Discovery and Evaluation of Thiazinoquinones as Anti-Protozoal Agents

Article abstract of DOI:10.3390/md11093472

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 A²⁽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.

Full text of DOI:10.3390/md11093472

Mar. Drugs 2013, 11, 3472-3499; doi:10.3390/md11093472
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Discovery and Evaluation of Thiazinoquinones as
Anti-Protozoal Agents
Cary F. C. Lam 1, A. Norrie Pearce 1, Shen H. Tan 1,†, Marcel Kaiser 2,3 and Brent R. Copp 1,*
1
School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New
Zealand; E-Mails: cf.lam@auckland.ac.nz (C.F.C.L.); n.pearce@auckland.ac.nz (A.N.P.);
shen.tan@rsc.anu.edu.au (S.H.T.)
2
Swiss Tropical and Public Health Institute, Socinstrasse 57, PO Box, Basel CH-4002, Switzerland;
E-Mail: marcel.kaiser@unibas.ch
3
University of Basel, Basel CH-4003, Switzerland
†
Current address: Research School of Chemistry, Institute of Advanced Studies, The Australian
National University, Canberra ACT 0200, Australia.
* Author to whom correspondence should be addressed; E-Mail: b.copp@auckland.ac.nz;
Tel.: +64-9-923-8284; Fax: +64-9-373-7422.
Received: 12 August 2013; in revised form: 20 August 2013 / Accepted: 30 August 2013 /
Published: 9 September 2013
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

Mar. Drugs 2013, 11
evaluation of a furan carboxylic acid analogue against P. berghei was undertaken,
3473
demonstrating 85.7% and 47% reductions in parasitaemia with ip or oral dosing respectively.
Keywords: marine natural products; protozoa; malaria; Plasmodium falciparum;
Trypanosoma brucei rhodesiense; quinone; dioxothiazine; alkaloid
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 [5–7].
As part of our own continuing search for new leads for the development of treatments for neglected
human diseases [8–12] 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) [13–15] (Figure 1).
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

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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
L. don. d
270
SI Pf g
clogP h
T. b. rhod. b
3.1
T. cruzi c
>290
180
>290
63
P. falc. K1 e
3.3
L6 f
170
140
180
24
1
2
2
50
56
300
26
37
10
14
72
10
15
17
31
33
3
−1.1 ± 1.1
−0.5 ± 0.9
−1.1 ± 1.0
0.3 ± 0.5
0.8 ± 0.6
2.3 ± 0.6
0.6 ± 0.5
0.9 ± 0.5
1.4 ± 0.4
2.1 ± 0.6
−0.6 ± 0.7
−0.9 ± 0.5
0.3 ± 0.8
0.8 ± 0.8
2.2 ± 0.8
0.8 ± 0.6
0.9 ± 0.7
1.3 ± 0.7
2.1 ± 0.7
−0.6 ± 0.8
−1.0 ± 0.9
−1.1 ± 1.0
−1.2 ± 0.7
3
6.6
31
2.5
3
4
4.0
190
0.60
0.94
0.62
1.1
4
7a
5.5
>280
29
5
7b
1.8
15
23
6
7c
3.9
15
48
12
7
7d
1.9
43
21
1.1
15
8
7e
2.4
140
27
160
1.5
110
13
9
7f
2.4
47
1.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
7g
3.4
41
83
1.6
24
7h
>150
120
3.7
53
170
2.4
41
7j
250
63
>260
>280
>270
53
3.4
110
23
8a
0.70
1.5
8b
3.6
48
4.8
6.5
34
8c
2.4
42
0.98
4.7
6
8d
4.2
160
140
59
>250
160
7
8e
0.67
5.9
6.5
52
8
8f
>240
150
1.2
6.5
4.9
50
5
8g
8h
2.5
42
1.1
4
10
150
140
160
160
230
1.7
29
67
57
93
8i
13
150
1.5
99
8j
35
220
1.8
100
110
8k
42
>280
1.2
Melarsoprol i
Benznidazole i
Miltefosine i
Chloroquine i
Podophyllotoxin i
0.005
1.8
0.53
0.28
0.019
a
b
IC50 values reported are the average of two independent assays. Assay protocols are described in [5]; Trypanosoma brucei
c
d
rhodesiense, STIB 900 strain, trypomastigotes stage; Trypanosoma cruzi, Tulahuen C4 strain, amastigotes stage; Leishmania
donovani, MHOM-ET-67/L82 strain, amastigote/axenic stage; e Plasmodium falciparum, K1 strain, IEF stage; f L6 rat skeletal myoblast
g
h
cell line; Selectivity index for P. falciparum = IC50 L6/IC50 Pf; cLogP calculated using ALOGPS 2.1, as described in [17,18];
i Melarsoprol, benznidazole, miltefosine, chloroquine and podophyllotoxin were used as positive controls.

