Antimalarial Dibenzannulated Medium-Ring Keto Lactams

Abstract

We discovered dibenzannulated medium-ring keto lactams (11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-diones) as a new antimalarial chemotype. Most of these had chromatographic LogD_7.4 values ranging from <0 to 3 and good kinetic solubilities (12.5 to >100 μg/mL at pH 6.5). The more polar compounds in the series (LogD_7.4 values of <2) had the best metabolic stability (CL_int values of <50 μL/min/mg protein in human liver microsomes). Most of the compounds had relatively low cytotoxicity, with IC₅₀ values >30 μM, and there was no correlation between antiplasmodial activity and cytotoxicity. The four most potent compounds had Plasmodium falciparum IC₅₀ values of 4.2 to 9.4 nM and in vitro selectivity indices of 670 to >12,000. They were more than 4 orders-of-magnitude less potent against three other protozoal pathogens (Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani) but did have relatively high potency against Toxoplasma gondii, with IC₅₀ values ranging from 80 to 200 nM. These keto lactams are converted into their poorly soluble 4(1H)-quinolone transannular condensation products in vitro in culture medium and in vivo in mouse blood. The similar antiplasmodial potencies of three keto lactam–quinolone pairs suggest that the quinolones likely contribute to the antimalarial activity of the lactams.

Graphical Abstract

Despite substantial investment in malaria control over the past 20 years, the disease is stubbornly persistent, particularly in Africa.¹ As antimalarial drug resistance is a constant threat, identifying new antimalarial chemotypes is a high priority.^2,3 Such chemotypes may also lead to the discovery of new antimalarial drug targets.³

With this in mind, we were interested in Witkop–Winterfeldt oxidative ring-expansion of cycloalkylindoles to the corresponding medium-ring keto lactams.^4,5 To this end, ozonolysis of dihydrobenzo[a]carbazole 1^6,7 afforded 11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (2)^6,7 (Figure 1). We discovered that 2 had promising antiplasmodial activity, with an IC₅₀ of 190 nM against the NF54 strain of Plasmodium falciparum, low cytotoxicity (IC₅₀ of 53 μM), and a good physicochemical/in vitro ADME profile, with a chromato-graphically determined LogD_7.4 of 1.9, aqueous solubility of 50–100 μg/mL, and acceptable metabolic stability (CL_int values of 20–40 μL/min/mg protein in human liver microsomes). To our surprise, we also found that 2 was converted to 4(1H)-quinolone 3 under the mild conditions (neutral pH values and 37 °C) employed in our in vitro and in vivo biological assays (vide infra); normally this transannular condensation reaction takes place only with a strong base (aq. NaOH) or high temperatures (180 °C).^4,6,7 We now report the initial structure–activity relationship (SAR) scoping of 2 by the synthesis and evaluation of analogs 4–37. This was driven by our efforts to maximize the structural diversity, improve antiplasmodial activity and selectivity, and optimize in vitro ADME.

Figure 1.

Witkop–Winterfeldt oxidation of indole 1 to medium-ring keto lactam 2 and conversion of 2 to 4(1H)-quinolone 3 by Camps transannular condensation.

RESULTS AND DISCUSSION

Several overarching trends are apparent from the physicochemical, in vitro ADME, and in vitro antiplasmodial and cytotoxicity data in Tables 1-4. First, the calculated polar surface area (PSA) values between 37 and 89 Ų would suggest that polarity is unlikely to limit membrane permeability and oral absorption.⁸ The exception is 19, with a LogD_7.4 value of less than zero; thus, permeability issues cannot be ruled out for this compound. Second, most of the compounds had LogD_7.4 values ranging from ~0.5 to 3 and had good to high kinetic solubilities (25 to >100 μg/mL at pH 6.5). Exceptions to this were 26 and 27, which had kinetic solubilities ≤12.5 μg/mL; these were also two of the three compounds with LogD_7.4 values >3. Third, and not surprisingly, the more polar compounds in the series (gLogD_7.4 values of <0 to 2) were the most metabolically stable (CL_int values of <46 μL/min/mg protein in human liver microsomes). Fourth, as assessed by growth inhibition of the rat skeletal myoblast L6 cell line, most compounds had relatively low cytotoxicity. with IC₅₀ values >30 μM; the exceptions were 18, 25, and 27. Fifth, there was no correlation (Pearson r of 0.2931, p = 0.1379) between the antiplasmodial activity and cytotoxicity. Sixth, the four most potent compounds (25–28), with P. falciparum IC₅₀ values of 4.2 to 9.4 nM, also had high in vitro selectivity indices of 670 to >12,000. That these same four compounds were more than 4 orders-of-magnitude less potent against three other protozoal pathogens (Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani (Table S1) confirms the high antimalarial selectivity of this chemotype.

Table 1.

Scaffold SAR: Physicochemical, In Vitro ADME, Antimalarial, and Cytotoxicity Data

Compd	gLogD7.4	PSA (Å2)a	Kinetic Sol6.5 (μg/mL)	h/m CLint(μL/min/mg protein)b	P. falciparumIC50 (μM)c	L6 IC50 (μM)d
2	1.9	46.2	50–100	20/39	0.19	53
3	2.4	29.1	1.6–3.1	390/380	0.16	>430
4	0.6	46.2	>100	13/7	19	>490
5	2.0	46.2	12.5–25	35/38e	>200	>400
6	1.9	46.2	>100	14/31	0.75	130
7	2.0	55.4	>100	c.n.c.e	21	31
8	2.1	46.2	25–50	<7/18	6.2	>380
9	1.9	46.2	>100	7/14	4.6	230

^aCalculated using ChemAxon JChem for Excel.

^bIn vitro intrinsic clearance measured in human (h) and mouse (m) liver microsomes.

^cChloroquine-sensitive NF54 P. falciparum isolate; mean from n ≥ 2 where individual measurements differed by less than 50%.

^dCytotoxicity determined against the rat myoblast L6 cell line; mean from n ≥ 2 where individual measurements differed by less than 50%.

^eApparent non-NADPH-mediated degradation to form the lactam hydrolysis product. c.n.c. = could not calculate.

Table 4.

Benzamide SAR: Physicochemical, In Vitro ADME, Antimalarial, and Cytotoxicity Data

Compd	LogD7.4	PSA (Å2)	Kinetic Sol6.5 (μg/mL)	h/m CLint(μL/min/mg protein)	P. falciparumIC50 (μM)	L6 IC50 (μM)
30	0.9	59.1	>100	19/18	9.4	160
31	0.9	59.1	>100	11/10	11	190
32	2.1	46.2	50-100	11/110	0.13	96
33	2.0	70.0	25–50	<7/160	0.39	150
34	1.1	66.4	25–50	61/84a	15	84
35	1.3	66.4	>100	16/60	5.1	210
36	2.1	46.2	>100	13/43	1.3	110
37	1.2	66.4	12.5–25	7/36	1.1	200

^aApparent non-NADPH-mediated degradation.

Data in Table 1 detail the scaffold SAR of 2. 4(1H)-quinolone 3, the Camps transannular condensation⁵ product of 2, was as potent as the latter but had very poor aqueous solubility and metabolic stability. The 100-fold loss of potency for 4 reveals that the benzamide phenyl group of 2 is required for high antiplasmodial activity. The more polar compound 4, however, had better metabolic stability and lower cytotoxicity than 2. As shown by inactive 5, ring-contraction resulted in a complete loss of activity. Ring-expansion of 2 led to various outcomes; 6 was only 4-fold less potent than 2, whereas 7 was >100-fold less potent. Keto lactam 8, a regioisomer of 6, was 33-fold less potent than 2 and had no measurable cytotoxicity. Similarly, keto lactam 9, a regioisomer of 2, was 12-fold less potent than the latter and had a low cytotoxicity. As measured by intrinsic clearance (CL_int) values, 6, 8, and 9 had a better metabolic stability than 2. These data reveal that 6, like 2, is also a promising antimalarial hit compound.

Data in Table 2 detail the keto lactam SAR of 2. All of these compounds were ≥2 orders of magnitude less potent than 2, indicating that its ketone and secondary lactam functional groups are required for high antiplasmodial activity. Compounds 14 and 15 show that N-alkylation was not tolerated, and both had significantly reduced metabolic stability compared to 2. Similarly, keto lactone 16 had low activity, but this more lipophilic derivative had good aqueous solubility and metabolic stability. Oxime 10, oxime ether 11, and secondary alcohol 12 had high aqueous solubility and metabolic stability comparable to those of 2. In contrast, 13, in which the ketone was fully reduced, had a lower metabolic stability.

Table 2.

Ketolactam SAR: Physicochemical, In Vitro ADME, Antimalarial, and Cytotoxicity Data

Compd	LogD7.4	PSA (Å2)	Kinetic Sol6.5 (μg/mL)	h/m CLint(μL/min/mg protein)	P. falciparumIC50 (μM)	L6 IC50 (μM)
10	1.8	64.5	>100	22/76	>190	>380
11	2.5	50.7	>100	11/54a	95	250
12	1.5	49.3	>100	11/27	83	>390
13	2.6	29.1	>25	62/81	100	190
14	2.4	37.4	>100	110/220a	77	>380
15	2.9	37.4	25–50	310/>870b	100	83
16	3.7	43.4	>100	14/16	100	210

^aApparent non-NADPH-mediated degradation detected.

^bRapid metabolic degradation, estimate only.

Data in Table 3 describe the SAR of the N-phenyl substructure of 2. Our initial focus was on 2-substituted derivatives of 2, which were derived from dihydrobenzo[a]carbazole precursors of defined regiochemistry obtained in Fischer indole reactions between para-substituted phenyl hydrazines and 1-tetralone. Methyl ether 17 and ethyl ester 18 were roughly 5-fold more potent than 2. In contrast, the more polar carboxylic acid 19 and carboxamides 20 and 21 were considerably less active than 2, but each had minimal cytotoxicity. Additionally, 19–21 had a higher metabolic stability than 2, whereas 17 and 18 were less stable. As anticipated, 18 underwent rapid ester hydrolysis to form 19. Although nitrile 22 was 3.7-fold less potent than 2, it had both good antiplasmodial selectivity and metabolic stability. In contrast, the more polar methylsulfone 23 was considerably less active and somewhat less stable to CYP450 metabolism than 2, even though it had high aqueous solubility. The monofluoro derivative 24 was slightly more potent but less stable metabolically than 2. The three derivatives with fluorine-containing substituents, trifluoromethyl 25, trifluoromethoxy 26, and pentafluorosulfanyl 27, had single-digit nanomolar IC₅₀ values and good antiplasmodial selectivity but poor metabolic stability. The 1,3-difluoro analogue 28 was similarly potent but had much better metabolic stability. Of these four potent derivatives, 26 and 28 had notably high in vitro antiplasmodial selectivity indices >10,000. For 22 and 24–28, non-NADPH-mediated degradation was observed in the metabolic stability experiments; in some instances, the lactam hydrolysis product was observed. Finally, pyridyl derivative 29 had relatively weak antiplasmodial activity, high aqueous solubility, and good metabolic stability.

Table 3.

N-Phenyl SAR: Physicochemical, In Vitro ADME, Antimalarial, and Cytotoxicity Data

Compd	LogD7.4	PSA (Å2)	Kinetic Sol6.5 (μg/mL)	h/m CLint(μL/min/mg protein)	P. falciparumIC50 (μM)	L6 IC50 (μM)
17	2.1	55.4	50–100	23/440a	0.037	47
18	2.6	72.5	>50	100/910a,b	0.041	15
19	<0	86.3	>100	12/<7	160	220
20	0.8	89.3	>100	<7/10	24	>340
21	1.3	75.7	>100	11/15	7.8	>270
22	2.1	70.0	25–50	23/20c	0.70	160
23	1.6	80.3	>100	46/19	5.3	>300
24	2.2	46.2	>100	38/74c	0.056	110
25	3.0	46.2	25–50	240/79c	0.0094	9.0
26	3.1	55.4	6.3–12.5	240/110c	0.0042	44
27	3.3	46.2	6.3–12.5	190/71b	0.0042	2.8
28	2.5	46.2	50–100	17/58b	0.0070	86
29	1.2	59.1	>100	13/26	8.9	60

^aRapid metabolic degradation, estimate only.

