Synthesis of a sugar-based thiosemicarbazone series and structure-activity relationship versus the parasite cysteine proteases rhodesain, cruzain, and Schistosoma mansoni cathepsin B1

Article abstract of DOI:10.1128/AAC.04601-14

The pressing need for better drugs against Chagas disease, African sleeping sickness, and schistosomiasis motivates the search for inhibitors of cruzain, rhodesain, and Schistosoma mansoni CB1 (SmCB1), the major cysteine proteases from Trypanosoma cruzi, Trypanosoma brucei, and S. mansoni, respectively. Thiosemicarbazones and heterocyclic analogues have been shown to be both antitrypanocidal and inhibitory against parasite cysteine proteases. A series of compounds was synthesized and evaluated against cruzain, rhodesain, and SmCB1 through biochemical assays to determine their potency and structure-activity relationships (SAR). This approach led to the discovery of 6 rhodesain, 4 cruzain, and 5 SmCB1 inhibitors with 50% inhibitory concentrations (IC₅₀s) of ≤10 μM. Among the compounds tested, the thiosemicarbazone derivative of peracetylated galactoside (compound 4i) was discovered to be a potent rhodesain inhibitor (IC₅₀ = 1.2 ± 1.0 μM). The impact of a range of modifications was determined; removal of thiosemicarbazone or its replacement by semicarbazone resulted in virtually inactive compounds, and modifications in the sugar also diminished potency. Compounds were also evaluated in vitro against the parasites T. cruzi, T. brucei, and S. mansoni, revealing active compounds among this series.

Full text of DOI:10.1128/AAC.04601-14

AAC Accepted Manuscript Posted Online 23 February 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.04601-14
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Synthesis of a sugar-based thiosemicarbazone series and structure-activity relationship versus the
parasite cysteine proteases: rhodesain, cruzain and Schistosoma mansoni cathepsin B1
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Nayara Cristina Fonseca,a Luana Faria da Cruz,b Filipe da Silva Villela,b Glaécia Aparecida do
Nascimento Pereira,b,c Jair Lage de Siqueira-Neto,d Danielle Kellar,e Brian M. Suzuki,e Debalina Ray,e
Thiago Belarmino de Souza,a Ricardo José Alves,a Policarpo Ademar Sales Júnior,f Alvaro José
Romanha,f,g Silvane Maria Fonseca Murta,f James H. McKerrow,d Conor R. Caffrey,e Renata Barbosa
de Oliveira,a Rafaela Salgado Ferreira b,#
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Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas
Gerais, Belo Horizonte, MG 31270-901, Brazila; Departamento de Bioquímica e Imunologia, Instituto
12 de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901,
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Brazilb; CAPES Foundation, Ministry of Education of Brazil, Brasília, DF 70040-020, Brazilc; Skaggs
School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, San Diego, CA
15 92093, USAd; Center for Discovery and Innovation in Parasitic Diseases and the Department of
Pathology, University of California San Francisco, San Francisco, CA 94158, USAe; Centro de
17 Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, MG 30190-002, Brazilf; Departamento de
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Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina, Florianópolis, SC
19 88040-900, Brazilg
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21 Running Head: Thiosemicarbazones as cysteine protease inhibitors
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#Address correspondence to Rafaela S. Ferreira, rafaelasf@icb.ufmg.br
N.C.F. and L.F.C. contributed equally to this work

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ABSTRACT
The pressing need for better drugs against Chagas disease, African sleeping sickness and
schistosomiasis motivates the search for inhibitors of cruzain, rhodesain and SmCB1, the major
27 cysteine proteases from Trypanosoma cruzi, Trypanosoma brucei and Schistosoma mansoni,
28 respectively. Thiosemicarbazones and heterocyclic analogues have been shown to be both
29
antitrypanocidal and inhibitory against parasite cysteine proteases. A series of compounds was
30 synthesized and evaluated against cruzain, rhodesain and SmCB1 through biochemical assays to
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determine their potency and structure-activity relationships (SAR). This approach led to the discovery
of 6 rhodesain, 4 cruzain and 5 SmCB1 inhibitors with IC50 ≤ 10 µM. Among the compounds tested, the
thiosemicarbazone derivative of peracetylated galactoside (4i) was discovered as a potent rhodesain
34 inhibitor (IC50= 1.2 ± 1.0 μM). The impact of a range of modifications was determined: removal of
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thiosemicarbazone or its replacement by semicarbazone resulted in virtually inactive compounds and
modifications in the sugar also diminished potency. Compounds were also evaluated in vitro against the
parasites T. cruzi, T. brucei and S. mansoni, revealing active compounds among this series.
39 Key words: thiosemicarbazone series, rhodesain, cruzain, Schistosoma mansoni cathepsin B1.

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INTRODUCTION
New drugs for parasitic diseases are urgently needed but these globally important infections are
often “neglected” because they most commonly afflict poor and marginalized communities. Current
therapies are limited by poor efficacy, toxicity, high costs and parasite resistance. Chagas disease,
African sleeping sickness and schistosomiasis are examples of diseases for which new therapies are
needed (1, 2). Among the most studied and exploited molecular targets for these diseases are cysteine
proteases. These enzymes have essential roles in parasite nutrition, immune evasion, host cell invasion
47 and metacyclogenesis (3–6). Indeed, the cysteine proteases cruzain, rhodesain and SmCB1 from
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Trypanosoma cruzi, Trypanosoma brucei and Schistosoma mansoni, respectively, are validated
molecular targets and have been the subject of numerous medicinal chemistry projects (7–17) that have
yielded trypanocidal inhibitors, both in parasite culture and in animal models of infection (13, 15, 18–
21).
The diverse inhibitors against these enzymes comprise compound classes which bind non-
covalently (11, 12) and scaffolds containing a warhead that binds covalently to the catalytic cysteine.
Within the latter category, vinylsulfones (8, 22–25), oxy-methyl ketones (7, 26), nitriles (16), epoxides
and thiosemicarbazones (13–15, 27–29) have been described. Thiosemicarbazones present as
advantages their low molecular weight, low cost of synthesis and their nonpeptidic nature (27).
Greenbaum and co-workers (13) synthesized and evaluated the cysteine protease inhibitory and anti-
parasitic activities of a library of thiosemicarbazones, with promising results. According to these
59 authors, the thiosemicarbazones are regarded as validated drug leads capable of killing different species
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of protozoan parasites (T. cruzi, P. falciparum, and T. brucei) via inhibition of cysteine proteases.
Heterocyclic thiazole derivatives are also of great importance in medicinal chemistry due to
their broad spectrum of biological activities (30–34). Also, many cysteine proteases inhibitors bearing
thiazole or isothiazolone ring systems have been described as promising compounds against parasitic
diseases (35, 36). Because of the versatile approach to the synthesis of the thiazole scaffold in

