Structure-activity relationship studies on thiaplidiaquinones A and B as novel inhibitors of Plasmodium falciparum and farnesyltransferase

Article abstract of DOI:10.1016/j.bmc.2017.06.029

Marine meroterpenoids, thiaplidiaquinones A and B and their respective non-natural dioxothiazine regioisomers have been shown to inhibit mammalian and protozoal farnesyltransferase (FTase) with the regioisomers exhibiting activity in the nanomolar range. In order to explore the structure-activity relationship (SAR) of this class of marine natural products, analogues of thiaplidiaquinones A and B and their regioisomers were synthesised, with variation in the number of isoprene units present in their side chains to afford prenyl and farnesyl analogues. The previously reported geranyl series of compounds were found to be the most potent FTase inhibitors closely followed by the novel farnesyl series. The prenyl series exhibited the most potent anti-plasmodial activity but the series was also the most cytotoxic. Overall, the farnesyl series exhibited moderate anti-plasmodial activity with one analogue, 14 also exhibiting low cytotoxicity, identifying it as a scaffold worthy of further exploration.

Full text of DOI:10.1016/j.bmc.2017.06.029

Accepted Manuscript
Structure-activity relationship studies on thiaplidiaquinones A and B as novel
inhibitors of Plasmodium falciparum and farnesyltransferase
Melissa M. Cadelis, Marie-Lise Bourguet-Kondracki, Joëlle Dubois, Marcel
Kaiser, Jean Michel Brunel, David Barker, Brent R. Copp
PII:
S0968-0896(17)30776-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.029
BMC 13812
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
10 April 2017
15 June 2017
16 June 2017
Please cite this article as: Cadelis, M.M., Bourguet-Kondracki, M-L., Dubois, J., Kaiser, M., Brunel, J.M., Barker,
D., Copp, B.R., Structure-activity relationship studies on thiaplidiaquinones A and B as novel inhibitors of
Plasmodium falciparum and farnesyltransferase, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.06.029
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Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Structure-activity relationship studies on thiaplidiaquinones A and B as novel
inhibitors of Plasmodium falciparum and farnesyltransferase
Melissa M. Cadelisa∗, Marie-Lise Bourguet-Kondrackib, Joëlle Duboisc, Marcel Kaiserd, Jean Michel
Brunele, David Barkera and Brent R. Coppa
aSchool of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
bLaboratoire Molécules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS, Muséum National d’Histoire Naturelle, 57 rue Cuvier (C.P.
54), 75005 Paris, France
cInstitut de Chimie des Substances Naturelles, CNRS UPR 2301, Centre de Recherche de Gif, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France
d Swiss Tropical and Public Health Institute, Socinstrasse 57, PO Box CH-4002, Basel, Switzerland, and The University of Basel, CH-4003 Basel, Switzerland
e Aix-Marseille Université, Centre de Recherches en Cancérologie de Marseille (CRCM), CNRS, UMR7258, 13385 Marseille, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Marine meroterpenoids, thiaplidiaquinones A and B and their respective non-natural
dioxothiazine regioisomers have been shown to inhibit mammalian and protozoal
Received in revised form
Accepted
farnesyltransferase (FTase) with the regioisomers exhibiting activity in the nanomolar range. In
order to explore the structure-activity relationship (SAR) of this class of marine natural products,
analogues of thiaplidiaquinones A and B and their regioisomers were synthesised, with variation
in the number of isoprene units present in their side chains to afford prenyl and farnesyl
analogues. The previously reported geranyl series of compounds were found to be the most
potent FTase inhibitors closely followed by the novel farnesyl series. The prenyl series exhibited
the most potent anti-plasmodial activity but the series was also the most cytotoxic. Overall, the
farnesyl series exhibited moderate anti-plasmodial activity with one analogue, 14 also exhibiting
low cytotoxicity, identifying it as a scaffold worthy of further exploration.
Available online
Keywords:
Thiaplidiaquinone
Natural Product
Biomimetic
FTase inhibitor
Anti-plasmodial
2017 Elsevier Ltd. All rights reserved
.
———
∗Corresponding author.
E-mail address: mcad006@aucklanduni.ac.nz (Melissa M. Cadelis).

1. Introduction
Prenylating enzymes, farnesyltransferase (FTase), geranylgeranyltransferase type I (GGTase I) and Rab geranylgeranyltranferase
(RabGGTase), are involved in posttranslational modification by farnesylation or geranylgeranylation of proteins which ensures
membrane association.1,2 Inhibitors of these enzymes are well known in the field of cancer drug discovery and have been recently
investigated as potential treatments for several neglected diseases.1,2 For example, Plasmodium falciparum, one of the causative agents
of malaria which claims the lives of around 1–3 million people every year1, expresses prenylating enzymes such as FTase whose
inhibition has been shown to impair the growth of the parasite.2 Given mammalian cells also utilise prenylating FTases, it is essential
that FTase-inhibiting anti-malarials exhibit selectivity towards the parasitic enzyme over the mammalian variant.1,2
Benzo[c]chromene-7,10-dione natural products, reported from both terrestrial and marine sources, exhibit a variety of biological
activities including the inhibition of FTases.3–6 For example, thiaplidiaquinones A (1) and B (2) (Figure 1), isolated from the ascidian
Aplidium conicum, have been shown to induce apoptosis in Jurkat cells by a mechanism involving the rapid production of reactive
oxygen species.7 A recent study by Harper et al. reported a more extensive biological evaluation of synthetically prepared
thiaplidiaquinones A (1) and B (2) and their respective non-natural dioxothiazine regioisomers 3 and 4 from which 1, 3 and 4 were
identified to be potent inhibitors of human and Trypanosoma brucei FTase, with 4 exhibiting activity in the nanomolar range (Table
1).8 In addition, both 3 and 4 exhibited moderate anti-plasmodial activity against P. falciparum (IC50 4.56 0.76 µM and 4.39 0.77 µM,
respectively) with 3 also exhibiting moderate anti-proliferative activity against melanoma cell lines leading to the conclusion that the
dioxothiazine regioisomers were more bioactive than their natural product counterparts.8
Modifications of the prenylated side chains of natural products have been shown to modulate biological activity.6,9 For example,
analogue studies conducted on mallotojaponin B showed that an increase in the number of prenyl units on the side chain met with an
increase in anti-plasmodial and FTase inhibitory activity while studies on tecomaquinone I have shown that such an increase leads to a
decrease in FTase activity.6,9 To expand on the previously reported SAR of the thiaplidiaquinones (1–4), a novel series of analogues
bearing a prenyl (7–10) or farnesyl (11–14) side chain were prepared and evaluated for their anti-plasmodial, anti-bacterial and FTase
inhibitory activity as well as their cytotoxicity against a rat skeletal muscle cell line. Herein we report the synthesis of a small set of
analogues to explore the influence of the geranyl side chain of 1–4 on their observed biological activities.
Figure 1 Structures of thiaplidiaquinones A (1) and B (2) and regioisomers 3 and 4.
2. Results and discussion
2.1. Synthesis
A biomimetic synthesis of thiaplidiaquinone A (1) and B (2) was reported by Khalil et al. using a strategy which also yielded their
respective regioisomers 3 and 4.10 This synthetic approach was based upon ring closure of tautomerised benzochromenediones 5 and/or
6 by a facile oxa-6π-electrocyclisation process to construct the benzo[c]chromene-7,10-dione scaffold, followed by addition of
hypotaurine to form the dioxothiazine ring.10 To confirm the reproducibility of this route, thiaplidiaquinones A (1) and B (2) and
regioisomers 3 and 4 were synthesised according to the procedure reported by Khalil et al..10 Reaction of geranyl benzoquinone (15)
with triethylamine for 2 minutes in CH2Cl2 followed by exposure to silica gel overnight afforded benzochromenediones 5 and 6 after
purification in lower yields than Khalil et al. (Scheme 1). Further investigation of minor products from this reaction that eluted with
increments of methanol identified trace amounts of the known benzochromenedione 1611 along with methoxy substituted
benzochromenediones 17 and 18, characterised as a 5:2 mixture. Benzochromenedione 16 was speculated to be a result of the reaction
of starting material 15 with benzoquinone while methoxy benzochromenediones 17 and 18 were speculated to arise from the solvolytic
incorporation of methanol onto benzochromenedione 6. It is not possible however to discern whether 16 is a result of the reaction with
benzoquinone, which was not detectable in samples of 15, or reaction with a further equivalent of geranyl benzoquinone (15) followed
by cleavage of the pendant geranyl group.
Reaction of benzochromenedione 5 with hypotaurine in CH3CN/EtOH for two days followed by purification by silica gel column
chromatography afforded thiaplidiaquinone A (1), as a red oil, and regioisomer 3, as a green oil, in yields of 25% and 38%,
respectively. Similarly, reaction of 6 with hypotaurine afforded thiaplidiaquinone B (2), as a purple oil, and regioisomer 4, as a blue oil,
in yields of 28% and 31%, respectively. Compounds 1 and 3 were synthesised in yields analogous to those reported by Khalil et al.
while 2 and 4 were obtained in higher yields than those reported and was speculated to be a result of the increase in the scale of the
reaction.
The prenyl series of analogues were prepared by reaction of prenyl benzoquinone (19) with triethylamine followed by exposure to
silica for 2 days to afford prenyl benzochromenediones 20 and 21 as purple solids in 1.4% and 2.5% yield, respectively (Scheme 2). In
addition, chromenol 22, a known sea squirt natural product,12 was also purified in trace amounts from the same reaction. As observed
2

for the geranyl series, minor reaction products, eluting with increments of methanol, comprising of inseparable mixtures of methoxy
substituted benzochromenediones 23 and 24 (5:2) and 25 and 26 (5:2) were also purified from the crude reaction product.
Prenyl benzochromenedione 20 was reacted with hypotaurine followed by purification to afford prenyl thiaplidiaquinone 7, as a pale
red oil, and the dioxothiazine regioisomer 8, as a grey oil, in yields of 36% and 44%, respectively. Similarly, reaction of prenyl
regioisomer 21 with hypotaurine afforded prenyl thiaplidiaquinone 9, as a purple oil, and regioisomer 10, as a blue oil, in yields of 32%
and 42%, respectively.
Scheme 1 Synthesis of geranyl benzochromenediones 5 and 6 from geranyl benzoquinone (15).
Reagents and conditions: (i) Et3N (5 eq.), CH2Cl2, 2 min, (ii) SiO2.
Scheme 2 Synthesis of prenyl thiaplidiaquinone analogues and regioisomers 7–10 from prenyl benzoquinone (19).
Reagents and conditions: (a) (i) Et3N (5 eq.), CH2Cl2, 2 min, (ii) SiO2; (b) hypotaurine (1 eq.), CH3CN:EtOH (1:1), H2O, 48 h.
Preparation of the farnesyl series of analogues proceeded in a similar manner. Reaction of farnesyl benzoquinone (27) with
triethylamine and subsequent exposure to silica overnight afforded the desired benzochromenedione analogues 28 and 29 as purple oils
in 3.8% and 2.3% yield, respectively. In addition, minor products including trace amounts of chromenol 30, a known natural product
isolated from Dictyopteris undulata,13 and a mixture of diastereomeric bischromenes 31A and 31B were also purified (Scheme 3).
Increasing the percentage of MeOH in the column chromatography eluent afforded farnesyl benzochromenedione 32 (0.8%), methoxy
benzochromenedione 33 (as a 1:1 mixture with 34), methoxy benzochromenedione 34 (0.4%) and benzochromene dimer 35 (0.2%).
The structures of chromenols 30 and 31 represent products arising from the cyclisation of starting material 27 and benzochromenedione
29, respectively, while products 32–35 appear to be a result of the reaction of starting material 27 with benzoquinone (32–34), as
observed in the geranyl series, or of 29 with benzoquinone (35).
Farnesyl benzochromenedione 28 was reacted with hypotaurine for 2 days (Scheme 4) to afford farnesyl thiaplidiaquinone 11, as a
red oil, and regioisomer 12, as a green oil, in yields of 12% and 15%, respectively. Similarly, reaction of benzochromenedione 29 with
hypotaurine afforded farnesyl thiaplidiaquinone 13, as a purple oil, and regioisomer 14, as a blue oil, in yields of 23% and 31%,
respectively.
3

