Strategies towards potent trypanocidal drugs: Application of Rh-catalyzed [2?+?2?+?2] cycloadditions, sulfonyl phthalide annulation and nitroalkene reactions for the synthesis of substituted quinones and their evaluation against Trypanosoma cruzi

Article abstract of DOI:10.1016/j.bmc.2020.115565

Rhodium-catalyzed [2 + 2 + 2] cycloadditions, sulfonyl phthalide annulations and nitroalkene reactions have been employed for the synthesis of 56 quinone-based compounds. These were evaluated against Trypanosoma cruzi, the parasite that causes Chagas disease. The reactions described here are part of a program that aims to utilize modern, versatile and efficient synthetic methods for the one or two step preparation of trypanocidal compounds. We have identified 9 compounds with potent activity against the parasite; 3 of these were 30-fold more potent than benznidazole (Bz), a drug used for the treatment of Chagas disease. This article provides a comprehensive outline of reactions involving over 120 compounds aimed at the discovery of new quinone-based frameworks with activity against T. cruzi.

Full text of DOI:10.1016/j.bmc.2020.115565

Journal Pre-proofs
Strategies towards potent trypanocidal drugs: application of Rh-catalyzed
[2+2+2] cycloadditions, sulfonyl phthalide annulation and nitroalkene reac‐
tions for the synthesis of substituted quinones and their evaluation against
Trypanosoma cruzi
James M. Wood, Nishikant S. Satam, Renata G. Almeida, Vinicius S.
Cristani, Dênis P. Lima, Luiza D. Pereira, Kelly Salomão, Rubem F.S.
Menna-Barreto, Irishi N.N. Namboothiri, John F. Bower, Eufrânio N. da Silva
Júnior
PII:
DOI:
Reference:
S0968-0896(20)30395-3
https://doi.org/10.1016/j.bmc.2020.115565
BMC 115565
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
26 March 2020
21 May 2020
22 May 2020
Please cite this article as: Wood, J.M., Satam, N.S., Almeida, R.G., Cristani, V.S., Lima, D.P., Pereira, L.D.,
Salomão, K., Menna-Barreto, R.F.S., Namboothiri, I.N.N., Bower, J.F., da Silva Júnior, E.N., Strategies towards
potent trypanocidal drugs: application of Rh-catalyzed [2+2+2] cycloadditions, sulfonyl phthalide annulation and
nitroalkene reactions for the synthesis of substituted quinones and their evaluation against Trypanosoma cruzi,
Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115565
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Strategies towards potent trypanocidal drugs: application of Rh-catalyzed [2+2+2]
cycloadditions, sulfonyl phthalide annulation and nitroalkene reactions for the
synthesis of substituted quinones and their evaluation against Trypanosoma cruzi
James M. Wood,a Nishikant S. Satam,b Renata G. Almeida,c Vinicius S. Cristani,d Dênis
P. Lima,d Luiza D. Pereira,e Kelly Salomão,e Rubem F. S. Menna-Barreto,e Irishi N. N.
Namboothiri,b* John F. Bowera* and Eufrânio N. da Silva Júniorc*
aSchool of Chemistry, University of Bristol, Bristol, BS8 1TS, UK; bDepartment of
Chemistry, Indian Institute of Technology Bombay, Mumbai, 400 076, India; cInstitute
of Exact Sciences, Department of Chemistry, Federal University of Minas Gerais, CEP
31270-901, Belo Horizonte, MG, Brazil; dInstitute of Chemistry, Federal University of
Mato Grosso do Sul, Campo Grande, MS, 79074-460, Brazil; eLaboratory of Cellular
Biology, IOC, FIOCRUZ, Rio de Janeiro, RJ, 21045-900, Brazil.
Corresponding authors: I.N.N. Namboothiri, E-mail: irishi@chem.iitb.ac.in; J.F. Bower,
E-mail: john.bower@bris.ac.uk and E.N. da Silva Júnior: E-mail: eufranio@ufmg.br
Abstract: Rhodium-catalyzed [2+2+2] cycloadditions, sulfonyl phthalide annulations
and nitroalkene reactions have been employed for the synthesis of 56 quinone-based
compounds. These were evaluated against Trypanosoma cruzi, the parasite that causes
Chagas disease. The reactions described here are part of a program that aims to utilize
modern, versatile and efficient synthetic methods for the one or two step preparation of
trypanocidal compounds. We have identified 9 compounds with potent activity against
the parasite; 3 of these were 30-fold more potent than benznidazole (Bz), a drug used for
the treatment of Chagas disease. This article provides a comprehensive outline of
reactions involving over 120 compounds aimed at the discovery of new quinone-based
frameworks with activity against T. cruzi.
Keywords: Quinones, Chagas disease, Rh-catalyzed [2+2+2] cycloadditions, annulation,
nitroalkenes.
1

1. Introduction
Chagas disease, caused by the hemoflagellate protozoan Trypanosoma cruzi, is a
neglected tropical disease endemic in Latin America, affecting approximately 6 million
individuals and leading to almost 10,000 deaths every year.1 Typically, this disease is
related to poverty, and low-income rural populations have been most severely affected.2,3
The intense immigratory flux of infected people to well-developed countries (USA,
Spain, Japan, Australia, among others) led to the occurrence of Chagas disease in non-
endemic areas.4,5 Due to successful control programmes, transfusional and vectorial
(triatomines) transmissions have progressively declined, and oral and congenital routes
have been associated with new cases.6,7 Clinically, Chagas disease is characterised by
acute and chronic phases. In the acute phase, which is frequently asymptomatic, a high
number of bloodstream parasites are detected. On the other hand, the chronic phase shows
an important reduction in parasitemia, but different clinical manifestations can be
observed: indeterminate (asymptomatic), cardiac (30% of the individuals) and digestive
forms (megasyndromes). More rarely, polyneuropathy is observed.8,9,10 Up to now, only
two trypanocidal drugs have been clinically employed, the 2-nitroimidazole benznidazole
(Bz) and the 5-nitrofuran nifurtimox; both are very effective for acute cases, but their
activity decreases with disease progression.11,12 Side-effects coupled with debatable
efficacy in chronic cases, justifies the search for alternative drugs/combinations for
Chagas disease.13
In the present manuscript, we describe our efforts to identify new lead compounds
with trypanocidal activity. Here, we outline the synthesis and trypanocidal evaluation of
56 compounds. The synthesis of these compounds involved the preparation of more than
120 substances; this work lays the foundations for the discovery of a new and versatile
family of trypanocidal compounds.
2. Results and Discussion
2.1 Chemistry
In this work we have prepared and evaluated the trypanocidal activity of three
families of quinones. The first group of compounds was generated via a [2+2+2]
cycloaddition-oxidation process using a Rh-catalyst. Next, we have explored a second
family of compounds accessed by the reaction of sulfonyl phthalide and 2-
2

nitrobenzofurans in an annulation type reaction. A third set of derivatives has been
prepared via reactions of lawsone and 2-aminonaphthoquinone with α-bromonitroalkenes
and nitroallylic acetates. Finally, three compounds were obtained via Hauser-Kraus
annulation of sulfonyl phthalide with Rauhut-Currier adducts of nitroalkenes.
2.1.1. Rh-Catalyzed [2+2+2] cycloaddition-oxidation process towards A-ring
substituted naphthoquinones
A series of 6,7-fused tricyclic naphthoquinones were prepared utilizing a Rh-
catalyzed [2+2+2] cycloaddition-oxidation protocol reported recently by our group.14
This methodology enabled efficient preparation of naphthoquinones with various A-ring
substitution patterns from simple 1,6-diynes and benzoquinones. Various metal catalysts
have been evaluated for this process, and these studies highlighted the efficiency of a
rhodium-based system.14 Dipropargyl malonate 2, which can be prepared in one step by
alkylation of dimethyl malonate (1),15 underwent Rh-catalysed cycloaddition with two
equivalents of benzoquinone (3) to afford indane-type naphthoquinone 4 in 70% yield
(Scheme 1). For the purpose of clarity, the numbering system shown in Scheme 1 is used
during the following discussion.
O
O
3
5
propargyl bromide
NaH
6
7
MeO2C
MeO2C
MeO2C
MeO2C
(200 mol%)
O
3
2
MeO2C
CO2Me
A
8
B
O
THF, rt, 18 h
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
DCE (0.1 M), 70 C, 18 h
1
2
(72%)
4
(70%)
Scheme 1. Synthesis of naphthoquinone 4.
Dipropargyl malonate 2 also underwent cycloaddition with a range of easily
accessible 2-mono- or 2,3-di-substituted benzoquinones to generate B-ring substituted
naphthoquinones, from which some trends in reactivity could be observed.
Benzoquinones with electron-donating substituents are generally more effective
substrates; 2-methoxy- and 2,3-dimethyl substituted systems provided naphthoquinones
14 and 15 in 78% and 91% yield, respectively (Scheme 2). Meanwhile, halogen
substituents on the benzoquinone ring were tolerated to varying degrees. For example,
chloride 16 was obtained in 60% yield, while bromide 17 and iodide 18 were obtained in
diminished yields of 32% and 22%, respectively. An aniline-substituted substrate 20 was
3

