Amino Acid Esters Substituted Phosphorylated Emtricitabine and Didanosine Derivatives as Antiviral...

Amino Acid Esters Substituted PhosphorylatedEmtricitabine and Didanosine Derivatives as Antiviraland Anticancer Agents

Kuruva Chandra Sekhar & Avilala Janardhan &

Yellapu Nanda Kumar & Golla Narasimha &

Chamarthi Naga Raju & S. K. Ghosh

Received: 27 November 2013 /Accepted: 21 April 2014 /Published online: 2 May 2014# Springer Science+Business Media New York 2014

Abstract Owing to the promising antiviral activity of amino acid ester-substituted phosphor-ylated nucleosides in the present study, a series of phosphorylated derivatives of emtricitabineand didanosine substituted with bioactive amino acid esters at P-atom were synthesized.Initially, molecular docking studies were screened to predict their molecular interactions withhemagglutinin-neuraminidase protein of Newcastle disease virus and E2 protein of humanpapillomavirus. The title compounds were screened for their antiviral ability against Newcastledisease virus (NDV) by their in ovo study in embryonated chicken eggs. Compounds 5g and9c exposed well mode of interactions with HN protein and also exhibited potential growth ofNDV inhibition. The remaining compounds exhibited better growth of NDV inhibition thantheir parent molecules, i.e., emtricitabine (FTC) and didanosine (ddI). In addition, the in vitroanticancer activity of all the title compounds were screenedagainst HeLa cell lines at 10 and100 μg/mL concentrations. The compounds 5g and 9c showed an effective anticancer activitythan that of the remaining title compounds with IC50 values of 40 and 60 μg/mL, respectively.The present in silico and in ovo antiviral and in vitro anticancer results of the title compoundsare suggesting that the amino acid ester-substituted phosphorylated FTC and ddI derivatives,especially 5g and 9c, can be used as NDV inhibitors and anticancer agents for the control andmanagement of viral diseases with cancerous condition.

Appl Biochem Biotechnol (2014) 173:1303–1318DOI 10.1007/s12010-014-0929-8

Electronic supplementary material The online version of this article (doi:10.1007/s12010-014-0929-8)contains supplementary material, which is available to authorized users.

K. C. Sekhar : C. N. Raju (*)Department of Chemistry, Sri Venkateswara University, Tirupati 517502, Indiae-mail: [email protected]

A. Janardhan : G. NarasimhaDepartment of Virology, Sri Venkateswara University, Tirupati 517502, India

Y. N. KumarDepartment of Zoology, Sri Venkateswara University, Tirupati 517502, India

S. K. GhoshBioorganic Division, Bhabha Atomic Research Centre, Mumbai 400085, India

http://dx.doi.org/10.1007/s12010-014-0929-8

Keywords PhosphorylatedderivativesofFTCandddI .Newcastlediseasevirus(NDV).Humanpapillomavirus (HPV) .Molecular docking . In ovo antiviral activity . Anticancer activity . HeLacell lines

Introduction

Newcastle disease virus (NDV) is an avian-enveloped single-stranded RNAvirus belonging tothe family of paramyxoviridae which contains two transmembrane glycoproteins—hemagglu-tinin-neuraminidase (HN) and fusion F protein [1]. In order to develop a treatment of NDVdisease, a number of drugs were invented, and, recently, we have developed new antiviralagents, dihydropyrimidine and 5H-thiazolo [3,2-a] pyrimidine derivatives, as NDV inhibitors[2]. Charles et al. established the analogues of S-adenosylhomocysteine (AdoHcy) derivativeswhich inhibit the NDV messenger RNA (guanine-7-)-methyltransferase. AdoHcy sulfoxide,AdoHcy sulfone, and N6-methyl-AdoHcy exhibited potent and selective inhibitory activities onthis RNA transmethylation, suggesting the possibility of designing specific inhibitors of thismethyltransferase for in vivo use [3]. It has been reported that amino acid ester pro-drug ofnucleoside analogue, especially L-valine, could be transported across the intestine mediated bythe intestinal proton-coupled peptide transporter for increasing oral absorption [4, 5], such asacyclovir and ganciclovir [6–9].

