Design, synthesis, molecular docking and biological evaluation of thiophen-2-iminothiazolidine derivatives for use against Trypanosoma cruzi

Accepted Manuscript

Design, synthesis, molecular docking and biological evaluation of thiophen-2-

iminothiazolidine derivatives for use against Trypanosoma cruzi

E.F. Silva-Júnior, E.P.S. Silva, P.H.B. França, J.P.N. Silva, E.O. Barreto, E.B.

Silva, R.S. Ferreira, C.C. Gatto, D.R.M. Moreira, J.L. Siqueira-Neto, F.J.B.

Mendonça-Júnior, M.C.A. Lima, J.H. Bortoluzzi, M.T. Scotti, L. Scotti, M.R.

Meneghetti, T.M. Aquino, J.X. Araújo-Júnior

PII:

S0968-0896(16)30505-3

DOI:

http://dx.doi.org/10.1016/j.bmc.2016.07.013

BMC 13126

Reference:

Bioorganic & Medicinal Chemistry

To appear in:

Received Date:

Revised Date:

Accepted Date:

14 June 2016

6 July 2016

8 July 2016

Please cite this article as: Silva-Júnior, E.F., Silva, E.P.S., França, P.H.B., Silva, J.P.N., Barreto, E.O., Silva, E.B.,

Ferreira, R.S., Gatto, C.C., Moreira, D.R.M., Siqueira-Neto, J.L., Mendonça-Júnior, F.J.B., Lima, M.C.A.,

Bortoluzzi, J.H., Scotti, M.T., Scotti, L., Meneghetti, M.R., Aquino, T.M., Araújo-Júnior, J.X., Design, synthesis,

molecular docking and biological evaluation of thiophen-2-iminothiazolidine derivatives for use against

Trypanosoma cruzi, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.07.013

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Bioorganic & Medicinal Chemistry

Design, synthesis, molecular docking and biological evaluation of thiophen-2-

iminothiazolidine derivatives for use against Trypanosoma cruzi

Silva-Júnior, E.F.^a, Silva, E.P.S.^a, França, P.H.B.^a, Silva, J.P.N.^b, Barreto, E.O.^b, Silva, E.B.^c, Ferreira,

R.S.^c, Gatto, C.C.^d, Moreira, D.R.M.^e, Siqueira-Neto, J.L.^f, Mendonça-Júnior, F.J.B.^g, Lima, M.C.A.^h,

Bortoluzzi, J.H.ⁱ, Scotti, M.T.^g, Scotti, L.^g, Meneghetti, M.R.ⁱ, Aquino, T.M.*^a, Araújo-Júnior, J.X.^a

^aMedicinal Chemistry Laboratory, Pharmacy and Nursing School, Federal University of Alagoas, Maceio, Brazil

^bCell Biology Laboratory, Federal University of Alagoas, Maceio, Brazil

^cBiochemistry and Immunology Department, Biological Sciences Institute, Federal University of Minas Gerais, Belo Horizonte, Brazil

^dInorganic Synthesis and Crystallography Laboratory, Institute of Chemistry, University of Brasilia, Federal District, Brazil

^eTissue Engineering and Immunopharmacology Laboratory, Oswaldo Cruz Foundation, Salvador, Bahia, Brazil

^fSkaggs School of Pharmacy and Pharmaceutical Sciences, California, San Diego La Jolla, United States of America

^gLaboratory of Drug Synthesis and Delivery, Biological Sciences Department, State University of Paraiba, Campus V, João Pessoa, Brazil

^hDrug Design and Synthesis Laboratory, National Science and Technology Institute for Pharmaceutical Innovation, Federal University of Pernambuco, Recife,

Brazil

ⁱCatalysis and Chemical Reactivity Group (GCaR), Institute of Chemistry and Biotechnology, Federal University of Alagoas, Maceio, Brazil

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

In this study, we designed and synthesized a series of thiophen-2-iminothiazolidine derivatives

from thiophen-2-thioureic with good anti-Trypanosoma cruzi activity. Several of the final

compounds displayed remarkable trypanocidal activity. The ability of the new compounds to

inhibit the activity of the enzyme cruzain, the major cysteine protease of T. cruzi, was also

explored. The compounds 3b, 4b, 8b and 8c were the most active derivatives against amastigote

form, with significant IC₅₀values between 9.7 and 6.03 μM. The 8c derivative showed the

highest potency against cruzain (IC₅₀= 2.4 μM). Molecular docking study showed that this

compound can interact with subsites S1 and S2 simultaneously, and the negative values for the

theoretical energy binding (Eb= -7.39 kcal∙mol-1) indicates interaction (via dipole-dipole)

between the hybridized sulfur sp3 atom at the thiazolidine ring and Gly66. Finally, the results

suggest that the thiophen-2-iminothiazolidines synthesized are important lead compounds for the

continuing battle against Chagas disease.

Available online

Keywords:

Thiophene

Thiazolidine

Docking

T. cruzi

Chagas disease

drugs, such as nifurtimox and benznidazole (Fig. 1), represents a

serious public health problem [2]. Also, these drugs present

disadvantages that limit their use: they produce active

metabolites which to interact with the DNA of the host leading to

deleterious effects, such as cancer [6,7], resistance, lack of

efficacy in the late-stage of the disease and a lack of pediatric

formulations [8]. The survival of T. cruzi in the cells of the host

is guaranteed by important enzymes (e.g. cruzain) which play a

role in different processes including absorption, penetration,

survival, infectivity, immune evasion, nutrition and growth [6].

1. Introduction

Neglected tropical diseases (NTDs) are a diverse group of

diseases that prevail in tropical and subtropical countries and are

responsible for causing illness in more than 1 billion people

around the world. It has been estimated that around 8 million of

these cases are associated with the parasite Trypanosoma cruzi,

which causes Chagas disease [1–3]. This disease is controlled at

present through the elimination of the vectors with the use of

insecticides and the serological screening of blood. Better

housing and educational campaigns are also fruitful approaches.

As with other parasitic diseases, this pathology is associated with

poverty and low educational levels [4]. Current chemotherapy for

Chagas disease is unsatisfactory due to its limited efficacy,

particularly in the chronic phase, with frequent side effects that

can lead to the discontinuation of treatment [5]. The development

of resistance by some strains of T. cruzi toward gold-standard

Cruzain is a cathepsin-L-like protease (also known as cysteine

protease) of the papain family. It is thought to be essential for the

infection of host cells and the replication and metabolism of the

parasite and it plays multiple roles in the disease pathogenesis

[4,5]. Furthermore, the selective inhibition of cysteine proteases

as a therapeutic target has transcended the laboratory to the

clinic. Recently, odanacatib (Fig. 1), a drug based on cysteine

protease inhibition, has shown high efficacy and a good safety

profile in Phase III clinical trials. Also, many studies in animal

models have validated the use of this enzyme for the control and

elimination of T. cruzi [8].

Figure 3. Some thiazolidine derivatives described in literature

which are active against Trypanosoma cruzi.

In this study, we synthesized new thiophene-thiazolidine hybrid

derivatives bearing various groups at the thiazolidine ring, with a

linker (imine group) between the thiophene and thiazolidine rings

(Fig. 4). All derivatives were tested for their ability to inhibit the

in vitro growth of amastigote and trypomastigote forms of

Trypanosoma cruzi and the activity of the enzyme cruzain. In

addition, we carried out theoretical studies involving molecular

docking simulations and pharmacophoric identification.

Figure 1. Structures of nifurtimox (1), benznidazole (2) and

odanacatib (3).

Many thiophene compounds have been described as very active

derivatives against T. cruzi (Fig. 2) and these are interesting

starting materials for new therapeutic agents [9]. The thiophene

aromatic ring is a hydrophobic site which binds to the

hydrophobic-pockets in some enzymes [10].

Figure 4. Molecular hybridization pathway for the synthesis of

the compounds. 1: thiophene ring; 2: thiazolidine ring; 3: hybrid

scaffold. Imine bond as linker shown in green.

2. Results and Discussion

2.1. Chemistry

The 2-aminothiophene analogues (1, 2) were readily prepared

via the Gewald reaction, a multicomponent synthesis involving

an aldehyde or ketone, an activated nitrile and elemental sulfur

[12–14]. The intermediates (3a-d) were then synthesized through

the treatment of 1 or 2 with substituted isothiocyanates (phenyl

and allyl). Spectral analysis for these compounds was carried out

according to methods described in the literature [13,15]. Finally,

thiophen-2-iminothiazolidines were prepared via thia-Michael

cyclization (7a-d) or substitution followed by intramolecular

cyclization (4a-d, 5c, d, 6a, 6c and 8a-d) between thiophen-2-

thiourea and dielectrophiles [16,17]. All of these reactions

proceeded well with refluxing overnight. The synthetic route is

shown in Scheme 1. Four compounds previously designed were

not included in this work (two cyclization using ethyl 2-chloro

acetate and two using 2-bromomalonate) due the formation of

many secondary products and impossibility of purification by

Figure 2. Some thiophene derivatives described in the literature

which are active against Trypanosoma cruzi.

The pharmacological activity of thiazolidine compounds is of

current interest. Thiazolidine and its hybrids have been reported

to possess promising anticancer and antiviral properties. In

addition, the thiazolidine scaffold is extremely important in the

design and synthesis of novel biologically active agents with

action against Trypanosoma spp. [11]. Many compounds with the

thiazolidine ring in their structure (Fig. 3) have been described in

the literature and they show promising anti-T. cruzi activity

[1,5,6,11].

recrystallization or flash column chromatography.

