Journal Pre-proof

Antitrypanosomal activity of new alkoxyhydroperoxides formed by cycloaddition of

ozone into allyl moiety of eugenol

Marcos Accioly Jr, Fernanda S. Ribeiro, Maiara Amaral, Erica V.C. Levatti, Andre G.
Tempone, João Henrique G. Lago, Miriam Uemi

PII: S0040-4020(24)00203-5
DOI: https://doi.org/10.1016/j.tet.2024.134023
Reference: TET 134023

To appear in: Tetrahedron

Received Date: 4 March 2024

Revised Date: 30 April 2024
Accepted Date: 3 May 2024

Please cite this article as: Accioly Jr M, Ribeiro FS, Amaral M, Levatti EVC, Tempone AG, Lago JHG,
Uemi M, Antitrypanosomal activity of new alkoxyhydroperoxides formed by cycloaddition of ozone into
allyl moiety of eugenol, Tetrahedron, https://doi.org/10.1016/j.tet.2024.134023.

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Ozonation of eugenol (1) leads to alkoxyhydroperoxides (2 – 4)
that showed antitrypanosomal activity.

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T. cruzi

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 1

1 Antitrypanosomal activity of new alkoxyhydroperoxides formed

2 by cycloaddition of ozone into allyl moiety of eugenol

5 Marcos Accioly Jra, Fernanda S. Ribeiroa, Maiara Amaralb, Erica V.C. Levattib,

6 Andre G. Temponeb, João Henrique G. Lagoc, Miriam Uemia*

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8

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a
9 Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal

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10

b
-p
University of São Paulo, 09972-270, São Paulo, SP, Brazil.

11 Laboratory of Pathophysiology, Butantan Institute, 05503-900, São Paulo, SP,

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12 Brazil.
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c
13 Center for Natural and Human Sciences, Federal University of ABC, 09210-
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14 580, Santo Andre, SP, Brazil.

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15
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16

17

18

19

20

21

22

23

24 *corresponding author.

25 E-mail address: [email protected]

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26 Abstract

27

28 Eugenol, a natural phenylpropanoid found in several plants, was exposed to

29 ozone atmosphere to afford three new alkoxyhydroperoxides and prevised

30 homovanillin. The effect of these compounds against the protozoan parasite

31 Trypanosoma cruzi resulted in EC50 values ranging from 7.4 to 10.5 μM, superior

32 to the standard drug benznidazole (EC50 = 18.7 μM). Based on these results, it is

33 possible to consider these compounds as scaffolds for the development of new

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34 drug candidates against Chagas disease.

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35

36 Keywords: eugenol, Criegee

-p mechanism, alkoxyhydroperoxides,
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37 antitrypanosomal activity.
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38
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39 1. Introduction

40

41 Eugenol [1,2], a component of essential oil found in several plants, such

42 as Syzygium aromaticum, Ocimum basilicum, and Cinnamomum verum [3,4],

43 exhibited different biological activities including anesthetic [5–7], antimicrobial [8–

44 11], and antiseptic [12–14]. These potentials are related to the structure of

45 eugenol since the phenolic group plays an important role in the antibacterial and

46 antifungal properties. As reported, this group interacts with the membrane cell of

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47 bacteria, generating the rupture of the cells leaving them to apoptosis. In fungi

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48 cells, this group influences the enzymatic system by a similar mechanism [4,15–

49
-p
17]. Due to eugenol versatility, this compound and several related derivatives
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50 have been studied for their antiparasitic proprieties, including antitrypanosomal
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51 activity. Caused by the parasite Trypanosoma cruzi, Chagas disease is a

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52 neglected tropical disease that has become a problem growing global population

53 in recent decades [18,19]. The World Health Organization (WHO) estimates that
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54 more than 8 million people worldwide are infected with T. cruzi both in rural and
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55 urban areas [18,19]. Current estimative indicates that less than 1% of infected

56 patients are treated, and 75 million people are at risk of contracting the disease

57 [19]. The chemotherapy for Chagas disease involves the use of toxic drugs such

58 as benznidazole and nifurtimox, discovery more than 60 years ago. Based on this

59 scenario, the discovery of new prototypes based on simple and widely distributed

60 natural products, such as eugenol, consists of an important approach for the

61 development of new drugs against Chagas disease [20].

