Design, Synthesis, Antibacterial Activity, andMolecular Docking Studies of Novel Hybrid 1,3-Thiazine-1,3,5-Triazine Derivatives as PotentialBacterial Translation Inhibitor

Udaya P. Singh1*, Manish Pathak1,Vaibhav Dubey1, Hans R. Bhat1, PrashantGahtori2 and Ramendra K. Singh3

1Department of Pharmaceutical Sciences, Sam HigginbottomInstitute of Agriculture Technology and Sciences, FormerlyAllahabad Agricultural Institute, Deemed to be University, 211007Allahabad, India2Faculty of Pharmacy, Uttarakhand Technical University, Dehradun,Uttarakhand 248007, India3Nucleic Acids Research Laboratory, Department of Chemistry,University of Allahabad, 211002 Allahabad, India*Corresponding author: Udaya P. Singh, udaysingh98@gmail.com

Some novel hybrid 1,3-thiazine-1,3,5-triazine deriv-atives were synthesized and tested for antibacte-rial activity. Compounds 8c and 8f were foundactive against Gram positive and Gram negativemicroorganisms. Molecular docking studies havebeen performed on eubacterial ribosomal decod-ing A site (Escherichia coli 16S rRNA A site) torationalize the probable mode of action, bindingaffinity, and orientation of the molecules at theactive site of receptor. The structures of all thesenewly synthesized compounds were confirmed bytheir elemental analyses and spectral data tech-niques viz. IR, 1H NMR, 13C NMR, and mass.

Key words: 1,3,5-Triazine, 1,3-thiazine, antibacterial, docking, trans-lation inhibition

Received 1 February 2012, revised 16 April 2012 and accepted for publi-cation 5 June 2012

Antibiotic resistance is all the time more recognized as a seriousand permanent public health concern and is usually considered tobe a consequence of wide use and misuse of antibiotics. Surveil-lance data for Streptococcus pneumoniae, a common cause of bac-terial respiratory tract infections, disclosed that 24% of isolateswere not susceptible to penicillin. Moreover, resistance to severalother antibacterial drugs is common; of which 1.5% of isolateswere resistant to cefotaxime (a third generation cephalosporin), andresistance to the newer fluoroquinolone antimicrobials has alreadybeen reported (1). In Europe, it is estimated that at least 25 000

people die each year from infections because of antibiotic-resistantbacteria, which also result in around 2.5 million extra hospital days(2). As a result, increased incidence of bacterial resistance to cur-rently available antibiotics altogether increases healthcare costassociated with it. According to recent study by Robert and cowork-ers, it has been calculated that annual costs of antibiotic-resistantinfections to the U.S. Healthcare system comes to be in excess of$20 billion (3).

In an our effort to design effective antimicrobial compound derivedfrom 1,3,5-triazine, we had earlier reported a novel heterocyclichybrid skeleton encompassing thiazole and 1,3,5-triazine connectedthrough –NH– linker (4,5). Structure-activity relationship suggestedthat, the thiazole amine pendant is well tolerated along with thepresence of electron withdrawing groups on phenyl amine on eitherside of 1,3,5-triazine core. Prompted by the results, we furtherdevised a series of analogues as potential antimicrobials by keepingthiazole fragment rigid and having diverse substitution pattern onboth wings of 1,3,5-triazine by amine and aromatic, aliphatic frag-ments tethered via amine and mercapto (-S-) bridge (6). Resultsrevealed that analogues having amine bridge were more effectivethan their respective mercapto equivalents and further explicatedthe critical structural requirement of electron withdrawing groups.

Over the past decade, bacterial ribosome is a key target for natu-rally occurring antibiotics, including the macrolides, tetracyclines,chloramphenicol, aminoglycosides, and the newly discovered syn-thetic oxazolidinones as well (7). Recently, 3,5-diamino-piperidinyltriazines (DAPT) having 1,3,5-triazine core scaffold, identified as anovel class of antibacterial agent have been found active in murinemodel, that target the bacterial decoding-site RNA in vitro and inhi-bit bacterial growth by a translation-dependent mechanism. Thesederivatives are considered to be as a mimetics of the natural ami-noglycoside antibiotics (8–10). Nevertheless, facts drawn throughmolinspiration program in our earlier studies allowed us to reportnuclear receptor binding domain for antibacterial action of 1,3,5-tri-azine derivatives (11).