On the other hand, human papilloma virus (HPV) was identified as an infectious agent incervical cancer in the development of cervical cytological abnormalities and precancerouslesions. This is due to papillomavirus E2 protein in HeLa cells prompts P53 causing arrest ofthe cell cycle and apoptosis [10, 11].

Emtricitabine (FTC) is a nucleoside, i.e., cytosine analogue, which have been worked asanti-HIV agent by inhibiting reverse transcriptase and indirectly increase the number ofimmune system cells. It was used for the treatment of HIV infections in adults and children.Due to lack of a 3-hydroxyl group, it does not support continued synthesis of the newly madeDNA strand and thus terminates the polymerization process catalyzed by the HIV reversetranscriptase [12]. FTC also exhibits significant histologic, virologic, and biochemical im-provement against the hepatitis B virus (HBV), but it has not been established [13]. Didan-osine (ddI) is also a nucleoside analogue of guanosine, which acts as an antiviral drugcommonly used in acquired immuno deficiency syndrome (AIDS) therapy [14, 15]. Thesedrugs undergo triphosphorylation to become active, with the initial monophosphorylationcatalyzed by cellular enzymes (kinases).

Phosphonic amide derivatives are used as prodrugs to improve the membrane permeabilityof drugs. Monophosphate nucleoside analogues are effective in intracellular delivery andrelaxed for the enzymatic phosphoramidate triester protide approach, especially in antiviraland anticancer path ways of many nucleoside analogues [16].

Based on the established studies of Charles et al. and the other literature studies, wedesigned and synthesized the phosphorylated derivatives of nucleoside drugs (FTCand ddI) by substitution with bioactive amino acid esters at P-atom. Initially, theirstructures were screened by molecular docking studies on NDV neuraminidase protein(PDB ID 1USX) and human papillomavirus E2 protein (PDB ID 1DHM) to predictthe binding affinities and orientations. The efficacy of the compounds was assayed byscreening their in ovo antiviral activity in NDV-infected embryonated chicken eggsand in vitro anticancer activities on HeLa cell lines. These assays acknowledged thetitle compounds as potential antiviral and anticancer agents and prompted them asvirtuous therapeutics against NDV and HPV.

1304 Appl Biochem Biotechnol (2014) 173:1303–1318

Materials and Methods

Chemistry

Chemicals procured from Sigma-Aldrich andMerck are used as such without further purification.All solvents used for spectroscopic and other physical studies were reagent grade andwere furtherpurified by standard methods [17]. Melting points (mp) were determined using a calibratedthermometer by Guna digital melting point apparatus and expressed in degrees centigrade (°C)and are uncorrected. Infrared spectra (IR) were recorded on a Perkin-Elmer Model 281-Bspectrophotometer. Samples were analyzed as potassium bromide (KBr) disks. Absorptions werereported in wave numbers (cm−1). 1H, 13C, and 31P NMR spectra were recorded in dimethylsulfoxide (DMSO)-d6 on a Bruker AMX 400 MHz spectrometer operating at 400 MHz for 1H,100 MHz for 13C, and 161.9 MHz for 31P NMR. The 1H and 13C chemical shifts were expressedin parts per million (ppm) with reference to tetramethylsilane (TMS) and 31P chemical shifts to85 % H3PO4. LCMS mass spectra were recorded on a Jeol SX 102 DA/600 Mass spectrometer.

Synthesis of [5-(4-Amino-5-Fluoro-2-Oxo-1,2-Dihydro-1-Pyrimidinyl)-1,3-Oxathiolan-2-yl]Methyl (2-Dihydrogenphosphato)(4-Nitrophenyl) Phosphate (5a)

