Discovery and Evaluation of Thiazinoquinones as Anti-Protozoal Agents

Abstract

Pure compound screening has identified the dioxothiazino-quinoline-quinone ascidian metabolite ascidiathiazone A (2) to be a moderate growth inhibitor of Trypanosoma brucei rhodesiense (IC₅₀ 3.1 μM) and Plasmodium falciparum (K1 dual drug resistant strain) (IC₅₀ 3.3 μM) while exhibiting low levels of cytotoxicity (L6, IC₅₀ 167 μM). A series of C-7 amide and Δ²⁽³⁾ analogues were prepared that explored the influence of lipophilicity and oxidation state on observed anti-protozoal activity and selectivity. Little variation in anti-malarial potency was observed (IC₅₀ 0.62–6.5 μM), and no correlation was apparent between anti-malarial and anti-T. brucei activity. Phenethylamide 7e and Δ²⁽³⁾-glycine analogue 8k exhibited similar anti-Pf activity to 2 but with slightly enhanced selectivity (SI 72 and 93, respectively), while Δ²⁽³⁾-phenethylamide 8e (IC₅₀ 0.67 μM, SI 78) exhibited improved potency and selectivity towards T. brucei rhodesiense compared to the natural product hit. A second series of analogues were prepared that replaced the quinoline ring of 2 with benzofuran or benzothiophene moieties. While esters 10a/10b and 15 were once again found to exhibit cytotoxicity, carboxylic acid analogues exhibited potent anti-Pf activity (IC₅₀ 0.34–0.035 μM) combined with excellent selectivity (SI 560–4000). In vivo evaluation of a furan carboxylic acid analogue against P. berghei was undertaken, demonstrating 85.7% and 47% reductions in parasitaemia with ip or oral dosing respectively.

1. Introduction

Natural products have historically played an important role in the discovery of new treatments for malaria [1]. From quinine and artemisinin [2,3] starting points, a diverse range of anti-malarials have been developed and have been the mainstay of frontline treatment for decades. Unfortunately with time has come loss of therapeutic efficacy due to the growing prevalence of drug resistant strains [4]. In the hunt for novel scaffolds from which to develop the next generation of anti-malarials, a structurally-diverse array of natural products, including those obtained from marine organisms, have been reported to exhibit activity towards Plasmodium falciparum [5,6,7].

As part of our own continuing search for new leads for the development of treatments for neglected human diseases [8,9,10,11,12] we have screened a library of synthesized and isolated marine natural products against a panel of four parasitic protozoa: Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum K1 dual drug-resistant strain, with concurrent counter-screening for toxicity towards the non-malignant L6 rat myoblast cell line. We recently disclosed details of the first hit from this screen, the previously reported anti-inflammatory polyamine diamide ascidian metabolite orthidine F (1) [13,14,15] (Figure 1).

Figure 1. Structures of orthidine F (1), ascidiathiazone A (2) and analogues 3 and 4.

Figure 1. Structures of orthidine F (1), ascidiathiazone A (2) and analogues 3 and 4.

A second series of hits identified in this screening program were ascidiathiazone A (2), also previously reported by us as an anti-inflammatory alkaloid from a New Zealand ascidian, and synthetic analogues 3 and 4 [16]. The anti-protozoal evaluation of 2 (Table 1, entry 1) established the natural product to be a moderately potent in vitro growth inhibitor of P. falciparum K1 strain (IC₅₀ 3.3 μM) and Trypanosoma brucei rhodesiense (IC₅₀ 3.1 μM) while being effectively inactive towards T. cruzi and Leishmania donovani and exhibiting low levels of cytotoxicity against a mammalian cell-line (L6, IC₅₀ 170 μM). Similar levels of potency and selectivity were observed for ester 3 (Table 1, entry 2), while Δ²⁽³⁾ analogue 4 (Table 1, entry 3) exhibited more potent anti-malarial activity (IC₅₀ 0.6 μM) with enhanced selectivity (SI Pf 300). Herein we report the results of a preliminary structure-activity relationship study investigating the influence of C-2 amide functionalization and thiazine-Δ²⁽³⁾ oxidation on the biological activity of 2. In addition, we report that novel furan and thiophene analogues of 2 exhibit potent in vitro anti-malarial activity and that one analogue exhibits in vivo activity towards P. berghei.

Table 1. Anti-protozoal, cytotoxic activities and clogP values of 2–4, 7a–h, j, 8a–k.

Entry	Compound	IC50 (μM) a					SI Pf g	clogP h
		T. b. rhod. b	T. cruzi c	L. don. d	P. falc. K1 e	L6 f
1	2	3.1	>290	270	3.3	170	50	−1.1 ± 1.1
2	3	6.6	180	31	2.5	140	56	−0.5 ± 0.9
3	4	4.0	>290	190	0.60	180	300	−1.1 ± 1.0
4	7a	5.5	63	>280	0.94	24	26	0.3 ± 0.5
5	7b	1.8	15	29	0.62	23	37	0.8 ± 0.6
6	7c	3.9	15	48	1.1	12	10	2.3 ± 0.6
7	7d	1.9	43	21	1.1	15	14	0.6 ± 0.5
8	7e	2.4	140	160	1.5	110	72	0.9 ± 0.5
9	7f	2.4	27	47	1.4	13	10	1.4 ± 0.4
10	7g	3.4	41	83	1.6	24	15	2.1 ± 0.6
11	7h	>150	53	170	2.4	41	17	−0.6 ± 0.7
12	7j	120	250	>260	3.4	110	31	−0.9 ± 0.5
13	8a	3.7	63	>280	0.70	23	33	0.3 ± 0.8
14	8b	3.6	48	>270	1.5	4.8	3	0.8 ± 0.8
15	8c	2.4	42	53	0.98	6.5	6	2.2 ± 0.8
16	8d	4.2	160	>250	4.7	34	7	0.8 ± 0.6
17	8e	0.67	140	160	6.5	52	8	0.9 ± 0.7
18	8f	5.9	59	>240	1.2	6.5	5	1.3 ± 0.7
19	8g	2.5	42	150	1.1	4.9	4	2.1 ± 0.7
20	8h	10	150	230	1.7	50	29	−0.6 ± 0.8
21	8i	13	140	150	1.5	99	67	−1.0 ± 0.9
22	8j	35	160	220	1.8	100	57	−1.1 ± 1.0
23	8k	42	160	>280	1.2	110	93	−1.2 ± 0.7
	Melarsoprol i	0.005
	Benznidazole i		1.8
	Miltefosine i			0.53
	Chloroquine i				0.28
	Podophyllotoxin i					0.019

^a IC₅₀ values reported are the average of two independent assays. Assay protocols are described in [5]; ^b Trypanosoma brucei rhodesiense, STIB 900 strain, trypomastigotes stage; ^c Trypanosoma cruzi, Tulahuen C4 strain, amastigotes stage; ^d Leishmania donovani, MHOM-ET-67/L82 strain, amastigote/axenic stage; ^e Plasmodium falciparum, K1 strain, IEF stage; ^f L6 rat skeletal myoblast cell line; ^g Selectivity index for P. falciparum = IC₅₀ L6/IC₅₀ Pf; ^h cLogP calculated using ALOGPS 2.1, as described in [17,18]; ⁱ Melarsoprol, benznidazole, miltefosine, chloroquine and podophyllotoxin were used as positive controls.

Table 1. Anti-protozoal, cytotoxic activities and clogP values of 2–4, 7a–h, j, 8a–k.

Entry	Compound	IC50 (μM) a					SI Pf g	clogP h
		T. b. rhod. b	T. cruzi c	L. don. d	P. falc. K1 e	L6 f
1	2	3.1	>290	270	3.3	170	50	−1.1 ± 1.1
2	3	6.6	180	31	2.5	140	56	−0.5 ± 0.9
3	4	4.0	>290	190	0.60	180	300	−1.1 ± 1.0
4	7a	5.5	63	>280	0.94	24	26	0.3 ± 0.5
5	7b	1.8	15	29	0.62	23	37	0.8 ± 0.6
6	7c	3.9	15	48	1.1	12	10	2.3 ± 0.6
7	7d	1.9	43	21	1.1	15	14	0.6 ± 0.5
8	7e	2.4	140	160	1.5	110	72	0.9 ± 0.5
9	7f	2.4	27	47	1.4	13	10	1.4 ± 0.4
10	7g	3.4	41	83	1.6	24	15	2.1 ± 0.6
11	7h	>150	53	170	2.4	41	17	−0.6 ± 0.7
12	7j	120	250	>260	3.4	110	31	−0.9 ± 0.5
13	8a	3.7	63	>280	0.70	23	33	0.3 ± 0.8
14	8b	3.6	48	>270	1.5	4.8	3	0.8 ± 0.8
15	8c	2.4	42	53	0.98	6.5	6	2.2 ± 0.8
16	8d	4.2	160	>250	4.7	34	7	0.8 ± 0.6
17	8e	0.67	140	160	6.5	52	8	0.9 ± 0.7
18	8f	5.9	59	>240	1.2	6.5	5	1.3 ± 0.7
19	8g	2.5	42	150	1.1	4.9	4	2.1 ± 0.7
20	8h	10	150	230	1.7	50	29	−0.6 ± 0.8
21	8i	13	140	150	1.5	99	67	−1.0 ± 0.9
22	8j	35	160	220	1.8	100	57	−1.1 ± 1.0
23	8k	42	160	>280	1.2	110	93	−1.2 ± 0.7
	Melarsoprol i	0.005
	Benznidazole i		1.8
	Miltefosine i			0.53
	Chloroquine i				0.28
	Podophyllotoxin i					0.019

^a IC₅₀ values reported are the average of two independent assays. Assay protocols are described in [5]; ^b Trypanosoma brucei rhodesiense, STIB 900 strain, trypomastigotes stage; ^c Trypanosoma cruzi, Tulahuen C4 strain, amastigotes stage; ^d Leishmania donovani, MHOM-ET-67/L82 strain, amastigote/axenic stage; ^e Plasmodium falciparum, K1 strain, IEF stage; ^f L6 rat skeletal myoblast cell line; ^g Selectivity index for P. falciparum = IC₅₀ L6/IC₅₀ Pf; ^h cLogP calculated using ALOGPS 2.1, as described in [17,18]; ⁱ Melarsoprol, benznidazole, miltefosine, chloroquine and podophyllotoxin were used as positive controls.

2. Results and Discussion

2.1. Chemistry

We undertook a preliminary structure-activity relationship study to explore the effect of carboxylic acid functionalization and thiazine ring oxidation state towards the observed anti-protozoal activity of 2. Efforts to directly prepare amide derivatives of 2 by reaction of the synthesized natural product [16] with various amines in the presence of peptide coupling reagents, led to the formation of complex product mixtures and low yields (data not shown). Instead we made use of a longer four step reaction sequence (Scheme 1). Commercially available 8-hydroxyquinoline-2-carboxylic acid was converted to amides 5a–5j by reaction with the appropriate amine using PyBOP as the coupling agent in DMF. Subsequent oxidation using PIFA (phenyliodine bis(trifluoroacetate)) in MeCN/H₂O yielded unstable quinones 6a–6j.

Scheme 1. General reaction sequence for the preparation of analogues 7a–j and 8a–k. Reagents and conditions: (i) PIFA (2–3 equiv.), MeCN/H₂O, 0 °C, 20 min; (ii) Hypotaurine (0.8 equiv.), CeCl₃·7H₂O, MeCN/EtOH, rt, 2 days; (iii) 2 N NaOH, DMF, rt, 2 h; (iv) SOCl₂, MeOH, 0 °C then rt, then 65 °C, 2 h, 93% yield.

Scheme 1. General reaction sequence for the preparation of analogues 7a–j and 8a–k. Reagents and conditions: (i) PIFA (2–3 equiv.), MeCN/H₂O, 0 °C, 20 min; (ii) Hypotaurine (0.8 equiv.), CeCl₃·7H₂O, MeCN/EtOH, rt, 2 days; (iii) 2 N NaOH, DMF, rt, 2 h; (iv) SOCl₂, MeOH, 0 °C then rt, then 65 °C, 2 h, 93% yield.

Previous studies by ourselves [16,19] and others [20,21] have found that hypotaurine addition to quinones typically yields a mixture of regio-isomeric thiazine adducts. In the present study, we found that slow addition of a dilute solution of hypotaurine in MeCN/EtOH at room temperature afforded, after filtration and washing, analogues 7a–7j in yields of 14%, 27%, 57%, 17%, 49%, 57%, 29%, 29%, 26% and 20%, respectively. The regio-isomeric identity of the product in each case was established by 2D-NMR data, particularly HMBC experiments optimized for 8.3 and 2.0 Hz, which showed correlations from H-9 to quinone C-10 (8.3 Hz) and from NH-4 to quinone C-5 (2.0 Hz) [16]. Reaction of each of 7a–7j with 2 N NaOH in DMF for 2 h [16] afforded the desired hydrolysed and autoxidised Δ²⁽³⁾-thiazine analogues 8a–8i and 8k in variable yield (30%–83%). In the specific case of the glycine methylester 7j the product of this reaction was the Δ²⁽³⁾-thiazino carboxylic acid 8k, methylation of which (SOCl₂, MeOH, 93% yield) afforded ester 8j.

Thiophene analogues of ascidiathiazone A were prepared (Scheme 2) starting from the literature quinone 9 [22]. Reaction with hypotaurine yielded two isomeric products 10a and 10b in a ratio of 1:0.3, as determined by NMR. Despite extensive attempts using chromatography, the isomers could not be separated and so were used as a mixture in the following steps. The regio-isomeric identity of 10a and 10b could not be established, as no relevant long range ¹H-¹³C correlations were observed in HMBC data. Acid-mediated ester hydrolysis afforded carboxylic acids 11a and 11b, again characterized as an inseparable 1:0.3 mixture. HMBC data obtained for this isomeric mixture however was able to establish that the major regio-isomer was 11a as shown. Thus correlations observed between the major isomer H-8 resonance (δ_H 7.84) to quinonoid resonance δ_C 171.7 (C-9) and from the thiazine NH (δ_H 9.31) to a second quinonoid resonance δ_C 173.1 (C-5) confirmed the identity of 11a. In the case of base hydrolysis/autoxidation, reaction of the isomeric mixture 10a/10b with 1N NaOH in a biphasic reaction in EtOAc, yielded the expected Δ²⁽³⁾ product 12.

