rational design and microwave assisted synthesis of some novel phenyl thiazolyl clubbed s triazine derivatives as antimalarial antifolate

Rational Design and Microwave Assisted Synthesis of Some Novel Phenyl ThiazolylClubbed s-Triazine Derivatives as Antimalarial Antifolate Jun Moni Kalita, Surajit Kumar Ghosh, Supriya Sah

Rational Design and Microwave Assisted Synthesis of Some Novel Phenyl Thiazolyl

Clubbed s-Triazine Derivatives as Antimalarial Antifolate

Jun Moni Kalita, Surajit Kumar Ghosh, Supriya Sahu, Mayurakhi Dutta

DOI: 10.1016/j.fjps.2016.09.004

Reference: FJPS 23

To appear in: Future Journal of Pharmaceutical sciences

Received Date: 12 May 2016

Revised Date: 15 August 2016

Accepted Date: 26 September 2016

Please cite this article as: Kalita JM, Ghosh SK, Sahu S, Dutta M, Rational Design and Microwave Assisted Synthesis of Some Novel Phenyl Thiazolyl Clubbed s-Triazine Derivatives as Antimalarial

Antifolate, Future Journal of Pharmaceutical sciences (2016), doi: 10.1016/j.fjps.2016.09.004.

This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain

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Rational Design and Microwave Assisted Synthesis of Some Novel Phenyl Thiazolyl

Clubbed s-Triazine Derivatives as Antimalarial Antifolate

Jun Moni Kalitaa*, Surajit Kumar Ghosha, Supriya Sahua, Mayurakhi Duttab

aDepartment of Pharmaceutical Sciences, Dibrugarh University

Dibrugarh, Assam,India

b

Department of Pharmaceutical Sciences, Assam University

Silchar, Assam,India

*Corresponding Author: Jun Moni Kalita,

Email: pjmk84@gmail.com

Phone: +91 9508980893

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Rational Design and Microwave Assisted Synthesis of Some Novel Phenyl Thiazolyl

Clubbed s-Triazine Derivatives as Antimalarial Antifolate

Abstract: Rational approach to drug design is the process to find new potent molecules on the basis of a known

target and available ligands for the target Compared to the traditional system of drug design and discovery, that

involves blind testing of different chemicals in vitro and in vivo in cultured cells and animals, rational approach

is totally based on the knowledge of the target and the pathway of action Recent developments in the field of rational approach to drug design can be credited to the development in the areas of computer science, molecular biology, biophysics, biotechnology and statistics Designing of new molecules based on the knowledge of receptor and the available ligands is well-known as Structure Based Drug Design (SBDD) The branch of rational approach that uses computer as a tool to design and screen design molecules is called as Computer Aided Drug Design (CADD) In this work computer was used to design and screen the designed molecules virtually Among the 60 designed molecules 10 were selected on the basis of their binding affinity to the

receptor molecule Synthesis of the selected molecules was done and In-vitro antimalarial activity was

evaluated

Keywords: Antifolate, Antimalarial, Docking, Phenylthiazole

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1 Introduction

Malaria is a mosquito born disease caused by a single celled organism known as protozoa Among the

five types of malaria, the disease caused by Plasmodium falciparum is the most common and virulent Although

malaria is less common in the developed countries, yet it is a life threatening infectious disease in the

developing Asian and African countries While there are numbers of antimalarial drugs available, today an

emergency occurred in the area of antimalarial drug search because of resistance occurred by the parasites against the available drugs [1, 2]

Resistance to antimalarial drugs has been reported for only two species of parasite among the five viz

P falciparum and p vivax Among the two species P falciparum acquired resistance to almost all the

antimalarial drugs available, however the extent of resistance varies from drug to drug The geographical distribution of resistant parasite depends upon the population movement from a resistant place to a nonresistant

one At present chloroquine resistant P.falciparum strain hass been reported everywhere throughout the world

[3-6]

Molecules containing thiazole nucleus as a part are reported to have a diverse activities such as antimicrobial [7-9], anticonvulsant [10], analgesic and anti- inflammatory[11, 12], antitubercular[13] and antican-cer[14-16] It is been also reported that thiazole containing molecules are easily metabolised inside the body without the production of any toxic biproducts[17] Molecules containing a thiazole ring attached with a substituted triazine nucleus were reported to have antimalarial activity as it can block DHFR (Dihydro Folate Reductase), which is a key enzyme responsible for metabolic activity in malarial parasites [18]