prepared by treatment of N-methylaniline (19) with excess benzoquinone 3.16
Cycloaddition of this substrate with diyne 2 afforded aniline-substituted naphthoquinone
21 in 58% yield. Benzoquinones with electron-withdrawing substituents were
investigated as substrates for this Rh-catalyzed cycloaddition methodology, but were
found to undergo decomposition.
MeO2C
O
OR4
OR1
O
O
2
R2
R3
R2
R2
R1
CAN, H2O
MeO2C
(100 mol%)
MeO2C
MeO2C
SiO2, CH2Cl2
rt, 30 min
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
DCE (0.1 M), 70 C, 18 h
R1
O
(200 mol%)
, R1 = R3 = R4 = H, R2 = OMe
, R1 = R4 = H, R2 = R3 = Me
, R1 = OMe, R2 = H (80%)
, R1 = R2 = Me (92%)
, R1 = Cl, R2 = H
, R1 = Br, R2 = H (49%)
, R1 = I, R2 = H (71%)
, R1 = OMe, R2 = H (78%)
, R1 = R2 = Me (91%)
, R1 = Cl, R2 = H (60%)
, R1 = Br, R2 = H (32%)
, R1 = I, R2 = H (22%)
5
6
9
14
10
11
12
13
15
16
17
18
, R1 = R4 = Me, R2 = Br, R3 = H
, R1 = R4 = Me, R2 = I, R3 = H
7
8
Me
MeO2C
MeO2C
N
O
H
O
O
2
MeO2C
MeO2C
(100 mol%)
19
AcOH-H2O
0 C, 15 min
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
DCE (0.1 M), 70 C, 18 h
N
N
Me
Me
O
O
O
(200 mol%)
(19%)
3
21
(58%)
20
Scheme 2. Synthesis of B-ring substituted naphthoquinones 14–18 and 21.
Aryl-substituted benzoquinone substrates were readily prepared from boronic
acids using a procedure developed by Fujiwara et al.17 The benzoquinones 26–29 thus
obtained underwent efficient Rh-mediated cycloaddition with diyne 2, and here it is
noteworthy that a less-activated aryl bromide 28 was well tolerated, delivering
naphthoquinone 32 in 56% yield (Scheme 3). The Rh-catalyzed cycloaddition conditions
could be extended to the synthesis of anthraquinones; cycloaddition of 1,4-
naphthoquinone (34) with diyne 2 afforded 35 in 62% yield (Scheme 4).
O
O
MeO2C
MeO2C
3
O
O
O
O
2
AgNO3 (20 or 40 mol%)
K2S2O8 (300 or 600 mol%)
R
R
(100 mol%)
MeO2C
MeO2C
R
HO
CH2Cl2, H2O, rt, 16 h
B
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
DCE (0.1 M), 70 C, 18 h
OH
(200 mol%)
22
23
24
25
26
27
28
29
30
31
32
33
, R = H
, R = H (69%)
, R = H (81%)
, R = o-OMe
, R = o-Br
, R = p-CF3
, R = o-OMe (96%)
, R = o-Br (90%)
, R = p-CF3 (76%)
, R = o-OMe (57%)
, R = o-Br (56%)
, R = p-CF3 (50%)
Scheme 3. Synthesis of aryl-substituted naphthoquinones 30–33.
4

MeO2C
MeO2C
O
O
O
O
2
(100 mol%)
MeO2C
MeO2C
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
DCE (0.1 M), 70 C, 18 h
(200 mol%)
34
35
(62%)
Scheme 4. Synthesis of anthraquinone 35.
As we were interested in evaluating the trypanocidal activity of naphthoquinones
with varying A-ring substituents, different diyne substrates were subjected to the Rh-
catalyzed cycloaddition with 1,4-benzoquinone (3). A range of 1,6-diyne tethers were
found to be compatible with the cycloaddition methodology; for example, ether- and
sulfonamide-linked diynes furnished 39 and 43 in 46% and 66% yield, respectively
(Scheme 5). Internal diynes featuring alkyl substituents were also well-tolerated,
however, efficient reaction of butyl-substituted diyne 42 required higher temperatures and
excess benzoquinone 3 to proceed. Nevertheless, preparation of naphthoquinone 44 was
achieved in 69% yield, and it is noteworthy that this product possesses a fully-decorated
A-ring.
O
O
3
(200 mol%)
O
X
X
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
DCE (0.1 M), 70 C, 18 h
O
36
37
, X = CH2
, X = O
38
39
, X = CH2 (50%)
, X = O (46%)
O
3
R
R
O
O
R
41
42
(200 mol% for
(500 mol% for
)
)
R
R
Br
O
TsNH2
40
TsN
TsN
acetone, reflux, 18 h
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
DCE (0.1 M), 18 h
41
42
43
44
, R = H (82%)
, R = n-Bu (74%)
, R = H (66%)
, R = n-Bu (69%)
41
70 C for , 70100 C for
42
Scheme 5. Synthesis of naphthoquinones 38, 39, 43 and 44.
Dimethyl malonate (1) provided a versatile starting point for the preparation of
differently substituted diynes (Scheme 6). Diynes 45 and 46, each possessing one internal
5

alkyne, underwent cycloaddition with benzoquinone 3 at the same reaction temperature
used for terminal diyne substrates. Silyloxymethyl-substituted naphthoquinone 48 was
produced in good yield (82%) and provides a synthetic handle for further
functionalization.
O
i) propargyl bromide
K2CO3
acetone, rt, 18 h
R
O
O
3
MeO2C
MeO2C
R
MeO2C
MeO2C
(200 mol%)
O
MeO2C
CO2Me
R
ii)
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
DCE (0.1 M), 70 C, 18 h
Br
K2CO3,
1
45
47
48
, R = n-Bu (42%)
, R = n-Bu (67%)
acetone, rt, 18 h
46
, R = CH2OTBS (35%)
, R = CH2OTBS (82%)
O
Me
O
O
Me
3
(500 mol%)
MeO2C
MeO2C
Me
Me
MeO2C
MeO2C
Br
THF, rt, 18 h
NaH
O
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
Me
DCE (0.1 M), 70100 C, 18 h
49
50
(77%)
(69%)
Scheme 6. Synthesis of 5- and 8-substituted naphthoquinones 47, 48 and 50.
Sonogashira reaction of diyne 2 with aryl iodides enabled expedient access to aryl-
substituted naphthoquinones 54–56 (Scheme 7). Electron-rich aryl substituted diynes are
more competent substrates in the rhodium-catalyzed cycloaddition, a trend which is
evidenced by the yields obtained for naphthoquinones 54–56.
O
ArI (240 mol%),
[PdCl2(PPh3)2] (4 mol%)
CuI (8 mol%)
Ar
O
O
3
MeO2C
MeO2C
Ar
Ar
MeO2C
MeO2C
MeO2C
MeO2C
(200 mol%)
O
i-Pr2NH (700 mol%)
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
THF, rt, 18 h
Ar
DCE (0.1 M), 70100 C, 18 h
2
51
52
53
, Ar = Ph (80%)
54
55
56
, Ar = Ph (52%)
, Ar = 4-MeOC6H4 (58%)
, Ar = 4-FC6H4 (30%)
, Ar = 4-MeOC6H4 (quant)
, Ar = 4-FC6H4 (99%)
Scheme 7. Synthesis of 5,8-diarylnaphthoquinones 54–56.
Indole and benzofuran-fused naphthoquinones 60 and 64 were accessed from
ynamide 59 and ethynyl phenyl ether 63 (Scheme 8). These N- and O- tethered alkynes
were in turn obtained from their respective aniline and phenol precursors by addition to
dichloroacetylene, generated in situ from trichloroethylene.18 Halogen-lithium exchange,
then elimination of the dichlorovinyl adducts provided the moderately unstable alkynes
6