Scheme 2. Preparation of thiophene analogues 10a/10b, 11a/11b and 12. Reagents and conditions: (i) Hypotaurine (1 equiv.), CeCl₃·7H₂O (1 equiv.), MeCN/EtOH, rt, 2 days, 18% yield (10a + 10b); (ii) conc. HCl, rt, 5 h, 57% yield (11a + 11b); (iii) 1 N NaOH, EtOAc, rt, 1 h, 78% yield.

Scheme 2. Preparation of thiophene analogues 10a/10b, 11a/11b and 12. Reagents and conditions: (i) Hypotaurine (1 equiv.), CeCl₃·7H₂O (1 equiv.), MeCN/EtOH, rt, 2 days, 18% yield (10a + 10b); (ii) conc. HCl, rt, 5 h, 57% yield (11a + 11b); (iii) 1 N NaOH, EtOAc, rt, 1 h, 78% yield.

Column chromatography in this case was successful in affording the major regio-isomeric product in pure form. HMBC data analysis, in particular the observation of correlations from H-2 (δ_H 6.57) and H-8 (δ_H 7.82) to the same quinonoid carbon resonance at δ_C 175.2 (C-9) established the dioxothiazine ring regiochemistry of 12 as shown.

A series of furan analogues were prepared in analogous fashion, this time starting from commercially available 7-methoxy-benzofuran-2-carboxylic acid ethyl ester 13 (Scheme 3). Oxidation using acidified ceric ammonium sulfate afforded quinone 14 in 85% yield. Slow addition of hypotaurine to the quinone afforded a single regio-isomer 15 of the expected dioxothiazine product (43% yield). As demonstrated earlier, acidic hydrolysis of ester 15 yielded the carboxylic acid 16 (63% yield), while biphasic 1N NaOH/EtOAc hydrolysis and autoxidation afforded the Δ²⁽³⁾ carboxylic acid 17 in 47% yield.

Scheme 3. Preparation of furan analogues 15–17. Reagents and conditions: (i) (NH₄)₄Ce(SO₄)₄·2H₂O, MeCN/H₂SO4, 60 °C, 90 min, 85% yield; (ii) Hypotaurine (1 equiv.), CeCl₃·7H₂O (0.5 equiv.), MeCN/EtOH, rt, 1 days, 43% yield; (iii) conc. HCl, 100 °C, 2 h, 63% yield; (iv) 1 N NaOH, EtOAc, rt, 2 h, 47% yield.

Scheme 3. Preparation of furan analogues 15–17. Reagents and conditions: (i) (NH₄)₄Ce(SO₄)₄·2H₂O, MeCN/H₂SO4, 60 °C, 90 min, 85% yield; (ii) Hypotaurine (1 equiv.), CeCl₃·7H₂O (0.5 equiv.), MeCN/EtOH, rt, 1 days, 43% yield; (iii) conc. HCl, 100 °C, 2 h, 63% yield; (iv) 1 N NaOH, EtOAc, rt, 2 h, 47% yield.

2.2. Biological Activities

2.2.1. In Vitro Biological Evaluation

The library of target analogues were screened against a set of four protozoa and for cytotoxicity towards the rat skeletal myoblast cell line L6 and the results are summarized in Table 1 (amide and oxidized analogues of 2) and Table 2 (thiophene and ester analogues). All of the amide analogues 7a–h,j evaluated were either equipotent or slightly more active against P. falciparum than the natural product 2. A similar observation was made for activities towards T. brucei rhodesiense, except for propargyl 7h (Table 1, entry 11) and glycyl ester 7j (Table 1, entry 12) both of which were significantly less active than 2. Notable in this series, unfortunately, was the lack of selectivity with most analogues exhibiting selectivity indices (SI) of 40 or less. Of this sub-set, only phenethyl amide 7e (Table 1, entry 8) exhibited anti-protozoal activity and cytotoxic selectivity similar to those observed for 2. The corresponding Δ²⁽³⁾ analogues 8a–k while being typically equipotent or slightly more active against P. falciparum, were on the whole more cytotoxic with low selectivity. Significant amongst the series were the Δ²⁽³⁾ phenethyl amide 8e (Table 1, entry 17), which was the most active anti-T. brucei rhodesiense analogue, and ether 8i, ester 8j and carboxylic acid 8k (Table 1, entries 21–23) which maintained the anti-Pf activity of 2 but with modestly enhanced selectivity. There was no apparent correlation between calculated logP and observed biological activity (Table 1).

Table 2. Anti-protozoal and cytotoxic activities of 10a/10b, 11a/11b, 12, 15–17.

Entry	Compound	IC50 (μM) a					SI Pf g	clogP h
		T. b. rhod.b	T. cruzic	L. don. d	P. falc. K1 e	L6 f
1	10a/10b	0.39	0.51	6.3	0.028	0.52	18	0.1 ± 0.8
2	11a/11b	5.4	>290	260	0.086	230	2700	−0.2 ± 0.7
3	12	2.2	210	>290	0.035	140	4000	−0.2 ± 0.7
4	15	1.1	4.7	40	0.11	5.1	46	−0.1 ± 0.8
5	16	7.5	>300	>300	0.12	290	2400	−0.8 ± 0.7
6	17	2.7	>300	120	0.34	190	560	−0.8 ± 0.9
	Melarsoprol i	0.01
	Benznidazole i		1.4
	Miltefosine i			0.53
	Chloroquine i				0.28
	Podophyllotoxin i					0.019

^a IC₅₀ values reported are the average of two independent assays. Assay protocols are described in [5]; ^b Trypanosoma brucei rhodesiense, STIB 900 strain, trypomastigotes stage; ^c Trypanosoma cruzi, Tulahuen C4 strain, amastigotes stage; ^d Leishmania donovani, MHOM-ET-67/L82 strain, amastigote/axenic stage; ^e Plasmodium falciparum, K1 strain, IEF stage; ^f L6 rat skeletal myoblast cell line; ^g Selectivity index for P. falciparum respectively = IC₅₀ L6/IC₅₀ Pf; ^h cLogP calculated using ALOGPS 2.1, as described in [17,18]; ⁱ Melarsoprol, benznidazole, miltefosine, chloroquine and podophyllotoxin were used as positive controls.

Table 2. Anti-protozoal and cytotoxic activities of 10a/10b, 11a/11b, 12, 15–17.

Entry	Compound	IC50 (μM) a					SI Pf g	clogP h
		T. b. rhod.b	T. cruzic	L. don. d	P. falc. K1 e	L6 f
1	10a/10b	0.39	0.51	6.3	0.028	0.52	18	0.1 ± 0.8
2	11a/11b	5.4	>290	260	0.086	230	2700	−0.2 ± 0.7
3	12	2.2	210	>290	0.035	140	4000	−0.2 ± 0.7
4	15	1.1	4.7	40	0.11	5.1	46	−0.1 ± 0.8
5	16	7.5	>300	>300	0.12	290	2400	−0.8 ± 0.7
6	17	2.7	>300	120	0.34	190	560	−0.8 ± 0.9
	Melarsoprol i	0.01
	Benznidazole i		1.4
	Miltefosine i			0.53
	Chloroquine i				0.28
	Podophyllotoxin i					0.019

^a IC₅₀ values reported are the average of two independent assays. Assay protocols are described in [5]; ^b Trypanosoma brucei rhodesiense, STIB 900 strain, trypomastigotes stage; ^c Trypanosoma cruzi, Tulahuen C4 strain, amastigotes stage; ^d Leishmania donovani, MHOM-ET-67/L82 strain, amastigote/axenic stage; ^e Plasmodium falciparum, K1 strain, IEF stage; ^f L6 rat skeletal myoblast cell line; ^g Selectivity index for P. falciparum respectively = IC₅₀ L6/IC₅₀ Pf; ^h cLogP calculated using ALOGPS 2.1, as described in [17,18]; ⁱ Melarsoprol, benznidazole, miltefosine, chloroquine and podophyllotoxin were used as positive controls.

Thiophene and furan analogues 10a/10b, 11a/11b, 12, and 15–17 were evaluated against the same selection of protozoa and for cytotoxicity (Table 2). Potent anti-Pf activity was observed for the thiophene examples, with the isomerically pure carboxylic acid 12 (Table 2, entry 3) showing a desirable combination of nanomolar potency (Pf IC₅₀ 35 nM) and excellent selectivity (SI Pf 4000). The furan analogues 15–17 (Table 2, entries 4–6) were slightly less active towards P. falciparum, exhibiting IC₅₀′s in the 110–340 nm range, with carboxylic acid 16 (Table 2, entry 5) exhibiting the best selectivity (SI Pf 2400). It is interesting to note the broad-range activities of esters 10a/10b (Table 2, entry 1) and 15 (Table 2, entry 4): such pan-panel activity suggests the presence of an underlying general cytotoxic mechanism for these analogues. Once again, there was no apparent correlation between biological activity and calculated logP values.

2.2.2. In Vivo Anti-Malarial Evaluation

Furan carboxylic acid analogue 16 was selected for preliminary proof-of-principle in vivo evaluation in Plasmodium berghei infected mice. Preliminary ip acute toxicity of 16 showed no toxicity up to the highest test dose of 150 mg/kg. Using a standard test protocol [23], a repeated ip dose of 50 (mg/kg)/day for four days led to an 85.7% reduction in parasitaemia, and an increase in mean survival time from 4–6 days (untreated control) to 9.6 days. Switching to an oral dosing experiment (100 mg/kg once per day for 4 days) yielded a 47% reduction of parasitaemia. Although not considered significant, these levels of activity for both ip and po dosing clearly identifies heterocyclic dioxothiazinoquinone carboxylic acids to be a novel anti-malarial drug scaffold warranting further structure-activity relationship studies.

3. Experimental Section

3.1. General

HRMS data were acquired on a Bruker micrOTOF-QII mass spectrometer. Infrared spectra were recorded on a Perkin-Elmer Spectrum 100 Fourier-transform IR spectrometer equipped with a universal ATR accessory. Melting points were obtained on an Electrothermal melting point apparatus and are uncorrected. NMR spectra were recorded using either a Bruker Avance DRX 300 or 400 spectrometer operating at 300 MHz or 400 MHz for ¹H nuclei and 75 MHz or 100 MHz for ¹³C nuclei. Resonance assignments were made by interpretation of 2D data. NMR assignments marked by an asterisk are interchangeable. Proto-deutero solvent signals were used as internal references (DMSO-d₆: δ_H 2.50, δ_C 39.52; CDCl₃: δ_H 7.25, δ_C 77.0; CD₃OD: δ_H 3.30, δ_C 49.05). Flash column chromatography was performed using reversed-phase Merck Lichroprep RP-18, or Kieselgel 60 PF silica gel. Thin layer chromatography used 0.2 mm thick plates of Kiesegel F₂₅₄ (Merck, Manakau, New Zealand). The syntheses of 2–4 [16] and 9 [22] have been reported previously.

3.2. Synthetic Procedures

3.2.1. General Procedure for the Preparation of 8-Hydroxyquinoline-2-carboxamides 5a–5j

To a solution of 8-hydroxyquinoline-2-carboxylic acid and PyBOP (1.25 equiv.) in dry DMF (3–6 mL), amine (1–2 equiv.) and triethylamine (1.25 equiv.) were added under N₂. The reaction mixture was then stirred under N₂ at rt for 12 h, after which time the mixture was dried in vacuo. The residue was purified by reversed-phase C₁₈ flash column chromatography (0%–80% MeOH in H₂O (0.05% TFA)) and silica gel column chromatography (0%–1% MeOH in CH₂Cl₂).

3.2.1.1. N-n-Butyl-8-hydroxyquinoline-2-carboxamide (5a)

From 8-hydroxyquinoline-2-carboxylic acid (100 mg, 0.529 mmol), PyBOP (330 mg, 0.64 mmol), n-butylamine (104 µL, 1.05 mmol) and triethylamine (88 µL, 0.632 mmol) in DMF (6 mL) to give 5a as a yellow oil (108 mg, 84% yield).

R_f = 0.68 (1% MeOH/CH₂Cl₂); IR ν_max (ATR) 3291, 1650, 1539, 1502, 1465, 1159 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ_H 8.34 (1H, d, J = 8.4 Hz, H-3), 8.29 (1H, d, J = 8.4 Hz, H-4), 8.01 (1H, br s, NH-2′), 7.86 (1H, br s, OH), 7.53 (1H, t, J = 7.8 Hz, H-6), 7.40 (1H, d, J = 8.4 Hz, H-5), 7.23 (1H, d, J = 8.0 Hz, H-7), 3.53 (2H, dt, J = 7.2, 7.2 Hz, H₂-3′), 1.63 (2H, p, J = 7.3 Hz, H₂-4′), 1.40 (2H, sex., J = 7.6 Hz, H₂-5′), 0.92 (3H, t, J = 7.6 Hz, H₃-6′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 164.1 (C-1′), 152.2 (C-8), 148.2 (C-2), 137.8 (C-4), 136.1 (C-8a), 129.7 (C-4a), 129.2 (C-6), 119.9 (C-3), 118.3 (C-5), 111.2 (C-7), 39.5 (C-3′), 31.8 (C-4′), 20.2 (C-5′), 13.8 (C-6′); (+)-ESIMS m/z 245 [M + H]⁺; (+)-HRESIMS m/z 245.1287 [M + H]⁺ (calcd. for C₁₄H₁₇N₂O₂, 245.1285).

3.2.1.2. N-n-Pentyl-8-hydroxyquinoline-2-carboxamide (5b)

From 8-hydroxyquinoline-2-carboxylic acid (50 mg, 0.26 mmol), PyBOP (165 mg, 0.32 mmol), n-pentylamine (61 µL, 0.53 mmol) and triethylamine (44 µL, 0.32 mmol) in DMF (3 mL) to give 5b as a colorless oil (65 mg, 97% yield).

