Antileishmanial activity of novel indolyl–coumarin hybrids: Design, synthesis, biological evaluation, molecular docking study and in silico ADME prediction

Accepted Manuscript Antileishmanial activity of novel indolyl-coumarin hybrids: Design, synthesis, biological evaluation, molecular docking study and in silico ADME prediction Jaiprakash N. Sangshetti, Firoz A. Kalam Khan, Abhishek A. Kulkarni, Rajendra H. Patil, Amol. M. Pachpinde, Kishan S. Lohar, Devanand B. Shinde PII: DOI: Reference:

S0960-894X(15)30398-X http://dx.doi.org/10.1016/j.bmcl.2015.12.085 BMCL 23446

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

2 September 2015 19 December 2015 24 December 2015

Please cite this article as: Sangshetti, J.N., Kalam Khan, F.A., Kulkarni, A.A., Patil, R.H., M. Pachpinde, Amol., Lohar, K.S., Shinde, D.B., Antileishmanial activity of novel indolyl-coumarin hybrids: Design, synthesis, biological evaluation, molecular docking study and in silico ADME prediction, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.12.085

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Antileishmanial activity of novel indolyl-coumarin hybrids: Design, synthesis, biological evaluation, molecular docking study and in silico ADME prediction Jaiprakash N. Sangshetti a,*, Firoz A. Kalam Khan a, Abhishek A. Kulkarni a, Rajendra H. Patil b, , Amol. M. Pachpinde c, Kishan S. Lohar d Devanand B. Shinde e a

Dr. Rafiq Zakaria Campus, Y.B. Chavan College of Pharmacy, Aurangabad 431001, (M.S.), India b Department of Biotechnology, Savitribai Phule Pune University, Pune 411007, (M.S.), India c Department of Chemistry, Jawahar Art Science and Commerce College, Andur, Osmanabad 413603, (M. S.), India d Materials Research Laboratory, Srikrishna Mahavidyalaya Gunjoti, Omerga, Osmanabad 413 613, (M. S.), India e Shivaji University, Vidyanagar, Kolhapur 416 004, (M.S.), India

*Corresponding author. Tel./fax: +91 240 2381129. E-mail address: [email protected] ABSTRACT In present work we have designed and synthesized total twelve novel 3-(3-(1H-indol-3-yl)-3phenylpropanoyl)-4-hydroxy-2H-chromen-2-one derivatives 13(a-l) using Ho3+ doped CoFe2O4 nanoparticles as catalyst and evaluated for their potential antileishmanial and antioxidant activities. The compounds 13a, 13d and 13h were found to possess significant antileishmanial activity (IC50 value= 95.50, 95.00 and 99.00 μg/mL, respectively) when compared to the standard sodium stibogluconate (IC50= 490.00 μg/mL). The compounds 13a (IC50= 12.40 μg/mL), 13d (IC50= 13.49 μg/mL), 13g (IC50= 13.24 μg/mL) and 13l (IC50= 13.74 μg/mL) had shown good antioxidant activity when compared with standards butylated hydroxy toluene (IC50= 16.5 μg/mL) and ascorbic acid (IC50= 12.8 μg/mL). After performing molecular docking studies, it was found that compounds 13a and 13d had potential to inhibit pteridine reductase 1 enzyme. In silico ADME pharmacokinetic parameters had shown promising results and none of the synthesized compounds had violated Lipinski’s rule of five. Thus, suggesting that compounds from the present series can serve as important gateway for the design and development of new antileishmanial as well as antioxidant agent. Keywords: Indolyl-coumarin hybrids; Ho3+ doped CoFe2O4 nanoparticles; Antileishmanial; Antioxidant; Molecular docking studies. 1

Leishmaniasis is a vector-borne fatal parasitic disease caused by different species of the genus Leishmania protozoan and transmitted to humans by the bite of female phlebotomine sand fly and mainly affects people living below poverty line. World Health Organization (WHO) classified leishmaniasis as a major tropical disease that ranks second after malaria.1-8 It is estimated that 12 million people worldwide are infected by over 20 different species of Leishmania and about 350 million people living in the endemic areas are at the risk to be infected.

