Anticancer and antileishmanial in vitro activity of gold(I) complexes with 1,3,4-oxadiazole-2(3H)-thione ligands derived from δ-D-gluconolactone

DR MAURO ALMEIDA (Orcid ID : 0000-0001-9669-0306)

PROFESSOR HEVELINE SILVA (Orcid ID : 0000-0003-3537-4961)

Article type : Research Article

Anticancer and antileishmanial in vitro activity of gold(I) complexes with 1,3,4-oxadiazole-

2

(3H)-thione ligands derived from δ-D-gluconolactone

1

2

Andrés Villaseñor Espinosa , Danilo de Souza Costa , Luiza Guimarães Tunes , Rubens L. do Monte-

2

1

3*

Neto , Richard Michael Grazul , Mauro Vieira de Almeida , Heveline Silva

Short running title: Bio-active gluconolactone gold complexes

¹Departamento de Química, ICE, Universidade Federal de Juiz de Fora, 36036-900, Juiz de Fora, MG,

Brazil.

²Instituto René Rachou-Fiocruz Minas, 30190-009, Belo Horizonte, MG, Brazil.

³Departamento de Química, ICEx, Universidade Federal de Minas Gerais, 31270-901, Belo

Horizonte, MG, Brazil.

*

Corresponding author:

phone: +55 (31) 34095700

e-mail: hevelinesilva@ufmg.br

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process, which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

1

0.1111/CBDD.13757

keywords: leishmania, antitumor, metal, synthesis

ABSTRACT

Four gold(I) complexes conceived as anticancer agents were synthesized by reacting [Au(PEt )Cl]

3

and [Au(PPh )Cl] with ligands derived from δ-D-gluconolactone. The ligands’ structure was designed

3

to combine desired biological properties previously reported for each group. Ligands were

synthesized from δ-D-gluconolactone via ketal protection and hydrazide formation followed by

cyclization with CS to produce the novel oxadiazolidine-2-thione 7 and 8. Increasing of the ligands’

2

lipophilicity via ketal protection proved useful since all four gold(I) complexes showed anticancer and

antileishmanial properties. The IC values are at low micromolar range, varying from 2-3 μM for the

5

0

most active compounds. The free D-gluconate 1,3,4 oxadiazole-derived ligands were neither toxic nor

presented anticancer or antileishmanial properties. Triethylphosphine-derived compounds 9 and 10

were more selective against B16-F10 melanoma cell line. Although similar in vitro antileishmanial

activity was observed for the gold(I) precursors themselves and their derived complexes, the latter

were three times less toxic for human THP-1 macrophage cell line; this result is attributed to an

isomeric variation of the D-gluconate ligand and the oxadiazole portion, which was one of the key

concepts behind this work. These findings should encourage further research on gold(I) complexes to

develop novel compounds with potential application in cancer and leishmaniasis chemotherapy.

1

INTRODUCTION

Gold(I) complexes with saccharide-based ligands have been extensively studied in recent

years, as they have shown significant in vitro cytotoxic activity against many cisplatin-resistant tumor

cell lines (Rigobello et al., 2008). Unlike cisplatin, which targets DNA to exert its cytotoxic effect,

gold(I) complexes interrupt intracellular protein signaling mechanisms (particularly thioredoxin

reductase), leading to cell apoptosis (da Silva Maia, Deflon, & Abram, 2014). The structure of ligands

used to synthesize gold(I) complexes has great influence on their biological activity (Yeo et al., 2013).

An important factor is the lipophilicity of the ligand, which directly impacts the ability of the complex

to penetrate through cell membranes. The insertion of a tertiary phosphine group in some gold(I)

complexes (as in auranofin) has consistently resulted in increased biological activity, when compared

to their analogous non-phosphorated compounds (Ott et al., 2009).

Despite the myriad ligand possibilities available for gold(I)-based metallodrug synthesis,

1

,3,4-oxadiazoles have become an important motif of interest for the design of new drugs due to a

plethora of biological properties they possess, including: anti-inflamatory, anticancer, antimalarial,

antibacterial, anticonvulsant, cytotoxic, tyrosinase inhibitory, angiogenesis inhibitory,

hypoglycemiant, analgesic, antitubercular, antiemetic, anti-allergic, hypoglycaemic, antiedema,

antifungal, diuretic, anti-hypertensive and anti-viral (Leite de Oliveira et al., 2012; Patel et al., 2014).

Oxadiazolidine-derived compounds belong to a broader group of heterocyclic molecules, particularly

because of their anticancer activity (J. D. S. Chaves et al., 2014). This makes them very appealing for

use in the synthesis of ligands. The intrinsic properties of these ligands could favor in vivo

performance by promoting a combinatory effect of different biological activities. The significance of

this work lies in the design and synthesis of novel ligands using multiple biologically active groups in

order to enhance the overall effectivity and selectivity of the gold(I) complexes. Our research group

already reported several studies describing gold(I) complexes with different ligands based on different

substrates, such as benzyl and benzoyl chloride (J. D. S. Chaves et al., 2014), alcohols and fatty acids

(

de Almeida et al., 2017), some monosaccharides (J. D. S. Chaves et al., 2015) and adamantane

Garcia et al., 2016); all of them showed selective cytotoxicity against tumor-derived cell lines and

(

were more effective than cisplatin. Some of these compounds also presented antileishmanial activity

against Leishmania infantum and Leishmania braziliensis intracellular amastigotes and axenic

promastigotes (O. A. Chaves et al., 2016). These Leishmania species are, respectively, the main

etiological agents of visceral and cutaneous leishmaniasis in Brazil (Falcão et al., 2016).