R_f = 0.65 (5% MeOH/CH₂Cl₂); IR ν_max (ATR) 3266, 2929, 1647, 1500 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ_H 8.46 (1H, t, J = 5.8 Hz, NH-2′), 8.33 (1H, d, J = 8.6 Hz, H-3), 8.24 (1H, d, J = 8.6 Hz, H-4), 7.49 (1H, t, J = 8.2 Hz, H-6), 7.34 (1H, d, J = 8.2 Hz, H-5), 7.20 (1H, d, J = 8.2 Hz, H-7), 3.46 (2H, dt, J = 7.4, 5.8 Hz, H₂-3′), 1.58 (2H, p, J = 7.4 Hz, H₂-4′), 1.30–1.18 (4H, m, H₂-5′/H₂-6′), 0.81 (3H, t, J = 7.4 Hz, H₃-7′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 164.4 (C-1′), 152.4 (C-8), 148.0 (C-2), 137.6 (C-4), 136.6 (C-8a), 129.7 (C-4a), 129.2 (C-6), 119.7 (C-3), 118.1 (C-5), 111.2 (C-7), 39.8 (C-3′), 29.4 (C-4′), 29.1 (C-5′), 22.3 (C-6′), 13.8 (C-7′); (+)-ESIMS m/z 281 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 281.1259 (calcd. for C₁₅H₁₈N₂NaO₂, 281.1260).

3.2.1.3. N-n-Octyl-8-hydroxyquinoline-2-carboxamide (5c)

From 8-hydroxyquinoline-2-carboxylic acid (50 mg, 0.26 mmol), PyBOP (165 mg, 0.32 mmol), n-octylamine (87 µL, 0.527 mmol) and triethylamine (44 µL, 0.32 mmol) in DMF (3 mL) to give 5c as a colorless oil (73 mg, 94% yield).

R_f = 0.80 (5% MeOH/CH₂Cl₂); IR ν_max (ATR) 3297, 2924, 1648, 1501 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ_H 8.69 (1H, t, J = 5.7 Hz, NH-2′), 8.33 (1H, d, J = 8.6 Hz, H-3), 8.22 (1H, d, J = 8.6 Hz, H-4), 7.48 (1H, t, J = 8.0 Hz, H-6), 7.32 (1H, d, J = 8.0 Hz, H-5), 7.18 (1H, d, J = 8.0 Hz, H-7), 3.45 (2H, dt, J = 7.2, 5.7 Hz, H₂-3′), 1.55 (2H, p, J = 7.2 Hz, H₂-4′), 1.27–1.09 (10H, m, H₂-5′/H₂-6′/H₂-7′/H₂-8′/H₂-9′), 0.80 (3H, t, J = 7.2 Hz, H₃-10′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 164.6 (C-1′), 152.5 (C-8), 147.8 (C-2), 137.6 (C-4), 136.6 (C-8a), 129.6 (C-4a), 129.2 (C-6), 119.6 (C-3), 118.1 (C-5), 111.2 (C-7), 39.9 (C-3′), 31.7 (C-6′*), 29.6 (C-4′), 29.2 (C-7′*), 29.1 (C-8′*), 27.0 (C-5′), 22.5 (C-9′*), 14.0 (C-10′); (+)-ESIMS m/z 323 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 323.1740 (calcd. for C₁₈H₂₄N₂NaO₂, 323.1730).

3.2.1.4. N-Benzyl-8-hydroxyquinoline-2-carboxamide (5d)

From 8-hydroxyquinoline-2-carboxylic acid (50 mg, 0.26 mmol), PyBOP (165 mg, 0.32 mmol), benzylamine (58 µL, 0.53 mmol) and triethylamine (44 µL, 0.32 mmol) in DMF (3 mL) to give 5d as a colorless oil (57 mg, 79% yield).

R_f = 0.72 (5% MeOH/CH₂Cl₂); IR ν_max (ATR) 3251, 3062, 1642, 1501 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ_H 8.90 (1H, br s, NH-2′), 8.29 (1H, d, J = 8.4 Hz, H-3), 8.20 (1H, d, J = 8.4 Hz, H-4), 7.48 (1H, t, J = 7.9 Hz, H-6), 7.33 (1H, d, J = 7.9 Hz, H-5), 7.25–7.13 (6H, m, H-7/2H-5′/2H-6′/H-7′), 4.61 (2H, br s, H₂-3′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 164.6 (C-1′), 152.4 (C-8), 147.5 (C-2), 137.9 (C-4′), 137.6 (C-4), 136.5 (C-8a), 129.7 (C-4a), 129.3 (C-6), 128.5 (C-5′), 127.7 (C-6′), 127.3 (C-7′), 119.7 (C-3), 118.1 (C-5), 111.3 (C-7), 43.6 (C-3′); (+)-ESIMS m/z 301 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 301.0949 (calcd. for C₁₇H₁₄N₂NaO₂, 301.0947).

3.2.1.5. N-Phenethyl-8-hydroxyquinoline-2-carboxamide (5e)

From 8-hydroxyquinoline-2-carboxylic acid (50 mg, 0.26 mmol), PyBOP (165 mg, 0.32 mmol), phenethylamine (66 µL, 0.53 mmol) and triethylamine (44 µL, 0.32 mmol) in DMF (3 mL) to give 5e as a colorless oil (65 mg, 86% yield).

R_f = 0.65 (5% MeOH/CH₂Cl₂); IR ν_max (ATR) 3288, 3073, 1643, 1501 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ_H 8.33 (1H, d, J = 8.5 Hz, H-3), 8.28 (1H, d, J = 8.5 Hz, H-4), 7.53 (1H, t, J = 7.8 Hz, H-6), 7.40–7.20 (7H, m, H-5/H-7/2H-6′/2H-7′/H-8′), 3.77 (2H, dt, J = 7.0, 6.8 Hz, H₂-3′), 2.96 (2H, t, J = 7.0 Hz, H₂-4′); ¹³C NMR (CDCl₃, 75 MHz) δ_C 164.1 (C-1′), 152.3 (C-8), 147.9 (C-2), 138.8 (C-5′), 137.8 (C-4), 136.5 (C-8a), 129.7 (C-4a), 129.3 (C-6), 128.8 (C-6′), 128.7 (C-7′), 126.7 (C-8′), 119.7 (C-3), 118.1 (C-5), 111.2 (C-7), 40.7 (C-3′), 35.8 (C-4′); (+)-ESIMS m/z 293 [M + H]⁺; (+)-HRESIMS m/z [M + H]⁺ 293.1292 (calcd. for C₁₈H₁₇N₂O₂, 293.1285).

3.2.1.6. N-(3-Phenylpropyl)-8-hydroxyquinoline-2-carboxamide (5f)

From 8-hydroxyquinoline-2-carboxylic acid (50 mg, 0.26 mmol), PyBOP (165 mg, 0.32 mmol), 3-phenylpropylamine (66 µL, 0.46 mmol) and triethylamine (44 µL, 0.32 mmol) in DMF (3 mL) to give 5f as a colorless oil (67 mg, 84% yield).

R_f = 0.65 (5% MeOH/CH₂Cl₂); IR ν_max (ATR) 3257, 2929, 1647, 1500 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ_H 8.64 (1H, br s, NH-2′), 8.32 (1H, d, J = 8.5 Hz, H-3), 8.20 (1H, d, J = 8.5 Hz, H-4), 7.49 (1H, t, J = 7.9 Hz, H-6), 7.33 (1H, d, J = 7.9 Hz, H-5), 7.22–7.00 (6H, m, H-7/2H-7′/2H-8′/H-9′), 3.53–3.46 (2H, m, H₂-3′), 2.57 (2H, t, J = 7.2 Hz, H₂-5′), 1.89 (2H, p, J = 7.2 Hz, H₂-4′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 164.6 (C-1′), 152.5 (C-8), 147.7 (C-2), 141.2 (C-6′), 137.6 (C-4), 136.5 (C-8a), 129.6 (C-4a), 129.2 (C-6), 128.2 (C-7′), 128.1 (C-8′), 125.8 (C-9′), 119.5 (C-3), 118.1 (C-5), 111.2 (C-7), 39.4 (C-3′), 33.2 (C-5′), 31.0 (C-4′); (+)-ESIMS m/z 329 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 329.1267 (calcd. for C₁₉H₁₈N₂NaO₂, 329.1260).

3.2.1.7. N-Geranyl-8-hydroxyquinoline-2-carboxamide (5g)

From 8-hydroxyquinoline-2-carboxylic acid (50 mg, 0.26 mmol), PyBOP (165 mg, 0.32 mmol), geranylamine (98 µL, 0.53 mmol) and triethylamine (44 µL, 0.32 mmol) in DMF (3 mL) to give 5g as a colorless oil (85 mg, 100% yield).

R_f = 0.66 (5% MeOH/CH₂Cl₂); IR ν_max (ATR) 3276, 2914, 1646, 1501 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ_H 8.38 (1H, t, J = 5.6 Hz, NH-2′), 8.33 (1H, d, J = 8.5 Hz, H-3), 8.23 (1H, d, J = 8.5 Hz, H-4), 7.49 (1H, t, J = 6.1 Hz, H-6), 7.34 (1H, dd, J = 6.1, 1.1 Hz, H-5), 7.19 (1H, dd, J = 6.1, 1.1 Hz, H-7), 5.27 (1H, t, J = 6.9 Hz, H-4′), 4.99 (1H, t, J = 6.9 Hz, H-8′), 4.12 (2H, dd, J = 6.3, 6.3 Hz, H₂-3′), 2.04–1.95 (2H, m, H₂-7′), 1.93–1.86 (2H, m, H₂-6′), 1.62 (6H, s, H₃-11′/H₃-12′), 1.53 (3H, s, H₃-10′); ¹³C NMR (CDCl₃, 75 MHz) δ_C 164.2 (C-1′), 152.4 (C-8), 148.0 (C-2), 139.7 (C-5′), 137.6 (C-4), 136.5 (C-8a), 131.6 (C-9′), 129.6 (C-4a), 129.2 (C-6), 123.7 (C-8′), 119.7 (C-4′), 119.6 (C-3), 118.1 (C-5), 111.2 (C-7), 39.4 (C-6′), 37.7 (C-3′), 26.3 (C-7′), 25.6 (C-12′), 17.6 (C-10′), 16.3 (C-11′); (+)-ESIMS m/z 325 [M + H]⁺; (+)-HRESIMS m/z [M + H]⁺ 325.1903 (calcd. for C₂₀H₂₅N₂O₂, 325.1911).

3.2.1.8. N-Propargyl-8-hydroxyquinoline-2-carboxamide (5h)

From 8-hydroxyquinoline-2-carboxylic acid (50 mg, 0.26 mmol), PyBOP (165 mg, 0.32 mmol), propargylamine (29 mg, 0.53 mmol) and triethylamine (44 µL, 0.32 mmol) in DMF (3mL) to give 5h as a colorless oil (47 mg, 80% yield).

R_f = 0.56 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3303, 3273, 1646, 1504 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 10.17 (1H, br s, OH-9), 9.99 (1H, t, J = 5.8 Hz, NH-2′), 8.49 (1H, d, J = 8.4 Hz, H-4), 8.14 (1H, d, J = 8.4 Hz, H-3), 7.56 (1H, t, J = 7.8 Hz, H-6), 7.46 (1H, d, J = 7.8 Hz, H-5), 7.18 (1H, d, J = 7.8, Hz, H-7), 4.23 (2H, dd, J = 5.8, 2.4 Hz, H₂-3′), 3.22 (1H, t, J = 2.4 Hz, H₂-5′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 163.5 (C-1′), 153.7 (C-8), 147.0 (C-2), 137.8 (C-4), 136.4 (C-8a), 129.6 (C-4a and C-6), 118.8 (C-3), 117.5 (C-5), 111.6 (C-7), 81.1 (C-4′), 73.3 (C-5′), 28.1 (C-3′); (+)-ESIMS m/z 227 [M + H]⁺; (+)-HRESIMS m/z [M + H]⁺ 227.0814 (calcd. for C₁₃H₁₁N₂O₂, 227.0815).

3.2.1.9. N-(2-Methoxyethyl)-8-hydroxyquinoline-2-carboxamide (5i)

From 8-hydroxyquinoline-2-carboxylic acid (100 mg, 0.529 mmol), PyBOP (330 mg, 0.64 mmol), 2-methoxyethylamine (92.6 µL, 1.07 mmol) and triethylamine (88 µL, 0.632 mmol) in DMF (6 mL) to give 5i as a colorless oil (106 mg, 82% yield).

R_f = 0.83 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3315, 1648, 1542, 1501, 1120 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ_H 8.68 (1H, br s, NH-2′), 8.38 (1H, br s, OH), 8.32 (1H, d, J = 8.4 Hz, H-3), 8.26 (1H, d, J = 8.4 Hz, H-4), 7.50 (1H, t, J = 8.0 Hz, H-6), 7.36 (1H, d, J = 8.4 Hz, H-5), 7.21 (1H, d, J = 7.6 Hz, H-7), 3.73 (2H, dt, J = 5.6, 5.2 Hz, H₂-3′), 3.61 (2H, t, J = 5.2 Hz, H₂-4′), 3.36 (3H, s, H₃-5′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 164.6 (C-1′), 152.6 (C-8), 147.9 (C-2), 137.6 (C-4), 136.6 (C-8a), 129.7 (C-4a), 129.3 (C-6), 119.7 (C-3), 118.1 (C-5), 111.3 (C-7), 71.3 (C-4′), 58.8 (C-5′), 39.5 (C-3′); (+)-ESIMS m/z 247 [M + H]⁺; (+)-HRESIMS m/z 247.1076 [M + H]⁺ (calcd. for C₁₃H₁₅N₂O₃, 247.1077).

3.2.1.10. N-Glycine(methylester)-8-hydroxyquinoline-2-carboxamide (5j)

From 8-hydroxyquinoline-2-carboxylic acid (100 mg, 0.529 mmol), PyBOP (330 mg, 0.64 mmol), glycine methyl ester hydrochloride (94.4 mg, 0.76 mmol) and triethylamine (220 µL, 1.58 mmol) in DMF (6 mL) to give 5j as a colorless oil (128 mg, 93% yield).