^bApparent non-NADPH-mediated degradation detected.

^cApparent non-NADPH-mediated degradation to form the lactam hydrolysis product.

Data in Table 4 describe the SAR of the benzamide substructure of 2. The two pyridyl analogs 30 and 31 were more polar, soluble, and metabolically stable than 2, but they had relatively modest antiplasmodial activity. The 9-fluoro (32) and 9-cyano (33) derivatives had antiplasmodial potency and selectivity similar to those of 2, and they were stable to human, but less so to mouse, liver microsomes. Although the 10-fluoro derivative 36 was less potent than its regioisomer 32, it was more resistant than the latter to degradation by mouse liver microsomes. Of the three phenol-containing derivatives 34, 35, and 37, the latter had the highest antiplasmodial potency, selectivity, and metabolic stability.

Next, we assessed the activity of the 10 compounds with submicromolar IC₅₀ values for P. falciparum against four other protozoal pathogens (Table S1). Against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani, IC₅₀ values for this set of compounds ranged from 12 to 190 μM, and there was no correlation between activity against any of these pathogens and P. falciparum (Pearson correlation coefficients ranging from 0.15 and 0.50, p values 0.14 to 0.68). Keto lactams 25–28, the four compounds with the highest antiplasmodial potency, were 4 orders-of-magnitude less potent against other protozoa but did have relatively high potency against Toxoplasma gondii,⁹ with IC₅₀ values ranging from 160 to 200 nM. Moreover, there was a positive correlation between activity against T. gondii and P. falciparum (Pearson correlation coefficient of 0.72, p = 0.012).

We then measured the activity of 18, an early and relatively potent derivative of 2, against a range of P. falciparum isolates (Table 5). Except for the atovaquone-resistant TM90C2B, the IC₅₀ values of 18 fell in the relatively narrow range of 11 to 41 nM. For comparison, the 4-aminoquinoline antimalarial drug chloroquine had potent activity against the NF54 clone but was considerably less active against the other P. falciparum isolates, whereas the semisynthetic artemisinin drug artesunate had single-digit nanomolar-level potency against every P. falciparum clone and isolate. These data indicate that 18 (and the chemotype it represents) may share a mechanism(s) of action or resistance with the antimalarial naphthoquinone drug atovaquone. In this respect, it is worth noting that prototype 2 and its 4(1H)-quinolone transannular condensation product 3 are essentially equipotent against Pseudomonas falciparum (Table 1). As atovaquone and numerous structurally diverse 4(1H)-quinolones possess high activity against multiple plasmodial life stages and inhibit the same parasite target cytochrome bc₁,^10-15 we wondered if 2 is converted to 3 in vitro or in vivo. Such a scenario might explain, in part, the lack of activity of 18 against the atovaquone-resistant P. falciparum clone.

Table 5.

Activity of 18 against Different P. falciparum Clones and Isolates

	P. falciparum IC50 (nM)a
Compd	NF54	K1	7G8	TM90C2B	RF12	Dd2
18	41 ± 7.4	30 ± 7.5	32 ± 9.9	1,000 ± 84	11 ± 0.86	30 ± 7.2
Chloroquine	9.1 ± 1.5	270 ± 41	57 ± 0.9	160 ± 22	350	180 ± 21
Artesunate	6.0 ± 0.9	2.8 ± 0.64	2.3 ± 0.15	7.4 ± 1.3	6.8 ± 1.4	6.1 ± 1.4

^aMean ± SD from n ≥ 2 independent experiments using the 72 h [³H] hypoxanthine incorporation assay.

To this end, we assessed the stability of three keto lactams in the in vitro activity test medium and mouse blood (Table 6). In assay medium, 2, 32, and 28 exhibited varying degrees of instability, with the formation of the corresponding 4(1H)-quinolone product. Compound 28 had the shortest half-life of 4.7 h, followed by 32 with a 25 h half-life and 2 which showed no loss over the incubation period of 6 h but did show increasing concentrations of the corresponding quinolone product, indicating some minor loss. Similar trends were seen in mouse blood, where loss of the parent lactam was seen with a corresponding increase in the quinolone over the course of a 6 h incubation period. The apparent degradation half-lives in blood were 1.9 h for 32, 3.9 h for 28, and 5.9 h for 2. The greater instability of 28 and 32 vs 2 in both media is likely due to the fluorine atoms accelerating the transannular condensation leading to the quinolones. In a control experiment, 2 and 32 were stable in 0.1 M PBS pH 7.4, with <5% loss observed over a 7 h incubation period. In sum, these data suggest that the 4(1H)-quinolones may contribute to the antiplasmodial activity of these keto lactams both in vitro and in vivo under conditions where conversion to the quinolone is likely. Indeed, the NF54 P. falciparum IC₅₀ values of 160, 44, and 2.2 nM for 3, 38, and 39 and 190, 130, and 7.0 nM for 2, 28, and 32 (Tables 1, 3, and 4) are not dissimilar.

Table 6.

In Vitro Apparent Degradation Half-Lives for 2, 28, and 32 in P. falciparum Activity Test Medium and Mouse Blood

	In Vitro Assay Medium		Mouse Blood
Compd	Apparent degradation t1/2 (h)a	Corresponding 4(1H)-quinolone detected	Apparent degradation t1/2 (h)a	Corresponding 4(1H)-quinolone detected
2	No loss detected over 6 h	3	5.9 (5.3–6.7)	3
28	4.7 (3.6–7.1)	39	1.9 (1.7–2.1)	39
32	25 (16–60)	38	3.9 (3.4–4.6)	38

^aValues in parentheses represent the 95% confidence intervals for the half-lives.

Since 28 was one of the more potent analogues, we determined its pharmacokinetic profile in mice. Plasma concentration versus time profiles for 28 following IV and PO dosing to mice are shown in Figure 2, and pharmacokinetic (PK) parameters are presented in Table S2. No adverse reactions or compound-related side effects were observed in any mice during the 24 h post-dose sampling period. After IV administration, 28 concentrations remained above the analytical limit of quantitation for only about 1 h, and plasma clearance was very high (147 mL/min/kg). Given the instability seen in mouse whole blood, the conversion of 28 to the corresponding 4(1H)-quinolone (39) likely represents a significant clearance pathway in vivo.

Figure 2.

Plasma concentrations of 28 in Swiss mice following IV and oral administration. Concentrations were below the limit of quantitation (LLQ) after 60 min following IV dosing. IV vehicle: 5% (v/v) DMSO in 0.9% (w/v) saline containing 20% (w/v) Trappsol; PO vehicle: 7% (v/v) Tween80 and 3% (v/v) ethanol in Milli-Q water.

Following oral administration, maximum plasma concentrations were observed at 0.25 h, indicating a rapid absorption. The apparent half-life of 3.5 h was estimated based on the last three time points only, and oral bioavailability was approximately 20%. The 4(1H)-quinolone 39 was detected after both IV and oral dosing, and profiles showed a very similar time course compared to the parent profiles, consistent with rapid in vivo conversion. Like many of the quinolone products, 39 had very poor solubility in organic solvents, precluding the preparation of a stock solution to allow quantitation of the product in the in vivo plasma samples.

Despite a C_max of approximately 500 ng/mL following oral dosing of 28 (Table S2), when female NMRI mice were infected with the GFP MRA-865 strain of Plasmodium berghei and treated with four 50 mg/kg daily oral doses of this keto lactam, the parasitemia of 49.0 ± 4.3% was not significantly different from that of 52.3 ± 2.6% for the untreated control animals.

Keto lactams 6–9 were synthesized in modest yields by ozonolysis of their respective cyclohepta[1,2-b]indoles (40, 42), oxepino[4,5-b]indole 41, and dihydrobenzo[c]carbazole 43 (Scheme 1). The latter were obtained by Fischer indole syntheses. Keto lactams 14 and 15 were furnished in low yields by ozonolysis of the corresponding N-alkylated dihydrobenzo[a]carbazoles 44 and 45 (Scheme 2). Keto lactone 16 was synthesized in high yield by the ozonolysis of dihydronaphtho-[1,2-b]benzofuran 46.

Scheme 1. Syntheses of 6–9^a.

^aReagents and conditions: (a) O₃, 4–5:1 DCM/MeOH, −78 °C, 5–25 min, then Me₂S, −78 °C to rt, 2 h.

Scheme 2. Syntheses of 14–16^a.

^aReagents and conditions: (a) see Scheme 1 (a).

Similarly, ozonolysis of dihydrobenzo[a]carbazoles 47–60 (obtained via Fischer indole synthesis) afforded keto lactams 17, 22–33, and 36 in 31–89% yields (Scheme 3). Ozonolysis of dihydrobenzo[a]carbazole ester 61 and its derivatives 62–64 afforded keto lactams 18–21 in 37–65% yields (Scheme 4). Dihydrobenzo[a]carbazole carboxylic acid 62 was obtained by hydrolysis of 61 in 89% yield, followed by conversion of the latter to amides 63 and 64 in good yields.

Scheme 3. Syntheses of 17, 22–33, and 36^a.

^aReagents and conditions: see Scheme 1 (a).

Scheme 4. Syntheses of 18–21^a.

^aReagents and conditions: (a) see Scheme 1 (a); (b) 1 N aq. NaOH, EtOH, 60 °C, 24 h; (c) HOBt, EDCl, DMA, rt, 24 h, then conc. NH₄OH, rt, 5 h; (d) morpholine, HOBt, EDCl, DMA, rt, 24 h.

Fischer indole synthesis of dihydrobenzo[a]carbazole phenols 65–67 followed by conversion to their carbonates 68–70, ozonolytic ring-expansion to 71–73, and carbonate hydrolysis afforded phenolic keto lactams 35, 34, and 37, respectively (Scheme 5).

Scheme 5. Syntheses of 34, 35, and 37^a.

^aReagents and conditions: (a) Boc anhydride, DMAP, DCM, rt, 12 h; (b) see Scheme 1 (a); (c) 1 M HCl, THF, rt, 12 h.

Conversion of lactam alcohol 12⁷ to its mesylate 74 (75%) followed by reduction with sodium borohydride in hot methanol/tert-butanol¹⁶ afforded 13 in 60% yield (Scheme 6). Condensation of keto lactam 2⁷ with hydroxylamine and methoxyamine furnished oxime 10 and oxime ether 11 in moderate yields.

Scheme 6. Syntheses of 10–13^a.

^aReagents and conditions: (a) methanesulfonyl chloride, Et₃N, DCM, rt, 12 h; (b) NaBH₄, 5:1 tert-BuOH/MeOH, reflux, 12 h; (c) hydroxylamine hydrochloride, pyridine, EtOH, rt, 24 h; (d) methoxyamine hydrochloride, pyridine, MeOH, rt, 24 h.

Exposure of keto lactams 32 and 28 to triethylamine or aq. NaOH in ethanol^5,17 at room temperature (rt) afforded quinolones 38 and 39 in yields of 77 and 80%, respectively (Table 6). Finally, the known compounds 2,^6,7,18 3,^6,7 4,^4,19,20 5,⁷ and 12⁷ were synthesized by published procedures. Other attempts to modify the structure of 2 by the Schmidt reaction,¹⁸ reductive amination, or conversion of the ketone to the gem-difluoride failed.

In summary, we identified dibenzannulated medium-ring keto lactams as a new antimalarial chemotype. The four most potent derivatives in this compound series have several attractive qualities, including a two-step synthesis from commercially available starting materials, no chiral centers, a MW range of 280–380, gLogD values ranging from 2.5 to 3.0, and two aromatic rings.^21,22 In contrast, the recently approved antimalarial drug tafenoquine and four leading clinical candidates in the MMV portfolio³ each have three aromatic rings. This chemotype does have several liabilities, however. These include cross-resistance with atovaquone, metabolic instability, and a facile conversion into the corresponding poorly soluble 4(1H)-quinolones, which limit the range of chemical reactions that can be employed in compound optimization. Nonetheless, these data from our SAR scoping of hit compound 2 suggest an alternative prodrug approach for potent but relatively insoluble antimalarial 4(1H)-quinolones^23,24 using their more polar and soluble keto amide^25,26 precursors. Finally, the fairly high potency of this chemotype against T. gondii, but not three other pathogenic protozoa, should help focus downstream mechanistic^27,28 experiments.