Scheme 3 Synthesis of farnesyl benzochromenediones 28 and 29 from farnesyl benzoquinone (27).
Reagents and conditions: (i) Et3N, CH2Cl2, 2 min, (ii) SiO2.
Scheme 4 Synthesis of farnesyl thiaplidiaquinone analogues and regioisomers 11–14.
Reagents and conditions: Hypotaurine (1 eq.), CH3CN:EtOH (1:1), H2O, 48 h.
2.2. Bioactivity of compounds
Biological testing was undertaken on prenyl and farnesyl thiaplidiaquinone analogues; 7–10 and 11–14 respectively, as well as
methoxy benzochromenediones 23–26, 33 and 34, benzochromenedione 32 and dimer 35. Methoxy analogues 23–26 were tested as
their respective mixtures. These compounds were tested for their ability to inhibit both human and parasitic FTase enzymes (Table 1)
4

and for whole cell/organism growth inhibitory properties against P. falciparum (FcM29 or NF54 strains), rat skeletal myoblast cells
(L6) and bacteria (S. aureus and S. intermedius) (Table 2).
The parasitic FTase used for the inhibition assays was from Trypanosoma brucei as it shows high sequence homology to P.
falciparum FTase1 and is more readily available while the human variant serves as a control to observe selectivity for parasitic FTase
over human FTase. FTase assays revealed farnesyl thiaplidiaquinone A 11 as the most active of all the new analogues against both
FTases with activity in the low micromolar range (Table 1). In addition, farnesyl thiaplidiaquinone 13 also exhibited potent inhibitory
activity against the human variant of the enzyme. In general, the farnesyl series (11–14) was more active than the prenyl series (7–10),
however, none of the analogues was as active as the geranyl series (1–4). None of the compounds in all three series exhibited good
selectivity for parasitic FTase over human FTase.
Anti-plasmodial assays against NF54 strain of chloroquinone-sensitive P. falciparum revealed both the prenyl and farnesyl series of
analogues (7–14) were more active than the geranyl series (1–4). The prenyl and farnesyl analogues exhibited moderate activity against
P. falciparum, with the exception of the most potent prenyl regioisomer 10 which exhibited activity in the low micromolar range (IC50
0.29 µM). Cytotoxic assays against an L6 rat skeletal myoblast cell line revealed the prenyl series to be more cytotoxic than the farnesyl
series with prenyl regioisomer 10 (IC50 0.4 µM) identified as the most toxic compound among all the analogues tested. Overall, the
farnesyl series exhibited greater selectivity towards malaria activity over cytotoxicity (SI = IC50 L6/IC50 P.f) with analogue 14
exhibiting 17-fold selectivity while the prenyl series exhibited no such selectivity. Previously reported cytotoxicity evaluation of 1–4 at
the NCI identified regioisomer 3 as the most active compound in the anti-cancer screening data, exhibiting moderate activity against
melanoma cell lines.8
The compounds were evaluated for anti-bacterial activity against a panel of Gram-negative (Pseudomonas aeruginosa (ATCC
27853) and Escherichia coli (ATCC 25922)) and Gram-positive (Staphylococcus aureus (ATCC 25923) and Staphylococcus
intermedius (1051997)) organisms. Benzochromenedione 32 inhibited Gram-positive bacteria potently while methoxy
benzochromenediones 23–26 and 34 inhibited the bacteria modestly and all other compounds were inactive against Gram-positive
bacteria. Furthermore, none of the compounds showed activity towards Gram-negative bacteria.
Table 1 Inhibitory activities of selected compounds against target enzymes, human and T. brucei FTase.
IC50 (µM)
Analogues
T. brucei
FTaseb
Human FTasea
0.78 0.17c
1.22 0.068c
0.14 0.0017c
0.054 0.005c
17.3 1.2
14.7 0.4
>22
0.74 0.20c
3.04 0.30c
0.22 0.034c
0.098 0.008c
>22
1
2
3
4
7
8
19.6 1.4
>22
9
10
3.1 0.5
2.3 0.4
0.35 0.009
2.9 0.5
1.0 0.05
5.2 1.3
8.5 1.1
5.4 0.6
3.3 0.6
>22.3
11
0.17 0.008
1.5 0.2
12
13
0.45 0.03
4.7 0.8
14
23 and 24
25 and 26
32
7.3 0.6
5.8 0.3
1.7 0.1
33 and 34
34
>22.3
>22.3
>22.3
35
1.0 0.06
1.6 0.2
a Human farnesyltransferase with FTI 276 as the positive control (IC50 0.015 0.004 µM) and values presented as the mean SEM (n = 3).
b T. brucei farnesyltransferase with FTI 276 as the positive control (IC50 0.010 0.002 µM) and values presented as the mean SEM (n = 3).
c Data taken from Harper et al..8
Table 2 Anti-plasmodial (P. falciparum), cytotoxicity (L6) and anti-bacterial (S. aureus and S. intermedius) activities of selected compounds.
IC50 (µM)
MIC (µM)
S. aureus (ATCC
25923)b
>200
Analogues
S. intermedius
(1051997)c
>200
P. falc
Cytotox L6a
1
>17d ,e
n.t.
2
>17d ,e
n.t.
>200
>200
3
4.56 0.76d ,e
4.39 0.77d ,e
8.0 0.5f
2.0 0.2f
4.8 0.4f
0.29 0.03f
4.0 0.2f
2.9 0.2f
n.t.
>200
>200
4
n.t.
>200
>200
7
12.8 0.9
4.2 1.0
5.2 1.4
0.4 0.07
30.6 6.3
16.7 1.7
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
8
9
10
11
12
5

3.4 0.2f
7.4 1.0f
n.t.g
25.8 6.9
126.4 5.5
n.t.
>200
>200
100
50
>200
>200
100
50
13
14
23 and 24
25 and 26
32
n.t.
7.2 0.2f
n.t.
23.5 2.5
n.t.
6.25
12.5
25
3.12
25
33 and 34
34
n.t.
20.4 0.8f
33.9 0.1f
34.3 11.8
50
35
53.2 2.1
>200
>200
a L6 rat skeletal myoblast cell line with podophyllotoxin as the positive control (IC50 0.012 µM) and values presented as the mean SEM (n = 2).
b S. aureus (ATCC 25923) with streptomycin (MIC 21.5 µM) and chloramphenicol (MIC 1.5–3 µM) used as positive controls and values presented as the mean
(n = 3).
c S. intermedius (1051997) with streptomycin (MIC 10.7 µM) and chloramphenicol (MIC 3–6 µM) used as positive controls and values presented as the mean (n
= 3).
d Data taken from Harper et al..8
e P. falciparum (FcM29-Cameroon strain) with chloroquine as the positive control (IC50 0.45 µM) and values presented as the mean SEM (n = 2).
f P. falciparum (NF54 strain, IEF stage) with chloroquine as the positive control (IC50 0.006 µM) and values presented as the mean SEM (n = 2).
g Not tested.
3. Conclusion
In conclusion, the synthesis of thiaplidiaquinones A (1) and B (2) and regioisomers 3 and 4 were successfully undertaken along with
their prenyl (7–10) and farnesyl (11–14) analogues utilising the reported biomimetic approach. Unexpectedly, during the synthesis of
the precursors, the formation of several side products was observed across all three series (prenyl, geranyl and farnesyl).
Evaluation of the FTase activity, identified the farnesyl series as better inhibitors than the prenyl series though none were as active as
the geranyl series. Both the prenyl and farnesyl series were more active in the anti-plasmodial assays than the geranyl series with prenyl
regioisomer 10 identified as the most potent compound. Prenyl analogue 10 however was also the most cytotoxic compound, with the
prenyl series observed to be more toxic than the farnesyl series. Of note was regioisomer 14, which exhibited nearly 20-fold selectivity
for anti-plasmodial activity over cytotoxicity identifying it as a suitable candidate for in vivo testing.
4. Experimental
4.1. General remarks
Infrared spectra were recorded on a Perkin-Elmer spectrometer. Mass spectra were acquired on a Bruker micrOTOF Q II
spectrometer. 1H and 13C NMR spectra were recorded at 298 K on Bruker AC300, AVANCE 400 or 500 spectrometer using standard
pulse sequences with TMS as an internal standard. Silica gel column chromatography was carried out using Davisil silica gel (40–60
µm) or Merck silica gel (15–40 µm). Thin layer chromatography was conducted on Merck DC-plastikfolien Kieselgel 60 F254 plates.
Quinones 15, 19 and 27 were prepared according to literature procedure.10,14,15
4.2. General procedure A: benzochromenedione synthesis
To a solution of quinone (1 eq.) in CH2Cl2 (20 mL) was added triethylamine (5 eq.) dropwise under an atmosphere of nitrogen.
Reaction was stirred for 2 mins and loaded onto a silica gel column (CH2Cl2). After elution with copious amounts of CH2Cl2, the
column was left overnight. Elution with CH2Cl2 afforded benzochromenediones.
4.3. General procedure B: hypotaurine addition
To a solution of benzochromenedione (1 eq.) in EtOH/MeCN (1:1) (4 mL) was added hypotaurine (1 eq.) in water (0.4 mL) at 0°C
and the reaction was stirred at room temperature for 48 h. Water (2 mL) was added, the aqueous layer was extracted with CH2Cl2 (2 x 5
mL) and the combined organic layers dried over anhydrous MgSO4. Solvent was removed under reduced pressure and the crude
products purified by silica gel column chromatography eluting with EtOAc/n-hexane (1:1) to afford thiaplidiaquinones.
4.4. Synthesis of compounds
4.4.1. Thiaplidiaquinone A (1)
Following general procedure B, benzochromenedione 5 (0.013 g, 0.03 mmol) was reacted with hypotaurine (0.003 g, 0.03 mmol)
and the crude product purified to afford the title compound as a red oil (0.004 g, 25%) as well as regioisomer 3 as a green oil (0.006 g,
38%). 1H NMR (CDCl3, 400 MHz) δ 7.57 (1H, d, J = 2.8 Hz, H-1), 6.72 (1H, d, J = 2.8 Hz, H-3), 6.65 (1H, br s, H-11), 6.13 (1H, d, J =
9.4 Hz, H-6), 5.28–5.21 (2H, m, H-2', H-1''), 5.12–5.06 (1H, m, H-6'), 4.96–4.92 (1H, m, H-5''), 4.11–4.08 (2H, m, H2-10), 3.32–3.30
(3H, m, H2-9, H2-1'A), 3.26–3.22 (1H, m, H2-1'B), 2.09–2.06 (2H, m, H2-5'), 2.03–1.99 (2H, m, H2-4'), 1.99–1.96 (2H, m, H2-4''), 1.96–
1.92 (2H, m, H2-3''), 1.94 (3H, br s, H3-9''), 1.68 (3H, s, H3-8'), 1.66 (3H, s, H3-10'), 1.60 (6H, s, H3-9', H3-7''), 1.51 (3H, s, H3-8''); (–)-
HRESIMS [M–H]– m/z 590.2564 (calcd for C34H40NO6S, 590.2582). 1H NMR data were in agreement with literature.10
4.4.2. Regioisomer 3
Green oil; yield: 38%; 1H NMR (CDCl3, 400 MHz) δ 7.84 (1H, d, J = 2.8 Hz, H-1), 6.79 (1H, d, J = 2.8 Hz, H-3), 6.57 (1H, br s, H-
8), 6.00 (1H, d, J = 9.3 Hz, H-6), 5.32–5.29 (1H, m, H-1''), 5.23–5.20 (1H, m, H-2'), 5.10–5.07 (1H, m, H-6'), 4.94–4.91 (1H, m, H-5''),
4.09–4.08 (2H, m, H2-9), 3.36–3.33 (3H, m, H2-10, H2-1'A), 3.23–3.19 (1H, m, H2-1'B), 2.09–2.06 (2H, m, H2-5'), 2.03–1.96 (4H, m, H2-
6