R_f = 0.86 (10% MeOH/CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz) δ_H 9.08 (1H, t, J = 6.1 Hz, NH-2′), 8.38 (1H, br s, OH), 8.03 (2H, s, H-3 and H-4), 7.43 (1H, t, J = 8.0 Hz, H-6), 7.23 (1H, dd, J = 8.4, 1.0 Hz, H-5), 7.13 (1H, dd, J = 7.6, 1.0 Hz, H-7), 4.27 (2H, d, J = 6.1 Hz, H₂-3′), 3.72 (3H, s, H₃-5′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 171.2 (C-4′), 165.0 (C-1′), 152.7 (C-8), 146.8 (C-2), 137.2 (C-4), 136.4 (C-8a), 129.6 (C-4a), 129.5 (C-6), 119.3 (C-3), 117.9 (C-5), 111.3 (C-7), 52.5 (C-5′), 41.3 (C-3′); (+)-ESIMS m/z 261 [M + H]⁺; (+)-HRESIMS m/z 261.0863 [M + H]⁺ (calcd. for C₁₃H₁₃N₂O₄, 261.0870).

3.2.2. General Procedure for Preparation of Quinones 6a–6j

A solution of PIFA (2–3 equiv.) in MeCN/H₂O (2:1 mL) was cooled to 0 °C, followed by the addition of the appropriate 8-hydroxyquinoline-2-carboxamide in CH₂Cl₂ (1 mL). The dark brown suspension was stirred for 20 min. at 0 °C before being poured into a mixture of CH₂Cl₂ (20 mL) and H₂O (30 mL). The organic phase was dried in vacuo and the crude product used in the subsequent reaction without further purification.

3.2.2.1. N-n-Butyl-5,8-dioxo-5,8-dihydroquinoline-2-carboxamide (6a)

From N-n-butyl-8-hydroxyquinoline-2-carboxamide (5a) (48 mg, 0.20 mmol), PIFA (254 mg, 0.59 mmol) to give 6a (44 mg, 85% yield) as a brown oil.

¹H NMR (CDCl₃, 400 MHz) δ_H 8.59 (1H, d, J = 8.0 Hz, H-3*), 8.56 (1H, d, J = 8.0 Hz, H-4*), 8.28 (1H, br s, NH-2′), 7.19 (1H, d, J = 10.4 Hz, H-7), 7.11 (1H, d, J = 10.4 Hz, H-6), 3.50 (2H, dt, J = 6.4, 6.4 Hz, H₂-3′), 1.64 (2H, p, J = 7.3 Hz, H₂-4′), 1.41 (2H, sex., J = 7.2 Hz, H₂-5′), 0.94 (3H, t, J = 7.6 Hz, H₃-6′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 183.8 (C-5), 182.6 (C-8), 162.4 (C-1′), 153.9 (C-2), 145.8 (C-8a), 139.3 (C-7*), 138.3 (C-6*), 136.5 (C-3*), 130.2 (C-4a), 126.2 (C-4*), 39.6 (C-3′), 31.6 (C-4′), 20.1 (C-5′), 13.8 (C-6′); (+)-ESIMS m/z 281 [M + Na]⁺; (+)-HRESIMS m/z 281.0893 [M + Na]⁺ (calcd. for C₁₄H₁₄N₂NaO₃, 281.0897).

3.2.2.2. N-n-Pentyl-5,8-dioxo-5,8-dihydroquinoline-2-carboxamide (6b)

From N-n-pentyl-8-hydroxyquinoline-2-carboxamide (5b) (38 mg, 0.15 mmol), PIFA (127 mg, 0.30 mmol) to give 6b (31 mg, 76% yield) as a brown oil.

¹H NMR (CDCl₃, 300 MHz) δ_H 8.57 (2H, s, H-3/H-4), 8.30 (1H, br s, NH-2′), 7.20 (1H, d, J = 10.3 Hz, H-7), 7.12 (1H, d, J = 10.3 Hz, H-6), 3.53–3.44 (2H, m, H₂-3′), 1.71–1.61 (2H, m, H₂-4′), 1.39–1.33 (4H, m, H₂-5′/H₂-6′), 0.90 (3H, t, J = 7.3 Hz, H₃-7′); ¹³C NMR (CDCl₃, 75 MHz) δ_C 183.7 (C-5*), 182.4 (C-8*), 162.4 (C-1′), 154.0 (C-2), 145.7 (C-8a), 139.2 (C-7), 138.2 (C-6), 136.3 (C-3*), 130.2 (C-4a), 126.1 (C-4*), 39.7 (C-3′), 29.2 (C-4′), 29.0 (C-5′), 22.2 (C-6′), 13.9 (C-7′); (+)-ESIMS m/z 295 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 295.1061 (calcd. for C₁₅H₁₆NaN₂O₃, 295.1053).

3.2.2.3. N-n-Octyl-5,8-dioxo-5,8-dihydroquinoline-2-carboxamide (6c)

From N-n-octyl-8-hydroxyquinoline-2-carboxamide (5c) (73 mg, 0.24 mmol), PIFA (127 mg, 0.30 mmol) to give 6c (64 mg, 85% yield) as a brown oil.

¹H NMR (CDCl₃, 300 MHz) δ_H 8.56 (2H, s, H-3/H-4), 8.23 (1H, br s, NH-2′), 7.18 (1H, d, J = 10.5 Hz, H-7), 7.10 (1H, d, J = 10.5 Hz, H-6), 3.48 (2H, dt, J = 6.7, 6.0 Hz, H₂-3′), 1.65 (2H, p, J = 7.2 Hz, H₂-4′), 1.42–1.20 (10H, br s, H₂-5′/H₂-6′/H₂-7′/H₂-8′/H₂-9′), 0.86 (3H, t, J = 7.1 Hz, H₃-10′); ¹³C NMR (CDCl₃, 75 MHz) δ_C 183.8 (C-5), 182.5 (C-8), 162.4 (C-1′), 154.1 (C-2), 145.0 (C-8a), 139.3 (C-7), 138.2 (C-6), 136.4 (C-3*), 130.3 (C-4a), 126.2 (C-4*), 39.9 (C-3′), 31.8 (C-6′*), 29.6 (C-4′), 29.2 (C-7′*), 29.1 (C-8′*), 27.0 (C-5′), 22.6 (C-9′*), 14.0 (C-10′); (+)-ESIMS m/z 337 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 337.1531 (calcd. for C₁₈H₂₂N₂NaO₃, 337.1523).

3.2.2.4. N-Benzyl-5,8-dioxo-5,8-dihydroquinoline-2-carboxamide (6d)

From N-benzyl-8-hydroxyquinoline-2-carboxamide (5d) (51 mg, 0.18 mmol), PIFA (127 mg, 0.30 mmol) to give 6d (44 mg, 84% yield) as a brown oil.

¹H NMR (CDCl₃, 400 MHz) δ_H 8.67 (1H, br s, NH-2′), 8.60 (1H, d, J = 8.1 Hz, H-3*), 8.55 (1H, d, J = 8.1 Hz, H-4*), 7.36–7.26 (5H, m, 2H-5′/2H-6′/H-7′), 7.14 (1H, d, J = 10.6 Hz, H-7), 7.09 (1H, d, J = 10.6 Hz, H-6), 4.69 (2H, d, J = 6.3 Hz, H₂-3′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 183.7 (C-5), 182.4 (C-8), 162.5 (C-1′), 153.8 (C-2), 145.8 (C-8a), 139.2 (C-7), 138.2 (C-6), 137.4 (C-4′), 136.4 (C-3*), 130.2 (C-4a), 128.7 (C-5′), 127.8 (C-6′), 127.5 (C-7′), 126.4 (C-4*), 43.6 (C-3′); (+)-ESIMS m/z 315 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 315.0748 (calcd. for C₁₇H₁₂N₂NaO₃, 315.0740).

3.2.2.5. N-Phenethyl-5,8-dioxo-5,8-dihydroquinoline-2-carboxamide (6e)

From N-phenethyl-8-hydroxyquinoline-2-carboxamide (5e) (58 mg, 0.20 mmol), PIFA (127 mg, 0.30 mmol) to give 6e (42 mg, 69% yield) as a brown oil.

¹H NMR (CDCl₃, 300 MHz) δ_H 8.57 (2H, s, H-3/H-4), 8.32 (1H, br s, NH-2′), 7.35–7.22 (5H, m, 2H-6′/2H-7′/H-8′), 7.17 (1H, d, J = 10.5 Hz, H-7), 7.10 (1H, d, J = 10.5 Hz, H-6), 3.75 (2H, dt, J = 7.7, 6.9 Hz, H₂-3′), 2.98 (2H, t, J = 7.7 Hz, H₂-4′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 183.7 (C-5), 182.3 (C-8), 162.4 (C-1′), 153.9 (C-2), 145.8 (C-8a), 139.3 (C-7), 138.2 (C-6), 137.4 (C-5′), 136.4 (C-3*), 130.2 (C-4a), 128.7 (C-6′*), 128.6 (C-7′*), 126.5 (C-8′), 126.1 (C-4*), 41.2 (C-3′), 35.8 (C-4′); (+)-ESIMS m/z 329 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 329.0904 (calcd. for C₁₈H₁₄N₂NaO₃, 329.0897).

3.2.2.6. N-(3-Phenpropyl)-5,8-dioxo-5,8-dihydroquinoline-2-carboxamide (6f)

From N-(3-phenylpropyl)-8-hydroxyquinoline-2-carboxamide (5f) (65 mg, 0.21 mmol), PIFA (127 mg, 0.30 mmol) to give 6f (45 mg, 67% yield) as a brown oil.

¹H NMR (CDCl₃, 400 MHz) δ_H 8.57 (2H, s, H-3/H-4), 8.34 (1H, br s, NH-2′), 7.33–7.19 (5H, m, 2H-7′/2H-8′/H-9′), 7.17 (1H, d, J = 10.4 Hz, H-7), 7.10 (1H, d, J = 10.4 Hz, H-6), 3.54 (2H, dt, J = 7.5, 6.8 Hz, H₂-3′), 2.72 (2H, t, J = 7.5 Hz, H₂-5′), 2.01 (2H, p, J = 7.5 Hz, H₂-4′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 183.7 (C-5), 182.4 (C-8), 162.4 (C-1′), 153.9 (C-2), 145.7 (C-8a), 139.2 (C-7), 138.2 (C-6), 137.4 (C-6′), 136.4 (C-3*), 130.2 (C-4a), 128.4 (C-7′), 128.3 (C-8′), 126.1 (C-9′), 125.9 (C-4*), 39.3 (C-3′), 33.2 (C-5′), 31.0 (C-4′); (+)-ESIMS m/z 343 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 343.1063 (calcd. for C₁₉H₁₆N₂NaO₃, 343.1053).

3.2.2.7. N-Geranyl-5,8-dioxo-5,8-dihydroquinoline-2-carboxamide (6g)

From N-geranyl-8-hydroxyquinoline-2-carboxamide (5g) (48 mg, 0.15 mmol), PIFA (127 mg, 0.30 mmol) to give 6g (35 mg, 69% yield) as a brown oil.

¹H NMR (CDCl₃, 400 MHz) δ_H 8.57 (1H, d, J = 8.1 Hz, H-3), 8.54 (1H, d, J = 8.1 Hz, H-4), 8.18 (1H, br s, NH-2′), 7.19 (1H, d, J = 10.5 Hz, H-7), 7.09 (1H, d, J = 10.5 Hz, H-6), 5.28 (1H, t, J = 5.6 Hz, H-4′), 5.06 (1H, t, J = 6.8 Hz, H-8′), 4.10 (2H, dd, J = 6.4, 5.6 Hz, H₂-3′), 2.11–2.05 (2H, m, H₂-7′), 2.04–1.99 (2H, m, H₂-6′), 1.72 (3H, s, H₃-11′), 1.65 (3H, s, H₃-12′), 1.58 (3H, s, H₃-10′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 183.8 (C-5), 182.5 (C-8), 162.2 (C-1′), 154.1 (C-2), 145.8 (C-8a), 140.0 (C-5′), 139.3 (C-7), 138.2 (C-6), 136.3 (C-3*), 131.7 (C-9′), 130.3 (C-4a), 126.2 (C-4*), 123.8 (C-8′), 119.4 (C-4′), 39.5 (C-6′), 37.7 (C-3′), 26.3 (C-7′), 25.6 (C-12′), 17.7 (C-10′), 16.4 (C-11′); (+)-ESIMS m/z 361 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 361.1526 (calcd. for C₂₀H₂₂N₂NaO₃, 361.1523).

3.2.2.8. N-Propargyl-5,8-dioxo-5,8-dihydroquinoline-2-carboxamide (6h)

From N-propargyl-8-hydroxyquinoline-2-carboxamide (5h) (45 mg, 0.20 mmol), PIFA (229 mg, 0.53 mmol) to give 6h (40 mg, 83% yield) as a brown oil.

¹H NMR (CDCl₃, 400 MHz) δ_H 8.57 (2H, br s, H-3/H-4), 7.19 (1H, d, J = 10.5 Hz, H-7), 7.12 (1H, d, J = 10.5 Hz, H-6), 4.30 (2H, dd, J = 5.6, 2.5 Hz, H₂-3′), 2.28 (1H, t, J = 2.5 Hz, H-5′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 183.6 (C-5), 182.4 (C-8), 162.3 (C-1′), 153.3 (C-2), 145.8 (C-8a), 139.3 (C-6*), 138.3 (C-7*), 136.5 (C-3*), 130.2 (C-4a), 126.4 (C-4*), 78.9 (C-4′), 71.8 (C-5′), 29.3 (C-3′); (+)-ESIMS m/z 263 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 263.0431 (calcd. for C₁₃H₈N₂NaO₃, 263.0427).