METHODS

Target Compound Characterization.

Melting points are uncorrected. ⁴H and ¹³C NMR spectra were recorded in CDCl₃, acetone-d₆, or DMSO-d₆ on 500 and 600 MHz spectrometers. All chemical shifts are reported in parts per million (ppm) and are relative to internal (CH₃)₄Si (0 ppm) for ¹H or CDCl₃ (77.0 ppm) and DMSO-d₆ (39.7 ppm) for ¹³C NMR. HPLC analysis confirmed that all target compounds possessed purities ≥95%. Starting materials were commercially available or were prepared according to known procedures.

6,7-Dihydrodibenzo[b,h]azecine-8,14(5H,13H)-dione (6).

5,6,7,12-Tetrahydrobenzo[6,7]cyclohepta[1,2-b]indole (40)²⁹ (1.4 g, 6.0 mmol) was dissolved in a mixture of DCM (40 mL) and MeOH (10 mL). The mixture was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. (CH₃)₂S (3 mL) was added, and the solvent was evaporated to give a crude residue, which was then digested in Et₂O (30 mL) and filtered and dried to give 6 as a white solid (500 mg, 31%). mp 205–206 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 10.60 (s, 0.65H), 10.33 (s, 0.35H), 7.58 (t, J = 7.4 Hz, 0.37H), 7.50 (t, J = 6.5 Hz, 1H), 7.32–7.45 (m, 1.74H), 7.18–7.29 (m, 2.28H), 7.13 (d, J = 4.2 Hz, 1.26H), 6.87–6.96 (m, 0.64H), 6.56 (d, J = 7.6 Hz, 0.63H), 3.25 (dd, J = 12.2, 15.5 Hz, 0.66H), 3.03 (td, J = 4.6, 13.2 Hz, 0.65H), 2.88 (d, J = 5.4 Hz, 1H), 2.81 (s, 1H), 2.67 (d, J = 12.9 Hz, 1H), 2.26 (q, J = 11.4 Hz, 1H), 2.14 (dd, J = 5.8, 15.6 Hz, 1H), 2.00–2.07 (m, 1H), 1.98 (d, J = 13.7 Hz, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ: 202.77, 199.88, 171.46, 171.25, 140.56, 139.10, 138.12, 137.40, 136.78, 136.54, 133.73, 133.54, 131.98, 131.77, 130.92, 130.77, 129.92, 129.59, 129.27, 128.65, 126.97, 126.74, 126.11, 126.04, 125.60, 124.74, 124.31, 37.07, 36.33, 30.95, 28.80, 27.57, 25.28. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₅NO₂Na 288.1000; Found 288.1009.

6,7-Dihydro-8H-dibenzo[b,f][1,5]oxazecine-8,14-(13H)-dione (7).

A mixture of 7,12-dihydro-6H-benzo[2,3]-oxepino[4,5-b]indole (41)²⁹ (470 mg, 2 mmol) in DCM/MeOH (5:1, 120 mL) was treated with ozone at −78 °C for 10 min and flushed with oxygen for 5 min. After addition of dimethyl sulfide (1.0 mL) the mixture was kept at rt for 2 h. Following solvent removal in vacuo, the crude product was purified by consecutive crystallizations from EtOH/H₂O 1:2 and ether to afford 7 (168 mg, 31%) as a colorless solid. mp 162–164 °C; ¹H NMR (500 MHz, CDCl₃) δ 10.52 (s, 1H), 8.20 (s, 1H), 7.81 (s, 1H), 7.59 (s, 2H), 7.48 (s, 1H), 7.26–7.34 (m, 2H), 7.19 (s, 1H), 4.72 (s, 2H), 3.00 (s, 2H); ¹³C NMR (126 MHz, CDCl₃) δ 204.42, 164.15, 157.78, 135.70, 134.58, 132.92, 131.49, 128.00, 126.35, 125.98, 125.72, 123.58, 75.75, 42.22. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₃NO₃Na 290.0793; Found 290.0795.

8,13-Dihydrodibenzo[b,f]azecine-6,14(5H,7H)-dione (8).

5,6,7,12-Tetrahydrobenzo[6,7]cyclohepta[1,2-b]indole (42)³⁰ (1.491 g, 6.4 mmol) was dissolved in a solution of DCM (40 mL) and MeOH (10 mL). The mixture was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. (CH₃)₂S (3 mL) was added, and the solvent evaporated to give a crude residue which was then digested in Et₂O (30 mL) and filtered and dried to give 8 as a tan solid (1.017 g, 60%). mp 227–228 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 10.42 (s, 1H), 7.56 (td, J = 1.0, 8.0 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H), 7.31 (t, J = 7.5 Hz, 1H), 7.16–7.25 (m, 3H), 7.12 (d, J = 7.8 Hz, 1H), 4.46 (s, 1H), 2.85–3.23 (m, 4H), 2.05 (s, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ: 197.68, 171.45, 138.56, 135.87, 134.65, 133.51, 132.10, 131.60, 130.03, 129.00, 126.47, 125.92, 125.30, 124.34, 41.37, 40.44, 31.17. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₅NO₂Na 288.1000; Found 288.0996.

7,8-Dihydro-5H-dibenzo[b,e]azonine-6,13-dione (9).

6,7-Dihydro-5H-benzo[c]carbazole (43)³¹ (0.416 g, 1.89 mmol) was dissolved in a mixture of DCM (40 mL) and MeOH (10 mL). The mixture was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min to remove excess ozone. (CH₃)₂S (3 mL) was added, and the solvent was extracted with water (3 × 25 mL) to remove DMSO. Following solvent removal in vacuo, the crude product was purified by crystallization from DCM/MTBE (1:6) to yield 9 (158 mg, 33%) as a light-yellow solid. mp 216–217 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.22 (s, 1H), 7.72–7.65 (m, 3H), 7.55 (t, J = 7.5 Hz, 1H), 7.48 (td, J = 7.5, 1.5 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.30–7.28 (m, 2H), 2.73–2.59 (m, 3H), 2.36–2.31 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 196.53, 171.90, 138.98, 138.94, 138.68, 136.35, 133.61, 132.28, 131.63, 130.02, 129.45, 128.58, 128.41, 127.21, 36.34, 28.34. HRMS (ESI/Q-TOf) m/z: [M+Na]⁺ Calcd for C₁₆H₁₃NO₂Na 274.0838; Found 274.0834.

13-(Hydroxyimino)-5,11,12,13-tetrahydro-6H-dibenzo[b,g]azonin-6-one (10).

A mixture of 2⁷ (0.5 g, 2.0 mmol), hydroxylamine hydrochloride (0.22 g, 2.5 mmol), and pyridine (1 mL) in EtOH (15 mL) was stirred at rt for 24 h, and then the solvent was removed in vacuo. The residue was dissolved in DCM (20 mL) and washed sequentially with water (10 mL), 1 M HCl (2 × 10 mL), and brine (10 mL). The organic layer was dried over MgSO₄ and the solvent was evaporated in vacuo. The crude product was crystallized from Et₂O to give 10 (0.28 g, 53%) as a white solid. mp 138–139 °C (dec.). ¹H NMR (500 MHz, CDCl₃) δ 12.09 (s, 1H), 10.24 (s, 1H), 7.14–7.06 (m, 4H), 6.99–7.05 (m, 1H), 6.93–6.99 (m, 1H), 6.88 (d, J = 7.8 Hz, 1H), 6.85 (d, J = 7.8 Hz, 1H), 3.94 (dd, J = 11.3, 5.4 Hz, 1H), 3.31 (td, J = 13.3, 2.0 Hz, 1H), 2.97 (ddd, J = 13.8, 6.2, 2.4 Hz, 1H), 2.03(td, J = 11.7, 5.0 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ: 176.08, 156.77, 138.75, 137.80, 135.31, 129.64, 129.29, 128.88, 128.53, 128.25, 127.90, 125.99, 125.66, 32.06, 30.94. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₄N₂O₂Na 289.0953; Found 289.0963.

(E)-13-(Methoxyimino)-5,11,12,13-tetrahydro-6H-dibenzo[b,g]azonin-6-one (11).

Keto lactam 2⁷ 823 mg, 3.275 mmol), methoxyamine hydrochloride (423 mg, 5.065 mmol), and pyridine (510 mg, 6.448 mmol) were stirred in MeOH (20 mL) for 24 h at rt. The solvent was removed in vacuo and the residue was dissolved in DCM (30 mL) and washed sequentially with water (50 mL), 1 M HCl (2 × 30 mL), and brine (50 mL). The organic layer was dried with MgSO₄, the solvent was evaporated in vacuo, and the residue was crystallized from Et₂O to give 11 (537 mg, 58%) as a white solid. mp 165–167 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.49 (s, 1H), 7.22–7.12 (m, 3H), 7.09 (s, 2H), 7.04 (d, J = 7.3 Hz, 1H), 7.02–6.93 (m, 2H), 4.00 (s, 3H), 3.73 (s, 1H), 3.09 (s, 1H), 2.92 (s, 1H), 2.00 (s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ: 173.78, 157.98, 137.44, 135.50, 135.12, 134.03, 129.53, 129.24, 129.14, 128.60, 128.48, 128.35, 126.18, 125.72, 62.06, 31.78, 30.89. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₆N₂O₂Na 303.1109; Found 303.1102.

5,11,12,13-Tetrahydro-6H-dibenzo[b,g]azonin-6-one (13).

Step 1: To a solution of 13-hydroxy-5,11,12,13-tetrahydro-6H-dibenzo[b,g]azonin-6-one (12)⁷ (600 mg, 2.37 mmol) in DCM (20 mL), TEA (479 mg, 4.74 mmol) and MsCl (545 mg, 4.74 mmol) were added and the solution was stirred at rt for 12 h. After solvent removal in vacuo, the residue was purified by sg chromatography, eluting with DCM/MeOH 20:1 to afford 6-oxo-6,11,12,13-tetrahydro-5H-dibenzo[b,g]-azonin-13-yl methanesulfonate (74) (589 mg, 75%) as a white solid. Step 2: To a stirring solution of 74 (200 mg, 0.60 mmol) in t-BuOH/MeOH 5:1 (12 mL) was added NaBH₄ (230 mg, 6.04 mmol), and the solution was refluxed for 12 h. Once the starting material was gone via TLC, the solvent was evaporated in vacuo, and the residue purified by sg chromatography eluting with hexane/EA 2:1 to afford 13 (86 mg, 60%) as a white solid. mp 173–174 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.56 (s, 1H), 7.10–6.99 (m, 4H), 6.99–6.89 (m, 4H), 3.02 (s, 1H), 2.86 (s, 2H), 2.77 (s, 1H), 2.34 (s, 1H), 1.61 (s, 2H). ¹³C NMR (125 MHz, CDCl₃) δ: 174.81, 141.97, 138.03, 135.63, 135.54, 130.28, 129.70, 129.20, 129.02, 128.67, 126.59, 125.65, 125.37, 34.44, 33.30, 31.75. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₅NONa 260.1051; Found 260.1050.

5-Methyl-11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (14).

11-Methyl-6,11-dihydro-5H-benzo[a]carbazole (44)³² (481 mg, 2.061 mmol) was dissolved in a mixture of DCM (40 mL) and MeOH (10 mL). The mixture was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. (CH₃)₂S (3 mL) was added, and the solvent was evaporated to give a crude residue which was then digested in Et₂O (30 mL) and filtered and dried to afford 14 as a white solid (162 mg, 16%). mp 182–184 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 7.40 (d, J = 7.7 Hz, 1H), 7.35 (td, J = 1.2, 7.4 Hz, 1H), 7.31 (dd, J = 1.1, 7.6 Hz, 1H), 7.18 (dd, J = 0.8, 7.3 Hz, 1H), 7.14 (d, J = 7.6 Hz, 1H), 7.09 (td, J = 0.9, 7.4 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 6.90 (d, J = 7.4 Hz, 1H), 3.29 (s, 3H), 3.03 (dt, J = 4.9, 13.2 Hz, 1H), 2.95–2.90 (m, 2H), 2.88–2.81 (m, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ: 206.35, 169.85, 142.16, 139.51, 137.37, 135.31, 131.04, 129.28, 128.90, 128.70, 127.76, 126.25, 125.82, 125.51, 44.39, 36.58, 30.94. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₅NO₂Na 288.1000; Found 288.0999.