Leishmaniasis is a worldwide neglected disease and a public health problem that urgently requires

new alternative chemotherapeutic agents for control and disease eradication. Curiously enough,

parasitic infections, including those caused by Leishmania parasites, present some similarities with

cancer (Oliveira, 2014). Both Leishmania and tumor cells present an elevated cell division rate that

cannot be controlled by the host; their energetic metabolism heavily relies on glycolysis; they device

strategies to evade the immune system and have multifactorial drug resistance mechanisms. For

instance, Miltefosine - originally designed as an antitumoral drug - was repurposed and it is currently

the only available oral antileishmanial agent to treat leishmaniasis (Sundar et al., 2002). Recently,

adamantane-gold(I) complexes were reported as an oral drug candidate to treat leishmaniasis (Tunes

et al., 2020). Gold(I)-based auranofin, which also shows anticancer activity, has also been reported as

an effective antileishmanial agent both in vitro and in vivo (Sharlow et al., 2014). These facts justify

continuous and concomitant research for anticancer and antileishmanial agents, which is the main

premise behind this work’s innovative scheme.

In this work, we explored the use of D-gluconate carbohydrate residues along with 1,3,4-

oxadiazoles as ligands. They presented remarkably low toxicity, and the extent of their biological

activity was readily distinguishable from that of other ligands (Yoshida et al., 1999). These findings

from previous research on ligand structure-activity relations led us to synthesize, characterize and

evaluate the anticancer and antileishmanial activities of two novel ligands and four novel gold(I)

complexes, which are described in this work.

2

MATERIALS AND METHODS

.1 Reagents and instrumentation

All reagents and solvents were used without further purification. [Au(PEt )Cl] is commercially

3

available and [Au(PPh )Cl] was synthesized from K[AuCl ] according to the literature (Baenziger,

3

4

Bennett, & Soborofe, 1976). Flash chromatography was performed on silica gel (Merck, 70–230

mesh) as the stationary phase. All reactions were monitored by TLC. IR spectra were acquired on a

-1

1

Perkin-Elmer 1720 FTIR spectrometer in the region of 4000–400 cm as KBr pellets. H NMR (500

1

3

31

MHz), C NMR (125 MHz) and P NMR (202 MHz) spectra were recorded as solutions in CDCl or

3

DMSO-d6 in an Avance III HD BRUKER 500 MHz spectrometer. Chemical shifts were expressed in

ppm. All NMR data were reported using the following syntaxes: chemical shift, multiplicity,

integration, followed by coupling constant values. High-resolution mass spectra were recorded on a

Shimadzu Biotech-Axima Performance mass spectrometer using Laser Desorption Ionization (LDI)

with a Time-Of-Flight (TOF) mass analyzer.

2

.2 Synthesis

Figure 1 shows the general synthetic route used in this work.

2

.2.1 Synthesis of compounds 3 and 4

δ-D-gluconolactone was used as the precursor to prepare the isopropylidene compounds 3 and 4,

according to methodology previously described in the literature (Regeling, de Rouville, & Chittenden,

1

987). In a 25 mL round-bottom flask, 10 mmol of δ-D-gluconolactone were dissolved in 10 mL of

acetone and 3 mL of methanol, followed by addition of 4 mL of 2,2-dimethoxypropane and 50 mg of

p-toluenesulfonic acid and 50 μL of concentrated H SO as catalysts. The reaction mixture was then

2

4

stirred for 48 hours at room temperature. Residual acid was removed by liquid-liquid extraction

dichloromethane and brine) and the isomers 3 and 4 were separated by column chromatography.

(

Methyl (3,4)/(5,6)-di-O-isopropylidene-D-gluconate 3 and its isomer methyl (2,3)/(5,6)-di-O-

isopropylidene-D-gluconate 4 were obtained in yields of 44 and 22%, respectively. Their spectral data

were in accordance with those reported in the literature (Regeling et al., 1987).

2

.2.2 Synthesis of Hydrazides 5 and 6

In a 10 mL round-bottom flask, 200 mg of the corresponding starting material were dissolved in 3 mL

of methanol and then 200 μL of 67% aqueous hydrazine solution were added. The mixture was stirred

for 8 hours at room temperature. Excess hydrazine was removed by liquid-liquid extraction using

dichloromethane and brine. The organic phase was evaporated under reduced pressure to yield the

desired hydrazides 5 and 6 in quantitative yield.

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Hydrazide 5: oil, µ: 68.95 mPa s, ρ: 1.341 g cm , IR (KBr) ν(cm ): 3500-3300 (O-H stretch), 3346

and 3315 (s, NH asymmetric stretch), 2985 and 2917 (m, CH asymmetric/symmetric stretch),

2

aliphatic

1

6

652 (s, C=O stretch), 1610 (m, NH scissoring), 1518 (m, NH bending), 1250, 1210, 1170, 1140,

2 2

1

085 (m, s, ketal C-C stretch). H NMR (500 MHz, CDCl ) δ (ppm): 8.84 (s, 1H), 5.60 (d, 1H, J =

3

.9), 4.27 (s, 1H), 4.22 (dd, 1H, J = 7.7 Hz, J = 1.8 Hz), 4.10 (ddd, 1H, J = 7.4 Hz, J = 6.3 Hz, J = 4.7

Hz), 4.05 (d, 1H, J = 8.3 Hz, J = 6.3 Hz), 3.99 (dd, 1H, J = 6.8 Hz, J = 1.7 Hz), 3.93 (t, 1H, J = 7.6),

1

3

.81 (dd, 1H, J = 8.3 Hz, J = 4.7 Hz), 1.33 (s, 3H), 1.31 (s, 3H), 1.27 (s, 3H), 1.26 (s, 3H). C NMR

(

125 MHz, CDCl ) δ (ppm): 170.0, 108.8, 108.7, 80.0, 76.4, 75.8, 69.5, 66.4, 27.0, 26.6 26.4, 25.0.

3

+

HRMS (LDI/TOF) m/z [M+H] calc.: 291.1556 found: 291.1039.