Mar. Drugs 2013, 11
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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.
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

Mar. Drugs 2013, 11
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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
1
10a and 10b could not be established, as no relevant long range H-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 (δH 7.84) to quinonoid resonance δC 171.7 (C-9) and from the
thiazine NH (δH 9.31) to a second quinonoid resonance δC 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.
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 (δH 6.57) and
H-8 (δH 7.82) to the same quinonoid carbon resonance at δC 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

Mar. Drugs 2013, 11
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(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.
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).

Mar. Drugs 2013, 11
Table 2. Anti-protozoal and cytotoxic activities of 10a/10b, 11a/11b, 12, 15–17.
3478
Entry
Compound
IC50 (μM) a
L. don. d
6.3
SI Pf g
clogP h
T. b. rhod. b
0.39
5.4
T. cruzi c
0.51
P. falc. K1 e
0.028
0.086
0.035
0.11
L6 f
0.52
230
140
5.1
1
2
3
4
5
6
10a/10b
11a/11b
18
0.1 ± 0.8
−0.2 ± 0.7
−0.2 ± 0.7
−0.1 ± 0.8
−0.8 ± 0.7
−0.8 ± 0.9
>290
210
260
2700
4000
46
12
2.2
>290
40
15
1.1
4.7
16
7.5
>300
>300
>300
120
0.12
290
190
2400
560
17
2.7
0.34
Melarsoprol i
Benznidazole i
Miltefosine i
Chloroquine i
Podophyllotoxin i
0.01
1.4
0.53
0.28
0.019
a
b
IC50 values reported are the average of two independent assays. Assay protocols are described in [5]; Trypanosoma brucei
c
d
rhodesiense, STIB 900 strain, trypomastigotes stage; Trypanosoma cruzi, Tulahuen C4 strain, amastigotes stage; Leishmania
donovani, MHOM-ET-67/L82 strain, amastigote/axenic stage; e Plasmodium falciparum, K1 strain, IEF stage; f L6 rat skeletal myoblast
g
h
cell line; Selectivity index for P. falciparum respectively = IC50 L6/IC50 Pf; 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.

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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:
δH 2.50, δC 39.52; CDCl3: δH 7.25, δC 77.0; CD3OD: δH 3.30, δC 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).
Rf = 0.68 (1% MeOH/CH2Cl2); IR νmax (ATR) 3291, 1650, 1539, 1502, 1465, 1159 cm−1; 1H NMR
(CDCl3, 400 MHz) δH 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) δC 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).

Mar. Drugs 2013, 11
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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).
1
Rf = 0.65 (5% MeOH/CH2Cl2); IR νmax (ATR) 3266, 2929, 1647, 1500 cm−1; H NMR (CDCl3,
400 MHz) δH 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
13
(3H, t, J = 7.4 Hz, H3-7′); C NMR (CDCl3, 100 MHz) δC 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).
1
Rf = 0.80 (5% MeOH/CH2Cl2); IR νmax (ATR) 3297, 2924, 1648, 1501 cm−1; H NMR (CDCl3,
400 MHz) δH 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′/
13
H2-8′/H2-9′), 0.80 (3H, t, J = 7.2 Hz, H3-10′); C NMR (CDCl3, 100 MHz) δC 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).
1
Rf = 0.72 (5% MeOH/CH2Cl2); IR νmax (ATR) 3251, 3062, 1642, 1501 cm−1; H NMR (CDCl3,
400 MHz) δH 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) δC 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).