2,3,8,9-Tetrahydro-1H-benzo[7,8]azonino[3,2,1-i,j]-quinoline-7,14-dione (15).

Step 1: 3,4-Dihydroquinolin-1(2H)-amine (2.044 g, 13.79 mmol), 3,4-dihydronaphthalen-1(2H)-one (2.058 g, 14.08 mmol), p-TsOH·H₂O (2.796 g, 14.7 mmol), and diglyme (10 mL) were combined in a 20 mL microwave vessel with stir-bar. The mixture was irradiated for 30 min at 200 °C. After cooling to rt, water (50 mL) was added, and the precipitate was filtered and dried to give crude 5,6,12,13-tetrahydro-4H-benzo[a]pyrido[3,2,1-jk]carbazole (45) as a tan foam (1.526 g, 43%) which was used in the next step without further purification. ¹H NMR (600 MHz, CDCl₃) δ 7.60 (d, J = 7.7 Hz, 1H), 7.38 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 7.4 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.91 (d, J = 7.0 Hz, 1H), 4.51 (t, J = 5.8 Hz, 2H), 3.00 (dt, J = 7.0, 19.0 Hz, 4H), 2.93 (dd, J = 6.1, 8.3 Hz, 2H), 2.25 (p, J = 5.9 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ: 137.80, 135.38, 133.62, 129.87, 128.63, 126.52, 126.13, 123.84, 122.03, 121.90, 119.46, 118.87, 116.24, 112.97, 44.86, 30.71, 24.92, 23.36, 20.27. Step 2: Indole 44 (1.526 g, 5.58 mmol) was dissolved in a mixture of DCM (40 mL) and MeOH (10 mL) and subjected to ozonolysis at −78 °C for 20 min and flushed with oxygen for 5 min. (CH₃)₂S (3 mL) was added, and the solution was washed with water (40 mL) and brine (60 mL) and then dried over MgSO₄. The solvent was evaporated in vacuo to give a crude residue, which was then digested in Et₂O (30 mL) and filtered and dried to give 15 as a yellow solid (200 mg, 12%). mp 162–164 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.69 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 7.3 Hz, 1H), 7.10 (td, J = 1.1, 7.5 Hz, 1H), 7.07 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.93 (t, J = 7.4 Hz, 1H), 6.68 (d, J = 7.5 Hz, 1H), 5.01 (ddd, J = 7.3, 9.2, 12.8 Hz, 1H), 3.83 (td, J = 8.1, 11.6 Hz, 1H), 3.57 (dd, J = 7.5, 14.4 Hz, 1H), 3.20 (ddd, J = 4.5, 8.8, 13.0 Hz, 1H), 3.07 (ddd, J = 8.3, 11.2, 14.3 Hz, 1H), 2.87–2.74 (m, 3H), 2.64 (td, J = 6.0, 13.2, 14.4 Hz, 1H), 2.47 (dddd, J = 3.0, 5.4, 9.1, 12.8 Hz, 2H), 1.88–1.76 (m, 1H). ¹³C NMR (125 MHz, CDCl₃) δ: 199.48, 171.73, 139.50, 137.69, 136.55, 135.79, 133.45, 131.99, 130.08, 129.12, 128.43, 126.69, 126.17, 124.13, 41.35, 38.22, 31.24, 27.19, 23.98. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₉H₁₇NO₂Na 314.1157; Found 314.1159.

11,12-Dihydrodibenzo[b,g]oxonine-6,13-dione (16).

5,6-Dihydronaphtho[1,2-b]benzofuran (46)³³ (247 mg, 1.12 mmol) was dissolved in a mixture of DCM (40 mL) and MeOH (10 mL). The mixture was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min to remove excess ozone. (CH₃)₂S (3 mL) was added, and the solvent was extracted with water (3 × 25 mL) to remove DMSO. The solvent was removed in vacuo, and the crude product was purified using sg chromatography in DCM/hexane (1:1) to yield 16 (243 mg, 86%) as a light yellow solid. mp 68–69 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.02 (dd, J = 7.7, 1.5 Hz, 1H), 7.94–7.92 (m, 1H), 7.69–7.63 (m, 2H), 7.52 (td, J = 7.5, 1.5 Hz, 1H), 7.44–7.36 (m, 3H), 3.35 (t, J = 6.5 Hz, 2H), 2.97 (t, J = 6.4 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ: 199.29, 165.64, 150.65, 144.47, 133.88, 133.23, 131.79, 131.35, 130.33, 130.13, 129.29, 127.48, 126.59, 123.86, 45.47, 31.09. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₂O₃Na 275.0684; Found 275.0692.

2-Methoxy-11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (17).

8-Methoxy-6,11-dihydro-5H-benzo[a]carbazole (47)³⁴ (0.824 g, 3.31 mmol) was dissolved in DCM (40 mL) and MeOH (10 mL). The mixture was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min to remove excess ozone. (CH₃)₂S (3 mL) was added, and the solvent was extracted with water (3 × 25 mL) to remove DMSO. The solvent was removed in vacuo, and the crude product was purified using sg chromatography eluting with 10–15% EA in DCM to yield 17 (300 mg, 32%) as a light-yellow solid. mp 124–125 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.36 (s, 1H), 7.14–7.11 (m, 1H), 7.08–7.03 (m, 4H), 6.96–6.95 (m, 1H), 6.86–6.85 (m, 1H), 3.83–3.77 (m, 1H), 3.71 (s, 3H), 3.40–.35 (m, 1H), 3.09–3.03 (m, 1H), 2.87–2.82 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ: 202.33, 174.23, 158.37, 138.56, 135.52, 135.12, 130.00, 129.84 129.40, 128.04, 126.80, 125.31, 118.82, 112.20, 55.52, 41.08, 31.22. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₅NO₃Na 304.0944; Found 304.0941.

Ethyl 6,13-Dioxo-6,11,12,13-tetrahydro-5H-dibenzo-[b,g]azonine-2-carboxylate (18).

A mixture of ethyl 6,11-dihydro-5H-benzo[a]carbazole-8-carboxylate (61)³⁵ (1.0 g, 3.4 mmol) in DCM/MeOH 5:1 (120 mL) was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. Dimethyl sulfide (1.0 mL) was added, and the mixture was kept cold for 1 h before warming to rt for 2 h. The solvent was then removed in vacuo, and the crude product was purified by crystallization from aq. EtOH to afford 18 (0.6 g, 54%) as a white solid. mp 189–190 °C (dec.) ¹H NMR (500 MHz, DMSO-d₆) δ 10.71 (br s, 1H), 8.07 (d, J = 2.1 Hz, 1H), 7.93–8.04 (m, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.10–7.25 (m, 2H), 6.97–7.09 (m, 1H), 6.80–6.96 (m, 1H), 4.27 (q, J = 7.2 Hz, 2H), 3.56–3.79 (m, 1H), 3.15–3.27 (m, 1H), 2.98–3.13 (m, 1H), 2.73–2.87 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 201.71 (br s), 172.35 (br s), 165.03, 141.31, 136.45 (br s), 133.16, 130.28, 129.98, 129.51, 128.75 (br s), 128.02, 127.04, 125.34 (br s), 61.53, 40.60, 30.85, 14.54. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₉H₁₇NO₄Na 346.1055; Found 346.1051.

6,13-Dioxo-6,11,12,13-tetrahydro-5H-dibenzo[b,g]-azonine-2-carboxylic Acid (19).

Step 1: To a solution of 61 ³⁵ (0.50 g) in EtOH (10 mL) was added 1 N NaOH (5 mL). The mixture was stirred at 60 °C for 24 h, and water (30 mL) was added. The mixture was acidified using 1 N HCl and the resulting precipitate was filtered to give 6,11-dihydro-5H-benzo[a]carbazole-8-carboxylic acid (62) (0.40 g, 89%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 12.43 (brs, 1H), 11.81 (s, 1H), 8.17 (d, J = 1.8 Hz, 1H), 7.73 (dd, J = 8.5, 1.7 Hz, 1H), 7.66 (dd, J = 7.1, 1.8 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.27–7.34 (m, 2H), 7.23–7.17 (m, 1H), 3.03 (t, J = 8.0 Hz, 2H), 2.95 (t, J = 8.0 Hz, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 168.82, 140.02, 136.50, 135.13, 129.03, 128.81, 127.20, 126.76, 123.36, 122.00, 121.52, 121.42, 112.34, 111.48, 29.26, 19.50. Step 2: A mixture of 62 (0.6 g, 2.3 mmol) in DCM/MeOH 5:1 (120 mL) was subjected to ozonolysis at −78 °C for 10 min and flushed with oxygen for 5 min. Dimethyl sulfide (1.0 mL) was added and the mixture was kept cold for 1 h before warming to rt for 2 h. Solvent removal in vacuo afforded a solid which was purified by crystallization from aq. EtOH to afford 19 as a white solid (0.25 g, 37%). mp >300 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 13.13 (br s, 1H), 10.66 (s, 1H), 8.04 (s, 1H), 7.96 (s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.10–7.25 (m, 2H), 6.97–7.09 (m, 1H), 6.81–6.96 (m, 1H), 3.61–3.76 (m, 1H), 3.14–3.26 (m, 1H), 2.98–3.13 (m, 1H), 2.71–2.86 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 201.96 (brs), 172.32 (brs), 166.58, 140.93 (brs), 136.35 (brs), 133.30, 130.27, 130.14, 129.47, 128.97, 128.57 (brs), 128.29, 127.02, 125.38, 40.91, 30.86. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₃NO₄Na 318.0742; Found 318.0750.

6,13-Dioxo-6,11,12,13-tetrahydro-5H-dibenzo[b,g]-azonine-2-carboxamide (20).

Step 1: To a mixture of 62 (0.53 g, 2.0 mmol) and HOBt (0.3 g, 2.2 mmol) in DMA (10 mL) at rt was added EDCI (0.5 g, 2.8 mmol). After 24 h, conc. aq. ammonia (1 mL) was added and the reaction was stirred for 5 h before quenching with water (20 mL). The resulting precipitate was filtered and recrystallized from CH₃CN to give primary amide 6,11-dihydro-5H-benzo[a]carbazole-8-carboxamide (63) (0.38 g, 72%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.68 (s, 1H), 8.13 (d, J = 2.0 Hz, 1H), 7.86 (br s, 1H), 7.63–7.69 (m, 2H), 7.39 (d, J = 8.5 Hz, 1H), 7.26–7.33 (m, 2H), 7.19 (td, J = 7.3, 1.4 Hz, 1H), 7.10 (brs, 1H), 2.99–3.05 (m, 2H), 2.90–2.97 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 169.44, 139.12, 136.43, 134.77, 129.21, 128.78, 127.31, 127.19, 126.61, 125.73, 121.96, 121.43, 119.10, 112.14, 111.11, 29.33, 19.67. Step 2: A mixture of 63 (1.0 g, 3.82 mmol) in 5:1 DCM/MeOH (120 mL) was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. Dimethyl sulfide (1.0 mL) was added and the mixture was kept cold for 1 h before warming to rt for 2 h. After solvent removal in vacuo, the residue was purified by crystallization from MeOH to afford 20 (0.60 g, 54%) as a white solid. mp >300 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 10.53 (s, 1H), 7.97–8.07 (m, 1H), 7.97 (s, 1H), 7.84–7.93 (m, 1H), 7.37–7.47 (m, 1H), 7.25–7.35 (m, 1H), 7.10–7.22 (m, 2H), 6.99–7.07 (m, 1H), 6.86–6.97 (m, 1H), 3.56–3.71 (m, 1H), 3.13–3.23 (m, 1H), 3.00–3.12 (m, 1H), 2.74–2.88 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 204.76, 172.12, 166.99, 139.26, 136.81, 136.68, 133.74, 131.52, 130.24, 129.41, 128.44, 128.14, 127.00, 125.41, 40.90, 30.94. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₄N₂O₃Na 317.0902; Found 317.0900.