59 and 63, which were used in the Rh-catalyzed cycloaddition reaction immediately after
isolation.
O
Cl
i) PhCCH
Cl
[PdCl2(PPh3)2], CuI
i-Pr2NH
THF, rt, 18 h
Cl
i) K2CO3,
O
O
Ph
Ph
3
I
(200 mol%)
O
DMF, 100 C, 18 h
ii) TsCl, pyridine
THF, reflux, 18 h
N
NH2
ii) PhLi
THF, 78 C, 4 h
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
DCE (0.1 M), 70 C, 18 h
NHTs
(93%)
N
Ts
Ts
57
58
59
60
(48%)
(35%)
O
Cl
i) PhCCH
Cl
Cl
[PdCl2(PPh3)2], CuI
i-Pr2NH
THF, 50 C, 3 h
O
Ph
3
(200 mol%)
I
I
O
KH
Cl
O
THF, rt, 18 h
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
DCE (0.1 M), 70 C, 18 h
O
ii) n-BuLi, Et2O,
78 C to 40 C, 3 h
OH
O
Cl
O
62
63
64
(76%)
(34%)
(50%)
61
Scheme 8. Synthesis of indole and benzofuran-fused naphthoquinones 60 and 64.
The anti-leishmanial natural product justicidone (68)19 was prepared in 42% yield
from diyne 67 and benzoquinone 9 (Scheme 9). Diyne 67 was prepared in high yield over
two steps by Sonogashira reaction of iodide 65 and propargyl alcohol, and then Steglich
esterification of alcohol 66 with propiolic acid. Interestingly, the pentasubstituted
benzenoid core of justicidone (68) was afforded as a single regioisomer, despite diyne 67
and benzoquinone 9 both being nonsymmetrical substrates.
O
O
O
O
propargyl alcohol (110 mol%)
PdCl2(PPh3)2 (5 mol%)
CuI (2.5 mol%)
OH
O
propiolic acid
DCC, DMAP
I
9
O
MeO
(300 mol%)
MeO
O
O
O
O
i-PrNH2 (700 mol%)
[Rh(coe)2Cl]2 (3.75 mol%)
(4-NCC6H4)3P (15 mol%)
DCE (0.1 M), 70 C, 3 h
CH2Cl2, 0 C, 2 h
O
THF, r.t., 4 h
O
O
O
O
O
65
66
67
(99%)
(89%)
68
Justicidone (
)
(42%, >15:1 r.r.)
Scheme 9. Synthesis of justicidone (68).
1,6-Diynes with heteroatom or electron-withdrawing substituents at C-1 or C-7
were not competent substrates in the [2+2+2] cycloaddition reaction; however, the
cycloadducts described above could be modified to provide a broader range of C5 and
C8-substituted naphthoquinones. Silyl ether 48 underwent desilylation and oxidation to
give carboxylic acid 69 when treated with Jones reagent (Scheme 10). Recently developed
conditions for carbonyl-directed Ru-catalyzed C-H oxidation20 enabled regioselective
transformation of naphthoquinone 14 to its C-5-hydroxylated product 70 in 81% yield. 5-
7

Hydroxynaphthoquinone 70 is activated towards further oxidation by PIFA,21 providing
5,8-dihydroxynaphthoquinone 71 in 75% yield under metal-free conditions.
TBSO
HO
O
O
O
O
O
MeO2C
MeO2C
MeO2C
MeO2C
Jones reagent
acetone, 0 C, 1 h
48
69
(58%)
OH
O
O
OH
O
O
O
O
[Ru(p-cymene)Cl2]2 (5 mol%)
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
PIFA (120 mol%)
PIFA
CH2Cl2, 80 C, 18 h
TFAA-TFA (98:2, 0.4 M)
MeO2C
OMe
OMe
OMe
80 C, 18 h
OH
14
70
71
(75%)
(81%)
Scheme 10. Derivatization of naphthoquinones 48 and 14.
The mechanism of the [2+2+2] cycloaddition presumably commences with
oxidative coupling of diyne to provide rhodacyclopentadiene. From here, cycloaddition
with benzoquinone occurs to provide intermediate 1’; this process could occur via either
a migratory insertion/reductive elimination sequence or a Diels-Alder pathway.22
Oxidation of 1’ to the respective 1,4-naphthoquinone occurs in situ and is enabled by
reduction of a sacrificial equivalent of benzoquinone (Scheme 11).
O
1,4-Naphthoquinone
R1
O
O
R1
O
R1
Rh(III)
R2
Rhodacyclopentadiene
H
H
R1
R2
Rh(I) (cat)
Oxidation
O
Z
Z
Z
Z
-Rh(I)
R2
R2
O
Diyne
Intermediate 1'
Scheme 11. Mechanistic outline for the oxidative [2+2+2] cycloaddition.
2.1.2. Annulation of sulfonyl phthalide with 2-nitrobenzofurans towards
naphthoquinones
The synthesis of the second family of compounds started with the preparation of
3-sulfonyl phthalide 75 in three steps following the procedure described by Ramström et
al.23 Commercially available 2-carboxybenzaldehyde 72 was reacted with methyl iodide
in acetone to generate methyl 2-formylbenzoate 73, which on treatment with thiophenol
in the presence of triethylamine in chloroform afforded 74. Compound 74 was then
oxidized with m-CPBA to provide 3-sulfonyl phthalide 75 in good yield (Scheme 12A).
8

Subsequently, 2-nitrobenzofurans were prepared on the basis of a protocol reported in the
literature.24 2-Hydroxy-β-nitrostyrenes 82–87 were obtained from commercially
available salicylaldehydes 76–81 by their reaction with nitromethane in the presence of
NH4OAc/AcOH at 90 ºC. Then, the formation of 2-nitrobenzofurans 88–93 was
performed in 2 steps. Conjugate reduction using NaBH4 in CHCl3-iPrOH at 0 °C was
followed by intramolecular cyclization and oxidation using PIDA and TBAI in the
presence of triethylamine and acetonitrile as solvent (Scheme 12B).
O
O
O
O
A
MeI, K2CO3
OH
H
OMe
H
PhSH
m-CPBA
O
O
acetone, rt, 12h
Et3N, 6 h
DCM, rt, 24 h
SO2Ph
75
(69%)
SPh
74
(80%)
O
72
O
73
(95%)
B
(a) NaBH4,
CHCl3-i-PrOH
0° C, 1 h
R1
R2
R1
R2
NO2
R1
R2
O
CH3NO2
NO2
O
OH
NH4OAc/AcOH
90 °C, 12 h
OH
(b) PhI(OAc)2
TBAI, Et3N, rt, 1 h
R3
R3
R3
, R1 = R2 = R3 = H (70%)
, R1 = R2 = R3 = H
, R1 = R2 = R3 = H (65%)
82
83
84
85
86
87
76
77
78
79
80
81
88
89
90
91
92
93
, R1 = R2 = H, R3 = OEt (79%)
, R1 = R2 = H, R3 = OEt
, R1 = OMe, R2 = R3 = H
, R1 = R2 = H, R3 = OMe
, R1 = Me, R2 = R3 = H
, R1 = R2 = Me, R3 = H
, R1 = R2 = H, R3 = OEt (79%)
, R1 = OMe, R2 = R3 = H (77%)
, R1 = R2 = H, R3 = OMe (76%)
, R1 = Me, R2 = R3 = H (65%)
, R1 = R2 = Me, R3 = H (61%)
, R1 = OMe, R2 = R3 = H (81%)
, R1 = R2 = H, R3 = OMe (78%)
, R1 = Me, R2 = R3 = H (75%)
, R1 = R2 = Me, R3 = H (84%)
Scheme 12. Synthesis of 3-sulfonyl phthalide 75 and 2-nitrobenzofurans 88–93.
With 2-nitrobenzofurans in hand, we decided to optimize the annulation reaction
of 3-sulfonyl phthalide 75 and 2-nitrobenzofuran 88 to enable subsequent studies (Table
1). Initially, we evaluated the use of Cs2CO3 in different solvents, such as DCM, MeCN,
toluene and THF (entries 1–4). The use of Cs2CO3 as base and THF as solvent allowed
us to prepare the desired product in 81% yield. Subsequent studies with different bases
offered no further improvements (entries 5–8).
9