4-Amino-5-fluoro-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)pyrimidin-2(1H)-one (FTC (1))(1 mmol) and N,N′,-dimethylpiperazine (DMP) (1 mmol) was dissolved in tetrahydrofuran(THF) (15 mL) and pyridine (5 mL). To this stirred solution of FTC, 4-nitrophenylphosphorodichloridate (2) (1 mmol) in 5 mL of THF was added dropwise at 0 °C. Aftercompletion of the addition, the reaction mixture was stirred for 2 h at 5–10 °C to get theintermediate, (5-(4-amino-5-fluoro-2-oxopyrimidin-1(2H)-yl)-1,3-oxathiolan-2-yl)methyl-4-nitrophenyl phosphorochloridate (3). The reaction progress was monitored by thin layerchromatography (TLC) using methanol/ethyl acetate (2:3). After completion of the reaction,it was filtered off to remove the salt of DMP HCl. Subsequently, monopotassium dihydrogenphosphate (4a) (1 mmol) in dry THF (10 mL) was added dropwise to the filtrate (3) at 0–5 °Cand stirring was continued for 2 h at 35 °C. The progress of the reaction wasmonitored by TLC using methanol/ethyl acetate (2:3). After completion of the reac-tion, it was filtered to remove KCl, and the filtrate was concentrated in a rota-evaporator to obtain crude product. The residue was dissolved in chloroform(10 mL) and washed with 1 M hydrochloric acid solution (2×15 mL), saturatedsodium bicarbonate (2×10 mL), and then water (3×15 mL). Then, the organic phasewas dried over MgSO4 and distilled under vacuum, and the residue was purified bycolumn chromatography on silica gel by eluting with 10 % methanol in ethylacetateto afford pure product of [5-(4-amino-5-fluoro-2-oxo-1,2-dihydro-1-pyrimidinyl)-1,3-oxathiolan-2-yl]methyl (2-dihydrogenphosphato)(4-nitrophenyl) phosphate (5a).

The same experimental procedure was adopted for the preparation of the remaining titlecompounds 5b-i (Scheme 1) by using various amines and amino acid esters.

Spectroscopic Analysis

[5-(4-Amino-5-Fluoro-2-Oxo-1,2-Dihydro-1-Pyrimidinyl)-1,3-Oxathiolan-2-yl]Methyl(2-Dihydrogenphosphato)(4-Nitrophenyl) Phosphate (5a)

Yield:74 %; mp: 192–194 °C; IR (KBr): υ1,217 and 1,207 (P=O), 1,677 (C=O), 3,279 (N-H),3,413 (O-H) cm−1; 1H NMR (400 MHz, DMSO-d6): δ 3.16 (2H, H-11), 3.71–3.79 (1H, H-9),

Appl Biochem Biotechnol (2014) 173:1303–1318 1305

4.52 (2H, H-12), 5.19–5.21 (1H, H-7), 7.32–7.95 (5H, Ar-H), 8.10 (2H, NH2), 10.3(br s, 2H, P-OH); 13C NMR (100 MHz, DMSO-d6): 37.1, 62.7, 79.6, 86.9, 98.3,120.8, 125.9, 126.9, 135.3, 137.2, 139.4, 153.4, 152.4, and 157.9; 31P NMR(161.9 MHz, DMSO-d6): δ 5.6, 11.3; LC MS (%): m/z 528.8 [M+H]+ (100 %); Anal.Calcd. for C14H15FN4O11P2S: C, 31.83; H, 2.86; and N, 10.61; Found: C, 31.71; H,2.74; and N, 10.45.

Spectroscopic data of the remaining compounds 5b-iwere presented in Supplementary data.

Synthesis of Phosphorylated Derivatives of Didanosine (9a-c)

Similarly, synthesis of phosphorylated didanosine derivatives (Scheme 2) was accom-plished. 9-(5-(Hydroxymethyl)tetrahydrofuran-2-yl)-1H-purin-6(9H)-one (ddI (6)) wastreated with 4-nitrophenyl phosphorodichloridate (2) in the presence of DMP intetrahydrofuran (THF) and pyridine at 0 °C. After the addition, the reaction mixturewas stirred for 2 h at 20 °C. The reaction progress was monitored by thin layerchromatography (TLC) (methanol/ethanol 2:3) to form the monochloride intermediate 7.Further, the monochloride was reacted with three different amino acid esters leucine ethyl ester(8a), cysteine ethyl ester (8b), and valine methyl ester (8c) to afford 9a-c in high yields. Thecompounds were purified by column chromatography usingmethanol/dichloromethane (1:2) aseluent (Scheme 2).