-3

-1

Hydrazide 6: oil, µ: 62.16 mPa s, ρ: 1.284 g cm , IR (KBr) ν(cm ): 3500-3300 (O-H stretch), 3330

(

s, NH asymmetric stretch), 2988 and 2937 (m, CH_a_l_i_p_h_a_t_i_casymmetric /symmetric stretch), 1661 (s,

2

1

C=O stretch), 1511 (m, NH bending), 1270, 1205, 1070 (s, ketal C-C stretch). H NMR (500 MHz,

2

CDCl ) δ (ppm): 8.03 (s, 1H), 4.48 (d, 1H, J = 8.2 Hz), 4.21 (dd, 1H, J = 8.2 Hz, J = 1.6 Hz), 4.03 (m,

3

4

H), 3.77 (dd, 1H, J = 7.0 Hz, J = 1.6 Hz), 3.31 (s, 1H), 1.42 (s, 3H), 1.39 (s, 3H), 1.37 (s, 3H), 1.30

1

3

(

s, 3H). C NMR (125 MHz, CDCl ) δ (ppm): 170.8, 111.0, 109.3, 78.7, 76.2, 74.9, 70.0, 66.7, 26.7,

3

+

2

6.6, 26.1, 25.3. HRMS (LDI/TOF) m/z [M+H] calc.: 291.1556 found: 291.0980.

2

.2.3 Synthesis of ligands 7 and 8

Ligands 7 and 8 were obtained by reacting the products 5 and 6 respectively with CS in basic

2

medium, as described by Manjunatha (Manjunatha, Poojary, Lobo, Fernandes, & Kumari, 2010). In a

1

0 mL round-bottom flask, 80 mg of NaOH were dissolved in 3 mL of ethanol, and then 300 mg of 5

or 6 were added along with 1 mL of CS . The reacting mixture was stirred at 45°C during 24 hours.

2

The solvent and excess of CS were evaporated under reduced pressure. The residue was extracted

2

using dichloromethane and brine, by dropwise addition of aqueous 0.1 M HCl until pH = 5. The

organic phase was dried over sodium sulfate, and then filtered. Compounds 7 and 8 were purified by

column chromatography and obtained in 58 and 78% yield, respectively.

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Ligand 1,3,4-Oxadiazolidine-2-thione 7: oil, µ: 72.56 mPa s, ρ: 1.354 g cm , IR (KBr) ν(cm ): 3500-

3

300 (br, O-H stretch), 3317 (m, NH stretch), 2990 and 2926 (m, CH_a_l_i_p_h_a_t_i_casymmetric/symmetric

1

stretch), 1652 (s, C=N stretch), 1260, 1210, 1160, 1140, 1080 (m, s, ketal C-C stretch). H NMR (500

MHz, CDCl ) δ(ppm): 11.27 (s, 1H), 4.93 (s, 1H), 4.28 (dd, 1H, J = 7.9 Hz, J = 2.4 Hz), 4.17 (dd, 1H,

3

J = 8.7 Hz, J = 6.0 Hz), 4.07 (ddd, 1H, J = 8.9 Hz, J = 6.0 Hz, J = 4.6 Hz), 3.98 (dd, 1H, J = 8.8 Hz,

J = 4.6 Hz), 3.94 (dd, 1H, J = 8.8 Hz, J = 8.0Hz), 3.49 (s, 1H), 1.42 (s, 3H), 1.39 (s, 3H), 1.38 (s, 3H),

1

3

1

6

.33 (s, 3H). C NMR (125 MHz, CDCl ) δ(ppm): 178.7, 162.8, 111.1, 110.4, 80.1, 76.9, 76.8, 67.8,

3

+

5.4, 27.1, 26.7, 26.5, 25.1. HRMS (LDI/TOF) m/z [M+H] calc.: 333.1120 found: 333.1031.

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-1

Ligand 1,3,4-Oxadiazolidine-2-thione 8: oil, µ: 60.11 mPa s, ρ: 1.276 g cm , IR (KBr) ν(cm ): 3445

(br, O-H stretch), 3161 (w, NH stretch), 2985 and 2934 (m, CH_a_l_i_p_h_a_t_i_casymmetric/symmetric stretch),

1

496 (s, C=N stretch), 1260, 1210, 1160, 1070 (s, m, ketal C-C stretch). H NMR (500 MHz, CDCl )

3

δ (ppm): 5.01 (d, 1H, J = 7.9 Hz), 4.63 (dd, 1H, J = 7.9 Hz, J = 3.6 Hz), 4.16 (m, 1H), 4.07 (m, 2H),

.64 (dd, 1H, J = 7.1 Hz, J = 3.6 Hz), 1.53 (s, 3H), 1.52 (s, 3H), 1.42 (s, 3H), 1.33 (s, 3H). ¹³C NMR

3

(

125 MHz, CDCl ) δ(ppm) 178.7, 161.2, 112.3, 109.9, 78.8, 75.8, 70.9, 70.0, 66.9, 26.7, 26.6, 26.0,

3

+

2

5.1. HRMS (LDI/TOF) m/z [M+H] calc.: 333.1120 found: 333.2360.

2

.2.4 Synthesis of gold complexes 9 to 12

In a 10 mL round-bottom flask were added 0.1 mmol of starting material (either 7 or 8) and then

dissolved in 4 mL of dichloromethane along with 0.1 mmol of NaHCO . After that, 0.1 mmol of

3

[

Au(PEt )Cl] (to produce 9 and 10) or [Au(PPh )Cl] (to produce 11 and 12) were added and the

3

reaction mixture was left under inert atmosphere and magnetic stirring for 24 hours, at room

temperature and protected from light. At the end of the reaction, the solvent was evaporated under

reduced pressure and the product was purified by column chromatography, whilst protected from

light. Compounds 9, 10, 11 and 12 were obtained in 85-90% yield and kept at -20 °C protected from

light.