Table 1. Selected optimization results.a
O
O
base
O
O2N
solvent, rt
12 h
O
O
SO2Ph
O
88
75
94
Entry
Base
Solvent
DCM
MeCN
Toluene
THF
THF
THF
THF
THF
Yield (%)b
1
2
3
4
5
6
7
8
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
KOtBu
LiOtBu
DBU
50
71
46
81
20
40
12
50
K2CO3
aReaction conditions: 3-sulfonyl phthalide (75, 1 equiv), 2-nitrobenzofuran (88, 1 equiv),
b
base (1 equiv) in 5-10 mL of solvent stirred at rt under N2 atmosphere. Yield after
purification by column chromatography.
With the optimized reaction conditions in hand, the scope of different 2-
nitrobenzofurans 88–93 was explored. Nitroalkenes with electron-neutral and electron-
rich substituents were treated with 3-sulfonyl phthalide 75 to afford the benzofuran fused
naphthoquinone derivatives 94–99 as summarized below (Scheme 13). The reaction with
parent 2-nitrobenzofuran 88 under the optimized reaction conditions delivered the desired
product 94 in 81% yield. 2-Nitrobenzofurans 89–91 containing a strongly electron
donating alkoxy groups underwent smooth reaction with phthalide 75 to afford the
benzofuran fused naphthoquinones 95–97 in excellent yields (84–87%). Notably,
annulations with nitroalkenes bearing one or two methyl groups, such as 92–93, provided
the corresponding benzofuran fused naphthoquinones 98–99 in 70-72% yields.
Compounds 94–99 are described here for the first time and have been characterized by
various analytical and spectroscopic techniques. In addition, compound 95 was analysed
by X-ray crystallography - the ORTEP-3 structure is shown in Figure 1.
10

O
O
O
Cs2CO3 (100 mol%)
THF, rt, 12 h
R
O
O2N
R
O
O
O
SO2Ph
75
88-93
O
O
O
O
O
O
O
O
O
O
94
95
96
(87%)
(81%)
(86%)
O
O
O
O
O
O
O
O
O
O
97
98
99
(72%)
(84%)
(70%)
Scheme 13. Synthesis of quinones 94–99 from 3-sulfonyl phthalide 75.
Figure 1. ORTEP-3 projection of the compound 95 with 50% probability displacement
ellipsoids.
The proposed mechanism of the annulation reaction is based on relevant literature,
including our recent work.25 3-Sulfonyl phthalide 75 functions as a 1,4-dipolar synthon
(donor and acceptor) in the Hauser-Kraus annulation. First, base-mediated deprotonation
of phthalide 75 generates stabilized carbanion I, which participates in Michael addition
with 2-nitrobenzofuran 88, resulting in the formation of Michael adduct II (Scheme 14).
This undergoes Thorpe-Ingold facilitated Dieckmann cyclization with the lactone
resulting in the formation of III. Intermediate III undergoes elimination of a sulfonyl
anion as well as the nitro group, which results in the formation of benzofuran-fused
naphthoquinone 94.
11

O
N
O
O
O
O
O
O
O
O
N
Base
88
O
O
X
O
Deprotonation
Intermolecular
Michael addition
X
X
75
X = SO2Ph
I
II
Dieckmann
cyclization
O
O
NO2
O
Elimination
O
-HNO2
-X
O
H
X
O
94
III
Scheme 14. Proposed mechanism for the formation of compounds 94–99.
2.1.3. Naphthoquinones via reactions of lawsone and 2-aminonaphthoquinone with
α-bromonitroalkenes
Using
a
protocol reported recently by our group,
a
series of
pyrrolonaphthoquinones and furanonaphthoquinones were prepared by reacting lawsone
and 2-aminonaphthoquinone with α-bromonitroalkenes.26 Initially, we accomplished the
reaction between substituted α-bromonitroalkenes and various N-arylated
aminonaphthoquinones to prepare compounds 100–112 in good to moderate yields
(Scheme 15A). In Scheme 15B we demonstrate the preparation of
furanonaphthoquinones 114–119. The methodology used here was also based on our
previous report26 and the method described by Zhang and co-workers.27 We used lawsone
(113) for reactions with α-bromonitroalkenes to obtain compounds 114–119 in good to
excellent yields (56–87%).
12

O
O
R
H
A
N
N
R
KOH (200 mol%)
THF, rt, 2-6 h
NO2
Ar
+
Br
Ar
O
O
, R1 = OMe, R2 = Me (86%)
, R1 = OMe, R2 = H (67%)
, R1 = R2 = OMe (47%)
R1
100
101
102
103
104
105
106
107
108
109
110
OCH3
nBu
O
, R1 = H, R2 = OMe (72%)
, R1 = Cl, R2 = OMe (29%)
, R1 = OMe, R2 = Cl (56%)
, R1 = OMe, R2 = F (35%)
, R1 = F, R2 = OMe (60%)
, R1 = 4-Br, R2 = Me (34%)
, R1 = 3-Br, R2 = Me (46%)
, R1 = R2 = Me (42%)
O
O
O
N
N
N
O
O
O
112
(44%)
111
(35%)
Cl
R2
O
O
B
O
O
OH
O
NO2 NaOAc, TBAB
R
+
Br
H2O, 70 ºC, 7 h
R
113
O
O
O
O
114
115
116
117
, R = F (87%)
, R = Br (73%)
, R = OMe (82%)
, R = SMe (56%)
O
O
O
NO2
O
O
S
118
119
(80%)
(75%)
R
Scheme 15. Synthesis of pyrrolonaphthoquinones 100–112 and furanonaphthoquinones
114–119.
2.1.4. Compounds prepared via Hauser-Kraus annulation of sulfonyl phthalide
with Rauhut-Currier adducts of nitroalkenes
The last series of compounds described in this manuscript was prepared using the
methodology recently described by the Namboothiri group.28 Here, Hauser-Kraus
annulation of phthalide 75 with α,β-disubstituted nitroalkenes led to the formation of
unsymmetrically substituted naphthoquinones 120–122 in good to high yield (64–75%)
(Scheme 16).
13

O
O
O
O
O
Cs2CO3 (150 mol%)
THF, rt, 9-12 h
+
O
R
R
NO2
SO2Ph
O
75
O
O
O
O
O
O
O
O
O
(70%)
OMe
F
120
121
122
(75%)
(64%)
Scheme 16. Synthesis of compounds 120–122 prepared via Hauser-Kraus annulation of
3-sulfonyl phthalide (75).
2.2. Biological Studies
In the past decade, the da Silva Júnior, de Castro, and Menna-Barreto groups have
evaluated the trypanocidal potential of quinones and heterocycles as part of a Medicinal
Chemistry program for the evaluation of compounds against the parasite Trypanosoma
cruzi.29 Our mission is to identify synthetic strategies for the discovery of compounds
with powerful trypanocidal activity and low cytotoxicity. This could enable the
establishment of a research base for the synthesis of efficient prototypes to combat the
parasite.
The search for novel compounds with features suitable for eventual progression
to the clinic is well known to be challenging.30 We are aware of the need to approach the
synthesis of trypanocidal substances via versatile and efficient synthetic routes. This kind
of approach can provide important details on structure-activity relationships.
The quinoidal framework presents dual reactivity, possessing both nucleophilic or
electrophilic behavior; the relative propensity of these characteristics depends on the
reaction medium and specific conditions used.31 Aware of these aspects, our research
group developed transition-metal catalyzed reactions that allow the direct modification of
the A-ring of naphthoquinones.32 Controlling reactions to modify A-ring quinonoid
systems allowed us to synthesize several series of compounds and evaluate their
trypanocidal activity. In this context, we assayed quinoidal molecules containing
oxygen,33 selenium,34 sulfur,35 iodine and aryl,36 triazole37 and alkene38 groups on the A-
ring. These studies clearly indicated that the substitution of this ring intensifies
trypanocidal activity - the substitution pattern is closely related to the ability of the
compound to kill the parasite and also to its cytotoxicity (Scheme 17).
14