Prompted by these findings, present study deals with the synthesisof hybrid analogues of 1,3-thiazine-1,3,5-triazine as core skeletontethered through –NH– linkage and its antibacterial activity deter-mination. To define binding mode of these constitutive compoundsas plausible bacterial translation inhibitor, molecular docking studies

1

Chem Biol Drug Des 2012

Research Article

ª 2012 John Wiley & Sons A/S

doi: 10.1111/j.1747-0285.2012.01430.x

were carried out on eubacterial ribosomal decoding A site (Escheri-chia coli 16S rRNA A site) receptor domain. The rationale behindthe present study was to optimize the substituent on pendant posi-tion, therefore, we expand the ring of earlier optimized substitutedphenyl thiazole-2-amine fragment by one carbon atom and introduc-tion of another substituted phenyl ring resulting in 4,6-substiututedphenyl-6H-1,3-thiazin-2-amine.

Experimental

Material and methodMelting points of compounds were determined in open capillarytubes Hicon melting point apparatus and are uncorrected. Thin layerchromatographic analysis was done to monitor the completion ofreaction as well as for identification and characterization of com-pounds. The different mobile phases were selected according to theassumed polarity of the products. The spots were visualized byexposure to iodine vapor and UV light. The structures of the inter-mediate compounds were established on the basis of spectral (FT-IR, 1H NMR, mass) and elemental analysis, whereas structures oftitle hybrid analogues on the basis of FT-IR, 1H NMR, 13C NMR,mass spectral, and elemental analysis data. FT-IR (in 2.0 per cm,flat, smooth, abex, KBr) spectra were recorded on Biored FTs spec-trophotometer. 1H NMR spectra were recorded on Bruker Avance II400 and 300 MHz NMR spectrometer in CDCl3-d6 and DMSO usingtetramethylsilane (TMS) as internal standard. 13C NMR spectrawere recorded on Bruker Avance II 300 NMR Spectrometer. Chemi-cal shifts are reported in parts per million (ppm, d) and signals aredescribed as s (singlet), d (doublet), t (triplet), q (quartet), and m(multiplet). The FAB mass spectrum was recorded on a THERMOFinnigan LCQ Advantage max ion trap mass spectrometer, sampleswere introduced into ESI source through Finnigan surveyor autosampler. The mobile phase MeOH ⁄ CAN:H2O (90:10) flowed at a rateof 250 lL ⁄ min by MS pump. Ion spray voltage was set at 5.3 KVand capillary voltage 34 V. The scan run upto 2.5 min and the spec-tra averaged over 10 scan at peak top in TLC. Elemental analysiswas carried out on a Vario EL III CHNOS elemental analyzer.

ChemistryThe synthesis of title analogues were carried out through followingsteps.

Step 1: Synthesis of 4,6-substituted di-phenyl1,3-thiazine-2-amineGeneral procedure for synthesis of substituted chalcone derivatives3(a–f). Dissolved substituted acetophenone, 1 (0.01 mol) andsubstituted benzaldehyde, 2 (0.01 mol) in 50 mL ethanolic solutionand stirred for 30 min, followed by dropwise addition of aqueoussodium hydroxide (0.05 mol), further stirring continued for 24 h.After completion of the reaction as reaction monitored by TLC usingthe mobile phase as n-butanol:acetic acid:water (4:3:1), a crudeproduct was obtained as substituted chalcone, 3(a–f). The resultantsolid was then filtered off, washed with water, dried, and re-crys-tallized from ethanol.

3-(2-Nitrophenyl)-1-phenylprop-2-en-1-one (3a). Yellow crystals;Yield: 76%; mp: 123–125 �C; Rf: 0.56; FT-IR (mmax; per cm KBr):1654 (C=O), 1592 (C=C), 1548.28–1446.06 (aromatic C=N); 1H NMR(400 MHz, CDCl3-d6, TMS): d 7.99–7.98 (m, 2H, H¢2 H¢6), 7.56–7.53(m, 3H, H¢3 H¢4 H¢5), 7.52 (d, 1H, J = 8.4 Hz H6), 7.51 (m, 3H, H3 H4

H5), 6.89 (d, 1H, J = 15.3 Hz Hb), 6.38 (d, 1H, J = 15.3 Hz Ha); Anal.Calcd. For C15H11NO3: C, 71.14; H, 4.38; N, 5.53. Found: C, 71.12;H, 4.39; N, 5.58.

