Synthesis and 2D-QSAR studies of neolignan-based diaryl-tetrahydrofuran and -furan analogues with remarkable activity against Trypanosoma cruzi and assessment of the trypanothione reductase activity

Accepted Manuscript

Synthesis and 2D-QSAR studies of neolignan-based diaryl-tetrahydrofuran and -furan

analogues with remarkable activity against Trypanosoma cruzi and assessment of the

trypanothione reductase activity

Ana Paula Hartmann, Marcelo Rodrigues de Carvalho, Lilian Sibelle Campos

Bernardes, Milene Hoehr de Mores, Eduardo Borges de Melo, Carla Duque Lopes,

Mario Steindel, João Santana da Silva, Ivone Carvalho

PII:

S0223-5234(17)30676-1

10.1016/j.ejmech.2017.08.064

EJMECH 9708

DOI:

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 11 July 2017

Revised Date: 7 August 2017

Accepted Date: 29 August 2017

Please cite this article as: A.P. Hartmann, M.R. de Carvalho, L.S.C. Bernardes, M.H. de Mores, E.B.

de Melo, C.D. Lopes, M. Steindel, Joã.Santana. da Silva, I. Carvalho, Synthesis and 2D-QSAR studies

of neolignan-based diaryl-tetrahydrofuran and -furan analogues with remarkable activity against

Trypanosoma cruzi and assessment of the trypanothione reductase activity, European Journal of

Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.08.064.

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ACCEPTED MANUSCRIPT

Graphical Abstract

ACCEPTED MANUSCRIPT

Synthesis and 2D-QSAR studies of neolignan-based diaryl-tetrahydrofuran and -furan

analogues with remarkable activity against Trypanosoma cruzi and assessment of the

trypanothione reductase activity

Ana Paula Hartmann,^aMarcelo Rodrigues de Carvalho,^aLilian Sibelle Campos Bernardes,^b

Milene Hoehr de Mores,^cEduardo Borges de Melo,^dCarla Duque Lopes,^eMario Steindel,^cJoão

Santana da Silva,^dIvone Carvalho^a*

^aSchool of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. Café s/n, Ribeirão

Preto, SP 14040-930, Brazil

^bDepartment of Pharmaceutical Sciences, Federal University of Santa Catarina, Florianopolis, 88040900

SC, Brazil

^cDepartment of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina,

Florianopolis, 88040900 SC, Brazil

^dSchool of Pharmacy, Western Parana State University, Rua Universitária, 2069, Cascavel, PR, 85819-

110, Brazil

^eRibeirão Preto Medical School, University of São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP

14049-900, Brazil

*Corresponding author: carronal@usp.br

Abstract

Two series of diaryl-tetrahydrofuran and -furan were synthesised and screened for anti-

trypanosomal activity against trypomastigote and amastigote forms of Trypanosoma cruzi, the

causative agent of Chagas disease. Based on evidence that modification of a natural product

may result in a more effective drug than the natural product itself, and using known neolignan

inhibitors veraguensin 1 and grandisin 2 as templates to synthesise simpler analogues,

remarkable anti-trypanosomal activity and selectivity were found for 3,5-dimethoxylated

diaryl-tetrahydrofuran 5c and 2,4-dimethoxylated diarylfuran 4e analogues with EC₅₀0.01 µM

and EC₅₀0.75 μM, respectively, the former being 260-fold more potent than veraguensin 1 and

150-fold better than benznidazole, the current available drugs for Chagas disease treatment.

The ability of the most potent anti-trypanosomal compounds to penetrate LLC-MK2 cells

infected with T. cruzi amastigotes parasite was tested, which revealed 4e and 5e analogues as

the most effective, causing no damage to mammalian cells. In particular, the majority of the

derivatives were non-toxic against mice spleen cells. 2D-QSAR studies show the rigid central

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core and the position of dimethoxy-aryl substituents dramatically affect the anti-trypanosomal

activity. The mode of action of the most active anti-trypanosomal derivatives was investigated

by exploring the anti-oxidant functions of Trypanothione reductase (TR). As a result,

diarylfuran series displayed the strongest inhibition, highlighting compounds 5d-e (IC₅₀19.2

and 17.7 μM) and 5f-g (IC₅₀8.9 and 7.4 μM), respectively, with similar or 2-fold higher than the

reference inhibitor clomipramine (IC₅₀15.2 μM).

Keywords: Trypanosoma cruzi, Chagas disease, natural neolignans, diaryl-tetrahydrofurans,

diarylfurans, trypanothione reductase, 2D-QSAR.

2. Introduction

Plant-derived natural products represent a valuable source of novel drug leads

because of the chemical diversity found in nature, giving rise to highly active compounds with

selective and specific mechanisms of action [1,2]. Despite the great interest of pharmaceutical

companies in combinatorial chemistry to access the synthetic compound library for HTS (High

Throughput Screen) approaches, the decline of new chemical entities approved by the FDA at

the beginning of the 21st century, has led the research community to reappraise plant-derived

natural product drug discovery as an important approach for the identification of novel

chemotherapeutics agents [3]. Furthermore, the extensive use of natural products by different

cultures for the treatment of various disease conditions make them a valuable supply of new

drugs [1,4,5].

Apart from that, several aspects preclude the successful research, development and

commercialisation of natural products, such as the supply constraints on bioactive

components, ecological and legal considerations for plant accessibility, the isolation processes,

variation of quality and composition (depend on species identity), harvest time, soil

composition, altitude, actual climate, and processing and storage conditions [3,6]. Even though

the resupply of natural products can be achieved by the application of plant cell and tissue

culture, heterologous production or semi-synthesis from isolated precursors. The limited

amount of isolated bioactive natural products available, and the total chemical synthesis of

complex structures containing several functional groups and chiral centers, slow down the

development process, which still requires optimisation of physicochemical properties using

chemical modification approaches [3,7]. Therefore, the chemical simplification and

derivatisation of natural products is an interesting alternative to generate a chemical library, as

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well as to understand the structure–activity relationship for pharmacophore identification. In

the last decade, several natural product extracts have been screened to find more active and

less toxic molecules against Chagas disease, a life-threatening illness caused by the protozoan

parasite Trypanosoma cruzi [8-10]. Chagas disease is endemic in Central and South American

countries, with an estimated 8 million people infected worldwide [11,12]. In 2011, Nakamura

reported 400 species related to approximately 100 families of plant extracts already tested

against different forms of T. cruzi, of which 10% were investigated and their corresponding

active components against the parasite were isolated and characterised, being around 136

compounds assessed [13]. Since then, a brief search at Web of Science [14] revealed about 33

active compounds from plant extracts against T. cruzi [15-26]. The phenotypic screening of

potential anti-trypanosomal compounds from natural products has shown a plethora of

chemical classes, illustrated by alkaloid, flavonoid, catechin and terpene derivatives, including

sesquiterpene lactones, xanthone, oxygenated hydrocarbons and lignans [13,27,28]. In

particular, lignans isolated from Lauraceae and Piperaceae families are of great interest since

the majority of the active components decrease parasite growth of both epimastigote and

trypomastigote forms [29]. Beside lignans, tetrahydrofuran neolignans such as veraguensin (1)

and grandisin (2), isolated from Virola surinamensis and Piper solmsianum, have been

identified as potent inhibitors of trypomastigote Y strain of T. cruzi, producing parasite lysis

with EC₅₀2.3 and 3.7 µM, respectively [30] (Figure 1). Notably, veraguensin (1) and grandisin

(2) possess 2,5-bis(3,4-dimethoxyphenyl)-ꢀandꢀ2,5-bis(3,4,5-trimethoxyphenyl)-tetrahydro-3,4-

dimethylfuran structures, respectively. In addition, 4-(furan-2-yl) benzenesulfonamide and

symmetrical non-substituted 2,5-bis(3,4-dimethoxyphenyl)furan have been further described

as T. cruzi amastigote growth inhibitors (70 and 65% inhibition at 20 µg/mL) [31]. Our previous

studies regarding the low water solubility of these neolignans have shown the importance of

the heterocycle core to the potency [32,33], considering the distinguished activity displayed by

dihydrofuran derivative 3, the most active of all series with EC₅₀1.5 µM [32], when comparing

the activities of 1-3 with open chain symmetrical and unsymmetrical diaryl diols and diaryl

diketones precursors, besides symmetrical acetylenic glycols, which preserve the diaryl

polymethoxylated pattern of natural products 1 and 2. These findings encouraged us to

investigate whether simplified unsymmetrical 2,5-diaryl-tetrahydrofuran and -furan derivatives

containing methoxyl substituents in a variable number and at different ring positions, such as

the set compounds 4 and 5, would possess enhanced activity against trypomastigote and

amastigote forms of T. cruzi. Further, 2D-QSAR studies were carried out to identify the

molecular descriptors that may encode structure information of the set molecules in order to

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predict the anti-parasitic responses of new hit derivatives [34-36]. Then, the most active anti-

trypanosomal derivatives were chosen to investigate whether they inhibit Trypanothione

reductase (TR), a key enzyme involved in the parasite redox metabolic pathways.