0.59 µM*
Bz: 9.68 µM*
Ref. 36
I
O
CO2Et
14.4 µM*
Bz: 103.6 µM*
Ref. 34
O
O
90.5 µM*
Bz: 103.6 µM*
Ref. 38
Se
O
O
O
[Rh]
Selenation
and
[Ru]
[Cu]
Alkenylation
S
O
H
O
O
12.4 µM*
Bz: 103.6 µM*
Ref. 35
[Rh] and [Cu]
Sulfenylation
1.24 µM*
Bz: 9.68 µM*
Ref. 36
[Rh] and [Pd]
Arylation
O
O
Oxygenation
[Ru]
[Pd]
and
O
Ph
[Rh]
Alkenylation
O
OH
O
O
0.19 µM*
Bz: 9.68 µM*
Ref. 36
105.0 µM*
Bz: 103.6 µM*
Ref. 33
Ph
N
N
N
O
O
O
86.7 µM*
Bz: 103.6 µM*
Ref. 37
Scheme 17. Trypanocidal A-ring substituted quinones. *IC50/24 h values for the lytic
activity on bloodstream trypomastigotes.
The first family of compounds described here was prepared via a direct and
modern strategy using [2+2+2] cycloaddition reactions to prepare A-ring modified
quinones. Our strategy has proved efficient, as we have identified eight compounds with
trypanocidal activity greater than benznidazole, a drug used against T. cruzi in the clinic.
Compound 4, prepared in only two synthetic steps, showed IC50/24 h = 51.5 µM and is
twofold more potent than benznidazole (Table 2). Similarly, derivatives 16, 18 and 32
were prepared from commercially available reactants in two steps and present outstanding
trypanocidal activity with IC50/24 h values = 56.6, 51.3 and 51.5 µM, respectively. The
synthesis of compound 38 was performed in only one step and has an IC50/24 h = 49.2
µM, which is around two times more active than benznidazole. Easy access to this
substance allows the planning of structural modifications to increase its antiparasitic
potential. Compound 47 was also prepared in two steps and presented IC50/24 h = 64.4
15

µM. This derivative was also more active than Bz. The most potent compounds described
here are derivatives 54, 55 and 56 with IC50 values lower than 3.5 µM. We also observed
that the addition of aryl groups to the A-ring increased trypanocidal activity. This effect
was most pronounced for electron rich aromatic units, as in the case of compound 55.
Arenes with electron withdrawing groups, such as compound 56, showed significant but
slightly attenuated trypanocidal activity (Scheme 18).
O
O
N
N
N
H
O
1,4-Naphthoquinone
94.4 µM*
NO2
Benznidazole
103.6 µM*
A-ring
modification
O
O
O
MeO2C
MeO2C
MeO2C
MeO2C
A
A
A
O
O
O
O
4
38
47
Substitution on the B-ring
Similar Trypanocidal Activity
51.5 µM*
49.2 µM*
64.4 µM*
O
O
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
Br
I
Cl
O
O
O
18
32
16
51.5 µM*
51.3 µM*
56.6 µM*
Electron donating
group increases
trypanocidal activity
Electron withdrawing
groups slightly
decreases activity
O
F
A
F
A-ring modified
naphthoquinoidal
compounds
O
O
O
O
O
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
A
O
A
MeO2C
O
55
54
56
2.3 µM*
2.5 µM*
3.3 µM*
Scheme 18. Trypanocidal A-ring modified quinones. *IC50/24 h values for the lytic
activity on bloodstream trypomastigotes.
Unfortunately, we did not observe significant trypanocidal activity for compounds
prepared by annulation reactions with 3-sulfonyl phthalide and 2-nitrobenzofurans, or for
16

those prepared in Section 2.1.3. Compounds 94–112 and 114–119 had IC50/24 h >500
µM and so were not active against T. cruzi.
The last series of compounds 120–122 was also evaluated against T. cruzi and we
identified two compounds with similar activity to benznidazole (Table 2). The
cytotoxicity of the compounds was also evaluated (Table 3). Compounds 120 and 121
with SI = 1.38 and 1.74, could be also considered important prototypes for subsequent
studies.
Table 2. IC50/24 h (µM) of quinones against the trypomastigote form of T. cruzi.a
Compounds
IC50/ 24 h
51.5 (± 13.2)
>500.0
Compounds
94
IC50/ 24 h
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
>500.0
109.2 (± 10.9)
113.9 (± 11.7)
>500
4
14
15
16
17
18
21
30
31
32
33
35
38
39
43
44
47
48
50
54
55
56
60
64
68
69
70
71
95
96
97
98
>500.0
56.6 (± 7.6)
149.5 (± 15.3)
51.3 (± 3.3)
>500.0
368.7 (± 32.4)
102.0 (± 9.7)
51.5 (± 5.2)
361.6 (± 45.5)
>500.0
49.2 (± 4.4)
192.9 (± 22.3)
>500.0
>500.0
64.4 (± 1.8)
>500.0
99
100
101
102
103
104
105
106
107
108
109
110
111
112
114
115
116
117
118
119
120
121
122
>500.0
2.5 (± 0.1)
2.3 (± 0.2)
3.3 (± 0.1)
122.4 (± 4.9)
247.3 (± 4.7)
>500.0
>500.0
>500.0
>500.0
aMean ± SD of at least three independent experiments, 5% of blood at 4 oC. IC50/24 h
for benznidazole = 103.6 (± 0.6).39
17

We also evaluated the cytotoxicity of 11 compounds that showed outstanding
trypanocidal activity (Table 3). In general, the compounds showed cytotoxicity against
mammalian cells, but still had a selectivity index near 1, as in the case of compounds 55
and 56. These results are promising and define these structures as special prototypes for
the preparation of new bioactive compounds that are efficient for combating parasites,
such as T. cruzi.
Table 3. IC50/24 h, LD50/24 h (µM) and Selectivity Index (SI) of most active quinones.
Compounds
IC50/24 ha
51.5 (± 13.2)
56.6 (± 7.6)
51.3 (± 3.3)
51.5 (± 5.2)
49.2 (± 4.4)
64.4 (± 1.8)
2.5 (± 0.1)
2.3 (± 0.2)
3.3 (± 0.1)
109.2 (± 10.9)
113.9 (± 11.7)
LD50/24 h
1.6 (± 0.7)
5.0 (± 0.8)
1.6 (± 0.3)
3.8 (± 1.5)
1.7 (± 0.3)
4.2 (± 1.7)
1.6 (± 0.5)
1.9 (± 0.3)
3.0 (± 1.2)
79.0 (± 4.7)
65.4 (± 6.5)
SI
4
16
18
32
38
47
54
55
56
120
121
0.03
0.09
0.03
0.07
0.03
0.07
0.60
0.81
0.89
1.38
1.74
a5% of blood at 4 oC.
Since the 1990s, Pinto and de Castro's groups have established the foundations for
trypanocidal studies involving quinones and heterocyclic compounds.40 The trypanocidal
assays have been standardized as: (a) 24h of incubation; (b) 4 °C (blood bank
temperature); and (c) presence of 5% mouse blood (due to the inactivation by serum
components).41 Trypanocidal activity is expressed as an IC50 value corresponding to the
concentration that lyses 50% of the parasites. It is well-known that low temperatures can
lead to low biological activity and so the values of IC50/24 h may be higher than values
obtained when the analyses are performed at 37 ºC (host temperature). Accordingly, we
decided to evaluate the trypanocidal activity of the three most active compounds 54-56 at
37 ºC in the absence of blood (Table 4). As expected, the activity of 54, 55 and 56
increased at least 5x, with IC50 values in the nanomolar range. Consequently, the SI values
increased and were 3.7, 13.9 and 9.3 for compounds 54, 55 and 56, respectively.
18