9, 10

No effective vaccine is available against leishmaniasis, so chemotherapy is the

only effective way to treat all forms of the disease. 11, 12 However, the drugs currently used for the treatment of human cutaneous and visceral leishmaniasis are toxic; having severe adverse reactions

(such

as

pancreatitis,

pancytopenia,

reversible

peripheral

neuropathy,

nephrotoxicity, cardiotoxicity, bone pain, myalgia etc.) and resistance growing towards them had also questioned their use.

13, 14

Therefore, development of novel, effective and safe

antileishmanial agents, with reduced side effects, is a major priority for health researchers. Free radicals can be defined as reactive chemical species having a single unpaired electron in an outer orbit. 15 The majority of free radicals that damages biological systems are oxygenfree radicals (reactive oxygen species), the by-products of aerobic organism’s cellular process which generally initiates autocatalytic reactions so that molecules to which they react are themselves converted again into free radicals which in turn mediates chain reaction to damage (oxidative stress) cell structures like proteins, nuclear substances etc.

16, 17

This

oxidative damage accumulates during the life cycle of humans and has been implicated in aging (neurodegeneration) and age related diseases such as cardiovascular disease, Alzheimer's disease, cancer, and other similar chronic conditions.

18-20

Thus, the discovery

and development of novel synthetic radical scavengers attained great importance in organic chemistry. Our present work entails the synthesis of such derivatives which has a skeleton that contains an orderly arrangement of coumarin and indole cores fused with chalcone like structure (Fig. 1). The rationale behind using these three scaffolds in our design is based on the diversity of these moieties in nature as well as in various pharmaceuticals (alone or in combination). For example: 2H-chromene-2-ones, the oxygenated heterocycles (natural coumarins) are found to be present in numerous natural products with wide range of biological activities.21, 22 Iranshahi et al. reported that a prenylated sesquiterpene coumarin Umbelliprenin (1), present as one of the components in the extract of Ferula szowitsiana (Apiaceae) roots, found to possess significant antileishmanial activity with an IC50 value of 2

13.3 µM against L. major promastigotes.

23

Other cores such as indole and chalcone were

also found to have antileishmanial properties. 24-26 Representative examples of them includes: an indole alkaloid Corynantheine (2), present in the bark of Corynanthe pachyceras (Rubiaceae) exhibited antileishmanial activity against L. major promastigotes with an IC50 value of about 3 µM, reported by Staerk et al.

27

Kharazmi et al. reported that Licochalcone

A (3), an oxygenated chalcone, inhibits in vitro growth of both L. major and L. donovani promastigote form. Its IC50 value was found to be in the range 2.5-4 µg/ml against L. major promastigotes.28 These scaffolds also have been well reported for antioxidant activity (Fig. 2). Wu et al. reported that they had studied antioxidant properties of coumarin ingredients present in the crude extract, fractions and ingredients of Cortex Fraxini (4) and the obtained results were found to be satisfactory. 29 Marques et al. reported that best antioxidant results were obtained for the indole derivatives such as tryptophan (5) and tryptamine (6) with IC50s of 3.50± 0.4 and 6.00± 0.60 µM, respectively.30 Butein (7), a naturally occurring substance having chalcone backbone is one of the major active components of Dalbergia odorifera T. Chen (Fabaceae) and also found in heartwood of Acacia, has been reported to have antioxidant properties. 31, 32

3

Figure 1. Natural compounds reported for antileishmanial activity and design of target compounds 13(a-l).

4

Figure 2. Various natural derivatives reported for antioxidant activity.