3.2.2.9. N-(2-Methoxyethyl)-5,8-dioxo-5,8-dihydroquinoline-2-carboxamide (6i)

From N-(2-methoxyethyl)-8-hydroxyquinoline-2-carboxamide (5i) (86 mg, 0.35 mmol), PIFA (300 mg, 0.70 mmol) to give 6i (78 mg, 86% yield) as a brown oil.

¹H NMR (CDCl₃, 400 MHz) δ_H 8.58 (2H, s, H-3/H-4), 8.49 (1H, br s, NH-2′), 7.19 (1H, d, J = 10.4 Hz, H-7), 7.10 (1H, d, J = 10.4 Hz, H-6), 3.72 (2H, dt, J = 5.6, 5.2 Hz, H₂-3′), 3.61 (2H, t, J = 5.2 Hz, H₂-4′), 3.40 (3H, s, H₃-5′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 183.8 (C-5), 182.3 (C-8), 162.7 (C-1′), 153.9 (C-2), 145.9 (C-8a), 139.3 (C-7*), 138.2 (C-6*), 136.4 (C-3*), 130.2 (C-4a), 126.2 (C-4*), 70.9 (C-4′), 58.9 (C-5′), 39.6 (C-3′); (+)-ESIMS m/z 283 [M + Na]⁺; (+)-HRESIMS m/z 283.0696 [M + Na]⁺ (calcd. for C₁₃H₁₂N₂NaO₄, 283.0689).

3.2.2.10. N-Glycine(methylester)-5,8-dioxo-5,8-dihydroquinoline-2-carboxamide (6j)

From N-glycine(methylester)-8-hydroxyquinoline-2-carboxamide (5j) (50 mg, 0.19 mmol), PIFA (132 mg, 0.31 mmol) to give 6j (40 mg, 77% yield) as a brown oil.

¹H NMR (CDCl₃, 400 MHz) δ_H 8.77 (2H, br t, J = 5.2 Hz, NH-2′), 8.59 (1H, d, J = 8.3 Hz, H-3), 8.55 (1H, d, J = 8.3 Hz, H-4), 7.20 (1H, d, J = 10.5 Hz, H-7), 7.13 (1H, d, J = 10.5 Hz, H-6), 4.31 (2H, d, J = 6.1 Hz, H₂-3′), 3.79 (3H, s, H₃-5′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 183.7 (C-5), 182.4 (C-8), 169.7 (C-4′), 163.1 (C-1′), 153.2 (C-2), 145.9 (C-8a), 139.4 (C-7), 138.3 (C-6), 136.5 (C-4), 130.2 (C-4a), 126.4 (C-3), 52.5 (C-5′), 41.4 (C-3′); (+)-ESIMS m/z 297 [M + Na]⁺; (+)-HRESIMS m/z 297.0477 [M + Na]⁺ (calcd. for C₁₃H₁₀N₂NaO₅, 297.0482).

3.2.3. General Procedure for the Preparation of Carboxamide Analogues 7a–7j

A solution of 5,8-dioxo-5,8-dihydroquinoline-2-carboxamide (6a–6j) was dissolved in MeCN/EtOH (1:1) before being cooled to 0 °C. In some cases, CeCl₃·7H₂O (1 equiv.) was also added to the reaction. Hypotaurine (0.8 equiv.) in H₂O was added dropwise over 3.5 h. The reaction mixture changed color from dark brown to dark orange, and was stirred at rt for 2 days. The product was purified either by filtration and washing with H₂O (3 × 20 mL) and MeOH (3 × 20 mL), or by reversed-phase C₁₈ flash column chromatography (0%–80% MeOH in H₂O (0.05% TFA)).

3.2.3.1. N-n-Butyl-5,10-dioxo-3,4,5,10-tetrahydro-2H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (7a)

From 6a (54 mg, 0.21 mmol) in MeCN/EtOH (1:1, 20 mL) and hypotaurine (16.0 mg, 0.15 mmol) in H₂O (3 mL). Filtration gave 7a as an orange powder (11.0 mg, 14% yield).

Mp 200 °C (decomp.); R_f = 0.49 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3300, 3237, 1682, 1594, 1580, 1508, 1336, 1280, 1170, 1107 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 9.35 (1H, br s, NH-4), 8.70 (1H, t, J = 6.4 Hz, NH-2′), 8.53 (1H, d, J = 8.2 Hz, H-9), 8.40 (1H, d, J = 8.2 Hz, H-8), 3.92–3.87 (2H, m, H₂-3), 3.43–3.36 (obscured by solvent, H₂-2 and H₂-3′), 1.55 (2H, p, J = 7.2 Hz, H₂-4′), 1.33 (2H, sex., J = 7.6 Hz, H₂-5′), 0.91 (3H, t, J = 7.6 Hz, H₃-6′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 176.2 (C-5), 173.4 (C-10), 162.5 (C-1′), 152.6 (C-7), 147.7 (C-4a), 145.3 (C-5a), 136.0 (C-9), 131.4 (C-9a), 126.5 (C-8), 110.7 (C-10a), 48.2 (C-2), 39.3 (obscured by solvent, C-3 and C-3′), 31.3 (C-4′), 19.6 (C-5′), 13.7 (C-6′); (+)-ESIMS m/z 386 [M + Na]⁺; (+)-HRESIMS m/z 386.0791 [M + Na]⁺ (calcd. for C₁₆H₁₇N₃NaO₅S, 386.0781).

3.2.3.2. N-n-Pentyl-5,10-dioxo-3,4,5,10-tetrahydro-2H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (7b)

From 6b (31 mg, 0.11 mmol), CeCl₃.7H₂O (37 mg, 98 µmol) in MeCN/EtOH (1:1, 14 mL) and hypotaurine (8.2 mg, 0.075 mmol) in H₂O (2 mL). Filtration gave 7b as a red-brown powder (11.0 mg, 27% yield).

Mp 200 °C (decomp.); R_f = 0.44 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3234, 2933, 1686, 1508 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 9.36 (1H, br s, NH-4), 8.72 (1H, t, J = 5.9 Hz, NH-2′), 8.53 (1H, d, J = 8.2 Hz, H-9), 8.40 (1H, d, J = 8.2 Hz, H-8), 3.90 (2H, br s, H₂-3), 3.43–3.33 (4H, obscured by H₂O, H₂-2, H₂-3′), 1.57 (2H, p, J = 6.8 Hz, H₂-4′), 1.34–1.27 (4H, m, H₂-5′/H₂-6′), 0.88 (3H, t, J = 6.8 Hz, H₃-7′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 176.2 (C-5), 173.5 (C-10), 162.5 (C-1′), 152.6 (C-7), 147.7 (C-4a), 145.4 (C-5a), 136.0 (C-9), 131.4 (C-9a), 126.5 (C-8), 110.7 (C-10a), 48.2 (C-2), 40.8 (C-3), 38.8 (C-3′), 28.8 (C-4′), 28.7 (C-5′*), 21.9 (C-6′*), 13.9 (C-7′); (+)-ESIMS m/z 378 [M + H]⁺; (+)-HRESIMS m/z [M + H]⁺ 378.1107 (calcd. for C₁₇H₂₀N₃O₅S, 378.1118).

3.2.3.3. N-n-Octyl-5,10-dioxo-3,4,5,10-tetrahydro-2H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (7c)

From 6c (32 mg, 0.10 mmol), CeCl₃·7H₂O (39 mg, 0.10 mmol) in MeCN/EtOH (1:1, 14 mL) and hypotaurine (9.7 mg, 0.089 mmol) in H₂O (2 mL). Filtration and solvent wash gave 7c as a red-brown powder (24.0 mg, 57% yield).

Mp 200 °C (decomp.); R_f = 0.44 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3240, 2925, 1669, 1521 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 9.36 (1H, br s, NH-4), 8.71 (1H, t, J = 6.1 Hz, NH-2′), 8.53 (1H, d, J = 8.0 Hz, H-9), 8.39 (1H, d, J = 8.0 Hz, H-8), 3.90 (2H, br s, H₂-3), 3.41 (2H, br t, J = 6.1 Hz, H₂-2), 3.32 (2H, obscured by H₂O, H₂-3′), 1.61–1.53 (2H, m, H₂-4′), 1.37–1.20 (10H, m, H₂-5′/H₂-6′/H₂-7′/H₂-8′/H₂-9′), 0.85 (3H, t, J = 6.7 Hz, H₃-10′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 176.1 (C-5), 173.4 (C-10), 162.4 (C-1′), 152.5 (C-7), 147.6 (C-4a), 145.2 (C-5a), 135.9 (C-9), 131.3 (C-9a), 126.4 (C-8), 110.6 (C-10a), 48.1 (C-2), 39.5 (C-3/C-3′), 31.1 (C-8′), 29.0 (C-4′), 28.6 (C-6′), 26.4 (C-5′), 22.0 (C-9′), 13.9 (C-10′); (+)-ESIMS m/z 420 [M + H]⁺; (+)-HRESIMS m/z [M + H]⁺ 420.1581 (calcd. for C₂₀H₂₆N₃O₅S, 420.1588).

3.2.3.4. N-Benzyl-5,10-dioxo-3,4,5,10-tetrahydro-2H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (7d)

From 6d (44 mg, 0.15 mmol), CeCl₃.7H₂O (55 mg, 0.15 mmol) in MeCN/EtOH (1:1, 14 mL) and hypotaurine (13.0 mg, 0.12 mmol) in H₂O (2 mL). Filtration and solvent wash gave 7d as a red-brown powder (10.0 mg, 17% yield).

Mp 200 °C (decomp.); R_f = 0.44 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3267, 1676, 1595, 1513 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 9.37 (1H, br s, NH-4), 9.28 (1H, t, J = 6.2 Hz, NH-2′), 8.55 (1H, d, J = 8.2 Hz, H-9), 8.43 (1H, d, J = 8.2 Hz, H-8), 7.37–7.23 (5H, m, 2H-5′/2H-6′/H-7′), 4.57 (2H, d, J = 6.4 Hz, H₂-3′), 3.90 (2H, br s, H₂-3), 3.41 (2H, t, J = 6.2 Hz, H₂-2); ¹³C NMR (DMSO-d₆, 75 MHz) δ_C 176.2 (C-5), 173.4 (C-10), 162.8 (C-1′), 152.5 (C-7), 147.7 (C-4a), 145.5 (C-5a), 139.3 (C-4′), 136.0 (C-9), 131.5 (C-9a), 128.4 (C-5′), 127.5 (C-6′), 126.9 (C-8), 126.7 (C-7′), 110.7 (C-10a), 48.2 (C-2), 42.7 (C-3′), 39.4 (C-3); (+)-ESIMS m/z 420 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 420.0618 (calcd. for C₁₉H₁₅N₃NaO₅S, 420.0625).

3.2.3.5. N-Phenethyl-5,10-dioxo-3,4,5,10-tetrahydro-2H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (7e)

From 6e (22.8 mg, 0.075 mmol), CeCl₃.7H₂O (51 mg, 0.14 mmol) in MeCN/EtOH (1:1, 14 mL) and hypotaurine (12.0 mg, 0.11 mmol) in H₂O (2 mL). Filtration and solvent wash gave 7e as a red-brown powder (15.0 mg, 49% yield).

Mp 240 °C (decomp.); R_f = 0.48 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3230, 1678, 1580, 1555 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 9.37 (1H, br s, NH-4), 8.77 (1H, t, J = 5.9 Hz, NH-2′), 8.54 (1H, d, J = 8.1 Hz, H-9), 8.40 (1H, d, J = 8.1 Hz, H-8), 7.33–7.19 (5H, m, 2H-6′/2H-7′/H-8′), 3.93–3.87 (2H, m, H₂-3), 3.60 (2H, dt, J = 7.7, 5.9 Hz, H₂-3′), 3.40 (2H, br t, J = 5.4 Hz, H₂-2), 2.90 (2H, t, J = 7.7 Hz, H₂-4′); ¹³C NMR (DMSO-d₆, 75 MHz) δ_C 176.2 (C-5), 173.4 (C-10), 162.5 (C-1′), 152.4 (C-7), 147.7 (C-4a), 145.3 (C-5a), 139.2 (C-5′), 136.0 (C-9), 131.4 (C-9a), 128.6 (C-6′), 128.4 (C-7′), 126.5 (C-8′), 126.2 (C-8), 110.7 (C-10a), 48.2 (C-2), 40.7 (C-3′), 38.6 (C-3), 35.1 (C-4′); (+)-ESIMS m/z 434 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 434.0768 (calcd. for C₂₀H₁₇N₃NaO₅S, 434.0781).

3.2.3.6. N-(3-Phenylpropyl)-5,10-dioxo-3,4,5,10-tetrahydro-2H-[1,4]thiazino[2,3-g] quinoline-7-carboxamide 1,1-Dioxide (7f)

From 6f (39.0 mg, 0.12 mmol), CeCl₃.7H₂O (51.0 mg, 0.14 mmol) in MeCN and EtOH (1:1, 14 mL) and hypotaurine (12.0 mg, 0.11 mmol) in H₂O (2 mL). Filtration and solvent wash gave 7f as a red-brown powder (29.0 mg, 57% yield).

Mp 204 °C (decomp.); R_f = 0.52 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3247, 2922, 1670, 1528 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 9.36 (1H, br s, NH-4), 8.77 (1H, t, J = 6.0 Hz, NH-2′), 8.54 (1H, d, J = 8.1 Hz, H-9), 8.40 (1H, d, J = 8.1 Hz, H-8), 7.31–7.15 (5H, m, H-7′/H-8′/H-9′), 3.90 (2H, t, J = 5.5 Hz, H₂-3), 3.44–3.35 (4H, m, H₂-2/H₂-3′), 2.64 (2H, t, J = 7.8 Hz, H₂-5′), 1.89 (2H, p, J = 7.8 Hz, H₂-4′); ¹³C NMR (DMSO-d₆, 75 MHz) δ_C 176.2 (C-5), 173.5 (C-10), 162.6 (C-1′), 152.6 (C-7), 147.7 (C-4a), 145.4 (C-5a), 141.6 (C-6′), 136.0 (C-9), 131.4 (C-9a), 128.3 (C-7′/C-8′), 126.6 (C-9′), 125.8 (C-8), 110.7 (C-10a), 48.2 (C-2), 39.1 (C-3), 38.8 (C-3′), 32.6 (C-5′), 30.8 (C-4′); (+)-ESIMS m/z 426 [M + H]⁺; (+)-HRESIMS m/z [M + H]⁺ 426.1119 (calcd. for C₂₁H₂₀N₃O₅S, 426.1118).