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Complex 9: oil, BP: ND, µ: 125.03 mPa s, ρ: 1.242 g cm , IR (KBr) ν(cm ): 3300 (br, O-H stretch),

2

1

4

990 and 2930 (m, CH_a_l_i_p_h_a_t_i_casymmetric/symmetric stretch), 1450 (s, P-C deformation), 1250, 1210,

1

165, 1085 (s, m, ketal C-C stretch). H NMR (500 MHz, CDCl ) δ(ppm): 4.99 (d, 1H, J = 2.4 Hz),

3

.30 (dd, 1H, J = 7.3 Hz, J = 2.6 Hz), 4.16 (dd, 1H, J = 8.6 Hz, J = 5.7 Hz), 4.06 (ddd, 1H, J = 8.7 Hz,

J = 5.6 Hz, J =4.8 Hz), 4.02 (dd, 1H, J = 8.6 Hz, J = 7.4 Hz), 3.96 (dd, 1H, J = 8.6 Hz, J = 4.7 Hz),

1

9

7

.90 (dq, 6H, J = 10.0 Hz, J = 7.6 Hz), 1.42 (s, 3H), 1.38 (s, 3H), 1.37 (s, 3H), 1.34 (s, 3H), 1.24 (dt,

1

3

H, J = 18. Hz, J = 7.6 Hz). C NMR (125 MHz, CDCl ) δ(ppm): 171.1, 164.9, 110.4, 110.0, 80.8,

3

1

7.0, 76.9, 67.9, 65.5, 27.1, 26.7, 26.5, 25.2, 17.9 (d, 3C, J = 34.2 Hz), 9.0 (s, 3C). P NMR (202

+

MHz, CDCl ) δ(ppm): 36.67. HRMS (LDI/TOF) m/z [M+H] calc.: 647.1618 found: 647.4000.

3

-3

-1

Complex 10: oil, BP: ND, µ: 104.95 mPa s, ρ: 1.240 g cm , IR (KBr) ν(cm ): 3350 (br, O-H stretch),

2

1

4

985 and 2935 (m, CH_a_l_i_p_h_a_t_i_casymmetric/symmetric stretch), 1450 (s, P-C deformation), 1255, 1215,

1

140, 1075 (s, m, ketal C-C stretch). H NMR (500 MHz, CDCl ) δ(ppm): 5.09 (d, 1H, J = 7.9 Hz),

3

.68 (dd, 1H, J = 7.8 Hz, J = 2.8 Hz), 4.06 (m, 3H), 3.58 (td, 1H, J = 7.3 Hz, J = 2.3 Hz), 2.73 (d, 1H,

J = 7.2 Hz), 1.89 (dq, 6H, J = 10.0 Hz, J = 7.6 Hz), 1.47 (s, 3H), 1.46 (s, 3H), 1.35 (s, 3H), 1.28 (s,

1

3

1

H), 1.24 (dt, 9H, J = 18.8 Hz, J = 7.6 Hz). C NMR (125 MHz, CDCl ) δ(ppm): 170.9, 163.6,

3

11.4, 109.4, 78.7, 76.0, 70.3, 70.0, 66.9, 26.8, 26.7, 26.2, 25.2, 17.9 (d, 3C, J = 34.3 Hz), 9.0 (s, 3C).

3

1

+

P NMR (202 MHz, CDCl ) δ(ppm): 36.63. HRMS (LDI/TOF) m/z [M+H] calc.: 647.1618 found:

3

6

47.3910.

-3

-1

Complex 11: oil, BP: ND, µ: 88.53 mPa s, ρ: 1.389 g cm , IR (KBr) ν(cm ): 3400 (br, O-H stretch),

060 (w, C-H_a_r_o_m_a_t_i_cstretch), 2990, 2940 (m, CH_a_l_i_p_h_a_t_i_casymmetric/symmetric stretch), 1460 (m,

3

aromatic C-C stretch), 1450 (m, P-C deformation), 1265, 1215, 1080 (s, m, ketal C-C stretch), 750 (s,

1

aromatic out-of-plane C-H bending), 537 (s, aromatic P-C stretch). H NMR (500 MHz, CDCl )

3

δ(ppm): 7.55 (m, 15H), 5.03 (d, 1H, J = 2.6 Hz), 4.34 (dd, 1H, J = 7.3 Hz, J = 2.6 Hz), 4.17 (dd, 1H,

J = 8.6 Hz, J = 5.7 Hz), 4.07 (ddd, 1H, J = 8.7 Hz, J = 5.7 Hz, J = 4.8 Hz), 4.03 (dd, 1H, J = 8.7 Hz,

J = 7.4 Hz), 3.97 (dd, 1H, J = 8.6 Hz, J = 4.7 Hz), 1.42 (s, 3H), 1.38 (s, 3H), 1.37 (s, 3H), 1.34 (s,

3

H). ¹³C NMR (126 MHz, CDCl ) δ(ppm): 171.5, 164.8, 134.2 (d, 6C, J = 13.8), 131.9 (d, 3C,

3

J = 2.4), 129.3 (d, 6C, J = 11.7), 128.8 (d, 3C, J = 58.9), 110.4, 110.1, 80.8, 77.1, 76.9, 67.9, 65.5,

3

1

+

2

7.1, 26.7, 26.5, 25.2. P NMR (202 MHz, CDCl ) δ(ppm): 37.63. HRMS (LDI/TOF) m/z [M+H]

3

calc.: 791.1618 found: 791.5861.