2-(Morpholine-4-carbonyl)-11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (21).

Step 1: To a mixture of 62 (0.52 g, 2.0 mmol), morpholine (0.34 g, 4.0 mmol), HOBt (0.3 g, 2.2 mmol), and TEA (1 mL) in DMA (10 mL) was added EDCI (0.45 g, 2.3 mmol). After stirring at rt for 24 h, the reaction was quenched with water (30 mL). The resulting precipitate was filtered and recrystallized from CH₃CN to give (6,11-dihydro-5H-benzo[a]carbazol-8-yl)-(morpholino)methanone (64) (0.50 g, 76%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.67 (s, 1H), 7.65 (d, J = 6.7 Hz, 1H), 7.56–7.60 (m, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.27–7.32 (m, 2H), 7.13–7.24 (m, 2H), 3.59–3.64 (m, 6H), 3.50–3.58 (s, 2H), 2.97–3.04 (m, 2H), 2.88–2.96 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 171.07, 137.98, 136.50, 134.81, 129.18, 128.79, 127.36, 127.18, 126.66, 126.57, 118.50, 111.78, 111.46, 66.70, 39.74, 29.33, 19.56. Step 2: Indole 64 (0.50 g, 1.5 mmol) was dissolved in a solution of DCM/MeOH 4:1 (50 mL) and ozonized at −78 °C for 10 min and flushed with oxygen for 5 min. (CH₃)₂S (1 mL) was added, and the solvent evaporated in vacuo to give a crude residue which was then digested in Et₂O (30 mL), filtered, and dried to give 21 (0.36 g, 65%) as a white solid. mp 132–133 °C (dec.). ¹H NMR (500 MHz, CDCl₃) δ 7.79 (brs, 1H), 7.63 (s, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.25 (d, J = 8.1 Hz, 1H), 7.19 (d, J = 6.5 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.03–7.10 (m, 1H), 6.91–7.01 (m, 1H), 3.65–3.89 (m, 6H), 3.51–3.65 (m, 2H), 3.38–3.50 (m, 1H), 3.20–3.34 (m, 1H), 3.07–3.19 (m, 1H), 2.80–2.95 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 200.96, 173.18, 168.01, 136.21, 135.37, 134.02, 133.60, 130.92, 129.70, 129.31, 128.86, 127.78, 127.54, 126.46, 124.91, 66.39, 66.21, 47.66, 42.19, 39.86, 30.48. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₂₁H₂₀N₂O₄Na 387.1321; Found 387.1317.

6,13-Dioxo-6,11,12,13-tetrahydro-5H-dibenzo[b,g]-azonine-2-carbonitrile (22).

Step 1: A mixture of 4-cyanophenylhydrazine hydrochloride (0.17 g, 1 mmol) and 3,4-dihydronaphthalen-1(2H)-one (0.15 g, 1 mmol) in ethylene glycol (5 mL) was irradiated via microwave at 250 °C for 20 min. The mixture was then added to ice water (20 mL) and the resulting precipitate was filtered to give 6,11-dihydro-5H-benzo[a]carbazole-8-carbonitrile (48) (0.13 g, 53%) as a pale-yellow solid. ¹H NMR (500 MHz, DMSO-d₆) δ 12.07 (s, 1H), (d, J = 1.8 Hz, 1H), 7.68 (dd, J = 8.1, 1.4 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 8.4, 1.6 Hz, 1H), 7.35–7.29 (m, 2H), 7.23 (td, J = 7.2, 1.4 Hz, 1H), 2.99–3.05 (m, 2H), 2.90–2.97 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 138.69, 136.26, 135.51, 128.41, 128.06, 127.45, 126.78, 126.50, 124.15, 123.96, 121.31, 120.75, 112.42, 111.40, 100.93, 28.60, 18.86. Step 2: Indole 48 (0.3 g, 1.2 mmol) dissolved in 4:1 DCM/MeOH (50 mL) was subjected to ozonolysis at −78 °C for 6 min and flushed with oxygen for 5 min. (CH₃)₂S (1 mL) was then added and the solvent evaporated in vacuo to give a crude residue, which was then digested in Et₂O (30 mL), filtered, and dried to give 22 as a white solid (0.16 g, 47%). mp 176–177 °C. ¹H NMR (500 MHz, CDCl₃) δ 9.05 (brs, 1H), 7.94 (d, J = 2.3 Hz, 1H), 7.67 (dd, J = 8.2, 2.1 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 6.5 Hz, 1H), 3.75–4.00 (m, 1H), 3.38–3.58 (m, 1H), 3.07–3.25 (m, 1H), 2.79–2.97 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ: 199.78, 173.74, 139.43, 136.68, 136.00, 135.52, 134.04, 133.84, 130.39, 128.40, 127.17, 125.30, 117.20, 111.38, 39.89, 30.86. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₂N₂O₂Na 299.0796; Found 299.0795.

2-(Methylsulfonyl)-11,12-dihydro-5H-dibenzo[b,g]-azonine-6,13-dione (23).

Step 1: (4-(Methylsulfonyl)-phenyl)hydrazine (1.041 g, 5.6 mmol), 3,4-dihydronaphthalen-1(2H)-one (842 mg, 5.8 mmol), p-TsOH·H₂O (1.075 g, 5.7 mmol), and diglyme (10 mL) were irradiated in a microwave for 40 min at 250 °C. After cooling to rt, water (50 mL) was added, and the precipitate was filtered and dried to give crude 8-(methylsulfonyl)-6,11-dihydro-5H-benzo[a]carbazole (49) as a tan foam (1.5 g, 82%) which was used in the next step without further purification. ¹H NMR (600 MHz, DMSO-d₆) δ 12.05 (s, 1H), 8.10 (s, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.56–7.63 (m, 2H), 7.32 (t, J = 7.2 Hz, 2H), 7.23 (t, J = 7.4 Hz, 1H), 3.17 (s, 3H), 3.00–3.05 (m, 2H), 2.95–2.99 (m, 2H). Step 2: Indole 49 (1.041 g, 5.58 mmol) was dissolved in a mixture of DCM (40 mL) and MeOH (10 mL). The mixture was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. (CH₃)₂S (3 mL) was added, and the solution was washed with water (40 mL) and brine (60 mL) and then dried over MgSO₄. The solvent was evaporated to give a crude residue, which was digested in Et₂O (30 mL) and filtered and dried to give 23 (932 mg, 56%) as a white solid. mp 238–240 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 10.83 (s, 1H), 8.00 (s, 2H), 7.49 (d, J = 8.3 Hz, 1H), 6.76–7.44 (m, 4H), 3.68 (s, 1H), 3.19 (s, 4H), 3.12 (s, 1H), 2.81 (s, 1H). ¹³C NMR (150 MHz, DMSO) δ 200.63, 171.69, 141.08, 135.95, 135.54, 130.66, 129.78, 129.10, 128.47, 127.64, 126.54, 124.79, 54.79, 43.13, 39.91, 30.18. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₅NO₄SNa 352.0619; Found 352.0612.

2-Fluoro-11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (24).

A mixture of 8-fluoro-6,11-dihydro-5H-benzo[a]carbazole (50)³⁶ (1.0 g, 4.22 mmol) in DCM/MeOH 5:1 (120 mL) was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. Dimethyl sulfide (1.0 mL) was added and the mixture was kept cold for 1 h before warming to rt for 2 h. After solvent removal in vacuo, the resulting solid was purified by crystallization from aq. EtOH to afford 24 (0.80 g, 70% yield) as a white solid. mp 163–164 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.70 (brs, 1H), 7.24 (d, J = 7.2 Hz, 1H), 7.15–7.22 (m, 2H), 7.03–7.14 (m, 3H), 7.00–6.95 (m, 1H), 3.70–3.81 (m, 1H), 3.43–3.36 (m, 1H), 3.33–3.44 (m, 1H), 2.84–2.94 (m, 1H). ¹³C NMR (126 MHz, DMSO) δ 202.82, 172.46, 160.24 (d, J = 241 Hz), 140.88, 136.84, 135.85, 132.39, 131.74, 130.02, 129.27, 126.99, 125.50, 119.04 (d, J = 22 Hz), 114.00 (d, J = 25 Hz), 42.35, 30.82. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₂NO₂FNa 292.0750; Found 292.0741.

2-(Trifluoromethyl)-11,12-dihydro-5H-dibenzo[b,g]-azonine-6,13-dione (25).

8-(Trifluoromethyl)-6,11-dihydro-5H-benzo[a]carbazole (51)³⁶ (1.1 g, 3.83 mmol) was dissolved in DCM (40 mL) and MeOH (10 mL). The mixture was subjected to ozonolysis at −78 °C for 25 min and flushed with oxygen for 5 min. (CH₃)₂S (3 mL) was added, and the resulting solution was washed with water (40 mL) and brine (60 mL) and then dried over MgSO₄. The solvent was evaporated in vacuo to give a crude residue that was recrystallized from hot CH₃CN (5 mL) and water (40 mL) to afford 25 as a tan solid, which was filtered and dried (635 mg, 52%). mp 207–209 °C. ¹H NMR (600 MHz, Acetone-d₆) δ 9.64 (s, 1H), 7.89 (s, 1H), 7.81 (d, J = 6.2 Hz, 1H), 7.58 (d, J = 8.2 Hz, 1H), 7.18 (s, 2H), 7.04 (d, J = 33.9 Hz, 2H), 3.82 (d, J = 17.6 Hz, 1H), 3.28 (s, 1H), 3.14 (s, 1H), 2.96–2.74 (m, 2H). ¹³C NMR (150 MHz, Acetone-d₆) δ 200.24, 171.59, 140.05, 136.99, 136.18, 135.91, 129.72, 128.95, 128.52 (d, J = 3.5 Hz), 127.59 (q, J = 32.8 Hz), 126.40, 125.53 (d, J = 3.6 Hz), 124.97, 123.56 (q, J = 271.5 Hz), 39.70, 30.41. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₂NO₂F₃Na 342.0718; Found 342.0709.

2-(Trifluoromethoxy)-11,12-dihydro-5H-dibenzo-[b,g]azonine-6,13-dione (26).

Step 1: (4-(Trifluoromethoxy)phenyl)hydrazine (1.197 g, 5.236 mmol), 3,4-dihydronaphthalen-1(2H)-one (782 mg, 5.349 mmol), p-TsOH·H₂O (1.153 g, 6.061 mmol), and diglyme (15 mL) were irradiated in a microwave oven for 40 min at 225 °C. Water (50 mL) was added, and the precipitate was filtered and dried to give crude 8-(trifluoromethoxy)-6,11-dihydro-5H-benzo[a]carbazole (52) as a brown foam (1.1 g, 69%) which was used for the next step without further purification. ¹H NMR (600 MHz, CDCl₃) δ: 8.31 (s, 1H), 7.38 (s, 1H), 7.34 (d, J = 8.6 Hz, 2H), 7.28 (t, J = 6.8 Hz, 2H), 7.20 (t, J = 7.4 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 3.07 (t, J = 7.7 Hz, 2H), 2.95 (t, J = 7.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ: 136.68, 135.14, 134.97, 128.63, 128.35, 127.66, 127.29, 126.73, 121.69, 119.99, 116.06, 112.82, 111.54, 111.19, 29.39, 19.57. Step 2: Indole 52 (1.1 g, 3.627 mmol) was dissolved in a solution of DCM (40 mL) and MeOH (10 mL). The mixture was subjected to ozonolysis at −78 °C for 25 min and flushed with oxygen for 5 min. (CH₃)₂S (3 mL) was added and the resulting solution was washed with water (40 mL), brine (60 mL) and then dried over MgSO₄. The solvent was evaporated to give a crude residue, which was crystallized from Et₂O to give 26 (900 mg, 74%) as a pale yellow solid. mp 109–111 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.59 (s, 1H), 7.43 (s, 1H), 7.26–7.15 (m, 3H), 7.13–7.02 (m, 2H), 6.96 (s, 1H), 3.84 (s, 1H), 3.42 (d, J = 12.9 Hz, 1H), 3.11 (s, 1H), 2.87 (s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ 200.69, 173.89, 147.81, 138.53, 135.57, 134.47, 133.75, 130.16, 129.86, 129.82, 127.02, 125.29, 124.66, 121.10, 120.18 (q, J = 258.7 Hz), 40.49, 31.02. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₂NO₃F₃Na 358.0667; Found 358.0660.