Here, in continuation of our work on synthesis of bioactive molecules,

33

we report the

design and synthesis of a series of of some new combi-hetrocycles 13(a-l) having combination of the coumarin, and indole linked via chalcone like chain and same were evaluated in vitro antileishmanial activity. Since, all the three cores were also found to possess antioxidant activities; we therefore performed their antioxidant screening too using DPPH-radical scavenging method. In order to clarify and understand the probable binding mode of synthesized compounds 13(a-l) for their antileishmanial activity, the compounds were docked for possible targets for antileishmanial activity. We had also predicted their ADME properties in silico to suggest the suitability of any of the new compounds for further drug development. The acetylated compound i.e. 3-acetyl-4-hydroxy-2H-chromen-2-one (9) was synthesized by refluxing commercially available 4-hydroxy-2H-chromen-2-one (8) (25 mmol) into acetic acid (133.3 mmol) in presence of phosphorous oxychloride (3 mL) at 250 oC for about 3.5 hours in an oil bath.

34

The 3-acetyl-4-hydroxy-2H-chromen-2-one (9) was then subjected for

reaction with different aromatic aldehydes 10(a-l) (both 0.01 mol) in presence of 40% NaOH using ethanol as a solvent to form different substituted chalcones 11(a-l). These chalcones 11(a-l) (0.01 mol) and indole 12 (0.01 mol) were used for synthesis of titled compounds 5

13(a-l) using 5 mol % of HoxCoFe2-xO4 (x= 0.05) as a novel magnetically recoverable and reusable catalyst (Scheme 1) in ethanol. Ho3+ doped CoFe2O4 nanoparticles were prepared and characterized by our group

35

(Supporting data, Fig. S1). In recent times, several

transition metal oxides in the form of nanoparticles were employed as recyclable catalysts for many reactions.

36

In general, nanomaterials with natural morphologies containing higher

surface area as reactive sites allow them to act as effective catalysts for organic synthesis.

37

These nanoparticles have provided a simplified isolation procedure for the product, with small amounts of catalyst, affording easy recovery and recyclability of the catalyst. In some cases, recovery of the nanoparticles from the reaction mixtures is so difficult that conventional techniques such as filtration or centrifugation are not enough for an efficient recovery. To overcome this issue, the use of magnetic nanoparticles has emerged as a viable solution; their insoluble and paramagnetic nature enables easy and efficient separation of the catalysts from the reaction mixture with an external magnet. Recently, we have reported the synthesis of 2,4,5 -triaryl-1H-imidazoles using Ho3+ doped CoFe2O4 nanoparticles. 38

Scheme 1. Snthesis of indolyl-coumarin hybrids 13(a-l). Reagents and conditions: (a) AcOH, POCl3, Reflux; (b) 40% NaOH, EtOH; (c) EtOH, 5 mol % of Ho xCoFe2-xO4 (x= 0.05) nanoparticles

6

To optimize the reaction conditions, we studied a model reaction of chalcone 11a (0.01 mol) and indole 12 (0.01 mol) in ethanol as solvent using Ho3+ doped CoFe2O4 nanoparticles as catalyst to synthesize compound 13a. We screened the nanoparticles at various loads such as 0, 5, 10, 15 and 20 mol % (Supporting data, Table S1). When no catalyst was added for model reaction, there was only small amount (yield 30 %) of product obtained after 180 min. The use of 5 mol % nanaoparticle gave the compound 13a with 97 % yield in short reaction time (45 min). The increase in amount of catalyst from 5 mol % to 10, 15 and 20 mol % did not show any change in yield and time of reaction (Supporting data, Table S1). Therefore, 5 mol % of the catalyst Ho3+ doped CoFe2O4 nanoparticles was assumed to ensure the best yield (97 %) in short reaction time (45 min). Thus, our results make the process under study more attractive and interesting from the viewpoint of economy and simplicity. The recovery and reusability of the catalyst was investigated in this reaction for model reaction (13a). After completion of reaction (monitored by TLC), catalyst recycling was achieved by fixing the catalyst magnetically at the bottom of the flask with a strong magnet, after which the solution was taken off with a pipette, the solid washed twice with acetone and the fresh substrates with solvent was introduced into the flask, allowing the reaction to proceed for the next run. The catalyst was consecutively reused three times without any noticeable loss of its catalytic activity (Cycle number and yield of 13a: 1, 97 %; 2, 97 %; 3, 93 %). So the catalyst was found to be recyclable and reusable. We further expanded our series and synthesized 12 novel derivatives of 3-(3-(1H-indol-3yl)-3-phenylpropanoyl)-4-hydroxy-2H-chromen-2-one

