Synthesis and Activity against Mycobacterium tuberculosis of Olivacine and Oxygenated Derivatives

The tetracyclic pyrido[4,3-b]carbazole olivacine and four of its oxygenated derivatives have been synthesized by a late-stage palladium-catalyzed Heck-type cyclization of the pyrrole ring as a key step. In a test for the inhibition of the growth of Mycobacterium tuberculosis, 9-methoxyolivacine showed the most significant inhibitory activity against Mycobacterium tuberculosis, with an MIC90 value of 1.5 μM.


Results and Discussion
For a convergent access to various A-ring substituted derivatives, we envisaged a late-stage B-ring construction of the pyrido [4,3-b]carbazole framework. Therefore, we applied the two-step sequence of palladium-catalyzed reactions developed by our group for carbazole assembly: synthesis of a diarylamine via Buchwald-Hartwig coupling of appropriate anilines 7 with a substituted isoquinoline 8 followed by oxidative cyclization to the pyrido[4,3-b]carbazoles 6 (Scheme 1) [11]. The isoquinoline 8 would be available by Bischler-Napieralski cyclization of the arylethylamine 9 via the corresponding acetamide. Henry reaction of an appropriately substituted benzaldehyde 10 and subsequent reduction should afford the arylethylamine 9. As the Bischler-Napieralski reaction works best on electron rich aromatic systems, we decided to start from the commercially available methoxy-substituted benzaldehyde 11 (Scheme 2) and to transform the methoxy group into a suitable leaving group at a later stage of our synthesis. Although 9-hydroxyolivacine (5) is the main derivative produced by the metabolic conversion of olivacine (1) [3], derivatives of olivacine (1) with A-ring substitution have been not described extensively in the literature [3,11,13,20]. This may be because the syntheses of pyrido [4,3-b]carbazoles usually involve the annulation of an isoquinoline or a pyridine at an indole or carbazole framework [8,10,11]. Thus, a facile variation of the substitution pattern at ring A is not easy to accomplish. Herein, we present a novel route for the synthesis of the tetracyclic pyrido [4,3-b]carbazole framework [21].

Results and Discussion
For a convergent access to various A-ring substituted derivatives, we envisaged a late-stage Bring construction of the pyrido [4,3-b]carbazole framework. Therefore, we applied the two-step sequence of palladium-catalyzed reactions developed by our group for carbazole assembly: synthesis of a diarylamine via Buchwald-Hartwig coupling of appropriate anilines 7 with a substituted isoquinoline 8 followed by oxidative cyclization to the pyrido[4,3-b]carbazoles 6 (Scheme 1) [11]. The isoquinoline 8 would be available by Bischler-Napieralski cyclization of the arylethylamine 9 via the corresponding acetamide. Henry reaction of an appropriately substituted benzaldehyde 10 and subsequent reduction should afford the arylethylamine 9. As the Bischler-Napieralski reaction works best on electron rich aromatic systems, we decided to start from the commercially available methoxysubstituted benzaldehyde 11 (Scheme 2) and to transform the methoxy group into a suitable leaving group at a later stage of our synthesis.

Total Synthesis
Starting from the commercial benzaldehyde 11, which can also be obtained in one step and 87% yield from the much cheaper m-anisaldehyde [22], amide 12 is prepared by a three-step sequence of Henry reaction, lithium aluminum hydride reduction, and N-acetylation (Scheme 2) [23]. Bischler-Napieralski cyclization using phosphorus oxychloride led to the corresponding dihydroisoquinoline, which was fully aromatized to 6-methoxy-1,5-dimethylisoquinoline (13) by dehydrogenation with palladium on charcoal in the presence of cyclohexene as additive. Cleavage of the methyl ether afforded the isoquinolinol, which on reaction with trifluoromethanesulfonic anhydride provided the known isoquinolinyl triflate 14 [24] in 58% yield over seven steps.

Total Synthesis
Starting from the commercial benzaldehyde 11, which can also be obtained in one step and 87% yield from the much cheaper m-anisaldehyde [22], amide 12 is prepared by a three-step sequence of Henry reaction, lithium aluminum hydride reduction, and N-acetylation (Scheme 2) [23]. Bischler-Napieralski cyclization using phosphorus oxychloride led to the corresponding dihydroisoquinoline, which was fully aromatized to 6-methoxy-1,5-dimethylisoquinoline (13) by dehydrogenation with palladium on charcoal in the presence of cyclohexene as additive. Cleavage of the methyl ether afforded the isoquinolinol, which on reaction with trifluoromethanesulfonic anhydride provided the known isoquinolinyl triflate 14 [24] in 58% yield over seven steps. Although 9-hydroxyolivacine (5) is the main derivative produced by the metabolic conversion of olivacine (1) [3], derivatives of olivacine (1) with A-ring substitution have been not described extensively in the literature [3,11,13,20]. This may be because the syntheses of pyrido [4,3-b]carbazoles usually involve the annulation of an isoquinoline or a pyridine at an indole or carbazole framework [8,10,11]. Thus, a facile variation of the substitution pattern at ring A is not easy to accomplish. Herein, we present a novel route for the synthesis of the tetracyclic pyrido [4,3-b]carbazole framework [21].