2-(Pentafluoro-λ⁶-sulfaneyl)-11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (27).

Step 1: Pentafluoro-(4-fluorophenyl)-λ⁶-sulfane (5.421 g, 24.4 mmol), hydrazine monohydrate (5.0 g, 99.880 mmol), and DMSO (20 mL) were heated at 95 °C for 12 h. After cooling to rt, to the reaction mixture was added to 1 N aq. NaOH (50 mL) and cooled to 0 °C to form a precipitate, which was filtered, washed with water, and dried in vacuo at rt to give (4-(pentafluoro-λ⁶-sulfaneyl)phenyl)hydrazine as a yellow solid (5.0 g, 88%). ¹H NMR (600 MHz, CDCl₃) δ: 7.58 (d, J = 9.1 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 5.52 (s, 1H), 3.63 (s, 2H). ¹³C NMR (150 MHz, CDCl₃) δ: 152.82, 144.78 (p, J = 17.1 Hz), 127.28 (p, J = 4.6 Hz), 110.40. Step 2: (4-(Pentafluoro-λ⁶-sulfaneyl)phenyl)hydrazine (2.236 g, 9.547 mmol), 3,4-dihydronaphthalen-1(2H)-one (1.425 g, 9.748 mmol), p-TsOH·H₂O (2.760 g, 14.509 mmol), and (20 mL) were irradiated in a microwave oven for 30 min at 150 °C. Water (50 mL) was added, and the precipitate was filtered and dried to give crude 8-(pentafluoro-λ⁶-sulfaneyl)-6,11-dihydro-5H-benzo[a]carbazole (53) as a yellow solid (2.14 g, 65%) which was used for the next step without further purification. ¹H NMR (600 MHz, CDCl₃) δ 8.42 (s, 1H), 7.96 (d, J = 1.5 Hz, 1H), 7.57 (dd, J = 1.7, 8.9 Hz, 1H), 7.36 (dd, J = 8.2, 12.7 Hz, 2H), 7.30 (d, J = 6.9 Hz, 1H), 7.28 (d, J = 7.5 Hz, 1H), 7.23 (t, J = 7.4 Hz, 1H), 3.09 (t, J = 7.7 Hz, 2H), 2.99 (t, J = 7.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ: 147.20 (p, J = 15.7 Hz), 137.27, 136.72, 135.35, 128.73, 127.97, 127.64, 126.80, 126.39, 120.04, 119.63 (t, J = 4.6 Hz), 117.31 (t, J = 4.6 Hz), 113.55, 110.35, 29.28, 19.46. Step 3: Indole 53 (2.14 g, 5.671 mmol) was dissolved in a mixture of DCM (40 mL) and MeOH (10 mL). The mixture was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. (CH₃)₂S (2 mL) was added, and the resulting solution was washed with water (40 mL) and brine (60 mL) and then dried over MgSO₄. The solvent was evaporated in vacuo to give a crude residue, which was digested in Et₂O (30 mL) and filtered and dried to give 27 as a white solid (1.777 g, 76%). mp 203–204 °C. ¹H NMR (600 MHz, CDCl₃) δ 9.20 (s, 1H), 8.06 (d, J = 2.4 Hz, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.23 (t, J = 7.4 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.09 (t, J = 6.9 Hz, 1H), 6.97 (s, 1H), (s, 1H), 3.49 (s, 1H), 3.16 (s, 1H), 2.87 (s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ: 199.57, 174.08, 151.89 (t, J = 19.8 Hz), 138.48, 135.92, 135.44, 133.92, 130.45, 130.20, 130.10 (t, J = 4.0 Hz), 127.93 (t, J = 4.5 Hz), 127.66, 127.21, 125.28, 39.15, 30.84. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₂NO₂SF₅Na 400.0407; Found 400.0398.

1,3-Difluoro-11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (28).

Step 1: A mixture of 3,5-difluorophenylhydrazine hydrochloride (720 mg, 4.0 mmol) and 3,4-dihydronaphthalen-1(2H)-one (584 mg, 4.0 mmol) in TFA (8 mL) was refluxed at 70 °C for 12 h. After the mixture was cooled to 0 °C, 1 M sodium hydroxide (20 mL) and water (50 mL) were successively added to the stirred reaction mixture. The resulting precipitate was filtered to give a crude product which was purified by sg chromatography with hexanes/EA 10:1 give 7,9-difluoro-6,11-dihydro-5H-benzo[a]carbazole (60) as a brown solid (746 mg, 65%). mp 200–201 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 11.86 (s, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.28 (t, J = 7.9 Hz, 1H), 7.18 (t, J = 7.4 Hz, 1H), 7.02 (dd, J = 9.5, 1.9 Hz, 1H), 6.77 (dt, J = 10.9, 1.8 Hz, 1H), 2.99 (s, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ 158.16 (dd, J = 236.4, 12.2 Hz), 155.60 (dd, J = 246.4, 15.4 Hz), 138.33 (t, J = 14.6 Hz), 135.37, 133.77 (d, J = 2.9 Hz), 128.23 (d, J = 8.1 Hz), 126.93, 126.71, 120.97, 112.29 (d, J = 19.9 Hz), 108.35, 94.50 (dd, J = 29.1, 23.5 Hz), 94.13 (dd, J = 25.7, 4.2 Hz). 28.77, 20.22. Step 2: Indole 60 (800 mg, 3.13 mmol) was dissolved in DCM/MeOH 4:1 (25 mL). The mixture was subjected to ozonolysis at −78 °C until the starting material was consumed, and then (CH₃)₂S (2 mL) was added. The solution was extracted by DCM (3 × 20 mL) and the combined organic extracts washed with water (40 mL) and brine (60 mL), and dried over MgSO₄. The solvent was removed in vacuo to give a crude residue, which was purified by sg chromatography eluting with DCM/MeOH 10:1 to afford 28 as a white solid (630 mg, 70%). mp >300 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.09 (s, 1H), 7.21 (td, J = 7.6, 1.3 Hz, 1H), 7.17 (d, J = 6.9 Hz, 1H), 7.10 (td, J = 7.5, 1.2 Hz, 1H), 7.04 (d, J = 7.4 Hz, 1H), 6.76 (dt, J = 8.3, 1.6 Hz, 1H), 6.63 (td, J = 8.8, 2.2 Hz, 1H), 3.16–3.11 (s, 1H), 3.10–3.09 (m, 1H), 3.08–3.03 (m, 1H), 3.02–2.90 (m, 1H). ¹³C NMR (150 MHz, CDCl₃) δ: 202.38, 172.31, 162.02 (dd, J = 253.5, 13.9 Hz), 158.22 (dd, J = 250.0, 14.0 Hz), 135.62, 135.32 (dd, J = 12.3, 8.7 Hz), 134.66, 129.48, 128.98, 128.26 (d, J = 22.1 Hz), 126.44, 125.25, 113.08 (dd, J = 22.3, 3.8 Hz), 103.74 (t, J = 25.4 Hz), 46.62, 30.64. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₁NO₂F₂Na 310.0656; Found 310.0653.

6,7-Dihydro-5H-benzo[g]pyrido[2,3-b]azonine-5,12-(13H)-dione (29).

A mixture 6,11-dihydro-5H-benzo[g]-pyrido[2,3-b]indole (57)³⁷ (1.0 g, 4.55 mmol) in DCM/MeOH 5:1 (120 mL) was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. Dimethyl sulfide (1.0 mL) was added and the mixture was kept cold for 1 h before warming to rt for 2 h. The solvent was then removed in vacuo to produce a crude solid that was crystallized from MeOH to afford 29 (0.52 g, 45%) as a white solid. mp 183–184 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 10.95 (s, 1H), 8.52 (d, J = 5.0 Hz, 1H), 7.92 (dd, J = 7.8, 2.0 Hz, 1H), 7.24–7.34 (m, 1H), 7.13–7.23 (m, 2H), 7.01–7.12 (m, 1H), 6.75–6.93 (m, 1H), 3.56–3.80 (m, 1H), 3.18–3.28 (m, 1H), 3.01–3.13 (m, 1H), 2.73–2.86 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 201.66, 172.28, 152.02, 149.55, 138.28, 136.61, 131.22, 130.20, 129.53, 127.04, 125.17, 122.39, 40.58, 30.79. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₅H₁₂N₂O₂Na 275.0796; Found 275.0792.

12,13-Dihydro-5H-benzo[b]pyrido[2,3-g]azonine-5,11(6H)-dione (30).

Step 1: 7,8-Dihydroquinolin-5(6H)-one (1.434 g, 9.74 mmol), phenylhydrazine (1.178 g, 10.9 mmol), p-TsOH·H₂O (1.877 g, 9.87 mmol), and diglyme (15 mL) were irradiated in a microwave oven for 20 min at 225 °C. After cooling to rt, water (50 mL) was added, and the precipitate was filtered and dried to give crude 6,11-dihydro-5H-pyrido[3,2-a]carbazole (58) as a yellow solid (2.083 g, 97%) which was used in the next step without further purification. ¹H NMR (600 MHz, DMSO-d₆) δ 11.55 (s, 1H), 8.29 (dd, J = 1.5, 4.9 Hz, 1H), 7.96 (dd, J = 1.6, 7.7 Hz, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.33 (dd, J = 5.0, 7.6 Hz, 1H), 7.13 (td, J = 1.1, 6.8 Hz, 1H), 7.03 (td, J = 0.7, 7.0 Hz, 1H), 3.16 (t, J = 7.8 Hz, 2H), 3.03 (t, J = 8.0 Hz, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ: 156.32, 145.61, 137.53, 131.18, 127.39, 126.26, 124.77, 122.12, 122.07, 119.10, 118.63, 111.37, 111.18, 31.36, 18.57. Step 2: Indole 58 (2.083 g, 9.456 mmol) was dissolved in DCM (40 mL) and MeOH (10 mL). MsOH (1.816 g, 18.9 mmol) was added, and the mixture was subjected to ozonolysis at −78 °C for 20 min and flushed with oxygen for 5 min. (CH₃)₂S (3 mL) was added, and the mixture was washed with sat. NaHCO₃ (70 mL), brine (100 mL) and dried over MgSO₄. The resulting residue was purified by sg chromatography (hexane to EA) and then crystallized from CH₃CN/Et₂O/hexane 1:10:10 (21 mL) to give 30 as yellow crystals (741 mg, 31%). mp 172–174 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.68 (s, 1H), 8.39 (d, J = 3.8 Hz, 1H), 7.38–7.29 (m, 3H), 7.20 (t, J = 7.4 Hz, 1H), 7.16 (d, J = 7.6 Hz, 1H), 6.98 (t, J = 5.5 Hz, 1H), 3.43–3.29 (m, 3H), 3.11 (s, 1H). ¹³C NMR (150 MHz, CDCl₃) δ: 204.58, 171.57, 154.82, 150.06, 141.00, 133.60, 133.18, 131.47, 130.90, 128.96, 128.21, 127.03, 121.39, 42.54, 34.18. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₅H₁₂N₂O₂Na 275.0796; Found 275.0790.

5,6-Dihydro-7H-benzo[b]pyrido[3,2-g]azonine-7,13-(12H)-dione (31).

6,11-Dihydro-5H-pyrido[2,3-a]carbazole (59)³⁸ (2.0 g, 9.08 mmol) was dissolved in a solution of DCM (40 mL) and MeOH (10 mL). The mixture was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. (CH₃)₂S (3 mL) was added, and the solvent was evaporated in vacuo to give a crude residue, which was then digested in Et₂O (30 mL), filtered, and dried to afford 31 as a tan solid (1.017 g, 44%). mp 211–213 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.75 (s, 1H), 8.25 (d, J = 4.3 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.41 (dd, J = 7.7, 17.9 Hz, 2H), 7.33 (d, J = 7.8 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.08 (dd, J = 4.8, 7.7 Hz, 1H), 3.95 (td, J = 6.4, 11.3 Hz, 1H), 3.44 (dt, J = 5.6, 14.2 Hz, 1H), 3.14–3.03 (m, 1H), 2.89 (dt, J = 6.0, 12.0 Hz, 1H). ¹³C NMR (150 MHz, CDCl₃) δ: 201.63, 171.36, 152.63, 147.77, 137.89, 136.67, 134.72, 133.06, 131.84, 129.07, 128.59, 127.57, 124.00, 40.02, 29.31. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₅H₁₂N₂O₂Na 275.0796; Found 275.0797.