Ethyl 4-Methyl-2-((4-Nitrophenoxy)((5-(6-Oxo-1H-Purin-9(6H)-yl)Tetrahydrofuran-2-yl)Methoxy)Phosphorylamino)Pentanoate (9a)

Yield: 65 %; mp: 198–200 °C; IR (KBr): υ 1,237 (P=O), 3,325 and 3,259 (N-H) cm−1; 1HNMR (400 MHz, DMSO-d6): δ 1.01 (6H, CH3), 1.31 (3H, CH3), 1.52 (1H, CH), 1.73 (2H,

5a

5c

N

NO

F

NH2

S

OOH

PO

Cl

Cl

N

NO

F

NH2

S

OOTHF,DMP, Py

5-15 oC

P

1FTC

NO2

O

Cl

NO2

THF, DMP, Py5-30 oC

N

NO

F

NH2

S

O

OP

O

R

NO2

O O

O

MeS

MeOOC

NHEtO

P

EtO

O

NH

5h

NH

COMe

O

HO

5i

H2NOEt

O

S

NHEtO

O

HO

NH

OMeO

NH

EtO

O

5e

5f

5g5d

Cl

N

Cl

R R RCompound Compound Compound

5b

HO

P

HO

O

O

2 3 5a-l4a-l

R-H

12

34

5

6

78 9

1011

12

13

14

Scheme 1 Synthesis of phosphorylated derivatives of emtricitabine (FTC)


CH2), 1.91–1.98 (2H, m, H-9), 2.33–2.45 (2H, m, H-8), 3.25–3.29 (1H, N-CH), 3.50–3.55(2H, m, H-11), 4.14–4.19 (1H, m, H-10), 4.32 (2H, O-CH2), 5.64 (1H, s, P-N-H), 6.25 (1H,dd, H-7), 7.59–8.78 (6H, m, Ar-H), 12.19 (1H, br, s, H-1); 13C NMR (100 MHz, DMSO-d6): δ16.2, 23.5, 25.2, 26.8, 31.5, 44.7, 45.2, 61.9, 65.4, 76.3, 81.4, 122.3, 123.8, 125.4, 135.9,141.1, 144.2, 145.8, 156.7, 157.3, and 170.3; 31P NMR(161.9 MHz, DMSO-d6): δ 11.8; LCMS (%): m/z 579.1 (72 %), 390 (40 %), 410 (100 %); Anal. Calcd. for C24H31N6O9P: C,49.83; H, 5.40; N, 14.53; Found: C, 49.75; H, 5.31; and N, 14.42.

Spectroscopic data of the compounds 9b and 9c were produced in the Supplementary data.

Molecular Modeling

All the in silico studies were carried out in the Molecular Operating Environment (MOE)software tool [18].

Protein Preparation and Processing

The three-dimensional X-ray crystallographic structures of PDB IDs 1USX and 1DHMwere retrieved from Protein Data Bank. The neuraminidase protein is a homotrimerconsisting of A, B, and C chains, chain A was considered for docking process. Thestructures were individually loaded into MOE working environment ignoring watermolecules and hetero atoms. Polar hydrogens were added to proteins and subjectedprotonation followed by energy minimization in the implicit solvated environment inMMFF94× force field at a gradient cutoff value of 0.05. Stabilized conformations ofproteins were obtained after energy minimization and they were used for moleculardocking process. All the ligands were effectively docked into both proteins individ-ually using alpha triangle placement methodology where the poses are generated bysuperposition of ligand atom triplets and triplets of receptor site points. A randomtriplet of ligand atoms and a random triplet of alpha sphere centers are used todetermine the binding pose at each interaction. The free binding energy of eachcompound from each pose generated after docking process is determined by LondondG scoring function. A total of 30 conformations were generated for each compoundand they were refined and rescored again using the same scoring function. The posewith lowest binding score was selected for further analysis and to analyze the binding modeorientations of the ligands.

HN

N N

N

O

O

OH

+

NO2

O

PO

ClCl

Pyridine, THF

DMP, 0 oC R-H, DMP

5-40 oC, THF

HN

N N

N

O

O O

NO2

O

PO

Cl

6 2 7 8 a-c 9 a-c

H2NOEt

O

S NH

OMeO

NH

EtO

O

9a 9b 9c

R' =

HN

N N

N

O

OO

NO2

OP

O R'

1 23

4

56

7

89

1011

12

13

14

15

16

17

Scheme 2 Synthesis of phosphorylated derivatives of didanosine (ddI)


Pharmacology

Antiviral Activity

Embryonated Chicken Eggs and Virus

Embryonated chicken eggs were obtained from the Poultry Division, Sri VenkateswaraVeterinary University, Tirupati and were incubated at 37 °C in an egg incubator. Lasota strainof NDV was obtained from the Department of Virology, Sri Venkateswara University, Tirupati.Titers of the NDV were determined by inoculating chick embryonating eggs and calculatedmedian egg infectious (EID50) of the virus as per the method of Young et al. [19]. From this,100 EID50/0.1 mL of the viral stock were prepared to proceed for the experiments. This viralstock was stored at −40 °C.