Scheme 1. Synthetic route for thiophen-2-thiourea and thiophen-

2-iminothiazolidine derivatives.

The structures of the new compounds synthesized were

established by ¹H and ¹³C 1D and 2D NMR analysis. For the final

compounds, in H NMR spectra, signs of the protons from

Scheme 2. Example of possible ambiguous synthetic route for

thiophen-2-iminothiazolidines 4a-d.

1

thiourea moiety of 3a-d signs were not displayed, and peaks

resonated at 154.48-158.49 and 158.32-163.94 ppm in the ¹³C

NMR spectra were respectively assigned to C-4 and C-2 of the

1

thiazolidine ring. For compounds 4a-d the H NMR spectra

revealed the presence of a singlet for thiomethylene protons at

3.66-3.90 ppm. In addition, ¹³C NMR spectra exhibited resonated

peaks for the same moiety at 33.39-34.97 ppm, as well as signals

at 155.86-158.49 and 161.99-163.60 ppm assigned to C=N and

C=O moieties, respectively. For compounds 5c and 5d, resonated

1

peaks at 2.57-2.60 ppm in the H NMR were attributed to

substituent methyl groups at C-4 of thiazolidine ring and whose

carbons appeared at 12.85-12.90 ppm in the ¹³C NMR spectra.

Compounds 6a, 6c, 7a-d and 8a-d may be distinguished by the

resonated peaks of SCH moiety, which appears as a singlet at

5.28-6.08 ppm in ¹H NMR of derivatives 6a and 6c. On the other

hand, for compounds 7a-d the ¹H NMR spectra exhibited

resonance assigned to the SCH group appearing as double

doublet at 4.39-4.53 ppm, due to the interaction with methylene

protons of the acetyl group substituent at C-5 of the 4-

thiazolidinone ring. For these compounds, the SCH moiety

ranges from 43.81 to 59.94 ppm in the ¹³C NMR. Lastly,

compounds 8a-d show resonated peaks for SCH further displaced

downfield, with ¹³C NMR signals being shown at 99.11-100.49

ppm.

Figure 5. Thiazolidine ring confirmation in 2D HMBC spectrum

for 4c.

Additionally, we performed a molecular modeling study using

the structures of the intermediary and transition state of the

synthetic route for compounds 3a to 4a (Scheme 3). A lower

transition state energy value and, consequently, a smaller energy

difference value for the intermediary of this route, verify that this

is a favorable synthetic route for obtaining the product formed

(Table 1). Also, the HOMO and LUMO energy gaps of both

intermediaries were calculated and the energy gap value for ethyl

2-{[N-[3-(1-ethoxyethenyl)-4,5,6,7-tetrahydro-1H-inden-2-yl]-

N'-phenylcarbamimidoyl]sulfanyl}acetate (385.52 kJ∙mol^-1) is

higher than that for ethyl 2-{[N'-[3-(1-ethoxyethenyl)-4,5,6,7-

tetrahydro-1H-inden-2-yl]-N-phenylcarbamimidoyl]sulfanyl}

actate (370.46 kJ∙mol^-1). Therefore, the HOMO-LUMO

interactions of the latter are stronger than those of the former,

corroborating with the results obtained for the transition state

energies and from the spectral data.

The mechanism of thiazolidine ring formation is not well

understood, which has led to erroneous proposal of structures

obtained through the reaction of thiourea analogues with α-halo

esters or acids in the presence of an inorganic base [18]. In the

synthesis of 7a-d, for example, it is accepted that this reaction

begins with a nucleophilic attack by the sulfur atom of the

thiourea on the C=C bond of maleic anhydride, followed by a

proton transfer and a nucleophilic attack of a nitrogen atom on

any of the two carbonyl groups, providing a five-membered 4-

oxo-1,3-thiazolidine ring. Finally, aminolysis of this product

occurs, giving the final compound with an acid function [19,20].

In ethanol reflux this reaction leads to many collateral products.

Therefore, an aprotic solvent, such as toluene, was employed and

satisfactory yields were obtained.

Scheme 3. Transition states of synthetic route for thiophen-2-

iminothiazolidines (4a-d).

The general mechanism suggested is that the sulfur atom (soft

nucleophile) of the thiourea will preferentially attack the α-halo

esters (soft electrophile) and the NH (hard nucleophile) will

attack the carbonyl center (hard electrophile) [18]. However, the

regioselectivity of the nucleophilic attack from the non-

equivalent nitrogen atoms of the thiourea still needs to be studied

[20]. For unsymmetrical thioureas (R1 ≠ R2), regiocontrol in the

cyclization step is typically influenced by electronic factors that

predispose electron-withdrawing substituents (i.e., aryl or

heteroaryl groups) to maintain conjugative stabilization with the

imine nitrogen [21].

Table 1. Energies of intermediaries and respective transition

states of possible ambiguous synthetic route for thiophen-2-

iminothiazolidines (4a-d).

Intermediary

I^a

TS^b

Δ= TS-I ^c

-5,410,603.0

-5,386,356.8

24,246.2

In the case of the thiophen-2-iminothiazolidines described

herein, the results of analysis using heteronuclear multiple bond

correlation (HMBC) (see Fig. 5 for 4c and supplementary data

for the other compounds containing an allyl group) techniques

support our conclusions regarding the position of the thiazolidine

ring and the possibility of the existence of an ambiguous

synthetic route is discarded (Scheme 2).

-5,410,565.8

-5,386,457.6

24,108.3

^a: Intermediary energy; ^b: Transition state energy; : Energy

c

difference.

In an attempt to improve the yield for the cyclization, the

synthesis of 4b and 8a were chosen, and sodium acetate was used

as a catalyst. However, it was observed that this did not lead to

the formation of the desired compound (Scheme 4), since the

base deprotonates the nitrogen neighboring to the phenyl group

F²

which, in turn, attacks the carbonyl of the ester group, closing the

ring and providing the monosodium salt of the corresponding

thienopyrimidine. Finally, thienopyrimidine salt reacts with the

electrophile, leading to 9 and 10 as final products. The reason for

this is that thienopyrimidines can be obtained from thiourea

derivatives using a base salt [22–24]. Compound 9 was obtained

as colorless crystals and its structure was confirmed by X-ray

crystallography (Fig. 6A; Table 2). Yellow crystal of the

compound 8b, obtained from cyclization without sodium acetate,

is shown in figure 6B, as well crystal data and structure

refinement are described in table 2.

Data/restraints/parameters 3993 / 0 / 246

5508 / 0 / 300

1.061

0.0875

0.1573

0.467 and

0.352

Goodness-of-fit on F²

R [I > 2σ(I)]

wR₂(all data)

0.925

0.0648

0.1856

Largest diff. peak and hole 0.210 and

-

(eÅ^-3)

0.216

R.M.S deviation from 0.050

mean (eÅ^-3)

0.043

Code number

9

10

(CCG_UFAL5) (CCG_UFAL7)

2.2. Cytotoxicity of thiophen-2-thiourea and thiophen-2-

iminothiazolidines

The cytotoxicity of the final compounds was evaluated using

the MTT assay. Good tolerance was observed for most of the

compounds when tested against the J774 macrophages cell line at

a concentration of 10 μM. Only 4b presented significant

cytotoxicity. The compounds 3b, 8b and 8c showed cell viability

(J774) above 50 % at 10 μM (Graphic 1).

Scheme 4. Synthetic routes for the compounds 9 and 10.

Graphic 1. Influence of all synthesized compounds on cell

viability after 24 hours of exposure. Each bar represents the mean

± standard deviation of triplicate experiments (10 and 100 μM,

respectively). Control bars represent cells exposed only to the

RPMI-1640 medium and vehicle refers to cells treated with

DMSO (0.01%).

2.3. Biological evaluation

Figure 6. ORTEP view of compounds 9 (A) and 8b (B).

Table 2. Crystal data and structure refinement for 9 and 8b.

The newly synthesized compounds were evaluated by in vitro

screening against the amastigote and trypomastigote forms of the

T. cruzi parasite [25]. A study on cruzain inhibition was carried

out using the method described by Ferreira et al [26].

Empirical formula

Formula weight

Temperature (K)

Wavelength (Å)

Crystal system

Space group

Unit cell dimensions

a (Å)

C₂₀H₂₀N₂O₃S₂

400.5

296(2)

0.71073

Orthorhombic

C₂₇H₂₆N₂O₂S₂

474.62

296(2)

0.71073

Monoclinic

2.3.1. Assessment of compound activity against

intracellular amastigotes

The amastigote stage was shown to be infective toward

phagocytic and non-phagocytic cells, an important process during

the cycle in different hosts. The amastigotes multiply by binary

fission in the cells of infected tissues. In addition, these

evolutionary forms are related to the chronic phase of Chagas

disease [27]. The results for activity against the intracellular

amastigotes forms are shown in Table 3. Benznidazole (BnZ),

with a low IC₅₀value (1.17 μM), was used as the positive control.

In general, the molecular hybridization strategy can be

considered satisfactory, due the cyclization of the compounds

increased their potency against the amastigote forms, as observed

on comparing 3b with 4b and 8b, and 3c with 8c. [28,29].