62 Thus, in the present work, eugenol was subjected to the reaction with

63 ozone in different solvents to afford three new alkoxyhydroperoxides: 4-(2-

 4

64 hydroperoxy-2-methoxyethyl)-2-methoxyphenol (2), 4-(2-ethoxy-2-

65 hydroperoxyethyl)-2-methoxyphenol (3), and 4-(2-hydroperoxy-2-

66 isopropoxyethyl)-2-methoxyphenol (4) as well as predictable product

67 homovanillin (5).[21] The formation of these compounds is proposed based on

68 the Criegee mechanism. Additionally, the effects of compounds 1 – 5 against

69 trypomastigote forms of T. cruzi were evaluated in vitro.

70

71 2. Results and discussion

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72

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73 2.1. Mechanism of ozonation of eugenol (1) to form

74
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alkoxyhydroperoxides 2 – 4 and homovanilin (5).
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75
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76 Ozonation of eugenol (1) followed the mechanism proposed by Criegee

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77 [22], that describes the formation of a primary ozonide (PO-Eug) by cycloaddition

78 [3+2] between O3 and the allyl moiety of eugenol, followed by cleavage of the 5-
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79 membered ring into carbonyl oxide (CO-Eug) and formaldehyde. The carbonyl
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80 oxide is the key intermediate of these reactions, which reacts differently according

81 to the media. The carbonyl oxide generated is susceptible to a nucleophilic

82 addition when the reaction is executed using alcohol as a solvent. After

83 prototropism, the tetrahedral intermediate (TI-Eug) leads to the

84 alkoxyhydroperoxides 2 – 4 were achieved (Scheme 1).

85
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86

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87

88 Scheme 1. Proposed mechanism for ozonation of eugenol (1) in ROH (R = Me,

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89
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Et or i-Pr) achieving the alkoxyhydroperoxides 2 – 4.
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90
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91 When using MeOH as a solvent with subsequent reduction in acetic acid

92 (AcOH) using powdered zinc (Zn), it generates compound 2 for further reduction
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93 to a hemiacetal (HA-Eug) and regeneration of the carbonyl, leading to

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94 homovanillin (5) (Scheme 2). Meanwhile, when performing the reaction with
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95 CH2Cl2, the 1,2,4-trioxolane of eugenol (1,2,4-TO-Eug) is formed by a [3+2]

96 cycloaddition between CO-Eug and formaldehyde and, after treatment with Zn in

97 AcOH, it is reduced to 5 (Scheme 2).

98
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99

100
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Scheme 2. Proposed mechanism for the ozonation of eugenol (1) in MeOH or
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101 CH2Cl2, with further reduction with Zn and AcOH, resulting in compound 5.
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102
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103 2.2. Chemical characterization of compounds 2 – 4.

104
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105 1
H NMR spectra of compounds 2 – 4 displayed coupling signals at δ 6.6 –
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106 6.9, corresponding to hydrogens from a 1,2,4-trisubstituted aromatic ring, as well

107 as two singlets at δ 3.9 and 5.6, assigned to one methoxy and one hydroxy group
13
108 at C-2 and C-1, respectively. In the C NMR spectra, signals for C-1/C-6 were

109 detected at ranging from δ 122 to 146, corresponding to those reported to the

110 aromatic ring of eugenol. However, these spectra lack the signals of the allyl

111 group, evidenced that the oxidation occurred in the double bond (Figures 1 and

112 2).

113
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-OMe
11 CHCl3

8 9 10 12 H6 H3 H5 H10 H12 H11

4
5 3 -OH
2 -OOH
6
H9 TMS
1
7
H8’ H8’’
D
H11
10 H6 H3 H5 H10
8
9
4 11
5 3 -OH
6 2
-OOH H9
1
H8’ H8’’
C 7

H10
8
9 H6 H3 H5
4 10
5 3

6 2 -OH
1
-OOH H9 H8’ H8’’
B 7

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10
H6 H3 H5

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8 9
4 -OH
5 3
2
H8
H10’ H10’’

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6 H9
1
A 7

114 11 10 9 8 7
-p 6 5 4 3 2 1 ppm
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115
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116 Figure 1. 1H NMR comparative spectra of compounds 1 (A), 2 (B), 3 (C), and 4
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117 (D), acquired at T = 273K, in CDCl3, TXI 5mm probe and TCI prodigy cryoprobe,
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118 using Avance III instrument, 500MHz, Bruker.

