Antileishmanial activity study and theoretical calculations for 4-amino-1,2,4-triazole derivatives

Accepted Manuscript Antileishmanial activity study and theoretical calculations for 4-amino-1,2,4triazole derivatives

Nevin Süleymanoğlu, Yasemin Ünver, Reşat Ustabaş, Şahin Direkel, Gökhan Alpaslan PII:

S0022-2860(17)30599-9

DOI:

10.1016/j.molstruc.2017.05.017

Reference:

MOLSTR 23758

To appear in:

Journal of Molecular Structure

Received Date:

07 April 2017

Revised Date:

05 May 2017

Accepted Date:

05 May 2017

Please cite this article as: Nevin Süleymanoğlu, Yasemin Ünver, Reşat Ustabaş, Şahin Direkel, Gökhan Alpaslan, Antileishmanial activity study and theoretical calculations for 4-amino-1,2,4triazole derivatives, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.05.017

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ACCEPTED MANUSCRIPT GRAPHICAL ABSTRACT

ACCEPTED MANUSCRIPT HIGHLIGHTS

 Theoretical calculations of 4-amino-1,2,4-triazole derivatives were performed by DFT method.  Structural parameters and spectral parameters of FT-IR and NMR were determined theoretically.  Compounds were tested against to Leishmania infantum promastigots.

ACCEPTED MANUSCRIPT

Antileishmanial activity study and theoretical calculations for 4-amino-1,2,4-triazole derivatives Nevin Süleymanoğlua*, Yasemin Ünverb, Reşat Ustabaşc, Şahin Direkeld, Gökhan Alpaslane

aTechnical

Sciences Vocational High School, Gazi University, 06374 Ostim, Ankara, Turkey

bDepartment

of Chemistry, Faculty of Sciences, Karadeniz Technical University, 61080

Trabzon, Turkey cDepartment

of Mathematics and Science Education, Educational Faculty, Ondokuz Mayıs

University, 55139 Kurupelit, Samsun, Turkey dDepartment

of Medical Microbiology, Faculty of Medicine, Giresun University, 28100

Giresun, Turkey eDepartment

of Medical Services and Techniques, Vocational School of Health Services,

Giresun University, 28200 Giresun, Turkey

ABSTRACT 4-amino-1,2,4-triazole derivatives; 4-amino-1-((5-mercapto-1,3,4-oxadiazole-2-yl)methyl)-3(thiophene-2-ylmethyl)-1H-1,2,4-triazole-5(4H)-one

(1)

and

4-amino-1-((4-amino-5

mercapto-4H-1,2,4-triazole-3-yl)methyl)-3-(thiophene-2-ylmethyl)-1H-1,2,4-triazole-5(4H)one (2) were studied theoretically by Density Functional Theory (DFT) method with 6311++G(d,p) basis set, structural and some spectroscopic parameters were determined. Significant differences between the experimental and calculated values of vibrational *Corresponding author. Tel: +903123548401 E-mail adress: [email protected] (N. Süleymanoğlu)

ACCEPTED MANUSCRIPT frequencies and chemical shifts were explained by the presence of intermolecular (S–H···O and S–H···N type) hydrogen bonds in structures.

The Molecular Electrostatic Potential

(MEP) maps obtained at B3LYP/6-311G++(d,p) support the existence of hydrogen bonds. Compounds were tested against to Leishmania infantum promastigots by microdilution broth assay with Alamar Blue Dye. Antileishmanial activity of 4-amino-1,2,4-triazole derivative (2) is remarkable. KEYWORDS 4-amino-1,2,4-triazole; DFT calculations; NMR chemical shifts; FT-IR vibrational frequencies; Antileishmanial activity.

1. Introduction

1,2,4-triazoles, an important group of heterocyclic compounds, exhibit important pharmacological activities such as anticonvulsant, anti-tubercular, antioxidant, antifungal, anticancer, anti-inflammatory, antimicrobial [1-3]. The other important heterocyclic system is 1,3,4-oxadiazole derivatives, which have a wide range of biological activities such as antimicrobial, anti-inflammatory, antioxidant,

antineoplastic, antituberculosis, anticancer,

antiviral and tyrosinase inhibitory activity [4-7]. In addition, 2,5 substituted oxadiazole derivatives possess anticonvulsant activity and antifungal activity [8]. Oxadiazole moiety is also used in material science in the field of liquid crystals and photo sensitizer [9]. Leishmaniasis is considered as one of the six most important infectious diseases by the World Health Organization (WHO) [10]. The disease is very common at the part of the Mediterranean basin in where Turkey is also located, in the Middle East, India and South America and in 88 countries; 350 million people are at risk [11]. Leishmaniasis, which is caused by protozoan parasites of the genus Leishmania and spread by the bite of certain types