P 21 21 21

P 1 21/n 1

4.9546(6)

13.3793(18)

29.523(5)

1957.1(5)

4

10.3078(2)

15.8286(4)

14.9372(4)

2437.02(10)

4

b (Å)

c (Å)

Volume (Å³)

Z

ρ_calc(g∙cm^-3)

µ (mm^-1)

1.359

0.295

1.294

0.245

F(000)

840

1000

Crystal size (mm)

0.060 x 0.130 x 0.420 x 0.600 x

0.590

0.630

Furthermore, is possible to observe that the combination of

ethyl and allyl groups at R₁and R₂, respectively, leads to lower

activity (IC₅₀value increases from 6.03 to 9.7 μM). Therefore,

these chemical groups can be considered as unfavorable in

relation to activity against these evolutionary forms.

Additionally, an i-propyl group at R₁and phenyl group at R₂

seems to be the best combination, increasing the activity, as

observed for 3b, 4b and 8b, with IC₅₀values between 8.78 and

θ range (º)

Reflections collected

Independent reflections

Observed reflections [I > 1568

2σ(I)]

R_int

Refinement method

1.38 to 26.39

10738

3993

1.87 to 27.37

24135

5508

3525

0.1086

Full-matrix

least-squares on least-squares on

0.0531

Full-matrix

6.03 μM. According to Kryshchyshyn et al. [6], this is related to

the hydrophobicity of the compounds, which leads to an increase

in the activity toward evolutionary forms of the T. cruzi parasite.

However, 8c, which has an allyl group at R₂(unfavorable),

showed a similar IC₅₀value when compared with 3b. This fact

can be attributed to the presence of a phenyl ring at R₅in the

structure of 8b, which increases the hydrophobic surface of this

compound.

6.7

9.0

16.7

6.4

5.1

7.7

5.8

4.5

5.1

6.1

100

0.01

N.D.

11.4

5d

6a

6c

7a

7b

7c

7d

8a

8b

8c

8d

Table 3. Anti-T. cruzi activity against intracellular amastigotes

for all compounds synthesized.

BnZ^a

DMSO+RP

MI^b

Code

3a

3b

3c

3d

4a

4b

4c

4d

5c

5d

6a

6c

7a

7b

7c

% Inhibition at 10 µM

47.0

IC₅₀Values^*

N.D.

8.78

N.D.

59.64

11.91

37.83

20.14

39.44

17.03

4.03

18.34

39.19

0.43

a

N.D.

6.96

*: values in µM; : Benznidazole, gold-standard drug employed

as positive control; ^b: Dimethyl sulfoxide and Roswell Park

Memorial Institute (RPMI-1640) medium employed as negative

control; N.D.: Not determined.

N.D.

6.03

2.3.3. Cruzain inhibition

Cruzain inhibition by thiophen-2-thiourea and thiophen-2-

iminothiazolidine derivatives was investigated as a possible

mechanism of trypanocidal activity against T. cruzi (Table 5). Of

the compounds which were most effective against amastigote and

trypomastigote forms of T. cruzi, 8c showed the highest potency

against cruzain (IC₅₀= 2.4 μM). Additionally, 5c, 7a, 7c and 7d

were also active against the enzyme cruzain, but with moderate

potencies. To determine whether the observed activity of the

thiophene derivatives 5c, 7a, 7c, 7d and 8c was due to

nonspecific binding, the assay was repeated in the absence and

presence of 0.01 and 0.1% Triton X-100 [30]. Compound 5c

showed a higher cruzain inhibition in the absence of the

detergent, possibly indicating aggregation. On the other hand,

the level of cruzain inhibition for compounds 7a, 7c, 7d and 8c

did not vary considerably in the presence of the different Triton

concentrations, suggesting that the enzyme inhibition is not due

to compound aggregation. Additionally, the IC₅₀value for 7c did

not increase in the presence of 2 mg∙mL^-1bovine serum albumin

(BSA), with 42 and 50% inhibition of the enzyme at 50 μM in

the presence and absence of BSA, respectively. This compound

was not sensitive to pre-incubation with BSA, which, at high

concentrations competes with the enzyme for colloid particles

(aggregates), preventing or reducing enzymatic inhibition due to

aggregation [26].

30.0

37.43

43.46

13.80

47.25

23.62

59.52

53.07

36.01

94.5

7d

8a

8b

8c

9.7

N.D.

1.17

8d

BnZ^a

DMSO+medium^b

1.7

N.D.

a

*: values in µM; : Benznidazole, gold-standard drug employed

as positive control; ^b: Dimethyl sulfoxide and Roswell Park

Memorial Institute (RPMI-1640) medium employed as negative

control; N.D.: Not determined.

2.3.2. Activity against Y strain trypomastigotes

Intracellular amastigotes transform into trypomastigotes and

then burst out of the cell and enter the bloodstream.

Trypomastigotes can infect other cells and transform into

intracellular amastigotes at new infection sites. The clinical

manifestations can result from this infective cycle [27].

Table 5. Summary of activities for thiophen-2-thiorea and

thiophen-2-iminothiazolidine derivatives against the enzyme

cruzain.

All results for anti-T. cruzi activity against Y strain

trypomastigote forms at a concentration of 25 µM are shown in

Table 4. Only 4d showed significant inhibition against these

parasite forms and its IC₅₀value was lower than that of BnZ

(10.3 and 11.4 μM, respectively).

Code

% Cruzain inhibition at 100

µM^a

% Cruzain

inhibition^c

without

pre-

incubatio

n

12 ± 7

43 ± 4

29 ± 3

29 ± 3.6

0 ± 0

33 ± 8

23 ± 8

45 ± 2

with pre-

incubatio

n

IC₅₀,

0.01%

Triton

0.1%

Triton

µM^b

Table 4. Anti-T. cruzi activity for all compounds against Y strain

trypomastigotes.

Code

3a

3b

% Inhibition at 25 µM

IC₅₀Values^*

N.D.

4 ± 0

74 ± 0.5

41 ± 1

34 ± 0.5

17 ± 0.6

72 ± 0.5

40 ± 0.6

70 ± 1

N.D.

3a

3b

3c

3d

4a

4b

4c

4d

1.6

5.8

N.D.

11.9

15.1

3.2

N.D.

3c

3d

4a

27.0

N.D.

4b

4.5

N.D.

4c

84.1

7.1

10.3

N.D.

4d

5c

34 ± 6

82 ± 0.5

85 ± 22

N.D.

33 ± 1 29 ± 0.6

5c

(100

μM)

N.D.

(100

μM)

N.D.

38 ± 5

(25μM)

46 ± 2

18 ± 0.5

66 ± 9

71 ± 0.5

(25μM)

66 ± 0.8

59 ± 1

5d

N.D.

121 ±

34

N.D.

5.4 ± 3

(100

N.D.

18 ± 4

(100

6a

6c

7a

86 ± 0.5

μM)

32 ± 9

64 ± 7

77 ± 1

89 ± 0.4

N.D.

7b

7c

50 ± 10 47 ± 2.5 44 ± 0.4

(50

(50 μM)

μM)

111 ± 9 53 ± 6.2

(100

52 ± 15

80 ± 0.7

52.7 ±

0.4

7d

μM)

(100

μM)

N.D.

Figure 7. 8c in complex with enzyme cruzain. Green dots

represent dipole-dipole interaction between hybridized sulfur sp³

atom at thiazolidine ring and Gly 66.

16 ± 2

(50μM)

13 ± 4

87 ± 5

40 ± 0.5

(50μM)

25 ± 1

N.D.

8a

N.D.

2.4 ±

1.2

N.D.

8b

8c

Normally, in a complex of hydrophobic compounds with the

enzyme cruzain, a phenyl ring (when present) is allocated to the

S2 subsite pocket and this is observed to be related to an increase

the binding affinity [8]. Based on this, the cruzain subsite

analysis shows that compound 8c can interact with subsites S1

and S2 simultaneously. For this compound, the phenyl ring is

97 ± 0.3

62 ± 3.2 55 ± 1.3

(6.25

μM)

N.D.

(6.25

μM)

N.D.

29 ± 4

100 ± 0

67 ± 0.5

100 ± 0

N.D.

0.4

8d

E-64^d

allocated

to

S2

(high

hydrophobicity)

and

the

^a: The results represent the average and standard error of two

independent experiments in triplicate. Errors are given as the

ratio between the standard deviation and the square root of the

number of measurements; : IC₅₀values represent the average

and standard error of two independent experiments which were

tetrahydrobenzothiophene bicycle is allocated to S1 (low

hydrophobicity) (Fig. 8). In addition, docking results showed that

the compounds 5c, 7a, 7c and 7d does not provided satisfatory E_b

values in comparison with 8c, and are not able to interact with

Gly66 amino acid.

b

determined based on at least 9 compound concentrations in

c

triplicate; : The compounds were tested at concentrations close

to the IC₅₀; ^d: Potent inhibitor employed as positive control; N.D.:

Not determined.

2.3.4. Molecular docking studies

Since 2008, computational methodologies such as

molecular docking and high-throughput screening (HTS) have

been employed to providing new scaffolds with high potency

and good tolerance [8]. Molecular docking models have been

shown to adequately predict the binding mode associated with

the binding of different anti-T. cruzi compounds to cruzain

[4]. Therefore, in order to gain a better insight into the nature

of the trypanocidal activity displayed by the novel thiophen-2-

iminothiazolidine reported herein, molecular docking studies

were performed with cruzain. The most active compound (8c)

against amastigote forms and the enzyme cruzain provided a

negative values for the theoretical energy binding (E_b= -7.39

kcal∙mol^-1). This indicates interaction (via dipole-dipole)

between the hybridized sulfur sp³atom at the thiazolidine ring

and Gly66 (Fig. 7) [5,29]. According to Martinez-Moyaorga

et al.[8], this amino acid is the main key residue related to

anti-T. cruzi activity, and interactions with water molecules

are not associated with inhibitory activity.