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66 of cysteine proteases. The covalent attachment of the thiosemicarbazone or thiazole unit and a
67 carbohydrate moiety was also designed to modulate solubility and interaction properties (for example,
68 by hydrogen bonding) with the molecular target (cysteine protease).
thiosemicarbazones, we synthesized and evaluated a series of thiazole analogues as potential inhibitors
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Here, we screened a series of thiosemicarbazones and cyclic analogues against rhodesain and
discovered an acetylated derivative of galactose as a potent inhibitor (IC50 = 1.2 ± 1.0 µM). This is the
first case of a sugar moiety being present in an inhibitor from this chemical class, encouraging further
SAR (structure-activity relationship) studies on these series. Herein we report their synthesis and
evaluation against the proteases, cruzain, rhodesain and SmCB1, and their in vitro bioactivities against
T. cruzi, T. brucei and S. mansoni.
MATERIALS AND METHODS
77 Chemistry. All melting points were determined on a Microquímica MQAPF 301 apparatus. The IR
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spectra were recorded using a PerkinElmer Spectrum One infrared spectrometer and absorptions are
reported as wave numbers (cm-1). The NMR spectra were recorded on a Bruker AVANCE DRX200 or
Bruker AVANCE DRX400 instrument, using tetramethylsilane (TMS) as the internal standard.
Chemical shifts are given in δ (ppm) scale and J values are given in Hz. All reagents of analytical grade
82 were obtained from commercial suppliers and used without further purification. Compounds 4c, 4h,
83 4n-q and 6a-f were synthesized according to the published procedure (36).
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85 General procedure A, for the synthesis of aryl glycosides bearing a formyl group (1-3). To a
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solution of vanillin (3 equiv.) in water containing 2.8 equimolar amounts of lithium hydroxide was
added the corresponding peracetylglycosyl bromide (1 equiv.) dissolved in acetone. The reaction
mixture was stirred at room temperature for 2h. The progress of the reaction was followed by thin-layer
chromatography (TLC) (1:1 hexane:ethyl acetate). The mixture was concentrated to remove acetone

90 and then diluted with water (10 mL) and washed with dichloromethane. The organic layer was
91 separated, and washed with 10 % (w/v) NaOH aqueous solution and water until pH 7. The resulting
92
organic phase was dried over sodium sulfate, filtered, and concentrated to dryness under reduced
93 pressure.
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4-Formyl-2-methoxyphenyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (1). Obtained from the
96 general procedure A as a white solid, yield 60 %; mp 123.1-123.8 ºC (123-124 ºC(37)); [α]D -8.1 (c
97 0,49, CH2Cl2); IR (ῡ/cm–1): 2988, 2901 (C-H sp3), 1752, 1740 (C=O), 1693 (C=O), 1590, 1514 (C=C),
98 1370 (C-H sp3); 1H NMR (400 MHz; CDCl3) δ 9.89 (s, 1H, CHO); 7.43-7.40 (m, 2H); 7.25 (d, 1H, J =
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101
8.4 Hz); 5.55 (t, 1H, J = 8.0 Hz); 5.46 (d, 1H, J = 2.8 Hz); 5.12 (dd, 1H, J = 8.0 Hz, J = 2.8 Hz); 5.05
(d, 1H, J = 8.0 Hz ); 4.23 (dd, 1H, J = 11.8 Hz, J = 6.8 Hz); 4.16 (dd, 1H, J = 11.8 Hz, J = 6.4 Hz);
4.07-4.03 (m, 1H); 3.90 (s, 3H, OCH3); 2.17-2.02 (4s, 12H,COCH3); 13C NMR (100 MHz; CDCl3) δ
102 190.89 (CHO); 170.33-169.35 (4C OCOCH3); 151.29; 150.97; 132.77; 125.39; 117.99; 110.78; 100.35;
103 71.28; 70.60; 68.48; 66.82; 61.31; 56.13 (OCH3); 20.69-20.58 (4C, COCH3).
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105
4-Formyl-2-methoxyphenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (2). Obtained from the general
106 procedure A as a white solid, yield 57 %; mp 136.1-137.3 ºC (135-137 ºC(38)); [α]D -39.2 (c 0,51,
107
108
109
110
111
CH2Cl2); IR (ῡ/cm–1): 2988, 2901 (C-H sp3), 1753, 1737 (C=O), 1694 (C=O), 1591, 1510 (C=C), 1378
1
(C-H sp3); H NMR (400 MHz; CDCl3) δ 9.89 (s, 1H, CHO); 7.43-7.40 (m, 2H); 7.21 (d, 1H, J = 8.0
Hz); 5.34-5.28 (m, 2H); 5.13 (t, 1H, J = 6.8 Hz); 5.09 (d, 1H, J = 6.4 Hz ); 4.27 (dd, 1H, J = 12.4 Hz, J
= 5.2 Hz); 4.18 (dd, 1H, J = 12.4 Hz, J = 2.4 Hz); 3.86 (s, 3H, OCH3); 3.85-3.70 (m, 1H); 2.07-2.04 (4s,
12H, COCH3); 13C NMR (100 MHz; CDCl3) δ 190.89 (CHO); 170.52-169.25 (4C OCOCH3); 151.11;
112 151.03; 132.86; 125.34; 118.23; 110.85; 99.73; 72.41; 72.28; 71.06; 68.28; 61.90; 56.12 (OCH3);
113 20.67-20.59 (4C, COCH3).