ACCEPTED MANUSCRIPT of sandflies [12], has an important clinical and epidemiological diversity. Pentavalent antimony derivatives, Sodium stibogluconate (Pentostam®) and megluminantimoniat (Glucantime®), is the conventional treatment; however, researchers have reported that resistance to these drugs has developed [13]. For this reason, new treatment approache such as amphotericin B and its lipid formulations, injectable paromomycin, and oral miltefosine have emerged [14]. There is no effective prophylactic vaccine against the disease. Due to the toxic effects of currently used drugs and the increasing resistance to these drugs, the discovery and development of new therapeutic agents becomes important [15]. In addition to other methods, colorimetric methods are also used to determine the viability and proliferation of promastigote forms of Leishmania species. Among the colorimetric methods, the alamar blue which has several advantages such as simple to apply, low cost, environmentally friendly and easily transferable is an applicable colorimetric indicator in drug screening test with Leishmania major promastigotes [16]. In this study, structural parameters of published compounds [17]; 4-amino-1-((5-mercapto1,3,4-oxadiazole-2-yl)methyl)-3-(thiophene-2-ylmethyl)-1H-1,2,4-triazole-5(4H)-one (1) and 4-amino-1-((4-amino-5-mercapto-4H-1,2,4-triazole-3-yl)methyl)-3-(thiophene-2-ylmethyl)1H-1,2,4-triazole-5(4H)-one (2), were obtained by theoretical calculations using the Density Functional Theory (DFT) method with 6-311++G(d,p). Theoretical parameters of Fourier Transform Infrared spectra (FT-IR) and, proton and carbon-13 Nuclear Magnetic Resonance (1H and

13

C NMR) spectra were also determined for both compounds and compared with

experimental ones to investigate the structural properties. Antileishmanial activity of 4-amino1,2,4-triazole derivatives (1 and 2) were tested by Leishmania infantum promastigotes in the axenic culture with the alamar blue microdilution method. Amphotericin B was used as the reference drug. No studies have been conducted on antileishmanial activity of these

ACCEPTED MANUSCRIPT compounds until now. So this article can be a guideline for the chemical substances that can be taken into account in drug development studies for specifically Leishmania infantum.

2. Experimental and computational method

2.1. Synthesis

The 4-amino-1-((5-mercapto-1,3,4-oxadiazole-2-yl)methyl)-3-(thiophene-2-ylmethyl)-1H1,2,4-triazole-5(4H)-one

(1)

and

4-amino-1-((4-amino-5-mercapto-4H-1,2,4-triazole-3-

yl)methyl)-3-(thiophene-2-ylmethyl)-1H-1,2,4-triazole-5(4H)-one (2) were published in literature [17]. The structures of the compounds are shown in Scheme 1.

Scheme 1 here 2.2. Computational procedures

All quantum chemical computations were carried out by using Gaussian 09W [18] packaged software and GaussView 5 [19] molecular visualization software. 4-amino-1,2,4triazole derivatives (1 and 2) were optimized by DFT/B3LYP (Becke’s three parameter hybrid functional using the LYP correlation functional) [20,21] method with 6-311++G(d,p) basis set. The same method and base set were used to calculate the vibration frequencies. Two scale factors used for the calculated frequencies are 0.983 and 0.958 for frequencies smaller than 1700 cm-1 and greater than 1700 cm-1, respectively [22,23]. For proton and carbon NMR chemical shifts calculations, standard GIAO/B3LYP/6-311++G(d,p) (Gauge-Independent Atomic Orbital) approach [24,25] was used. The 1H NMR and

13C

NMR chemical shifts in

TMS scale were described by subtraction of the calculated absolute chemical shielding of

ACCEPTED MANUSCRIPT TMS in 6-311G++(d,p) values from values 31.9681 and 184.0184 ppm for 1H NMR and

13C

NMR, respectively. The equation d = R0–R enables us to calculate TMS values, where d is the chemical shift, R is the absolute shielding and R0 is the absolute shielding of TMS.