Figure 8. Cruzain binding site showing the subsites S1', S1, S2

and S3. 8c allocated to S2 and S1 subsites.

3. Conclusion

In this study new thiazolidine based conjugates with the

thiophene moiety in position 2 are described using accessible

methods. The assay to determine the trypanocidal activity of

synthesized compounds allowed us to identify thiophene-thiourea

hybrid 3b and thiophene-thiazolidine hybrids 4b, 8b and 8c,

which were found to be the most active derivatives against

amastigote form, with significant IC₅₀values between 9.7 and

6.03 μM. In addition, 8c showed excellent ability to inhibit the

cruzain enzyme, with an IC₅₀value of 2.4 µM. The results of the

in silico studies corroborated the in vitro cruzain inhibition,

showing that the most stable molecule is also the most potent

cruzain inhibitor. Modifications to improve the potency of the

most potent derivatives are currently under progress in our

laboratory.

4.13 (2H, q, J= 7.1, CH₂CH₃), 7.33-7.39 (3H, m, ArH), 7.46-7.50

(2H, m, ArH), 7.98 (1H, br s, NH), 12.17 (1H, br s, NH). ¹³C

NMR (100 MHz, CDCl₃) δ 14.19 (CH₃), 22.86 (CH₂), 22.97

(CH₂), 24.32 (CH₂), 26.31 (CH₂), 60.39 (OCH₂), 113.12 (Cq),

125.62 (CH Ar), 126.86 (Cq), 127.74 (CH Ar), 130.01 (CH Ar),

130.89 (Cq), 135.87 (Cq Ar), 149.78 (Cq), 166.31 (C=O), 176.17

(C=S).

4. Experimental section

4.1. Chemistry

All reagents were commercial grade and purified according to

the established procedures. Melting points (m.p.) were detected

with open capillaries using MSTecnopon® PFMII Digital

Melting point, and are incorrect. NMR analyses were performed

on Bruker^®Avance DRX 400 MHz Spectrometer by using

tetramethylsilane (TMS) as internal standard. The chemical shifts

were reported in δ units, and coupling constants (J) were

measured in hertz. The peaks are presented as s (singlet), d

(doublet), t (triplet), q (quartet), qi (quintet), sex (sextet), sep

(septet), br s (broad singlet), dd (double doublet), ddt (double

doublet of triplet), m (multiplet). The reactions were monitored

by TLC (Merck^®, silica gel, type 60, 0.25 mm).

4.1.2.2. Isopropyl 2-(3-phenylthioureido)-4,5,6,7-

tetrahydrobenzo[b]thiophene-3-carboxylate (3b)

Pale brown amorphous solid. Yield: 60%, m.p. 194-195 ºC.

1H NMR (400 MHz, CDCl₃) δ 1.24 (6H, d, J= 6.2, CH₃), 1.74-

1.79 (4H, m, CH₂), 2.63-2.65 (2H, m, CH₂), 2.71-2.73 (2H, m,

CH₂), 5.01 (1H, sep, J= 6.2, CH), 7.33-7.38 (3H, m, ArH), 7.45-

7.49 (2H, m, ArH), 7.92 (1H, br s, NH), 12.30 (1H, br s, NH).

¹³C NMR (100 MHz, CDCl₃) δ 21.89 (CH₃), 22.91 (CH₂), 22.97

(CH₂), 24.34 (CH₂), 26.42 (CH₂), 68.09 (CH), 113.43 (Cq),

125.50 (CH Ar), 126.84 (Cq), 127.62 (CH Ar), 129.95 (CH Ar),

130.79 (Cq), 135.94 (Cq Ar), 149.77 (Cq), 166.09 (C=O), 176.19

(C=S).

4.1.1. Procedure for Synthesis of 2-aminothiophene

(1,2)

A mixture of cyclohexanone (1 mmol), an activated nitrile (1

eq.), morpholine (1 eq.) and elemental sulfur (1 eq.) in 5 ml of

ethanol was stirred at 0 ºC overnight. After, the solvent was

evaporated at reduced pressure and the crude product was

extracted with ethyl acetate thrice. The organic layer was treated

with anhydrous sodium sulphate and evaporated again. Finally

the final compounds were purified by recrystallization from

MeOH/water.

4.1.2.3. Ethyl 2-(3-allylthioureido)-4,5,6,7-

tetrahydrobenzo[b]thiophene-3-carboxylate (3c)

Pale brown amorphous solid. Yield: 80%, m.p. 173-174 ºC. ¹H

NMR (400 MHz, CDCl₃) δ 1.38 (3H, t, J= 7.1, CH₃), 1.76-1.81

(4H, m, CH₂), 2.62-2.65 (2H, m, CH₂), 2.75-2.77 (2H, m, CH₂),

4.13-4.18 (2H, m, CH₂CH=CH₂), 4.32 (2H, q, J= 7.1, CH₂CH₃),

5.26 (1H, dd, J= 0.9 and 10.2, CH₂CH=CH₂), 5.36 (1H, d, J=

17.1, CH₂CH=CH₂), 5.92 (1H, ddt, J= 5.8, 10.2 and 17.1,

CH₂CH=CH₂), 6.34 (1H, br s, NH), 12.14 (1H, br s, NH). ¹³C

NMR (100 MHz, CDCl₃) δ 14.28 (CH₃), 22.88 (CH₂), 22.98

(CH₂), 24.31 (CH₂), 26.37 (CH₂), 46.62 (CH₂CH=CH₂), 60.66

(OCH₂), 112.03 (Cq), 118.22 (CH₂CH=CH₂), 126.26 (Cq),

130.52 (Cq), 131.90 (CH₂CH=CH₂), 151.04 (Cq), 167.44 (C=O),

176.93 (C=S).

4.1.1.1. Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]

thiophene-3-carboxylate (1)

1

Yellow crystalline solid. Yield: 98%, m.p. 134-135 ºC. H

NMR (400 MHz, DMSO-d₆) δ 1.20 (3H, t, J= 7.1, CH₃), 1.60-

1.65 (4H, m, CH₂), 2.35-2.37 (2H, m, CH₂), 2.55-2.56 (2H, m,

CH₂), 4.11 (2H, q, J= 7.3, CH₂CH₃), 7.14 (2H, br s, NH₂). ¹³C

NMR (100 MHz, DMSO-d₆) δ 14.18 (CH₃), 22.34 (CH₂), 22.76

(CH₂), 23.88 (CH₂), 26.49 (CH₂), 58.71 (OCH₂), 103.16 (Cq),

115.96 (Cq), 131.50 (Cq), 163.19 (Cq), 165.32 (C=O).

4.1.2.4. Isopropyl 2-(3-allylthioureido)-4,5,6,7-

tetrahydrobenzo[b]thiophene-3-carboxylate (3d)

4.1.1.2. Isopropyl 2-amino-4,5,6,7-tetrahydrobenzo

[b]thiophene-3-carboxylate (2)

Pale brown amorphous solid. Yield: 80%, m.p. 140-141 ºC. ¹H

NMR (400 MHz, CDCl₃) δ 1.36 (6H, t, J= 6.3, CH₃), 1.77-1.79

(4H, m, CH₂), 2.62-2.64 (2H, m, CH₂), 2.74-2.77 (2H, m, CH₂),

4.12-4.18 (2H, m, CH₂CH=CH₂), 5.19 (1H, sep, J= 6.3, CH),

5.26 (1H, dd, J= 1.0 and 10.2, CH₂CH=CH₂), 5.36 (1H, d, J=

17.1, CH₂CH=CH₂), 5.92 (1H, ddt, J= 5.8, 10.2 and 17.1,

CH₂CH=CH₂), 6.28 (1H, br s, NH), 12.19 (1H, br s, NH). ¹³C

NMR (100 MHz, CDCl₃) δ 22.06 (CH₃), 22.96-23.01 (CH₂),

24.35 (CH₂), 26.51 (CH₂), 46.64 (CH₂CH=CH₂), 68.37 (CH),

112.38 (Cq), 118.22 (CH₂CH=CH₂), 126.28 (Cq), 130.56

(CH₂CH=CH₂), 132.00 (Cq), 150.88 (Cq), 166.99 (C=O), 176.97

(C=S).

Yellow amorphous solid. Yield: 70%, m.p. 67-68 ºC. ¹H NMR

(400 MHz, DMSO-d₆) δ 1.20 (6H, d, J= 6.2, CH₃), 1.60-1.65

(4H, m, CH₂), 2.35-2.38 (2H, m, CH₂), 2.53-2.55 (2H, m, CH₂),

4.96 (1H, sep, J= 6.2, CH), 7.11 (2H, br s, NH₂). ¹³C NMR (100

MHz, DMSO-d₆) δ 22.32 (CH₃), 22.83 (CH₂), 23.17 (CH₂), 24.30

(CH₂), 27.01 (CH₂), 66.43 (CH), 103.43 (Cq), 116.16 (Cq),

131.85 (Cq), 162.78 (Cq), 165.21 (C=O).

4.1.2. Procedure for Synthesis of intermediates

Thiophen-2-thiourea (3a-d)

A mixture of 2-aminothiophene (1 mmol) and isothiocyanate

(1.1 eq) in 15 ml ethanol was stirred until reflux overnight. After,

the solvent was evaporated at reduced pressure and the crude

product was extracted with ethyl acetate thrice. The organic layer

was treated with anhydrous sodium sulphate and evaporated

again. Subsequently, was performed a flash chromatography

using hexane/ethyl acetate (8:2) as eluent. Finally, the product

was recrystallized from MeOH/water.