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CDCl3
11 C7 C12
C5 C8
8 9 12 C10
10
C6 C3 C9 C11
4
5 3
C2 C4
6 2 C1
1
7
D
C6 C
3
10 C10 C7 C8 C11
8
9
C5
4 11
3 C C9
2 C1 2 C4
1
7
C
C6 C
3
C5 C8
8
9 C7
5
4 10 C9 C10
3
C C4
2 C1 2
1
B 7

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C5 C6 C7
8 9
4 C9 C3 C8
3
C4 C10
2 C1C2

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A 7

119 200 180 160 140 120

-p 100 80 60 40 20 ppm
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120
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121 Figure 2. 13C NMR comparative spectra of compounds 1 (A), 2 (B), 3 (C), and 4
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122 (D), acquired at T = 273K, in CDCl3, TXI 5mm probe and TCI prodigy cryoprobe,

123 using Avance III instrument, 125 MHz, Bruker.

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124
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125 Ozonation of eugenol (1) in MeOH, EtOH, or i-PrOH without reduction

126 afforded compounds 2 – 4, respectively. 1H NMR spectrum of 2 displays two

127 simplets, one for the hydroperoxide group at δ 8.12, and the other for H-10 at δ

128 3.51 of the methoxy group, as well as one triplet for H-9 at δ 4.87 (J = 5.8 Hz). In

129 addition, the hydrogens H-8’ and H-8’’ were observed as two dd at δ 3.32 to 3.01

130 with J = 14.3 and 5.8 Hz. HSQC spectrum exhibited cross-peaks between H-9 (δ

131 4.87) and C-9 (δ 109.1) as well as between H-10 (δ 3.51) and C-10 (δ 55.8).

132 HMBC spectrum displayed correlations between H-8’/H-8’’ (δ 3.01/2.90) with C-

133 9 (δ 109.1), H-10 (δ 3.51) with C-9 (δ 109.1) and H-9 (δ 4.87) with C-10 (δ 109.1).
 9

134 Finally, ESI-HRMS spectrum showed the [M + Na]+ ion peak m/z 237.0733

135 confirming the structure of 2 as 4-(2-hydroperoxy-2-methoxyethyl)-2-

136 methoxyphenol.
1
137 H NMR spectra of 3 and 4 showed several similarities with that recorded

138 to 2 being one singlet for the hydroperoxide group at δ 8.35/8.15 and one triplet

139 for H-9 at δ 4.93/4.98 (J = 5.8 Hz). In the case of 3 was observed one multiplet

140 for H-10 at δ 3.53/3.92 and one triplet for H-11 at δ 1.22 (J = 7.0 Hz). It is important

141 to mention that the multiplet for H-10 at δ 3.87–3.92 is overlapped by the singlet

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142 H-7 of the methoxy group at δ 3.87. Otherwise, in the 1H NMR spectrum of 4 one

multiplet for H-10 at δ 3.92 and two doublets (J = 6.0 Hz) for H-11 and H-12 at δ

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143

144
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1.28 and 1.03, respectively, were observed. Similarly to compound 2,
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145 assignments of 1H and 13
C NMR data for 3 and 4 were confirmed by analysis of
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146 HSQC and HMBC spectra. Finally, ESI-HRMS spectral analysis showed the [M
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147 + Na]+ ion peaks at m/z 251.0890 and 265.1046, confirming the structure of 3 and

148 4 as 4-(2-ethoxy-2-hydroperoxyethyl)-2-methoxyphenol and 4-(2-hydroperoxy-2-

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149 isopropoxyethyl)-2-methoxyphenol, respectively.

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150 The main product obtained for the ozonation of eugenol, with either CH2Cl2

151 or MeOH, and further reduction afforded compound 5. Analysis of the 1H NMR

152 spectrum indicated the presence of one aldehyde group due to the presence of

153 one triplet (J = 2.5 Hz) at δ 9.73 (H-9), confirmed by HSQC spectrum by direct

154 correlation of this signal with that at δ 200.0 (C-9). The peak assigned to H-8 was

155 observed as one doublet at δ 3.64 (J = 2.5 Hz), resulting from the coupling

156 between H-8 and H-9. Analyzing the HMBC spectrum, it was observed a

157 correlation between H-9 with C-8 and C-4 at δ 50.8 and 123.2, respectively.
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158 Comparison with data reported in the literature allowed the identification of 5 as

159 2-(4-hydroxy-3-methoxyphenyl)acetaldehyde – homovanillin.