3. Pharmacology

3.1. Preparation of Leishmania infantum Promastigotes

In antiparasitic activity study, the axenic standard Leishmania infantum promastigotes MON-183 (Montpellier system) were propagated in RPMI-1640 (Roswell Park Memorial Institute) (R8758 Sigma Aldrich USA) medium which contains 10% Fetal Bovine Serum (FBS F4135 Sigma-Aldrich USA), 1% Penicillin (P3032 Sigma-Aldrich USA)

and

Streptomycin (S9137 Sigma-Aldrich USA) (100.000 units penicillin and 10 mg streptomycin). 20 mL promastigotes transferred to sterile falcon tubes from this medium was centrifuged at 1000 g for 10 min. Then, the supernatants in the tubes were removed; 10 mL sterile Phosphate-buffered saline (PBS) was added and shaken. This mixture was centrifuged at 1000 g for 10 min. The process was repeated three times. Finally, promastigotes were diluted with RPMI-1640 to a cell number of 2.5x107 cells/mL using hemocytometer [26].

3.2. In Vitro Antileishmanial Activity Test

4-amino-1,2,4-triazole derivatives (1 and 2) were dissolved in DMSO/H2O (10%). The stock solutions of compounds were prepared at concentration of 40 mg/mL by adding RPMI1640 medium without phenol red containing heat-inactivated 10% Fetal Bovine Serum (FBS), and sterilized at 0.45-μm membrane filter (Millipore, USA). Firstly, 112.5 μL RPMI-1640

ACCEPTED MANUSCRIPT medium was added in all wells from second to 10th. Secondly, 225 μL of stock solution of 4amino-1,2,4-triazole derivative (1) was added to the first well, and then half of it was transferred to the second well. From the second well to the tenth well, dilutions were made from 20 mg/mL to 39 μg/mL. Finally, 112.5 μL standard parasites were added to the wells. The 11th well containing only 225 μL solution as negative control and the 12th well containing only 225 μL parasites as positive control were used. The process was repeated for 4-amino-1,2,4-triazole derivative (2). After 20 hours of incubation at 27 ° C, 25 μL alamar blue was added. Microplates were evaluated after 24, 48 and 72 hours [26]. Amphotericin B was used as the standard control drug. Each test was repeated twice.

4. Results and discussion

4.1. Optimized geometries

By means of DFT/B3LYP method with 6-311++G(d,p) basis set, 4-amino-1,2,4-triazole derivatives (1 and 2) were optimized. Obtained geometrical parameters and theoretical geometric structures are given in Table 1 and Fig. 1, respectively. 4-amino-1,2,4-triazole derivative (1) has thiophene, triazole and oxadiazole rings, whereas 4-amino-1,2,4-triazole derivative (2) has thiophene and two triazole rings. Comparing the bond lengths and bond angles given in Table 1, it can be seen that the geometrical parameters of the same rings in both optimized molecules have too close values. The calculated S16–C14 and S16–C19 bond lengths of thiophene rings in 4-amino-1,2,4triazole derivatives (1 and 2) are [1.7473/1.7474 Å] and [1.7342/1.7343 Å], respectively. These bond lengths for some similar structures are reported as [1.712(3)/1.700(4) Å] [27], [1.725(3)/1.709(3) Å] [28] and [1.724(6)/1.740(7) Å] [29]. The calculated bond lengths of

ACCEPTED MANUSCRIPT single bonded N6–N7 and double bonded N6=C2 in triazole rings of compounds are [1.3861/1.3856 Å], respectively and [1.2971/1.2969 Å], respectively. The bond lengths obtained for some similar structures characterized by X-ray diffraction method are [1.353(2)/1.331 Å] [30], [1.379(6)/1.303(6) Å] [31] and [1.387(3)/1.321(3) Å] [32], respectively. The N6–N7–C8=120.91/120.74o bond angle of both compounds are given as 120.2(4)o [31] for similar structures in literature, and some values reported for S16–C14–C11 = 121.52/121.51o bond angle are 122.8(2) [29] and 122.21(18)o [28].

As a result of

comparing with the literature, it can be stated that the optimized geometries of both compounds reflect the molecular structures very well, and thus it can be deduced that the spectral parameters can be accurately determined.