4.1.3. Procedure for Synthesis of Thiophen-2-

thiazolidine (4a-d, 5c,d, 6a, 6c, 7a-d and 8a-d)

A

mixture of thiophen-2-thiourea (1 mmol) and the

respectively dielectrophile (1.0 eq) in 15 ml ethanol (for

synthesis of 7a-d was utilized toluene) was stirred until reflux

overnight. After, the solvent was evaporated at reduced pressure

and the crude product was extracted with ethyl acetate thrice. The

organic layer was treated with anhydrous sodium sulphate and

evaporated again. Finally the final compounds were purified by

flash chromatography using hexane/ethyl acetate (8:2) as eluent.

4.1.2.1. Ethyl 2-(3-phenylthioureido)-4,5,6,7-

tetrahydrobenzo[b]thiophene-3-carboxylate (3a)

1

White amorphous solid. Yield: 98%, m.p. 199-200 ºC. H

NMR (400 MHz, CDCl₃) δ 1.25 (3H, t, J= 7.1, CH₃), 1.74-1.79

(4H, m, CH₂), 2.63-2.66 (2H, m, CH₂), 2.71-2.74 (2H, m, CH₂),

4.1.3.1. (Z)-Ethyl 2-((4-oxo-3-phenylthiazolidin-2-

ylidene)amino)-4,5,6,7-tetrahydrobenzo[b]thiophen

-3-carboxylate (4a)

J= 7.1, CH₃), 1.74-1.82 (4H, m, CH₂), 2.57 (3H, s, CH₃), 2.65-

2.68 (2H, m, CH₂), 2.74-2.77 (2H, m, CH₂), 4.24 (2H, q, J= 7.1,

CH₂), 4.25 (2H, q, J= 7.1, CH₂), 4.69 (2H, d, J= 5.2,

CH₂CH=CH₂), 5.15 (1H, dd, J= 0.8 and 17.1, CH₂CH=CH₂),

5.21 (1H, dd, J= 0.9 and 10.3, CH₂CH=CH₂), 5.94 (1H, ddt, J=

5.2, 10.4 and 15.6, CH₂CH=CH₂). ¹³C NMR (100 MHz, CDCl₃) δ

12.85 (CH₃), 14.37 (CH₃), 14.47 (CH₃), 22.80 (CH₂), 23.17

(CH₂), 25.20 (CH₂), 26.32 (CH₂), 47.13 (CH₂CH=CH₂), 59.74

(CH₂CH₃), 60.96 (CH₂CH₃), 102.46 (C=C), 117.65

1

White amorphous solid. Yield: 95%, m.p. 195-196 ºC. H

NMR (400 MHz, CDCl₃) δ 1.31 (3H, t, J= 7.1, CH₃), 1.79-1.91

(4H, m, CH₂), 2.75-2.78 (2H, m, CH₂), 2.93-2.96 (2H, m, CH₂),

3.85 (2H, s, SCH₂), 4.22 (2H, q, J= 7.1, CH₂CH₃), 7.32-7.35 (2H,

m, ArH), 7.54-7.56 (3H, m, ArH). ₁₃C NMR (100 MHz, CDCl₃) δ

14.27 (CH₃), 22.27 (CH₂), 22.98 (CH₂), 25.11 (CH₂), 25.35

(CH₂), 34.97 (SCH₂), 61.88 (CH₂CH₃), 119.31 (Cq), 129.10 (CH

Ar), 129.83 (CH Ar), 130.12 (CH Ar), 131.71 (Cq), 131.98 (Cq),

135.50 (Cq Ar), 155.66 (Cq), 158.49 (C=N), 161.99 (NC=O),

168.48 (C=O).

(CH₂CH=CH₂),

118.92

(Cq), 127.25

(Cq),

131.44

(CH₂CH=CH₂), 134.24 (Cq), 146.29 (C=C), 153.96 (Cq), 156.71

(C=N), 161.72 (C=O), 164.28 (C=O).

4.1.3.6. (Z)-Ethyl 3-allyl-2-((3-(isopropoxycarbonyl

)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)imino)-

4-methyl-2,3-dihydrothiazole-5-carboxylate (5d)

4.1.3.2. (Z)-Isopropyl 2-((4-oxo-3-phenylthiazolidin

-2-ylidene)amino)-4,5,6,7-tetrahydrobenzo[b]thio-

phene-3-carboxylate (4b)

1

Dark brown oil. Yield: 75%. H NMR (400 MHz, CDCl₃) δ

1.29 (6H, d, J= 6.2, CH₃), 1.33 (3H, t, J= 7.16, CH₂CH₃), 1.77-

1.84 (4H, m, CH₂), 2.60 (3H, s, CH₃), 2.63-2.69 (2H, m, CH₂),

2.71-2.77 (2H, m, CH₂), 4.27 (2H, q, J= 7.12, CH₂CH₃), 4.71-

4.72 (2H, m, CH₂CH=CH₂), 5.18 (1H, sep, J= 6.2, CH), 5.20

(1H, dd, J= 1.2 and 10.1, CH₂CH=CH₂), 5.94 (1H, ddt, J= 6.2,

10.1 and 17.0, CH₂CH=CH₂). ¹³C NMR (100 MHz, CDCl₃) δ

12.90 (CH₃), 14.39 (CH₃), 22.13 (CH₃), 22.86 (CH₂), 23.21

(CH₂), 25.22 (CH₂), 26.35 (CH₂), 47.05 (CH₂CH=CH₂), 60.95

(CH₂CH₃), 102.20 (C=C), 119.18 (Cq), 127.13 (Cq), 131.55

(CH₂CH=CH₂), 134.07 (Cq), 146.29 (C=C), 154.03 (Cq), 156.36

(C=N), 161.87 (C=O), 163.94 (C=O).

1

Dark yellow oil. Yield: 60%. H NMR (400 MHz, CDCl₃) δ

1.20 (6H, d, J= 6.0, CH₃), 1.80-1.89 (4H, m, CH₂), 2.60-2.68

(2H, m, CH₂), 2.76-2.93 (2H, m, CH₂), 3.84 (2H, s, SCH₂), 5.85

(1H, sep, J= 6.2, CH), 7.30-7.32 (2H, m, ArH), 7.50-7.56 (3H, m,

ArH) . ¹³C NMR (100 MHz, CDCl₃) δ 22.00 (CH₃), 22.27 (CH₂),

22.98 (CH₂), 25.11 (CH₂), 25.35 (CH₂), 34.66 (SCH₂), 61.61

(CH), 119.33 (Cq), 129.11 (CH Ar), 129.83 (CH Ar), 130.13 (CH

Ar), 131.72 (Cq), 132.01 (Cq), 135.46 (Cq Ar), 155.49 (Cq),

158.46 (C=N), 161.99 (NC=O), 168.95 (C=O).

4.1.3.3. (Z)-Ethyl 2-((3-allyl-4-oxothiazolidin-2-

ylidene)amino)-4,5,6,7-tetrahydrobenzo[b]thio-

phene-3-carboxylate (4c)

4.1.3.7. (Z)-Ethyl 2-((3-(ethoxycarbonyl)-4,5,6,7-

tetrahydrobenzo[b]thiophen-2-yl)imino)-4-oxo-3-

phenylthiazolidine-5-carboxylate (6a)

1

Pale orange amorphous solid. Yield: 68%, m.p. 94-95 ºC. H

NMR (400 MHz, CDCl₃) δ 1.29 (3H, t, J= 7.1, CH₃), 1.75-1.86

(4H, m, CH₂), 2.67 (2H, t, J= 6.0, CH₂), 2.76 (2H, t, J= 6.1, CH₂),

3.90 (2H, s, SCH₂), 4.24 (2H, q, J= 7.1, CH₂CH₃), 4.46 (2H, d,

J= 6.0, CH₂CH=CH₂), 5.24 (1H, d, J= 10.1, CH₂CH=CH₂), 5.34

(1H, d, J= 17.1, CH₂CH=CH₂), 5.95 (1H, ddt, J= 6.1, 10.1 and

16.9, CH₂CH=CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 14.44 (CH₃),

22.61 (CH₂), 23.02 (CH₂), 25.0 (CH₂), 26.18 (CH₂), 33.39

(SCH₂), 45.50 (CH₂CH=CH₂), 60.04 (OCH₂), 119.06

Pale orange amorphous solid. Yield: 60%, m.p. 130-131 ºC.

¹H NMR (400 MHz, CDCl₃) δ 1.30 (6H, t, J= 7.1, CH₃), 1.80-

1.89 (4H, m, CH₂), 2.75-2.78 (2H, m, CH₂), 2.92-2.95 (2H, m,

CH₂), 4.25 (4H, q, J= 7.0, CH₂CH₃), 5.28 (1H, SCH), 7.33-7.34

(2H, m, CH Ar), 7.35-7.54 (3H, m, CH Ar). ¹³C NMR (100 MHz,

CDCl₃) δ 14.05 (CH₃), 22.24 (CH₂), 22.96 (CH₂), 25.11 (CH₂),

25.32 (CH₂), 54.00 (SCH), 62.87 (CH₂CH₃), 119.51 (Cq), 129.10

(CH Ar), 129.97 (CH Ar), 130.33 (CH Ar), 130.94 (Cq), 131.17

(Cq), 132.32 (Cq), 135.05 (Cq Ar), 154.48 (C=N), 158.32

(NC=O), 161.59 (C=O), 165.49 (C=O).