Rational drug design is also sometimes referred as drug design or rational design In the era of modern drug design and discovery, computer aided drug design played a major rule In contrast to the traditional method

of drug discovery, which relies on the trial and error testing rational drug design begins with a hypothesis that modulation in a specific target can give a desired pharmacological activity With the advancement in the

technology, it is now possible to simulate in vitro as well as in vivo condition within a computer using any

sophisticated software Accordingly molecules can be virtually screened for their activities as well as probable toxicities prior to a real laboratory work This type of virtual screening enables the proper use of time and resources [19-22]

In the last two decades microwave assisted synthesis become very popular in pharmaceutical and academic areas because of its technology enabling a fast and steady chemical synthesis Further advancement has been achieved in case of Enhanced Microwave Synthesis (EMS), where the reaction vessel is simultaneously cooled during the reaction time Short reaction time and a wide range of reaction scope have

enabled microwave assisted synthesis very popular among the researchers and industrial persons [23, 24]

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2 Materials and Methods

2.1 Insilico studies:

After a thorough literature review, 60 molecules were designed and these designed molecules were

tested for their probable molecular property and expected toxicity Properties of the molecules were calculated

by feeding the structures in an online java based program molinspiration property calculator (http://www.molinspiration.com/cgi-bin/properties) Molecular properties such as miLogP, Total Polar Surface

Area (TPSA), No of Atoms, Molecular Weight (MW), No of Hydrogen Bond Donor (HBD), No of Hydrogen Bond Acceptor (HBA), No of Rotatable Bonds, Molecular Volume were calculated Cut off for these properties

were kept according to the lipinski’s rule of five and number of violations was calculated Molecules with any

violation were discarded from the study Table 1

Table 1 Estimated property of the designed molecules

Molecule

No

miLogP TPSA

(c Å)

No of atoms

MW (Dalton)

No

of

O, N

No of

OH,

NH

No of rot bonds

Volume (c Å)

Violations

66B 3.08 104.883 26.0 388.888 8 4 4 319.725 0 67B 3.08 104.883 26.0 388.888 8 4 4 319.725 0 68B 2.791 121.873 24.0 363.834 8 5 6 292.197 0 69B 3.043 116.322 29.0 432.941 9 4 6 361.728 0 72B 2.628 115.641 21.0 319.781 7 5 3 249.462 0 78B 4.425 88.094 27.0 403.899 8 2 5 333.982 0 79B 3.023 113.672 25.0 376.877 8 5 7 313.143 0 81B 3.875 90.887 27.0 402.915 8 3 5 337.4 0 82B 4.471 82.098 28.0 416.942 8 2 5 354.343 0 84B 3.839 102.326 30.0 446.968 9 3 7 379.403 0 Cut off values for the properties: miLogP: 5, TPSA: 400 c Å, MW: 500 Dalton, No of O, N: 10,

No of OH, NH: 5, Volume: 800 c Å

After the end of the first property calculation, the qualified designed molecules were further passed through another virtual filter Here different probable toxicities like Mutagenicity, Carcinogenicity, Tumorogenicity and Teratogenicity of the molecules were calculated using another online java based program called Osiris Property Explorer (http://www.organic-chemistry.org/prog/peo/) (Table 2) Molecules reported with good score by both the filter were retained and were considered for docking studies

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Table 2 Estimated toxicological properties of the designed molecules

Molecule Mutagenic Irritant Reproductive

effective 66B

67B 68B 69B 72B 78B 79B 81B 82B 84B

Not toxic moderately toxic highly toxic

2.1.1 Preparation of protein:

The crystal structure of wild type Pf-DHFR-TS complex was obtained from protein data bank using

Accelrys’ Discovery studio version 2.5 (PDB entry code: 1J3I) Water molecules, co-crystallized ligand (WR99210) were removed and cofactors NADPH and dUMP were allowed to retain Protein was cleaned to remove any extra conformation and binding site was analysed Finally, protein was prepared according to the requirements of the docking protocol