Table 1 here Figure 1 here

4.2. Vibrational spectra and NMR spectra

GIAO 1H and

13C

chemical shift values (regarding TMS) were determined by employing

DFT/B3LYP/6-311++G(d,p) method. Experimental [17] and calculational results are presented in Tables 2 and 3, respectively. As compared experimental [17] and calculated vibrational frequencies in Table 2, for both compounds, it can be seen the remarkable differences for especially the N–H2 stretching modes, the C = O and S–H stretching vibrations. The differences of the N–H2 stretching modes for 4-amino-1,2,4-triazole derivative (1) are 89 and 125 cm-1 as asymmetric and symmetric, respectively. These differences for 4-amino-1,2,4-triazole derivative (2) are 91 and 63 cm-1, for other N–H2 stretching modes are 116 and 74 cm-1, respectively. The

ACCEPTED MANUSCRIPT differences obtained for the C = O and S–H stretching vibrations are 48 and 198 cm-1 for 4amino-1,2,4-triazole derivative (1), 13 and 181 cm-1 for 4-amino-1,2,4-triazole derivative (2), respectively. Due to these differences, it can be suggested the existence of the S–H···O and S–H···N type intermolecular hydrogen bonds in the molecular structure of the compounds.

Table 2 here Table 3 here

As can be seen in Table 3, The S–H signals observed as 14.00 ppm for 4-amino-1,2,4triazole derivative (1) and 13.63 ppm for 4-amino-1,2,4-triazole derivative (2) is calculated at 5.10 and 4.85 ppm, respectively. In addition, The N–H2 signals recorded as 5.39 ppm for 4amino-1,2,4-triazole derivatives (1 and 2) were determined as 3.94 and 3.46 ppm for 4-amino1,2,4-triazole derivative (1); 3.94 and 3.52 ppm for 4-amino-1,2,4-triazole derivative (2). Especially the significant differences observed in S–H signals (8.90 and 8.78 ppm for 4amino-1,2,4-triazole derivatives (1 and 2), respectively) can point out strong hydrogen bonds. Figs 2 and 3 show possible hydrogen bonds suggested for these compounds, respectively. On the other hand, intramolecular hydrogen bonds for compounds 1 and 2 can also be discussed. If intramolecular hydrogen bonds, N4–H29···O5, are present, then these protons are not equivalent and therefore the chemical shift values of the protons H30 and H29 must be different. However, for both compound 1 and 2, these protons were observed as singlet at 5.39 ppm (Table 3), and additionally there is no significant difference in the calculated chemical shift values. For this reason, it could not be suggested the presence of intramolecular hydrogen bonds in compounds 1 and 2.

ACCEPTED MANUSCRIPT

In order to support the existence of hydrogen bonds in molecular structures of 4-amino1,2,4-triazole derivatives (1 and 2), the molecular electrostatic potentials at the B3LYP/6311++G(d,p) of optimized geometries were computed. The MEP maps of the compounds are shown in Fig. 4. As expected, The MEP maps (Fig. 4) reveal the around O5 atoms as the negative (red and yellow) regions for both compounds and; the around N4 atom for 4-amino1,2,4-triazole derivative (1) and especially the around N31 atom for 4-amino-1,2,4-triazole derivative (2) as the positive (blue) regions.

Figure 4 here

4.3. The results of Antileishmanial Activity in vitro

The antileishmanial activity of 4-amino-1,2,4-triazole derivatives (1 and 2) were evaluated by using microdilution alamar blue method as that change of from dark blue to bright pink of the color indicated the production of parasite in 96-well microplates (Fig. 5). In the wells, PC is positive control which the color changes from blue to pink and NC is negative control, no the color change. The Minimal Inhibitor Concentration (MIC) values of compounds and amphotericin B used as a standard drug are given in the Table 4. According to obtained results, the most effective substance is 4-amino-1,2,4-triazole derivative (2) (MIC = 625 µg/mL) (Table 4) and even, 4-amino-1,2,4-triazole derivative (1) (MIC = 2500 µg/mL) can be evaluated as antiparasitic. These values are much bigger than the MIC value of the standard drug (˂39 µg/mL). However, considering the side effects of standard drugs, these compounds obtained as antiparasitic can be considered as remarkable contents in future drug development studies. In order to be able to use the compounds as a

ACCEPTED MANUSCRIPT drug, the in vitro activity of the compounds should also be determined against Leishmania amastigotes in macrophage culture and control studies should be performed in vivo animal models.