Figure 1. Neolignans veraguensin (1) and grandisin (2), the synthetic derivative 3 previously

described and target simplified structures of neolignan-based diaryl-tetrahydrofuran and -

furan analogues (4, 5).

2. Results and Discussion

2.1 Chemistry

As shown in scheme 1, the condensation of the dianion of racemic 1-phenylprop-2-yn-

1-ol (6) with a diverse set of commercially available aryl aldehydes 7a-g bearing methoxy

groups at different positions of the aromatic ring afforded key intermediates 1,4-diphenyl-2-

butyn-1,4-diol (8a-g) in good to moderate yields (30-82%) (Scheme 1) [33,37]. Several attempts

to improve the yields led to the protection of the hydroxyl group of precursor carbinol 6 to

prevent the dianion formation under basic conditions. Typically, the treatment of 6 with tert-

butyldimethylsilyl chloride promoted the protection of both hydroxyl and acetylenic groups,

whereas dihydropyran (DHP) under acid conditions [38] gave the mono-O-protected

intermediate in 45% yield. Despite the low to moderate yields obtained during the

condensation reaction between the O-DHP carbinol derivative 6 with a panel of aldehydes 7a-

g, the removal of the O-DHP protecting group using several protocols (p-TsOH, Dowex®50WX4-

50, trifluoro-acetic and acetic acids and 1M HCl) [39-42], proved to be difficult. Therefore, we

performed the condensation reactions with unprotected carbinol 6 for formation of the

1

diastereoisomeric mixtures of 8a-g, which were assessed by H NMR analysis with two

characteristic singlets or doublets around 5.50 ppm, related to the hydrogen attached to the

carbon-linked hydroxyl group (except for the symmetrical intermediate 8a). Furthermore, ¹³C

NMR revealed clearly the duplicity of signals at 85.0 ppm related to acetylene carbons.

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Subsequent one-pot isomerisation of 1,4-diphenyl-2-butyn-1,4-diol (8a-g)

intermediates with palladium complex Pd₂(dba₃CHCl₃) [tris(dibenzylideneacetone)dipalladium]

and triphenylphosphine [43] was not convenient to afford the corresponding diaryl-diketone

derivative 9a-g, since the yields were very low (6%) and time-consuming (70 h). On the other

hand, an appropriate two-step procedure, involving the alkyne reduction of 8a-g intermediates

followed by hydroxyl group oxidation, was accomplished to produce both 1,4-diphenylbutane-

1,4-diol derivatives 10a-g and 1,4-diphenylbutane-1,4-dione 9a-g intermediates, crucial for the

generation of target diaryl-tetrahydrofurans 4a-g and -furans 5a-g, respectively. As a result,

alkyne reduction with platinum oxide at 10 atm [44] proved suitable for formation of 10a in

92% yield, whilst hydroxyl oxidation with IBX (2-iodoxybenzoic acid) [45] led to the diaryl-

diketone 9a formation in 98% yield, superior to the reaction initially performed with PCC

(pyridinium chlorochromate, 49%) [46]. Therefore, the conditions established for sequential

conversion of 8a into compounds 10a and 9a were successfully applied to produce the

remaining set of compounds.

Scheme 1. Synthesis of 2,5-diaryl-tetrahydrofuran 4a-g and -furan 5a-g derivatives, comprising

methoxyl substituents at different positions of the aromatic ring, starting from the

condensation reaction of the racemic 1-phenylprop-2-yn-1-ol (6) with a diverse set of

commercially available aryl aldehydes 7a-g.

Owing to their significant biological activities [47] and natural occurrence [29,30],

tetrahydrofuran syntheses have been addressed by a wide variety of approaches, the

intramolecular S_N2 reaction between a hydroxyl group and a leaving group being a convenient

and practical strategy to afford functionalised cyclic ethers [48]. Therefore, a straightforward

synthesis of a mixture of an equal amount of cis- and trans-2,5-diphenyl-tetrahydrofurans 4a-g

was achieved by cyclisation/dehydration reaction of the diaryl-dihydroxyl derivatives 10a-g,

according to a well-established protocol using trifluoroacetic acid in chloroform [49]. Apart

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from the known compounds 4a [50-55], 4d [56-57] and 4g [51,58-60], obtained by different

procedures, the novel diastereomers were isolated by column chromatography as a single spot

in moderate yield (30–65%) and assigned based on ¹H NMR spectra, which showed

characteristic overlapping signals at 1.80–2.50 and 4.80–5.20 ppm related, respectively, to H-

3/H-4 and H-2/H-5 tetrahydrofuran stereoisomers. Additionally, the ¹³C NMR also revealed the

tetrahydrofuran methylene and methine carbons at 35.0 and 80.0 ppm.

Despite the availability of several metal-catalysed furan procedures [61], the synthesis

of 2,5-diphenylfurans 5a-g was achieved by microwave-assisted Pal-Knorr classical reaction[62]

via cyclisation of 1,4-diphenylbutane-1,4-dione intermediates 9a-g using triflic acid in 20 min,

rather than 68 h previously described [63]. The target furan structures 5c-e were confirmed by

¹H and ¹³C NMR, which showed the signals of the furan ring at 6.30–6.80 ppm and 104.0–

107.0/150.0 ppm, respectively, and the NMR spectra of the known compounds 5a [64,65], 5b

[66], 5f [65] and 5g [66] were in accordance with the literature.

After preparing 2,5-diaryl-tetrahydrofuran 4a-g and -furan derivatives 5a-g, we next

evaluated their anti-trypanosomal activity on trypomastigote forms of Tulahuen using

colorimetric assay as a preliminary screening [67]. Thereafter, we assessed the anti-

trypanosomal activity of the most potent compounds against the infective Y parasite strain,

besides their ability to penetrate the host LLC-MK2 cells and kill the intracellular and

replicative T. cruzi amastigote form [68,69]. The natural compounds veraguensin (1) and

grandisin (2) and the drug benznidazole, currently used to treat Chagas disease, were also

assessed for comparative analysis.

2.2 Anti-trypanosomal activity and cytotoxicity

Trypomastigote forms of T. cruzi Tulahuen strain incubated with monkey kidney cells

were used to measure parasite viability in the presence of the synthesised compounds

(concentrations ranging from 3.75 to 500 µM). Compound concentrations corresponding to

50% anti-trypanosomal activity were calculated based on dose response curves and expressed

as EC₅₀. Furthermore, cytotoxicity assays were performed in mice spleen cells and the

synthesised compounds were tested at concentrations from 1.50 to 250 µM, using Tween 80

as cell death control. The anti-trypanosomal and cytotoxic activities were compiled in Table 1,

besides the selectivity index (SI).

According to Table 1, in the 2,5-diaryl tetrahydrofuran 4a-g series, presenting closer

similarity to veraguensin (1) and grandisin (2), remarkable anti-trypanosomal activity on

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Tulahuen strain and selectivity was achieved by derivative 4e (2,4-OMe) with EC₅₀0.75 μM,

about twice as high as the activity showed by benznidazole (EC₅₀1.65 μM). The activity of 4e,

up to 50-fold more potent than the corresponding unsubstituted 4a (EC₅₀37.31 µM), revealed

a favourable effect of the 2,4-dimethoxy substitution pattern on the aromatic ring in improving

anti-trypanosomal activity. The efficacy also displayed by compound 4f (3-OMe) (EC₅₀2.29 μM)

and the ability of this series to not damage host cells make compounds 4e and 4f very

promising since they fulfill the TDR criteria for antichagasic agents, based on EC₅₀of <4.0 μM

and SI of ≥50. Moreover, the remaining methoxylated derivatives were deemed moderately

active (EC₅₀between 26.21 and 9.07 µM). Interestingly, despite the common 3,4-OMe

substituents that resemble veraguensin (1, EC₅₀2.67 µM), compound 4d displayed only

moderate efficacy against the trypomastigote parasite (EC₅₀24.63 µM), being one of the less

favourable modifications introduced in the tetrahydrofuran series, along with the

unsubstituted counterpart 4a (EC₅₀37.31 µM). A similar effect was achieved for grandisin (2,

EC₅₀28.59 µM) when compared to the corresponding derivative 4b bearing the 3,4,5-OMe

group, with closer parasite inhibition (EC₅₀26.21 µM).

Based on the promising results obtained with derivatives 4e and 4f against Tulahuen

strains, we also investigated their activities against the more infective T. cruzi Y strain and

found compound 4e preserves high anti-trypanosomal activity (EC₅₀1.05 µM) while 4f

exhibited only EC₅₀34.22 µM. Therefore, the maintenance of a single methoxylated phenyl

ring in the set of compounds 4, rather than two methoxylated phenyl rings, besides the

absence of the 3,4-dimethyl tetrahydrofuran substituents as observed in natural products 1

and 2, has proved to be an interesting simplification strategy applied to this class of

neolignans.