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4-Formyl-2-methoxyphenyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-β-D-
glucopyranoside (3). Obtained from the general procedure A as a white solid, yield 52 %; mp 120.0-
122.1 ºC (121-124 ºC(39)); [α]D -12.5 (c 0.48, CH2Cl2); IR (ῡ/cm–1): 2942 (C-H sp3), 1741 (C=O),
1
1687 (C=O), 1592, 1507 (C=C), 1424, 1370 (C-H sp3); H NMR (400 MHz; CDCl3) δ 9.88 (s, 1H,
CHO); 7.42-7.39 (m, 2H); 7.17 (d, 1H, J = 8.0 Hz); 5.35 (d, 1H, J = 8.0 Hz); 5.32 (t, 1H, J = 8.8 Hz);
120 5.22 (t, 1H, J = 8.8 Hz ); 5.14-5.08 (m, 2H); 4.97 (dd, 1H, J = 12.0 Hz, J = 3.2 Hz); 4.53-4.51 (m, 2H);
4.17-4.06 (m, 3H); 3.93-3.89 (m, 2H); 3.88 (s, 3H); 3,79-3.75 (m, 1H); 2.15-1.97 (s, 21H); 13C NMR
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122 (100 MHz; CDCl3) δ 190.89 (CHO); 170.38-169.10 (7C COCH3); 151.16; 150.93; 132.72; 125.37;
123
124
125
117.87; 110.77; 101.13; 99.35; 76.05; 73.04; 72.47; 71.33; 70.94; 70.79; 69.14; 66.64; 61.84; 60.84;
56.11 (OCH3); 20.79-20.50 (7C, COCH3).
126 General procedure B, for the synthesis of thiosemicarbazones. To a suspension of 1 equiv. of
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thiosemicarbazide and 1 equiv. of the corresponding aldehyde or ketone in ethanol were added 3 drops
of glacial acetic acid. The reaction mixture was kept under reflux and magnetic stirring for 2 hours.
129 Then, the resulting suspension was vacuum filtered and washed with cold distilled water.
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131 2-Phenylmethylenehydrazinecarbothioamide (4a). Obtained from the general procedure B as a white
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solid (91% yield). Mp 157.3-158.6 ºC (lit. 157-159 ºC(40)). IR, (ῡ/cm–1): 3418 (NH), 1589 (C=N),
1539, 1447 (C=C aromatic). 1H NMR (200 MHz, DMSO-d6), δ/ppm: 11.43 (1 H, s, NH); 8.20 (1 H, s,
134 NH2); 8.05 (1 H, s, CH=N); 7.99 (1 H, s, NH2); 7.78 (2 H, m, ArH); 7.39 (3 H, m, ArH).
135
136
137
2-[(4-methylphenyl)methylene]hydrazinecarbothioamide (4b). Obtained from the general procedure B
as a white solid (82% yield). Mp 168.5-169.8 ºC (lit. 162-163 °C(40)). IR, (ῡ/cm–1): 3398 (NH), 1596

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(C=N), 1509, 1462 (C=C aromatic).
2-(4-pyridinylmethylene)hydrazinecarbothioamide (4d). Obtained from the general procedure B, as a
141 pale yellow solid (80% yield). Mp 235-236 ºC (lit. 240 ºC(41)). IR, (ῡ/cm–1): 3420 (NH), 1591 (C=N),
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142
143
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1536 (C=C aromatic). H NMR (200 MHz, DMSO-d6), δ/ppm: 11.69 (1 H, s, NH); 8.58 (2 H, d, H-2
pyridine); 8.04 (1 H, s, NH2); 8.21 (1 H, s, NH2); 8.00 (1 H, s, CH=N); 7.76 (2 H, d, H-3 pyridine).
2-(1H-pyrrol-2-ylmethylene)hydrazinecarbothioamide (4e). Obtained from the general procedure B, as
a solid (66 % yield). Mp 191.9-193.6 ºC (lit. 195-197 ºC(42)). IR, (ῡ/cm–1): 3445 (NH), 1583 (C=N),
1530, 1550 (C=C aromatic).
1-Cyclopentylidenethiosemicarbazide (4f). Obtained from the general procedure B, as a solid (51 %
150 yield). Mp 155.3-157 ºC (lit. 152-154 ºC(43)). IR, (ῡ/cm–1): 3375 (NH), 1586 (C=N), 1508, 1448 (C=C
1
151
152
153
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aromatic). H NMR (200 MHz, CDCl3), δ/ppm: 8.61 (1 H, s, NH); 7.33 (1 H, s, NH2); 6.67 (1 H, s,
NH2); 2.36 (4 H, m, CH2); 1.85 (4 H, m, CH2).
1-Cyclohexylidenethiosemicarbazide (4g). Obtained from the general procedure B, as a solid (65 %
155 yield). Mp 160.2-161.1 ºC (lit. 154-155 ºC(43)). IR, (ῡ/cm–1): 3375 (NH), 1583 (C=N), 1505, 1461
156
157
158
159
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(C=C). 1H NMR (200 MHz, CDCl3), δ/ppm: 8.93 (1 H, s, NH); 7.30 (1 H, s, NH2); 6.60 (1 H, s, NH2);
2.32 (4 H, m, CH2); 1.67 (6 H, m, CH2).
2-[[3-methoxy-4-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]phenyl]methylene]
hydrazinecarbothioamide (4i). Obtained from the general procedure B as a solid, yield 76%; mp 127.2-
161 129.9 ºC; [α]D -18.9 (c 0,53, EtOH); IR (ῡ/cm–1): 3454 (NH), 1743 (C=O), 1597 (C=N), 1504, 1450
(C=C), 1068 (C-O); 1H NMR (200 MHz; CDCl3) δ/ppm: 10.27 (1H, s, NH); 7.90 (1H, s, CH=N); 7.27-
162