160

161 2.3. Antitrypanosomal activity of compounds 1 - 5.

162

163 The antitrypanosomal activity of precursor eugenol (1), hydroperoxides 2

164 – 4, and homovanilin (5) was evaluated against trypomastigote forms of T. cruzi.

165 As observed in Table 1, compound 1 was inactive, as previously observed in the

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166 literature. However, the substitution of one carbon from allyl moiety by one

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167 carbonyl to form homovanillin (5), played an important role in the antiparasitic

168
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activity, resulting in a trypanocidal effect with an EC50 value of 23.4 μM.
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169 Otherwise, compounds 2 – 4 displayed higher potency against the parasite with
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170 EC50 values of 10.5, 8.8, and 7.4 μM, superior to that determined for standard
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171 drug benznidazole (EC50 = 18.7 μM). These findings suggested that the structural

172 differences between compounds 2 – 4 were not important to the effect against
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173 trypomastigotes of T. cruzi. Considering the mammalian cytotoxicity for murine

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174 fibroblasts (NCTC cells), it was observed that compounds 1 and 5, including the

175 drug benznidazole, were non-cytotoxic to the highest tested concentration of 200

176 μM. However, the hydroperoxides displayed moderate toxicity, resulting in CC50

177 values of 166.4 μM (2), 142.8 μM (3), and 147.9 μM (4) with selectivity index

178 values of 15.8, 16.2, and 20.0, respectively. Taken together, the obtained results

179 suggested that hydroperoxide compounds, prepared from the oxidation of

180 inactive natural products eugenol, can be important scaffolds for the search for

181 hit compounds for Chagas disease.

182
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183 Table 1. Anti-T. cruzi activity (trypomastigote forms) and mammalian

184 cytotoxicity (NCTC cells) of compounds 1 – 5 and positive control benznidazole.

185

compound EC50 / μMa CC50 / μMb SIc

1 > 100 > 200 -

2 10.5 ± 1.7 166.4 ± 17.6 15.8

3 8.8 ± 0.5 142.8 ± 23.8 16.2

4 7.4 ± 0.4 147.9 ± 11.8 20.0

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5 23.4 ± 3.4 > 200 > 8.5

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benznidazole 18.7 ± 4.1 > 200 > 10.7

186 a
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EC50: 50% Effective Concentration; b CC50: 50% Cytotoxic Concentration; c SI:
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187 Selectivity Index.
188
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189 3. Conclusion
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190

191 In this study, we describe the synthesis of one known aldehyde (2) and three
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192 new alkoxyhydroperoxides (2 – 4) by ozonation of eugenol (1). The mechanism

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193 for the formation of these compounds involves the cycloaddition of O3 to the allyl

194 moiety of eugenol, followed by a cleavage of C-C and O-O bonds, leading to a

195 carbonyl oxide and an aldehyde. Solvolysis using MeOH, EtOH or i-PrOH

196 afforded compounds 2 – 5 which were characterized by extensive analysis of

197 NMR, IR, and ESI-MS data. In vitro evaluation of the antitrypanosomal activity of

198 compounds 1 – 5 against trypomastigote forms of T. cruzi indicated that

199 alkoxyhydroperoxides 2 – 4 exhibited EC50 values ranging from 7.4 to 10.5 μM,

200 superior to that determined for standard drug benznidazole (EC50 = 18.7 μM), on

201 contrary to eugenol (1), which showed no activity to the highest tested
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202 concentration (100 μM). However, new alkoxyhydroperoxides 2 – 4 displayed

203 moderate toxicity against NCTC cells to afford selectivity index (SI) values higher

204 than 15. Taking together, the obtained results suggest that using a simple method

205 to preparation of hydroperoxides from eugenol, it is possible to obtain potent

206 compounds against trypomastigote forms of T. cruzi that can be used as scaffolds

207 for the development of new drug candidates against T. cruzi.