The influence of the number and position of the methoxyl group on the aromatic ring

was also evident in the 2,5-diaryl-furan series 5a-g. Among them, the 3,5-OMe derivative 5c

showed the strongest anti-trypanosomal activity of the current study, with EC₅₀0.01 µM, 75-

fold better than the corresponding compound 4e (2,4-OMe), the most potent in the

tetrahydrofuran series (EC₅₀0.75 μM), and more than a 150-fold better than benznidazole

(EC₅₀1.65 µM); Table 1. Besides the nanomolar activity, the cytotoxicity of 5c (CC₅₀) was not

detectable until 250 µM, resulting in a highly selective agent (SI>250) for further optimisation

and development. In addition, the significant inhibitory effect on the parasite growth displayed

by compounds 5d (3,4-OMe) and 5e (2,4-OMe), with EC₅₀values of 1.34 and 1.12 µM,

respectively, also revealed the importance of the dimethoxylated substitution pattern on the

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aromatic ring in the 2,5-diaryl-furan series. On the other hand, the trimethoxylated 5b (29.10

µM) and unsubstituted counterparts 5a (21 µM) of this 2,5-diaryl-furan series led to decrease

in inhibitory activity, being less active than the monomethoxylated products 5f (3.34 µM) and

5g (4.75 µM), bearing monomethoxylated groups at the 3 and 4 positions, respectively. The

tests carried out with the more active diarylfurans 5c-g against T. cruzi Y strain confirm the

better profile of compound 5c (EC₅₀4.07 µM) compared to 5d-g (19.16-35.87 µM), despite the

lower than nanomolar range found with Tulahuen strain (0.01 µM) and reference benznidazole

(0.04 µM) against the same Y strain.

In order to investigate the impact of the tetrahydrofuran/furan central cores on anti-

trypanosomal activities, we also evaluated the anti-trypanosomal and cytotoxic activities of

the precursors related to rigid compounds (8a-g, 9a-g) and flexible (10a-g) open chain

intermediates, having different connection pattern between the substituted and unsubstituted

aromatic rings that can adopt different spatial orientations (Table 1). It was found that among

1,4-diaryl-2-butyn-1,4-diol precursors (8a-g) bearing a rigid alkynyl link, derivatives having

close methoxyl aryl substitution (3,4,5-OMe, 8b; 3,5-OMe 8c and 3,4-OMe, 8d) to grandisin (2)

and veraguensin (1), just in one ring, showed significant activities of 4.88, 4.98 and 8.98 µM,

respectively, whereas others were moderately active (39.11-13.97 µM). This set of compounds

was not cytotoxic in the tested concentrations up to 250 μM. Compared to the distinguished

activities displayed by some rigid 1,4-diaryl-2-butyn-1,4-diol precursors (8b-d), the anti-

trypanosomal activity induced by the corresponding reduced 1,4-diarylbutane-1,4-diol set

(10a-g), containing a flexible central chain linking both aromatic rings, was significantly lower

(19.88 to 193.10 µM). In addition to the decreased activities displayed by 10b (3,4,5-OMe) and

10e (2,4-OMe) derivatives, it was also accompanied by increased toxicity. Oxidation of these

intermediates (10a-g) rendered the constrained 1,4-diarylbutane-1,4-diketone analogues 9a-g,

which highlights 9c (3,5-OMe) and 9e (2,4-OMe), with EC₅₀1.69/15.91 and 7.79/1.76 µM for

Tulahuen/Y strains, respectively, as the most active of this series, despite lower activities than

those observed for the same methoxylated pattern required for higher activity in the furan (5c)

and tetrahydrofuran (4e) series. Therefore, taking into account the highest potency exhibited

by series 4, 5, 8 and 9, compared to the 1,4-diarylbutane-1,4-diol 10, it should be inferred that

a preferential constrained central core is essential for anti-trypanosomal activity, since in the

1,4-diketones series a specific rigid spatial arrangement might be adopted owing to the

conjugated dienol tautomer formation. Besides, this diketone series does not display

cytotoxicity at concentrations lower than 250 µM.

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Table 1. Anti-trypanosomal activities of novel diaryl-tetrahydrofuran (4a-g) and -furan (5a-g)

derivatives of natural products veraguensin (1) and grandisin (2) and the corresponding

precursors, 8-10(a-g), against T. cruzi Trypomastigote Tulahuen strain, and the most active

compounds against Y strain.

Trypoma Amastigo

Trypomastigote Cytotox

stigote

form of

Y strain

EC₅₀(µM)

te form

of

Tulahuen

strain

SI

CC₅₀/

EC₅₀)

form of

Tulahuen

strain

icity

CC₅₀

(µM)

Compound

EC₅₀(µM)

R₁

R₂

H

R₃

H

R₄

H

4a

H

37.31±24.1

26.21±7.3

12.09±1.9

24.63±0.3

0.75±0.6

NC

-

4b OMe OMe OMe

H

-

4c

OMe

H

OMe

H

4d OMe OMe

H

NC

-

1.05±

0.05

19.77±

0.3

H

OMe

H

OMe

4e

4f

34.22±

0.1

125.7±

2.0

NC

-

OMe

H

2.29±1.6

9.07±3.7

4g

OMe

-

193.60

±62.3

9.2

-

5a

5b

5c

5d

5e

5f

H

21.00±2.4

29.10±5.1

0.01±0.007

1.34±0.9

-

OMe OMe OMe

NC

-

4.08±

0.3

32.63±

0.6

35.87±

2.6

34.22± 125.60±

0.6 0.2

133.10±

4.0

125.8±

3.2

19.00±

2.7

OMe

H

OMe

H

-

OMe OMe

H

-

H

OMe

H

OMe

H

1.12±0.2

OMe

3.34±2.6

9

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19.16± 127.00±

-

5g

H

OMe

H

4.75±1.4

NC

0.6

4.5

8a

8b

8c

8d

8e

8f

H

18.67 ± 2.6

4.88 ± 1.5

NC

-

97.11±

5.3

OMe OMe OMe

NC

78.6

±6.4

OMe

H

OMe

H

4.98 ±1.5

8.98 ± 4.5

15.8

-

NC

OMe OMe

-

H

OMe

H

OMe 37.87 ± 0.4

OMe

H

39.11 ±6.4

13.97±2.7

NC

-

8g

OMe

198.60±

12.1

219.90

±12.7

9a

1.10

H

-

OMe OMe OMe

H

-

9b

9c

9d

9e

9f

36.87±3.3

1.69±0.05

35.96±5.7

7.79±0.8

NC

-

15.91±

3.9

OMe

H

OMe

OMe OMe

H

OMe

H

-

1.76±

1.9

H

OMe

H

OMe

H

18.31±0.02

15.87±7.2

-

9g

OMe

H

10

ACCEPTED MANUSCRIPT

165.90±

22.2

115.20

±38.20

5.0

1.5

-

10a

H

33.16±4.31

77.50±7.9

-

10b OMe

10c OMe

OMe OMe

H

OMe

H

67.62±0.8

NC

83.12±11.7

-

OMe

10d

236.40±

51.9

19.88±6.0

11.9

10e

10f

10g

H

OMe

H

OMe

H

OMe

H

-

193.10±8.8

68.07±6.7

NC

-

OMe

H

NC: Not Cytotoxicity until 250 µM; EC₅₀: Effective Concentration of compounds causing 50% parasite

death, being veraguensin (1): EC₅₀2.67± 0.5 µM, grandisin (2): EC₅₀28.59± 0.4 µM and benznidazole:

EC₅₀1.65± 0.3 µM, using trypomastigote T. cruzi Tulahuen strain. CC₅₀: Cytotoxic Concentration of

compounds causing death to 50% of viable spleen cells (isolated from C57BL/6 mice). SI: Selective Index:

relation between CC₅₀(µM) (Cytotoxic Concentration of compounds) and EC₅₀(µM) (Effective

Concentration of compounds against Trypomastigote form of Tulahuen Strain).

To find out whether hit compounds (4e, 4f, 5c-g) might affect not only trypomastigote

forms, but also the replicative T. cruzi amastigote form located inside the host cell, we

performed additional assays using infected LLC-MK2 cells. As a result, the 2,4-dimethoxy-

diaryltetrahydrofuran 4e and 2,4-dimethoxy-diarylfuran 5e were the most potent of the

selected compounds when compared to the benznidazole drug (EC₅₀9.78 µM), with similar

EC₅₀values of 19.77 and 19 µM, respectively. These findings are in agreement with the

activities established in the tetrahydrofuran series against trypomastigote parasite, in which

compound 4e was also the most active. Conversely, the most active furan derivative 5c against

T. cruzi trypomastigote displayed only weak activity against the amastigote (EC₅₀133.1 µM),

about 13-fold lower than benznidazole, while the most potent 5e, identified in the amastigote

assay, was the second most potent against T. cruzi trypomastigote tests (Figure 2).

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100

80

60

40

20

0

100

5c

5d

5e

5f

4e

80

4f

8b

60

9c

5g

BZ

40

20

0

9e

BZ

0

1

2

3

0

1

2

3

log[C]

Figure 2. Biological evaluation of the most active compounds identified in the trypomastigote

assays towards amastigote form of T. cruzi, Tulahuen strain.

2.3 2D-QSAR studies

Five molecular descriptors were selected by OPS-PLS methodology[70] and the model

(1) was obtained using 29 compounds with no outliers. Based on three mutually orthogonal

variables with 63.445% of information (LV1: 34.497%; LV2: 14.066%; LV3: 14.882%), obtained

from these descriptors, partial least squares (PLS) was conveniently applied to the model [71].