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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).
1
Rf = 0.65 (5% MeOH/CH2Cl2); IR νmax (ATR) 3288, 3073, 1643, 1501 cm−1; H NMR (CDCl3,
300 MHz) δH 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) δC 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).
1
Rf = 0.65 (5% MeOH/CH2Cl2); IR νmax (ATR) 3257, 2929, 1647, 1500 cm−1; H NMR (CDCl3,
400 MHz) δH 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′),
13
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′); C NMR
(CDCl3, 100 MHz) δC 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).
1
Rf = 0.66 (5% MeOH/CH2Cl2); IR νmax (ATR) 3276, 2914, 1646, 1501 cm−1; H NMR (CDCl3,
300 MHz) δH 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) δC 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).

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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).
Rf = 0.56 (10% MeOH/CH2Cl2); IR νmax (ATR) 3303, 3273, 1646, 1504 cm−1; 1H NMR (DMSO-d6,
400 MHz) δH 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,
13
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′); C NMR
(DMSO-d6, 100 MHz) δC 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).
1
Rf = 0.83 (10% MeOH/CH2Cl2); IR νmax (ATR) 3315, 1648, 1542, 1501, 1120 cm−1; H NMR
(CDCl3, 400 MHz) δH 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) δC 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).
1
Rf = 0.86 (10% MeOH/CH2Cl2); H NMR (CDCl3, 400 MHz) δH 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) δC 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).

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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) δH 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,
13
J = 7.6 Hz, H3-6′); C NMR (CDCl3, 100 MHz) δC 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) δH 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′),
13
1.39–1.33 (4H, m, H2-5′/H2-6′), 0.90 (3H, t, J = 7.3 Hz, H3-7′); C NMR (CDCl3, 75 MHz) δC 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) δH 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) δC 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).

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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) δH 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) δC 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) δH 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,
13
6.9 Hz, H2-3′), 2.98 (2H, t, J = 7.7 Hz, H2-4′); C NMR (CDCl3, 100 MHz) δC 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) δH 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,
13
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′); C NMR (CDCl3,
100 MHz) δC 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) δH 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

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(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,
13
H3-10′); C NMR (CDCl3, 100 MHz) δC 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) δH 8.57 (2H, br s, H-3/H-4), 7.19 (1H, d, J = 10.5 Hz, H-7), 7.12 (1H,
13
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′); C NMR
(CDCl3, 100 MHz) δC 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) δH 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) δC 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) δH 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,
13
d, J = 6.1 Hz, H2-3′), 3.79 (3H, s, H3-5′); C NMR (CDCl3, 100 MHz) δC 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).

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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.); Rf = 0.49 (10% MeOH/CH2Cl2); IR νmax (ATR) 3300, 3237, 1682, 1594,
1
1580, 1508, 1336, 1280, 1170, 1107 cm−1; H NMR (DMSO-d6, 400 MHz) δH 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) δC 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.); Rf = 0.44 (10% MeOH/CH2Cl2); IR νmax (ATR) 3234, 2933, 1686,
1508 cm−1; 1H NMR (DMSO-d6, 400 MHz) δH 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) δC 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).

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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.); Rf = 0.44 (10% MeOH/CH2Cl2); IR νmax (ATR) 3240, 2925, 1669,
1521 cm−1; 1H NMR (DMSO-d6, 400 MHz) δH 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,
13
H2-5′/H2-6′/H2-7′/H2-8′/H2-9′), 0.85 (3H, t, J = 6.7 Hz, H3-10′); C NMR (DMSO-d6, 100 MHz) δC
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.); Rf = 0.44 (10% MeOH/CH2Cl2); IR νmax (ATR) 3267, 1676, 1595,
1513 cm−1; 1H NMR (DMSO-d6, 300 MHz) δH 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) δC 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.); Rf = 0.48 (10% MeOH/CH2Cl2); IR νmax (ATR) 3230, 1678, 1580,
1555 cm−1; 1H NMR (DMSO-d6, 300 MHz) δH 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) δC 176.2 (C-5), 173.4 (C-10), 162.5 (C-1′),