Preparation of Inoculum (Virus/Compound mixture)

A 1:2v/v dilution of the 100 EID50/0.1 mL of virus with predetermined compound concen-trations were made to put the final concentration of extract in the virus/compound mixture at10 mg/mL. The virus/compound mixtures were kept at 4 °C for 1 h to react.

Antiviral Assay

In ovo antiviral testing of the title compounds was carried out in developing chickembryos. Nine-day-old embryonated chicken eggs were labeled according to thecompounds used. The eggs were swabbed with 70 % alcohol and transferred intosterile trays. The swabbed eggs were placed in the microsafety cabinet where theywere punched and inoculated with the compound/virus mixture via the allantoic route.The compound/virus mixture is prepared by suspending 0.1 mL of NDV in 0.1 mL of1 mg/mL compound in DMSO. Virus dissolved in saline solution without compoundswere used as controls. This mixture (inoculum) is incubated for 1 h at 4 °C beforeusing for inoculation.

A suspension of 0.1 mL of NDV was treated with 0.1 mL of 1 mg/mL of thecompound which was dissolved in DMSO. The treated viruses were incubated at 4 °Cfor about 1 h. The treated viruses and controls were then inoculated via CAM and theyolk sac of 9- to 11-day-old chick embryos for NDV. Saline solution and the viruswithout treatment were used as controls. Triplicate tests were carried out for eachcompound against each of the two viruses; the results were compared to the samplewithout treatment. The eggs were sealed with molten wax and incubated at 37 °C.Allantoic fluid from treated eggs was collected for spot test and hemagglutination testto detect NDV in the eggs. Antiviral activity results of title compounds against NDVviruses were presented in Fig. 3.

Haemagglutination (HA) Test

Preparation of Red Blood Cells Suspension

Dead embryos that have been chilled were brought out of the refrigerator and kept at roomtemperature for 30 min. The eggs were swabbed and placed in the biosafety cabinet. The shellof each egg was opened to reveal the air space. Blood was collected into an equal volume of


Alsever’s solution. The blood was then clarified by centrifugation at 1,000 rpm for 10 min, thesupernatant was discarded, and equal volume of sterile NS was added to the packed red bloodcells (RBCs) and then centrifuged at 1,000 rpm for 10 min. This procedure was repeated threetimes, and packed cells were diluted to 1 % for use in HA test.

HA Test Procedure

Placed in all wells of the U-bottomed microtiter plastic plates with the aid of a multichannelpipette was 0.025 mL of PBS. Twofold serial dilutions of the virus were made. Volumes of0.025 mL containing 1 % suspension of RBCs were added to each well. The end results wereread after 30 min of incubation at room temperature [20].

Screening of Anticancer Activity by MTT Assay

Stock Preparation

Cervical cancer-derived HeLa cell lines were cultured in 96-well plate at a density of 1,000cells per well and incubated at 37 °C, 5 % CO2 for 24 h on day 1. On day 2, the compoundswere added to the plate and incubated further at 37 °C, 5 % CO2 for 24 h to expose the cells tothe compounds. On day 3, compounds were removed from all the wells and added 100 μL ofmedia and 20 μL of MTT to each of the well. The culture was incubated further at 37 °C, 5 %CO2 for 4 h. After 4 h of incubation, 100 μL of DMSO was added to each of the well todissolve the Formazan crystals.