4', H2-4''), 1.95–1.93 (2H, m, H2-3''), 1.91 (3H, br s, H3-9''), 1.68 (3H, s, H3-8'), 1.65 (3H, s, H3-10'), 1.59 (6H, s, H3-9', H3-7''), 1.51 (3H,
s, H3-8''); (–)-HRESIMS [M–H]– m/z 590.2570 (calcd for C34H40NO6S, 590.2582). 1H NMR data were in agreement with literature.10
4.4.3. Thiaplidiaquinone B (2)
Following general procedure B, benzochromenedione 6 (0.024 g, 0.05 mmol) was reacted with hypotaurine (0.005 g, 0.05 mmol)
and the crude product purified to afford the title compound as a purple oil (0.008 g, 28%) as well as regioisomer 4 as a blue oil (0.009 g,
31%). 1H NMR (CDCl3, 500 MHz) δ 7.72 (1H, s, H-1), 6.70 (1H, s, H-4), 6.63 (1H, br s, H-11), 6.08 (1H, d, J = 9.7 Hz, H-6), 5.30–
5.29 (2H, m, H-2', H-1''), 5.09–5.05 (1H, m, H-6'), 4.96–4.94 (1H, m, H-5''), 4.11–4.08 (2H, m, H2-10), 3.33–3.31 (4H, m, H2-9, H2-1'),
2.13–2.10 (2H, m, H2-5'), 2.09–2.07 (2H, m, H2-4'), 2.01–1.98 (2H, m, H2-4''), 1.95–1.92 (2H, m, H2-3''), 1.93 (3H, d, J = 1.0 Hz, H3-
9''), 1.74 (3H, s, H3-10'), 1.69 (3H, s, H3-8'), 1.60 (6H, s, H3-9', H3-7''), 1.52 (3H, s, H3-8''); (+)-HRESIMS [M+Na]+ m/z 614.2539 (calcd
for C34H41NNaO6S, 614.2547). 1H NMR data were in agreement with literature.10
4.4.4. Regioisomer 4
Blue oil; yield: 31%; 1H NMR (CDCl3, 500 MHz) δ 7.99 (1H, s, H-1), 6.66 (1H, s, H-4), 6.55 (1H, br s, H-8), 5.96 (1H, d, J = 9.5
Hz, H-6), 5.35–5.34 (1H, m, H-1''), 5.30–5.26 (1H, m, H-2'), 5.09–5.06 (1H, m, H-6'), 4.95–4.93 (1H, m, H-5''), 4.08–4.07 (2H, m, H2-
9), 3.32–3.31 (4H, m, H2-10, H2-1'), 2.12–2.10 (2H, m, H2-5'), 2.09–2.07 (2H, m, H2-4'), 2.01–1.98 (2H, m, H2-4''), 1.96–1.93 (2H, m,
H2-3''), 1.88 (3H, br s, H3-9''), 1.70 (3H, s, H3-10'), 1.68 (3H, s, H3-8'), 1.60 (6H, s, H3-9', H3-7''), 1.52 (3H, s, H3-8''); (+)-HRESIMS
[M+Na]+ m/z 614.2535 (calcd for C34H41NNaO6S, 614.2547). 1H NMR data were in agreement with literature.10
4.4.5. 6-((E)-2,6-Dimethylhepta-1,5-dien-1-yl)-4-((E)-3,7-dimethylocta-2,6-dien-1-yl)-2-hydroxy-6H-benzo[c]chromene-7,10-
dione (5)
Following general procedure A, quinone 15 (0.76 g, 3 mmol) was reacted with triethylamine (1.57 g, 15 mmol), loaded onto a silica
gel column (CH2Cl2) and left overnight. Elution with CH2Cl2 afforded the title compound as a red oil (0.013 g, 1.7%) as well as
benzochromenedione 6 as a purple oil (0.024 g, 3.2%). Elution with CH2Cl2/MeOH (9:1) afforded benzochromenedione 16 in trace
amounts and methoxy benzochromenediones 17 and 18 as a 5:2 mixture (0.002 g). 1H NMR (CDCl3, 400 MHz) δ 7.66 (1H, d, J = 3.0
Hz, H-1), 6.74 (1H, d, J = 3.0 Hz, H-3), 6.73 (2H, s, H-8, H-9), 6.04 (1H, d, J = 9.4 Hz, H-6), 5.33–5.30 (1H, m, H-1''), 5.26–5.22 (1H,
m, H-2'), 5.11–5.07 (1H, m, H-6'), 4.95–4.90 (1H, m, H-5''), 4.53 (1H, br s, OH), 3.26–3.23 (2H, m, H2-1'), 2.10–2.06 (2H, m, H2-5'),
2.03–2.00 (2H, m, H2-4'), 2.00–1.97 (2H, m, H2-4''), 1.95–1.92 (2H, m, H2-3''), 1.92 (3H, s, H3-9''), 1.68 (3H, s, H3-8'), 1.66 (3H, s, H3-
10'), 1.59 (6H, br s, H3-9', H3-7''), 1.51 (3H, s, H3-8''); (+)-HRESIMS [M+Na]+ m/z 509.2659 (calcd for C32H38NaO4, 509.2662). 1H
NMR data were in agreement with literature.10
4.4.6. 6-((E)-2,6-Dimethylhepta-1,5-dien-1-yl)-3-((E)-3,7-dimethylocta-2,6-dien-1-yl)-2-hydroxy-6H-benzo[c]chromene-7,10-
dione (6)
Purple oil; yield: 3.2%; 1H NMR (CDCl3, 400 MHz) δ 7.81 (1H, s, H-1), 6.72 (2H, s, H-8, H-9), 6.71 (1H, s, H-4), 5.99 (1H, d, J =
9.5 Hz, H-6), 5.36–5.34 (1H, m, H-1''), 5.31–5.28 (1H, m, H-2'), 5.10–5.06 (1H, m, H-6'), 4.95–4.91 (1H, m, H-5''), 4.81 (1H, br s, OH),
3.34–3.31 (2H, m, H2-1'), 2.13–2.11 (2H, m, H2-5'), 2.11–2.07 (2H, m, H2-4'), 2.02–1.97 (2H, m, H2-4''), 1.96–1.92 (2H, m, H2-3''), 1.92
(3H, d, J = 1.2 Hz, H3-9''), 1.73 (3H, s, H3-10'), 1.69 (3H, s, H3-8'), 1.60 (3H, s, H3-7''), 1.59 (3H, s, H3-9'), 1.51 (3H, s, H3-8''); (+)-
HRESIMS [M+Na]+ m/z 509.2671 (calcd for C32H38NaO4, 509.2662). 1H NMR data were in agreement with literature.10
4.4.7. (E)-6-(2,6-Dimethylhepta-1,5-dien-1-yl)-2-hydroxy-6H-benzo[c]chromene-7,10-dione (16)
Red oil; trace; 1H NMR (CDCl3, 400 MHz) δ 7.85 (1H, d, J = 2.8 Hz, H-1), 6.85 (1H, dd, J = 9.0, 2.8 Hz, H-3), 6.81 (1H, d, J = 9.0
Hz, H-4), 6.74 (2H, s, H-8, H-9), 6.00 (1H, d, J = 9.4 Hz, H-6), 5.33 (1H, d, J = 9.4 Hz, H-1'), 4.95–4.90 (1H, m, H-5'), 4.65 (1H, br s,
OH), 2.01–1.98 (2H, m, H2-4'), 1.96–1.94 (2H, m, H2-3'), 1.92 (3H, br s, H3-9'), 1.59 (3H, s, H3-7'), 1.51 (3H, s, H3-8'); (–)-HRESIMS
[M–H]– m/z 349.1449 (calcd for C22H21O4, 349.1445). 1H NMR data were in agreement with literature.11
4.4.8. 6-((E)-2,6-Dimethylhepta-1,5-dien-1-yl)-3-((E)-3,7-dimethylocta-2,6-dien-1-yl)-2-hydroxy-9-methoxy-6H-
benzo[c]chromene-7,10-dione (17)
Purple oil; trace; Rf (CH2Cl2) 0.17; IR (ATR) vmax 3415, 2928, 2853, 1639, 1598, 1424, 1224, 1005, 849 cm-1; 1H NMR (CDCl3, 400
MHz) δ 7.81 (1H, s, H-1), 6.69 (1H, s, H-4), 6.01 (1H, d, J = 9.5 Hz, H-6), 5.89 (1H, s, H-8), 5.36–5.34 (1H, m, H-1''), 5.32–5.28 (1H,
m, H-2'), 5.10–5.06 (1H, m, H-6'), 4.95–4.92 (1H, m, H-5''), 4.91 (1H, s, OH), 3.84 (3H, s, OMe), 3.34–3.31 (2H, m, H2-1'), 2.13–2.11
(2H, m, H2-5'), 2.11–2.07 (2H, m, H2-4'), 2.02–1.97 (2H, m, H2-4''), 1.96–1.92 (2H, m, H2-3''), 1.91 (3H, br s, H3-9''), 1.73 (3H, s, H3-
10'), 1.69 (3H, s, H3-8'), 1.59 (6H, br s, H3-9', H3-7''), 1.51 (3H, s, H3-8''); 13C NMR (CDCl3, 125 MHz, deduced from HSQC and
HMBC) δ 184.6 (C-7), 181.4 (C-10), 158.1 (C-9), 148.7 (C-4a), 148.4 (C-2), 143.8 (C-2''), 138.7 (C-3'), 134.6 (C-6a), 132.9 (C-3),
131.9 (C-7'), 131.6 (C-6''), 128.6 (C-10a), 124.2 (C-6'), 123.7 (C-5''), 120.7 (C-2'), 118.5 (C-4), 118.1 (C-1''), 115.5 (C-10b), 114.8 (C-
1), 106.9 (C-8), 67.5 (C-6), 56.5 (OMe), 39.8 (C-4', C-3''), 29.7 (C-1'), 26.6 (C-5'), 26.3 (C-4''), 25.8 (C-8'), 25.7 (C-7''), 17.8 (C-9', C-
8''), 17.4 (C-9''), 16.3 (C-10'); (+)-HRESIMS [M+Na]+ m/z 539.2764 (calcd for C33H40NaO5, 539.2768).
4.4.9. 6-((E)-2,6-Dimethylhepta-1,5-dien-1-yl)-3-((E)-3,7-dimethylocta-2,6-dien-1-yl)-2-hydroxy-8-methoxy-6H-
benzo[c]chromene-7,10-dione (18)
Purple oil; trace; Rf (CH2Cl2) 0.27; IR (ATR) vmax 3415, 2928, 2853, 1639, 1598, 1424, 1224, 1005, 849 cm-1; 1H NMR (CDCl3, 400
MHz) δ 7.85 (1H, s, H-1), 6.69 (1H, s, H-4), 6.03 (1H, d, J = 9.3 Hz, H-6), 5.89 (1H, s, H-9), 5.36–5.34 (1H, m, H-1''), 5.32–5.28 (1H,
m, H-2'), 5.10–5.06 (1H, m, H-6'), 4.95–4.92 (1H, m, H-5''), 4.88 (1H, s, OH), 3.83 (3H, s, OMe), 3.34–3.31 (2H, m, H2-1'), 2.13–2.11
(2H, m, H2-5'), 2.11–2.07 (2H, m, H2-4'), 2.02–1.97 (2H, m, H2-4''), 1.96–1.92 (2H, m, H2-3''), 1.91 (3H, br s, H3-9''), 1.73 (3H, s, H3-
10'), 1.69 (3H, s, H3-8'), 1.59 (6H, br s, H3-9', H3-7''), 1.51 (3H, s, H3-8''); 13C NMR (CDCl3, 125 MHz, deduced from HSQC and
HMBC) δ 186.8 (C-10), 178.9 (C-7), 158.1 (C-8), 149.6 (C-4a), 149.2 (C-2), 144.6 (C-2''), 138.7 (C-3'), 133.5 (C-3, C-6a), 131.9 (C-
7'), 131.6 (C-6''), 131.3 (C-10a), 124.2 (C-6'), 123.7 (C-5''), 120.7 (C-2'), 118.5 (C-4), 118.1 (C-1''), 115.5 (C-10b), 115.4 (C-1), 107.9
7