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Complex 12: oil, BP: ND, µ: 95.68 mPa s, ρ: 1.261 g cm , IR (KBr) ν(cm ): 3300 (br, O-H stretch),

060 (w, aromatic C-H stretch), 2985, 2935 (m, s CH_a_l_i_p_h_a_t_i_casymmetric/symmetric stretch), 1440 (s,

3

aromatic C-C stretch), 1265, 1215, 1075 (m, ketal C-C stretch), 735 (s, aromatic out-of-plane C-H

1

bending), 540 (s, aromatic P-C stretch). H NMR (500 MHz, CDCl ) δ(ppm): 7.54 (m, 15H), 5.15 (d,

3

1

H, J = 7.8 Hz), 4.74 (dd, 1H, J = 7.8 Hz, J = 2.8 Hz), 4.05 (m); 3.63 (d, 1H, J = 4.8 Hz), 2.48 (s,

1

3

H), 1.50 (s, 3H), 1.45 (s, 3H), 1.36 (s, 3H), 1.30 (s, 3H). C NMR (126 MHz, CDCl ) δ(ppm):

3

70.6, 163.7, 134.2 (d, 6C, J = 13.7 Hz), 131.9 (d, 3C, J = 2.5 Hz), 129.3 (d, 6C, J = 11.6 Hz), 128.8

3

1

(

d, 3C, J = 58.9 Hz), 111.5, 109.5, 78.7, 76.0, 70.5, 70.1, 66.9, 26.8, 26.7, 26.2, 25.3. P NMR (202

+

MHz, CDCl ) δ(ppm): 37.65. HRMS (LDI/TOF) m/z [M+H] calc. : 791.1618 found: 791.5796.

3

2

.3 Biological activity assays

2

.3.1 Cytotoxicity assay against tumor and non-tumor cell lines

Cytotoxic activity was tested against the following tumor cell lines: 4T1 (murine mammary

carcinoma) and B16-F10 (mouse metastatic skin melanoma). Baby hamster kidney BHK-21, was used

as non-tumoral cell line. Drug activity was measured by assessing the inhibitory concentration that led

to apoptosis of 50% of growing cells (IC ). Cell lines were axenically propagated in complete RPMI

5

0

1

640 culture medium at pH 7.4, supplemented with 10% of heat-inactivated fetal bovine serum

(

FBS), 4 mM HEPES,14 mM NaHCO , 0.27 mM ampicillin, and 0.06 mM streptomycin in 5% (v/v)

3

CO humid atmosphere. Cells were rinsed with PBS/EDTA pH 7.4 buffer and harvested with trypsin

2

0

.02% (v/v). Trypsin was then inactivated by adding 1.5 mL of complete RPMI where cells were

3

seeded in 96-well tissue culture plates at the following densities: 0.5x10 (BHK-21), 1.5x10 (B16-

3

F10) and 2.0x10 (4T1) cells/well/100 μL and incubated at 37 °C in a humidified atmosphere

containing 5% CO during 24 hours for complete adherence.

2

DMSO solutions of the tested compounds were serially diluted (from 100 to 0.1 μM) in RPMI

cell culture medium (< 1% DMSO) in quadruplicate. The negative control was obtained with cells

exposure in RPMI 1640 medium supplemented with 10% FBS. After 72 h drug exposure, cells were

incubated with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT (Sigma-

Aldrich #M2128) (5 μg/10 μL/well) for 4 h at 37 °C in 5% CO humid atmosphere. The supernatant

2

was then removed by aspiration and 100 μL of DMSO/well were added. Cell viability was determined

by absorbance at 570 nm (using a plate spectrophotometer) of the obtained solution which contains

formazan salts, a product of mitochondrial reduction of MTT by viable cells (Mosmann, 1983).

2

.3.2 Antileishmanial assays against intracellular amastigotes

Leishmania (Viannia) braziliensis (strain MHOM/BR/1994/H3227) promastigotes were maintained in

minimum essential culture medium (α-MEM) (Gibco, Invitrogen NY, USA) supplemented with 10%

(v/v) heat inactivated FBS (Cultilab, Campinas, SP, Brazil), 100 mg/mL kanamycin, 50 mg/mL

ampicillin, 2 mM L-glutamine, 5 mg/mL hemin, 5 mM biopterin (Sigma-Aldrich #B2517), at pH 7.0

and incubated at 25°C. L. braziliensis (MHOM/BR/94/H3227) and L. infantum

(MHOM/MA/67/ITMAP-263) parasites expressing firefly luciferase as a reporter gene

(pSP72αHYGαLUC1.2) – LbrLUC and LinLUC. Transfections were performed as previously

described (Roy et al., 2000). Human monocyte-derived macrophage cell line THP-1 was maintained

in RPMI 1640 medium supplemented with 10 % FBS. Cells were plated in blank 96-well tissue

5

culture plate at 8x10 cells/mL and differentiated by incubating with 20 ng/mL of Phorbol 12-

myristate 13-acetate - PMA (SigmaAdrich, #P8139) at 37 °C in a 5 % CO containing humid

2

atmosphere for 3 days. Cells were washed with RPMI medium and subsequently infected with

LbrLUC or LinLUC at a parasite/macrophage ratio of 10∶1 for 3 h. Non-internalized parasites were

removed by three washes with HEPES/NaCl buffer (20 mM HEPES, 0.15 M NaCl, 10 mM glucose,

pH 7.2). After 3 days RPMI was aspirated and the luciferase activity assessed by adding 20 μL of

reconstituted One-Glo™ Luciferase Assay System solution as enzyme substrate, following

manufacturer’s instructions (Promega, Madison, WI, USA #E6110). Luciferase activity was measured

by luminescence detection in a luminometer SpectraMax M5 (Molecular Devices, Sunnyvale, CA,

USA) using 1-second integration/well. Non-infected THP-1 macrophages were considered as signal

background while non-treated infected THP-1 cells were used as a control for growth comparison.