Results and Discussion

Chemistry

The title compounds 5a-i and 9a-c were synthesized by the above reported methods.The structures of the synthesized compounds were characterized by the IR, NMR (1H,13C, 31P) mass spectral data, and elemental analysis. The IR spectra of 5a-i showedthe bands at 3,272–3,295 and 1,207–1,237 cm−1 for NH2and P=O stretching vibra-tions, respectively [21]. Amino acid ester C=O bands fell in the 1,769–1,742 cm−1

region for the compounds 5d-i and 9a-c. 1H NMR signals and proton counting of thetitle compounds were observed in their expected region. The NH proton attached toP=O was identified as singlet at δ 6.85–6.43 ppm except for 5a, 5c, and 5e. All 13Csignals of phosphorylated FTC and ddI derivatives were observed in their expectedregions which are correlated with the literature values [22]. 31P NMR signals appearedin the range of 5.4 to 11.8 as expected for the P=O group of the title compounds[23]. The compounds 5a and 5b gave two different P=O signals in 31P NMR.

Molecular Docking

A total of 30 binding pose conformations were generated for each compound with each proteinfrom docking simulations using MOE dock system. The free energy of binding of each ligandwas ranked and assessed by London dG scoring function. The binding energies and hydrogenbond interactions of each receptor-ligand complexes were studied, and the information istabulated in Tables 1 (NDV) and 2 (HPV).


Table 1 Molecular docking of the title compounds (5a-i and 9a-c) into the neuraminidase protein

Ligand Docking score(Kcal/mol)

No. H-bonds Interacting residues H-bond length (Å)

5a −16.35 3 Arg363 2.9

Gly468 2.5

Arg498 2.4

Arene-cationic interactions Arg363

Arg416

5b −15.97 8 Arg363 2.9

Arg363 2.9

Arg363 3.1

Arg363 3.1

Arg416 2.8

Thr467 1.7

Ser452 2.0

Val466 2.2

5c −14.08 Nil

5d −13.32 2 Arg363 3.2

Glu401 2.5


5e −14.12 8 His199 2.8

Ser237 2.7

Glu258 2.9

Arg416 2.0

Arg416 3.0

Arg416 3.0

Arg498 2.7

Tyr526 3.1


5f −13.97 3 Arg174 2.4

Ser237 3.2

Tyr317 3.0


5g −14.45 6 Arg174 2.3

Arg174 3.2

Thr467 3.0

Gln496 2.6

Arg498 2.8

Tyr526 3.3


5h −14.32 3 Arg174 2.9

Arg174 3.4

Tyr526 2.2


5i −15.41 11 Gly451 3.1

Gln496 2.9


Neuraminidase Protein

The best lowest docking score −16.85 Kcal/mol was observed for the compound 9b and thenext lowest docking scores of −16.35 and −15.97 Kcal/mol were observed for 5a and 5b. Theligand structures of the compounds 5i (−15.41 Kcal/mol) > 9c (−14.94 Kcal/mol) > 5g (−14.45Kcal/mol) > 5h (−14.32 Kcal/mol) > 5e (−14.12 Kcal/mol) were showed better docking scoresindicating the good affinity levels between the receptor and compounds. The remainingcompounds are also showing improved docking scores indicating the good affinity levelsbetween the receptor and compounds. The binding energy is mainly due to the presence of

Table 1 (continued)

Ligand Docking score(Kcal/mol)

No. H-bonds Interacting residues H-bond length (Å)

Arg416 3.4

Arg416 2.8

Arg416 3.1

Arg416 2.7

Arg416 2.5

Arg498 2.5

Arg498 2.9

Tyr526 2.4

Tyr526 2.2


9a −12.83 5 Arg363 3.0

Arg363 3.0

Arg416 3.4

Arg416 2.3

Arg498 2.5


9b −16.85 4 Ser237 2.8

Arg363 3.0

Glu401 2.9

Arg416 2.4


Arg363

Arg416

9c −14.94 5 Ser237 2.7

Gly468 2.6

Thr467 2.7

Tyr526 2.9

Tyr526 2.9


Arg498

FTC −11.53 2 Arg363 2.7

Arg416 3.1

ddI −11.25 1 Tyr317 2.6


hydrogen bonds and arene cationic interactions. The binding mode of orientation of 5i isforming the highest number of hydrogen bonds (11) and, consequently, 5b and 5e areexhibiting eight hydrogen bonds and the next 5g is six, and 9a and 9c are five; accordingly,their docking scores are observed as 15.41, 15.97, 14.12, 14.45, 12.83, and 14.94 Kcal/mol,respectively. Maximum number of ligands formed the hydrogen bonds with Arg363 andArg416 of HN protein. Especially, amino acid ester-substituted ligands exhibited hydrogenbonds with Arg416. Though they have less number of hydrogen bonds, 5a, 9b, and 9c ligand-receptor complexes are showing better docking score due to the presence of two, three, andtwo arene cationic interactions, respectively. The remaining ligand-receptor complexes alsoexhibited one arene cationic interaction for each except 5b. The ligand-receptor complexes 5d,5e, 5f, 5h, 5i, 9a, 9b, and 9c are showing the hydrophobic interactions essentially at P=O,