(CH₂CH=CH₂),

120.12

(Cq), 129.52

(Cq),

130.55

(CH₂CH=CH₂), 134.50 (Cq), 151.82 (Cq), 155.86 (C=N), 163.60

(NC=O), 171.00 (C=O).

4.1.3.8. (Z)-Ethyl 3-allyl-2-((3-(ethoxycarbonyl)-

4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)imino)-4-

oxothiazolidine-5-carboxylate (6c)

4.1.3.4. (Z)-Isopropyl 2-((3-allyl-4-oxothiazolidin-

2-ylidene)amino)-4,5,6,7-tetrahydrobenzo[b]thio-

phene-3-carboxylate (4d)

1

Pale orange amorphous solid. Yield: 50%, m.p. 80-81 ºC. H

Pale yellow amorphous solid. Yield: 55%, m.p. 133-134 ºC.

¹H NMR (400 MHz, CDCl₃) δ 1.04 (6H, d, J= 6.2, CH₃), 1.53-

1.61 (4H, m, CH₂), 2.42-2.45 (2H, m, CH₂), 2.50-2.53 (2H, m,

CH₂), 3.66 (2H, s, SCH₂), 4.23 (2H, d, J= 6.02, CH₂CH=CH₂),

4.91 (1H, sep, J= 6.25, CH), 5.01 (1H, dd, J= 1.2 and 10.1,

CH₂CH=CH₂), 5.12 (1H, dd, J= 1.2 and 17.1, CH₂CH=CH₂),

5.71 (1H, ddt, J= 6.1, 10.1 and 16.2, CH₂CH=CH₂). ¹³C NMR

(100 MHz, CDCl₃) δ 22.27 (CH₃), 22.87 (CH₂), 23.25 (CH₂),

25.22 (CH₂), 26.44 (CH₂), 33.52 (SCH₂), 45.72 (CH₂CH=CH₂),

67.54 (CH), 119.40 (CH₂CH=CH₂), 120.61 (Cq), 129.73 (Cq),

130.83 (CH₂CH=CH₂), 134.60 (Cq), 151.88 (Cq), 156.17 (C=N),

163.17 (NC=O), 171.19 (C=O).

NMR (400 MHz, CDCl3) δ 1.30 (6H, t, J= 7.2, CH₃), 1.77-1.84

(4H, m, CH₂), 2.67-2.70 (2H, m, CH₂), 2.77-2.80 (2H, m, CH₂),

4.27 (4H, q, J= 7.2, CH₂), 4.54 (2H, d, J= 6.0, CH₂CH=CH₂),

4.98 (1H, dd, J= 1.6 and 17.0, CH₂CH=CH₂), 5.13 (1H, dd, J=

1.2 and 10.4, CH₂CH=CH₂), 5.96 (1H, ddt, J= 5.1, 10.2 and 17.1,

CH₂CH=CH₂), 6.08 (1H, s, SCH). ¹³C NMR (100 MHz, CDCl₃) δ

14.59 (CH₃), 14.69 (CH₃), 23.04 (CH₂), 23.42 (CH₂), 25.44

(CH₂), 26.54 (CH₂), 47.31 (CH₂CH=CH₂), 59.94 (SCH), 61.16

(CH₂CH₃), 117.87 (CH₂CH=CH₂), 119.05 (Cq), 127.34 (Cq),

131.72 (CH₂CH=CH₂), 134.46 (Cq), 146.47 (Cq), 154.46 (Cq),

156.77 (C=N), 161.99 (NC=O), 164.54 (C=O), 166.11 (C=O).

4.1.3.9. (Z)-2-(2-((3-(Ethoxycarbonyl)-4,5,6,7-

tetrahydrobenzo[b]thiophen-2-yl)imino)-4-oxo-3-

phenylthiazo-lidin-5-yl)acetic acid (7a)

4.1.3.5. (Z)-Ethyl 3-allyl-2-((3-(ethoxycarbonyl)-

4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)imino)-4-

methyl-2,3-dihydrothiazole-5-carboxylate (5c)

1

Red amorphous solid. Yield: 80%, m.p. 99-100 ºC. H NMR

1

Pale orange amorphous solid. Yield: 40%, m.p. 60-61 ºC. H

(400 MHz, CDCl₃) δ 1.24 (3H, t, J= 7.4, CH₃), 1.74-1.81 (4H, m,

CH₂), 2.61-2.65 (2H, m, CH₂), 2.69-2.74 (2H, m, CH₂), 3.10 (1H,

NMR (400 MHz, CDCl₃) δ 1.29 (3H, t, J= 7.1, CH₃), 1.31 (3H, t,

dd, J= 8.9, 17.6, CHa), 3.35 (1H, dd, J= 3.2, 17.6, CHb), 4.17

(2H, q, J= 7.4), 4.53 (1H, dd, J= 3.2, 8.9, SCH), 7.38-7.43 (3H,

m, ArH), 7.49-7.53 (2H, m, ArH). ¹³C NMR (100 MHz, CDCl₃) δ

14.40 (CH₃), 22.58 (CH₂), 22.97 (CH₂), 24.95 (CH₂), 26.23

(CH₂), 26.30 (CH₂), 37.50 (CH₂CO), 43.86 (SCH), 60.24

(CH₂CH₃), 119.33 (Cq), 127.92 (CH Ar), 129.11 (CH Ar),

129.26 (CH Ar), 129.55 (Cq), 134.28 (Cq Ar), 134.40 (Cq),

152.66 (Cq), 157.07 (C=N), 163.72 (NC=O), 173.03 (C=O),

174.13 (C=O).

1.84 (4H, m, CH₂), 2.65-2.68 (2H, m, CH₂), 2.73-2.76 (2H, m,

CH₂), 4.14 (2H, q, J= 7.2, CH₂), 6.22 (1H, s, SCH), 7.07-7.25

(7H, m, CH Ar), 7.30-7.36 (3H, m, CH Ar). ¹³C NMR (100 MHz,

CDCl₃) δ 14.33 (CH₃), 22.69 (CH₂), 23.08 (CH₂), 25.06 (CH₂),

26.28 (CH₂), 59.34 (CH₂CH₃), 100.49 (SCH), 117.99 (Cq),

128.38 (CH Ar), 128.45 (CH Ar), 128.54 (CH Ar), 128.60 (CH

Ar), 128.73 (CH Ar), 128.85 (CH Ar), 128.93 (CH Ar), 128.99

(CH Ar), 129.10 (CH Ar), 129.32 (CH Ar), 130.11 (Cq), 134.19

(Cq), 140.42 (Cq Ar), 164.50 (C=O).

4.1.3.10. (Z)-2-(2-((3-(Isopropoxycarbonyl)-

4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)imino)-4-

oxo-3-phenyl-thiazolidin-5-yl)acetic acid (7b)

4.1.3.14. (Z)-Isopropyl 2-((3,4-diphenylthiazol-

2(3H)-ylidene)amino)-4,5,6,7-tetrahydrobenzo

[b]thiophene-3-carboxylate (8b)

1

Dark orange oil. Yield: 70%. H NMR (400 MHz, CDCl₃) δ

Yellow crystalline solid. Yield: 80%, m.p. 140-141 ºC. H

1.22 (6H, d, J= 6.3, CH₃), 1.74-1.80 (4H, m, CH₂), 2.61-2.62

(2H, m, CH₂), 2.69-2.72 (2H, m, CH₂), 3.08 (1H, dd, J= 9.2, 17.9,

CHa), 3.34 (1H, dd, J= 3.5, 17.9, CHb), 4.52 (1H, dd, J= 3.5,

9.2, SCH), 5.09 (1H, sep, J= 6.3, CH), 7.38-7.44 (3H, m, ArH),

7.47-7.51 (2H, m, ArH). ¹³C NMR (100 MHz, CDCl₃) δ 22.02

(CH₃), 22.05 (CH₃), 22.63 (CH₂), 22.98 (CH₂), 24.94 (CH₂),

26.30 (CH₂), 37.64 (CH₂CO), 43.81 (SCH), 67.49 (CH), 119.43

(Cq), 127.89 (CH Ar), 129.01 (CH Ar), 129.21 (CH Ar), 129.34

(Cq), 134.25 (Cq Ar), 134.27 (Cq), 152.70 (Cq), 157.01 (C=N),

163.14 (NC=O), 173.05 (C=O), 173.80 (C=O).

NMR (400 MHz, CDCl₃) δ 1.19 (6H, d, J= 6.2, CH₃), 1.76-1.82

(4H, m, CH₂), 2.63-2.66 (2H, m, CH₂), 2.72-2.75 (2H, m, CH₂),

5.09 (1H, sep, J= 6.2, CH), 6.23 (1H, s, SCH), 7.07-7.09 (2H, m,

CH Ar), 7.18-7.34 (8H, m, CH Ar). ¹³C NMR (100 MHz,

CDCl₃) δ 21.97 (CH₃), 22.80 (CH₂), 23.16 (CH₂), 25.05 (CH₂),

26.33 (CH₂), 67.01 (CH), 99.46 (SCH), 117.84 (Cq), 126.86

(Cq), 127.95 (CH Ar), 128.31 (CH Ar), 128.38 (CH Ar), 128.62

(CH Ar), 128.64 (CH Ar), 128.90 (CH Ar), 131.01 (Cq), 134.14

(Cq), 137.00 (Cq Ar), 139.98 (Cq Ar), 152.36 (Cq), 161.67 (Cq),

163.81 (C=O).