Results and Discussion
For a convergent access to various A-ring substituted derivatives, we envisaged a late-stage Bring construction of the pyrido[4,3-b]carbazole framework. Therefore, we applied the two-step sequence of palladium-catalyzed reactions developed by our group for carbazole assembly: synthesis of a diarylamine via Buchwald-Hartwig coupling of appropriate anilines 7 with a substituted isoquinoline 8 followed by oxidative cyclization to the pyrido[4,3-b]carbazoles 6 (Scheme 1) [11]. The isoquinoline 8 would be available by Bischler-Napieralski cyclization of the arylethylamine 9 via the corresponding acetamide. Henry reaction of an appropriately substituted benzaldehyde 10 and subsequent reduction should afford the arylethylamine 9. As the Bischler-Napieralski reaction works best on electron rich aromatic systems, we decided to start from the commercially available methoxysubstituted benzaldehyde 11 (Scheme 2) and to transform the methoxy group into a suitable leaving group at a later stage of our synthesis.

Total Synthesis
Starting from the commercial benzaldehyde 11, which can also be obtained in one step and 87% yield from the much cheaper m-anisaldehyde [22], amide 12 is prepared by a three-step sequence of Henry reaction, lithium aluminum hydride reduction, and N-acetylation (Scheme 2) [23]. Bischler-Napieralski cyclization using phosphorus oxychloride led to the corresponding dihydroisoquinoline, which was fully aromatized to 6-methoxy-1,5-dimethylisoquinoline (13) by dehydrogenation with palladium on charcoal in the presence of cyclohexene as additive. Cleavage of the methyl ether afforded the isoquinolinol, which on reaction with trifluoromethanesulfonic anhydride provided the known isoquinolinyl triflate 14 [24] in 58% yield over seven steps.  The cyclization reaction of the diarylamine 18a with catalytic amounts of palladium(II) acetate in the presence of P(tBu)3·HBF4 and K2CO3 in DMA at 110 °C or in DMF at 120 °C [31,32] proceeded very slowly and gave the product in only moderate yields after 1-2 days ( Table 1, entries 1 and 4). Hydrodehalogenation leading to compound 16 was the major side reaction. Using only slightly higher temperatures (130-140 °C), the reaction proceeded much faster, and the yields for olivacine (1) were significantly better (Table 1, entries 2, 5, and 6). Finally, using larger amounts of the catalyst combined with shorter reaction times, olivacine (1) was obtained in 71% yield. The structure of 1 was confirmed by an X-ray crystal structure determination ( Figure 3).   The cyclization reaction of the diarylamine 18a with catalytic amounts of palladium(II) acetate in the presence of P(tBu) 3 ·HBF 4 and K 2 CO 3 in DMA at 110 • C or in DMF at 120 • C [31,32] proceeded very slowly and gave the product in only moderate yields after 1-2 days ( Table 1, entries 1 and 4). Hydrodehalogenation leading to compound 16 was the major side reaction. Using only slightly higher temperatures (130-140 • C), the reaction proceeded much faster, and the yields for olivacine (1) were significantly better (Table 1, entries 2, 5, and 6). Finally, using larger amounts of the catalyst combined with shorter reaction times, olivacine (1) was obtained in 71% yield. The structure of 1 was confirmed by an X-ray crystal structure determination ( Figure 3).  The cyclization reaction of the diarylamine 18a with catalytic amounts of palladium(II) acetate in the presence of P(tBu)3·HBF4 and K2CO3 in DMA at 110 °C or in DMF at 120 °C [31,32] proceeded very slowly and gave the product in only moderate yields after 1-2 days ( Table 1, entries 1 and 4). Hydrodehalogenation leading to compound 16 was the major side reaction. Using only slightly higher temperatures (130-140 °C), the reaction proceeded much faster, and the yields for olivacine (1) were significantly better (Table 1, entries 2, 5, and 6). Finally, using larger amounts of the catalyst combined with shorter reaction times, olivacine (1) was obtained in 71% yield. The structure of 1 was confirmed by an X-ray crystal structure determination ( Figure 3).   Application of these conditions to the cyclization of the diarylamines 18b and 18c provided 8-methoxyolivacine (19b) and 9-methoxyolivacine (19c) in 65% and 62% yield, respectively (Scheme 4). The structure of 8-methoxyolivacine (19b) was additionally confirmed by an X-ray analysis of single crystals ( Figure 4). 9-Methoxyolivacine (19c) is a natural product that was isolated in 1967 from the bark of the coastal Venezuelan tree Aspidosperma vargasii A. DC. [33], and has been synthesized previously [3,13,20]. Interestingly, the 11bH-pyrido [3,4-c]carbazoles 20a-c containing a quaternary carbon atom were obtained as byproducts of the cyclization reactions of the diarylamines 18a-c in up to 12% yield. The structural assignments for the 11bH-pyrido[3,4-c]carbazoles 20a-c were supported by two-dimensional (2D) NMR (COSY, HMBC, HSQC, NOESY) spectroscopic studies (see Supplementary Materials). The compounds 20a-c result from an attack at the C5 carbon atom of the isoquinoline moiety. Cleavage of the methyl ether of 19b and 19c provided 8-hydroxyolivacine (4) and 9-hydroxyolivacine (5) [3] in 84% and 70% yield, respectively. For biological testing, the products were additionally purified by HPLC. Application of these conditions to the cyclization of the diarylamines 18b and 18c provided 8methoxyolivacine (19b) and 9-methoxyolivacine (19c) in 65% and 62% yield, respectively (Scheme 4). The structure of 8-methoxyolivacine (19b) was additionally confirmed by an X-ray analysis of single crystals ( Figure 4). 9-Methoxyolivacine (19c) is a natural product that was isolated in 1967 from the bark of the coastal Venezuelan tree Aspidosperma vargasii A. DC. [33], and has been synthesized previously [3,13,20]. Interestingly, the 11bH-pyrido [3,4-c]carbazoles 20a-c containing a quaternary carbon atom were obtained as byproducts of the cyclization reactions of the diarylamines 18a-c in up to 12% yield. The structural assignments for the 11bH-pyrido [3,4-c]carbazoles 20a-c were supported by two-dimensional (2D) NMR (COSY, HMBC, HSQC, NOESY) spectroscopic studies (see Supplementary Materials). The compounds 20a-c result from an attack at the C5 carbon atom of the isoquinoline moiety. Cleavage of the methyl ether of 19b and 19c provided 8-hydroxyolivacine (4) and 9hydroxyolivacine (5) [3] in 84% and 70% yield, respectively. For biological testing, the products were additionally purified by HPLC.