Table 2 Molecular docking of the title compounds (5a-i and 9a-c) into the human Papillomavirus E2 protein

Ligand Docking score (Kcal/mol) No.H-bonds Interacting residues H-bond length (Å)

5a −11.76 2 Val33 2.8

Arg20 2.7

5b −9.43 3 Lys27 2.2

Lys27 2.9

Tyr30 3.3

5c −8.41 Nil

5d −8.36 1 Arg20 2.8

5e −10.11 Nil

5f −10.01 4 Lys25 2.3

Tyr26 2.7

Tyr26 3.1

Gln28 2.9

5g −10.73 2 Lys27 3.1

Val33 2.5

5h −9.79 1 Lys25 2.6

5i −9.20 3 Arg20 2.1

Arg20 2.3

Arg20 3.0

9a −9.52 3 Arg20 2.6

Tyr21 2.6

Val33 3.0

9b −10.15 3 Lys17 2.8

Lys17 2.8

Tyr36 2.0

9c −10.48 3 Arg20 2.7

Arg20 3.0

Tyr21 2.9

FTC −8.19 1 Arg20 2.6

ddI −9.06 3 Arg20 2.6

Arg20 2.8

Arg20 3.0



fluoro, ester, nitro, phenyl positions of the ligand structures. These results are concludes thatthe phosphorylated derivatives of FTC- and ddI-carrying substitution with bioactive aminoacid esters at P-atom improves the binding capacity with HN protein and encouraged to screentheir antiviral activity against Newcastle disease virus. Molecular docking complex of 5a, 5g,5i, and 9c with neuraminidase protein of NDV are produced in Fig. 1.

Human Papillomavirus E2 Protein

Among all the docking conformations, 5a presented the best lowest docking score of −11.76Kcal/mol by forming two hydrogen bonds with Val33 with protein. Furthermore, the com-pounds 5g and 9c exhibited a better docking score of −10.73 and −10.48 Kcal/mol with two(Lys27 and Val33) and three (Arg20 and Tyr21) hydrogen bonds, respectively. 5e showed adocking score of −10.11 Kcal/mol without forming any hydrogen bonds and arene-cationic interactions. All the remaining compounds exhibited moderate binding energyvalues. None of the ligands formed arene-cationic interactions with E2 protein except ddI. 5a,5b, 5d, 5i, and 9c are exhibited hydrophobic interactions with the E2 protein of HPV.Moleculardocking complex of 5g and 9c with the E2 protein of human papillomavirus are presentedin Fig. 2.

5a 5i

5g 9c

Fig. 1 Molecular docking complex of 5a, 5g, 5i, and 9c with neuraminidase protein


Pharmacology

Two studies were performed to achieve this assignment. Firstly, in ovo antiviral activity of thetitle compounds was carried out in NDV-infected embryonated chicken eggs. Secondly, theanticancer activity of the title compounds was carried out by MTT assay, and the best two, 5gand 9c, were selected to find out the IC50 value of the HeLa cell lines.

Antiviral Assay

All eggs inoculated with the virus control, 10 mg/mL of the title compounds (5a-i and 9a-c)/NDV suspension, died between 48 and 72 h of inoculation and showed positive hemagglu-tination by spot testing. Survival of NDV-infected eggs were compared to control animals for4 days of post-infection; on the other hand, the highest survival percentage was observedamong the embryonated eggs inoculated with the compound 5g/NDV suspension up to 96 hpost-inoculation. There were varying percentages of embryo survivability among the remain-ing title compounds/NDV suspension groups. Compounds 5d/NDV-, 5e/NDV-, 5i/NDV-, and9c/NDV-treated group eggs showed 60 % survivability. NDV infected and treated with the