Figure 5 here Table 4 here

5. Conclusions

4-amino-1,2,4-triazole derivatives; 4-amino-1-((5-mercapto-1,3,4-oxadiazole-2-yl)methyl)3-(thiophene-2-ylmethyl)-1H-1,2,4-triazole-5(4H)-one

(1)

and

4-amino-1-((4-amino-5-

mercapto-4H-1,2,4-triazole-3-yl)methyl)-3-(thiophene-2-ylmethyl)-1H-1,2,4-triazole-5(4H)one (2), were optimized by theoretical calculations using Density Functional Theory (DFT) method with 6-311++G(d,p) basis set. Spectral parameters; vibrational frequencies and chemical shifts, and experimental data obtained from literature were compared. Because of considerable differences of the experimental and calculated data, intermolecular (S–H···O and S–H···N type) hydrogen bonds in molecular structures of 4-amino-1,2,4-triazole derivatives (1 and 2) can be suggested. Molecular Electrostatic Potential (MEP) maps obtained at B3LYP/6-311G++(d,p) support the presence of hydrogen bonds. The in vitro study against to Leishmania infantum (MON-183) by microdilution broth assay with Alamar Blue Dye shows that both compounds are antiparasitic and especially 4-amino-1,2,4-triazole derivative (2) can be evaluated in future drug development studies due to the antileishmanial activity.

ACCEPTED MANUSCRIPT Acknowledgement

The authors are grateful to Prof. Dr. Seray TÖZ and Prof. Dr. Yusuf ÖZBEL of Ege University, Faculty of Medicine, Medical Parasitology Department, providing Leishmania parasites.

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ACCEPTED MANUSCRIPT N N

N N CH2 N

N S

CH2

N

O

O

CH2

SH N

N S

CH2

O

N

NH2

NH2

(1)

(2)

N

SH

NH2

Scheme 1. The structures of 4-amino-1-((5-mercapto-1,3,4-oxadiazole-2-yl)methyl)-3(thiophene-2-ylmethyl)-1H-1,2,4-triazole-5(4H)-one

(1)

and

4-amino-1-((4-amino-5-

mercapto-4H-1,2,4-triazole-3-yl)methyl)-3-(thiophene-2-ylmethyl)-1H-1,2,4-triazole-5(4H)one (2).

ACCEPTED MANUSCRIPT

Fig. 1. The molecular conformations obtained by DFT/B3LYP/6-311G++(d,p) method; a) 4amino-1,2,4-triazole derivative (1), b) 4-amino-1,2,4-triazole derivative (2).

ACCEPTED MANUSCRIPT N N N S

N

O

S

NH2

H O

N NH2

N

O N

S

N

O HS N N

a

N N N S

N NH2

N

O

S

H

S

H N

O

H

N O

N

O

b

HS

N

N N

Fig. 2. The hydrogen bonds suggested for 4-amino-1,2,4-triazole derivative (1); a) S27– H28···O5 strong intermolecular hydrogen bond, b) S27–H28···N4 weak intermolecular hydrogen bond.

ACCEPTED MANUSCRIPT N N N

H2N

H

NH2

N

S

S

N

N

NH2

N

O

O

S

N

H2N

N

N HS

a

N

N

N N N S

N NH2

N

N

S

H

S

H N

NH2

H

O

N O

H2N

b

N

N

HS

N

N N

N N N S

N NH2

N

N

S H

NH2

H

O H

N N

HS

c

NH2

O N

N N

N N

S

Fig. 3. The hydrogen bonds suggested for 4-amino-1,2,4-triazole derivative (2); a) S27– H28···O5 strong intermolecular hydrogen bond, b) S27–H28···N4 weak hydrogen bond, c) S27–H28···N31 weak intermolecular hydrogen bond.

intermolecular

ACCEPTED MANUSCRIPT

Fig. 4. The molecular electrostatic potential maps computed at B3LYP/6-311G++(d,p); a) 4amino-1,2,4-triazole derivative (1), b) 4-amino-1,2,4-triazole derivative (2).

ACCEPTED MANUSCRIPT

Fig. 5. Antileishmanial activity in vitro against to the axenic standard MON-183 Leishmania promastigotes; a) 4-amino-1,2,4-triazole derivative (1), b) 4-amino-1,2,4-triazole derivative (2), c) Amphotericin B, and dilution concentrations from 20 mg/mL to 39 μg/mL.