(C-9), 67.7 (C-6), 56.4 (OMe), 39.8 (C-4', C-3''), 29.8 (C-1'), 26.6 (C-5'), 26.3 (C-4''), 25.8 (C-8'), 25.7 (C-7''), 17.8 (C-9', C-8''), 17.4
(C-9''), 16.3 (C-10'); (+)-HRESIMS [M+Na]+ m/z 539.2764 (calcd for C33H40NaO5, 539.2768).
4.4.10. 2-Hydroxy-4-(3-methylbut-2-en-1-yl)-6-(2-methylprop-1-en-1-yl)-6H-benzo[c]chromene-7,10-dione (20)
Following general procedure A, quinone 19 (1.06 g, 6 mmol) was reacted with triethylamine (3.05 g, 30 mmol) and loaded onto a
silica gel column. After two days, elution with CH2Cl2 afforded the title compound as a purple solid (0.015 g, 1.4%) as well as
benzochromenedione 21 as a purple solid (0.026 g, 2.5%). Elution with CH2Cl2/MeOH (9:1) afforded chromenol 22 in trace amounts
and methoxy benzochromenediones 23 and 24 as a 5:2 mixture as well as 25 and 26 as a 5:2 mixture. m.p. 169–171°C; Rf (CH2Cl2)
0.13; IR (ATR) vmax 3376, 1717, 1469, 1363, 1223, 1132, 956 cm-1; 1H NMR (CDCl3, 400 MHz) δ 7.66 (1H, d, J = 3.0 Hz, H-1), 6.74
(1H, d, J = 3.0 Hz, H-3), 6.73 (2H, s, H-8, H-9), 6.03 (1H, d, J = 9.5 Hz, H-6), 5.34–5.31 (1H, m, H-1''), 5.24–5.20 (1H, m, H-2'), 4.52
(1H, s, OH), 3.30–3.25 (1H, m, H2-1'A), 3.21–3.15 (1H, m, H2-1'B), 1.94 (3H, d, J = 1.0 Hz, H3-3''), 1.71 (3H, s, H3-4'), 1.68 (3H, s, H3-
5'), 1.66 (3H, d, J = 1.0 Hz, H3-4''); 13C NMR (CDCl3, 100 MHz) δ 187.0 (C-10), 185.2 (C-7), 149.8 (C-2), 147.0 (C-4a), 140.9 (C-2''),
137.2 (C-8/C-9), 135.8 (C-8/C-9), 134.9 (C-6a), 133.2 (C-3'), 132.3 (C-4), 130.8 (C-10a), 121.8 (C-2'), 120.2 (C-3), 118.6 (C-1''), 117.7
(C-10b), 112.5 (C-1), 67.4 (C-6), 28.6 (C-1'), 26.1 (C-4''), 25.9 (C-4'), 19.0 (C-3''), 17.9 (C-5'); (–)-HRESIMS [M–H]– m/z 349.1457
(calcd for C22H21O4, 349.1445).
4.4.11. 2-Hydroxy-3-(3-methylbut-2-en-1-yl)-6-(2-methylprop-1-en-1-yl)-6H-benzo[c]chromene-7,10-dione (21)
Purple solid; yield: 2.5%; m.p. 170–172°C; Rf (CH2Cl2) 0.31; IR (ATR) vmax 3007, 1710, 1422, 1359, 1220, 1093, 905 cm-1; 1H
NMR (CDCl3, 500 MHz) δ 7.81 (1H, s, H-1), 6.72 (2H, s, H-8, H-9), 6.70 (1H, s, H-4), 5.98 (1H, d, J = 9.6 Hz, H-6), 5.38 (1H, dq, J =
9.6, 1.0 Hz, H-1''), 5.31–5.28 (1H, m, H-2'), 4.89 (1H, s, OH), 3.32 (2H, t, J = 6.0 Hz, H2-1'), 1.92 (3H, d, J = 1.0 Hz, H3-3''), 1.77 (3H,
s, H3-4'), 1.74 (3H, s, H3-5'), 1.67 (3H, d, J = 1.0 Hz, H3-4''); 13C NMR (CDCl3, 125 MHz) δ 187.1 (C-10), 185.1 (C-7), 149.2 (C-4a),
149.0 (C-2), 140.7 (C-2''), 137.0 (C-8/C-9), 135.9 (C-8/C-9), 135.3 (C-3'), 133.7 (C-6a), 133.4 (C-3), 130.2 (C-10a), 120.8 (C-2'), 118.6
(C-4), 118.4 (C-1''), 115.6 (C-10b), 115.0 (C-1), 67.7 (C-6), 29.7 (C-1'), 26.2 (C-4''), 26.0 (C-4'), 19.0 (C-3''), 18.0 (C-5'); (–)-
HRESIMS [M–H]– m/z 349.1432 (calcd for C22H21O4, 349.1445).
4.4.12. 2,2-Dimethyl-2H-chromen-6-ol (22)
Red oil; trace; 1H NMR (CDCl3, 400 MHz) δ 6.65 (1H, d, J = 8.6 Hz, H-8), 6.58 (1H, dd, J = 8.6, 3.0 Hz, H-7), 6.49 (1H, d, J = 3.0
Hz, H-5), 6.25 (1H, d, J = 9.8 Hz, H-4), 5.63 (1H, d, J = 9.8 Hz, H-3), 1.40 (6H, s, H3-1'); (–)-HRESIMS [M–H]– m/z 175.0763 (calcd
for C11H11O2, 175.0765). 1H NMR data were in agreement with literature.16
4.4.13. 2-Hydroxy-9-methoxy-4-(3-methylbut-2-en-1-yl)-6-(2-methylprop-1-en-1-yl)-6H-benzo[c]chromene-7,10-dione (23)
Purple oil; Rf (CH2Cl2) 0.04; IR (ATR) vmax 3278, 2927, 1717, 1640, 1366, 1325, 1225, 1206 cm-1; 1H NMR (CDCl3, 500 MHz) δ
7.67 (1H, d, J = 3.0 Hz, H-1), 6.73 (1H, d, J = 3.0 Hz, H-3), 6.05 (1H, d, J = 9.2 Hz, H-6), 5.90 (1H, s, H-8), 5.32–5.30 (1H, m, H-1''),
5.24–5.21 (1H, m, H-2'), 4.66 (1H, s, OH), 3.84 (3H, s, OMe), 3.30–3.25 (1H, m, H2-1'A), 3.21–3.16 (1H, m, H2-1'B), 1.94 (3H, d, J =
0.9 Hz, H3-3''), 1.71 (3H, s, H3-4'), 1.68 (3H, s, H3-5'), 1.66 (3H, d, J = 0.9 Hz, H3-4''); 13C NMR (CDCl3, 125 MHz) δ 185.0 (C-7),
181.3 (C-10), 158.6 (C-9), 149.8 (C-2), 146.6 (C-4a), 140.9 (C-2''), 135.7 (C-6a), 133.2 (C-3'), 132.3 (C-4), 129.2 (C-10a), 121.9 (C-2'),
119.8 (C-3), 118.6 (C-1''), 117.6 (C-10b), 112.3 (C-1), 106.9 (C-8), 67.5 (C-6), 56.6 (OMe), 28.6 (C-1'), 26.1 (C-4''), 25.9 (C-4'), 19.0
(C-3''), 17.9 (C-5'); (+)-HRESIMS [M+Na]+ m/z 403.1526 (calcd for C23H24NaO5, 403.1516).
4.4.14. 2-Hydroxy-8-methoxy-4-(3-methylbut-2-en-1-yl)-6-(2-methylprop-1-en-1-yl)-6H-benzo[c]chromene-7,10-dione (24)
Purple oil; Rf (CH2Cl2) 0.04; IR (ATR) vmax 3278, 2927, 1717, 1640, 1366, 1325, 1225, 1206 cm-1; 1H NMR (CDCl3, 500 MHz) δ
7.71 (1H, d, J = 3.0 Hz, H-1), 6.75 (1H, d, J = 3.0 Hz, H-3), 6.07 (1H, d, J = 8.8 Hz, H-6), 5.90 (1H, s, H-9), 5.32–5.30 (1H, m, H-1''),
5.24–5.21 (1H, m, H-2'), 4.66 (1H, s, OH), 3.83 (3H, s, OMe), 3.30–3.25 (1H, m, H2-1'A), 3.21–3.16 (1H, m, H2-1'B), 1.94 (3H, d, J =
0.9 Hz, H3-3''), 1.71 (3H, s, H3-4'), 1.68 (3H, s, H3-5'), 1.65 (3H, d, J = 0.9 Hz, H3-4''); 13C NMR (CDCl3, 125 MHz) δ 187.1 (C-10),
178.5 (C-7), 158.6 (C-8), 149.9 (C-2), 147.7 (C-4a), 140.9 (C-2''), 133.3 (C-3'), 133.21 (C-4/C-6a), 133.15 (C-4/C-6a), 130.8 (C-10a),
121.8 (C-2'), 120.4 (C-3), 118.4 (C-1''), 117.8 (C-10b), 113.0 (C-1), 108.0 (C-9), 67.6 (C-6), 56.4 (OMe), 28.6 (C-1'), 26.1 (C-4''), 25.9
(C-4'), 19.0 (C-3''), 17.9 (C-5'); (+)-HRESIMS [M+Na]+ m/z 403.1526 (calcd for C23H24NaO5, 403.1516).
4.4.15. 2-Hydroxy-9-methoxy-3-(3-methylbut-2-en-1-yl)-6-(2-methylprop-1-en-1-yl)-6H-benzo[c]chromene-7,10-dione (25)
Purple oil; Rf (CH2Cl2) 0.11; IR (ATR) vmax 3430, 2974, 1717, 1648, 1364, 1223, 1132, 953, 741 cm-1; 1H NMR (CDCl3, 500 MHz)
δ 7.81 (1H, s, H-1), 6.70 (1H, s, H-4), 6.00 (1H, d, J = 9.5 Hz, H-6), 5.89 (1H, s, H-8), 5.38–5.35 (1H, m, H-1''), 5.32–5.29 (1H, m, H-
2'), 4.94 (1H, s, OH), 3.84 (3H, s, OMe), 3.33–3.31 (2H, m, H2-1'), 1.92 (3H, d, J = 1.0 Hz, H3-3''), 1.77 (3H, s, H3-4'), 1.74 (3H, s, H3-
5'), 1.67 (3H, d, J = 1.0 Hz, H3-4''); 13C NMR (CDCl3, 125 MHz) δ 185.0 (C-7), 181.4 (C-10), 158.5 (C-9), 148.8 (C-4a), 148.7 (C-2),
140.7 (C-2''), 135.2 (C-3'), 134.4 (C-6a), 132.9 (C-3), 128.8 (C-10a), 120.9 (C-2'), 118.6 (C-4), 118.3 (C-1''), 115.5 (C-10b), 114.8 (C-
1), 106.9 (C-8), 67.8 (C-6), 56.5 (OMe), 29.7 (C-1'), 26.2 (C-4''), 26.0 (C-4'), 19.0 (C-3''), 18.0 (C-5'); (+)-HRESIMS [M+Na]+ m/z
403.1520 (calcd for C23H24NaO5, 403.1516).
4.4.16. 2-Hydroxy-8-methoxy-3-(3-methylbut-2-en-1-yl)-6-(2-methylprop-1-en-1-yl)-6H-benzo[c]chromene-7,10-dione (26)
Purple oil; Rf (CH2Cl2) 0.11; IR (ATR) vmax 3430, 2974, 1717, 1648, 1364, 1223, 1132, 953, 741 cm-1; 1H NMR (CDCl3, 500 MHz)
δ 7.86 (1H, s, H-1), 6.70 (1H, s, H-4), 6.02 (1H, d, J = 9.7 Hz, H-6), 5.89 (1H, s, H-9), 5.38–5.35 (1H, m, H-1''), 5.32–5.29 (1H, m, H-
2'), 4.92 (1H, s, OH), 3.83 (3H, s, OMe), 3.33–3.31 (2H, m, H2-1'), 1.92 (3H, d, J = 1.0 Hz, H3-3''), 1.77 (3H, s, H3-4'), 1.74 (3H, s, H3-
5'), 1.66 (3H, d, J = 1.0 Hz, H3-4''); 13C NMR (CDCl3, 125 MHz) δ 187.2 (C-10), 178.4 (C-7), 158.5 (C-8), 149.8 (C-4a), 149.2 (C-2),
140.7 (C-2''), 135.2 (C-3'), 133.7 (C-3, C-6a), 131.0 (C-10a), 120.8 (C-2'), 118.6 (C-4), 118.3 (C-1''), 115.5 (C-10b), 115.4 (C-1), 107.9
(C-9), 67.9 (C-6), 56.4 (OMe), 29.6 (C-1'), 26.2 (C-4''), 26.0 (C-4'), 19.0 (C-3''), 18.0 (C-5'); (+)-HRESIMS [M+Na]+ m/z 403.1520
(calcd for C23H24NaO5, 403.1516).
8