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7.12 (4H, m, ArH); 6.64 (1H, s, NH2); 5.57-5.44 (m, 2H); 5.13 (1H, dd, J = 10.4 Hz, J = 3.2 Hz); 4.97
(1H, d, J = 8.0 Hz); 4.29-4.00 (3H, m); 3.86 (3H, s, OCH3); 2.17-2.02 (12H, 4s, COCH3); 13C NMR (50
165 MHz; CDCl3) δ/ppm: 177.55 (C=S); 170.02-169.09 (4C, OCOCH3); 150.38; 147.96; 143.33; 128.81;
166 121.22; 118.59; 109.59; 100.33; 70.65; 70.19; 68.10; 66.40; 60.86; 55.73 (OCH3); 20.34-20.25 (4C,
167 COCH3); HRMS (m/z) 556.1590 [M+H]+, calcd 556.1596 C23H30N3O11S+.
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169
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2-[[3-methoxy-4-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]phenyl]methylene]
hydrazinecarbothioamide (4j). Obtained from the general procedure B as a solid, yield 84%; mp 119.4-
171 122.9 ºC; [α]D -28.6 (c 0,28, MeOH); IR (ῡ/cm–1): 3278 (NH), 1739 (C=O), 1597 (C=N), 1504, 1450
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174
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(C=C), 1030 (C-O); 1H NMR (200 MHz; CDCl3) δ/ppm: 10.37 (1H, s, NH); 7.91 (1H, s, CH=N); 7.21-
7.10 (4H, m, ArH); 6.72 (1H, s, NH2); 5.31-5.00 (m, 4H); 4.32-4.15 (2H, m); 3.84 (4H, m, OCH3 + H-
5); 2.07-2.04 (12H, 4s, COCH3); HRMS (m/z) 556.1591 [M+H]+, calcd 556.1596 C23H30N3O11S+.
2-[[-3-methoxy-4-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-β-D-
glucopyranosyl)oxy]phenyl]methylene]hydrazinecarbothioamide (4k). Obtained from the general
178 procedure B as a solid, yield 71%; mp 220.1-121.8 ºC; [α]D -12.0 (c 0.5, MeOH); IR (ῡ/cm–1): 3469
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179
(NH), 1739 (C=O), 1594 (C=N), 1528, 1505 (C=C), 1046 (C-O); H NMR (200 MHz; CDCl3) δ/ppm:
180 10.0 (1H, s, NH); 7.86 (1H, s, CH=N); 7.27-7.08 (4H, m, ArH); 6.60 (1H, s, NH2); 5.37-4.98 (7H, m);
4.53 (2H, d, J = 7.8 Hz); 4.16-3.77 (8H, m); 2.16-1.97 (s, 21H); 13C NMR (50 MHz; CDCl3) δ 178
181
182 (C=S); 170.33-169.05 (7C COCH3); 150.72; 148.20; 143.60; 129.08; 121.63; 118.88; 109.92; 100.99;
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99.75; 75.97; 72.84; 72.36; 71.25; 70.85; 70.62; 69.01; 66.53; 61.72; 60.72; 56.07 (OCH3); 20.74-20.45
(7C, COCH3); HRMS (m/z) 844.2423 [M+H]+, calcd 844.2441 C35H46N3O19S+.
General procedure C, for the synthesis of thiazole derivatives. To a solution of 1 equiv. of
thiosemicarbazone in isopropyl alcohol was added 1 equiv. of 2-bromoacetophenone, and the resulting

188
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190
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192
193
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mixture was kept under reflux and magnetic stirring. The completion of the reaction was monitored by
TLC (approximately 2 hours). After cooling to room temperature, the formed precipitate was filtered,
washed with saturated solution of NaHCO3 followed by cold distilled water. The final product was
recrystallized in ethanol.
Benzaldehyde-2-(4-phenyl-2-thiazolyl)hydrazone (5a). Obtained from the general procedure C as a
pale solid, yield 63%; mp 187.6-188.8 ºC (lit. 186-187 ºC(44)). IR, (ῡ/cm–1): 3306 (NH), 1557 (C=N),
1
1482, 1428 (C=C). H NMR (200 MHz, DMSO-d6), δ/ppm: 12.16 (1 H, s, NH); 7.88 (2 H, broad s.,
196 ArH); 7.61-7.08 (9 H, m, 8 x ArH and CH=N); 6.85 (1 H, s, H-thiazole).
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200
201
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204
4-Methylbenzaldehyde 2-(4-phenyl-2-thiazolyl)hydrazone (5b) .Obtained from the general procedure C
as a pale solid, yield 73%; mp 192.4-194.3 ºC (lit. 195-196 ºC(44)). IR, (ῡ/cm–1): 3279 (NH), 1553
(C=N), 1509, 1480 (C=C).
4-N,N-dimethylbenzaldehyde-2-(4-phenyl-2-thiazolyl)hydrazone (5c). Obtained from the general
procedure C as a solid, yield 81 %; mp 203.4-204.4 ºC (lit. 207-208 ºC(45)). IR, (ῡ /cm–1): 3288 (NH),
1603 (C=N), 1520, 1480 (C=C). 1H NMR (200 MHz, DMSO-d6), δ/ppm: 11.85 (1 H, s, NH); 7.92-7.86
205 (3 H, m, ArH); 7.48-7.24 (6 H, m, ArH and CH=N); 6.74 (2 H, broad s, ArH and H-thiazole).
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4-Pyridinylcarbaldehyde-2-(4-phenyl-2-thiazolyl)hydrazone (5d). Obtained from the general procedure
C as an orange solid, yield 53%; mp 240.1-242.2 ºC (lit. 250-252 ºC (46)). IR, (ῡ/cm–1): 3450 (NH),
1
1571 (C=N), 1482, 1441 (C=C). H NMR (200 MHz, DMSO-d6), δ/ppm: 12.57 (1 H, broad s., NH);
8.60 (1 H, broad s., ArH); 8.00 (1 H, s, CH=N); 7.85 (2 H, d, ArH); 7.61 (2 H, broad s., ArH); 7.38 (4
H, m, ArH and H-thiazole).