The obtained and predicted values of each compound are reported in the Supplementary

Material (Table S1).

pEC_50ꢀ=ꢀ16.937ꢀ−ꢀ6.916(SpMin3_Bh(e))ꢀ−ꢀ0.878(VE1_B(s))ꢀ+ꢀ61.221(JGI6)ꢀ−ꢀ4.261(MATS3m)ꢀ+ꢀ0.1

52(CATS2D_05_AL) (1)

nꢀ=ꢀ29; R²ꢀ= ꢀ0.679; RMSECꢀ= 0.354; Fꢀ= 17.627; Q²_LOOꢀ= 0.507; RMSECVꢀ= 0.439.

The internal validation shows that the model explains and predicts some information,

and fulfills the requirements of literature (R²> 0.6; Q²_LOO> 0.5), and shows, by the result of the

F test (F_critical= 2.991, pꢀ= 3, and nꢀ–ꢀpꢀ–ꢀ1ꢀ= 25, αꢀ= 0.05), that the model is reliable and

significant. Moreover, the difference between R²and Q²(0.172) [72] suggests the model

LOO

does not undergo data over fitting. The model was also approved in the internal quality

robustness tests (LNO cross-validation) and chance correlation y-randomisation (Figure 3) [72-

74].

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Figure 3. Plots of LNO cross-validation (A) and y-randomisation tests (B).

Regarding the external validation, it indicates that Model 1 is realistic and applicable

for prediction purposes. Despite the lower correlation in the cross-validation process, the

external validation of the model showed a good external predictive power, with R²_predꢀ= 0.848,

2

RMSEPꢀ= 0.190, average r_m(LOO)-scaledꢀ= 0.753, Δr_m(LOO)-scaledꢀ= 0.062, |R₀-R'₀|= 0.004,

kꢀ= 0.965 and k'ꢀ= 1.036 [34,75]. Besides the better values of R²_predin comparison with Q²_LOO, it

is important to highlight that there is no relationship between internal and external

predictability (i.e., a model with low internal predictability may present high external

predictability, and vice versa); a paradox previously described by Kubinyi [76,77]. The results of

Golbraikh and Tropsha statistics and the RmSquare metrics also confirm the good external

predictability of the model [34,72].

All five selected descriptors are topological descriptors, which encode molecular

information such as size, shape, symmetry, branching, and rigidity, besides atom type and

bond multiplicity [78]. The order of importance of each descriptor is based on absolute values

of their autoscaled coefficients, (|0.647|ꢀxꢀSpMin3_Bh(e) > |0.398|ꢀxꢀVE1_B(s) > |0.369|ꢀxꢀJGI6

> |0.364|ꢀxꢀCATS2D_05_AL > |0.264|ꢀxꢀMATS3m). The meaning of each descriptor and their

corresponding values are shown in Tables S2 and S3, respectively, in the Supplementary

Material.

It is worth noting that the two most important descriptors, (SpMin3_Bh(e) and

VE1_B(s)) are related to molecular electronegativity and intrinsic electropological state [79].

Both may influence the anti-trypanosomal activity according to the position of the methoxyl

group on the aromatic ring, even in those with the same number of methoxyl groups, such as

compounds 8d (3,4-OMe) and 8e (2,4-OMe), 10d (3,4-OMe) and 10e (2,4-OMe). Taking into

account that SpMin3_Bh (e) is obtained based on Burden matrices (B(w)), where the diagonal

elements are atomic carbon-scaled properties (w_i/w_C),[79] a large number of electronegative

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atoms in the molecule may lead to a decrease of the descriptor value in the model since

trimethoxylated derivatives showed lower activity than the remaining products, with the

exception of 8b (3,4,5-OMe). Alternatively, the electronic effect (σ) of the methoxyl group,

with electron-donating effect at the para-position and electron-withdrawing at the meta-

position[80] may contribute to the high and moderate activities of compounds 4f (3-OMe), 5d

(3,4-OMe), 5f (3-OMe) e 9c (3,5-OMe), along with the most active of the series 5c (3,5-OMe),

containing two methoxyl groups at the meta-position, indicating that the decreased electron

density on the ring is actually relevant for the biological activity, but to a certain extent, which

can be evidenced by the negative sign of the descriptor SpMin3_Bh(e). Similarly, VE1_B(s) is

also obtained from Burden matrix, with close interpretation, and from the sum of the number

of vertices (i.e., atoms) present in an H-depleted molecular graph [79]. This indicates that the

more structures are branched (i.e., the larger the number of methoxyl groups) the greater the

tendency to be less active.

It was also evident that the influence of the number of methoxyl groups on the activity

is not enough to explain the entire model. The position of the substituent also plays an

important role due to the combination of electronic effects on the aromatic ring, as described

by the third descriptor (JGI6), which encodes the total charge transfer between atoms placed

at topological distance 6 (aromatic ring topology). Furthermore, the fourth descriptor

(CATS2D_05_AL) comprises topological distances formed by five bonds (lags) between

hydrogen bond acceptors and lipophilic chemical groups (i.e., two main characteristics of the

methoxyl group). The positive signal of this descriptor in model 1 means the more this

topological feature is present, the greater the tendency of the molecule to belong to the group

of the most active derivatives. Amongst the most active products, it is important to highlight

the prevalence of a methoxyl substitution pattern of R₁and R₃, such as in compounds 9c and

5c, or R₂and R₄, such as in9e, 4e, 5e and 10e, which is in agreement with the biological results.

Finally, the descriptor (MATS3m) involving atomic mass can be considered the least

important. An increase of the absolute value of this descriptor tends to increase the activity of

derivatives, indicating the tendency of the higher molecular weight compounds to be slightly

more active. In fact, unsubstituted and monosubstituted compounds (9a, 5a, 10a, 4a, 8f, 10g,

10f) represent 41% of the less active compounds (pEC₅₀< 4.729), while the most active (4e, 4f,

5f, 5g, 9f) make up 29% (pEC₅₀> 4.729).

2.4 Trypanothione reductase activity

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Although the phenotypic screening of diaryltetrahydrofuran and diarylfuran against the

trypomastigote and amastigote forms of T. cruzi (Tulahuen and Y strains) revealed potent

inhibitors, the identification of the corresponding molecular target are still essential for

subsequent lead optimization process and drug development [12].

Thus, the role played by furan [31] and nitrofuran derivatives, in particular, the redox-

cycling drugs nitrofurazone and nifurtimox [81,82] and the subversive substrates related to

guanidine-containing nitro-furans (under aerobic conditions) [83,84], on inhibition of T. cruzi

Trypanothione reductase (TR) motivated us to investigate the effect of the most active anti-

trypanosomal compounds from our series (IC₅₀< 12 μM) on this target, taking into account the

unique metabolic pathway and anti-oxidant functions of TR that provides a cellular reducing

environment to protect the parasite against oxidative stress [85].

(A)

(B)

Trypanothione reductase assay

5d

5f

100

50

0

30

20

10

0

5e

5g

Clo

Concentration (µM)

Figure 4. (A) Inhibition of trypanothione reductase by compounds 5d-g and comparison of their

IC₅₀values with clomipramine (Clo) (B) Corresponding curves of inhibition.

The assessment of TR activity was conducted using a continuous colorimetric

microplate assay (time-dependent TR inhibition of 30 min) based on the capacity of the

enzyme to reduce the trypanothione disulphide substrate (TS₂) to the di-thiol product T(SH)₂

in the presence of the NAPH cofactor. After incubation, the newly formed T(SH)₂is re-oxidized

back to the original substrate (TS₂) by the addition of 5,5'-dithiobis-(2-nitrobenzoic acid)

(DTNB), which releases the chromophore 2-nitro-5-mercaptobenzoic acid (TNB), detected at

412 nm [86]. Thus, the assessment of the prospective diarylfurans and diaryl-tetrahydrofurans

TR inhibitors (50,0 μM concentration) revealed the diarylfuran series as the most active

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inhibitors, highlighting compounds 5d-e (IC₅₀19.2 and 17.7 μM) and 5f-g (IC₅₀8.9 and 7.4 μM),

respectively, with similar or 2-fold higher than clomipramine (IC₅₀15.2 μM) (Figure 4). It was

evident that a monomethoxylated pattern of the diarylfuran core (at either -para or -meta

position) had a significant impact on the TR inhibition.

Unexpectedly, compound 5c that displayed the strongest activity against Tulahuen and

Y strains of T. cruzi proved to be inactive, suggesting that its mechanism of action does not

involve TR activity inhibition. Furthermore, similar results were found for the tetra-hydrofuran

derivatives that showed no TR inhibition at tested concentrations and additional studies are

necessary to elucidate their mechanism of action.

3. Conclusions

Based on natural neolignan products, a series of fourteen novel diaryl-tetrahydrofuran

(4a-g) and -furan derivatives (5a-g) were synthesised as a simplified approach for the

development of more potent anti-trypanosomal agents. The cyclisation/dehydration

intramolecular S_N2 reaction between a hydroxyl group and a leaving group was pursued to

produce functionalised cyclic ethers as a mixture of equal amounts of cis- and trans-2,5-

diphenyl-tetrahydrofurans 4a-g. In addition, microwave-assisted acceleration of the Pal-Knorr

classical reaction allowed the preparation of 2,5-diphenylfurans 5a-g in good to moderate

yields.