Table 4. IC50/24 h, LD50/24 h (µM) and Selectivity Index (SI) of most quinones 54-56.
Compounds
IC50/24 ha
0.44 (± 0.04)
0.14 (± 0.04)
0.32 (± 0.04)
LD50/24 h
1.6 (± 0.5)
1.9 (± 0.3)
3.0 (± 1.2)
SI
3.7
13.9
9.3
54
55
56
a0% of blood at 37 oC. IC50/24 h for benznidazole = 8.8 (± 1.1) µM, LD50/24h >1000 µM.
2.3. Structure Activity Relationships: Some general aspects
To consider further the potential of the quinones described here as trypanocidal
compounds, it is important to establish details that enable the understanding of the
relationship between structure and activity. In total 56 compounds were evaluated against
T. cruzi in this work. Although the compounds have in common the quinonoid nucleus,
the library is diverse and can be broadly divided into 5 subgroups, according to the
substitution in the A- and B-rings in comparison with 1,4-naphthoquinone (34). We
categorize the molecules into: (1) A-ring functionalized quinones; (2) A- and B-ring
functionalized quinones; (3) B-ring modified quinones with oxygenated heterocycles; (4)
B-ring modified quinones with nitrogenated heterocycles and; (5) B-ring functionalized
quinones.
1. A-ring functionalized quinones: The strategy for obtaining new trypanocidal
compounds was based on the structural modification of the basic nucleus of 1,4-
naphthoquinone (34). Here it is important to note that we are discussing the design of new
bioactive molecules and not synthetic routes for the preparation of new compounds,
which can be prepared from 1,2-benzoquinones or other quinoidal derivatives. The
structure of 34 is composed of two rings, which we define as the A- and B-rings. Studies
previously published by our group have described that the simple modification of these
rings can cause either an increase or decrease in trypanocidal activity.34-38 We observed
that A-ring modified compounds containing a fused cyclopentane were highly active
against T. cruzi. The appendage of substituted aryl groups also enhances the trypanocidal
activity. On the other hand, when the A-ring was substituted by O- or N-heterocyclic
compounds we observed that trypanocidal activity was only moderate. The substitution
of the A-ring with electron donating groups, for instance methyl groups, delivers
compounds that are inactive against T. cruzi (see Table 5 for more details).
19

2. A- and B-ring functionalized quinones: The compound library included 13 A-
and B-ring modified quinones. Three molecules from this group were highly active
against T. cruzi with IC50 in the range of 51.3 and 56.6 µM. In general, we observed that
the presence of chlorine and iodine on the B-ring did not cause a significant change in the
trypanocidal activity of the respective compounds 16 and 18, and did not decrease the
ability of the compounds to kill the parasite. On the contrary, the presence of bromine on
the B-ring decreased activity against the parasite. Aryl bromides on the B-ring did not
cause a change in trypanocidal activity, but aryl groups without substitution or with other
substitution, such as methoxy or trifluoromethyl groups, decreased trypanocidal activity.
An important observation is that electron donating substituents on B-ring, such as
methoxy, methyl and arylamino groups, deactivate the compound’s ability to eliminate
the parasite (for more details, see Table 6).
3. B-ring modified quinones with oxygenated heterocycles and 4. B-ring modified
quinones with nitrogenated heterocycles: Unfortunately, all compounds with these
modifications were inactive against T. cruzi (compounds 94-112 and 114-119). To gain a
better understanding of the biological aspects, studies with derivatives of this series of
these compounds are in progress in our laboratories and will be published in due course.
5. B-ring functionalized quinones: Compounds 120-122 with substitution only on
the B-ring were also prepared and evaluated against T. cruzi. Quinones 120 and 121
showed moderate activity against the parasite, showing that different patterns of
substitution on the A-ring are important for trypanocidal activity. In general, the
trypanocidal activity of quinones is intrinsically related to the generation of ROS. The
mechanism of pharmacological action of the compounds described here has not yet been
established, but studies previously published in the literature demonstrate that
functionalization of benzenoid or quinoidal rings can cause changes in the redox system
with direct consequences on the parasitic activity observed.29
20

Table 5. A-ring functionalized quinones.
A-ring functionalized quinones
Highly active compounds
(55) 2.3 µM*
MeO2C
(47) 64.4 µM*
(54) 2.5 µM*
MeO2C
(56) 3.3 µM*
MeO2C
Ar
A
O
O
(4) 51.5 µM*
(38) 49.2 µM*
A
Ar
A
O
O
Ar
A
O
O
R
A
O
O
O
O
O
O
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
A
Ar
Ar
Ar
Ar = Ph
Ar = 4-FC6H4
R = -Bu
n
Ar = 4-MeOC6H4
- A-ring modification can increase trypanocidal activity
- Addition of aryl groups onto the A-ring increases trypanocidal activity strongly
- Electron donating aryl groups are slightly more activite against T. cruzi
Moderately active compounds
Inactive compounds
(60) 122.4 µM*
Ph
(64) 247.3 µM*
>500 µM*
TsN
(39) 129.9 µM*
R
O
(69) >500 µM*
HO
(48) >500 µM*
(50) >500 µM*
O
O
O
Me
O
O
TBSOH2C
O
O
O
O
Ph
A
O
O
A
R
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
O
A
A
A
A
A
O
MeO2C
N
O
43, R = H
44, R = -Bu
Ts
Me
O
n
O
- Addition of heterocycles onto the A-ring decreases trypanocidal activity
- Electron donating groups, for instance n-Bu and Me groups, makes the compounds inactive against the T. cruzi
*IC50/24 h values for the lytic activity on bloodstream trypomastigotes.
21

Table 6. A- and B-ring functionalized quinones.
A- and B-ring functionalized quinones
Highly active compounds
Moderately active compounds
(16) 56.6 µM*
(18) 51.3 µM*
(32) 51.5 µM*
MeO2C
(17) 149.5 µM*
MeO2C
O
O
O
O
O
R
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
A
B
O
A
B
O
A
B
O
B
O
A
30, R = H - 368.7 µM*
A
B
O
MeO2C
Br
MeO2C
MeO2C
Br
31, R = -OMe - 102.0 µM*
o
I
Cl
33, R = -CF3 - 361.6 µM*
p
- Chlorine and iodine substituents on the B-ring did not cause a significant change in trypanocidal activity
- A bromine substituent on the B-ring decreased activity against the parasite
- Aryl bromide substituents on the B-ring did not cause a change in trypanocidal activity
- Aryl groups without substitution, or with OMe or CF3 groups decreased trypanocidal activity
Inactive compounds
OH
O
(21) >500 µM*
(14) >500 µM*
(15) >500 µM*
(35) >500 µM*
MeO2C
MeO2C
O
O
O
O
A
R
B
O
Me
Me
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
MeO2C
OMe
C
A
A
B
O
B
O
A
B
O
A
B
O
OMe
N
70, R = H - >500 µM*
71, R = OH - >500 µM*
Me
- The compounds were inactive against T. cruzi when methoxy, methyl or arylamino groups are inserted on B-ring
- The presence of an aromatic C-ring also caused the loss of trypanocidal activity
*IC50/24 h values for the lytic activity on bloodstream trypomastigotes.
22

3. Conclusions
Effective methods for the synthesis of quinones have been described. The
strategies discussed here allowed the preparation of bioactive molecules in a fast, simple
and direct manner. This synthetic efficiency underpinned the evaluation of 56 compounds
and the identification of various trypanocidal derivatives more active than benznidazole,
a drug used in the treatment of Chagas disease. For instance, we identified 3 compounds
that are around 30-fold more active than Bz. After evaluating the cytotoxicity of these
compounds in mammalian cells, the selectivity index for these substances was close to 1.
In general terms, we describe here a set of substances with potent trypanocidal activity
that offer high potential for further optimization. Our ongoing aim is to target more active
compounds with lower cytotoxicity. Additionally, studies into the mechanism of
pharmacological action of the more active compounds are ongoing in our laboratories and
will be published in due course.
4. Experimental Section
4.1. Chemistry
4.1.1. General experimental details
Starting materials sourced from commercial suppliers (Acros, Aldrich, Alfa Aesar,
Fluorochem, TCI) were used as received unless otherwise stated. Anhydrous 1,2-
dichloroethane was sourced from Aldrich and used as received. Other dry solvents, where
necessary, were obtained by distillation using standard procedures or by passage through
a column of anhydrous alumina using equipment from Anhydrous Engineering. Petrol
refers to the fraction of petroleum ether boiling in the range of 40–60 °C. The removal of
solvents in vacuo was achieved using both a Büchi rotary evaporator (bath temperatures
up to 45 °C) at a pressure of either 4 mbar (diaphragm pump) or 1 mbar (oil pump), as
appropriate, and a high vacuum line at room temperature. Reactions requiring anhydrous
conditions were run under an atmosphere of dry nitrogen or argon; glassware was either
flame dried immediately prior to use or placed in an oven (200 °C) for at least 2 h and
allowed to cool either in a desiccator or under an atmosphere of nitrogen or argon; liquid
reagents, solutions or solvents were added via syringe through rubber septa; solid reagents
were added via Schlenk type adapters. Commercially available Merck Kieselgel 60F254
aluminium backed plates were used for TLC analysis. Visualisation was achieved by
23