213
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215
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218
219
220
Pyrrole-2-carboxaldehyde-(4-phenyl-1,3-thiazol-2-yl)hydrazone (5e). Obtained from the general
procedure C as a dark solid, yield 99%; mp 126 ºC (lit. 125 ºC(47)). IR, (ῡ/cm–1): 3304 (NH), 1623
(C=N), 1498, 1422 (C=C).
2-[(2-Cyclopentylmethylene)hydrazino]-4-phenyl-thiazole (5f). Obtained from the general procedure C
as a solid, yield 98%, mp 170.2-172.4 ºC (lit. 156-157 ºC(48)). IR, (ῡ/cm–1): 3438 (NH), 1626 (C=N),
1
1560, 1481 (C=C). H NMR (200 MHz, CDCl3), δ/ppm: 7.73 (2 H, dd, J = 7.8 Hz; J = 2.0 Hz, ArH);
7.51-7.36 (3H, m, ArH and NH); 6.73 (1H, s, H-thiazole); 2.61-2.48 (4H, m, CH2); 1.99-1.79 (4H, m,
221 CH2).
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224
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2-[(2-Cyclohexylmethylene)hydrazino]-4-phenyl-thiazole (5g). Obtained from the general procedure C
as a solid, yield 51%, mp 149-151 ºC (lit. 148-149 ºC(48)). IR, (ῡ/cm–1): 3050 (NH), 1610 (C=N),
1476, 1431 (C=C).
2-[(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methylene]hydrazino-4-phenyl-1,3-thiazole
(5h).
Obtained from the general procedure C as a solid, yield 29 %, mp 152.4-154.2 ºC. IR, (ῡ/cm–1): 3363
1
229 (NH), 1615 (C=N), 1495, 1470 (C=C). H NMR (200 MHz, DMSO-d6), δ/ppm: 7.87-7.69 (3H, m,
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ArH); 7.47-7.29 (4H, m, ArH, CH=N and NH); 7.26 (1H, s, H-thiazole); 5.97 (1H, s, C=CH); 2.93-2.85
(1H, m, CH); 2.48-2.33 (3H, m, CH2, CH); 2.14 (1H, m, CH2); 1.33 (3H, s, CH3); 1.13-1.03 (1H, m,
232 CH2); 0.78 (3H, s, CH3). HRMS (m/z) 324.1530 [M+H]+, calcd 324.1529 C19H22N3S+.
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234 2-[[3-methoxy-4-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]phenyl]methylene]hydrazino-4-
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phenyl-thiazole (5i). Obtained from the general procedure C as a solid, yield 41%; mp 111.4-113.7 ºC;
[α]D -36 (c 0,5, MeOH); IR (ῡ/cm–1): 2952 (NH), 1748 (C=O), 1602 (C=N), 1567, 1510 (C=C), 1071
237 (C-O); 1H NMR (200 MHz; CDCl3) δ/ppm: 7.80 (2H, d, J = 7.0 Hz, ArH); 7.44-7.29 (4H, m, ArH and

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N=CH); 7.05-6.97 (3H, m, ArH); 6.81-6.75 (2H, m, H-thiazole and NH); 5.31-4.96 (4H, m); 4.33-4.13
(2H, m); 3.82 (4H, m, OCH3 and H-5); 2.08-2.04 (12H, 4s, COCH3); HRMS (m/z) 656.1905 [M+H]+,
240 calcd 656.1909 C31H34N3O11S+.
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Enzyme expression and purification. Recombinant enzymes cruzain, rhodesain and SmCB1 have
been expressed and purified as previously described (11, 49–51).
Assay against cruzain, rhodesain and SmCB1. Cruzain, rhodesain and SmCB1 activity were
measured by monitoring the cleavage of the fluorogenic substrate Z-Phe-Arg-aminomethylcoumarin
(Z-FR-AMC) in a Synergy 2 (Biotek), from the Center of Flow Cytometry and Fluorimetry at the
248 Biochemistry and Immunology Department (UFMG). All assays were performed in triplicate using
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0.1M sodium acetate, pH 5.5, in the presence of 1 mM beta-mercaptoethanol and 0.01% Triton X-100.
The final concentrations of cruzain and rhodesain were 0.5 nM, and the substrate concentration was 2.5
μM (Km = 1 μM). For assays with SmCB1, the enzyme concentration was 8 nM and substrate
concentration was 5 μM. Enzyme kinetic was followed by continuous reading for 5 min at 12s
intervals, in the case of cruzain and rhodesain, and for 30 min at 23s intervals, in the case of SmCB1.
Activity was calculated based on initial velocity rates, compared to a DMSO control. For evaluation of
time-dependent inhibition, percentages of enzyme inhibition by a compound with or without pre-
incubation with enzyme for 10 min were compared. Firstly, the inhibitory activity for all enzymes were
screening at 100 μM of compound. When the inhibition was higher than 80%, the IC50 was determined
based on at least two IC50 curves. Each curve was determined based on at least seven compound
concentration, each in triplicate, and the data analyzed with GraphPad Prism 5.0, employing a
nonlinear regression analysis of “log (inhibitor) vs response with variable slope – four parameters”
(Figure S1). The reported values on Tables 1-3 refer to the average and standard deviation between the
262 values obtained for at least two curves.

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T. brucei brucei 221 maintenance. The parasites were cultured in HMI-9 medium supplemented with
20% of heat-inactivated Fetal Bovine Serum (FBS Gibco) starting at a density of 2 x 104 parasites/ml
and sub-culturing every other day.
267 Compound plates preparation. All compounds were stored in powder. Solutions at 10 mM in neat
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DMSO were prepared a few hours before assay experimentation, and seeded in the row A of 384 wells
plate (Greiner 784201). Serial dilution with two-fold factor was prepared until the row P (from 10mM
to 305nM). The compounds were pinned and 50nl of each well was transferred to the assay plate
(Greiner 781091) containing 25µl of HMI-9 media supplemented with 20% FBS.
T. brucei brucei screening assay. Five thousand (5 x 103) parasites were seeded in a volume of 25 µl
in the 384 wells plate already containing the compounds in serial dilution, being the highest
concentration tested 10 μM. After 72 hours incubation at 37C and 5% CO2, each well received 12.5 μl
of Sybr Green in lysis solution (30 mM Tris pH 7.5, 7.5 mM EDTA, 0.012% saponin, 0.12% Triton X-
100 and 0.3μl/ml of Sybr Green). After addition of lysis solution the plates were sealed with plastic
film and the mixture was vortexed for 45 seconds at 1700 rpm (MixMate). The mixture was incubated
for 1 hour at room temperature and the plate was read in Flexstation (Molecular Devices) to detect the
fluorescence signal corresponding to parasite viability (Ex 485nm/Em 530nm). Raw viability data
consists of values of relative fluorescence unit (R.F.U.) obtained from the reading of Sybr Green that
binds to the viable parasite's DNA, and includes max and min controls and measured values.
283 Thymerasol (2 μM) was used as the reference drug EC100. The activity normalization was done based
284 on the non-treated (negative control, 0% activity) and the reference drug at the EC100 concentration
285 (positive control, 100% activity), with at least 16 wells for each control per plate.
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287 T. cruzi screening assay. The assay was performed using T. cruzi (Tulahuen strain) expressing the