Preliminary biological results suggested that derivatives with aryl-methoxy groups

linked by a rigid central core, as observed in series 4, 5, 8 and 9, may adopt a favourable

orientation for target interactions in order to contribute with a beneficial entropic effect,

which is not achieved by similar aryl methoxyl groups connected by a flexible link (series 10).

Based on the significant impact of the number and variable positions of the aryl methoxyl

groups on the anti-trypanosomal activities, it should be inferred that the presence of a

dimethoxy substituent pattern in all series had a stronger anti-trypanosomal effect than the

corresponding monomethoxylated ones, which in turn, were better inhibitors than the

trimethoxylated (with exception of compound 8b) and unsubstituted counterparts.

2D-QSAR studies reinforce that the electronic effect of the methoxyl groups and their

corresponding substitution pattern (number and position) on the aromatic ring have a

significant influence on the overall electronic distribution in the final structure, affecting anti-

trypanosomal activities. These findings open up the possibility of further chemical

modifications to obtain more active derivatives, keeping, for instance, methoxyl group at R₁

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and varying R₂with similar molecular weight and electron donating groups, such as fluoro,

cyano, trifluoromethyl and chlorine groups.

The identification of the most potent anti-trypanosomal non-chiral derivative in the

diarylfuran series (5c) suggests that the configuration of the 2,5-tetrahydrofuran carbons in

series 4 or even in the corresponding carbons of the rigid alkynyl open chain 8 are not crucial

for parasite growth inhibition. Additionally, the simplified approach that maintained a single

methoxylated aromatic ring rather than two, besides the elimination of two stereogenic

centres (3,4-dimethyl groups of this class of neolignans natural products 1 and 2) afforded

more active derivatives against both Tulahuen and Y strains, along with strong effects on

trypomastigote and amastigote forms of T. cruzi.

Finally, the assessment of trypanothione reductase inhibition using the most active

anti-trypanosomal derivatives, highlight the potential of some diarylfurans to inhibit parasite

growth by targeting its defense mechanism against oxidative stress through TR inhibition. In

summary, this study demonstrates the relevance of simplified neolignan-based derivatives to

act as stronger anti-trypanosomal agents than the parent natural product and, some of them,

as trypanothione reductase inhibitors.

4. Experimental

4.1. General information

All chemicals were purchased as reagent grade and used without further purification and

solvents were dried according to standard procedures [87]. Reactions were monitored by thin

layer chromatography (TLC) on pre-coated silica gel aluminum plates (60 GF254, Merck) with

the indicated eluents. Compounds were visualized under UV light (254 nm) and/or dipping in

ethanol–sulfuric acid (95:5, v/v), followed by heating the plate for a few minutes. Column

chromatography was performed on silica gel 60 (20–63 µm) or on a Biotage Horizon High-

Performance FLASH Chromatography system using 12 mm or 25 mm flash cartridges with the

1

indicated eluents. H and ¹³C Nuclear magnetic resonance spectra were recorded on Bruker

Advance DRX 300 (300 MHz), DPX 400 (400 MHz) or DPX 500 (500 MHz) spectrometers and

chemical shifts (δ) were expressed in parts per million (ppm), using tetramethylsilane (TMS) as

internal standard. Accurate mass electrospray ionization mass spectra (ESI-HRMS) were

obtained using positive or negative ionization modes on a Bruker Daltonics MicroOTOF II ESI-

TOF mass spectrometer.

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4.2 Synthesis

4.2.1 General procedure for synthesis of derivatives 2,5-diphenyl-tetrahydrofuran, 4a-g.

A solution of 1,4-diphenylbutane-1,4-diol derivatives (10a-g) in CHCl₃was cooled at 0 ºC and

trifluoracetic acid solution (5 eq) in CHCl₃was added drop wise and stirred for 2 h. After that,

the temperature was raised to ambient temperature for complete consumption of starting

material. The reaction mixture was quenched into saturated aqueous NaOH 10% solution and

extracted with dichloromethane. The organic layer was dried over anhydrous MgSO₄. The

solvent was removed under reduced pressure and the mixture was purified by flash column

chromatography (silica gel), hexane/ethyl acetate (9:1 v/v).

2,5-diphenyl-tetrahydrofuran (4a)[50-55]

Following procedure described in section 4.2.4, the reaction of 1,4-diphenylbutane-1,4-diol

(10a) (20.3 mg, 0.085 mmol) in CHCl₃(2.0 mL) with TFA (32 µL, 0.425 mmol) in CHCl₃(1.0 mL)

gave the product 4a as a yellow oil, (8.06 mg, 0.036 mmol, 42%). ¹H NMR (400 MHz, CDCl₃): δ:

1.79-1.97 (4H, m, H-8, H-9); 4.68-4.79 (2H, m, H-7, H-10); 7.24-7.30 (2H, m, H-4); 7.30-7.39 (8H,

m, H-2, H-3, H-5, H-6). ¹³C NMR (100 MHz, CDCl₃): δ: 35.2, 35.9 (C-8, C-9); 74.3, 74.7 (C-7, C-

10); 125.8 (C-2, C-6); 127.5, (C-4); 128.5 (C-3, C-5); 144.5, 144.6 (C-1, C-1’). ESI-HRMS: calcd for

C₁₆H₁₇O [M⁺H]⁺225.1279; found 225.1242.

2-phenyl-5-(3,4,5-trimethoxyphenyl)tetrahydrofuran (4b)

Following procedure described in section 4.2.4, the reaction of 1-phenyl-4-(3,4,5-

trimethoxyphenyl)butane-1,4-diol (10b) (25.2 mg, 0.076 mmol) in CHCl₃(2.0 mL) with TFA

(29.1 µL, 0.380 mmol) in CHCl₃(1.0 mL) gave the product 4b as a yellow oil, (12.25 mg, 0.039

1

mmol, 52%). H NMR (400 MHz, CDCl₃): δ: 1.95-2.08 (2H, m, H-8a, H-9a); 2.40-2.54 (2H, m, H-

8b, H-9b); 3.84 (6H, s, OCH₃); 3.88 (3H, s, OCH₃); 5.00-5.12 (1H, m, H-7 or H-10); 5.17-5.30 (1H,

m, H-7 or H-10); 6.65 (2H, d, J 3.1 Hz, H-2’, H-6’); 7.26-7.31 (1H, m, H-4); 7.34-7.48 (4H, m, H-2,

H-3, H-5, H-6). ). ¹³C NMR (100 MHz, CDCl₃) ): δ:35.5 (C-8,C-9), 56.0 (C11, C13), 60.8 (C12), 81.3

(C7-C10), 102.6 (C1´, C5´), 124.9 (C1-C5), 127.2 (C3), 127.4 (C2-C4), 137.2 (C3´), 139.3 (C6´),

143.6 (C6), 153,3 (C2´, C4´). ESI-HRMS: calcd for C₂₁H₂₅NaO₄[M⁺Na]⁺337.1416; found

337.1412.

2-(3,5-dimethoxyphenyl)-5-phenyltetrahydrofuran (4c)

18

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Following procedure described in section 4.2.4, the reaction of 1-(3,5-dimethoxyphenyl)-4-

phenylbutane-1,4-diol (10c) (20.7 mg, 0.068 mmol) in CHCl₃(2.0 mL) with TFA (26.5 µL, 0.342

1

mmol) in CHCl₃(1.0 mL) gave the product 4c as a yellow oil, (5.7 mg, 0.020 mmol, 30%). H

NMR (400 MHz, CDCl₃): δ: 1.85-1.97 (2H, m, H-8a, H-9a); 2.29-2.42 (2H, m, H-8b, H-9b); 3.71

(3H, s, OCH₃); 3.73 (3H, s, OCH₃); 4.91-5.02 (1H, m, H-7 or H-10); 5.11-5.22 (1H, m, H-7 or H-

10); 6.30 (1H, q, J 2.3 Hz, H-4’); 6.51 (1H, d, J 2.3 Hz, H-2’ or H5’); 6.53 (1H, d, J 2.3 Hz, H-2’ or

H5’); 7.21-7.40 (5H, m, H-2, H-3, H-4, H-5, H-6). ¹³C NMR (100 MHz, CDCl₃): δ: 34.0, 34.3, 35.4

(C-8, C-9); 55.3 (OCH₃); 81.1, 81.2, 81.3, 81.4 (C-7, C-10); 99.1 (C-4’); 103.3, 103.9 ( C-2’, C-6’);

125.6, 126.0 (C-2, C-6); 127.2, 127.3 (C-4); 128.3, 128.4 (C-3, C-5); 145.7 (C-1, C-1’); 160.8 (C-3’,

C-5’). ESI-HRMS: calcd for C₁₈H₂₁O₃[M⁺H]⁺285.1491; found 285.1487.