13(a-l).

The

reaction

preceded

smoothly under mild reaction conditions (stirring at room temperature). The isolated yields of synthesized compounds were in the range of 80-97 % and reactions were completed in about 45-60 min (monitored by TLC). Melting points were determined in open capillary tubes and are uncorrected. The physical data for the compounds 13(a-l) are presented in Table 1. The formation of synthesized compounds was confirmed by IR, 1H NMR, spectral analysis.

7

13

C NMR and Mass

Table 1 Physical data for 3-(3-(1H-indol-3-yl)-3-phenylpropanoyl)-4-hydroxy-2H-chromen-2-ones 13(a-l)

Entry

R

Molecular formula

Reaction

%

(mw)

time (min)

Yield

M.P.*

Rf # value

13a

H

C26H19NO4 (409)

45

97

72-74

0.74

13b

2-Cl

C26H18ClNO4 (444)

60

83

76-78

0.66

13c

4-Cl

C26H18ClNO4 (444)

60

85

118-120

0.68

13d

2-Cl, 6-Cl

C26H17Cl2NO4 (478)

55

95

124-126

0.61

13e

4-F

C26H18FNO4 (427)

50

92

78-80

0.63

13f

4-OCH3

C27H21NO5 (439)

45

97

74-76

0.77

13g

2-OCH3, 4-OCH3

C28H23NO6 (470)

60

97

118-120

0.79

13h

2-OCH3, 5-OCH3

C28H23NO6(470)

45

97

128-130

0.78

13i

3-OCH3, 4-OCH3

C28H23NO6 (470)

50

96

126-128

0.83

13j

4-OH

C26H19NO5 (425)

45

91

58-60

0.69

13k

3-OH, 4-OH

C26H19NO6 (441)

60

89

92-94

0.65

13l

4-CN

C27H18N2O4 (434)

60

87

80-82

0.70

* Melting point uncorrected; # Solvent system: n-Hexane: Ethyl acetate (8:2).

The title compounds 13(a-l) were tested for their in vitro antileishmanial activity against a culture of Leishmania donovani promastigotes (NHOM/IN/80/DD8). Parasite viability was evaluated using a modified 3-(4,5-dimethylthiazol-2 yl)-2,5-diphenyl tetrazolium bromide (MTT) assay wherein the amount of formazan produced is directly proportional to the number of metabolically active cells.39 The concentration that decreased cell growth by 50% (IC50) was determined by graphic interpolation and data obtained depicted in Table 2. Sodium 8

stibogluconate and pentamidine were used as standard drugs. Compounds 13(a-l) showed varying degrees of antileishmanial activities with IC50 ranging between 95.00 to 287.50 μg/mL. Amongst all tested compounds 13a, 13d and 13h were found to be most promising compounds showing IC50 value of 95.50 μg/mL, 95.00 μg/mL and 99.00 μg/mL, respectively when compared with sodium stilbogluconate. All the synthesized compounds showed better activity than standard sodium stibogluconate (IC50 = 490 μg/mL) against L. donovani promastigotes.