4.4.17. 2-Hydroxy-4-(3-methylbut-2-en-1-yl)-6-(2-methylprop-1-en-1-yl)-10,11-dihydrobenzo[3,4]isochromeno[7,6-
b][1,4]thiazine-7,12(6H,9H)-dione 8,8-dioxide (7)
Following general procedure B, benzochromenedione 20 (0.009 g, 0.027 mmol) was reacted with hypotaurine (0.003 g, 0.027 mmol)
and the crude product purified to afford the title compound as a pale red oil (0.004 g, 36%) as well as regioisomer 8 as a grey oil (0.006
g, 44%). Rf (EtOAc) 0.64; IR (ATR) vmax 3420, 1717, 1654, 1364, 1225, 1030 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.55 (1H, d, J = 3.0
Hz, H-1), 6.72 (1H, d, J = 3.0 Hz, H-3), 6.67 (1H, br s, H-11), 6.10 (1H, d, J = 9.5 Hz, H-6), 5.27–5.24 (1H, m, H-1''), 5.22–5.19 (1H,
m, H-2'), 4.64 (1H, br s, OH), 4.11–4.10 (2H, m, H2-10), 3.35–3.30 (2H, m, H2-9), 3.30–3.26 (1H, m, H2-1'A), 3.18–3.13 (1H, m, H2-
1'B), 1.93 (3H, d, J = 1.0 Hz, H3-3''), 1.70 (3H, s, H3-4'), 1.67 (3H, s, H3-5'), 1.64 (3H, d, J = 1.0 Hz, H3-4''); 13C NMR (CDCl3, 125
MHz) δ 179.3 (C-12), 175.3 (C-7), 149.7 (C-2), 146.2 (C-4a), 143.8 (C-11a), 142.1 (C-2''), 138.9 (C-6a), 133.3 (C-3'), 132.6 (C-4),
128.2 (C-12a), 121.7 (C-2'), 120.1 (C-3), 117.3 (C-1''), 116.8 (C-12b), 111.6 (C-1), 110.7 (C-7a), 67.6 (C-6), 48.8 (C-9), 40.1 (C-10),
28.5 (C-1'), 26.2 (C-4''), 25.9 (C-4'), 19.1 (C-3''), 17.9 (C-5'); (+)-HRESIMS [M+Na]+ m/z 478.1284 (calcd for C24H25NNaO6S,
478.1295).
4.4.18. 2-Hydroxy-4-(3-methylbut-2-en-1-yl)-6-(2-methylprop-1-en-1-yl)-9,10-dihydrobenzo[3,4]isochromeno[6,7-
b][1,4]thiazine-7,12(6H,8H)-dione 11,11-dioxide (8)
Grey oil; yield: 44%; Rf (EtOAc) 0.74; IR (ATR) vmax 3388, 2974, 1717, 1470, 1364, 1225, 1032, 953 cm-1; 1H NMR (CD3OD, 500
MHz) δ 7.69 (1H, d, J = 2.8 Hz, H-1), 6.70 (1H, d, J = 2.8 Hz, H-3), 5.99 (1H, d, J = 9.5 Hz, H-6), 5.34–5.32 (1H, m, H-1''), 5.22–5.18
(1H, m, H-2'), 3.99–3.95 (2H, m, H2-9), 3.40–3.34 (2H, m, H2-10), 3.27–3.23 (1H, m, H2-1'A), 3.13–3.09 (1H, m, H2-1'B), 1.93 (3H, d, J
= 1.0 Hz, H3-3''), 1.70 (3H, s, H3-4'), 1.68 (3H, s, H3-5'), 1.67 (3H, d, J = 1.0 Hz, H3-4''); 13C NMR (CD3OD, 125 MHz) δ 178.8 (C-12),
177.8 (C-7), 152.7 (C-2), 148.3 (C-4a), 146.6 (C-7a), 141.7 (C-2''), 134.5 (C-12a), 133.5 (C-6a/C-3'), 133.4 (C-6a/C-3'), 132.9 (C-4),
123.3 (C-2'), 122.0 (C-3), 120.1 (C-1''), 119.4 (C-12b), 114.7 (C-11a), 114.6 (C-1), 68.4 (C-6), 49.9 (C-10), 40.7 (C-9), 29.4 (C-1'), 26.0
(C-4''), 25.9 (C-4'), 18.8 (C-3''), 17.8 (C-5'); (+)-HRESIMS [M+Na]+ m/z 478.1288 (calcd for C24H25NNaO6S, 478.1295).
4.4.19. 2-Hydroxy-3-(3-methylbut-2-en-1-yl)-6-(2-methylprop-1-en-1-yl)-10,11-dihydrobenzo[3,4]isochromeno[7,6-
b][1,4]thiazine-7,12(6H,9H)-dione 8,8-dioxide (9)
Following general procedure B, benzochromenedione 21 (0.014 g, 0.042 mmol) was reacted with hypotaurine (0.005 g, 0.042 mmol)
and the crude product purified to afford the title compound as a purple oil (0.006 g, 32%) as well as regioisomer 10 as a blue oil (0.008
g, 42%). Rf (EtOAc) 0.69; IR (ATR) vmax 3388, 2979, 1715, 1653, 1364, 1225, 1033, 953 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.70 (1H,
s, H-1), 6.71 (1H, s, H-4), 6.67 (1H, br s, H-11), 6.06 (1H, d, J = 9.8 Hz, H-6), 5.32–5.27 (2H, m, H-2', H-1''), 4.95 (1H, br s, OH),
4.11–4.08 (2H, m, H2-10), 3.33–3.31 (4H, m, H2-9, H2-1'), 1.92 (3H, d, J = 0.9 Hz, H3-3''), 1.77 (3H, s, H3-4'), 1.75 (3H, s, H3-5'), 1.66
(3H, d, J = 0.9 Hz, H3-4''); 13C NMR (CDCl3, 125 MHz) δ 179.4 (C-12), 175.3 (C-7), 148.9 (C-2), 148.3 (C-4a), 143.7 (C-11a), 142.2
(C-2''), 137.6 (C-6a), 135.7 (C-3'), 133.1 (C-3), 127.9 (C-12a), 120.6 (C-2'), 118.9 (C-4), 117.1 (C-1''), 114.8 (C-12b), 114.3 (C-1),
110.5 (C-7a), 67.9 (C-6), 48.9 (C-9), 40.1 (C-10), 29.9 (C-1'), 26.3 (C-4''), 26.0 (C-4'), 19.2 (C-3''), 18.1 (C-5'); (+)-HRESIMS [M+Na]+
m/z 478.1282 (calcd for C24H25NNaO6S, 478.1295).
4.4.20. 2-Hydroxy-3-(3-methylbut-2-en-1-yl)-6-(2-methylprop-1-en-1-yl)-9,10-dihydrobenzo[3,4]isochromeno[6,7-
b][1,4]thiazine-7,12(6H,8H)-dione 11,11-dioxide (10)
Blue oil; yield: 42%; Rf (EtOAc) 0.71; IR (ATR) vmax 3375, 2974, 1715, 1470, 1364, 1225, 1032, 953 cm-1; 1H NMR (CD3OD, 500
MHz) δ 7.86 (1H, s, H-1), 6.57 (1H, s, H-4), 5.93 (1H, d, J = 9.6 Hz, H-6), 5.36–5.34 (1H, m, H-1''), 5.31–5.28 (1H, m, H-2'), 3.98–
3.95 (2H, m, H2-9), 3.40–3.35 (2H, m, H2-10), 3.28–3.27 (2H, m, H2-1'), 1.90 (3H, d, J = 1.1 Hz, H3-3''), 1.75 (3H, s, H3-4'), 1.70 (3H,
s, H3-5'), 1.67 (3H, d, J = 1.1 Hz, H3-4''); 13C NMR (CD3OD, 125 MHz) δ 179.0 (C-12), 178.5 (C-7), 151.2 (C-2), 150.8 (C-4a), 146.7
(C-7a), 141.5 (C-2''), 136.8 (C-3), 134.3 (C-12a/C-3'), 134.2 (C-12a/C-3'), 131.9 (C-6a), 122.6 (C-2'), 120.0 (C-1''), 118.9 (C-4), 117.0
(C-12b), 116.0 (C-1), 114.1 (C-11a), 68.5 (C-6), 49.8 (C-10), 40.7 (C-9), 29.5 (C-1'), 26.0 (C-4''), 25.9 (C-4'), 18.8 (C-3''), 17.8 (C-5');
(+)-HRESIMS [M+Na]+ m/z 478.1279 (calcd for C24H25NNaO6S, 478.1295).
4.4.21. 2-Hydroxy-4-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)-6-((1E,5E)-2,6,10-trimethylundeca-1,5,9-trien-1-yl)-6H-
benzo[c]chromene-7,10-dione (28)
Following general procedure A, quinone 27 (1.42 g, 4.5 mmol) was reacted with triethylamine (2.30 g, 22.7 mmol), loaded onto a
silica gel column and left overnight. Elution with CH2Cl2 afforded the title compound as a purple oil (0.054 g, 3.8%) as well as
benzochromenedione 29 as a purple oil (0.032 g, 2.3%). Elution with CH2Cl2/MeOH (9:1) afforded benzochromenedione 32 (0.008 g,
0.8%), methoxybenzochromene 33 (as a 1:1 mixture with 34) (0.006 g), 34 (0.004 g, 0.4%) and dimer 35 (0.003 g, 0.2%). Rf (CH2Cl2)
0.42; IR (ATR) vmax 3056, 1712, 1363, 1221, 737, 729, 702 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.66 (1H, d, J = 3.0 Hz, H-1), 6.74
(1H, d, J = 3.0 Hz, H-3), 6.73 (2H, s, H-8, H-9), 6.04 (1H, d, J = 9.5 Hz, H-6), 5.33 (1H, dq, J = 9.5, 1.1 Hz, H-1''), 5.25 (1H, t, J = 7.4
Hz, H-2'), 5.12–5.08 (2H, m, H-6', H-10'), 5.05–5.02 (1H, m, H-9''), 4.95–4.92 (1H, m, H-5''), 4.54 (1H, s, OH), 3.24 (2H, d, J = 7.4 Hz,
H2-1'), 2.10–2.07 (2H, m, H2-5'), 2.04–2.00 (4H, m, H2-4', H2-9'), 2.00–1.96 (6H, m, H2-4'', H2-8', H2-8''), 1.96–1.92 (2H, m, H2-3''),
1.94 (3H, s, H3-14''), 1.89–1.86 (2H, m, H2-7''), 1.67 (6H, s, H3-11'', H3-12'), 1.66 (3H, s, H3-15'), 1.59 (6H, s, H3-13', H3-14'), 1.57 (3H,
s, H3-12''), 1.50 (3H, s, H3-13''); 13C NMR (CDCl3, 125 MHz) δ 187.0 (C-10), 185.2 (C-7), 149.8 (C-2), 147.0 (C-4a), 144.4 (C-2''),
137.1 (C-8/C-9), 137.0 (C-3'), 135.8 (C-8/C-9), 135.6 (C-6''), 135.2 (C-7'), 134.9 (C-6a), 132.3 (C-4), 131.5 (C-11', C-10''), 130.5 (C-
10a), 124.5 (C-10'), 124.4 (C-6'), 124.2 (C-9''), 123.4 (C-5''), 121.6 (C-2'), 120.2 (C-3), 118.3 (C-1''), 117.6 (C-10b), 112.5 (C-1), 67.3
(C-6), 39.91 (C-4'/C-3''), 39.86 (C-4'/C-3''), 39.82 (C-8'/C-7''), 39.78 (C-8'/C-7''), 28.3 (C-1'), 26.9 (C-5'/C-9'), 26.8 (C-5'/C-9'), 26.7 (C-
8''), 26.3 (C-4''), 25.8 (C-12', C-11''), 17.8 (C-13', C-12''), 17.4 (C-14''), 16.3 (C-15'), 16.2 (C-13''), 16.1 (C-14'); (–)-HRESIMS [M–H]–
m/z 621.3947 (calcd for C42H53O4, 621.3949).
4.4.22. 2-Hydroxy-3-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)-6-((1E,5E)-2,6,10-trimethylundeca-1,5,9-trien-1-yl)-6H-
benzo[c]chromene-7,10-dione (29)
9