208

209 4. Experimental section

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210

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211 4.1. General information

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213 Eugenol, CDCl3, sodium sulfate, powder zinc, n-hexane, EtOAc, CH2Cl2,
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214 MeOH, EtOH, and i-PrOH were purchased from Sigma Aldrich (Steinheim,
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215 Germany). Oxygen (> 99% purity) was obtained from Air Liquide (São Paulo,

216 Brasil). Ozone was generated using an Aquazone instrument plus (Red Sea Fish
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217 Pharm Ltd., Eilat, Israel). Silica gel 60 (230-400 mesh) and silica gel 60 F254
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218 plates were acquired from Macherey-Nagel (Düren, Germany). Acetic acid from

219 Labsynth (São Paulo, Brazil). The water used in the experiments was treated with

220 the MilliQ Reference equipment, Merck Millipore (Darmstadt, Germany). NMR

221 analyses were performed on a Bruker-Biospin Ascend 500 instrument, Avance III

222 series, (Rheinstetten, Germany), operating at 11.7 T. The instrument was

223 equipped with 5-mm trinuclear, inverse detection probe with z-gradient (TXI). The

224 temperature was controlled by a BCU I accessory. All chemical shifts were

225 expressed in ppm relative to the deuterium solvent or TMS, and data were

226 acquired and processed using TOPSPIN 3.2 (Bruker-Biospin, Rheinstetten,

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227 Germany). 1H NMR (500.13 MHz) and 13

C NMR (125.77 MHz) spectra were

228 recorded at 0°C, using CDCl3 as solvent. 1H NMR spectra were acquired with a

229 spectral width of 14.45 ppm and 64 K data points, providing a digital resolution of

230 0.10 Hz. For 13C NMR spectra, a spectral width of 270.4 ppm and 64 K data points

231 were set, providing a digital resolution of 0.11 Hz. For 2D spectra, a spectral width

232 was the same as mentioned above for 1H and 13

C nuclei, using 2K and 512 or

233 400 data points for direct and indirect dimensions, respectively. Electrospray

234 ionization-high resolution mass spectrometry (ESI-HRMS) experiments were

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235 performed using a microTOF-Q spectrometer (Bruker Daltonics Inc., Billerica,

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236 MA). For the analysis, 1 mg of each compound was dissolved in 1 mL of MeOH

237
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or i-PrOH and injected directly through the instrument at a flow rate 180 µL/h. The
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238 flow rate of drying gas was kept at 5.0 L/min, and the nebulizing gas pressure
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239 was maintained at 2.0 Bar. The capillary potential was set to 4.5 kV, the dry gas
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240 temperature was set to 180°C, and the capillary current was set to 2512 nA. IR

241 analysis was conducted using an IR spectrometer, model Prestige 21 (Shimadzu,

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242 Tokyo, Japan) using 1 mg of each sample dissolved in 30 µL of CHCl3 and

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243 dropped into a KBr disk.

244

245 4.2. Procedure A for ozonation of eugenol (1) – preparation of

246 alkoxyhydroperoxides 2 – 4.

247

248 The ozonation of eugenol (1, 106 mg, 0.65 mmol) was carried out in 5 mL of

249 adequate alcohol (MeOH, EtOH, or i-PrOH) for 20 min, under a flow of 50 mL/min

250 of ozone (44.7 mmol) and at 4 ºC. After remotion of the solvent under reduced

251 pressure, the residue was purified by flash column chromatography using silica
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252 gel 60 eluted with mixtures of n-hexane:EtOAc (95:5 to 75:25) to afford the

253 alkoxyhydroperoxides 2 – 4.

254

255 4-(2-Hydroperoxy-2-methoxyethyl)-2-methoxyphenol (2). Yield: 31.6 mg

256 (21%), yellow oil. IR (KBr) max 3399, 2932, 2845, 1607, 1518, 1435, 1368, 1236,

257 1032 cm-1; ESI-HRMS m/z 237.0733 [M+Na]+ (calcd. for C10H14O5Na, 237.0729);

258 1
H NMR (500 MHz, CDCl3) δ 8.12 (s, 1H), 6.87 (d, J = 7.9 Hz, 1H), 6.79–6.75

259 (m, 2H), 5.59 (s, OH), 4.87 (t, J = 5.8 Hz, 1H), 3.90 (s, 3H), 3.51 (s, 3H), 3.01 (dd,

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260 J = 14.3 and 5.8 Hz, 1H), 2.90 (dd, J = 14.3 and 5.8 Hz, 1H); 13C NMR (125 MHz,

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261 CDCl3) δ 146.2, 144.1, 127.8, 122.0, 114.1, 111.8, 109.1, 56.4, 55.8, 37.7.