2-(3,4-dimethoxyphenyl)-5-phenyltetrahydrofuran (4d)[56,57]

Following procedure described in Section 4.2.4, the reaction of 1-(3,4-dimethoxyphenyl)-4-

phenylbutane-1,4-diol (10d) (30.8 mg, 0.102 mmol) in CHCl₃(3.0 mL) with TFA (39.0 µL, 0.510

1

mmol) in CHCl₃(1.0 mL) gave the product 4d as a yellow oil, (18.75 mg, 0.066 mmol, 65%). H

NMR (300 MHz, CDCl₃): δ: 1.96-2.06 (2H, m, H-8a, H-9a); 2.40-2.51 (2H, m, H-8b, H-9b); 3.84

(3H, s, OCH₃); 3.88 (3H, s, OCH₃); 4.99-5.11 (1H, m, H-7 or H-10); 5.24 (1H, dt, J 6.8 Hz, J 7.1 Hz,

H-7 or H-10); 6.65 (2H, d, J 3.1 Hz, H-2’, H4’); 7.26-7.32 (1H, m, H-6’); 7.32-7.45 (5H, m, H-2, H-

3, H-4, H-5, H-6). ¹³C NMR (75 MHz, CDCl₃): δ: 34.2, 35.5, 35.6 (C-8, C-9); 55.8, 55.9 (OCH₃);

81.1, 81.2 (C-7, C-10); 109.0, 109.5, 111.0 (C-2’, C-5’); 117.8, 118.1 (C-6’); 125.5, 125.9 (C-2, C-

6); 127.1, 127.2 (C-4); 128.3 (C-3, C-5); 136.1, 135.6, 142.5, 143.7 (C-1, C-1’); 148.2, 149.0 (C-3’,

C-4’). ESI-HRMS: calcd for C₁₈H₂₁O₃[M⁺H]⁺285.1491; found 285.1488.

2-(2,4-dimethoxyphenyl)-5-phenyltetrahydrofuran (4e)

Following procedure described in section 4.2.4, the reaction of 1-(2,4-dimethoxyphenyl)-4-

phenylbutane-1,4-diol (10e) (9.8 mg, 0.032 mmol) in CHCl₃(1.0 mL) with TFA (13.0 µL, 0.160

1

mmol) in CHCl₃(1.0 mL) gave the product 4e as a yellow oil, (3.98 mg, 0.014 mmol, 45%). H

NMR (400 MHz, CDCl₃): δ: 1.82-2.00 (2H, m, H-8a, H-9a); 2.34-2.53 (2H, m, H-8b, H-9b); 3.81

(3H, s, OCH₃); 3.82 (3H, s, OCH₃); 5.00 (0.5 H, t, J 7.0 Hz, H-7 or H-10); 5.26 (1H, t, J 6.7 Hz, H-7

or H-10); 5.46 (0.5 H, t, J 6.7 Hz, H-7 or H-10); 6.45-6.53 (2H, m, H-3’, H-5’); 7.27-7.53 (6H, m,

H-2, H-3, H-4, H-5, H-6, H-6’). ¹³C NMR (100 MHz, CDCl₃): δ: 33.1, 33.9, 34.3, 35.5 (C-8, C-9),

55.3, 55.4 (OCH₃), 80.8, 80.9 (C-7, C-10), 98.3, 98.4 (C-3’), 103.8 (C-5’), 124.0, 124.6 (C-1’),

125.6 (C-2, C-6), 126.4, 126.7 (C-6’), 127.0, 127.2 (C-4), 128.3 (C-3, C-5), 143.2, 144.0 (C-1),

19

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157.2, 157.3, 159.8, 159.9 (C-2’, C-4’). ESI-HRMS: calcd for C₁₈H₂₁O₃[M⁺H]⁺285.1491; found

285.1488.

2-(3-methoxyphenyl)-5-phenyltetrahydrofuran (4f)

Following procedure described in section 4.2.4, the reaction of 1-(3-methoxyphenyl)-4-

phenylbutane-1,4-diol (10f) (36.9 mg, 0.135 mmol) in CHCl₃(3.0 mL) with TFA (51.8 µL, 0.677

1

mmol) in CHCl₃(1.0 mL) gave the product 4f as a yellow oil, (13.7 mg, 0.054 mmol, 40%). H

NMR (400 MHz, CDCl₃): δ: 1.86-1.96 (2H, m, H-8a, H-9a); 2.33-2.42 (2H, m, H-8b, H-9b); 3.74

(3H, s, OCH₃); 4.98 (1H, J 3.0 Hz, J, 4.0 Hz, J 7.0 Hz, H-7 or H-10); 5.16-5.21 (1H, m, H-7 or H-10);

6.72-6.77 (1H, m, H-6’); 6.89-6.97 (2H, m, H-2’, H-4’); 7.18-7.23 (2H, m, H-4, H-5’); 7.26-7.40

(4H, m, H-2, H-3, H-5, H-6). ¹³C NMR (100 MHz, CDCl₃): δ: 34.3, 34.4, 35.6 (C-8, C-9); 55.2, 55.3

(OCH₃); 81.1, 81.2, 81.3, 81.4 (C-7, C-10); 111.0, 111.6 (C-2’); 112.6, 112.7 (C-4’); 117.9, 118.3

(C-6’); 125.6, 126.0 (C-2, C-6); 127.2, 127.3 (C-4); 128.4 (C-3, C-5); 129.4 (C-5’); 142.9, 143.6,

144.8,145.5 (C-1, C-1’); 159.7 (C-3’). ESI-HRMS: calcd for C₁₇H₁₈NaO₂[M⁺Na]⁺277.1204; found

277.1236.

2-(4-methoxyphenyl)-5-phenyltetrahydrofuran (4g)[51,58-60]

Following procedure described in Section 4.2.4, the reaction of 1-(4-methoxyphenyl)-4-

phenylbutane-1,4-diol (10g) (42.2 mg, 0.155 mmol) in CHCl₃(3.0 mL) with TFA (59.3 µL, 0.775

1

mmol) in CHCl₃(1.0 mL) gave the product 4g as a yellow oil, (26.43 mg, 0.104 mmol, 67%). H

NMR (400 MHz, CDCl₃): δ: 1.84-1.95 (2H, m, H-8a, H-9a); 2.25-2.42 (2H, m, H-8b, H-9b); 3.72

(3H, s, OCH₃); 4.89-4.97 (1H, m, H-7 or H-10); 5.10-5.19 (1H, m, H-7 or H-10); 6.80-6.84 (2H, m,

H-3’, H-5’); 7.15-7.21 (1H, m, H-4); 7.25-7.37 (6H, m, H-2, H-3, H-5, H-6, H-2’, H-6’). ¹³C NMR

(100 MHz, CDCl₃): δ: 34.3, 34.6, 35.6, 35.7 (C-8, C-9); 55.3 (OCH₃); 81.1, 81.2 (C-7, C-10); 113.8

(C-3’, C-5’); 125.6, 126.0 (C-2, C-6); 127.0, 127.2 (C-4); 127.3, 127.4 (C-2’, C-6’); 128.4 (C-3, C-5);

134.9, 135.6 (C-1’); 143.1, 143.8 (C-1); 158.9, 159.0 (C-4’). ESI-HRMS: calcd for C₁₇H₁₈NaO₂

[M⁺Na]⁺277.1204; found 277.1218.

4.2.2 General procedure for synthesis of derivatives 2,5-diphenylfuran, 5a-g.

A solution of 1,4-diphenylbutane-1,4-dione derivatives (9a-g) in MeCN was treated with triflic

acid (1.1 equiv). The reaction mixture was stirred for 20 min at 80 ºC (sealed microwave tube)

under microwave irradiation (100 W). After that, the reaction was quenched into saturated

NaHCO₃solution. The organic and aqueous phases were separated and the aqueous phase

extracted with EtOAc (3 x 2.0 mL). The organic layer was dried over anhydrous MgSO₄.

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Removal of the solvent under reduced pressure followed by purification by flash column

chromatography (silica gel), hexane/ethyl acetate (9:1 v/v) afforded products (5a-g)

2,5-diphenylfuran (5a)[64,65]

Following procedure described in section 4.2.5, the reaction of 1,4-diphenylbutane-1,4-dione

(9a) (31.5 mg, 0.132 mmol) in MeCN (2.0 mL) with TfOH (12.8 µL, 0.145 mmol) gave the

1

product 5a as a white solid, (21.35 mg, 0.097 mmol, 74%). H NMR (400 MHz, CDCl₃): δ: 6.74

(2H, s, H-8, H-9); 7.27 (2H, ddt, J 1.3 Hz, J 1.9 Hz, J 7.4 Hz, H-4); 7.41 (4H, ddt, J 1.3 Hz, J 1.9 Hz,

J 7.4 Hz, H-3, H-5); 7.75 (4H, ddt, J 1.2 Hz, J 1.9 Hz, J 7.3 Hz, H-2, H-6). ¹³C NMR (100 MHz,

CDCl₃): δ: 107.2 (C-8, C-9); 123.7 (C-2, C-6); 127.3 (C-4); 128.7 (C-3, C-5); 130.8 (C-1); 153.4 (C-

7, C-10). ESI-HRMS: calcd for C₁₆H₁₂KO [M⁺K]⁺259.0525; found 259.0727.