Table 2 Evaluation of Antileishmanial and antioxidant activities. Entry

Antileishmanial activity IC50

Antioxidant activity IC50

Pteridine reductase 1

(μg/mL)

(μg/mL)

Binding energy (kcal/mol)

13a

95.50

12.40

-83.39

13b

102.00

39.62

-79.01

13c

125.00

46.35

-78.42

13d

95.00

13.49

-84.82

13e

155.50

47.35

-76.98

13f

174.00

16.21

-76.84

13g

249.50

13.24

-73.60

13h

99.00

19.02

-82.77

13i

212.00

63.16

-76.49

13j

287.50

58.91

-72.15

13k

231.00

71.08

-74.63

13l

101.75

13.74

-80.55

STD1

490.00

*

*

STD2

5.50

*

*

STD3

*

16.50

*

STD4

*

12.80

*

IC50 represents the mean values of three replicates; standard errors were all within 10% of the mean; (*) denotes not performed; STD1: Sodium stibogluconate; STD2: Pentamidine; STD1: Butylated hydroxytoluene; STD4: Ascorbic acid

9

Structural-activity relationship (SAR) revealed that coumarin, indolyl and chalcone like cores are important for antilishmanial activity (Table 2). Modification of the parent compounds with various substituents on phenyl ring such as halogen, hydroxyl, methoxyl, amino and cyano were performed to explore the SAR of these synthesized compounds 13(al). Compound 13a with no substitution on phenyl ring showed good antileishmanial activity (IC50=95.50 μg/mL). Introduction of 2-Cl 13b (IC50=102.00 μg/mL) and 4-Cl 13c (IC50=125.00 μg/mL) on phenyl ring showed significant antileishmanial activity. Intoduction of two chloro groups i.e. 2,6-dichloro 13d (IC50=95.00 μg/mL) on phenyl ring further increased antileishmanial potency and found to be most promising from the synthesized series. Furthermore, phenyl ring containing 4-F 13e (IC50=155.50 μg/mL) was found to reduce activity when compared with 13a, 13b, 13c and 13d. Therefore, there may be chances of interference of electronegativity of atoms and position of atoms with the activity. Introduction of 4-OCH3 13f (IC50=174.00 μg/mL) on phenyl ring was found to reduce the activity. Replacement of 4-OCH3 13f with 4-OH 13j (IC50=287.00 μg/mL) led to decrease in antileishmanial activity about 2 folds. Compounds 13g (IC50=249.50 μg/mL) and 13i (IC50=212.00 μg/mL) with 2,4-dimethoxy and 3,4-dimethoxy substitutions on phenyl ring, respectively further reduced the activity. But introduction of 2,5-dimethoxy 13h (IC50=99.00 μg/mL) on phenyl ring was found to be increase in activity by 2 folds when compared with other methoxy substituted derivatives 13f, 13g and 13i. The replacement of 3,4-dimethoxy 13i with 3,4-dihydroxy 13k (IC50=231.00 μg/mL) did not lead to any significant change in activity. The introduction of 4-CN group on phenyl ring had also shown good activity (IC50=101.75 μg/mL) as compared to most promising compounds 13a, 13d and 13h. As observed from activity data, compounds 13b, 13c, 13d, 13e and 13l with electron withdrawing groups substituents at phenyl ring are more effective than compounds 13f, 13g, 13i, 13j and 13k with electron donating substituent at phenyl ring. Antioxidant activities of the synthesized compounds 13(a-l) were measured using the 2,2diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay.40 The percent of inhibition (I %) of free radical production from DPPH was calculated by equation: % of scavenging= [(A control- A sample)/A blank] ×100. Where ‘A control’ is the absorbance of the control reaction (containing all reagents except the test compound) and ‘A sample’ is the absorbance of the test compound. DPPH radical scavenging activity is the most commonly used method for screening the antioxidant activities of various natural as well as synthetic antioxidants. Butylated hydroxytoluene (BHT) and ascorbic acid (AA) have been used as standard drugs 10