ACCEPTED MANUSCRIPT Table 1 Some optimized parameters obtained DFT method with 6-311++G(d,p) basis set. 4-amino-1,2,4-triazole derivatives (1) (2) Bond Lengths (Å) C1–N3 C1–N7 C1–O5 N3–N4 N3–C2 N6–C2 N6–N7 C14–C15 C14–S16 C15–C17 C17–C19 C19–S16 C22–N25 C22–O26 C22–N26 N24–N25 N24–C23 C23–O26 C23–N26 N26–N31 C22–S27

1.4003 1.3763 1.2170 1.3952 1.3805 1.2971 1.3861 1.3676 1.7473 1.4272 1.3644 1.7342 1.2902 1.3619 ----1.4046 1.2839 1.3711 --------1.7523

1.4008 1.3752 1.2177 1.3952 1.3803 1.2969 1.3856 1.3676 1.7474 1.4274 1.3644 1.7343 1.3029 ----1.3779 1.3926 1.3018 -----1.3782 1.3975 1.7639

120.91 125.09 129.94 117.45 ----116.92 ----113.22 --------111.60 110.56 121.52 113.50

120.74 125.04 127.23 ----122.98 ----121.67 ----110.43 130.03 111.59 110.54 121.51 113.52

64.98 -104.64 177.45 -78.28 179.26 -----179.98 -----

-65.03 104.49 -177.50 77.36 -----179.23 -----179.92

Bond Angles (°) N6–N7–C8 N4–N3–C1 C8–C23–N24 C8–C23–O26 C8–C23–N26 O26–C22–S27 N26–C22–S27 O26–C22–N25 N26–C22–N25 N31–N26–C22 S16–C19–C17 S16–C14–C15 S16–C14–C11 C17–C15–C14

Torsion Angles (°) S16–C14–C11–C2 C14–C11–C2–N6 C11–C2–N6–N7 N6–N7–C8–C23 C8–C23–O26–C22 C8–C23–N26–C22 C23–O26–C22–S27 C23–N26–C22–S27

ACCEPTED MANUSCRIPT N31–N26–C22–S27 N4–N3–C1–O5

-----3.80

-0.33 4.63

Table 2 Comparison of the experimental and calculated vibrational frequencies (cm-1).

Assignments

4-amino-1,2,4-triazole derivatives (1) (2) * * Experimental Calculated Experimental Calculated

ν(H29-N4-H30)as

3305

3394

3301

3392

ν(H29-N4-H30)s

3199

3324

3260

3323

ν(H32-N31-H33)as

-----

-----

3301

3417

ν(H32-N31-H33)s

-----

-----

3260

3334

ν(S27-H28)

2768

2570

2753

2572

ν(C1=O5)

1667

1715

1699

1712

ν(C2=N6)

1581

1603

1622

1602

ν(C23-O26-C22)

1162

1150

-----

-----

ν, Stretching; s, symmetric; as, asymmetric *Referred [17]

ACCEPTED MANUSCRIPT Table 3 Theoretical and experimental 1H and

13

C isotropic chemical shifts (with respect to TMS, all

values in ppm) in DMSO-d6.

C1 C2 C8 C11 C14 C15 C17 C19 C22 C23 H9 H10 H12 H13 H18 H20 H21 H28 H29 H30 H32 H33

4-amino-1,2,4-triazole derivatives (1) (2) Experimental* Calculated Experimental* Calculated 153.23 158.91 153.42 159.45 148.10 155.95 147.98 155.38 40.51 43.37 40.57 43.92 25.39 30.60 25.44 30.53 137.38 152.52 137.62 152.78 127.04 131.15 126.95 131.62 125.63 129.85 125.55 129.91 127.37 137.03 127.34 137.24 178.47 172.97 166.84 161.47 159.49 171.79 147.38 160.03 5.02(s,2H) 4.88 4.92(s,2H) 5.16 5.02(s,2H) 5.31 4.92(s,2H) 4.91 4.09(s,2H) 3.98 4.07(s,2H) 4.49 4.09(s,2H) 4.49 4.07(s,2H) 3.96 7.36(s,1H) 7.25 7.36(s,1H) 7.31 6.94(s,2H) 7.14 6.93(s,2H) 7.11 6.94(s,2H) 7.38 6.93(s,2H) 7.42 14.00(s,1H) 5.10 13.63(s,1H) 4.85 5.39(s,2H) 3.94 5.39(s,4H) 3.94 5.39(s,2H) 3.46 5.39(s,4H) 3.52 ---------5.39(s,4H) 4.14 ---------5.39(s,4H) 4.08

*Referred

[17]

Atom

ACCEPTED MANUSCRIPT Table 4 The Minimal Inhibitor Concentration (MIC) values obtained against to the axenic standard MON-183 Leishmania promastigotes. 4-amino-1,2,4-triazole derivatives (1) (2) Amphotericin B

MIC values (µg/mL) 2500 625 ˂39