2.1.2 Preparation of ligand:

Structures of the designed ligands were prepared by Marvin sketch tool as supported by Sanjeevani online program Then the 3D structures of the ligands were imported to Discovery Studio workplace and energy minimization was done by applying CharmM forcefield Further possible ligand conformations were generated

by considering an in-silico PH of 7-7.4 Ligand with lowest energy was selected and docked at the active site of the enzyme protein

2.1.3 Molecular Docking:

To validate the docking protocol, all atoms RMSD (Root Mean Square Deviation) of the docked ligand with respect to the co-crystallized ligand was calculated The RMSD value for LigandFit protocol was found to be 0.2426 Ao which is less than that of 2 Ao Figure 1

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Figure 1 All atoms RMSD (co-crystallized ligand in dark blue colour and docked ligand shown in elemental

colour)

Then the Prepared Ligands were docked at the active site of the prepared protein using LigandFit protocol in Discovery Studio 2.5 package At last binding energy of ligand protein complex was calculated with

in situ ligand minimization and non-bond list radius of 14.0 Å by using calculate binding energy protocol

Binding pose and ligand orientation at the active site was studied and the molecules were ranked according to

their estimated binding energy

2.1 Synthesis

Reactions were carried out using dry, freshly distilled solvents under anhydrous conditions, unless otherwise noted Melting points were determined by open capillary tubes using Buchi M-560 melting point apparatus and were uncorrected FTIR spectra of the powdered final compounds were recorded using ATR with

a Bruker FTIR spectrophotometer 1H NMR spectra were recorded on a Bruker Advance II spectrophotometer using TMS as an internal reference (Chemical shift represented in δ ppm) Mass spectra were recorded on MS ZQMAA255 System Purity of the compounds was checked on TLC plates using silica gel G as stationary phase and was visualized using iodine vapors

2.1.1 Synthesis of substituted phenyl thiazole

Synthetic scheme for the preparation of 4-(4-chlorophenyl) thiazol-2-amine is depicted in Scheme 1 4

chloro acetophenone was reacted with thiourea in the presence of strong oxidising agents like sulfuryl chloride

And then the reaction mixture was allowed to reflux for 3 hrs [25]

Colour: Light yellow, Nature: Amorphous powder, Yield: 84%, m.p 166o C; FTIR (cm -1 ): 3433.60

and 3244.40 (NH2), 3114.41 (Ar-H), 1 H NMR (400 MHz, DMS0): δ 4.0 (NH2) 6.6 (CH thiazole) 7.32, 7.48 (CH aromatic) 13 C NMR (DMSO) δ 100.0, 127.0, 128.8, 129.0, 148.2, 168.4 MS (EI) m/z 209 (M +1)

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N S

Cl

H2N

H 3 C O

Cl

S

sulfuryl chloride

105 o C 3 hr

Scheme 1 Synthesis of p-chloro phenyl thiazole amine

2.1.2 Nucleophillic substitution at triazine ring:

Nucleophillic substitution substitution of different selected amines was carried out in three steps as

shown in Scheme 2 The first chlorine of cyanuric chloride was substituted at a temperatue of 0-5º C taking

ether as solvent The second chlorine was substituted at a temperature of 40º C with the help of microwave synthesizer where acetone as solvent, whereas the third chlorine atom of cyanuric chloride was substituted by at

110 º C with microwave irradiation taking dioxane as solvent [25-27] Structure and physicochemical properties

of synthesized molecules are shown in Table 3

2.2 Chemistry

N2-(4-(4-chlorophenyl) thiazol-2-yl)-6-(piperazin-1-yl)-1, 3, 5-triazine-2, 4-diamine (66B)

FTIR (cm -1 ) 3368.34, 3359.67 (N-H primary, Str.); 1640.02, 851.83(N-H primary, Bend.); 2949.05

(C-H Str.); 1279.57, 1185.64 (CN Aro.) 1 H NMR (CDCl 3 ): δ,ppm: 2.76(t 4H, piperazine ring); 3.67 (m, 4H,

piperazine ring); 7.12 (s, 1H, thiazole ring); 7.50-7.70 (m, 8H, CH2, phenyl ring) 13 CNMR (CDCl 3 ): δ,ppm:

34.21, 45.15, 46.02-46.31, 101.62 , 127.38, 128.56, 134.67, 142.30, 150.73 MS (EI) m/z 387.12 (M +1)

N2-(4-(4-chlorophenyl) thiazol-2-yl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazine-2,4-diamine (67B)

FTIR (cm -1 ) 3369.11, 3359.52 (N-H primary, Str.); 1640.22, 850.83(N-H primary, Bend.); 2950.15,

2889.00 (C-H Str.); 1279.12, 1184.64 (CN Aro.) 1 H NMR (CDCl 3 ): δ,ppm: 2.39 (s, 3H, CH3); 2.66 (t, 4H, piperazine ring); 3.2(m, 4H, pierazine ring); 7.04 (s, 1H, thiazole ring); 7.52-7.71 (m, 8H, phenyl ring)

13 CNMR (CDCl 3 ): δ,ppm: 44.20, 46.10, 54.32, 125.10, 127.38, 130.61, 134.56, 142.30, 148.73, 167.23 MS (EI) m/z 401.322 (M +1)

2-(4-amino-6-(4-(4-chlorophenyl)thiazol-2-ylamino)-1,3,5-triazin-2-ylamino)ethanol (68B)

FTIR (cm -1 ) 3442.69 (O-H, Str.); 3369.01, 3339.52 (N-H primary, Str.) 1600.92, 842.97 (N-H

primary, Bend.); 2953.74 (C-H Str.); 1353.17, 1304.3891 (C-N Ar, Str.) 1 H NMR (MeOD): δ,ppm: 3.48(t, 4H,

amino ethanol); 7.32 (s, 1H, thiazole ring); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13 CNMR (MeOD):

δ,ppm: 46.34, 57.80, 102.71 , 127.74, 128.60, 134.62, 148.11, 160.56 MS (EI) m/z 362.274 (M +1)

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2-(4-(4-amino-6-(4-(4-chlorophenyl)thiazol-2-ylamino)-1,3,5-triazin-2-yl)piperazin-1-yl)ethanol (69B)

FTIR (cm -1 ) 3424.91, 3353.66, 3289.60 (N-H primary, Str.); 1562.70, 844.93 (N-H primary, Bend.);

1512.49 (N-H secondary, Bend.); 2857.27(C-H Str.); 1198.18, 1331.91 (CN Aro., Str.) 1 H NMR (MeOD):

δ,ppm: 2.61 (t, 4H, piperazine); 2.71(t, 2H,ethanol); 3.58(m, 6H, piperazine and ethanol); 7.24 (s, 1H, thaizole);

7.48-7.62 (m, 4H, phenyl) 13 CNMR (MeOD): δ,ppm: 42.03, 50.01, 53.36, 62.20, 100.93, 129.25, 129.07,

160.70, 165.70 MS (EI) m/z 431.01 (M +1)

N2-(4-(4-chlorophenyl)thiazol-2-yl)-1,3,5-triazine-2,4,6-triamine (72B)

FTIR (cm -1 ) 3364.43, 3306.57 (N-H primary, Str.); 1530 (N-H secondary, Bend.); 2857.51(C-H Str.);

1352.94, 1254.06 (C-N Aro., Str.), 1 H NMR (MeOD): δ,ppm: 4.26 (s, 4H, NH); 7.59 (d, 2H, phenyl); 7.69 (d,

2H, phenyl) 13 C NMR (MeOD): δ,ppm: 102.36 , 127.66 , 129.56, 135.23 151.10, 165.92 MS (EI) m/z

318.015 (M +1)

N2-(4-(4-chlorophenyl) thiazol-2-yl)-N4-methyl-6-morpholino-1,3,5-triazine-2,4-diamine (78B)

FTIR (cm -1 ) 3324.24(N-H secondary, Str.); 1532.17(N-H secondary, Bend.); 2942.45, 2851.74(C-H

Str.); 1301.66, 1272.60 (CN Aro., Str.), 1 H NMR (MeOD): δ,ppm: 2.62 (s, 3H, methyl); 3.52-3.64 (sextet, 8H,

morpholine); 7.22 (s, 1H, thiazole); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13 CNMR (MeOD): δ,ppm:

27.56, 43.07, 64.84,102.54, 127.71, 128.58, 166.56, 167.613 MS (EI) m/z 402.362 (M +1)

N2-(2-aminoethyl)-N4-(4-(4-chlorophenyl)thiazol-2-yl)-N6-methyl-1,3,5-triazine-2,4,6-triamine (79B)

FTIR (cm -1 ) 3367.34, 3378.29 (N-H, Str), 1563.39, 770.62 (N-H primary, Bend.); 3294.28 (N-H

secondary, Str.); 1514.41(N-H secondary, Bend.); 2928.51, 2863.89 (C-H Str.); 1332.94, 1253.72 (CN Aro., Str.), 1 H NMR (MeOD):δ,ppm: 2.62 (s, 3H, methyl); 2.82 (t, 2H, ethylamine); 3.27 (t, 2H, ethylamine); 7.22 (s,

1H, thiazole); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13 CNMR (MeOD): δ,ppm: 28.31, 42.52, 57.93, 61.32,

102.69, 126.00, 127.73, 128.61, 165.35 MS (EI) m/z 374.812 (M +1)

N2-(4-(4-chlorophenyl)thiazol-2-yl)-N4,N4-dimethyl-6-(piperazin-1-yl)-1,3,5-triazine-2,4-diamine (81B)

FTIR (cm -1 ) 3366.34 (N-H secondary, Str.); 1640.02 (N-H Bend.); 2949.05 (C-H Str.); 1279.57,

1185.64 (CN Aro) 1 H NMR (MeOD): δ,ppm: 2.82 (t, 4H, piperazine); 3.22 (t, 4H, piperazine); 7.24 (s, 1H,

thiazole); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13 CNMR (MeOD): δ,ppm: 35.95, 45.35, 52.74, 60.38,

102.55, 127.98, 128.64, 150.44 MS (EI) m/z 415.181 (M +1)

N2-(4-(4-chlorophenyl)thiazol-2-yl)-N4-methyl-6-(4-methylpiperazin-1-yl)-1,3,5-triazine-2,4-diamine (82B)

FTIR (cm -1 ) 3289.82(N-H secondary, Str.); 1514.93(N-H secondary, Bend.); 2921.71, 2853.30

(C-H Str.); 1353.86, 1290.74(CN Aro.,tr.), 1 H NMR (MeOD):δ,ppm: 2.41 (s, 3H, CH3, piperazine); 2.45 (t, 4H, piperazine); 2.61(s, 3H, methyl); 3.56 (t, 4H, piperazine); 7.24 (s, 1H, thiazole); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13 CNMR (MeOD): δ,ppm: 35.97,41.67, 52.39, 60.41, 102.59, 127.91, 128.65, 150.41, 165.29

MS (EI) m/z 414.827 (M +1)

2-(4-(4-(4-(4-chlorophenyl)thiazol-2-ylamino)-6-(methylamino)-1,3,5-triazin-2-yl)piperazin-1-yl)ethanol (84B)

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FTIR (cm -1 ) 3326.52(N-H secondary, Str.); 1534.93(N-H secondary, Bend.); 2821.75, 2842.38

(C-H Str.); 1341.72, 1291.95(CN Aro.,Str.), 1 H NMR (MeOD): δ,ppm: 2.60 (s, 3H, methyl); 2.77 (t, 2H, ethyl);

3.58(m, 6H, piperazine and ethanol); 7.24 (s, 1H, thiazole); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13 CNMR (MeOD): δ,ppm: 27.22, 45.72, 57.31, 62.91, 102.57 , 127.81, 128.74, 150.14, 165.35 MS (EI) m/z 445.019 (M

+1)

Table 3 Structures and physic-chemical properties of the synthesized molecules

TLC*

% Yield Melting point !

66B

N N N

NH 2

N

HN N S

Light yellow

67B

N N N

NH 2

N

HN N S

68B

N N N

NH2

NH

HN N S Cl

HO

Brownish yellow

69B

N N N

NH 2

N HN N S

OH

72B

N N N

NH 2

NH 2

HN N S Cl

Brownish yellow

78B

N N N NH

N HN N S

79B

N N N NH

NH

HN N S

Light yellow

81B

N N N HN

N HN N S

82B

N N N NH

N

HN N S