methyl 9-Methoxyphenazine-l-carboxylate.-9-Methoxy-phenazine- l-carboxylic acid was esterified with methanoland concentrated sulphuric acid (2 h under reflux). Non-acidic material was chromatographed (preparative t.1.c. ;5 MeOH in CHCl,) . Methyl 9-methoxyphenazine- 1-carboxylate was isolated as the major product (68),m.p. 114-116" (from aq. EtOH); vmx. (KCl) 1728 cm-l; Lx. (EtOH) 263 (log E 4.82), 346 (3.95), 364 (4.09), and 410nm (3.73); m/e 268 (M+), 267, 253, 239, 235, 222, 208, 179,and 176; 6 (CF,*CO,H), 9.23 (1 H, dd, J 1.0 and 7.0 Hz,C-2 H), 9.09 (1 H, dd, J 1.0 and 9.0 Hz, C-4 H), 8.5-8.2(3 H, m, C-3, -6, and -7 H), 7.84 (1 H, four lines, the outerones being separated by 8.5 Hz, C-8 H), 4.43 (3 H, s), and4.32 (3 H, s) (Found: C, 67.45; H, 4.6; N, 10.5.C15H12-N,O, requires C, 67.1; H, 4.5; N, 10.45).9-Hydroxy+henazine- 1-carboxylic Acid (4) .-9-Methoxy-phenazine- l-carboxylic acid (0.047 g) was suspended in drybenzene (10 ml) . The mixture was refluxed with anhydrousaluininium chloride (0.10 g) for 5 h. The benzene wasevaporated off and the residue was treated with ice-waterand extracted with chloroform to remove unchanged start-ing material. The aqueous solution was basified (10 ml ofSw-NaOH) and heated at 100 "C for 30 min. Acidificationprecipitated the 9-hydroxyphenazine- l-carboxylic acid (0.021g, 47y0), m.p.290" (from aq. EtOH) ; vmK (KC1) 1 720 cm-l;Lax. (EtOH) 270.5 and 372 nm; wz/e 240.053 40 (C,,H8N,0,requires M , 240.052 96), 222 (M+ - 18, m* 205), 195(Mf - 18) - 28, m* 1691, and 196 (M+ - 44) (Found: C,60.4; H, 4.2; N, 10.4. C,,H,N203,H20 requires C, 60.5;H, 3.9; N, 10.9).Methyl 6-Methoxy-N-( 2-nitrophenyl)anthranilate ( 15) .-Methyl 2-amino-6-methoxybenzoate prepared from 2-methoxy-6-nitrobenzoic acid 21 by esterification 22 followedby hydrogenation in ethanol over platinum (0.10 g), o-iodonitrobenzene (0.217 g), potassium carbonate (0.10 g),and copper powder (0.001 g) were thoroughly mixed and21 T. Takahashi and Y . Hamada, J . Pharm. Soc. Japan, 1955,75, 1434 (Chem. Abs., 1956, 50, 10,096).heated at 200 "C (bath temp.) for 15 min.The productwas extracted into chloroform and purified by chromato-graphy (benzene). The anthranilate (15) (0.055 g, 33) wasobtained as orange plates, m.p. 130-131" (from ethanol);vmx. (KCl) 1712 cm-1 (Found: C, 59.2; H, 5.00; N, 9.6.Cl5HI4N2O5 requires C, 59.6; H, 4.65; N, 9.3).Methyl 2-Methoxyphenaziae- l-carboxylate ( 16) .-Theanthranilate (15) (2.3 g ) was hydrogenated in ethanol overplatinum oxide (0.2 8). The amine (14) obtained afterevaporation of solvent was used without purification for thenext step.A solution of this amine in nitrobenzene was refluxedunder nitrogen for 162 h. Removal of the nitrobenzeneleft the crude product, which was chromatographed (ben-zene and 1 : 1 benzene-ether). The methyl 2-methoxy-phenazine-1-carboxylate obtained was recrystallised fromlight petroleum; yield 0.464 g (23); m.p.129-130';vmax (KCl) 1737 cm-1; (EtOH) 215 (log E 4.47), 257(4.87), 363 (3.94), and 398nm (3.75); 6 (CDC1,) 8.31 (1 H, d,J 9 Hz, C-4 H), 8.3-8.1 (2 H, m), 7.9-7.8 (2 H, m), 7.83(1 H, d, J 9 Hz, C-3 H), 4.13 (3 H, s), and 4.10 (3 H, s)(Found: C, 67.05; H, 4.45; N, 10.65. CI,Hl2N2O3 re-quires C, 67.1; H, 4.5; N, 10.45), identical (t.l.c., ix.,u.v., and 1H n.m.r. spectra, and m.p. and mixed m.p.) withthat obtained by methylation (ethereal diazomethane intetrahydrof uran) of 2-hydroxyphenazine- 1 -carboxylic acidobtained from Pseudomonas azareofaciens cultures.2-Hydroxyphenazine-l-ca~boxylic A cid (2) .-TO methyl2-methoxyphenazine- 1-carboxylate (5 mg) in dichlorome-thane at 0 "C was added a cooled (- 70 "C) solution of borontrichloride (0.2 ml) in dichloromethane (1 ml) . The reactionwas kept at 0 "C for 10 min, then aqueous sodium acetatewas added followed by aqueous sodium hydroxide. Thesolution was acidified and extracted with chloroform. Theextracts were dried and evaporated leaving a residue whichhad properties consistent with 2-methoxyphenazine- 1-carboxylic acid m.p. 193-195'; v,, (CHCl,) 1 716cm-11 .Treatment of this material with boron trichloride asabove gave 2-hydroxyfihenazine- 1-carboxylic acid (overall56) which was recrystallised from benzene after prepam-tive t.1.c. (10 MeOH in CHCl,). This material was identi-cal (t.l.c., i.r. and U.V. spectra, and melting behaviour)with that obtained from Ps. aureofaciens cultures (Found :C, 64.85; H, 3.35; N, 11.95. C13HsN20, requires C, 66.0;H, 3.35; N, 11.7).We are indebted to Mr. T. Etherington for technicalWe thank the S.R.C. for grants (to P. K. B.,6/620 Received, 31sl March, 1976122 H. Sirai and N. Oda, Bull. Nagoya City Univ. Pharm. School,assistance.S. R. C., M. E. F.,andP. N. I.).1956, 4, 30 (Cltenz. Abs., 1957, 51, 9522)