Purple oil; yield: 2.3%; Rf (CH2Cl2) 0.56; IR (ATR) vmax 3493, 1710, 1420, 1361, 1222, 1090, 735 cm-1; 1H NMR (CDCl3, 400 MHz)
δ 7.80 (1H, s, H-1), 6.71 (2H, s, H-8, H-9), 6.69 (1H, s, H-4), 5.99 (1H, d, J = 9.6 Hz, H-6), 5.36 (1H, d, J = 9.6 Hz, H-1''), 5.32–5.28
(1H, m, H-2'), 5.11–5.07 (2H, m, H-6', H-10'), 5.07–5.01 (1H, m, H-9''), 4.96–4.93 (1H, m, H-5''), 4.86 (1H, s, OH), 3.33 (2H, t, J = 7.8
Hz, H2-1'), 2.13–2.10 (2H, m, H2-5'), 2.10–2.05 (4H, m, H2-4', H2-9'), 2.04–2.01 (2H, m, H2-4''), 2.01–1.97 (4H, m, H2-8', H2-8''), 1.96–
1.93 (2H, m, H2-3''), 1.92 (3H, d, J = 1.3 Hz, H3-14''), 1.91–1.86 (2H, m, H2-7''), 1.74 (3H, s, H3-15'), 1.67 (3H, s, H3-11''), 1.66 (3H, s,
H3-12'), 1.60 (6H, s, H3-13', H-14'), 1.57 (3H, s, H3-12''), 1.51 (3H, s, H3-13''); 13C NMR (CDCl3, 100 MHz) δ 187.1 (C-10), 185.1 (C-
7), 149.2 (C-4a), 149.1 (C-2), 144.0 (C-2''), 139.0 (C-3'), 137.0 (C-8/C-9), 135.9 (C-8/C-9), 135.65 (C-7'/C-6''), 135.58 (C-7'/C-6''),
133.7 (C-3), 133.4 (C-6a), 131.5 (C-11'/C-10''), 131.4 (C-11'/C-10''), 130.2 (C-10a), 124.6 (C-10'), 124.4 (C-6'), 123.9 (C-9''), 123.5 (C-
5''), 120.7 (C-2'), 118.6 (C-4), 118.1 (C-1''), 115.7 (C-10b), 115.1 (C-1), 67.6 (C-6), 39.9 (C-4', C-3''), 39.81 (C-8'/C-7''), 39.76 (C-8'/C-
7''), 29.7 (C-1'), 26.9 (C-5', C-9'), 26.6 (C-8''), 26.2 (C-4''), 25.8 (C-12', C-11''), 17.8 (C-13', C-12''), 17.4 (C-14''), 16.4 (C-15'), 16.19
(C-14'/C-13''), 16.15 (C-14'/C-13''); (–)-HRESIMS [M–H]– m/z 621.3951 (calcd for C42H53O4, 621.3949).
4.4.23. (E)-2-(4,8-Dimethylnona-3,7-dien-1-yl)-2-methyl-2H-chromen-6-ol (30)
Red oil; trace; 1H NMR (CDCl3, 400 MHz) δ 6.64 (1H, d, J = 8.6 Hz, H-8), 6.57 (1H, dd, J = 8.6, 2.9 Hz, H-7), 6.48 (1H, d, J = 2.9
Hz, H-5), 6.27 (1H, d, J = 9.9 Hz, H-4), 5.60 (1H, d, J = 9.9 Hz, H-3), 5.12–5.06 (2H, m, H-3', H-7'), 4.40 (1H, s, OH), 2.11–2.07 (2H,
m, H2-2'), 2.06–1.99 (2H, m, H2-6'), 1.97–1.93 (2H, m, H2-5'), 1.89–1.85 (2H, m, H2-1'), 1.67 (3H, s, H3-9'), 1.59 (6H, s, H3-10', H3-11'),
1.37 (3H, s, H3-9); HRESIMS [M+Na]+ m/z 335.1992 (calcd for C21H28NaO2, 335.1982). 1H NMR data were in agreement with
literature.17
4.4.24. 10-((E)-4,8-Dimethylnona-3,7-dien-1-yl)-10-methyl-5-((1E,5E)-2,6,10-trimethylundeca-1,5,9-trien-1-
yl)benzo[c]pyrano[2,3-g]chromene-1,4(5H,10H)-dione (31A and 31B)
Purple oil; trace; Rf (CH2Cl2) 0.89; IR (ATR) vmax 2976, 1738, 1378, 1264, 730, 704 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.79 (1H, s,
H-12), 6.71 (2H, s, H-9, H-10), 6.53 (1H, s, H-5), 6.29 (1H, d, J = 10.0 Hz, H-4), 5.98 (1H, d, J = 9.6 Hz, H-7), 5.73 (1H, d, J = 10.0
Hz, H-3), 5.36 (1H, dq, J = 9.6, 1.0 Hz, H-1''), 5.12–5.06 (2H, m, H-3', H-7'), 5.06–5.04 (1H, m, H-9''), 4.96–4.94 (1H, m, H-5''), 2.17–
2.03 (4H, m, H2-2', H2-6'), 2.03–1.99 (2H, m, H2-4''), 1.99–1.94 (4H, m, H2-5', H2-8''), 1.94–1.89 (5H, m, H2-3'', H3-14''), 1.89–1.86 (2H,
m, H2-7''), 1.70–1.62 (11H, m, H2-1', H3-9', H3-11', H3-11''), 1.59 (3H, s, H3-10'), 1.57 (3H, s, H3-12''), 1.50 (3H, s, H3-13''), 1.40 (3H, br
s, H3-13); 13C NMR (CDCl3, 125 MHz, deduced from HSQC and HMBC) δ 186.9 (C-11), 186.2 (C-8), 148.8 (C-5a), 147.9 (C-12a),
144.1 (C-2''), 137.1 (C-9/C-10), 135.9 (C-9/C-10), 135.8 (C-4', C-6''), 133.7 (C-3, C-7a), 131.5 (C-8', C-10''), 128.4 (C-11a), 124.5 (C-
7'), 124.4 (C-3', C-9''), 123.6 (C-5''), 122.5 (C-4, C-4a), 118.3 (C-1''), 117.0 (C-11b), 115.9 (C-12), 114.8 (C-5), 78.5 (C-2), 67.6 (C-7),
41.2 (C-1'), 39.8 (C-5', C-3'', C-7''), 26.9 (C-6', C-8''), 26.8 (C-13), 26.2 (C-4''), 25.8 (C-9', C-11''), 22.8 (C-2'), 17.8 (C-10', C-12''), 17.3
(C-14''), 16.1 (C-11', C-13''); HRESIMS [M+Na]+ m/z 643.3740 (calcd for C42H52NaO4, 643.3758).
Purple oil; trace; Rf (CH2Cl2) 0.83; IR (ATR) vmax 2976, 1738, 1378, 1264, 730, 704 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.78 (1H, s,
H-12), 6.72 (2H, s, H-9, H-10), 6.53 (1H, s, H-5), 6.29 (1H, d, J = 9.8 Hz, H-4), 5.98 (1H, d, J = 9.4 Hz, H-7), 5.72 (1H, d, J = 9.8 Hz,
H-3), 5.36 (1H, dq, J = 9.4, 1.0 Hz, H-1''), 5.12–5.06 (2H, m, H-3', H-7'), 5.06–5.04 (1H, m, H-9''), 4.96–4.94 (1H, m, H-5''), 2.17–2.03
(4H, m, H2-2', H2-6'), 2.03–1.99 (2H, m, H2-4''), 1.99–1.94 (4H, m, H2-5', H2-8''), 1.94–1.89 (7H, m, H2-1', H2-3'', H3-14''), 1.89–1.86
(2H, m, H2-7''), 1.67 (3H, br s, H3-9'), 1.66 (3H, br s, H3-11''), 1.59 (6H, s, H3-10', H3-11'), 1.57 (3H, s, H3-12''), 1.51 (3H, s, H3-13''),
1.41 (3H, br s, H3-13); 13C NMR (CDCl3, 125 MHz, deduced from HSQC and HMBC) δ 186.9 (C-11), 186.2 (C-8), 148.8 (C-5a), 147.9
(C-12a), 144.2 (C-2''), 137.1 (C-9/C-10), 135.9 (C-9/C-10), 135.8 (C-4', C-6''), 133.7 (C-3, C-7a), 131.6 (C-8', C-10''), 128.4 (C-11a),
124.5 (C-7'), 124.4 (C-3', C-9''), 123.6 (C-5''), 122.5 (C-4, C-4a), 119.7 (C-1''), 117.0 (C-11b), 115.9 (C-12), 114.8 (C-5), 78.5 (C-2),
67.6 (C-7), 39.8 (C-5', C-3'', C-7''), 38.5 (C-1'), 26.9 (C-8''), 26.8 (C-6'), 26.7 (C-13), 26.2 (C-4''), 25.8 (C-9', C-11''), 22.6 (C-2'), 17.8
(C-10', C-12''), 17.4 (C-14''), 16.1 (C-11', C-13''); HRESIMS [M+Na]+ m/z 643.3740 (calcd for C42H52NaO4, 643.3758).
4.4.25. 2-Hydroxy-6-((1E,5E)-2,6,10-trimethylundeca-1,5,9-trien-1-yl)-6H-benzo[c]chromene-7,10-dione (32)
Red oil; yield: 0.8%; Rf (CH2Cl2) 0.21; IR (ATR) vmax 3233, 2916, 1638, 1599, 1335, 1315, 1276, 1229, 1200, 1000, 842, 827, 766
cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.86 (1H, d, J = 2.9 Hz, H-1), 6.85 (1H, dd, J = 8.6, 2.9 Hz, H-3), 6.81 (1H, d, J = 8.6 Hz, H-4),
6.74 (2H, s, H-8, H-9), 6.01 (1H, d, J = 9.5 Hz, H-6), 5.34 (1H, dq, J = 9.5, 1.1 Hz, H-1'), 5.05–5.02 (1H, m, H-9'), 5.01 (1H, br s, OH),
4.95–4.93 (1H, m, H-5'), 2.04–1.98 (2H, m, H2-4'), 1.98–1.94 (4H, m, H2-3', H2-8'), 1.93 (3H, d, J = 1.1 Hz, H3-14'), 1.89–1.86 (2H, m,
H2-7'), 1.66 (3H, s, H3-11'), 1.57 (3H, s, H3-12'), 1.50 (3H, s, H3-13'); 13C NMR (CDCl3, 125 MHz) δ 187.0 (C-10), 185.1 (C-7), 150.4
(C-2), 149.0 (C-4a), 144.5 (C-2'), 137.1 (C-8/C-9), 135.8 (C-8/C-9), 135.6 (C-6'), 134.9 (C-6a), 131.5 (C-10'), 130.0 (C-10a), 124.4 (C-
9'), 123.4 (C-5'), 119.9 (C-3), 118.7 (C-4), 117.8 (C-10b, C-1'), 115.0 (C-1), 67.6 (C-6), 39.8 (C-3'), 39.7 (C-7'), 26.8 (C-8'), 26.1 (C-4'),
25.8 (C-11'), 17.8 (C-12'), 17.4 (C-14'), 16.2 (C-13'); HRESIMS [M+Na]+ m/z 441.2043 (calcd for C27H30NaO4, 441.2036).
4.4.26. 2-Hydroxy-8-methoxy-6-((1E,5E)-2,6,10-trimethylundeca-1,5,9-trien-1-yl)-6H-benzo[c]chromene-7,10-dione (33)
Purple oil; Rf (CH2Cl2) 0.11; IR (ATR) vmax 3412, 2966, 2915, 2852, 1636, 1602, 1569, 1441, 1382, 1313, 1225, 1192, 1131, 1002,
824, 808 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.93 (1H, d, J = 2.9 Hz, H-1), 6.86 (1H, dd, J = 8.9, 2.9 Hz, H-3), 6.80 (1H, d, J = 8.9 Hz,
H-4), 6.04 (1H, d, J = 9.5 Hz, H-6), 5.91 (1H, s, H-9), 5.34 (1H, dq, J = 9.5, 1.1 Hz, H-1'), 5.06–5.02 (2H, m, H-9'), 4.96–4.93 (1H, m,
H-5'), 3.84 (3H, s, OMe), 2.02–2.00 (2H, m, H2-4'), 1.99–1.94 (4H, m, H2-3', H2-8'), 1.93 (3H, d, J = 1.1 Hz, H3-14'), 1.89–1.86 (2H, m,
H2-7'), 1.66 (3H, s, H3-11'), 1.57 (3H, s, H3-12'), 1.50 (3H, s, H3-13'); 13C NMR (CDCl3, 125 MHz) δ 187.3 (C-10), 179.9 (C-7), 158.2
(C-8), 150.5 (C-2), 149.3 (C-4a), 144.3 (C-2'), 135.6 (C-6'), 133.3 (C-6a), 131.5 (C-10'), 130.4 (C-10a), 124.5 (C-9'), 123.4 (C-5'),
120.2 (C-3), 118.7 (C-4), 117.9 (C-10b), 117.7 (C-1'), 115.5 (C-1), 108.0 (C-9), 67.8 (C-6), 56.5 (OMe), 39.9 (C-3'), 39.7 (C-7'), 26.9
(C-8'), 26.1 (C-4'), 25.8 (C-11'), 17.8 (C-12'), 17.4 (C-14'), 16.2 (C-13'); (–)-HRESIMS [M–H]– m/z 447.2185 (calcd for C28H31O5,
447.2177).
4.4.27. 2-Hydroxy-9-methoxy-6-((1E,5E)-2,6,10-trimethylundeca-1,5,9-trien-1-yl)-6H-benzo[c]chromene-7,10-dione (34)
10