9-Fluoro-11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (32).

Step 1: Conc. HCl (2.5 mL) was added to a solution of phenylhydrazine (0.54 g, 5 mmol) and 6-fluoro-3,4-dihydronaphthalen-1(2H)-one (0.80 g, 5 mmol) in EtOH (20 mL) at rt. The reaction mixture was then heated to reflux at 80 °C for 12 h. After cooling to rt, ice water (50 mL) was added, and the resulting precipitate was filtered to afford 3-fluoro-6,11-dihydro-5H-benzo[a]carbazole (54) (0.60 g, 51%) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.16 (brs, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.26–7.32 (m, 1H), 7.22 (td, J = 7.8, 1.3 Hz, 1H), 7.13–7.19 (m, 1H), 6.94–7.06 (m, 2H), 3.08 (t, J = 7.9 Hz, 2H), 3.00 (t, J = 7.9 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 161.68 (d, J = 238 Hz), 139.15 (d, J = 8.8 Hz), 136.92, 132.39, 127.41, 125.24 (d, J = 3.8 Hz), 122.39, 121.01 (d, J = 7.6 Hz), 120.02, 118.74, 115.90 (d, J = 22.7 Hz), 113.16 (d, J = 21.4 Hz), 112.00, 111.09, 29.75, 19.50. Step 2: Indole 54 (0.8 g, 3.38 mmol) in 5:1 DCM/MeOH (120 mL) was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. Dimethyl sulfide (1.0 mL) was added and the mixture was kept cold for 1 h before warming to rt for 2 h. After solvent removal in vacuo, the reaction residue was purified by crystallization from EtOH to afford 32 (0.6 g, 66%) as a white solid. mp 168–169 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.50 (brs, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.17–7.27 (m, 2H), 6.97 (s, 1H), 6.83 (dd, J = 9.3, 2.6 Hz, 1H), 6.75 (t, J = 9.1 Hz, 1H), 3.71–3.85 (m, 1H), 3.35–3.46 (m, 1H), 3.02–3.16 (m, 1H), 2.82–2.94 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 202.37, 173.18, 162.72 (d, J = 249 Hz), 138.91, 137.65, 134.94, 132.53, 131.18 (d, J = 3.8 Hz), 128.80, 128.12, 127.69, 127.49, 117.05 (d, J = 21.4 Hz), 113.92 (d, J = 22.7 Hz), 40.92, 31.10. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₂NO₂FNa 292.0750; Found 292.0750.

6,13-Dioxo-6,11,12,13-tetrahydro-5H-dibenzo[b,g]-azonine-9-carbonitrile (33).

Step 1: A mixture of phenylhydrazine (0.54 g, 5.0 mmol) and 5-oxo-5,6,7,8-tetrahydronaphthalene-2-carbonitrile (0.85 g, 5.0 mmol) was dissolved in stirring EtOH (20 mL). Conc. HCl (2.5 mL) was then added, and the reaction mixture was heated to reflux at 80 °C for 12 h. After cooling to rt, ice-cold water (50 mL) was added to give a precipitate that was filtered to afford 6,11-dihydro-5H-benzo[a]carbazol-2-carbonitrile (55) (1.1 g, 90%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.62 (s, 1H), 7.73–7.79 (m, 2H), 7.69–7.73 (m, 1H), 7.55 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 3.05 (t, J = 7.7 Hz, 2H), 2.94 (t, J = 7.7 Hz, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 138.22, 137.26, 133.93, 131.93, 131.76, 131.54, 126.74, 123.49, 121.65, 119.82, 119.57, 114.60, 112.14, 108.42, 28.80, 19.34. Step 2: Indole 55 (1.1 g, 4.51 mmol) in DCM/MeOH 5:1 (120 mL) was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. Dimethyl sulfide (1.0 mL) was added and the mixture was kept cold for 1 h before warming to rt for 2 h. After solvent removal in vacuo and crystallization from EtOH, 33 (1.1 g, 89%) was afforded as a white solid. mp 156–157 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 10.55 (s, 1H), 7.71 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H), 7.25 (d, J = 7.9 Hz, 1H), 7.23–7.15 (m, 2H), 3.42–3.55 (m, 1H), 3.11–3.22 (m, 1H), 3.02–3.11 (m, 1H), 2.82–2.93(m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 203.60, 170.74, 141.46, 139.02, 137.87, 135.31, 133.90, 132.49, 130.87, 129.27, 127.93, 127.64, 126.79, 118.57, 111.93, 41.99, 30.34. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₇H₁₂N₂O₂Na 299.0796; Found 299.0796.

9-Hydroxy-11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (34).

Step 1: A mixture of 6,11-dihydro-5H-benzo[a]carbazol-3-ol (66) (1.2 g, 5 mmol), di(tert-butyl) decarbonate (1.45 g, 6.6 mmol), and DMAP (1.2 g, 10 mmol) in DCM (20 mL) was stirred at rt for 12 h. Water (80 mL) was added, and the resulting precipitate was filtered to give tert-butyl (6,11-dihydro-5H-benzo[a]carbazol-3-yl)carbonate (69) (1.7 g, 99%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.45 (s, 1H), 7.65 (d, J = 8.2 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.16–7.07 (m, 3H), 7.06–6.97 (m, 1H), 2.98–3.04 (m, 2H), 2.93–2.87 (m, 2H), 1.51 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 151.89, 149.52, 138.05, 137.54, 132.89, 127.46, 127.09, 122.18, 122.12, 121.89, 119.96, 119.45, 118.88, 111.81, 111.14, 83.61, 29.39, 27.75, 19.52. Step 2: A mixture of 69 (1.0 g, 3.0 mmol) in 5:1 DCM/MeOH (120 mL) was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. Dimethyl sulfide (1.0 mL) was added and the mixture was kept cold for 1 h before warming to rt for 2 h. After solvent removal in vacuo, the resulting solid was purified by crystallization from EtOH to afford tert-butyl (6,13-dioxo-6,11,12,13-tetrahydro-5H-dibenzo[b,g]azonin-9-yl) carbonate (72) (0.60 g, 60%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 10.39 (s, 1H), 7.47–7.52 (m, 1H), 7.39–7.46 (m, 1H), 7.15–7.26 (m, 2H), 6.99–7.05 (m, 1H), 6.93–6.99 (m, 1H), 6.83–6.90 (m, 1H), 3.49–3.59 (m, 1H), 3.10–3.06 (m, 2H), 2.83–2.78 (m, 1H), 1.45 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 203.35, 171.90, 151.20, 138.20, 132.57, 128.92, 128.20, 127.85, 127.23, 126.89, 122.77, 121.83, 120.78, 120.46, 119.92, 83.88, 41.46 30.76, 27.68. Step 3: A mixture of 72 (0.30 g, 0.8 mmol) in 1 M HCl in THF (20 mL) was stirred at rt for 12 h. After solvent removal in vacuo, the resulting solid was purified by crystallization from aq. EtOH to afford 34 (0.16 g, 73%) as a white solid. mp 158–159 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 10.13 (br s, 1H), 9.58 (brs, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.39–7.48 (m, 1H), 7.13–7.28 (m, 2H), 6.65–6.81 (m, 1H), 6.47–6.56 (m, 1H), 6.33–6.46 (m, 1H), 3.89–4.13 (m, 1H), 3.01–3.16 (m, 1H), 2.84–2.98 (m, 1H), 2.66–2.79 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 203.31, 157.82, 137.60, 137.16, 137.06, 132.57, 128.69, 128.43, 127.87, 127.19, 126.91, 116.75, 113.67, 41.01, 31.02. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₃NO₃Na 290.0793; Found 290.0793.

10-Hydroxy-11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (35).

Step 1: A mixture of 6,11-dihydro-5H-benzo[a]carbazol-4-ol (65)³⁹ (0.5 g, 2.1 mmol), di(tert-butyl) dicarbonate (0.7 g, 3.2 mmol) and DMAP (0.6 g, 5 mmol) in DCM (20 mL) was stirred at rt for 12 h. Water (80 mL) was added, and the resulting precipitate was filtered to give tert-butyl (6,11-dihydro-5H-benzo[a]carbazol-4-yl)carbonate (68) (0.67 g, 94%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.50 (s, 1H), 7.57 (d, J = 7.0 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 6.98–7.04 (m, 2H), 2.87–2.95 (m, 2H), 2.80–2.86 (m, 2H), 1.51 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 151.50, 148.96, 137.69, 132.71, 131.17, 127.89, 126.95, 122.52, 120.72, 119.55, 119.23, 119.04, 111.90, 111.52, 83.77, 27.69, 22.38, 18.87. Step 2: A mixture of 68 (1.0 g, 3.0 mmol) in DCM/MeOH 5:1 (120 mL) was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. Dimethyl sulfide (1.0 mL) was added and the mixture was kept cold for 1 h before warming to rt for 2 h. After solvent removal in vacuo, the reaction residue was purified by crystallization from EtOH to afford tert-butyl (6,13-dioxo-6,11,12,13-tetrahydro-5H-dibenzo[b,g]azonin-10-yl)carbonate (71) (0.50 g, 45%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 10.37 (s, 1H), 7.44–7.34 (m, 2H), 7.24–7.15 (m, 2H), 7.08 (t, J = 7.8 Hz, 1H), 7.00 (d, J = 7.9 Hz, 1H), 6.86 (d, J = 7.3 Hz, 1H), 3.49–3.59 (m, 1H), 3.14–3.07 (m, 1H), 2.84–2.72 (m, 2H), 1.50 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ 204.11, 171.24, 151.38, 148.79, 139.53, 138.82, 135.40, 132.03, 129.17, 128.31, 128.24, 127.50, 127.40, 123.53, 123.02, 84.15, 41.10, 27.71, 23.88. Step 3: A mixture of 71 (0.30 g, 0.8 mmol) in 1 M HCl in THF (20 mL) was stirred at rt for 12 h. After solvent removal in vacuo, the reaction residue was purified by crystallization from aq. EtOH to afford 35 (0.12 g, 54%) as a white solid. mp >300 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 10.08 (brs, 1H), 9.63 (brs, 1H), 7.27–7.35 (m, 2H), 7.11–7.19 (m, 2H), 6.81 (t, J = 7.8 Hz, 1H), 6.60 (d, J = 8.1 Hz, 1H), 6.35 (d, J = 7.5 Hz, 1H), 3.26–3.35 (m, 1H), 3.17 (m, 1H), 2.80–2.89 (m, 1H), 2.51–2.60 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 206.08, 172.38, 154.93, 141.37, 138.59, 134.86, 131.11, 129.62, 127.68, 127.35, 126.50, 122.24, 116.18, 115.32, 42.36, 23.63. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₃NO₃Na 290.0793; Found 290.0799.

8-Fluoro-11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (36).

