Synthesis of phenazine-2,8-dicarboxylates

Phenazine is a tricyclic heteroarene that forms the core of diverse functional molecules including DNA intercalators. However, 2,8-disubstituted phenazines are rare

Recently, as part of a medicinal chemistry project focusing on DNA intercalators we desired to secure phenazine-2,8-dicarboxylates 5 and 6 ( Figure 1). We therefore sought to identify, and describe herein, a reliable and regioselective method for accessing phenazine-2,8-dicarboxylates. We also describe a simple NMR-based method for distinguishing 2,8-from 2,7-disubstituted phenazines, to hopefully forestall any future structural misassignments.

Results and Discussion
We considered several potential synthetic strategies at the outset of this work (Scheme 1). Murdock's reductive cyclization approach (Scheme 1a) is known to successfully afford 2,8-dimethyl phenazine, but only a limited substrate scope for this process has been reported. 7 A double cross-coupling approach, such as that developed by Laha, 23 is another validated option (Scheme 1b), but this method suffers from a lack of regioselectivity. We therefore decided to focus on two further approaches which offered the possibility of delivering our target compounds with appropriate regiocontrol and functional group tolerance. The first of these is a reductive cyclization approach based on the work of Holliman 24,25 (Scheme 1c); and the second is a stepwise cross-coupling approach loosely based on the work of Emoto 26 (Scheme 1d). The latter two approaches (Scheme 1c-d) have previously been employed to generate a variety of substituted phenazines, but never with the 2,8-disubstitution pattern. We investigated Holliman's reductive cyclization approach first (Scheme 2). An intramolecular Buchwald-Hartwig cross-coupling reaction between aryl bromide 13 and aniline 14 successfully delivered the diarylamine 15, which upon hydrolysis gave the diacid 10. However, when diacid 10 was exposed to Holliman's reductive cyclization conditions, a complex mixture of products was formed, and separation of the mixture indicated that no cyclized product had formed. Previous explorations of the substrate scope of this type of cyclization reaction have indicated that while the reaction is robust to substituent variation on the nitrobenzene ring, substituent variation on the alternate ring can influence reactivity towards cyclization. 24 We speculated that in our system, cyclization was too slow and could therefore have been outcompeted by alternative pathways such as over-reduction of the nitro group. We briefly explored a strategy that involved installing the bridging amine para to the carboxylate groups first (not shown); however, cyclization attempts yielded similar results.
Accordingly, we turned our attention to the strategy based on Emoto's stepwise cross-coupling approach (Scheme 3). Bromonitrobenzoate 11 and bromoaniline 12 underwent an intermolecular Buchwald-Hartwig Narylation, which delivered the diarylamine 17 in 78% yield despite the possible competitive homocoupling of bromoaniline 12, which was not observed. The next task was to selectively reduce the nitro group of 17 to an aniline while avoiding dehalogenation. Reduction attempts using Pd/C and hydrazine-hydrate as an in situ hydrogen source resulted in accompanying dehalogenation; however, iron powder (-325 mesh) in the presence of hydrochloric acid successfully furnished the desired product (18) in good yield. A number of conditions were important in order to effectively mix the heterogenous reaction mixture, manage the use of ferromagnetic iron powder and avoid degradation of the product. These included vigorous stirring, moderate reaction scales (~1 g), the use of fine iron powder and short reaction times. The final step in the synthesis of dimethyl phenazine-2,8-dicarboxylate (5) involved an intramolecular Buchwald-Hartwig N-arylation and concurrent oxidation of the piperazine ring to form the phenazine core, which proceeded smoothly in good yield (Scheme 3). X-ray crystallographic analysis confirmed the assigned structure of the 2,8-diester (5), and a detailed discussion of the crystal structure and packing is supplied in the Supplementary Material. The bismethyl ester 5 was also hydrolyzed to the corresponding diacid 6, thereby furnishing another 2,8-disubstituted phenazine with versatile synthetic handles suitable for future elaboration. As an example of such elaborations, diacid 6 was coupled with N 1 ,N 1 -dimethylethylenediamine to furnish diamide 19.  Although there is no ambiguity about the structures of compounds 5, 6 and 19, we wish to describe a simple and broadly applicable spectroscopic method to distinguish the 2,8-from the 2,7-phenazine disubstitution pattern. 1 H and 13 C NMR-based methods are of limited utility for this purpose, because the 2,8and 2,7-disubstituted phenazines both contain analogous units of symmetry and there are no 3 JCH throughbond couplings that extend between the nitrogen-separated units (Figure 2a). In contrast, 15 N NMR spectroscopy reduces the problem to a trivial exercise. The 1 H-15 N HMBC plot of 5 ( Figure 2b) reveals that there are two nitrogen environments; this is consistent with structure 5 and inconsistent with structure 20 (Figure 2c). Also notable in the 1 H-15 N HMBC plot of 5 is that each nitrogen atom correlates with just one proton environment; again, this is consistent with structure 5 and inconsistent with structure 20. This simple analysis should also be more broadly capable of distinguishing 2,8-from 2,7-disubstituted phenazines even if the two substituents were different and units of symmetry were not equivalent: for any 2,8-disubstituted phenazine, N 5 should correlate exclusively with wide doublets (green) and N 10 should correlate exclusively with narrow doublets (red); whereas for any 2,7-disubstituted phenazine, N 5 and N 10 should each couple to one wide and one narrow doublet. © AUTHOR(S) a) 1 H-NMR multiplicities and unit of symmetry for 5 and 20

Conclusions
We have described the synthesis of phenazine-2,8-dicarboxylic acid (6), its bis-methyl ester (5) and a representative diamide (19) through a modified version of Emoto's successive palladium-catalyzed N-arylation approach. We have also highlighted the ease with which 15 N NMR analysis can be employed to specifically identify 2,8-disubstituted phenazines, which have previously been confused with the 2,7-disubstitution pattern. The target molecules produced in this work constitute a rare substitution pattern for the phenazine class, and they pave the way for the expanded utility of these chromophores through novel decorations and applications.