Purple oil; yield: 0.4%; Rf (CH2Cl2) 0.08; IR (ATR) vmax 3412, 2966, 2915, 2852, 1636, 1602, 1569, 1441, 1382, 1313, 1225, 1192,
1131, 1002, 824, 808 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.86 (1H, d, J = 2.8 Hz, H-1), 6.83 (1H, dd, J = 8.8, 2.8 Hz, H-3), 6.80 (1H,
d, J = 8.8 Hz, H-4), 6.03 (1H, d, J = 9.5 Hz, H-6), 5.90 (1H, s, H-8), 5.32 (1H, dq, J = 9.5, 1.0 Hz, H-1'), 5.06–5.02 (2H, m, H-9'), 4.95–
4.93 (1H, m, H-5'), 4.83 (1H, br s, OH), 3.84 (3H, s, OMe), 2.04–2.00 (2H, m, H2-4'), 1.99–1.95 (4H, m, H2-3', H2-8'), 1.92 (3H, d, J =
1.0 Hz, H3-14'), 1.89–1.86 (2H, m, H2-7'), 1.66 (3H, s, H3-11'), 1.57 (3H, s, H3-12'), 1.50 (3H, s, H3-13'); 13C NMR (CDCl3, 125 MHz) δ
184.8 (C-7), 181.2 (C-10), 158.5 (C-9), 150.2 (C-2), 148.7 (C-4a), 144.5 (C-2'), 135.7 (C-6a), 135.6 (C-6'), 131.5 (C-10'), 128.7 (C-
10a), 124.4 (C-9'), 123.4 (C-5'), 119.5 (C-3), 118.7 (C-4), 117.9 (C-1'), 117.8 (C-10b), 114.9 (C-1), 106.9 (C-8), 67.7 (C-6), 56.6
(OMe), 39.9 (C-3'), 39.8 (C-7'), 26.9 (C-8'), 26.1 (C-4'), 25.8 (C-11'), 17.8 (C-12'), 17.4 (C-14'), 16.2 (C-13'); (–)-HRESIMS [M–H]–
m/z 447.2189 (calcd for C28H31O5, 447.2177).
4.4.28. 5,14-Dihydroxy-1,8-bis((1E,5E)-2,6,10-trimethylundeca-1,5,9-trien-1-yl)benzo[c]chromeno[3,4-h]chromene-9,12(1H,8H)-
dione (35)
Purple oil; yield: 0.2%; Rf (CH2Cl2) 0.34; IR (ATR) vmax 3351, 2916, 2853, 1647, 1567, 1420, 1383, 1304, 1274, 1234, 1195, 1092,
1000, 839, 816, 760 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.84 (1H, d, J = 2.9 Hz, H-6), 7.73 (1H, s, H-13), 6.81 (1H, d, J = 8.6 Hz, H-
3), 6.76 (1H, s, H-10/H-11), 6.75 (1H, s, H-10/H-11), 6.70 (1H, dd, J = 8.6, 2.9 Hz, H-4), 6.14 (2H, d, J = 9.3 Hz, H-1, H-8), 5.32 (1H,
d, J = 9.3 Hz, H-1'), 5.24 (1H, d, J = 9.3 Hz, H-1''), 5.04–4.99 (2H, m, H-9', H-9''), 4.96–4.93 (1H, m, H-5'), 4.85–4.82 (1H, m, H-5''),
4.80 (1H, br s, 14-OH), 4.48 (1H, br s, 5-OH), 2.02–1.99 (2H, m, H2-4'), 1.97–1.91 (6H, m, H2-3', H2-8', H2-8''), 1.94 (3H, d, J = 0.9 Hz,
H3-14'), 1.91–1.88 (2H, m, H2-4''), 1.88–1.84 (4H, m, H2-3'', H2-7'), 1.87 (3H, d, J = 1.0 Hz, H3-14''), 1.81–1.78 (2H, m, H2-7''), 1.65
(3H, s, H3-11'/ H3-11''), 1.64 (3H, s, H3-11'/ H3-11''), 1.56 (6H, s, H3-12', H3-12''), 1.50 (3H, s, H3-13'), 1.43 (3H, s, H3-13''); 13C NMR
(CDCl3, 125 MHz) δ 186.8 (C-12), 184.9 (C-9), 149.6 (C-5), 147.2 (C-2a), 146.6 (C-6c), 145.3 (C-14/C-2''), 145.2 (C-14/C-2''), 142.2
(C-2'), 137.2 (C-10/C-11), 135.9 (C-10/C-11), 135.6 (C-6'), 135.5 (C-6''), 134.8 (C-8a, C-14a), 131.3 (C-10', C-10''), 130.1 (C-12a),
128.4 (C-6b), 124.59 (C-9'/C-9''), 124.56 (C-9'/C-9''), 123.6 (C-5'), 123.3 (C-5''), 121.6 (C-6a), 120.6 (C-1'), 118.4 (C-3), 117.9 (C-1''),
117.3 (C-12b), 116.1 (C-4), 115.6 (C-6), 115.1 (C-13), 69.0 (C-8), 67.4 (C-1), 39.82 (C-3'/C-3''), 39.79 (C-3'/C-3''), 39.7 (C-7''), 39.6
(C-7'), 26.8 (C-8', C-8''), 26.2 (C-4'), 26.1 (C-4''), 25.8 (C-11', C-11''), 17.8 (C-12', C-12''), 17.5 (C-14''), 17.1 (C-14'), 16.2 (C-13'), 16.0
(C-13''); HRESIMS [M+Na]+ m/z 751.3969 (calcd for C48H56NaO6, 751.3969).
4.4.29. 2-Hydroxy-4-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)-6-((1E,5E)-2,6,10-trimethylundeca-1,5,9-trien-1-yl)-
10,11-dihydrobenzo[3,4]isochromeno[7,6-b][1,4]thiazine-7,12(6H,9H)-dione 8,8-dioxide (11)
Following general procedure B, benzochromenedione 28 (0.045 g, 0.072 mmol) was reacted with hypotaurine (0.008 g, 0.072 mmol)
and the crude product purified to afford the title compound as a red oil (0.006 g, 12%) as well as regioisomer 12 as a green oil (0.008 g,
15%). Rf (EtOAc) 0.79; IR (ATR) vmax 3423, 2927, 1708, 1677, 1537, 1462, 1394, 1378, 1349, 1326, 1115, 957, 822, 786, 746, 727,
713 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.56 (1H, d, J = 2.6 Hz, H-1), 6.72 (1H, br s, H-11), 6.71 (1H, d, J = 2.6 Hz, H-3), 6.11 (1H, d,
J = 9.6 Hz, H-6), 5.27–5.21 (2H, m, H-2', H-1''), 5.12–5.08 (2H, m, H-6', H-10'), 5.06–5.03 (1H, m, H-9''), 4.96–4.94 (1H, m, H-5''),
4.10–4.07 (2H, m, H2-10), 3.34–3.28 (2H, m, H2-9), 3.28–3.24 (1H, m, H2-1'A), 3.21–3.17 (1H, m, H2-1'B), 2.11–2.08 (2H, m, H2-5'),
2.07–2.03 (2H, m, H2-9'), 2.03–2.00 (2H, m, H2-4'), 2.00–1.96 (4H, m, H2-8', H2-8''), 1.96–1.90 (4H, m, H2-3'', H2-4''), 1.94 (3H, d, J =
0.9 Hz, H3-14''), 1.90–1.86 (2H, m, H2-7''), 1.67 (3H, s, H3-11''), 1.66 (3H, s, H3-12'), 1.65 (3H, s, H3-15'), 1.59 (6H, br s, H3-13', H3-
14'), 1.57 (3H, s, H3-12''), 1.50 (3H, s, H3-13''); 13C NMR (CDCl3, 125 MHz) δ 179.2 (C-12), 175.3 (C-7), 149.7 (C-2), 146.2 (C-4a),
145.6 (C-2''), 143.9 (C-11a), 138.9 (C-6a), 137.2 (C-3'), 135.6 (C-6''), 135.2 (C-7'), 132.5 (C-4), 131.5 (C-10''), 131.4 (C-11'), 128.2 (C-
12a), 124.5 (C-6', C-10'), 124.2 (C-9''), 123.6 (C-5''), 121.4 (C-2'), 120.0 (C-3), 116.9 (C-1''), 116.8 (C-12b), 111.7 (C-1), 110.6 (C-7a),
67.6 (C-6), 48.8 (C-9), 40.1 (C-10), 39.92 (C-4', C-3''), 39.86 (C-8'), 39.8 (C-7''), 28.2 (C-1'), 26.9 (C-5', C-9'), 26.7 (C-8''), 26.3 (C-4''),
25.8 (C-12', C-11''), 17.8 (C-13', C-12''), 17.6 (C-14''), 16.3 (C-14'), 16.2 (C-15'), 16.1 (C-13''); (+)-HRESIMS [M+Na]+ m/z 750.3808
(calcd for C44H57NNaO6S, 750.3799).
4.4.30. 2-Hydroxy-4-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)-6-((1E,5E)-2,6,10-trimethylundeca-1,5,9-trien-1-yl)-9,10-
dihydrobenzo[3,4]isochromeno[6,7-b][1,4]thiazine-7,12(6H,8H)-dione 11,11-dioxide (12)
Green oil; yield: 15%; Rf (EtOAc) 0.86; IR (ATR) vmax 3427, 2927, 1708, 1677, 1535, 1462, 1390, 1377, 1329, 1113, 823, 784, 743,
727, 713 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.74 (1H, d, J = 3.0 Hz, H-1), 6.71 (1H, d, J = 3.0 Hz, H-3), 6.62 (1H, br s, H-8), 5.91
(1H, d, J = 9.5 Hz, H-6), 5.23 (1H, dq, J = 9.5, 0.9 Hz, H-1''), 5.15–5.13 (1H, m, H-2'), 5.04–5.00 (2H, m, H-6', H-10'), 4.98–4.96 (1H,
m, H-9''), 4.89–4.86 (1H, m, H-5''), 4.05–4.01 (2H, m, H2-9), 3.31–3.28 (2H, m, H2-10), 3.18–3.13 (1H, m, H2-1'A), 3.11–3.06 (1H, m,
H2-1'B), 2.04–1.97 (4H, m, H2-5', H2-9'), 1.96–1.93 (2H, m, H2-4'), 1.91–1.86 (8H, m, H2-8', H2-3'', H2-4'', H2-8''), 1.83 (3H, d, J = 0.9
Hz, H3-14''), 1.82–1.80 (2H, m, H2-7''), 1.60 (3H, s, H3-11''), 1.59 (3H, s, H3-12'), 1.58 (3H, s, H3-15'), 1.52 (6H, br s, H3-13', H3-12''),
1.50 (3H, s, H3-14'), 1.44 (3H, s, H3-13''); 13C NMR (CDCl3, 125 MHz) δ 177.7 (C-12), 177.5 (C-7), 150.4 (C-2), 148.4 (C-4a), 144.8
(C-2''), 143.7 (C-7a), 137.1 (C-3'), 135.7 (C-6''), 135.3 (C-7'), 133.6 (C-12a), 132.2 (C-4), 131.6 (C-10''), 131.4 (C-11'), 130.8 (C-6a),
124.5 (C-6'/C-10'), 124.4 (C-6'/C-10'), 124.2 (C-9''), 123.4 (C-5''), 122.1 (C-3), 121.4 (C-2'), 118.2 (C-1''), 117.5 (C-12b), 113.8 (C-1),
111.3 (C-11a), 67.1 (C-6), 49.1 (C-10), 40.2 (C-9), 39.9 (C-4'), 39.85 (C-3''), 39.79 (C-8'/C-7''), 39.75 (C-8'/C-7''), 28.2 (C-1'), 26.9 (C-
5'/C-9'), 26.8 (C-5'/C-9'), 26.7 (C-8''), 26.3 (C-4''), 25.8 (C-12', C-11''), 17.8 (C-13', C-12''), 17.4 (C-14''), 16.3 (C-14'), 16.2 (C-15', C-
13''); (+)-HRESIMS [M+Na]+ m/z 750.3820 (calcd for C44H57NNaO6S, 750.3799).
4.4.31. 2-Hydroxy-3-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)-6-((1E,5E)-2,6,10-trimethylundeca-1,5,9-trien-1-yl)-
10,11-dihydrobenzo[3,4]isochromeno[7,6-b][1,4]thiazine-7,12(6H,9H)-dione 8,8-dioxide (13)
Following general procedure B, benzochromenedione 29 (0.022 g, 0.035 mmol) was reacted with hypotaurine (0.004 g, 0.035 mmol)
and the crude product purified to afford the title compound as a purple oil (0.006 g, 23%) as well as regioisomer 14 as a blue oil (0.008
g, 31%). Rf (EtOAc) 0.74; IR (ATR) vmax 3427, 2927, 1708, 1677, 1534, 1462, 1392, 1378, 1350, 1116, 822, 783, 746, 729, 711 cm-1;
1H NMR (CDCl3, 500 MHz) δ 7.70 (1H, s, H-1), 6.69 (2H, br s, H-4, H-11), 6.07 (1H, d, J = 9.6 Hz, H-6), 5.31–5.28 (2H, m, H-2', H-
1''), 5.10–5.08 (2H, m, H-6', H-10'), 5.07–5.03 (1H, m, H-9''), 4.98–4.95 (1H, m, H-5''), 4.11–4.07 (2H, m, H2-10), 3.34–3.29 (4H, m,
H2-9, H2-1'), 2.14–2.10 (2H, m, H2-5'), 2.10–2.06 (2H, m, H2-4'), 2.06–2.03 (2H, m, H2-9'), 2.01–1.97 (4H, m, H2-8', H2-8''), 1.97–1.90
11