2-phenyl-5-(3,4,5-trimethoxyphenyl)furan (5b)[66]

Following procedure described in section 4.2.5, the reaction of 1-phenyl-4-(3,4,5-

trimethoxyphenyl)butane-1,4-dione (9b) (15.0 mg, 0.046 mmol) in MeCN (1.0 mL) with TfOH

1

(5.0 µL, 0.051 mmol) gave the product 5b as a yellow solid, (4.30 mg, 0.014 mmol, 31%). H

NMR (300 MHz, CDCl₃): δ: 3.89 (3H, s, OCH₃); 3.96 (6H, s, OCH₃); 6.68 (1H, d, J 3.5 Hz, H-8 or H-

9); 6.74 (1H, d, J 3.5 Hz, H-8 or H-9); 6.96 (2H, s, H-2’, H-6’); 7.30 (1H, m, H-4), 7.42 (2H, t_app, J

7.5 Hz, H-3, H-5), 7.74 (2H, m, H-2, H-6). ¹³C NMR (75 MHz, CDCl₃): δ:56.2 (C11, C13), 61.0

(C12), 101.1 (C8, C9), 107.3, 107.0 (C1´, C5´), 123.7 (C1, C5), 126.5 (C3), 127.4 (C6), 128.7 (C2,

C4), 130.7 (C6´),137.7 (C3´),153.2 (C8, C9), 153.6 (C2´, C4´). ESI-HRMS: calcd for C₁₉H₁₉O₄[M⁺H]⁺

311.1283; found 311.1265.

2-(3,5-dimethoxyphenyl)-5-phenylfuran (5c) [65]

Following procedure described in section 4.2.5, the reaction of 1-(3,5-dimethoxyphenyl)-4-

phenylbutane-1,4-dione (9c) (27.2 mg, 0.091 mmol) in MeCN (1.5 mL) with TfOH (8.9 µL, 0.100

mmol) gave the product 5c as a yellow solid, (13.16 mg, 0.047 mmol, 52%). ¹H NMR (400 MHz,

CDCl₃): δ: 3.87 (6H, s, OCH₃); 6.41 (1H, t, J 2.3 Hz, H-4’); 6.73 (1H, d, J 3.5 Hz, H-8 or H-9); 6.74

(1H, d, J 3.5 Hz, H-8 or H-9); 6.91 (2H, d, J 2.3 Hz, H-2’, H-6’); 7.28 (1H, dt, J 1.6 Hz, J 7.4 Hz, H-

4); 7.41 (2H, t, J 7.4 Hz, H-3, H-5); 7.74 (2H, dd, J 1.6 Hz, J 7.4 Hz, H-2, H-6). ¹³C NMR (100 MHz,

CDCl₃): δ: 55.4 (OCH₃); 99.6 (C-4’); 102.0 (C-2’, C-6’); 107.2, 107.8 (C-8, C-9); 123.8 (C-2, C-6);

127.4 (C-4); 128.7 (C-3, C-5); 130.7 (C-1); 132.5 (C-1’); 153.1, 153.4 (C-7, C-10); 161.1 (C-3’, C-

5’). ESI-HRMS: calcd for C₁₈H₁₆NaO₃[M⁺Na]⁺303.0997; found 303.0993.

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2-(3,4-dimethoxyphenyl)-5-phenylfuran (5d)

Following procedure described in section 4.2.5, the reaction of 1-(3,4-dimethoxyphenyl)-4-

phenylbutane-1,4-dione (9d) (21.8 mg, 0.073 mmol) in MeCN (1.5 mL) with TfOH (7.1 µL, 0.080

mmol) gave the product 5d as a yellow solid, (15.41 mg, 0.055 mmol, 75%). ¹H NMR (300 MHz,

CDCl₃): δ: 3.85 (3H, s, OCH₃); 3.93 (3H, s, OCH₃); 6.54 (1H, d, J 2.4 Hz, H-2’); 6.60 (1H, dd, J 8.6

Hz, J 2.4 Hz, H-6’); 6.74 (1H, d, J 3.4 Hz, H-8 or H-9); 6.88 (1H, d, J 3.4 Hz, H-8 or H-9); 7.24 (1H,

ddt, J 1.2 Hz, J 1.7 Hz, J 7.4 Hz, H-4); 7.39 (2H, t_app, J 7.4 Hz, H-3, H-5), 7.74 (2H, m, H-2, H-6);

7.89 (1H, d, J 8.6 Hz, H-5’). ¹³C NMR (75 MHz, CDCl₃): δ: 56.0 (OCH₃); 106.0, 107.3 (C-8, C-9);

111.5 (C-2’); 116.7 (C-5’); 123.6 (C-2, C-6, C-6’); 124.1 (C-1’); 127.2 (C-4); 128.7 (C-3, C-5); 130.9

(C-1); 148.8, 149.2 (C-3’, C-4’); 152.9, 153.4 (C-7, C-10). ESI-HRMS: calcd for C₁₈H₁₆NaO₃[M⁺Na]⁺

303.0997; found 303.0999.

2-(2,4-dimethoxyphenyl)-5-phenylfuran (5e)

Following procedure described in section 4.2.5, the reaction of 1-(2,4-dimethoxyphenyl)-4-

phenylbutane-1,4-dione (9e) (22.0 mg, 0.074 mmol) in MeCN (1.5 mL) with TfOH (7.2 µL, 0.081

mmol) gave the product 5e as a yellow solid, (14.40 mg, 0.050 mmol, 68%). ¹H NMR (300 MHz,

CDCl₃): δ: 3.92 (3H, s, OCH₃); 3.98 (3H, s, OCH₃); 6.62 (1H, d, J 3.5 Hz, H-8 or H-9); 6.72 (1H, d, J

3.5 Hz, H-8 or H-9); 6.91 (1H, d, J 8.4 Hz, H-5’), 7.22-7.29 (2H, m, H-4, H-3’), 7.32 (1H, dd, J 8.4

Hz, J 2.0 Hz, H-6’), 7.40 (2H, t_app, J 7.5 Hz, H-3, H-5); 7.73 (2H, dd, J 7.5 Hz, J 1.3 Hz, H-2, H-6).

¹³C NMR (75 MHz, CDCl₃): δ: 55.4 (OCH₃); 98.7 (C-3’); 104.8, 107.3 (C-8, C-9); 110.2 (C-5’); 113.3

(C-1’); 123.6 (C-2, C-6); 126.6 (C-6’); 126.9 (C-4); 128.6 (C-3, C-5); 131.0 (C-1); 150.0; 151.6 (C-7;

C-10); 156.7, 160.0 (C-2’, C-4’). ESI-HRMS: calcd for C₁₈H₁₆NaO₃[M⁺Na]⁺303.0997; found

303.0994.

2-(3-methoxyphenyl)-5-phenylfuran (5f)

Following procedure described in section 4.2.5, the reaction of 1-(3-methoxyphenyl)-4-

phenylbutane-1,4-dione (9f) (26.4 mg, 0.098 mmol) in MeCN (1.5 mL) with TfOH (7.2 µL, 0.081

mmol) gave the product 5f as a yellow solid, (15.75 mg, 0.063 mmol, 64%). ¹H NMR (400 MHz,

CDCl₃): δ: 3.88 (3H, s, OCH₃); 6.74 (2H, s, H-8, H-9); 6.83 (1H, dt, J 2.5 Hz, J 8.4 Hz, H-4’); 7.24-

7.34 (4H, m, H-4, H-2’, H-5’, H-6’); 7.40 (2H, t_app, J 7.3 Hz, H-3, H-5); 7.75 (2H, dt, J 1.3 Hz, J 7.3

Hz, H-2, H-6). ¹³C NMR (100 MHz, CDCl₃): δ: 55.3 (OCH₃); 107.2, 107.6 (C-8, C-9); 109.2 (C-2’);

112.9 (C-4’); 116.4 (C-6’); 123.7 (C-2, C-6); 127.4 (C-4); 128.7 (C-3, C-5); 129.8 (C-5’); 130.7,

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132.0 (C-1, C-1’); 153.1, 153.4 (C-7, C-10); 159.9 (C-3’). ESI-HRMS: calcd for C₁₇H₁₅O₂[M⁺H]⁺

251.1072; found 251.1066.

2-(4-methoxyphenyl)-5-phenylfuran (5g) [66]

Following procedure described in section 4.2.5, the reaction of 1-(4-methoxyphenyl)-4-

phenylbutane-1,4-dione (9g) (24.3 mg, 0.091 mmol) in MeCN (1.5 mL) with TfOH (8.8 µL, 0.100

1

mmol) gave the product 5g as a yellow solid, (12.0 mg, 0.048 mmol, 53%). H NMR (300 MHz,

CDCl₃): δ: 3.84 (3H, s, OCH₃); 6.60 (1H, d, J 3.5 Hz, H-8 or H-9); 6.71 (1H, d, J 3.5 Hz, H-8 or H-9);

6.94 (2H, d, J 8.9 Hz, H-3’, H-5’); 7.25 (1H, ddt, J 1.2 Hz, J 1.9 Hz,J 7.5 Hz, H-4); 7.39 (2H, t_app, J

7.5 Hz, H-3, H-5); 7.68 (2H, dt, J 1.5 Hz, J 8.9 Hz, H-2’, H-6’); 7.73 (2H, m, H-2, H-6). ¹³C NMR (75

MHz, CDCl₃): δ: 55.3 (OCH₃); 105.6, 107.2 (C-8, C-9); 114.2 (C-3’, C-5’); 123.5 (C-2, C-6); 123.9

(C-1’); 125.2 (C-2’, C-6’); 127.1 (C-4); 128.7 (C-3, C-5); 130.9 (C-1); 152.7, 153.4 (C-7, C-10);

159.0 (C-4’). ESI-HRMS: calcd for C₁₇H₁₅O₂[M⁺H]⁺251.1072; found 251.1056.