Biological Activity
A weak inhibitory activity against Mycobacterium tuberculosis was described in early reports for some simple tricyclic carbazole alkaloids [34][35][36]. Based on that work, we investigated the inhibitory activity of a range of oxygenated carbazole alkaloids and their derivatives, and found very promising results for several compounds [37][38][39]. Therefore, we also tested olivacine (1) and its oxygenated derivatives 4, 5, 19b, and 19c for their inhibition of M. tuberculosis (Table 2).  4 These compounds showed no significant inhibition in a

Biological Activity
A weak inhibitory activity against Mycobacterium tuberculosis was described in early reports for some simple tricyclic carbazole alkaloids [34][35][36]. Based on that work, we investigated the inhibitory activity of a range of oxygenated carbazole alkaloids and their derivatives, and found very promising results for several compounds [37][38][39]. Therefore, we also tested olivacine (1) and its oxygenated derivatives 4, 5, 19b, and 19c for their inhibition of M. tuberculosis (Table 2). In a preliminary activity test against M. tuberculosis, only two of the five pyrido [4,3-b]carbazoles, namely olivacine (1) and 9-methoxyolivacine (19c), showed significant effects and have been studied further. The minimum concentrations effecting a 90% inhibition of growth (MIC 90 ) of the M. tuberculosis strain H 37 Rv were determined by the microplate Alamar blue assay (MABA) [40,41]. The in vitro cytotoxicity towards mammalian (vero) cells was determined as described previously [40,42].
The MIC 90 value for 3-methoxy-2-methylcarbazole-1,4-quinone served as a benchmark for comparison with the inhibitory activities of carbazoles that were found in our previous studies [39].
Although olivacine (1) shows an activity comparable to our benchmark compound, the SI value is considerably lower (SI = 3.8) due to its toxicity. However, 9-methoxyolivacine (19c) exhibits a strong inhibition of M. tuberculosis (MIC 90 = 1.5 µM) combined with a lower cytotoxicity towards mammalian cells, which leads to a very good selectivity index (SI = 16.3).

General
All of the reactions were carried out in oven-dried glassware using anhydrous solvents under an argon atmosphere, unless stated otherwise. CH 2 Cl 2 , THF, and toluene were dried using a for Windows [46] (University of Glasgow, Glasgow, UK) were used as software.
Supplementary Materials: The following data are available online. Copies of the 1 H-NMR, 13 C-NMR and 2D NMR spectra.
Author Contributions: U.S. and H.-J.K. conceived and designed the experiments; U.S. and G.T. performed the chemical synthesis and characterized the compounds; A.J. and O.K. performed the X-ray analyses; B.W. and S.G.F. designed and performed the study for inhibition of M. tuberculosis; U.S. and H.-J.K. wrote the paper.
Funding: This research received no external funding.