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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.); Rf = 0.52 (10% MeOH/CH2Cl2); IR νmax (ATR) 3247, 2922, 1670,
1528 cm−1; 1H NMR (DMSO-d6, 300 MHz) δH 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,
13
J = 7.8 Hz, H2-4′); C NMR (DMSO-d6, 75 MHz) δC 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.); Rf = 0.45 (10% MeOH/CH2Cl2); IR νmax (ATR) 3230, 3076, 1693,
1561 cm−1; 1H NMR (DMSO-d6, 300 MHz) δH 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,
13
s, H3-12′), 1.56 (3H, s, H3-10′); C NMR (DMSO-d6, 75 MHz) δC 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

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reversed-phase C18 flash column chromatography to give 7h as a bright yellow powder (15.0 mg,
29% yield).
Mp 280 °C (decomp.); Rf = 0.52 (10% MeOH/CH2Cl2); IR νmax (ATR) 3369, 3255, 2936, 1667,
1595 cm−1; 1H NMR (DMSO-d6, 300 MHz) δH 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) δC 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.); Rf = 0.54 (10% MeOH/CH2Cl2); IR νmax (ATR) 3546, 3251, 1673, 1594,
1
1581, 1556, 1339, 1122 cm−1; H NMR (DMSO-d6, 400 MHz) δH 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,
13
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′); C NMR
(DMSO-d6, 100 MHz) δC 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.); Rf = 0.46 (10% MeOH/CH2Cl2); IR νmax (ATR) 3576, 3335, 1748, 1666,
1
1594, 1581, 1557, 1346, 1271, 1212, 1164, 1115 cm−1; H NMR (DMSO-d6, 400 MHz) δH 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) δC 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).

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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.); Rf = 0.53 (10% MeOH/CH2Cl2); IR νmax (ATR) 3402, 3058, 1714, 1653,
1
1632, 1527, 1503, 1318, 1125 cm−1; H NMR (DMSO-d6, 400 MHz) δH 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,
13
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′); C NMR
(DMSO-d6, 100 MHz) δC 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.); Rf = 0.44 (10% MeOH/CH2Cl2); IR νmax (ATR) 3319, 3057, 1710, 1635,
1528 cm−1; 1H NMR (DMSO-d6, 300 MHz) δH 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,
13
H2-5′/H2-6′), 0.88 (3H, t, J = 7.5 Hz, H3-7′); C NMR (DMSO-d6, 75 MHz) δC 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).

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Mp 280 °C (decomp.); Rf = 0.41 (10% MeOH/CH2Cl2); IR νmax (ATR) 3289, 2924, 1635, 1527
1
cm−1; H NMR (DMSO-d6, 300 MHz) δH 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
13
(10H, m, H2-5′/H2-6′/H2-7′/H2-8′/H2-9′), 0.85 (3H, t, J = 6.8 Hz, H3-10′); C NMR (DMSO-d6, 75
MHz) δC 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.); Rf = 0.47 (10% MeOH/CH2Cl2); IR νmax (ATR) 3213, 1706, 1634,
1
1513 cm−1; H NMR (DMSO-d6, 400 MHz) δH 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,
13
H-2), 4.58 (2H, d, J = 6.3 Hz, H2-3′); C NMR (DMSO-d6, 100 MHz) δC 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.); Rf = 0.47 (10% MeOH/CH2Cl2); IR νmax (ATR) 3103, 3067, 1714, 1678,
1
1512 cm−1; H NMR (DMSO-d6, 300 MHz) δH 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) δC 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).