for the comparison of antioxidant activity and the observed results are summarized in Table 2. According to the DPPH assay, compounds 13a (IC50= 12.40 μg/mL), 13d (IC50= 13.49 μg/mL), 13f (IC50= 16.21 μg/mL), 13g (IC50= 13.24 μg/mL) and 13l (IC50= 13.74 μg/mL) showed promising radical scavenging activities when compared with synthetic antioxidant BHT (IC50= 16.5 μg/mL). The compound 13a (IC50= 12.40 μg/mL) showed equipotent activity as that of natural antioxidant ascorbic acid (IC50= 12.80 μg/mL). Compounds 13b (IC50= 39.62 μg/mL), 13c (IC50= 46.35 μg/mL), 13e (IC50= 47.35 μg/mL), 13h (IC50= 19.02 μg/mL), 13i (IC50= 63.16 μg/mL), 13j (IC50= 58.19 μg/mL) and 13k (IC50= 71.08 μg/mL) were found to be inactive when compared with standard antioxidants. From the antioxidant activity data (Table 2), the synthesized compounds had shown moderate to good antioxidant activity. The compound 13a (IC50= 12.40 μg/mL) with no substitution on phenyl ring was found most potent for antioxidant properties when compared to standard drugs BHT (IC50= 16.50 μg/mL) and ascorbic acid (IC50= 12.80 μg/mL). The introduction of 2-Cl 13b (IC50=39.62 μg/mL) and 4-Cl 13c (IC50=46.32 μg/mL) on phenyl ring led to reduced in antioxidant activity by 3-4 fold when compared with compound 13a. But introduction of 2,6-dichloro 13d (IC50=13.49 μg/mL) led to increase in activity and was more potent when compared with standard BHT. Replacement of 4-Cl with 4-F 13e (IC50=47.35 μg/mL) did not show any change in antioxidant activity. The methoxyl (-OCH3) substituted compounds 13f (4-OCH3, IC50= 16.21 μg/mL), 13g (2,4-OCH3, IC50= 13.24 μg/mL) and 13h (2,5-OCH3, IC50= 19.02 μg/mL) was found to be show good antioxidant activity except compound with 3,4-OCH3 13i (IC50=16.50 μg/mL) when compared with standard drugs. The replacement of 4-CH3 13f with 4-OH 13j (IC50= 58.91 μg/mL) led to decrease in antioxidant activity by 3 folds. The compound 13k with 3,4-OH on phenyl ring was found to most inactive compounds of the synthesized series with IC 50= 71.08 μg/mL. The 4-CN substitution on phenyl ring (compound 13l) showed remarkable antioxidant activity (IC50=13.74 μg/mL) more than that of standard BHT (IC50= 16.50 μg/mL) and nearly same activity as that of ascorbic acid (IC50=12.80 μg/mL). Cytotoxic study was performed on HeLa cell line and evaluated by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. 41 The cells were seen under the microscope (Ziess, Germany) at 10X magnification. The compound 13d from the synthesized series was found to be most promising showing good antileishmanial (IC50= 95.00 μg/mL) and antioxidant (IC50= 13.49 μg/mL) activities. Therefore, cytotoxic study against HeLa cell

11

line of compound 13d was carried out at concentration of 100 μg/mL. The compound 13d had shown no cytotoxicity effect against HeLa cell lines to its tested concentration (Fig. 3).

13d dd

Figure 3. Cytotoxic study against HeLa cell line of most promising compound 13d.