3.2.3.7. (E)-N-(3,7-Dimethylocta-2,6-dien-1-yl)-5,10-dioxo-3,4,5,10-tetrahydro-2H-[1,4]thiazino [2,3-g]quinoline-7-carboxamide 1,1-Dioxide (7g)

From 6g (26.6 mg, 0.079 mmol), CeCl₃.7H₂O (31 mg, 0.083 mmol) in MeCN/EtOH (1:1, 14 mL) and hypotaurine (7.2 mg, 0.066 mmol) in H₂O (2 mL). Filtration and solvent wash gave 7g as a dark orange powder (10.0 mg, 29% yield).

Mp 200 °C (decomp.); R_f = 0.45 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3230, 3076, 1693, 1561 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 9.35 (1H, br s, NH-4), 8.73 (1H, t, J = 5.7 Hz, NH-2′), 8.53 (1H, d, J = 8.1 Hz, H-9), 8.41 (1H, d, J = 8.1 Hz, H-8), 5.27 (1H, t, J = 6.2 Hz, H-4′), 5.07 (1H, t, J = 6.2 Hz, H-8′), 3.97 (2H, dd, J = 6.2, 5.7 Hz, H₂-3′), 3.90 (2H, br t, J = 5.5 Hz, H₂-3), 3.41 (2H, t, J = 5.5 Hz, H₂-2), 2.09–2.02 (2H, m, H₂-7′), 2.01–1.95 (2H, m, H₂-6′), 1.71 (3H, s, H₃-11′), 1.62 (3H, s, H₃-12′), 1.56 (3H, s, H₃-10′); ¹³C NMR (DMSO-d₆, 75 MHz) δ_C 176.2 (C-5), 173.4 (C-10), 162.3 (C-1′), 152.6 (C-7), 147.7 (C-4a), 145.3 (C-5a), 137.5 (C-5′), 136.0 (C-9), 131.4 (C-9a), 130.9 (C-9′), 126.5 (C-8), 123.9 (C-8′), 121.0 (C-4′), 110.7 (C-10a), 48.2 (C-2), 40.3 (C-3), 38.6 (C-6′), 37.1 (C-3′), 25.9 (C-7′), 25.5 (C-12′), 17.5 (C-10′), 16.1 (C-11′); (+)-ESIMS m/z 466 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 466.1395 (calcd. for C₂₂H₂₅N₃NaO₅S, 466.1407).

3.2.3.8. N-(Prop-2-yn-1-yl)-5,10-dioxo-3,4,5,10-tetrahydro-2H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (7h)

From 6h (35 mg, 0.15 mmol), CeCl₃.7H₂O (46.5 mg, 0.12 mmol) in MeCN/EtOH (1:1, 14 mL) and hypotaurine (9.5 mg, 0.087 mmol) in H₂O (2 mL). The crude reaction mixture was purified by reversed-phase C₁₈ flash column chromatography to give 7h as a bright yellow powder (15.0 mg, 29% yield).

Mp 280 °C (decomp.); R_f = 0.52 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3369, 3255, 2936, 1667, 1595 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 9.38 (1H, t, J = 5.4 Hz, NH-4), 9.09 (1H, t, J = 6.0 Hz, NH-2′), 8.55 (1H, d, J = 8.1 Hz, H-9), 8.41 (1H, d, J = 8.1 Hz, H-8), 4.13 (2H, dd, J = 6.0, 2.4 Hz, H₂-3′), 3.93–3.87 (2H, m, H₂-3), 3.40 (2H, obscured by water, H₂-2), 3.12 (1H, t, J = 2.4 Hz, H-5′); ¹³C NMR (DMSO-d₆, 75 MHz) δ_C 176.1 (C-5), 173.3 (C-10), 162.6 (C-1′), 152.0 (C-7), 147.7 (C-4a), 145.5 (C-5a), 136.0 (C-9), 131.5 (C-9a), 126.7 (C-8), 110.7 (C-10a), 80.9 (C-4′), 72.8 (C-5′), 48.2 (C-2), 39.4 (C-3), 28.7 (C-3′); (+)-ESIMS m/z 368 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 368.0294 (calcd. for C₁₅H₁₁N₃NaO₅S, 368.0312).

3.2.3.9. N-(2-Methoxyethyl)-5,10-dioxo-3,4,5,10-tetrahydro-2H-[1,4]thiazino[2,3-g] quinoline-7-carboxamide 1,1-Dioxide (7i)

From 6i (31 mg, 0.12 mmol) in MeCN/EtOH (1:1, 20 mL) and hypotaurine (7.8 mg, 0.072 mmol) in H₂O (3 mL). The crude reaction mixture was purified by reversed-phase C₁₈ flash column chromatography to give 7i as an orange powder (11.2 mg, 26% yield).

Mp 200 °C (decomp.); R_f = 0.54 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3546, 3251, 1673, 1594, 1581, 1556, 1339, 1122 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 9.37 (1H, br s, NH-4), 8.64 (1H, t, J = 5.4 Hz, NH-2′), 8.54 (1H, d, J = 8.0 Hz, H-9), 8.41 (1H, d, J = 8.0 Hz, H-8), 3.92–3.88 (2H, m, H₂-3), 3.56–3.51 (4H, m, H₂-3′ and H₂-4′), 3.43–3.39 (2H, m, H₂-2), 3.29 (3H, s, H₃-5′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 176.2 (C-5), 173.4 (C-10), 162.5 (C-1′), 152.2 (C-7), 147.7 (C-4a), 145.4 (C-5a), 136.1 (C-9), 131.4 (C-9a), 126.5 (C-8), 110.7 (C-10a), 70.3 (C-4′), 57.9 (C-5′), 48.2 (C-2), 39.2 (C-3), 38.7 (C-3′); (+)-ESIMS m/z 388 [M + Na]⁺; (+)-HRESIMS m/z 388.0566 [M + Na]⁺ (calcd. for C₁₅H₁₅N₃NaO₆S, 388.0574).

3.2.3.10. Methyl 2-(1,1-Dioxido-5,10-dioxo-3,4,5,10-tetrahydro-2H-[1,4]thiazino[2,3-g] quinoline-7-carboxamido)acetate (7j)

From 6j (50 mg, 0.18 mmol) in MeCN/EtOH (1:1, 20 mL) and hypotaurine (11.9 mg, 0.11 mmol) in H₂O (3 mL). Reversed-phase C₁₈ flash column chromatography gave 7j as a bright red powder (13.8 mg, 20% yield).

Mp 200 °C (decomp.); R_f = 0.46 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3576, 3335, 1748, 1666, 1594, 1581, 1557, 1346, 1271, 1212, 1164, 1115 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 9.40 (1H, br t, J = 3.4 Hz, NH-4), 9.08 (1H, t, J = 6.1 Hz, NH-2′), 8.56 (1H, d, J = 8.0 Hz, H-9), 8.42 (1H, d, J = 8.0 Hz, H-8), 4.15 (2H, d, J = 6.1 Hz, H-3′), 3.92–3.88 (2H, m, H₂-3), 3.67 (3H, s, H₃-5′), 3.43–3.40 (2H, m, H₂-2); ¹³C (DMSO-d₆, 100 MHz) δ_C 176.2 (C-5), 173.4 (C-10), 170.0 (C-4′), 163.0 (C-1′), 151.7 (C-7), 147.7 (C-4a), 145.6 (C-5a), 136.1 (C-9), 131.6 (C-9a), 126.6 (C-8), 110.8 (C-10a), 51.9 (C-5′), 48.2 (C-2), 41.2 (C-3′), 39.2 (C-3); (+)-ESIMS m/z 380 [M + H]⁺; (+)-HRESIMS m/z 380.0538 [M + H]⁺ (calcd. for C₁₅H₁₄N₃O₇S, 380.0547).

3.2.4. General Procedure for Preparation of Δ²⁽³⁾ Analogues 8a–8i, 8k

Thiazine-quinoline-carboxamide (7a–7j) in DMF (1–3 mL) was stirred in 2 N NaOH (3 mL) at rt for 2 h. HCl (10% vol) was added dropwise until the reaction mixture was pH 5 and the mixture was then purified by reversed-phase C₁₈ flash column chromatography (0%–10% MeOH (0.05% TFA)) to give the desired product.

3.2.4.1. N-n-Butyl-5,10-dioxo-5,10-dihydro-4H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (8a)

From 7a (34.0 mg, 0.094 mmol) using the general procedure to give 8a as a yellow solid (13.0 mg, 38% yield).

Mp 200 °C (decomp.); R_f = 0.53 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3402, 3058, 1714, 1653, 1632, 1527, 1503, 1318, 1125 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 11.42 (1H, br s, NH-4), 8.76 (1H, t, J = 6.0 Hz, NH-2′), 8.58 (1H, d, J = 8.0 Hz, H-9), 8.43 (1H, d, J = 8.0 Hz, H-8), 7.17 (1H, d, J = 9.0 Hz, H-3), 6.62 (1H, d, J = 9.0 Hz, H-2), 3.38 (2H, dt, J = 6.9, 6.9 Hz, H₂-3′), 1.56 (2H, p, J = 7.0 Hz, H₂-4′), 1.33 (2H, sex., J = 7.5 Hz, H₂-5′), 0.91 (3H, t, J = 7.5 Hz, H₃-6′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 177.7 (C-10), 175.5 (C-5), 162.4 (C-1′), 153.3 (C-7), 145.5 (C-5a), 141.4 (C-4a), 136.1 (C-9), 130.6 (C-9a), 130.5 (C-3), 126.4 (C-8), 115.2 (C-10a), 112.0 (C-2), 38.6 (C-3′), 31.3 (C-4′), 19.6 (C-5′), 13.7 (C-6′); (+)-ESIMS m/z 384 [M + Na]⁺; (+)-HRESIMS m/z 384.0632 [M + Na]⁺ (calcd. for C₁₆H₁₅N₃NaO₅S, 384.0625).

3.2.4.2. N-n-Pentyl-5,10-dioxo-5,10-dihydro-4H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (8b)

From 7b (10.0 mg, 0.027 mmol) using the general procedure to give 8b as a yellow solid (4.0 mg, 40% yield).

Mp 280 °C (decomp.); R_f = 0.44 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3319, 3057, 1710, 1635, 1528 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 11.44 (1H, br s, NH-4), 8.78 (1H, t, J = 6.1 Hz, NH-2′), 8.58 (1H, d, J = 8.1 Hz, H-9), 8.43 (1H, d, J = 8.1 Hz, H-8), 7.17 (1H, d, J = 8.9 Hz, H-3), 6.62 (1H, d, J = 8.9 Hz, H-2), 3.40–3.34 (2H, m, H₂-3′), 1.58 (2H, p, J = 7.5 Hz, H₂-4′), 1.34–1.27 (4H, m, H₂-5′/H₂-6′), 0.88 (3H, t, J = 7.5 Hz, H₃-7′); ¹³C NMR (DMSO-d₆, 75 MHz) δ_C 177.7 (C-10), 175.5 (C-5), 162.4 (C-1′), 153.3 (C-7), 145.6 (C-5a), 141.4 (C-4a), 136.1 (C-9), 130.6 (C-9a), 130.5 (C-3), 126.4 (C-8), 115.2 (C-10a), 112.0 (C-2), 38.8 (C-3′), 28.9 (C-4′), 28.7 (C-5′), 21.9 (C-6′), 14.0 (C-7′); (+)-ESIMS m/z 398 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 398.0776 (calcd. for C₁₇H₁₇N₃NaO₅S, 3798.0781).

3.2.4.3. N-n-Octyl-5,10-dioxo-5,10-dihydro-4H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (8c)

From 7c (12.0 mg, 0.029 mmol) using the general procedure to give 8c as a yellow solid (6.0 mg, 50% yield).

Mp 280 °C (decomp.); R_f = 0.41 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3289, 2924, 1635, 1527 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 11.44 (1H, d, J = 5.3 Hz, NH-4), 8.79 (1H, t, J = 5.8 Hz, NH-2′), 8.58 (1H, d, J = 8.0 Hz, H-9), 8.43 (1H, d, J = 8.0 Hz, H-8), 7.17 (1H, dd, J = 8.8, 5.3 Hz, H-3), 6.63 (1H, d, J = 8.8 Hz, H-2), 3.37 (obscured by solvent, H₂-3′), 1.60–1.52 (2H, m, H₂-4′), 1.31–1.23 (10H, m, H₂-5′/H₂-6′/H₂-7′/H₂-8′/H₂-9′), 0.85 (3H, t, J = 6.8 Hz, H₃-10′); ¹³C NMR (DMSO-d₆, 75 MHz) δ_C 177.7 (C-10), 175.5 (C-5), 162.4 (C-1′), 153.3 (C-7), 145.6 (C-5a), 141.4 (C-4a), 136.1 (C-9), 130.6 (C-9a), 130.5 (C-3), 126.4 (C-8), 115.2 (C-10a), 112.0 (C-2), 38.9 (C-3′), 31.3 (C-8′), 29.2 (C-4′), 28.8 (C-6′), 28.7 (C-7′), 26.5 (C-5′), 22.1 (C-9′), 14.0 (C-10′); (+)-ESIMS m/z 440 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 440.1232 (calcd. for C₂₀H₂₃N₃NaO₅S, 440.1251).

3.2.4.4. N-Benzyl-5,10-dioxo-5,10-dihydro-4H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (8d)

From 7d (10.0 mg, 0.025 mmol) using the general procedure to give 8d as a yellow solid (3.0 mg, 30% yield).