Amphotericin B and miltefosine were added as a positive control.

2

.3.3 Cytotoxicity assay against human macrophage cell line

In vitro cytotoxicity activity was evaluated by quantifying the ability of living cells to reduce the

yellow dye MTT (Sigma-Aldrich #M2128) to a purple formazan product (Mosmann, 1983).

Cytotoxicity (cell viability) was evaluated on human monocyte-derived THP-1 macrophages cell

5

lines. For macrophage differentiation, 5 x 10 precursor monocytes/well were seeded in 96-well plates

treated with 20 ng/mL phorbol 12-myristate 13-acetate PMA (Sigma-Aldrich #P8139) and incubated

4

8 h at 37 °C in 5 % CO humid atmosphere. Stock solutions of gold(I) complexes were prepared in

2

DMSO and serially diluted in RPMI medium (<1 % DMSO). After 72 h of drug exposure at 37 °C

and 5 % CO , cells were incubated with MTT (10 mM in water solution – 10 µL/well) for 4 h at 37 °C

2

and 5 % CO . MTT is metabolized by viable cells resulting in a purple product that after being

2

solubilized in 100 µL of DMSO, can be quantified through colorimetric assay using a plate reader

(absorbance at 570 nm). Negative control was performed considering untreated cells in RPMI 1640.

2

.3.4 Statistical analyses

IC and CC values were calculated based on concentration-response curves applying a sigmoidal

5

0

50

concentration-response equation with variable slope carried out using the software GraphPad Prism

version 6.0 (GraphPadSoftware Inc., San Diego, CA, USA). Selectivity index was estimated by the

ratio CC /IC When applied, data were analyzed analysis of variance (ANOVA) followed by

5

0

50.

Tukey’s multicomparisons test. A p value < 0.05 was considered statistically significant.

3

RESULTS AND DISCUSSION

3

.1 Synthesis of gold(I) complexes

1

In the H NMR spectra of ligand 7, we observe the presence of the signal corresponding to the

1

heterocyclic NH hydrogen at 11.27 ppm, although this signal is not readily observable in the H NMR

1

spectra of 8, due to its characteristic broadness. Also, the signals previously present in the H NMR

spectra of the starting materials 5 and 6, at 8.84 and 4.27 ppm, and at 8.03 and 3.31 ppm respectively,

corresponding to the hydrazide group hydrogens, are no longer present, thus suggesting the formation

1

3

of the desired compounds 7 and 8. Also, we observe in the C NMR spectra of 7 and 8 that, instead

of signals at 170.0 and 170.8 ppm (corresponding to the carbonyl carbons), respectively, we observe

new signals at 178.7 and 162.8 ppm for 7 and at 178.7 and 161.2 ppm for 8, corresponding to the

C=N and the C=S carbons, respectively. Also important, the IR spectra of 5 and 6 differ of 7 and 8 in

many points such as some variation in the absorption bands corresponding to NH stretch, replaced

2

-1

instead by the single NH stretching band around 3300 cm . These observations, along with mass

spectrometry, provide evidence for the formation of 7 and 8.

1

In the H NMR spectra of 9 and 10, we observe at 1.90 and 1.24 ppm (for 9) and at 1.89 and

1

.24 ppm (for 10) the signals for the methylene and methyl hydrogens, respectively, corresponding to

1

3

the phosphine moiety of the molecule. We can also observe in the C NMR spectra of 9 and 10 the

signals of the methylene and methyl carbons adjacent to the phosphorous atom, namely at 17.97 and

9

.09 ppm for 9 and at 17.93 and 9.09 ppm for 10. Furthermore, the IR spectra of 9 and 10 both show

-1

the characteristic deformation band of the P-C bond, at 1450 cm . These results are expected for the

1

synthesized compounds. Finally, if we pay close attention to the H NMR spectra of 9 and 10 and

compare it to that of its precursors 7 and 8, we can readily observe that after coordination with

gold(I), the relative positions of hydrogens 6’ and 4 have been interchanged; i.e. hydrogen 6’ was

1

originally located further down than hydrogen 4 in the H NMR spectra of the ligands, whereas the

opposite is true for the complexes.

1

In the H NMR spectra of 11 and 12, we observe the multiplets corresponding to the aromatic

1

3

hydrogens at 7.55 and 7.54 ppm respectively. We can also observe in the C NMR spectra of 11 and

2 the signals of the aromatic carbons between 134.2 and 128.8 ppm in both cases. Also, the IR

1

-1

spectra of 11 and 12 both show the characteristic stretch band of the C-C aromatic bond, at 1450 cm ,

-1

as well as the characteristic out-of-plane bending of aromatic C-H at 750 cm . In a similar manner as

with complexes 9 and 10, we observe that after coordination with gold(I), the relative positions of

1

hydrogens 6’ and 4 between the H NMR spectra of the ligands and the complexes are interchanged.

3

.2 Gold(I) complexes presented selective anticancer activity

All compounds showed higher selectivity indexes when compared to cisplatin (Table 1). None of the

ligands (compounds 7 and 8) showed cytotoxicity below 100µM. In the case of skin melanoma-

derived cell line B16-F10, results show that complexes 9 and 10 (triethylphosphine derivatives) are

more cytotoxic and selective than the triphenylphosphine analogues compounds 11 and 12, presenting

IC values of 2.0 ± 0.2 and 3.2 ± 0.2 μM, with selectivity indexes of 10.6 and 6.9, respectively.