Molecular docking study of the synthesized compounds 13(a-l) was performed to clarify understand the probable binding interactions with various antileishmanial targets using standard protocol implemented in VLife MDS 4.3 package. 42 With this purpose, crystal structures of various antileishmanial targets including, Pteridine reductase 1 (PDB ID: 2XOX),43 Adenine phosphoribosyltransferase (PDB ID: 1QCD),44 UMP synthase (PDB ID: 3QW4) 45 and MapK (PDB ID: 4QNY) 46 were obtained from the Protein Data Bank in order to prepare protein for docking study. The docking study was performed using Vlife MDS 4.3 software by GRIP method. The compounds 13(a-l) have shown some significant binding energy but did not show any binding affinity in the active site of Adenine phosphoribosyl transferase and UMP synthase enzymes. The compounds have shown poor binding energy with MapK enzyme (Supporting data, Table S2). But in turn, the compounds showed good binding energy (-84.82 to -72.15 kcal/mol) toward Pteridine reductase 1 and docked well into active pocket against this enzyme (Table 2). Pteridine reductase 1 (PTR1), a Leishmania enzyme responsible for salvage of pteridines is potential target for chemotherapeutic intervention.47 The binding mode of compounds 13a and 13d in active pocket of Pteridine reductase 1 is shown in Fig. 4.

12

13a

13d d

Figure 4. Docking study of compounds 13a and 13d with Pteridine reductase 1 (PDB ID: 2XOX). Ligands are shown in red color. Hydrogen bonds are shown in green color. The binding energy of the compounds 13(a-l) and their antileishmanial activity showed the corresponding results. The active compounds 13a and 13d showed lowest interaction energy that is -83.39 kcal/mol and -84.82 kcal/mol, respectively. In case of compound 13a, the 13

compound was held in active site by forming various interactions with amino acid residues like, THR12, GLY13, ALA15, LYS16, ARG17, HIS36, TYR37, HIS38, ARG39, SER40, LEU66, ASN109, and SER111. The amino acid GLY13 (1.73 Å) had formed hydrogen bonding with nitrogen of indole ring, and amino acids HIS38 (2.47 Å), ARG39 (1.58 Å) and SER40 (1.92 Å) had formed hydrogen bondings with oxygen of chalcone like chain. In case compound 13d, the compound was held in active site by forming various interactions with amino acid residues like, THR12, SER14, ARG39, LEU66, SER67, ASN109, ALA110, ASN147, SER111, LEU143, SER146, ASN147, ALA150, PRO151, and LEU188. The amino acids SER146 (1.28 Å) and ASN147 (2.58 Å) had formed hydrogen bond with –OH of coumarin ring. The chloro substituent of phenyl ring was held in active pocket by forming van der waals interactions with amino acids SER111. On the basis of activity data and docking result, it was found the compounds 13a and 13d had potential to inhibit pteridine reductase 1 enzyme. An in silico study of synthesized compounds 13(a-l) was performed for prediction of ADME properties (Table 3). In this study, we calculated molecular volume (MV), molecular weight (MW), logarithm of partition coefficient (miLogP), number of hydrogen bond acceptors (n-ON), number of hydrogen bonds donors (n-OHNH), topological polar surface area (TPSA), number of rotatable bonds (n-ROTB) and Lipinski’s rule of five

48

using

Molinspiration online property calculation toolkit. 49 Absorption (% ABS) was calculated by: % ABS= 109-(0.345×TPSA).50 From all these parameters (Table 3), it can be observed that all titled compounds exhibited a good % ABS (66.30–80.26 %). it was observed that none of the compounds violated Lipinski's rule of five, suggesting that the compounds 13(a-l) may be developed as good drug candidates. A molecule likely to be developed as an orally active drug candidate should show no more than one violation of the following four criteria: logP (octanol-water partition coefficient) ≤5, molecular weight ≤500, number of hydrogen bond acceptors ≤10 and number of hydrogen bond donors ≤5. 51 Since all the synthesized compounds 13(a-l) followed this criteria for orally active drug and therefore can be developed as oral drug candidates.