Step 1: Conc. HCl (5 mL) was added to a solution of phenylhydrazine (1.08 g, 10 mmol) and 7-fluoro-3,4-dihydronaphthalen-1(2H)-one (1.64 g, 10 mmol) in EtOH (20 mL) at rt. The reaction mixture was then heated to reflux at 80 °C for 12 h. After the mixture cooled to rt, ice water (50 mL) was added, and the resulting precipitate was filtered and further purified by crystallization from aq. EtOH to give 2-fluoro-6,11-dihydro-5H-benzo[a]carbazole (56) (1.3 g, 55%) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.24 (brs, 1H), 7.58 (dd, J = 7.5, 0.5 Hz, 1H), 7.42 (dd, J = 8.1, 0.8 Hz, 1H), 7.23 (td, J = 8.1, 1.3 Hz, 2H), 7.16 (td, J = 8.1, 1.2 Hz, 1H), 7.05 (dd, J = 9.3, 2.6 Hz, 1H), 6.87 (td, J = 8.5, 2.6 Hz, 1H), 3.08–2.96 (m, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 161.96 (d, J = 244 Hz), 137.15, 132.31, 131.85 (d, J = 2.5 Hz), 130.50 (d, J = 8.1 Hz), 129.60 (d, J = 8.8 Hz), 127.25, 122.88, 120.08, 119.02, 113.75, 112.77 (d, J = 21.4 Hz), 111.23, 107.00 (d, J = 22.7 Hz), 28.84, 19.78. Step 2: Indole 56 (1.2 g, 5.0 mmol) in DCM/MeOH 5:1 (120 mL) was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. Dimethyl sulfide (1.0 mL) was added and the mixture was kept cold for 1 h before warming to rt for 2 h. After solvent removal in vacuo, the reaction residue was purified by crystallization from EtOH to afford 36 (1.0 g, 74%) as a white solid. mp 205–206 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.27 (brs, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.43 (t, J = 7.2 Hz, 1H), 7.19–7.29 (m, 2H), 7.08 (dd, J = 8.5, 5.2 Hz, 1H), 6.86 (td, J = 8.6, 2.7 Hz, 1H), 6.73–6.67 (m, 1H), 3.70–3.81 (m, 1H), 3.32–3.41 (m, 1H), 3.06–3.16 (m, 1H), 2.83–2.93 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ: 202.62, 172.09, 161.04 (d, J = 249 Hz), 137.85, 136.41 (d, J = 6.6 Hz), 134.54, 132.58, 131.95 (d, J = 7.6 Hz), 131.73, 128.72, 128.31, 127.88, 116.50 (d, J = 21.0 Hz), 112.62 (d, J = 22.9 Hz), 41.26, 30.40. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₂NO₂FNa 292.0750; Found 292.0753.

8-Hydroxy-11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-dione (37).

Step 1: A mixture of 6,11-dihydro-5H-benzo[a]carbazol-2-ol (67) (0.5 g, 2.1 mmol), di(tert-butyl) dicarbonate (0.7 g, 3.2 mmol), and DMAP (0.6 g, 5 mmol) in DCM (20 mL) was stirred at rt for 12 h. Water (80 mL) was then added, and the resulting precipitate was filtered to give tert-butyl (6,11-dihydro-5H-benzo[a]carbazol-2-yl)carbonate (70) (0.56 g, 79%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.44 (s, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.45 (s, 1H), 7.38 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.96 (dd, J = 8.1, 2.6 Hz, 1H), 2.97–3.02 (m, 2H), 2.88–2.96 (m, 2H), 1.53 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ 151.93, 150.04, 137.63, 133.80, 132.78, 130.69, 129.55, 126.97, 122.51, 119.53, 119.40, 119.09, 114.46, 112.05, 111.88, 83.63, 28.85, 27.76, 19.70. Step 2: Indole 70 (0.7 g, 2.1 mmol) in 5:1 DCM/MeOH (120 mL) was subjected to ozonolysis at −78 °C for 15 min and flushed with oxygen for 5 min. Dimethyl sulfide (1.0 mL) was added and the mixture was kept cold for 1 h before warming to rt for 2 h. After solvent removal in vacuo, the reaction residue was purified by crystallization from EtOH to afford tert-butyl (6,13-dioxo-6,11,12,13-tetrahydro-5H-dibenzo[b,g]azonin-8-yl)carbonate (73) (0.40 g, 52%) as a white solid. ¹H NMR (500 MHz, DMSO-d6) δ ¹H NMR (500 MHz, DMSO-d₆) δ 10.37 (s, 1H), 7.42–7.58 (m, 1H), 7.37–7.45 (m, 1H), 7.14–7.30 (m, 3H), 6.96 (d, J = 7.9 Hz, 1H), 6.80 (s, 1H), 3.46–3.56 (m, 1H), 3.03–3.11 (m, 2H), 2.77–2.85 (m, 1H), 1.43 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 203.64, 171.18, 151.19, 149.24, 138.60, 137.88, 135.87, 133.71, 132.39, 131.41, 129.24, 127.97, 127.37, 122.06, 118.48, 83.79, 41.96, 27.66, 25.60. Step 3: Indole 73 (0.3 g, 0.8 mmol) in 1 M HCl in THF (20 mL) was stirred at rt for 12 h. After solvent removal in vacuo, the reaction solid was purified by crystallization from aq. EtOH to afford 37 (0.10 g, 45%) as a white solid. mp 119–120 °C (dec.). ¹H NMR (500 MHz, DMSO-d₆) δ 10.24 (br s, 1H), 9.36 (br s, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.38–7.46 (m, 1H), 7.13–7.26 (m, 2H), 6.89 (d, J = 8.8 Hz, 1H), 6.50 (d, J = 8.5 Hz, 1H), 6.24 (s, 1H), 3.49–3.58 (m, 1H), 2.96–3.05 (m, 1H), 2.86–2.96 (m, 1H), 2.68–2.78 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 203.54, 172.30, 155.87, 137.90, 137.63, 136.50, 132.51, 131.34, 128.92, 128.23, 127.11, 126.23, 116.10, 112.10, 41.68, 30.20. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₃NO₃Na 290.0973; Found 290.0973.

2-Fluoro-5,11-dihydro-10H-indeno[1,2-b]quinolin-10-one (38).

A mixture of 32 (0.14 mg, 0.5 mmol) and 1 N aq. NaOH (1 mL) in EtOH (5 mL) was stirred at rt for 24 h. Water (20 mL) was added, and the precipitate was filtered and dried to afford 38 as a white solid (0.10 g, 77%). mp 236–237 °C (dec.). ¹H NMR (500 MHz, DMSO-d₆) δ 12.44 (br s, 1H), 8.21 (d, J = 8.1 Hz, 1H), 8.15 (dd, J = 8.4, 5.1 Hz, 1H), 7.66–7.74 (m, 2H), 7.57 (dd, J = 8.8, 2.6 Hz, 1H), 7.33–7.45 (m, 2H), 3.80 (s, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 173.48, 163.19 (d, J = 245.0 Hz), 148.40, 147.51 (d, J = 9.0 Hz) 140.23, 132.59, 131.32, 125.38, 125.01, 122.84, 122.77, 119.51, 118.32, 114.51 (d, J = 22.5 Hz), 113.09 (d, J = 22.5 Hz), 33.00. HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₁₀FNONa 274.0644; Found 274.0650.

7,9-Difluoro-5,11-dihydro-10H-indeno[1,2-b]-quinolin-10-one (39).

A mixture of 28 (200 mg, 0.70 mmol) and 1 N aq. NaOH (1 mL) in EtOH (10 mL) was stirred at rt for 24 h. Water (20 mL) was added and the precipitate was filtered and dried to afford 39 (0.15 g, 80%) as a white solid. mp 236–237 °C dec. ⁴H NMR (500 MHz, DMSO-d₆) δ 12.53 (brs, 1H), 8.05 (dd, J = 6.6, 2.4 Hz, 1H), 7.72–7.67 (m, 1H), 7.58–7.49 (m, 2H), 7.23 (d, J = 9.6 Hz, 1H), 7.09 (t, J = 10.8 Hz, 1H), 3.72 (s, 2H). HRMS (ESI/Q-TOF) m/z: [M+Na]⁺ Calcd for C₁₆H₉F₂NONa 292.0550; Found 292.0542.

Antiprotozoal Assays.

In vitro activity data against a panel of protozoan parasites and cytotoxicity data were generated using the methods of Snyder et al.⁴⁰ and Orhan et al.⁴¹ Toxoplasma gondii assays were performed as described by Sanford et al.⁴² In vivo antimalarial activity data was generated following the methods of Ridley et al.⁴³

Physicochemical and In Vitro ADME.

LogD_7.4 values (gLogD_7.4) were estimated by correlation of compound chromatographic retention properties using gradient HPLC. The method was based on that originally published by Lombardo⁴⁴ with modifications as described previously.⁴⁵ Briefly, LogD_7.4 calibration standards (Table S3) or test compounds were prepared in DMSO (1–10 mg/mL) and diluted to 100–250 μg/mL with 50% acetonitrile in water (standards) or 50% isopropranolol in 50 mM ammonium acetate buffer, pH 7.4 (test compounds). Samples were injected onto a Waters 2795 HPLC instrument with a Phenomenex Synergi Hydro-RP column (4 μm, 30 × 2 mm) and eluted with a mobile phase consisting of aqueous buffer (50 mM ammonium acetate, pH 7.4) and acetonitrile with the acetonitrile concentration being varied from 0 to 100% using a linear gradient over 10 min. The flow rate was 0.4 mL/min and the injection volume was 5 μL. UV detection was conducted at 220 and 254 nm using a Waters 2487 dual channel UV detector. Solubility was estimated using a turbidimetric method based on that published previously.⁴⁶ Compounds in DMSO were spiked into pH 6.5 phosphate buffer (final DMSO concentration of 1%) and allowed to stand at ambient temperature for 30 min, after which they were analyzed by nephelometry to determine a solubility range. The minimum value was the last concentration for which there was no precipitation evident, and the maximum value was the concentration at which precipitation was present. In vitro intrinsic clearance was assessed over 60 min at 37 °C using human and mouse liver microsomes (Sekisui XenoTech) with a protein concentration of 0.5 mg/mL and a substrate concentration of 0.5–1 μM.⁴⁷

Stability in P. falciparum Assay Medium and Mouse Blood.

Aliquots of medium or mouse blood were spiked with test compound individually at a nominal concentration of 1 μM and mixed gently (by inverting tubes 4–5 times). Initial aliquots were immediately transferred into fresh micro centrifuge tubes and snap frozen in dry ice. The remaining volume was maintained at 37 °C/4% CO₂ (for medium) or 37 °C/7.5% CO₂ (for blood) over the 6 h incubation period to maintain the target pH. At designated time points, n = 3 aliquots were taken and snap-frozen in dry ice before transferring to −80 °C freezer for storage until analysis. On the day of analysis, samples were thawed, internal standard was added, and proteins were precipitated using a 3-fold volume excess of acetonitrile. Calibration standards were prepared in blank medium or mouse blood and processed in the same manner. Samples were analyzed by LC-MS/MS as described below.

In Vivo Pharmacokinetics in Mice.

In vivo studies were conducted using established procedures in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and the study protocols were reviewed and approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee. The pharmacokinetics of 28 were studied in non-fasted male Swiss outbred mice weighing 24.0–33.2 g. Mice had access to food and water ad libitum throughout the pre- and post-dose sampling period. Compound 28 was dosed by bolus injection into the lateral tail vein (2 mL/kg) and by oral gavage (10 mL/kg), and blood samples were collected up to 24 h (n = 3 mice per time point) with a maximum of three samples from each mouse via submandibular bleed (approximately 120 μL; conscious sampling). Blood was collected into polypropylene Eppendorf tubes containing heparin as anticoagulant and centrifuged immediately. Supernatant plasma was removed and snap-frozen in dry ice, and subsequently stored frozen (−80 °C) until analysis by LC-MS. On the day of analysis, plasma samples were thawed, spiked with internal standard (diazepam) and proteins precipitated using a 2-fold volume excess of acetonitrile. Calibration standards were similarly prepared in blank mouse plasma.

Bioanalysis.

Sample supernatant (3–7 μL) following protein precipitation was injected onto the LC-MS/MS system consisting of a Waters Xevo TQD mass spectrometer coupled to a Waters Acquity UPLC. The column was either a Supelco Ascentis Express RP C8 (50 × 2.1 mm, 2.7 μm) or a Kinetex PFP (50 × 2.1 mm, 2.6 μm), and the mobile phase consisted of 0.05% formic acid in water and 0.05% formic acid in acetonitrile delivered by gradient elution over a 2–4 min cycle time with a flow rate of 0.4–0.8 mL/min. Detection was by positive electrospray ionization for all blood and plasma samples (except for 28 and 32 in blood samples for which negative electrospray ionization was used) with multiple-reaction monitoring using the highest abundance product ion with the least interference from the matrix for quantitation. Accuracy, precision, matrix effects, lower limit of quantification, and linearity over the calibration range were confirmed.

ABBREVIATIONS

EA

ethyl acetate

EDCI

1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide

HOBt

hydroxybenzotriazole

MeOH

methanol

Sg

silica gel

TEA

triethylamine