5

0

Similar evidence was previously found in the literature, where triethylphosphine derivatives have

shown better activity, possibly because of steric factors, a result that is consistent with past works of

other leading authors (J. D. S. Chaves et al., 2015; J. D. S. Chaves et al., 2014; O. A. Chaves et al.,

2

016; de Almeida et al., 2017; Garcia et al., 2016). Furthermore, in the case of skin melanoma (B16-

F10), if we compare the results for compounds 9 and 10, we see that compound 9 is slightly more

active than compound 10 (with IC of 2 ± 0.2, µM compared to 3.2 ± 0.2 µM from compound 10).

5

0

On the other hand, results obtained from the mammary carcinoma (4T1) tests are quite scattered and

do not show any immediate pattern that could allow us to establish a satisfying structure-activity

relation. It is very important to notice that the ligands showed cytotoxicity only after coordination

with gold(I), and also that the starting gold material alone already presented some activity, yet it

became more selective and more active after complexation with some of the gluco-derived moieties.

3

.3 Gold(I) complexes are antileishmanial agents less toxic than their metal precursors

Gold(I) complexes 9, 10, 11 and 12 were able to impair the proliferation of L. braziliensis

intramacrophagic amastigotes in vitro with mean IC values of 3 µM, the same range as those shown

5

0

by their gold(I) precursors AuPEt Cl – gold(I) triethylphosphine chloride – and AuPPh Cl – gold(I)

3

triphenylphosphine chloride (Table 2 and Fig. 2). Considering their cytotoxicity against THP-1 host

macrophages, the gold(I) complexes were 4 to 6 times more selective towards the parasite than their

metal precursor counterparts alone, which showed SI values around 2 (Table 2). This is consistent

with the fact that all four gold(I) complexes were around 3 times less toxic to the host macrophage

than the free AuPEt Cl and AuPPh Cl (Table 2 and Figure 2). Complexes 10 and 12, derived from

3

methyl (2,3)/(5,6)-di-O-isopropylidene-D-gluconate 4 – were both less toxic to the host macrophages

when compared to complexes 9 and 11, synthesized from the ligand isomer methyl (3,4)/(5,6)-di-O-

isopropylidene-D-gluconate 3 (Table 2). This hexose-driven differential cytotoxicity is quite

intriguing, since THP-1 human macrophages do express different forms of glucose transporters (Fu,

Maianu, Melbert, & Garvey, 2004). Additionally, as intracellular parasites, it is expected that a good

antileishmanial agent should be able to pass through the macrophage’s membrane to become available

against Leishmania inside the cell. It is no surprise that the most widely used drugs against

leishmaniasis are antimony-derived sugar-based molecules: sodium stibogluconate and meglumine

antimoniate (El Fadili et al., 2008; Frézard, Demicheli, & Ribeiro, 2009). Inspired by this facts,

efforts have been made to employ this ‘Trojan horse’ strategy in antileishmanial metallodrug-based

chemotherapy (C. S. Ferreira et al., 2010; W. A. Ferreira et al., 2014). Thus, these four less toxic D-

gluconate-derived gold(I) complexes represent potential antileishmanial agents that deserve attention

in further drug development studies, including sugar-mediated drug delivery strategies. Although no

difference in terms of antileishmanial activity was observed between the gold(I) complexes and their

precursors, the former’s synthesis is justified by their lesser toxicity against THP-1 human

macrophages, which directly impact drug selectivity.

Unlike the anticancer profile presented in this study – where triethylphosphine-substituted

gold(I) complexes 9 and 10 were more active against metastatic skin melanoma derived cell line B16-

F10 than triphenylphosphine-based complexes 11 and 12 (Table 1) – the structure of the phosphine

group used, either triphenylphosphine (PPh ) or triethylphosphine (PEt ), had no impact on the

3

structure-activity relationship when tested against intramacrophagic L. braziliensis amastigotes (Table

and Figure 2). This was also a surprise for the rest of our research team, since we had been

observing this PPh /PEt -dependent biological response in gold(I) complexes with different ligands (J.

2

3

D. Chaves et al., 2015; O. A. Chaves et al., 2016; Garcia et al., 2016). Instead, we found that a slight

isomeric variation in the D-gluconate ligand was enough to drive a higher cytotoxic effect. This also

highlights the importance of using different cell types to infer about biological activities in drug

discovery and drug development studies.

The complexes’ antileishmanial activity at low micromolar range against L. braziliensis raises

the question on how would they behave against other Leishmania species, and also opens the

possibility for them be considered as a potential solution against both cutaneous and visceral

leishmaniasis. In fact, we recently observed that gold(I) complexes show very similar antileishmanial

activity among different Leishmania species (O. A. Chaves et al., 2016). Gold(I)-containing

heterocyclic complexes were also active against L. infantum, the main etiological agent of visceral

leishmaniasis (Zhang et al., 2018). A previous study demonstrates that gold(I)-based compound

auranofin presented antileishmanial activity both in vitro and in vivo against cutaneous leishmaniais-

causing species L. amazonensis and L. major (Sharlow et al., 2014). In this context, we had expected

that gold(I)-derivatives were actually active in a wide spectrum against different Leishmania spp.

4

CONCLUSIONS

In this work two ligands and four gold(I) complexes were synthesized, characterized and evaluated

for anticancer and antileishmanial properties. All tested gold(I) complexes are potential anticancer

and antileishmanial drug candidates that should be further investigated. For both biological activities

explored in this work, the complexation of the ligands with gold(I) somehow improved drug activity

(or reduced toxicity against control cell), since neither ligand was biologically active by itself. We

thus reinforce the fact that mechanistic approach studies are encouraged in order to further support

drug development follow-up. Such studies could help to demonstrate the potential application of

oxadiazolidine-based gold(I) complexes as anticancer and antileishmanial drugs. Since we observed

an anticancer property dependent on the triethylphosphine substitution, the evaluation of compounds

with different phosphine ligands should help us establish a more consistent structure-activity relation,

aimed towards higher efficacy and selectivity. Additionally, these novel gold(I) complexes could be

considered for combined chemotherapy in anticancer and antileishmanial treatment, because both the

1

,3,4-oxadiazole and gold(I) itself can act as anti-inflammatory agents and help immune response to

tackle both tumor cells and Leishmania parasites.