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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.); Rf = 0.41 (10% MeOH/CH2Cl2); IR νmax (ATR) 3059, 2930, 1653,
1511 cm−1; 1H NMR (DMSO-d6, 300 MHz) δH 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) δC 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).
1
Mp 280 °C (decomp.); Rf = 0.44 (10% MeOH/CH2Cl2); H NMR (DMSO-d6, 400 MHz) δH 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) δC 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.); Rf = 0.46 (10% MeOH/CH2Cl2); IR νmax (ATR) 3310, 3058, 1636,
1
1509 cm−1; H NMR (DMSO-d6, 300 MHz) δH 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) δC 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

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(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.); Rf = 0.54 (10% MeOH/CH2Cl2); IR νmax (ATR) 3250, 3057, 1633, 1508,
1
1278, 1097 cm−1; H NMR (DMSO-d6, 400 MHz) δH 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) δC 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).
Rf = 0.20 (10% MeOH/CH2Cl2); IR νmax (ATR) 3582, 3250, 3057, 1748, 1634, 1508, 1279,
1127 cm−1; 1H NMR (DMSO-d6, 400 MHz) δH 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,
13
J = 8.8 Hz, H-2), 4.07 (2H, d, J = 6.0 Hz, H2-3′); C NMR (DMSO-d6, 100 MHz) δC 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).
Rf = 0.49 (10% MeOH/CH2Cl2); IR νmax (ATR) 3247, 3056, 1753, 1635, 1508, 1273, 1128 cm−1; 1H
NMR (DMSO-d6, 400 MHz) δH 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

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13
(1H, d, J = 8.8 Hz, H-2), 4.16 (2H, d, J = 6.2 Hz, H2-3′), 3.68 (3H, s, H3-5′); C NMR (DMSO-d6,
100 MHz) δC 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.); Rf = 0.36 (10% MeOH/CH2Cl2); IR νmax (ATR) 3222, 3003, 1726, 1683,
1
1579 cm−1; H NMR (DMSO-d6, 400 MHz) δH 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 δC 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.); Rf = 0.25 (10% MeOH/CH2Cl2); IR νmax (ATR) 3357, 3230, 1674, 1577; 1H
NMR (DMSO-d6, 300 MHz) δH 9.42 (br s, NH minor isomer), 9.31 (1H, br s, NH), 7.93 (s, H-8 minor
13
isomer), (7.84 (1H, s, H-8), 3.86 (2H, br s, H2-3), 3.40–3.34 (2H, br m, H2-2); C NMR (DMSO-d6,
75 MHz) 11a δC 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).

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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.); Rf = 0.27 (10% MeOH/CH2Cl2); IR νmax (ATR) 3227, 3068, 1689, 1637,
1
1510; H NMR (DMSO-d6, 300 MHz) δH 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) δC 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.
1
IR νmax (ATR) 3570, 2955, 1752, 1726, 1534, 1475, 1367, 1187, 1160, 1139 cm−1; H NMR
(DMSO-d6, 75 MHz) δH 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; Rf = 0.36 (10% MeOH/CH2Cl2); IR νmax (ATR) 3225, 1733, 1695, 1566 cm−1; 1H NMR
(DMSO-d6, 300 MHz) δH 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) δC 172.2 (C-9), 167.8 (C-5), 157.1 (C-1′), 149.2 (C-5a), 148.7 (C-7), 147.4

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(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.); Rf = 0.23 (10% MeOH/CH2Cl2); IR νmax (ATR) 3234, 3093, 1635, 1694,
1
1561 cm−1; H NMR (DMSO-d6, 300 MHz) δH 9.35 (1H, br s, NH-4), 7.47 (1H, s, H-8), 3.87–3.82
13
(2H, m, H2-3), 3.37–3.31 (2H, m, H2-2); C NMR (DMSO-d6, 75 MHz) δC 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.); Rf = 0.36 (10% MeOH/CH2Cl2); IR νmax (ATR) 3223, 3072, 1677, 1577,
1516 cm−1; 1H NMR (DMSO-d6, 400 MHz) δH 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) δC 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

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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.
Acknowledgments
We acknowledge funding from AMRF (81547) and the University of Auckland. We thank M. Cal,
C. Braghiroli and G.Riccio (Swiss TPH) for parasite assay results, M. Schmitz for assistance with
NMR data acquisition, and Ms. R. Imatdieva and N. Lloyd for MS data.
Conflict of Interest
The authors declare no conflict of interest.
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