5g 9c

Fig. 2 Molecular docking complex of 5g and 9c with human papillomavirus E2 protein

Fig. 3 Survival percentage of NDV-infected eggs treated with title compounds


remaining title compounds showed moderate survivability, but a few are better than FTC andddI. These results are presented in Fig. 3. The hemagglutination test was carried out toassociate embryo mortality with viral activity. The results of mortalities and HA spot testingare shown in Fig. 4. None of the groups of eggs inoculated with title compounds/NDVsuspension had a dead embryo at 24 h post-inoculation. This study has evaluated the potentialsof the title compounds. The results of the virus propagation showed that amino acid ester-substituted phosphorylated FTC and ddI derivatives 5g, 5d, 5e, 5i, and 9c compoundsinhibited efficiently the growth of NDV in embryonated chicken eggs than FTC and ddI. Inconclusion, this work has shown that the amino acid ester substituted phosphorylated FTC andddI derivatives have direct antiviral activity against NDV.

Anticancer Activity by MTT Assay

The docking results of the title compounds with E2 protein of HPV with stable ligand poseinteractions in the docking sphere were encouraged for screening of their anticancer activityagainst HeLa cell lines. MTT assay was accomplished for the all title compounds (5a-i and 9a-c) against HeLa cell lines at 10 and 100 μg/mL. In 96-well plates, 1,000 cells per well werecultured, and two different concentrations in 10 and 100 μg/mL of each compound 5a-i and9a-c were treated; after treating with these compounds, the MTT [(3-(4,5-dimethylthiazol-2-

Fig. 4 HA test results of all title compounds

0102030405060708090

100

5a 5b 5c 5d 5e 5f 5g 5h 5i 9a 9b 9c

% o

f ce

ll v

iab

ilit

y

Compounds

10µg/mL 100µg/mL

Fig. 5 Anticancer activity of test compounds against HeLa cell lines by MTT assay


yl)-2,5-diphenyltetrazolium bromide)] was added to each well. The formed formazan crystalswere dissolved in DMSO, and the percentage of live and dead cells were counted at 570 nm onBio-Rad, microtiter plate reader. The title compounds gave good results (Fig. 5); 5g and 9cespecially show virtuous effects. The compounds 5g and 9c gave favorable docking score withHPV E2 protein and also good anticancer activity results against HeLa cell lines at 10 and100 μg/mL. Based on these results, the compounds 5g and 9c, with better docking score and

Fig. 6 Percent of living cells at different concentrations of 5g and 9c

A B

C D

Fig. 7 a HeLa cell without compound showing 88% live cells. bHeLa cells with the compound 5g at 10 μg/mL.c HeLa cells with the compound 5g at 40 μg/mL (IC50). d HeLa cells with the compound 5g at 160 μg/mL


good anticancer activity than the remaining lead compounds, were chosen for screening theirIC50 values against the HeLa cell lines.

MTT assay was performed to find out the IC50 value of compounds 5g and 9c on HeLa celllines. In this connection, the compounds 5g and 9c were screened by MTT assay at differentconcentrations in micrograms per milliliter (0, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, and200). The graph was plotted between concentration of drug on x-axis and the percentage oflive cells on y-axis. The IC50 were obtained at 40 and 60 μg/mL concentrations for 5g and 9c,respectively. These results are expressed in Figs. 6 and 7.

Conclusion

In conclusion, it is found that the phosphorylation of the nucleosides improves the bindingenergy with the target protein. Predominantly, amino acid esters as substituents to thephosphorylated nucleosides (FTC and ddI) are playing a vital role in terminating the NDVvirus. Among the various amino acid esters, particularly, the valine methyl ester-substitutedphosphylated nucleosides (FTC and ddI) derivatives (5g and 9c) are predominantly exhibitinga promising effect on NDV and HPV. These results engrossed us for screening anticanceractivity of the title compounds on HeLa cell lines where an effective inhibition was observedwith IC50 values of 40 and 60 μg/mL, respectively. These results conclusively explain thetherapeutic value of 5g and 9c and could prompt them as effective antiviral and anticanceragents.

Acknowledgments The authors are grateful to BRNS (DAE) research project (2012/37C/21/BRNS/785),Mumbai, India for providing financial assistance through the project.

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