4.3 Biological evaluation

Trypomastigote assay

To evaluate the anti-trypanosomal activity, cell-derived trypomastigote forms of T. cruzi,

Tulahuen strain stably expressing the β-galactosidase gene from Escherichia coli (Tulahuen-

lacZ), 5×10⁵trypomastigotes, were cultured in 96-well plates and incubated for 4 days at 37°C

with the synthesized compounds (products 4-5a-g and intermediates 8-10a-g), natural

products veraguensin (1) and grandisin (2) and benznidazole drug (Sigma-Aldrich) at serial

concentrations in a range between 500 to 3.75 µM. Thereafter, 50 μL of PBS containing 0.5%

of Triton X-100 and 100 μM chlorophenol Red-β-D-galactoside (CPRG-Sigma) were added.

Plates were incubated at 37°C for 4 h and absorbance was read at 570 nm.[67] Results of

parasite viability were measured based on the catalysis of CPRG by β-galactosidase and the

experiments were performed in duplicates. To test the compounds against T. cruzi Y strain,

trypomastigotes were platted in 96 well flat bottom plate at concentration of 1×10⁶parasites

mL^-1and the more active products on trypomastigote parasite, 4e-f and 5c-g and

intermediates 8b, 9c, were platted in serial concentrations (500 to 3.75 µM). After 24 h, the

parasites were removed and counted in Neubauer chamber as previously described.[68,69]

Amastigote assay

Anti-trypanosomal activities against amastigote form of T. cruzi Tulahuen Lac-Z strain were

performed in vitro. LLC-MK2 cells were resuspended in RPMI 1640 without phenol red (Gibco)

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supplemented with 5% FBS and antibiotics. LLC-MK2 cells were harvested at 2 x 10⁴cell ×mL^-1

in 96 wells plate for 24 h and infected in a parasite/cell ratio 3:1 with the trypomastigote of T.

cruzi Tulahuen strain at 37°C. After 4 hours post-infection, cells were hard washed to remove

the extracellular parasites. The most active compounds against the trypomastigote forms

(products 4e-f, 5c-g and intermediates 8b and 9c), and veraguensin (1) and benznidazole drug

(Sigma-Aldrich) were added at concentrations of 500 µM to 0.24 µM, twenty-four hours after

infection and cultivated at 37°C. After 4 days of culture, the cells were extensive washed and

added 50µL of PBS containing 0.5% of Triton X-100 and 100µM chlorophenol red-β-D-

galactosidase (CPRG, Sigma Aldrich). Plates were incubated at 37°C for 4 h and absorbance was

measured at 570 nm. Results of parasite viability were determined at the base of the catalysis

of CPRG by β-galactosidase. Amastigotes without treatment was considered negative control

and infected cells treated with benznidazole, positive control.

Cytotoxicity assay

The cytotoxicity of compounds (products 4-5a-g and intermediates 8-10a-g), natural products

veraguensin (1) and grandisin (2) and benznidazole drug (Sigma-Aldrich) was evaluated on

spleen cells isolated from C57BL/6 mice at a concentration range of 250 to 1.50 µM. Spleens

were macerated in RPMI 1640 medium (Gibco-BRL Life Technologies, Grand Island, NY) and

incubated for 5 min with red blood cell lysis buffer (one part of 0.17M Tris–HCl and nine parts

of 0.16 M ammonium chloride). The isolated cells were centrifuged at 1500 rpm for 10 min and

resuspended in RPMI medium containing 5% fetal bovine serum (Life Technologies Inc.,

Bethesda, MD) and antibiotics (Sigma Chemical Co., St. Louis). The spleen cells were seeded

flat-bottom 96-well plates at 6.5×10⁶/mL cells/well with different concentrations of the

synthesized compounds at 37°C for 24 h [88,89]. Tween 20 at 1% was used as cell death

positive control and benznidazole drug (Roche) was used as a reference drug. After 24 h, the

cells were incubated with 10 µg/mL propidium iodide (Sigma) and acquired using a

FACSCantoII (Becton-Dickinson Immunocytometry System Inc., San Jose, CA, USA). Data

analysis was performed using FlowJo software (Ashland, Oregon, USA). The experiments were

also performed in triplicates.

Trypanothione reductase assay

Recombinant trypanothione reductase from T. cruzi (TcTR) was expressed in

Escherichia coli BL21DE3 and purified by affinity chromatography. TR assays were performed

as described by Hamilton[86] in 96-well flat bottom micro plates (final volume = 240 µL),

containing TcTR (5 m-unit), HEPES (40 mM pH 7.5), NADPH (Sigma-Aldrich) (0.15 mM) and

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EDTA (1 mM), TS₂(Sigma-Aldrich) (1 μM) and tested compounds (50 µM), diluted in DMSO.

The reaction mixture was pre-incubated at 27ºC for 30 min and after 10μL of DNTB was added,

absorbance was read at 412 nm for 30 min in 5 min interval at 27ºC in a TECAN Infinite M200

micro plate reader. Clomipramine and DMSO 1% were used as positive and negative controls,

respectively. Compounds that inhibited > 50% of TR activity were tested again in serial dilution

(50μM -1.5μM) and IC₅₀values were calculated by a non-linear regression using the GraphPad

Prism program. All assays were done in triplicate and repeated three times [86].

4.4 Quantitative structure-activity relationships studies

Because most of the compounds are racemic mixtures, a set of more simple molecular

descriptors (as constitutional, topological and molecular descriptors) were obtained using only

the Simplified Molecular Input Line Entry System (SMILES) in the Dragon 6. The absolute

stereochemistry of the derivatives was not considered for generation of the strings. Next, it

was carried out a variable reduction step, to eliminate descriptors with irrelevant or redundant

information. Thus, the following features found in some descriptors were excluded: (i)

constant values; (ii) near-constant values; (iii) standard deviation of less than 0.001; (iv) at least

one missing value; (v) a pair correlation larger than or equal to 0.90; and (vi) absolute

Pearson’s correlation coefficient (|r|) values with the biological activity vector y (in the –log

EC₅₀, or pEC₅₀, format) less than 0.15. The first steps were also carried out in Dragon 6, and the

last step in 2D-QSAR Modeling [90]. A final verification was carried out by visual inspection,

using a resultant matrix with 104 descriptors. Variable selection was performed using the

ordered predictor selection (OPS) algorithm.[49] The models were constructed based on the

partial least squares (PLS). Statistical tools to validate the quality of the fit, significance, and

predicting power of the models were applied in order to evaluate the capacity of the models

to generate reliable predictions of the biological activity under study to find out new active

derivatives [91]. The validation approach comprised the internal and external steps, using 29

and 5 compounds, respectively. The set compounds selected for external validation was

chosen according to their biological activities encompassing a range of 2.423 logaritimic units.

The external validation results were obtained from the Xternal Validation Metric Calculator

program [92]. In order to maintain the quality of the model, compound 5c was removed from

the study for presenting a pEC₅₀value outside the range of the biological activity variation,

higher than 1.875 logaritimic units in relation to the second more active derivative 4e.

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Acknowledgments

This work was supported by Grants from FAPESP (Fundação de Amparo à Pesquisa do

Estado de São Paulo), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico),

CAPES (Comissão de Aperfeiçoamento do Pessoal de Nível Superior) and FAPPR (Fundação

Araucária) in Brazil. We are grateful to Vinícius Palaretti and José Carlos Tomaz for NMR and

ESI-HRMS analyses, respectively.

Supplementary data

Supplementary data (synthesis of precursors 8-10 (a-g), 1H, 13C NMR, DEPT-135,

HMQC, HMBC and HRMS (ES+) spectra for all compounds) associated with this article can be

found, in the online version, at ... These data include MOL files of the most important

compounds described in this article.

Rerefences

Acknowledgments

This work was supported by Grants from FAPESP (Fundação de Amparo à Pesquisa do

Estado de São Paulo), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico),

CAPES (Comissão de Aperfeiçoamento do Pessoal de Nível Superior) and FAPPR (Fundação

Araucária) in Brazil. We are grateful to Vinícius Palaretti and José Carlos Tomaz for NMR and

ESI-HRMS analyses, respectively.

Supplementary data

Supplementary data (synthesis of precursors 8-10 (a-g), 1H, 13C NMR, DEPT-135,

HMQC, HMBC and HRMS (ES+) spectra for all compounds) associated with this article can be

found, in the online version, at ... These data include MOL files of the most important

compounds described in this article.

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Article Doi

Synthesis and 2D-QSAR studies of neolignan-based diaryl-tetrahydrofuran and -furan analogues with remarkable activity against Trypanosoma cruzi and assessment of the trypanothione reductase activity

DOI: 10.1016/j.ejmech.2017.08.064

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