262
-p
4-(2-Ethoxy-2-hydroperoxyethyl)-2-methoxyphenol (3). Yield: 25.2 mg
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263 (17%), yellow oil. IR (KBr) max 3393, 2974, 2932, 2847, 1605, 1516, 1464, 1452,
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264 1371, 1269 cm-1; ESI-HRMS m/z 251.0890 [M+Na]+ (calcd. for C11H16O5Na,
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265 251.0893); 1H NMR (500 MHz, CDCl3) δ 8.35 (s, 1H), 6.86 (d, J = 8.0 Hz, 1H),

266 6.80 (d, J = 1.8 Hz, 1H), 6.76 (dd, J = 8.0 and 1.8 Hz, 1H), 5.62 (s, OH), 4.93 (t,
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267 J = 5.8 Hz, 1H), 3.92–3.87 (m, 4H), 3.60–3.52 (m, 1H), 3.00 (dd, J = 14.1 and 6.0
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268 Hz, 1H), 2.89 (dd, J = 14.1 and 6.0 Hz, 1H), 1.22 (t, J = 7.0 Hz, 3H). C NMR

269 (125 MHz, CDCl3) δ 146.1, 144.0, 127.9, 122.1, 114.0, 111.9, 108.0, 65.0, 55.7,

270 38.1, 15.2.

271 4-(2-Hydroperoxy-2-isopropoxyethyl)-2-methoxyphenol (4). Yield: 46.2 mg

272 (29%), yellow oil. IR (KBr) max 3404, 2972, 2930, 1607, 1516, 1460, 143, 1373,

273 1271 cm-1; ESI-HRMS m/z 265.1046 [M+Na]+ (calcd. for C12H18O5Na, 265.1051);

274 1
H NMR (500 MHz, CDCl3) δ 8.15 (s, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.79 (d, J =

275 1.8 Hz, 1H), 6.76 (dd, J = 8.0, 1.8 Hz, 1H), 5.61 (s, OH), 4.98 (t, J = 5.5 Hz, 1H),

276 3.94–3.89 (m, 4H), 3.00 (dd, J = 14.1, 5.5 Hz, 1H), 2.86 (dd, J = 14.1, 5.5 Hz,
 15

277 1H), 1.28 (d, J = 6.0 Hz, 3H), 1.03 (d, J = 6.0 Hz, 3H). 13C NMR (125 MHz, CDCl3)

278 δ 146.1, 144.0, 128.1, 122.2, 114.0, 112.1, 106.6, 71.7, 55.8, 23.2, 22.2, 15.2.

279 2-(4-Hydroxy-3-methoxyphenyl)acetaldehyde - homovanillin (5). Yield:

280 50.7 mg (47%), yellow oil. IR (KBr) max 3426, 2932, 2843, 1746, 1601, 1516,

281 1464, 1431, 1367, 1269 cm-1; 1H NMR (500 MHz, CDCl3) δ 9.73 (t, J = 2.5 Hz,

282 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.74 (dd, J = 8.0 and 1.9 Hz, 1H), 6.69 (d, J = 1.9

283 Hz, 1H), 5.65 (s, 1H), 3.90 (s, 1H), 3.64 (d, J = 2.5 Hz, 2H); 13C NMR (125 MHz,

284 CDCl3) δ 200.0, 146,7, 144.8, 123.2, 122.4, 114.7, 111.6, 55.8, 50.2.

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285

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286 4.3. Procedure B for ozonation of eugenol (1) – preparation of

287 homovanillin (5).