4.1.3.11. (Z)-2-(3-Allyl-2-((3-(ethoxycarbonyl)-

4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)imino)-4-

oxothiazolidin-5-yl)acetic acid (7c)

4.1.3.15. (Z)-Ethyl 2-((3-allyl-4-phenylthiazol-

2(3H)-ylidene)amino)-4,5,6,7-

tetrahydrobenzo[b]thiophene-3-carboxylate (8c)

1

Dark red oil. Yield: 86%. H NMR (400 MHz, CDCl₃) δ 1.29

Green oil. Yield: 78%. H NMR (400 MHz, CDCl₃) δ 1.30

(3H, t, J= 7.1, CH₃), 1.78-1.80 (4H, m, CH₂), 2.64-2.65 (2H, m,

CH₂), 2.72-2.75 (2H, m, CH₂), 2.91 (1H, dd, J= 9.8, 17.9, CHa),

3.33 (1H, dd, J= 3.5, 17.9, CHb), 4.24 (2H, q, J= 7.1, CH2), 4.39

(1H, dd, J= 3.5, 9.8, SCH), 4.45 (2H, d, J= 5.8, CH₂CH=CH₂),

5.23 (1H, d, J= 10.2, CH₂CH=CH₂), 5.33 (1H, d, J= 17.1,

CH₂CH=CH₂), 5.93 (1H, ddt, J= 6.1, 10.1 and 16.9,

CH₂CH=CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 14.44 (CH₃),

22.60 (CH₂), 23.01 (CH₂), 25.01 (CH₂), 26.18 (CH₂), 37.53

(CH₂CO), 44.01 (SCH), 45.61 (CH₂CH=CH₂), 60.18 (OCH₂),

119.01 (CH₂CH=CH₂), 120.26 (Cq), 129.72 (Cq), 130.42

(CH₂CH=CH₂), 134.49 (Cq), 151.75 (Cq), 154.85 (C=N), 163.71

(NC=O), 174.51 (C=O), 174.60 (C=O).

(3H, t, J= 7.1, CH₃), 1.75-1.86 (4H, m, CH₂), 2.69 (2H, t, J= 6.0,

CH₂), 2.78 (2H, t, J= 6.0, CH₂), 4.26 (2H, q, J= 7.1, CH₂CH₃),

4.54 (2H, d, J= 4.8, CH₂CH=CH₂), 4.98 (1H, dd, J= 10.3 and

17.2, CH₂CH=CH₂), 5.13 (1H, dd, J= 10.3 and J= 17.1,

CH₂CH=CH₂), 5.95 (1H, ddt, J= 5.2, 10.3 and 17.1,

CH₂CH=CH₂), 6.08 (1H, s, SCH), 7.37-7.40 (2H, m, Ar H), 7.43-

7.45 (3H, m, Ar H). ¹³C NMR (100 MHz, CDCl₃) δ 14.44 (CH₃),

22.87 (CH₂), 23.22 (CH₂), 25.22 (CH₂), 26.40 (CH₂), 48.55

(CH₂CH=CH₂), 59.72 (CH₂CH₃), 99.11 (SCH), 117.64

(CH₂CH=CH₂), 123.08 (Cq), 126.68 (Cq), 128.67 (CH Ar),

129.21 (CH Ar), 129.54 (CH Ar), 130.88 (Cq Ar), 134.21 (Cq),

138.06 (Cq), 140.51 (Cq), 161.45 (Cq), 164.45 (C=O).

4.1.3.12. (Z)-2-(3-Allyl-2-((3-(isopropoxycarbonyl

)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)imino)-

4-oxothiazolidin-5-yl)acetic acid (7d)

4.1.3.16. (Z)-Isopropyl 2-((3-allyl-4-phenylthiazol-

2(3H)-ylidene)amino)-4,5,6,7-

tetrahydrobenzo[b]thiophene-3-carboxylate (8d)

1

Pale orange amorphous solid. Yield: 90%, m.p. 139-140 ºC.

¹H NMR (400 MHz, CDCl₃) δ 1.28 (6H, dd, J= 2.0 and 6.3,

CH₃), 1.76-1.83 (4H, m, CH₂), 2.64-2.67 (2H, m, CH₂), 2.72-2.75

(2H, m, CH₂), 2.90 (1H, dd, J= 9.9, 17.9, CHa), 3.34 (1H, dd, J=

3.5, 17.9, CHb), 4.40 (1H, dd, J= 3.5, 9.9, SCH), 4.46 (2H, d, J=

5.9, CH₂CH=CH₂), 5.14 (1H, sep, J= 6.3, CH), 5.24 (1H, dd, J=

1.0 and 10.2, CH₂CH=CH₂), 5.34 (1H, dd, J= 1.24 and 17.1,

CH₂CH=CH₂), 5.93 (1H, ddt, J= 6.1, 10.1 and 16.3,

CH₂CH=CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 22.05 (CH₃),

22.08 (CH₃), 22.63 (CH₂), 23.01 (CH₂), 25.00 (CH₂), 26.20

(CH₂), 37.57 (CH₂CO), 43.98 (SCH), 45.57 (CH₂CH=CH₂),

67.55 (CH), 119.21 (CH₂CH=CH₂), 120.60 (Cq), 129.74 (Cq),

130.44 (CH₂CH=CH₂), 134.29 (Cq), 151.54 (Cq), 154.80 (C=N),

163.28 (NC=O), 172.63 (C=O), 174.70 (C=O).

Dark red oil. Yield: 65%. H NMR (400 MHz, CDCl₃) δ 1.28

(6H, d, J= 6.4, CH₃), 1.75-1.84 (4H, m, CH₂), 2.67-2.70 (2H, m,

CH₂), 2.75-2.78 (2H, m, CH₂), 4.62 (2H, s, CH₂CH=CH₂), 5.01

(1H, d, J= 16.9, CH₂CH=CH₂), 5.15 (1H, d, J= 16.8,

CH₂CH=CH₂), 5.19 (1H, m, CH), 5.94 (1H, ddt, J= 14.9, 6.8 and

7.4, CH₂CH=CH₂), 6.14 (1H, s, SCH), 7.36-7.40 (2H, m, CH Ar),

7.41-7.47 (3H, m, CH Ar). ¹³C NMR (100 MHz, CDCl₃) δ 14.46

(CH₃), 22.87 (CH₂), 23.23 (CH₂), 25.23 (CH₂), 26.43 (CH₂),

48.44 (CH₂CH=CH₂), 59.60 (CH), 98.40 (SCH), 117.49

(CH₂CH=CH₂), 121.68 (Cq), 123.11 (Cq), 123.70 (Cq), 125.02

(Cq), 128.53 (CH Ar), 128.68 (CH Ar), 128.88 (CH Ar), 129.21

(CH Ar), 129.55 (CH Ar), 130.09 (Cq Ar), 130.88

(CH₂CH=CH₂), 132.38 (Cq), 134.23 (Cq), 140.55 (Cq), 164.46

(C=O).

4.1.3.13. (Z)-Ethyl 2-((3,4-diphenylthiazol-2(3H)-

ylidene)amino)-4,5,6,7-

tetrahydrobenzo[b]thiophene-3-carboxylate (8a)

4.2. Crystallography

Crystallographic data were collected on

diffractometer at 296 K. The frames were integrated with the

Bruker SAINT software package using narrow-frame

algorithm. Data reduction and empirical absorption corrections

a

Bruker

Pale yellow amorphous solid. Yield: 65%, m.p. 120-121 ºC.

a

¹H NMR (400 MHz, CDCl₃) δ 1.17 (3H, t, J= 7.2, CH₃), 1.75-

were carried out using the multi-scan method (SADABS). The

structure was solved by direct methods and refined with

SHELXS97 [31]. All non-H-atoms were refined anisotropically

and H-atoms were constrained at their estimated positions using a

riding model. The thermal ellipsoid diagram was generated with

ORTEP3 [32]. The crystals were isolated from a tetrahydrofuran

and hexane mixture at room temperature.

into five different concentrations, adjusted to 25 µM and added to

the respective wells. The plate was then incubated for 24 hours at

37 ºC in 5% CO₂. In the next step, propidium iodide (400 ng∙mL^-

¹) was added and the test samples were incubated for 5 minutes.

Aliquots of each well were collected and the number of viable

parasites, based on parasite motility, was assessed in a Neubauer

chamber. The percentage of inhibition was calculated in relation

to untreated cultures. The IC₅₀was calculated using non-linear

regression with the Prism 4.0 GraphPad software. Benznidazole

was used as the reference drug [1,5].

The fractional atomic coordinates and isotropic temperature

parameters (Å²), the interatomic bond distances (Å) and angles

(º) and the view of the packing showing the inter- and

intramolecular interactions that stabilize the crystal structures for

9 and 8b can be found in the supplementary material.

4.4.3. Cruzain inhibition

Procruzain truncated at the C-terminal was expressed and

purified using a modified protocol (Lee, Balouch and Craik,

unpublished results). A 0.5 mg/mL solution of procruzain (in 100

mM sodium acetate pH 5.5, 10 mM EDTA, 5mM DTT and 1M

NaCl, pH adjusted to dialyzed in 10 mM Tris buffer pH 7.5,

inhibited with the covalent reversible inhibitor methyl

methanethiosulfonate (MMTS), and dialyzed again in the same

buffer. Finally, the protein was purified in a MONO-Q anion

exchange column, using 0 to 500 mM NaCl gradient in 10 mM

Tris buffer (pH 7.5) [35].