Mp 280 °C (decomp.); R_f = 0.47 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3213, 1706, 1634, 1513 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 11.41 (1H, d, J = 5.6 Hz, NH-4), 9.33 (1H, t, J = 6.3 Hz, NH-2′), 8.59 (1H, d, J = 8.2 Hz, H-9), 8.46 (1H, d, J = 8.2 Hz, H-8), 7.38–7.30 (4H, m, 2H-5′/2H-6′), 7.27–7.23 (1H, m, H-7′), 7.17 (1H, dd, J = 8.9, 5.6 Hz, H-3), 6.61 (1H, d, J = 8.9 Hz, H-2), 4.58 (2H, d, J = 6.3 Hz, H₂-3′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 177.6 (C-10), 175.4 (C-5), 162.7 (C-1′), 153.1 (C-7), 145.6 (C-5a), 141.3 (C-4a), 139.2 (C-4′), 136.1 (C-9), 130.7 (C-9a), 130.5 (C-3), 128.3 (C-5′), 127.5 (C-6′), 126.9 (C-8), 126.6 (C-7′), 115.2 (C-10a), 112.0 (C-2), 42.7 (C-3′); (+)-ESIMS m/z 418 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 418.0470 (calcd. for C₁₉H₁₃N₃NaO₅S, 418.0468).

3.2.4.5. N-Phenethyl-5,10-dioxo-5,10-dihydro-4H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (8e)

From 7e (11.0 mg, 0.027 mmol) using the general procedure to give 8e as a yellow solid (4.0 mg, 36% yield).

Mp 290 °C (decomp.); R_f = 0.47 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3103, 3067, 1714, 1678, 1512 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 11.45 (1H, d, J = 5.6, NH-4), 8.85 (1H, t, J = 6.1 Hz, NH-2′), 8.58 (1H, d, J = 8.1 Hz, H-9), 8.44 (1H, d, J = 8.1 Hz, H-8), 7.34–7.22 (5H, m, 2H-6′/2H-7′/H-8′), 7.17 (1H, dd, J = 8.7, 5.6 Hz, H-3), 6.62 (1H, d, J = 8.7 Hz, H-2), 3.61 (2H, dt, J = 6.9, 6.1 Hz, H₂-3′), 2.90 (2H, t, J = 6.9 Hz, H₂-4′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 177.7 (C-10), 175.5 (C-5), 162.4 (C-1′), 153.0 (C-7), 145.6 (C-5a), 141.4 (C-4a), 139.3 (C-5′), 136.2 (C-9), 130.7 (C-9a), 130.5 (C-3), 128.7 (C-6′), 128.5 (C-7′), 126.4 (C-7′), 126.2 (C-8), 115.2 (C-10a), 112.0 (C-2), 40.8 (C-3′), 35.1 (C-4′); (+)-ESIMS m/z 432 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 432.0618 (calcd. for C₂₀H₁₅N₃NaO₅S, 432.0625).

3.2.4.6. N-(3-Phenylpropyl)-5,10-dioxo-5,10-dihydro-4H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (8f)

From 7f (20.0 mg, 0.047 mmol) using the general procedure to give 8f as a yellow solid (6.0 mg, 30% yield).

Mp 230 °C (decomp.); R_f = 0.41 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3059, 2930, 1653, 1511 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 11.45 (1H, br s, NH-4), 8.86 (1H, t, J = 6.1 Hz, NH-2′), 8.58 (1H, d, J = 8.2 Hz, H-9), 8.44 (1H, d, J = 8.2 Hz, H-8), 7.31–7.15 (6H, m, H-3/2H-7′/2H-8′/H-9′), 6.63 (1H, d, J = 9.1 Hz, H-2), 3.40 (2H, dt, J = 7.5, 6.1 Hz, H₂-3′), 2.64 (2H, t, J = 7.5 Hz, H₂-5′), 1.89 (2H, p, J = 7.5 Hz, H₂-4′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 177.7 (C-10), 175.5 (C-5), 162.5 (C-1′), 153.3 (C-7), 145.6 (C-5a), 141.6 (C-6′), 141.4 (C-4a), 136.1 (C-9), 130.6 (C-9a), 130.5 (C-3), 128.3 (C-7′/C-8′), 126.5 (C-9′), 125.8 (C-8), 115.2 (C-10a), 112.0 (C-2), 38.9 (C-3′), 32.7 (C-5′), 30.8 (C-4′); (+)-ESIMS m/z 446 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 446.0790 (calcd. for C₂₁H₁₇N₃NaO₅S, 446.0781).

3.2.4.7. (E)-N-(3,7-Dimethylocta-2,6-dien-1-yl)-5,10-dioxo-5,10-dihydro-4H-[1,4]thiazino[2,3-g] quinoline-7-carboxamide 1,1-Dioxide (8g)

From 7g (10.0 mg, 0.023 mmol) using the general procedure to give 8g a yellow solid (5.0 mg, 50% yield).

Mp 280 °C (decomp.); R_f = 0.44 (10% MeOH/CH₂Cl₂); ¹H NMR (DMSO-d₆, 400 MHz) δ_H 11.44 (1H, d, J = 5.7 Hz, NH-4), 8.81 (1H, t, J = 6.2 Hz, NH-2′), 8.58 (1H, d, J = 7.8 Hz, H-9), 8.44 (1H, d, J = 7.9 Hz, H-8), 7.17 (1H, dd, J = 8.6, 5.7 Hz, H-3), 6.62 (1H, d, J = 8.6 Hz, H-2), 5.27 (1H, t, J = 6.6 Hz, H-4′), 5.07 (1H, t, J = 7.0 Hz, H-8′), 3.98 (2H, dd, J = 6.2, 5.8 Hz, H₂-3′), 2.07–2.03 (2H, m, H₂-6′), 2.01–1.96 (2H, m, H₂-7′), 1.71 (3H, s, H₃-11′), 1.62 (3H, s, H₃-12′), 1.56 (3H, s, H₃-10′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 177.6 (C-10), 175.5 (C-5), 162.2 (C-1′), 153.2 (C-7), 145.6 (C-5a), 141.4 (C-4a), 137.6 (C-5′), 136.1 (C-9), 131.0 (C-9a), 130.6 (C-9′), 130.5 (C-3), 126.4 (C-8), 123.9 (C-8′), 121.1 (C-4′), 115.2 (C-10a), 112.0 (C-2), 38.9 (C-6′), 37.1 (C-3′), 26.0 (C-7′), 25.5 (C-12′), 17.6 (C-10′), 16.2 (C-11′); (+)-ESIMS m/z 464 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 464.1254 (calcd. for C₂₂H₂₃N₃NaO₅S, 464.1251).

3.2.4.8. N-(Prop-2-yn-1-yl)-5,10-dioxo-5,10-dihydro-4H-[1,4]thiazino[2,3-g]quinoline-7-carboxamide 1,1-Dioxide (8h)

From 7h (8.0 mg, 0.023 mmol) using the general procedure to give 8h as a yellow solid (6.0 mg, 76% yield).

Mp 230 °C (decomp.); R_f = 0.46 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3310, 3058, 1636, 1509 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 11.45 (1H, d, J = 5.5 Hz, NH-4), 9.16 (1H, t, J = 6.2 Hz, NH-2′), 8.60 (1H, d, J = 8.2 Hz, H-9), 8.45 (1H, d, J = 8.2 Hz, H-8), 7.17 (1H, dd, J = 8.8, 5.5 Hz, H-3), 6.62 (1H, d, J = 8.8 Hz, H-2), 4.15 (2H, dd, J = 5.9, 2.5 Hz, H₂-3′), 3.13 (1H, t, J = 2.5 Hz, H-5′); ¹³C NMR (CDCl₃, 100 MHz) δ_C 177.6 (C-10), 175.4 (C-5), 162.5 (C-1′), 152.7 (C-7), 145.8 (C-5a), 141.4 (C-4a), 136.1 (C-9), 130.8 (C-9a), 130.5 (C-3), 126.6 (C-8), 115.3 (C-10a), 112.0 (C-2), 80.9 (C-4′), 72.9 (C-5′), 28.7 (C-3′); (+)-ESIMS m/z 366 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 366.0151 (calcd. for C₁₅H₉N₃NaO₅S, 366.0155).

3.2.4.9. N-(2-Methoxyethyl)-5,10-dioxo-5,10-dihydro-4H-[1,4]thiazino[2,3-g] quinoline-7-carboxamide 1,1-Dioxide (8i)

From 7i (18.8 mg, 0.052 mmol) using the general procedure to give 8i as a yellow solid (15.6 mg, 83% yield).

Mp 200 °C (decomp.); R_f = 0.54 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3250, 3057, 1633, 1508, 1278, 1097 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 11.44 (1H, s, NH-4), 8.70 (1H, t, J = 5.6 Hz, H-2′), 8.59 (1H, d, J = 8.0 Hz, H-9), 8.45 (1H, d, J = 8.0 Hz, H-8), 7.17 (1H, d, J = 9.0 Hz, H-3), 6.62 (1H, d, J = 9.0 Hz, H-2), 3.59–3.50 (4H, m, H₂-3′ and H₂-4′), 3.29 (3H, s, H₃-5′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 177.6 (C-10), 175.4 (C-5), 162.4 (C-1′), 152.8 (C-7), 145.5 (C-5a), 141.3 (C-4a), 136.2 (C-9), 130.7 (C-9a), 130.5 (C-3), 126.3 (C-8), 115.3 (C-10a), 112.0 (C-2), 70.2 (C-4′), 57.9 (C-5′), 38.4 (C-3′); (+)-ESIMS m/z 364 [M + H]⁺; (+)-HRESIMS m/z 364.0606 [M + H]⁺ (calcd. for C₁₅H₁₄N₃O₆S, 364.0598).

3.2.4.10. 2-(1,1-Dioxido-5,10-dioxo-5,10-dihydro-4H-[1,4]thiazino[2,3-g]quinoline-7-carboxamido)acetic Acid (8k)

From 7j (13.8 mg, 0.036 mmol) using the general procedure to give carboxylic acid 8k as a yellow oil (8.2 mg, 62% yield).

R_f = 0.20 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3582, 3250, 3057, 1748, 1634, 1508, 1279, 1127 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 11.95 (1H, br s, NH-4), 9.00 (1H, t, J = 6.0 Hz, NH-2′), 8.60 (1H, d, J = 8.0 Hz, H-9), 8.45 (1H, d, J = 8.0 Hz, H-8), 7.18 (1H, d, J = 8.8 Hz, H-3), 6.62 (1H, d, J = 8.8 Hz, H-2), 4.07 (2H, d, J = 6.0 Hz, H₂-3′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 177.6 (C-10), 175.5 (C-5), 170.9 (C-4′), 162.7 (C-1′), 152.4 (C-7), 145.7 (C-5a), 141.6 (C-4a), 136.2 (C-9), 130.8 (C-9a), 130.6 (C-3), 126.4 (C-8), 115.2 (C-10a), 112.0 (C-2), 41.3 (C-3′); (−)-ESIMS m/z 362 [M − H]⁻; (−)-HRESIMS m/z 362.0083 [M − H]⁻ (calcd. for C₁₄H₈N₃O₇S, 362.0088).

3.2.5. Methyl 2-(1,1-dioxido-5,10-dioxo-5,10-dihydro-4H-[1,4]thiazino[2,3-g] quinoline-7-carboxamido)acetate (8j)

Thionyl chloride (8.4 µL, 0.116 mmol) was added to a solution of 8k (7.0 mg, 0.019 mmol) in dry MeOH (3 mL) at 0 °C. The reaction mixture was stirred at that temperature for 20 min, then heated to 65 °C and stirred for an additional 2 h. The solution was then cooled to rt and loaded directly onto a C₁₈ reversed-phase chromatography column. The crude material was washed with two column volumes of H₂O and the product eluted with 100% MeOH (+0.05% TFA) to afford 8j as a yellow oil (6.8 mg, 93% yield).

R_f = 0.49 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3247, 3056, 1753, 1635, 1508, 1273, 1128 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 11.46 (1H, br d, J = 5.2 Hz, NH-4), 9.14 (1H, t, J = 6.2 Hz, NH-2′), 8.61 (1H, d, J = 8.0 Hz, H-9), 8.45 (1H, d, J = 8.0 Hz, H-8), 7.17 (1H, dd, J = 8.8, 5.2 Hz, H-3), 6.63 (1H, d, J = 8.8 Hz, H-2), 4.16 (2H, d, J = 6.2 Hz, H₂-3′), 3.68 (3H, s, H₃-5′); ¹³C NMR (DMSO-d₆, 100 MHz) δ_C 177.6 (C-10), 175.4 (C-5), 170.0 (C-4′), 162.9 (C-1′), 152.3 (C-7), 145.7 (C-5a), 141.4 (C-4a), 136.2 (C-9), 130.9 (C-9a), 130.4 (C-3), 126.4 (C-8), 115.3 (C-10a), 112.0 (C-2), 51.9 (C-5′), 41.3 (C-3′); (+)-ESIMS m/z 400 [M + Na]⁺; (+)-ESIMS m/z 400.0222 [M + Na]⁺ (calcd. for C₁₅H₁₁N₃NaO₇S, 400.0210).

3.2.6. Methyl 5,9-Dioxo-3,4,5,9-tetrahydro-2H-thieno[2′,3′:4,5]benzo[1,2-b][1,4]thiazine-7-carboxylate 1,1-Dioxide (10a) and Methyl 5,9-Dioxo-2,3,5,9-tetrahydro-1H-thieno[3′,2′:4,5]benzo [1,2-b][1,4]thiazine-7-carboxylate 4,4-Dioxide (10b)

A solution of methyl 4,7-dioxo-4,7-dihydrobenzo[b]thiophene-2-carboxylate (9) [22] (74 mg, 0.33 mmol) and CeCl₃·7H₂O (124 mg, 0.33 mmol) in MeCN (10 mL) and EtOH (10 mL) was cooled to 0 °C. Hypotaurine (36 mg, 0.33 mmol) in H₂O (2 mL) was added dropwise to the mixture leading to a color change from yellow to orange. The reaction was stirred at rt for 2 days. The residue was filtered and washed with H₂O (3 × 20 mL) and MeOH (3 × 20 mL), to give a mixture of regioisomers (10a/10b, 1:0.3 ratio determined by NMR)) as an orange solid (20 mg, 18% yield).