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Escherichia coli β-galactosidase as reporter gene (52, 53). Infective trypomastigote forms were
obtained through culture in monolayers of mouse L929 fibroblasts in RPMI-1640 medium (pH 7.2-7.4)
without phenol red (Gibco BRL) plus 10% fetal bovine serum and 2 mM glutamine. For the bioassay,
4,000 L929 cells in 80 μl of supplemented medium were added to each well of a 96-well microtiter
plate. After an overnight incubation, 40,000 trypomastigotes in 20 μl were added to the cells and
incubated for 2 h. The medium containing extracelullar parasites was replaced with 200 μl of fresh
medium and the plate was incubated for an additional 48 h to establish the infection. For IC50
determination, the cells were exposed to active samples at serial decreasing dilutions starting at 1000
μM in DMSO (less than 1% in RPMI-1640 medium), and the plate was incubated for 96 h. After this
period, 50 μl of 500 μM chlorophenol red β-D-galactopyranoside (CPRG) in 0.5% Nonidet P40 was
added to each well, and the plate was incubated for 16 to 20 h, after which the absorbance at 570 nm
was measured. Controls with uninfected cells, untreated infected cells, infected cells treated with
benznidazole at 1 μg/ml = 3.8 μM (positive control) or DMSO 1% was used (Faria J et al, manuscript
in press). The results were expressed as the percentage of T. cruzi growth inhibition in compound-tested
302 cells as compared to the infected cells and untreated cells. IC50 values were calculated by linear
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interpolation. Tetraplicates were run in the same plate, and the experiments were repeated at least once.
In vitro assay for analysis of cell viability. The active compounds were tested in vitro for cytotoxicity
306 versus L-929 cells using the alamarBlue® dye. Were used the same cell number, time of the cell
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development and time of compound exposure used for the beta-galactosidase assay. The cells were
exposed to compounds at increasing concentrations starting at the IC50 value of T. cruzi. The
compounds were tested in tetraplicate. After 96 hours of compound exposure, alamarBlue® was added
and the absorbance at 570 and 600 nm measured after 4-6 h. The cell viability was expressed as the
percentage of difference in the reduction between treated and untreated cells (53). IC50 values were
calculated by linear interpolation and the Selectivity Index (SI) was determined by the ratio between

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the cytotoxic concentration (CC50) and the IC50 value against the parasite for each compound.
S. mansoni screening assay. The acquisition, preparation and in vitro maintenance of newly
316 transformed S. mansoni schistosomula (derived from infective stage cercariae) and adult parasites have
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been described by us (54, 55). We employ a Puerto Rican isolate of S. mansoni that is cycled between
Biomphalaria glabrata snails and female Golden Syrian hamsters (infected at 4-6 weeks of age) as
intermediate and definitive hosts, respectively. Maintenance and handling of small mammals are
carried out in accordance with a protocol approved by the Institutional Animal Care and Use
Committee (IACUC) of the University of California San Francisco. For schistosomula, 200-300
parasites are incubated in 96w flat-bottomed plates (Corning Costar 3599) containing 200 µL in Basch
medium 169 (56) supplemented with 2.5% FBS and 1x penicillin-streptomycin solution in a 5% CO2
atmosphere at 37 ˚C. For adult parasites, 5 male worms are incubated in 2 ml of the above medium in
24w flat-bottomed plates (Corning Costar 3526) under the same conditions. Compound, 100% DMSO,
is added at the final concentrations indicated in Table 4. Controls employ the equivalent volume of
DMSO at a final concentration never exceeding 0.5%. Parasite responses to chemical insult are
adjudicated visually each day using a constrained descriptive nomenclature (54, 57). The types and
number of phenotypic responses recorded are then converted into a ‘severity score’ ranging from 0 (no
effect) to 4 (severely compromised). Thus, for schistosomula and adults, alterations in shape (e.g.,
‘rounding’), motility (‘slow’ or ‘overactive’) and density (‘darkening’) are each awarded a score of 1
up to the maximum of 4. In addition, for adults, the inability to adhere to the bottom of the well is
awarded a score of 1: damage to the integrity of the outer surface (tegument) is considered lethal to the
parasite and is awarded the maximum score of 4.
RESULTS AND DISCUSSION
Chemistry