(4H, m, H2-3'', H2-4''), 1.93 (3H, d, J = 0.9 Hz, H3-14''), 1.90–1.86 (2H, m, H2-7''), 1.75 (3H, s, H3-15'), 1.67 (3H, s, H3-11''), 1.66 (3H,
s, H3-12'), 1.59 (6H, br s, H3-13', H3-14'), 1.58 (3H, s, H3-12''), 1.51 (3H, s, H3-13''); 13C NMR (CDCl3, 125 MHz) δ 179.3 (C-12), 175.3
(C-7), 149.1 (C-4a), 148.3 (C-2), 145.5 (C-2''), 143.7 (C-11a), 139.5 (C-3'), 137.6 (C-6a), 135.8 (C-6''), 135.6 (C-7'), 133.0 (C-3, C-11'),
131.4 (C-10''), 127.9 (C-12a), 124.5 (C-6', C-10'), 123.8 (C-9''), 123.6 (C-5''), 120.5 (C-2'), 118.9 (C-4), 116.8 (C-1''), 114.9 (C-12b),
114.4 (C-1), 110.7 (C-7a), 67.8 (C-6), 48.8 (C-9), 40.1 (C-10), 40.0 (C-3''), 39.9 (C-4'), 39.80 (C-8'/C-7''), 39.76 (C-8'/C-7''), 29.8 (C-
1'), 26.9 (C-5'/C-9'), 26.8 (C-5'/C-9'), 26.6 (C-8''), 26.2 (C-4''), 25.8 (C-12', C-11''), 17.8 (C-13', C-12''), 17.6 (C-14''), 16.4 (C-14'), 16.2
(C-15'), 16.1 (C-13''); (+)-HRESIMS [M+Na]+ m/z 750.3814 (calcd for C44H57NNaO6S, 750.3799).
4.4.32. 2-Hydroxy-3-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)-6-((1E,5E)-2,6,10-trimethylundeca-1,5,9-trien-1-yl)-9,10-
dihydrobenzo[3,4]isochromeno[6,7-b][1,4]thiazine-7,12(6H,8H)-dione 11,11-dioxide (14)
Blue oil; yield: 31%; Rf (EtOAc) 0.88; IR (ATR) vmax 3431, 2923, 1710, 1675, 1539, 1466, 1390, 1375, 1120, 819, 783, 746, 730,
716 cm-1; 1H NMR (CDCl3, 500 MHz) δ 7.98 (1H, s, H-1), 6.65 (1H, s, H-4), 6.56 (1H, br s, H-8), 5.96 (1H, d, J = 9.7 Hz, H-6), 5.36
(1H, dq, J = 9.7, 0.9 Hz, H-1''), 5.30–5.27 (1H, m, H-2'), 5.11–5.09 (1H, m, H-6'), 5.09–5.06 (1H, m, H-10'), 5.06–5.04 (1H, m, H-9''),
4.97–4.95 (1H, m, H-5''), 4.09–4.05 (2H, m, H2-9), 3.33–3.30 (4H, m, H2-10, H2-1'), 2.13–2.08 (2H, m, H2-5'), 2.08–2.04 (4H, m, H2-4',
H2-9'), 2.04–2.00 (2H, m, H2-4''), 2.00–1.93 (6H, m, H2-8', H2-3'', H2-8''), 1.93–1.89 (2H, m, H2-7''), 1.89 (3H, d, J = 0.9 Hz, H3-14''),
1.71 (3H, s, H3-15'), 1.67 (3H, s, H3-11''), 1.66 (3H s, H3-12'), 1.59 (6H, br s, H3-13', H3-14'), 1.58 (3H, s, H3-12''), 1.52 (3H, s, H3-13'');
13C NMR (CDCl3, 125 MHz) δ 177.8 (C-12), 177.2 (C-7), 150.7 (C-4a), 149.4 (C-2), 144.5 (C-2''), 143.6 (C-7a), 138.9 (C-3'), 136.1 (C-
3), 135.7 (C-6''), 135.6 (C-7'), 133.3 (C-12a), 131.6 (C-10''), 131.4 (C-11'), 129.5 (C-6a), 124.5 (C-6'/C-10'), 124.4 (C-6'/C-10'), 124.0
(C-9''), 123.4 (C-5''), 120.3 (C-2'), 118.5 (C-4), 117.9 (C-1''), 116.0 (C-1), 115.6 (C-12b), 111.4 (C-11a), 67.3 (C-6), 49.1 (C-10), 40.1
(C-9), 39.9 (C-4'), 39.80 (C-3''), 39.76 (C-8', C-7''), 29.5 (C-1'), 26.83 (C-5'/C-9'), 26.81 (C-5'/C-9'), 26.6 (C-8''), 26.2 (C-4''), 25.9 (C-
12', C-11''), 17.8 (C-13', C-12''), 17.3 (C-14''), 16.4 (C-14'), 16.18 (C-15'/C-13''), 16.17 (C-15'/C-13''); (+)-HRESIMS [M+Na]+ m/z
750.3812 (calcd for C44H57NNaO6S, 750.3799).
4.5 In vitro anti-plasmodial assays
In vitro activity against the erythrocytic stage of P. falciparum was determined using a 3H-hypoxanthine incorporation assay,18 using
a strain susceptible to known antimalarial drugs (P. falciparum NF54) and all the test compounds were compared for activity with the
standard drug chloroquine. Compounds were dissolved in DMSO at 10 mg/mL and added to parasite cultures incubated in RPMI 1640
medium without hypoxanthine, supplemented with HEPES (5.94 g/L), NaHCO3 (2.1 g/L), neomycin (100 U/mL), AlbumaxR (5 g/L)
and washed human red cells Aþ at 2.5% haematocrit (0.3% parasitaemia). Serial drug dilutions of eleven 3-fold dilution steps covering
a range from 100 to 0.002 mg/mL were prepared. The 96-well plates were incubated in a humidified atmosphere at 37 ºC; 4% CO2, 3%
O2, 93% N2. After 48 h 50 mL of 3H-hypoxanthine (¼0.5 mCi) was added to each well of the plate. The plates were incubated for a
further 24 h under the same conditions. The plates were then harvested with a Betaplate cell harvester (Wallac, Zurich, Switzerland),
and the red blood cells transferred onto a glass fibre filter then washed with distilled water. The dried filters were inserted into a plastic
foil with 10 mL of scintillation fluid, and counted in a Betaplate liquid scintillation counter (Wallac, Zürich, Switzerland). IC50 values
were calculated from sigmoidal inhibition curves by linear regression using Microsoft Excel.19
4.6 In vitro cytotoxicity against L6
Assays were performed in 96-well microtiter plates, each well containing 100 µL of RPMI 1640 medium supplemented with 1% L-
glutamine (200 mM) and 10% fetal bovine serum, and 4 × 104 L6 cells (a primary cell line derived from rat skeletal myoblasts). Serial
drug dilutions of seven 3-fold dilution steps covering a range from 90 to 0.123 µg/mL were prepared. After 72 h of incubation, the
plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. Alamar Blue solution (10
µL) was then added to each well and the plates incubated for another 2 h. Then the plates were read with a Spectramax Gemini XS
microplate fluorometer using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. Data were analysed using the
microplate reader software Softmax Pro. Podophyllotoxin was the reference drug used.20
4.7 In vitro FTase inhibition assays
4.7.1 Inhibition of human FTase
Assays were conducted on 96-well plates and read on a Wallac Victor fluorimeter from Perkin-Elmer. To each well, 20µL of
farnesyl pyrophosphate (10 µM) was added to 180 µL of a solution containing 2 µL of varied concentrations of potential inhibitors
(dissolved in DMSO) and 178 µL of a solution composed by 5 µL of partially purified human recombinant FTase (1.5 mg/mL) and 1.0
mL of dansyl-GCVLS peptide in the following buffer: 5.8 mM DTT, 6 mM MgCl2, 12 µM ZnCl2 and 0.18% (w/v) octyl-D-
glucopyranoside, 53 mM Tris/HCl, pH 7.5.21 Fluorescence development was recorded for 15 min (0.7 seconds per well, 15 repeats) at
30 °C with an excitation filter at 340 nm and an emission filter at 486 nm. Each measurement was performed in triplicate.
4.7.2 Inhibition of T. brucei FTase
T. brucei assays were conducted on 96-well plates, as described for human FTase with the dansylated peptide dansyl-GCAIM and
the solution contains 15 µL of partially purified recombinant TbFTase (1.0 mg/mL) in 1 mL peptide solution.22
12

4.8 In vitro anti-bacterial assays
4.8.1 Strains
Reference strains used were Escherichia coli ATCC 25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 25923 and were
purchased from Pasteur Institute (France). S. Intermedius 1051997 was provided by VIRBAC Company (Carros, France).
4.8.2 Anti-bacterial evaluation
Anti-bacterial activity of the compounds was studied by determination of minimal inhibitory concentrations (MIC) according to the
NCCLS guidelines M7-A3 using the microbroth dilution methods. The bacterial strains were grown on trypticase soy agar (Becton
Dickinson) at 37 °C for 24 h. Inocula were prepared in Mueller-Hinton broth by measuring the turbidity at 623 nm to obtain 2-6 105
CFU/mL.
Anti-bacterial activities of the compounds were determined by using a broth microdilution method performed in sterile 96-well
microplates. All compounds were solubilized in DMSO at a concentration of 5 mg/mL and were transferred to each microplate well (in
all cases concentrations of the desired molecules in DMSO do not exceed 2% of the total proportion), to obtain a two-fold serial dilution
in 100 µL of broth and 100 µL of inoculum containing 2-6 105 CFU of each bacteria and yeast were added to each well. Several wells
were reserved for positive controls, inoculum viability and solvent effect. After 24 h incubation, MIC was defined for each agent from
duplicate observations as the lowest concentration of compound allowing no visible growth.23
Acknowledgments
We thank Dr. Michael Schmitz and Tony Chen for their assistance with the NMR and mass spectrometric data.
Supplementary Material
Supplementary data (comprised of copies of 1H and 13C NMR spectra) associated with this article can be found, in the online version,
at http://
References and notes
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