either UV fluorescence, basic KMnO4 solution and heat. Flash column chromatography
was performed using silica gel (Aldrich 40-63 μm, 230-400 mesh). The crude material
was applied to the column as a solution in CH2Cl2 or by pre-adsorption onto silica, as
appropriate. Melting points were determined using a Reichert melting point table and
temperature controller and are uncorrected. Infrared spectra were recorded in the range
4000-600 cm-1 on a Perkin Elmer Spectrum either as neat films or solids compressed onto
a diamond window. NMR spectra were recorded using either a Varian 400-MR, or Bruker
Nano 400. Chemical shifts (δ) are quoted in parts per million (ppm), coupling constants
(J) are given in Hz to the nearest 0.5 Hz. 1H and 13C NMR spectra were referenced to the
appropriate residual solvent peak. Mass spectra were determined using a Shimadzu
GCMS QP2010+ (EI+ mode), Brüker Daltonics FT-ICRMS Apex 4e 7.0T FT-MS (ESI+
mode), Thermo Scientific Orbitrap Elite (APCI mode).
4.1.2. Rh-Catalyzed [2+2+2] cycloaddition-oxidation process towards A-ring
substituted naphthoquinones
General procedure for the [2+2+2] cycloaddition reactions was based on our
previous report.14 A flame-dried reaction tube, fitted with a magnetic stirrer, was
charged with [Rh(coe)2Cl]2 (3.75 µmol, 2.69 mg, 3.75 mol%), (4-NCC6H4)3P (15 µmol,
5.06 mg, 15 mol%) and the appropriate quinone substrate (0.4 mmol, 200 mol% or 1.0
mmol, 500 mol%, as specified). The tube was flushed with argon, then fitted with a rubber
septum, placed under a balloon of argon, and DCE (0.25 mL) was added via syringe. The
reaction tube was placed in a preheated heating block at 70 °C, then the appropriate diyne
substrate (0.1 mmol, 100 mol%) in DCE (0.75 mL) was added dropwise by syringe pump
over 2 h. After the addition of diyne was complete, the reaction mixture was stirred at 70
°C or 100 °C (as specified) for 16 h, then cooled to room temperature and concentrated
in vacuo. The crude reaction mixture was purified by flash column chromatography on
silica gel using either a petroleum ether/ethyl acetate or acetone/toluene eluent system to
yield the target naphthoquinones.
24

4.1.3. Annulation of sulfonyl phthalide with 2-nitrobenzofurans towards
naphthoquinones
General procedure for the synthesis of annulated quinones. To a stirred solution of 3-
sulfonyl phthalide 75 (0.2 mmol, 54 mg, 1.0 equiv) and 2-nitrobenzofurans 88–93 (0.2
mmol, 1.0 equiv) in THF (5.0 ml) was added Cs2CO3 (0.2 mmol, 66 mg, 1.0 equiv) at
room temperature until completion of the reaction (overnight, 12 h, monitored by TLC).
The solvent was evaporated in vacuo. The residue was then subjected to silica gel column
chromatography by gradient elution with ethyl acetate/hexane (2:98) affording the
respective quinones 94–99.
Naphtho[2,3-b]benzofuran-6,11-dione (94). Yellow solid; Yield 40 mg, 81%, mp 231–
233 ºC; IR (neat, cm-1) 3077 (vw), 2915 (vw), 2847 (vw), 1660 (br vs), 1563 (m), 1486
(w), 1223 (m), 1179 (m), 987 (m), 747 (s), 713 (s); 1H NMR (500 MHz, CDCl3) δ 7.50
(t, J = 7.8 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.75–7.84 (m, 2H),
8.20–8.28 (m, 2H), 8.31 (d, J = 7.8 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 113.1, 122.9,
124.2, 126.3, 127.0, 127.1, 129.8, 132.5, 133.5, 134.1, 134.4, 153.7, 156.6, 175.7, 181.6;
HRMS (ES+) calcd for C16H9O3 (MH+) 249.0544, found 249.0546.
4-Ethoxynaphtho[2,3-b]benzofuran-6,11-dione (95). Orange solid; Yield 50 mg, 86%,
mp 224–226 ºC; IR (neat, cm-1) 3080 (w), 2974 (m), 2920 (m), 1673 (br vs), 1284 (m),
1241 (m), 780 (vs), 710 (vs); 1H NMR (500 MHz, CDCl3) δ 1.54 (t, J = 7.0 Hz, 3H), 4.30
(q, J = 7.0 Hz, 2H), 7.03 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.75–7.79 (m, 2H),
7.84 (d, J = 8.0 Hz, 1H), 8.21–8.25 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 14.8, 65.0,
112.1, 115.3, 124.4, 124.5, 126.8, 126.9, 127.0, 132.4, 133.3, 133.8, 134.1, 145.6, 146.4,
153.4, 175.2, 181.5; HRMS (ES+) calcd for C18H12O4Na (MNa+) 315.0628, found
315.0628.
2-Methoxynaphtho[2,3-b]benzofuran-6,11-dione (96). Yellow solid; Yield 48 mg,
87%, mp 211–213 ºC; IR (neat, cm-1) 2937 (vw), 1670 (vs), 1559 (w), 1490 (m), 1454
(w), 1357 (w), 1277 (m), 1266 (m), 1245 (s), 1204 (m), 1026 (m), 710 (m); 1H NMR (500
MHz, CDCl3) δ 3.91 (s, 3H), 7.14 (dd, J = 9.1, 2.5 Hz, 1H), 7.45 (d, J = 9.1 Hz, 1H), 7.63
(d, J = 2.5 Hz, 1H), 7.73–7.79 (m, 2H), 8.17–8.23 (m, 2H); 13C NMR (125 MHz, CDCl3)
δ 56.2, 104.1, 113.8, 120.3, 123.6, 124.3, 126.9, 127.1, 132.6, 133.4, 134.0, 134.3, 151.7,
154.1, 158.6, 175.4, 181.7; HRMS (ES+) calcd for C17H11O4 (MH+) 279.0652, found
279.0657.
25