Experimental Section
General. Unless otherwise stated, reagents and solvents were purchased from commercial suppliers and used without further purification. Iron powder was purchased from Sigma-Aldrich (-325 mesh, 97% purity, catalogue number 209309). The preparations of starting materials 11-14 are detailed in the Supplementary Material. When used as reaction solvents, toluene, dichloromethane and DMF were obtained from a solvent purification system and further dried over 4 Å molecular sieves. Unless otherwise stated, reactions were performed under an atmosphere of nitrogen, and moisture-sensitive reactions in oven-dried glassware. Reactions were monitored by thin layer chromatography (TLC) where appropriate, using Merck aluminumbacked 60 F254 0.2 mm silica plates. Plates were visualized by UV light (254 nm) and/or ninhydrin or potassium permanganate stains with heating. Purification by flash column chromatography was performed manually using Davisil 40-63 mesh silica gel. NMR spectra were obtained using Bruker Avance III instruments at 300, 400 and 600 MHz and 298 K, and calibrated using residual solvent signals as internal references. 27 Molecular connectivities were assigned using 2D NMR experiments ( 1 H-13 C HSQC, 1 H-13 C HMBC, 1 H-15 N HMBC and 1 H-1 H COSY) where possible, and by comparison of coupling constants. Chemical shifts are recorded in ppm. Multiplicities are designated as: s = singlet; br s = broad singlet; d = doublet; dd = doublet of doublets; app dt = apparent doublet of triplets; m = multiplet. IR spectra were recorded on neat samples using an Agilent Technologies Cary 630 FTIR spectrometer with ATR attachment, and spectra were processed using MicroLab FTIR processing software. Characteristic peaks are reported and assigned based on reported ranges. 28 Abbreviations include s = strong, m = medium, w = weak, br = broad, str. = stretch. Melting points were measured using an OptiMelt MPA100 automated melting point apparatus (Stanford Research Systems). HRMS results were recorded at the Bioanalytical Mass Spectrometry Facility (BMSF) at UNSW Sydney using an Orbitrap LTQ XL ion trap MS in positive ion mode with an electrospray (ESI) ion source. Methods detailed are of selected replicates of the procedure, and yields (mol%) refer to isolated products for that replicate.  0 mg, 40.3 mol), and Cs2CO3 (246 mg, 756 mol) were suspended in dry toluene (6 mL) and heated to reflux for 2 h, whereupon TLC indicated the consumption of the starting material. The reaction mixture was cooled to r.t., diluted with EtOAc (20 mL) and water (15 mL) added. HCl (2 M) was added to achieve pH2. The biphasic mixture was filtered to remove a black residue, which was washed with EtOAc (10 mL). The phases of the filtrate were separated, and the aqueous phase extracted with EtOAc (3  30 mL). The combined organic extracts were washed with brine (20 mL), dried (MgSO4), filtered, and the filtrate concentrated under reduced pressure to afford a black and yellow crude solid. Purification of this material by flash chromatography (25-50% EtOAc in hexane) afforded the title compound as a yellow crystalline solid (29.6 mg, 65%); Rf 0.31 (30% EtOAc in hexane); mp 204-206 C (from EtOAc, yellow plates and needles); IR (neat) max 1710 (s, C=O (8 mL), and heated to reflux for 18 h. The reaction mixture was cooled to r.t., diluted with EtOAc (20 mL) and water (20 mL) added. The biphasic mixture was filtered to remove a black residue, which was washed with EtOAc (10 mL). The filtrate phases were separated, and organic phase washed with HCl (2 M, 2  20 mL). The combined aqueous phases were extracted with EtOAc (2  20 mL) and the organic extracts were combined, washed with brine (20 mL), dried (MgSO4), filtered and the filtrate concentrated under reduced pressure to afford a red and black 127.0 (C1), 125.1 (C5), 120.4 (C4'), 117.7 (C6), 116.3 (C2), 114.4 (C6'), 109.6 (C2'), 51.9 (2C, 1-CO2CH3 and 4'-CO2CH3); HRMS (ESI, +ve) C16H16BrN2O4 + [M + H] + requires m/z 379.0288, 381.0268, found 379.0281, 381.0258. N 2 ,N 8 -Bis(2-(dimethylamino)ethyl)phenazine-2,8-dicarboxamide (19). To a stirring solution of 6 (100 mg, 0.373 mmol) in DMF (8 mL) at 50 C was added CDI (544 mg, 3.35 mmol). The resultant yellow suspension was stirred for 1 h at 50 C and was observed to nearly turn transparent before further yellow precipitate formed. After cooling to r.t., EtOAc (1 mL) was added and the precipitate collected by vacuum filtration and washed with EtOAc and hexane. Residual solvent was removed under reduced pressure, to yield a bisimidazolyl intermediate, phenazine-2,8-diylbis((1H-imidazol-1-yl)methanone), as a fluffy yellow solid (122 mg, 89%). A portion of this material (54 mg, 0.15 mmol) was resuspended in DMF (2 mL), and N 1 ,N 1dimethylethylenediamine (48 L, 0.44 mmol) added. The reaction mixture was stirred for 1 h at r.t., then placed under vacuum to reduce the volume by ~half. Diethyl ether (2 mL) was added and the resultant precipitate was collected by vacuum filtration (drawing excessive air or additional solvent over the solid was avoided) to yield the title compound as a powdery yellow solid (40 mg, 67%); IR (neat) max 3296 (w, N-H str.