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The sugar derivatives 1, 2 and 3 were synthesized by reaction of vanillin with the corresponding
peracetylglycosyl bromide using lithium hydroxide as a base, according to a method previously
described (Figure 1) (58). A series of thiosemicarbazones was synthesized by classical methods from
an aldehyde or ketone and thiosemicarbazide with yields in the range of 51-95 %. Then, the
342 thiosemicarbazones obtained were subjected to reaction with α-bromo-acetophenone, giving the
343
corresponding thiazole heterocycles (yield 29-98 %) (Figure 2). The stereochemistry at the C=N bond
1
344 of the thiosemicarbazone derivatives was established by H NMR spectroscopy. The value of the
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chemical shift of the NH (9–12 ppm) is indicative of (E) configuration (59). Some semicarbazones
were also synthesized according to a previously described procedure (60), for comparison of the
activity of semicarbazones with that of the thiosemicarbazones.
Fifteen thiosemicarbazones and nine thiazole analogues were prepared containing an aryl or
349 heteroaryl, and/or cycloalkyl or glycosyl moieties (Figure 3). The introduction of the carbohydrate
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moiety (compounds 4i-4k and 5i) was designed to modulate solubility and interaction properties (for
example, by hydrogen bonding) with the molecular target (cysteine protease).
Discovery of a potent rhodesain and SmCB1 inhibitor
An initial screen of 18 compounds, mostly thiosemicarbazones, was performed against
355 rhodesain. Compounds were screened in two conditions, that is with and without a 10 min pre-
356 incubation with the enzyme (Table 1). A clear time-dependence was observed for the active
357 compounds, as expected based on the formation of a covalent bond between rhodesain and the
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inhibitor. To verify if the differences in percentage of inhibition observed were statistically significant,
we have applied an unpaired t-test comparing the results of assays with and without pre-incubation.
This analysis reveals that, for all compounds which inhibited rhodesain by more than 80% in the
screen, the percentage of inhibition is higher if the enzyme is pre-incubated with the compound, and
this difference is statistically significant, with p-values <0.0002 (Table S1). Therefore all subsequent

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assays were performed with a 10 minute pre-incubation against the enzymes; the SAR discussion refers
to the inhibition observed under this condition.
Eleven compounds inhibited rhodesain by at least 50% at 100 µM; IC50 values were determined
for five of them. Several trends were observed based on this initial screen. Comparison between 4a and
4g indicates the importance of the aromatic ring. Addition of a methyl substituent at the para position
does not affect inhibition (4b), whereas a dimethylamine at the same position results in a more potent
compound (4c, IC50 = 3.0 ± 0.8 µM), as does the replacement of the phenyl by a pyridine (4d, IC50
=
370 3.3 ± 0.9 µM) or by an imidazol (4e, IC50 = 4.0 ± 1.8 µM). Three cyclic analogues were less soluble,
371 and in a few cases could not be evaluated at 100 µM. They were therefore assayed at 50 µM (5c) or 75
372 µM (5a and 5b). Assay results for several pairs of compounds suggested that replacement of the
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thiosemicarbazones by a cyclized analogue decreases potency against the enzyme (4d vs. 5d, 4h vs. 5h,
4e vs. 5e, 4j vs. 5i), except for 4a vs. 5a, which showed similar potency. This initial screen resulted in
375 the discovery of a potent inhibitor, 4i, with an IC50 of 1.2 ± 1.0 µM. Comparison to 4j shows the
importance of the sugar, since replacement of the acetylated galactose by an acetylated glucose,
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representing a difference in only one chiral center, results in a 20-fold decrease in potency (4j, IC50
=
26.2 ± 1.5 µM).
The compounds were also evaluated against the T. cruzi and S. mansoni cysteine proteases,
cruzain and SmCB1. Like rhodesain, cruzain is a cathepsin L-like protease, and only two active
residues are different between both proteins. Similarity between the cathepsin B-like SmCB1 and
rhodesain active sites is lower, however common inhibitors have been reported for these enzymes (19).
The SAR for the three enzymes showed several similarities and also interesting differences. The
importance of the aromatic ring was confirmed (4a vs. 4g), and as observed for rhodesain both the
addition of a dimethylamine in this ring and its replacement by a pyrol increased potency against
cruzain (4c, IC50= 6.6 ± 3.2 μM, and 4e, IC50 =9.7 ± 5.2 μM) and SmCB1 (4c, IC50= 1.5 ± 0.4 μM, and
4e, IC50 = 6.8 ± 2.1 μM). However, in contrast to what was measured for rhodesain, against cruzain and

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SmCB1, the pyridine analogue (4d) was not as potent as the compound containing a phenyl ring (4a).
Interestingly, although addition of the methyl substituent neither influenced the inhibition of cruzain
nor rhodesain, potency against SmCB1 was increased ten-fold (4a, IC50= 22.4 ± 3.1 μM vs 4b, IC50=
2.5 ± 1.9 μM). Overall, the thiosemicarbazones 4c and 4e were the most potent inhibitors of the three
enzymes.
The most significant difference in potency was observed for 4i. Although this compound had
low micromolar potency against rhodesain (IC50 = 1.2 ± 1.0 μM), it was approximately 35-fold less
potent against cruzain (IC50 = 37.7 ± 9.8 μM) and essentially inactive against SmCB1 (IC50 could not
be determined). Despite the similarity of the active sites of cruzain and rhodesain, the bottom of the S2
pocket in cruzain and SmCB1 contains a glutamate (Glu208 and Glu316, respectively), whereas
rhodesain has an alanine in the equivalent position. The S2 pocket is therefore considerably more open
in rhodesain, possibly providing an explanation for the ability of this enzyme to bind larger scaffolds.
The epimer 4j showed lower potency against cruzain and SmCB1, when compared to rhodesain.
Nevertheless, against these two enzymes we observed that both compounds (4i and 4j) where only
modest inhibitors.
SAR
The SAR for 4i was exploited based on the impact of removing or modifying the sugar (Table
2), and removing the thiosemicarbazone or modifying it to a semicarbazone (Table 3). Significant
differences were observed in SAR for the three enzymes regarding modifications in the sugar. Removal
of the sugar moiety decreased potency against rhodesain by at least 5-fold (4l, IC50 = 7.3 ± 4.0 μM; 4m,
IC50 = 6.2 ± 2.7 μM) or more so depending on the pattern of phenyl substitution (4n, 4o). On the other
hand, against cruzain this modification increased potency by 6-fold, and both 4l and 4m had IC50
values of approximately 10 µM. It is worth noting that in the analogues which do not contain a sugar
(4l-4o) the potencies against cruzain and rhodesain are similar.