Mp 280 °C (decomp.); R_f = 0.36 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3222, 3003, 1726, 1683, 1579 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 9.36 (1H, br s, NH), 8.03 (1H, s, H-8 minor isomer), 7.93 (1H, s, H-8), 3.91 (3H, s, H₃-2′), 3.89 (3H, s, H₃-2′ minor isomer), 3.88–3.84 (2H, m, H₂-3), 3.37 (2H, obscured by water, H₂-2); ¹³C NMR (DMSO-d₆, 75 MHz) 10a δ_C 173.1 (C-5), 171.5 (C-9), 160.8 (C-1′), 148.0 (C-4a), 142.6 (C-5a*), 141.8 (C-8a*), 141.1 (C-7), 130.2 (C-8), 109.6 (C-9a), 53.3 (C-2′), 48.1 (C-2), 39.2 (C-3); (+)-FABMS m/z 328 [M + H]⁺; (+)-HRFABMS m/z [M + H]⁺ 327.9950 (calcd. for C₁₂H₁₀NO₆S₂, 327.9950).

3.2.7. 5,9-Dioxo-3,4,5,9-tetrahydro-2H-thieno[2′,3′:4,5]benzo[1,2-b][1,4]thiazine-7-carboxylic Acid 1,1-Dioxide (11a) and 5,9-Dioxo-2,3,5,9-tetrahydro-1H-thieno[3′,2′:4,5]benzo[1,2-b][1,4] thiazine-7-carboxylic Acid 4,4-Dioxide (11b)

Methyl ester (as a mixture of regioisomers) 10a/10b (20.0 mg, 0.061 mmol) was dissolved in conc. HCl (3 mL), and stirred at rt for 5 h, after which time, the mixture was heated to 100 °C and stirred for a further 2 h. The crude reaction mixture was subjected to reversed-phase C₁₈ column chromatography (0%–10% MeOH/H₂O (0.05% TFA)) to give 11a/11b as a mixture of regioisomers (1:0.3, 11.0 mg, 57% yield) as a bright orange solid.

Mp 200 °C (decomp.); R_f = 0.25 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3357, 3230, 1674, 1577; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 9.42 (br s, NH minor isomer), 9.31 (1H, br s, NH), 7.93 (s, H-8 minor isomer), (7.84 (1H, s, H-8), 3.86 (2H, br s, H₂-3), 3.40–3.34 (2H, br m, H₂-2); ¹³C NMR (DMSO-d₆, 75 MHz) 11a δ_C 173.1 (C-5), 171.7 (C-9), 161.8 (C-1′), 147.9 (C-4a), 144.2 (C-7), 142.8 (C-8a), 141.2 (C-5a), 129.6 (C-8), 109.6 (C-9a), 48.1 (C-2), 39.2 (C-3); (+)-ESIMS m/z 336 [M + Na]⁺; (+)-HRESIMS m/z [M + Na]⁺ 335.9605 (calcd. for C₁₁H₇NNaO₆S₂, 335.9607).

3.2.8. 5,9-Dioxo-5,9-dihydro-4H-thieno[2′,3′:4,5]benzo[1,2-b][1,4]thiazine-7-carboxylic Acid 1,1-Dioxide (12)

Thiophene methyl ester (10a/10b) (20.0 mg, 0.061 mmol) was dissolved in hot EtOAc (2 mL), followed by the addition of 1 N NaOH (1 mL). The biphasic mixture was stirred at rt for 1.5 h. HCl (10% vol) was added dropwise until the reaction mixture turned acidic. The crude mixture was subjected to reversed-phase C₁₈ column chromatography (0%–10% MeOH/H₂O (0.05% TFA)) to give 12 (single regio-isomer) (15 mg, 78% yield) as a bright orange solid.

Mp 290 °C (decomp.); R_f = 0.27 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3227, 3068, 1689, 1637, 1510; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 11.41 (1H, br s, NH), 7.82 (1H, s, H-8), 7.13 (1H, d, J = 8.9 Hz, H-3), 6.57 (1H, d, J = 8.9 Hz, H-2); ¹³C NMR (DMSO-d₆, 75 MHz) δ_C 175.2 (C-9), 172.5 (C-5), 161.7 (C-1′), 147.4 (C-7), 141.5 (C-5a*), 141.4 (C-4a*), 141.2 (C-8a*), 130.3 (C-3), 128.1 (C-8), 114.5 (C-9a), 112.1 (C-2); (+)-FABMS m/z 312 [M + H]⁺; (+)-HRFABMS m/z [M + H]⁺ 311.9642 (calcd. for C₁₁H₆NO₆S₂, 311.9637).

3.2.9. Methyl 4,7-Dihydroxybenzo[b]thiophene-2-carboxylate (14)

Commercially available 7-methoxy-benzofuran-2-carboxylic acid ethyl ester (13) (105 mg, 0.477 mmol) in MeCN/4 N H₂SO₄ (20 mL/5 mL) was stirred at rt, before addition of (NH₄)₄Ce(SO₄)₄·2H₂O (1.80 g, 3.02 mol) in 4 N H₂SO₄ (25 mL). The reaction mixture was heated to 60 °C for 90 min. changing the colour from orange to yellow as well as inducing the formation of a white precipitate. The reaction was cooled, filtered, and the filtrate was extracted repeatedly with CH₂Cl₂ (5 × 50 mL). The combined organic phases were then dried (MgSO₄) and the solvent removed in vacuo to give 14 as a yellow solid (89 mg, 85% yield). The product was used immediately in the next step without further purification.

IR ν_max (ATR) 3570, 2955, 1752, 1726, 1534, 1475, 1367, 1187, 1160, 1139 cm⁻¹; ¹H NMR (DMSO-d₆, 75 MHz) δ_H 7.49 (1H, s, H-3), 6.82 (2H, s, H-5/H-6), 4.45 (2H, q, J = 7.2 Hz, H₂-3′), 1.42 (3H, t, J = 7.2 Hz, H₃-4′); EIMS m/z 220 [M]⁺; (+)-HREIMS m/z [M]⁺ 220.0369 (calcd. for C₁₁H₈O₅, 220.0372).

3.2.10. Ethyl 5,9-Dioxo-3,4,5,9-tetrahydro-2H-benzofuro[5,6-b][1,4]thiazine-7-carboxylate 1,1-Dioxide (15)

A solution of quinone 14 (100 mg, 0.45 mmol) and CeCl₃.7H₂O (78 mg, 0.21 mmol) in MeCN (10 mL) and EtOH (10 mL) was cooled to 0 °C. Hypotaurine (49 mg, 0.45 mmol) in H₂O (1 mL) was added dropwise to the reaction mixture, changing the colour from yellow to orange. The reaction was stirred at rt for 24 h. The residue was filtered and washed with H₂O (3 × 20 mL) and MeOH (3 × 20 mL), to give 15 (62 mg, 43% yield) as a red solid.

Mp 277 °C; R_f = 0.36 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3225, 1733, 1695, 1566 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 9.38 (1H, br s, NH), 7.58 (1H, s, H-8), 4.37 (2H, q, J = 7.1 Hz, H₂-3′), 3.85 (2H, dt, J = 5.7, 5.7 Hz, H₂-3), 3.34 (2H, t, J = 5.7 Hz, H₂-2), 1.33 (3H, t, J = 7.1 Hz, H-4′); ¹³C NMR (DMSO-d₆, 75 MHz) δ_C 172.2 (C-9), 167.8 (C-5), 157.1 (C-1′), 149.2 (C-5a), 148.7 (C-7), 147.4 (C-4a), 130.1 (C-8a), 114.2 (C-8), 108.9 (C-9a), 62.0 (C-3′), 48.1 (C-2), 39.2 (C-3), 14.0 (C-4′); (+)-FABMS m/z 326 [M + H]⁺; (+)-HRFABMS m/z [M + H]⁺ 326.0341 (calcd. for C₁₃H₁₂NO₇S, 326.0335).

3.2.11. 5,9-Dioxo-3,4,5,9-tetrahydro-2H-benzofuro[5,6-b][1,4]thiazine-7-carboxylic Acid 1,1-Dioxide (16)

Ethyl ester 15 (64 mg, 0.20 mmol) was dissolved in conc. HCl (3 mL), and the mixture was heated to 100 °C and stirred for 2 h. The crude reaction mixture was purified by reversed-phase C₁₈ column chromatography (0%–10% MeOH/H₂O (0.05% TFA)), to give 16 (37 mg, 63% yield) as a bright red solid.

Mp 210 °C (decomp.); R_f = 0.23 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3234, 3093, 1635, 1694, 1561 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ_H 9.35 (1H, br s, NH-4), 7.47 (1H, s, H-8), 3.87–3.82 (2H, m, H₂-3), 3.37–3.31 (2H, m, H₂-2); ¹³C NMR (DMSO-d₆, 75 MHz) δ_C 172.5 (C-9), 167.8 (C-5), 158.5 (C-1′), 150.3 (C-5a*), 149.0 (C-7*), 147.4 (C-4a), 130.3 (C-8a), 113.5 (C-8), 108.9 (C-9a), 48.1 (C-2), 39.5 (C-3); (+)-ESIMS m/z 298 [M + H]⁺; (+)-HRESIMS m/z [M + H]⁺ 298.0009 (calcd. for C₁₁H₈NO₇S, 298.0016).

3.2.12. 5,9-Dioxo-5,9-dihydro-4H-benzofuro[5,6-b][1,4]thiazine-7-carboxylic Acid 1,1-Dioxide (17)

Ethyl ester 15 (15.0 mg, 0.046 mmol) was dissolved in hot EtOAc (2 mL), followed by the addition of 1 N NaOH (1 mL). The biphasic mixture was stirred at rt for 2 h. HCl (10% vol) was added dropwise until the reaction mixture turned acidic. The crude product was purified by reversed-phase C₁₈ column chromatography (0%–10% MeOH/H₂O (0.05% TFA)), to give 17 (6.4 mg, 47% yield) as a red solid.

Mp 280 °C (decomp.); R_f = 0.36 (10% MeOH/CH₂Cl₂); IR ν_max (ATR) 3223, 3072, 1677, 1577, 1516 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ_H 11.42 (1H, br s, NH-4), 7.47 (1H, s, H-8), 7.12 (1H, d, J = 8.8 Hz, H-3), 6.58 (1H, d, J = 8.8 Hz, H-2); ¹³C NMR (DMSO-d₆, 75 MHz) δ_C 176.1 (C-9), 167.4 (C-5), 158.5 (C-1′), 152.0 (C-7), 149.2 (C-5a), 140.4 (C-4a), 130.2 (C-3), 129.1 (C-8a), 113.9 (C-9a), 112.3 (C-2 and C-8); (+)-FABMS m/z 296 [M + H]⁺; (+)-HRFABMS m/z [M + H]⁺ 295.9861 (calcd. for C₁₁H₆NO₇S, 295.9865).

3.3. Biological Assays

3.3.1. In Vitro Anti-Protozoal Activity

The in vitro activities against the protozoan parasites T.b. rhodesiense, T. cruzi, L. donovani, and P. falciparum and cytotoxicity assessment against L6 cells were determined as reported elsewhere [5]. The following strains, parasite forms and positive controls were used: T.b. rhodesiense, STIB900, trypomastigote forms, melarsoprol, IC₅₀ of 0.01 μM (4 ng/mL); T. cruzi, Tulahuen C2C4, amastigote forms in L6 rat myoblasts, benznidazole, IC₅₀ of 1.4 μM (0.352 μg/mL); L. donovani, MHOM/ET/67/L82, axenic amastigote forms, miltefosine, IC₅₀ of 0.5 μM (0.213 μg/mL); P. falciparum, K1 (chloroquine and pyrimethamine resistant), erythrocytic stages, chloroquine, IC₅₀ of 0.20 μM (0.065 μg/mL) and L6 cells, rat skeletal myoblasts, podophyllotoxin, IC₅₀ of 0.01 μM (0.004 μg/mL).

3.3.2. In Vivo Anti-Malarial Efficacy Studies

In vivo anti-malarial activity was assessed as previously described [23]. Groups of three female NMRI mice (20–22 g) were intravenously infected with 2 × 10⁷ parasitized erythrocytes on day 0 with GFP-transfected P. berghei strain ANKA [24]. Compounds were formulated in 100% DMSO, diluted 10-fold in distilled water and administered intraperitoneally in a volume of 10 ml kg⁻¹ on four consecutive days (4, 24, 48 and 72 h post infection). Control experiments used DMSO-H₂O vehicle alone. Parasitemia was determined on day 4 post infection (24 h after last treatment) by FACS analysis. Activity was calculated as the difference between the mean per cent parasitaemia for the control (n = 5 mice) and treated groups expressed as a per cent relative to the control group. The survival of the animals was usually monitored up to 30 days: a compound was considered curative if the animal survived to day 30 after infection with no detectable parasites. In vivo efficacy studies in mice were conducted according to the rules and regulations for the protection of animal rights (“Tierschutzverordnung”) of the Swiss “Bundesamt für Veterinärwesen”. They were approved by the veterinary office of Canton Basel-Stadt, Switzerland.

4. Conclusions

The dioxothiazinoquinone marine natural product ascidiathiazone A (2) has been identified as a moderate in vitro growth inhibitor of Trypanosoma brucei rhodesiense and Plasmodium falciparum. A series of C-7 amide and Δ²⁽³⁾ analogues were prepared that explored the influence of lipophilicity and oxidation state on observed anti-protozoal activity and selectivity. Little variation in anti-malarial potency was observed (IC₅₀ 0.62–6.5 μM), and no correlation was apparent between anti-malarial and anti-T. brucei activity. Changing the quinoline-based structure of 2 to incorporate benzofuran or benzothiophene moieties yielded particularly potent anti-malarials. The finding of ip and oral dosing anti-malarial activity for benzofuran carboxylic acid 16 is highly encouraging, suggesting that future studies should be directed at exploring this novel antiprotozoal pharmacophore.