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288
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289 The ozonation of eugenol (1, 106 mg, 0.65 mmol) was carried out in 3 mL
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290 of CH2Cl2 for 10 minutes under a flow of 100 mL/min of O3 (44.7 mmol) and at 4

291 ºC. After solvent removal under reduced pressure, the reactional mixture was
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292 stirred with 100 mg of powder zinc and 5.5 mL of acetic acid/water (20:1, v/v) for
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293 30 min at room temperature. Then, the product was filtered, diluted with 10 mL of

294 CH2Cl2, and washed with 3 x 10 mL of H2O. Subsequently, the organic phase

295 was dried over with anhydrous Na2SO4 and the solvent was removed under

296 reduced pressure. The residue was purified by flash column chromatography

297 using silica gel 60 eluted with mixtures of n-hexane and EtOAc (from 95:5 to

298 75:25) to afford compound 5.

299

300

301
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302 4.4. Parasites and mammalian cells

303

304 In vitro assays were performed with trypomastigotes of Trypanosoma cruzi

305 (Y strain) in Rhesus monkey kidney cells (LLC-MK2-ATCC CCL 7) using RPMI-

306 1640 (Roswell Park Memorial Institute) medium supplemented with 2% bovine

307 serum at 37 ºC in a 5% CO2 incubator (Tada et al., 1986). Murine conjunctive

308 cells (NCTC clone 929, ATCC) and LLC-MK2 were maintained in RPMI-1640

309 supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5%

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310 CO2.

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311

312 4.5.
-p
Evaluation of antitrypanosomal activity
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313
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314 Trypomastigotes of T. cruzi (Y strain) were counted in a Neubauer

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315 hemocytometer and deposited on a microplate (96 wells; 1 × 106 cells/well).

316 Subsequently, compounds 1 – 5 and positive control benznidazole were added

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317 to the cells in different concentrations (100 - 1.56 µM) and incubated for 24 h at
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318 37 ºC in 5% CO2 for calculation of 50% effective concentration (EC50).

319 Trypomastigote activity was based on the conversion of the soluble tetrazolium

320 salt 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) into the

321 insoluble formazan by mitochondrial enzymes [23]. The extraction of formazan

322 was performed for 18 h at 24 ºC with sodium dodecyl sulfate (SDS) 10% v/v (100

323 mL/well) [24].

324
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325 4.6. Cytotoxicity against mammalian cells

326

327 In vitro cytotoxic concentrations 50% (CC50) were determined using NCTC

328 cells-clone L929, which were seeded at 6 × 104/well in 96 well microplates and

329 incubated with compounds 1 – 5 and benznidazole (200 - 1.56 µM) diluted in

330 RPMI-1640 for 48 h at 37º C in 5% CO2. The viability of the cells was determined

331 by the MTT colorimetric assay and the optical density was determined in

332 FilterMax F5 (Molecular Devices) at 570 nm [24]. The respective selectivity

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333 indexes (SI) were determined using the following equation: CC50 against NCTC

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334 cells/EC50 against parasites [25].

335
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336 4.7. Statistical analysis
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337
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338 The data obtained represent the mean and standard deviation of duplicate

339 samples from two independent assays. The EC50 or CC50 values were calculated
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340 using sigmoid dose-response curves in GraphPad Prism 5.0 software, and the
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341 95% confidence intervals are included in parentheses. The ANOVA test was used

342 for the significance p-value.

343

344 Conflicts of interest

345

346 The authors declare that they have no known competing financial interests

347 or personal relationships that could have appeared to influence the work reported

348 in this paper.

349
 18

350 Data availability

351

352 Data will be made available on request.

353

354 Acknowledgements

355

356 This work was funded by grants and fellowships provided by Fundação de

357 Amparo à Pesquisa do Estado de São Paulo (FAPESP – 2014/14801-8 and

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358 2023/12447-1) and Conselho Nacional de Desenvolvimento Científico e

Tecnológico (CNPq – 405691/2021-1 and 484628/2013-5). We also thank

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359

360
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CAPES (financial code 001), CNPq scientific research award to JHGL and AGT
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361 as well as to FINEP for financial support of the NMR instrument.
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362
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363 Credit author statement

364
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365 Marcos Accioly Jr: Methodology, Validation, Investigation, Writing – Original

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366 Draft; Fernanda S. Ribeiro: Investigation; Validation; Maiara Amaral, Erica V.C.

367 Levatti, Andre G. Tempone, João Henrique G. Lago: Investigation, Writing –

368 Review & Editing; Miriam Uemi: Conceptualization, Methodology, Supervision,

369 Resources, Writing – reviewing, Funding acquisition.

370

371 References

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐ The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

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