4-Methoxynaphtho[2,3-b]benzofuran-6,11-dione (97). Yellow solid; Yield 41 mg,
84%, mp 227–229 ºC; IR (neat, cm-1) 2950 (w), 1665 (br vs), 1596 (vs), 1502 (m), 1376
(s), 1331 (s), 1278 (s), 1246 (s), 1075 (m), 943 (s), 778 (m), 711 (m); 1H NMR (500 MHz,
CDCl3) δ 4.06 (s, 3H), 7.04 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.70–7.80 (m,
2H), 7.85 (d, J = 7.8 Hz, 1H), 8.21–8.25 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 56.5,
111.3, 115.6, 124.5, 124.6, 127.0, 127.1, 127.2, 132.6, 133.5, 134.0, 134.3, 146.3, 146.4,
153.7, 175.3, 181.6; HRMS (ES+) calcd for C17H11O4 (MH+) 279.0652, found 279.0651.
2-Methylnaphtho[2,3-b]benzofuran-6,11-dione (98). Yellow solid; Yield 37 mg, 70%,
mp 251–253 ºC; IR (neat, cm-1) 3107 (w), 2915 (m), 1643 (br s), 1598 (vs), 1422 (m),
1351 (m), 1263 (s), 1045 (m), 966 (m), 821 (m), 736 (vs); 1H NMR (500 MHz, CDCl3) δ
2.52 (s, 3H), 7.39 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.75–7.81 (m, 2H), 8.09
13
(s, 1H), 8.21–8.26 (m, 2H); C NMR (125 MHz, CDCl3) δ 21.6, 112.5, 123.0, 123.6,
124.2, 127.0, 127.1, 131.4, 132.6, 133.5, 134.0, 134.3, 136.4, 153.8, 155.2, 175.7, 181.8;
HRMS (ES+) calcd for C17H11O3 (MH+) 263.0703, found 263.0702.
2,3-Dimethylnaphtho[2,3-b]benzofuran-6,11-dione (99). Yellow solid; Yield 39 mg,
72%, mp 225–227 ºC; IR (neat, cm-1) 3055 (w), 2922 (m), 1672 (vs), 1659 (vs), 1591 (m),
1574 (s), 1560 (s), 1456 (m), 1251 (s), 1189 (vs), 987 (s), 712 (s); 1H NMR (500 MHz,
CDCl3) δ 2.41 (s, 3H), 2.43 (s, 3H), 7.45 (s, 1H), 7.74–7.80 (m, 2H), 8.04 (s, 1H), 8.20–
13
8.26 (m, 2H); C NMR (125 MHz, CDCl3) δ 20.3, 21.3, 113.1, 120.7, 123.6, 124.4,
126.9, 127.0, 132.7, 133.5, 134.0, 134.2, 135.8, 140.3, 153.2, 155.9, 175.6, 181.9; HRMS
(ES+) calcd for C18H13O3 (MH+) 277.0859, found 277.0858.
4.1.4. Naphthoquinones via reactions of lawsone and 2-aminonaphthoquinone with
α-bromonitroalkenes
General procedure for the synthesis of pyrrolonaphthoquinones 100–112 was based
on our previous report.26 To a stirred solution of 2-aminonaphthoquinone (0.5 mmol,
1.0 equiv) in THF (4 mL), KOH (56 mg, 1.0 mmol, 2 equiv) was added and the reaction
mixture was stirred at room temperature under N2 atmosphere. After 5 min, α-
bromonitroalkene (0.75 mmol, 1.5 equiv) in THF (2 mL) was added dropwise to the
reaction mixture and continued stirring at room temperature. After completion of the
reaction (as evidenced by TLC), the solvent was removed in vacuo and water (4 mL) was
added to the crude residue. The aqueous phase was extracted with ethyl acetate (3 × 10
mL) and the combined organic layers were washed with brine (10 mL), dried over
26

anhydrous sodium sulfate and concentrated under reduced pressure. The residue on
purification by column chromatography (neutral alumina) using ethyl acetate/petroleum
ether (5:95 to 10:90) yielded the respective pyrrolonaphthoquinones. General procedure
for the synthesis of furanonaphthoquinones 114–119 was based on our previous
report.26 To a stirred solution of lawsone 113 (26 mg, 0.15 mmol) and α-
bromonitroalkene (0.3 mmol, 2 equiv) in water (1 mL) were added NaOAc (15 mg, 0.18
mmol, 1.2 equiv) and tetrabutylammonium bromide (TBAB, 10 mg, 0.03 mmol, 20
mol%). The reaction mixture was heated at 70 °C for 7 h. After completion of the reaction,
the crude product was isolated by filtration and washed with water. The product was
further purified by recrystallization from EtOAc.
4.1.5. Compounds prepared via Hauser-Kraus annulation of sulfonyl phthalide
with Rauhut-Currier adducts of nitroalkenes
General procedure for the synthesis of compounds 120–122 was based on our
previous report.28 To a stirred solution of 3-sulfonyl phthalide 75 (90 mg, 0.33 mmol,
1.1 equiv) in THF (4 mL), Cs2CO3 (146 mg, 0.45 mmol, 1.5 equiv) was added. After 5
min, the RC adduct of nitroalkene (0.3 mmol, 1 equiv) was added and the reaction mixture
was stirred until the completion of reaction. The solvent was removed in vacuo and the
crude residue was directly subjected to silica gel column chromatography and the product
was isolated by gradient elution with ethyl acetate/petroleum ether (10:90 to 18:82).
4.2. Crystallographic data
X-ray diffraction data collection for three compounds was performed on an Enraf-
Nonius Kappa-CCD diffractometer (95 mm CCD camera on κ-goniostat) using graphite
monochromated MoK_radiation (0.71073 Å), at room temperature. Data collection was
carried out using the COLLECT software42 up to 50° in 2θ. Integration and scaling of the
reflections, correction for Lorentz and polarization effects were performed with the HKL
DENZO-SCALEPACK system of programs.43 The structure of the compounds was
solved by direct methods with SHELXS-97.44 The models were refined by full-matrix
least squares on F2 using the SHELXL-97.45 The program ORTEP-346 was used for
graphic representation and the program WINGX47 to prepare materials for publication.
All H atoms were located by geometric considerations (C-H = 0.93-0.97; O-H = 0.82 Å)
27

and refined as riding with Uiso(H) = 1.5Ueq(C-methyl) or 1.2Ueq(other).
Crystallographic data for the structures were deposited in the Cambridge Crystallographic
Data Centre, with numbers CCDC 1985681.
4.3. Animals
Albino Swiss mice were employed for the trypanocidal and cytotoxicity assays,
in accordance to the guidelines of the Colégio Brasileiro de Experimentação Animal
(COBEA), and these were performed under biosafety conditions. All animal
experimentation procedures were approved by the Comissão de Ética em Experimentação
Animal (CEUA/Fiocruz), license L-005/2017.
4.4. Trypanocidal Assay
The experiments were performed with the Y strain of T. cruzi.48 Stock solutions
of the compounds were prepared in dimethylsulfoxide (DMSO), with the final
concentration of the latter in the experiments never exceeding 0.1%. Preliminary
experiments showed that at concentrations of up to 0.5%, DMSO has no deleterious effect
on the parasites.49 Bloodstream trypomastigotes were obtained from infected Albino
Swiss mice at the peak of parasitemia by differential centrifugation. The parasites were
resuspended to a concentration of 10x106 parasites/mL in DMES medium. This
suspension (100 μL) was added to the same volume of each of the compounds, which had
been previously prepared at twice the desired final concentrations. The incubation was
performed in 96-well microplates (Nunc Inc., Rochester, USA) at 4 °C for 24 h.
Benznidazole (Lafepe, Brazil), the standard drug for treatment of chagasic patients, was
used as control. Cell counts were performed in a Neubauer chamber, and the activity of
the compounds corresponding to the concentration that led to 50% lysis of the parasites
was expressed as the IC50/24 h.
4.5. Cytotoxicity to mammalian cells
28

The cytotoxicity assays were performed using primary cultures of peritoneal
macrophages obtained from Albino Swiss mice. For the experiments, 2.5 x 104 cells in
200 µL of RPMI-1640 medium (pH 7.2 plus 10% foetal bovine serum and 2 mM
glutamine) were added to each well of a 96-well microtiter plate and incubated for 24 h
at 37°C. The treatment of the cultures was performed in fresh supplemented medium (200
µL/well) for 24 h at 37 °C. After this period, 110 µL of the medium was discarded and
10 µL of PrestoBlue (Invitrogen) was added to complete the final volume of 100 µL.
Thus, the plate was incubated for 2 h and the measurement was performed at 560 and 590
nm, as recommended by the manufacturer. The results were expressed as the difference
in the percentage of reduction between treated and untreated cells being the LC50/24 h
value, corresponding to the concentration that leads to damage of 50% of the mammalian
cells.50
Appendix A. Supplementary data
Supplementary data related to this article can be found at DOI
Notes
The authors declare no competing financial interest.
Acknowledgements
We thank the Royal Society (CHG\R1\170009 and URF) CNPq (404466/2016-8 and PQ
305741/2017-9), FAPERJ, FAPEMIG (PPM-00638-16 and PPM-00635-18), CAPES and
INCT-Catálise for support. ENSJ also thanks Return Fellowship of the Alexander von
Humboldt Foundation and Royal Society of Chemistry for the Research Fund grant (R19-
9781). RFSMB, JFB and ENSJ thank Worldwide Universities Network (WUN) -
Research Development Fund 2019. INNN thanks SERB, India for financial support.
References
Research highlights
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