CONFLICT OF INTEREST

There are no conflicts of interest of any of the parts involved in this work.

ACKNOWLEDGMENTS

Thanks to Dr. Silvia Uliana, University of São Paulo, Brazil, who kindly provide the L. braziliensis

H3227 expressing luciferase as reporter. The authors wish to thank Fapemig, CNPq and UFJF for

financial support. This work was financed in part by the Coordenação de Aperfeiçoamento de Pessoal

de Nível Superior - Brasil (CAPES) - Finance Code 88881.133591/2016-01. This study was also

funded by Fapemigl – APQ-03059-16. The authors also thank the Organization of American States

for providing the scholarship that made this work possible.

DATA AVAILABILITY STATEMENT

I confirm that my article contains a Data Availability Statement. Detailed transcripts are included in

the article so that analyses can be validated. For additional data contact the correspondence author.

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Table 1 Anticancer activity profile of gold(I) complexes expressed as 50% inhibitory concentration

IC ) of compounds 7-12 against 4T1 (murine mammary carcinoma), B16-F10 (mouse metastatic

(

5

0

skin melanoma), along with their selectivity index (SI) compared to non-tumor cell BHK-21 (baby

hamster kidney). IC₅₀are presented in μM and SD-standard deviation of three independent

experiments.

Tumor cells

Non-tumor cell

a

Compound

IC (μM ±SD )

5

0

50

B16-F10

>100

SI^b

ND

4T1

SI^b

ND

5.4

8.5

6.2

6.1

2.9

2.4

BHK-21

>100

7

8

9

>100

2.0 ± 0.2

3.2 ± 0.2

8.3±0.1

7.1 ± 0.5

6.4 ± 1.0

6.6 ± 0.1

2.3 ± 0.5

10.6

6.9

2.7

3.3

2.8

3.4

9.7

3.9 ± 0.3

2.6 ± 0.2

3.6 ± 0.3

3.9 ± 0.1

6.2 ± 2.3

10.3 ± 2.3

9.5 ± 1.1

21.2 ± 0.3

22.2 ± 0.9

22.3 ± 0.4

23.6 ± 0.8

18.1 ± 3.0

23.0 ± 0.3

22.5 ± 0.2

1

0

1

2

Cisplatin

AuPPh Cl

3

AuPEt Cl

3

^aSD: standard deviation;

b

SI: selectivity index estimated by the ratio: IC tumor/IC non-tumor

5

0-

50

ND: not determined

Table 2 Antileishmanial activity and selectivity index of gold(I) complexes based on oxadiazolidine-

-thione and D-gluconate ligands against Leishmania braziliensis intracellular amastigotes and THP-1

human macrophage cell line. IC and CC are presented in μM and 95% CI- confidence interval.

2

5

0

50

IC μM (95% CI)

CC μM (95% CI)

5

0

50

Compound

L. braziliensis

SI

THP-1 macrophages

intracellular amastigotes

> 10

7

8

9

ND

4.9

5.9

4

> 10

ND

3.09 (2.78-3.42)

3.3 (2.98-3.65)

2.96 (2.39-3.67)

3.27 (2.34-4.57)

2.83 (2.57-3.12)

3.31 (2.79-3.93)

6.68 (5.55-8.03)

0.12 (0.10-0.15)

15.1 (13.7-16.66)

19.44 (17.52-21.57)

11.77 (10.87-12.75)

19.3 (17.58-21.17)

4.98 (4.64-5.33)

5.43 (5.08-5.79)

39.08 (37.65-40.57)

12 (11.43-12.59)

1

0

1

2

6

AuPPh Cl

1.7

1.6

5.85

100

3

AuPEt Cl

3

Miltefosine

AmB

9

5% CI: 95% confidence interval;

SI: selectivity index estimated by the ratio CC /IC ;

5

0

50

ND: not determined;

AmB: amphotericin B

Figure captions

Figure 1 Scheme representing the synthetic route to obtain gold(I) complexes 9-12

Figure 2 Antileishmanial and cytotoxic activity of gold(I) complexes with oxadiazolidine-2-thione and D-

gluconate ligands. A) The IC against Leishmania braziliensis intracellular amastigotes was calculated based

5

0

on concentration-response curves and shown here to compare antileishmanial activity among the gold(I)

complexes and their precursors AuPEt Cl and AuPPh Cl. B) The cytotoxic concentration that kills 50% of

3

THP-1 human macrophages (CC ) was compared; highlighting that the four gold(I) complexes were 3x less

5

0

toxic than their free metal precursor. Complexes 10 and 12– derived from methyl (3,4)/(5,6)-di-O-

isopropylidene-D-gluconate 3– were either less toxic when compared to complexes 9 and 11, synthesized

from the ligand isomer methyl (2,3)/(5,6)-di-O-isopropylidene-D-gluconate 4. AuPEt Cl: gold(I)

3

triethylphosphine chloride; AuPPh Cl: gold(I) triphenylphosphine chloride. Statistical differences were

3

calculated using ANOVA one-way followed by Tukey’s multiple comparisons test. *** p = 0.001; **** p =

0

.0001.

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Anticancer and antileishmanial in vitro activity of gold(I) complexes with 1,3,4-oxadiazole-2(3H)-thione ligands derived from δ-D-gluconolactone

DOI: 10.1111/cbdd.13757

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Article abstract of DOI:10.1111/cbdd.13757

Full text of DOI:10.1111/cbdd.13757

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