4.3. Cytotoxicity assay (MTT)

The cytotoxicity was evaluated using the enzymatic reduction

of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) assay to produce formazan crystals [33,34]. Cells

(Macrophages J774) were seeded at 1.5 x 10⁵cells per well in

96-well plates in RPMI-1640 medium containing 10% fetal

bovine serum for 16 hours. Cells were exposed to different

concentrations of thiophene derivatives (10-100 µmol∙L^-1)

dissolved in the vehicle (0.01%, DMSO) and maintained in

culture for 24 hours at 37 ºC. At the end of the incubation in the

presence of the samples, the cells were washed three times with

PBS pH 7.4 and incubated with 100 μL of MTT solution (5

mg∙mL^-1in RPMI) for 4 h at 37 °C. Following this incubation

period, the supernatant was discarded and DMSO (150 μL/well)

was added to each well to dissolve the formazan crystals. After

15 minutes at room temperature the absorbance was

spectrophotometrically measured at 540 nm. Three individual

wells were assayed per treatment and the results were expressed

as percentage of cell viability ([absorbance of treated

cells/absorbance of untreated cells] × 100).

Cruzain activity was measured by monitoring the cleavage of

the fluorescent substrate Z-Phe-Arg-aminomethylcoumarin (Z-

FR-AMC), in a Synergy 2 (Biotek) fluorimeter, at the Centre for

Flow Cytometry Fluorimetry at the Department of Biochemistry

and Immunology (UFMG). All assays were performed in the 96-

well plate format, with a final volume of 200 μL, in a buffer

solution of 0.1 M sodium acetate pH 5.5 in the presence of 0.1

mM of betamercaptoethanol, 0.01% of Triton X-100, 0.5 nM of

cruzain and 2.5 µM of substrate [26]. The assay was performed

with and without the pre-incubation of the compounds with the

enzyme. The initial screening was performed with 100 μM of the

inhibitors, except for compounds 5d and 8a, which were assayed

at 25 and 50 μM, respectively, due to solubility limitations. For

each assay, two independent experiments were performed, each

in triplicate and monitored for 5 minutes. Enzymatic activity was

calculated based on a comparison with a DMSO control,

considering the initial reaction rate. E64 (100 μM) was used in

the assay as a positive control. Since most compounds were

observed to be time-dependent, which is consistent with a

covalent mode of inhibition, all subsequent assays were

performed with 10 minutes of pre-incubation. The IC₅₀curves

were determined in two independent experiments, each involving

at least nine compound concentrations, in triplicate. IC₅₀curves

were determined with GraphPad Prism. The most potent

compounds were further evaluated for detergent-sensitivity at

different concentrations (0.01 and 0.1% Triton X-100) at a

concentration close to its IC₅₀. The compound 7c was also pre-

incubated in the presence of 2 mg∙mL^-1BSA to evaluate the

formation of aggregates [26].

4.4. Biological evaluation

4.4.1. Assessment of compound activity against

intracellular amastigotes

Trypanosoma cruzi CA-I/72 strain trypomastigotes were

collected from the supernatant of infected C2C12 mice myocytes

in T.75 culture flasks 7 days after the initial infection. The

trypomastigotes were counted in a hemacytometer and their

density was determined. A mixture containing 1.5 x 10⁵parasites

and 1.0 x 10⁴C2C12 cells per milliliter of culture media was

obtained and 50 uL was seeded per well in a 384-well plate with

a clear bottom for microscopic imaging. Immediately after

seeding the mixture of cells and parasites (1:15) we added 50 nL

of the compounds, starting with a 10 mM stock solution, serially

diluted. After three days of incubation the plate was fixed with

the addition of 50 uL of 8% paraformaldehyde solution. We

waited at least 1 hour to aspirate the well contents and added 0.5

µg∙mL^-1of DAPI for the staining of nucleic acid. After at least 3

hours of staining the plates were read in an ImageXpress Micro

XL system (Molecular Devices) and images were analyzed using

a dedicated algorithm developed in the MetaXpress software.

Antiparasitic activity was normalized based on negative controls

(untreated wells) and positive controls (10 µM benznidazole).

The host cell viability was based on the total number of host cells

(C2C12) in each well relative to the average number of host cells

from untreated wells (in percentage terms) [25].

4.5. Molecular docking

For docking purposes, the three-dimensional structure of

cruzain (PDB code: 1U9Q) was obtained from the RCSB protein

databank. Ligands that were already present within the receptor

in bound form were removed for the docking protocol. All of the

ligands were prepared and docked for this study in flexible

docking mode and atoms located within a range of 5.0 Å from

the amino acid residues were included in the active site. The

docking calculations and minimization were carried out in the

ArgusLab® module, with the Merck Molecular Force Field

(MMFF), and most of the parameters had a set default with

10.000 cycles per molecule for the active site cavity.

4.4.2. Toxicity toward Y strain trypomastigotes

Trypomastigotes collected from the supernatant of LLC-MK2

cells were dispensed into 96-well plates at a cell density of 4.0 x

10⁵cells/well. Test inhibitors, dissolved in DMSO, were diluted

Terrazas-Alvares, D. E. Drug Discov Today 2015, 20, 890.

For the preparation of the macromolecule, all heteroatoms,

including water molecules, were deleted. Polar hydrogen atoms

were added to the cruzain tree-dimensional structure. The partial

atomic charges of the cruzain and thiophen-2-thiourea and 2-

iminothiazolidine derivatives were then calculated using Kollman

and Gasteiger-Marsili methods, respectively. The cruzain was

placed inside a box with the number of center grid points in X x

Y x Z directions being 2.77 x 10.289 x 6.45 and a grid spacing of

0.375 Å. Lamarckian genetic algorithms were employed to

perform the docking calculations. The number of genetic

algorithm runs and the number of evaluations were set to 100 and

2.5 million, respectively. For all other parameters default settings

were used. For each of the docking cases, the lowest energy

docked conformation, according to the AutoDock scoring

function, was selected as the binding mode. Molecular models

were built to discuss the binding modes by docking using an

AutoDock Tools program.

9.

Saravanan, J.; Mohan, S.; Roy, J. J. Eur J Med Chem 2010, 45,

4365.

10.

Neres, J.; Brewer, M. L.; Ratier, L.; Botti, H.; Buschiazzo, A.;

Edwards, P. N.; Mortenson, P. N.; Charlton, M. H.; Alzari, P. M.;

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Lesyk, R. Eur J Med Chem 2014, 85, 245.

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Gewald, K.; Schinke, E.; Böttcher, H. Chem Ber 1966, 99, 94.

Arhin, F.; Bélanger, O.; Ciblat, S.; Dehbi, M.; Delorme, D.;

Dietrich, E.; Dixit, D.; Lafontaine, Y.; Lehoux, D.; Liu, J.; McKay,

G. a.; Moeck, G.; Reddy, R.; Rose, Y.; Srikumar, R.; Tanaka, K. S.

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Acknowledgments

This work was supported by the Conselho Nacional de

Desenvolvimento

Científico

e

Tecnológico

(CNPq),

Coordernação de Aperfeiçoamento de Pessoal de Nível Superior

(CAPES) and Fundação de Amparo à Pesquisa do Estado de

Alagoas (FAPEAL).

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Graphical Abstract

Design, synthesis, molecular docking and

biological evaluation of thiophen-2-

iminothiazolidine derivatives for use against

Trypanosoma cruzi

Leave this area blank for abstract info.

Silva-Júnior, E.F.^a, Silva, E.P.S.^a, França, P.H.B.^a, Silva, J.P.N.^b, Barreto, E.O.^b, Silva, E.B.^c, Ferreira, R.S.^c,

Gatto, C.C.^d, Moreira, D.R.M.^e, Siqueira-Neto, J.L.^f, Mendonça-Júnior, F.J.B.^g, Lima, M.C.A.^h, Bortoluzzi,

J.H.ⁱ, Scotti, M.T.^g, Scotti, L.^g, Meneghetti, M.R.ⁱ, Aquino, T.M.*^a, Araújo-Júnior, J.X.^a

^aMedicinal Chemistry Laboratory, Pharmacy and Nursing School, Federal University of Alagoas, Maceio,

c

Brazil. ^bCell Biology Laboratory, Federal University of Alagoas, Maceio, Brazil. Biochemistry and

Immunology Department, Biological Sciences Institute, Federal University of Minas Gerais, Belo Horizonte,

Brazil. ^dInorganic Synthesis and Crystallography Laboratory, Institute of Chemistry, University of Brasilia,

Federal District, Brazil. ^eTissue Engineering and Immunopharmacology Laboratory, Oswaldo Cruz

Foundation, Salvador, Bahia, Brazil. ^fSkaggs School of Pharmacy and Pharmaceutical Sciences, California,

San Diego La Jolla, United States of America. ^gLaboratory of Drug Synthesis and Delivery, Biological Sciences

Department, State University of Paraiba, Campus V, João Pessoa, Brazil. ^hDrug Design and Synthesis

Laboratory, National Science and Technology Institute for Pharmaceutical Innovation, Federal University of

Pernambuco, Recife, Brazil. ⁱCatalysis and Chemical Reactivity Group (GCaR), Institute of Chemistry and

Biotechnology, Federal University of Alagoas, Maceio, Brazil

Article Doi

Design, synthesis, molecular docking and biological evaluation of thiophen-2-iminothiazolidine derivatives for use against Trypanosoma cruzi

DOI: 10.1016/j.bmc.2016.07.013

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Authors:

Article abstract of DOI:10.1016/j.bmc.2016.07.013

Full text of DOI:10.1016/j.bmc.2016.07.013

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