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Despite the sugar not being essential for binding, it could drastically affect inhibition. For
example, addition of another sugar monomer (4k) resulted in a compound inactive against both cruzain
and rhodesain, but active against SmCB1 (4k, IC50 = 7.7 ± 1.7 μM).
Removal of the thiosemicarbazone (galactosyl 1 and lactosyl 3) or its replacement by
semicarbazone (6a vs. 4n, 6b vs. 4m, 6c vs. 4l, 6d vs. 4o, 6f vs. 4h) resulted in compounds inactive
418 against the three enzymes, the only exception being 6f against SmCB1 (IC50 = 5.2 ± 2.8 μM). This
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effect has also been reported for a related compound series (27) and can be explained by the more
electrophilic nature of the thiosemicarbazones, and the mechanism of cysteine protease inhibition by
these compounds.
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Assays against parasites in vitro
Compounds were evaluated against T. brucei, T. cruzi and S. mansoni. To assess the anti-
parasitic activity of the 24 compounds against the bloodstream form of T. b. brucei, we used a viability
assay based on the fluorescence of the parasite’s nucleic acid (Faria J et al, manuscript in press).
Parasites were co-incubated for 72 h with compound two-fold serially diluted from 10 µM to 305 pM.
At 10 µM, none of the 24 tested compounds showed more than 60% bioactivity, defined as the
reduction in parasite number compared to untreated control (Table S2).
For T. cruzi, compounds were tested against both amastigote and trypomastigote forms of the
Tulahuen strain (52). Weak trypanocidal activity was observed for this class and five compounds
432 generated IC50 values under 100 µM. Based on the difference between the IC50 values for parasites and
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the L929 mouse fibroblast cell line, a Selectivity Index (SI) of each compound could be determined.
The SI ranged from 1.3 to 10.7 (Table S2). Importantly, even though the trypanocidal IC50 values were
high, the trypanocidal concentration was not toxic to fibroblasts, and in the case of compounds 4b and
436 4h, the IC50 values for fibroblasts were an order of magnitude greater than those for T. cruzi (Table S2).

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Although those compounds with SI values ≥ 10 might be considered prototypes for new trypanocidal
drugs, they are not recommended for in vivo tests as the SI values do not cross a decision gate threshold
439 of 50 (53).
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442
For S. mansoni, screens were performed against post-invasive larvae (schistosomula) and adult
parasites, as previously described (54, 55). Parasite responses to chemical insult were adjudicated
visually each day using a constrained nomenclature (54, 57) that accounts for changes in shape,
443 motility and density. The types and number of phenotypic responses recorded are then converted into a
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‘severity score’ ranging from 0 (no effect) to 4 (severely compromised). Five compounds (5a, 5b, 5c,
5d, 4h) showed activity against schistosomula. Among these compounds only 5d was active against
adult worms.
CONCLUSION
Here we report the discovery of 4i, a sugar-containing thiosemicarbazone which showed low
450 micromolar potency against rhodesain (IC50 = 1.2 ± 1.0 μM) and modest potency against cruzain (IC50
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= 37.7 ± 9.8 μM). Synthesis of a series of analogues allowed determination of the SAR in this series,
and resulted in six rhodesain, four cruzain and five SmCB1 inhibitors with IC50 ≤ 10 µM. Only three
453 thiosemicarbazones (4c, 4e and 4m) showed similar potencies against the rhodesain, cruzain, and
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SmCB1, a result that demonstrates that considerable differences in the SAR for the three enzymes exist.
In a few cases, using larger scaffolds, higher potency was observed against rhodesain. Direct assays of
the most potent inhibitors against the parasites T. cruzi, T. brucei and S. mansoni showed some anti-
parasitic activity but also suggested that further SAR modifications will be needed to produce lead
compounds.
ACKNOWLEGDMENTS
RSF acknowledges CNPq (Grant 477435/2012-2), Capes (Grants A118/2013 and Edital

462
Biocomputacional AUXPE 3379/2013) and FAPEMIG (Grant PPM-00357-14) and RBO acknowledges
463 CNPq (Grant 4041130/2012-0) for financial support. GANP acknowledges a postdoctoral fellowship
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from CAPES Grant A118/2013. JHM and JLdeSN received funding from the European Community’s
7th Framework Programme (602777) Project Kindred. Research by CRC is supported in part by NIH-
466 NIAD R21AI107390 and R01AI089896 awards. The authors also thank the Center of Flow Cytometry
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and Fluorimetry at the Biochemistry and Immunology Department (UFMG) and the Program for
Technological Development of Tools for Health-PDTIS-FIOCRUZ for use of its facilities and
Plataforma de Bioprospecção RPT10A-PDTIS-CPqRR-Fiocruz for HRMS measurements.

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FIGURE LEGENDS
Figure 1 – Synthesis of the glycosides 1, 2 and 3.
651
652
653
Figure 2 – General synthetic route for preparation of thiosemicarbazones and their corresponding
thiazole heterocycles.
Figure 3 – Chemical structure of the thiosemicarbazones 4a-o and cyclic analogues 5a-i synthesized.

654
655
Table 1. Inhibition of rhodesain, cruzain and SmCB1 by a series of thiosemicarbazones and cyclic
analogues.
656
Rhodesain
%
Cruzain
SmCB1
%
%
b
b
b
inhibition IC50 inhibition IC50 inhibition IC50
Compound
R1
at 100
μMa
(μM)
at 100
μMa
(μM)
at 100
μMa
(μM)
72.5 ±
12.7
22.4
± 3.1
2.5 ±
1.9
4a
4b
4c
4d
Ph
ND 53.7 ± 1.2 ND 84.4 ± 8.5
87.2 ±
77.6 ± 2.7 ND 41.9 ± 1.1 ND
11.9
100.0 ±
0.0
100.0 ±
0.0
3.0 ±
0.8
6.6 ±
3.2
1.5 ±
0.4
91.9 ± 0.2
3.3 ±
0.9
73.1 ±
23.4
42.4
± 2.6
6.8 ±
2.1
81.7 ± 2.1
60.8 ± 4.4 ND
4.0 ±
1.8
9.7 ±
97.2 ± 0.1
5.2
4e
4f
100 ± 0.0
1.8 ± 9.3
0.0 ± 0.0
86.1 ± 4.4
0.0 ± 0.0
0.0 ± 0.0
ND
ND
0.0 ± 0.0
0.0 ± 0.0
ND
ND
ND
ND
4g