14

Table 3 Pharmacokinetic parameters important for good oral bioavailability of synthesized compound 13(a-l). Entry

%ABS

Rule

n-ON n-OHNH acceptors donors <10 <5

Lipinski’s violations ≤1

nROTB -

MV

MW

miLogP

-

TPSA (A2) -

-

<500

≤5

13a

80.26

83.30

5

361.01

409.44

4.78

5

2

0

13b

80.26

83.30

5

374.55

443.89

5.41

5

2

1

13c

80.26

83.30

5

374.55

443.89

5.46

5

2

1

13d

80.26

83.30

5

388.08

478.33

6.04

5

2

1

13e

80.26

83.30

5

365.94

427.43

4.95

5

2

0

13f

77.08

92.53

6

386.56

439.47

4.84

6

2

0

13g

73.89

101.77

7

412.10

469.49

4.83

7

2

0

13h

73.89

101.77

7

412.10

469.49

4.83

7

2

0

13i

73.89

101.77

7

412.10

469.49

4.43

7

2

0

13j

73.28

103.53

5

369.03

425.44

4.30

6

3

0

13k

66.30

123.76

5

377.05

441.44

3.81

7

4

0

13l

72.05

107.09

5

377.87

435.45

4.54

6

2

0

% ABS: percentage absorption, TPSA: topological polar surface area, n-ROTB: number of rotatable bonds, MV: molecular volume, MW: molecular weight, milogP: logarithm of partition coefficient of compound between n-octanol and water, n-ON acceptors: number of hydrogen bond acceptors, n-OHNH donors: number of hydrogen bonds donors.

In conclusion, design, synthesis and biological of a novel series of 3-(3-(1H-indol-3-yl)-3phenylpropanoyl)-4-hydroxy-2H-chromen-2-one 13(a-l) has been presented. Use of 5 mol % of Ho3+ doped CoFe2O4 nanoparticles as a catalyst helps in fast conversion of substituted coumarin chalcones to desired products therefore proving its advantage. Good yields and reusability of catalyst imparts further advantage of using it in the reaction. Thus, our results made the process under study more attractive and interesting from the viewpoint of economy and simplicity. Newly synthesized compounds were evaluated for both antileishmanial as well as antioxidant activities. Based on the activity data, SAR for the series has been 15

developed. Compounds 13a (IC50= 95.50 μg/mL), 13d (IC50= 95.00 μg/mL) and 13h (IC50= 99.00 μg/mL) were found to possess significant antileishmanial activity when compared with standard sodium stibogluconate (IC50= 490.00 μg/mL). Compound 13a (IC50=12.40 μg/mL) with no substitution on phenyl ring was found to be most potent antioxidant when compared to standard drugs BHT (IC50= 16.50 μg/mL) and ascorbic acid (IC50= 12.80 μg/mL). After performing docking studies, on the basis of activity data and docking result, it was found the compounds 13a and 13d had potential to inhibit pteridine reductase 1 enzyme. In silico physicochemical pharmacokinetic parameters for synthesized compounds had shown promising results. Thus, suggesting that the compounds from the present series can serve as important gateway for the design and development of new antileishmanial agents along with antioxidant activity. Acknowledgements The authors are thankful to Mrs. Fatma Rafiq Zakaria, Chairman, Maulana Azad Educational Trust and Dr. Zahid Zaheer, Principal, Y.B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Aurangabad 431001 (MS), India for providing the laboratory facility. The authors are also thankful Department of Biotechnology, Savitribai Phule Pune University, Pune (MS), India for performing antileishmanial and antioxidant screening. The authors are also thankful to SAIF, Punjab University, Chandigarh, India for providing NMR spectra.

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Figure captions Figure 1. Natural compounds reported for antileishmanial activity and design of target compounds 13(a-l). Figure 2. Various natural derivatives reported for antioxidant activity. Figure 3. Cytotoxic study of most promising compound 13d against HeLa cell line. Figure 4. Docking study of compounds 13a and 13d with Pteridine reductase 1 (PDB ID: 2XOX). Ligands are shown in red color. Hydrogen bonds are shown in green color.

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Graphical Abstract

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