3-(2-Nitrophenyl)-1-(4-nitrophenyl)prop-2-en-1-one (3b). Light Yellowcrystals; Yield: 85%; mp: 133–134 �C; Rf: 0.64; FT-IR (mmax; per cmKBr): 1656 (C=O), 1594 (C=C), 1548.28–1446.06 (aromatic C=N); 1HNMR (400 MHz, CDCl3-d6, TMS): d 7.97–7.96 (m, 2H, H¢2 H¢6),7.54–7.53 (m, 2H, H¢3 H¢5), 7.51 (d, 1H, J = 8.3 Hz H6), 7.51 (m, 3H,H3 H4 H5), 6.89 (d, 1H, J = 15.3 Hz Hb), 6.38 (d, 1H, J = 15.3 HzHa); Anal. Calcd. For C15H10N2O5: C, 60.41; H, 3.38; N, 9.39. Found:C, 60.32; H, 3.36; N, 9.40.

1-(4-Hydroxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (3c). Brown crys-tals; Yield: 83%; mp: 129–130 �C; Rf: 0.54; FT-IR (mmax; per cm KBr):1659 (C=O), 1595 (C=C), 1546.28–1448.06 (aromatic C=N); 1H NMR(400 MHz, CDCl3-d6, TMS) d ppm: 7.97–7.98 (m, 2H, H¢2 H¢6), 6.94–6.96 (m, 2H, H¢3 H¢5), 5.28 (s, 1H, Ar-OH), 7.51 (d, 1H, J = 8.2 HzH6), 7.50 (m, 3H, H3 H4 H5), 6.89 (d, 1H, J = 15.3 Hz Hb), 6.38 (d,1H, J = 15.3 Hz Ha); Anal. Calcd. For C15H11NO4: C, 66.91; H, 4.12;N, 5.20. Found: C, 66.90; H, 4.14; N, 5.20.

3-(2-Chlorophenyl)-1-(4-nitrophenyl)prop-2-en-1-one (3d). Dark Yel-low crystals; Yield: 87%; mp: 142–143 �C; Rf: 0.68; FT-IR (mmax; percm KBr): 1652 (C=O), 1590 (C=C), 1546–1448 (aromatic C=N), 810,754; 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.97–7.98 (m, 2H,H¢2 H¢6), 7.53–7.51 (m, 2H, H¢3 H¢5), 7.48 (d, 1H, J = 8.3 Hz H6),7.48–749 (m, 3H, H3 H4 H5), 6.89 (d, 1H, J = 15.3 Hz Hb), 6.37 (d,1H, J = 15.3 Hz Ha); Anal. Calcd. For C15H10ClNO3: C, 62.62; H,3.50; N, 4.87. Found: C, 62.65; H, 3.53; N, 4.86.

1-(4-Chlorophenyl)-3-(4-nitrophenyl)prop-2-en-1-one (3e). Brown yellowcrystals; Yield: 79%; mp: 138–139 �C; Rf: 0.47; FT-IR (mmax; per cmKBr): 1658 (C=O), 1594 (C=C), 1543–1441 (aromatic C=N), 1230, 880;1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.95–7.96 (m, 2H, H¢2H¢6), 7.54–7.53 (m, 2H, H¢3 H¢5), 7.51 (m, 3H, H3 H4 H5), 6.89 (d, 1H,J = 15.3 Hz Hb), 6.38 (d, 1H, J = 15.3 Hz Ha); Anal. Calcd. ForC15H10ClNO3: C, 62.62; H, 3.50; N, 4.87. Found: C, 62.66; H, 3.52;N, 4.88.

1,3-Bis(4-nitrophenyl)prop-2-en-1-one (3f). Orange crystals; Yield:64%; mp: 153–154 �C; Rf: 0.78; FT-IR (mmax; per cm KBr): 1654(C=O), 1596 (C=C), 1545–1443 (aromatic C=N), 1240, 880, 749; 1HNMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.97–7.98 (m, 2H, H¢2 H¢6),7.53–7.52 (m, 2H, H¢3 H¢5), 8.01–8.03 (m, 2H, H2,H6), 8.17 (m, 2H,H3 H5), 6.88 (d, 1H, J = 15.3 Hz Hb), 6.37 (d, 1H, J = 15.3 Hz Ha);Anal. Calcd. For C15H10N2O5: C, 60.41; H, 3.38; N, 9.39. Found: C,60.43; H, 3.38; N, 9.38.

General procedure for synthesis of substituted 1,3-thiazine from cor-responding chalcone derivative 3(a–f). The corresponding substi-tuted chalcone derivatives 3(a–f) (0.01 mol) and thiourea (0.01 mol)was dissolved in ethanol (50 mL) and refluxed the mixture, while a

Singh et al.

2 Chem Biol Drug Des 2012

solution of potassium hydroxide (0.05 mol) in water (10 mL) addedportion-wise for 2 h. Then again refluxed it for further 6 h and afterthat resultant mixture was poured into ice-cold water. A solid prod-uct 4(a–f) thus obtained was filtered off, dried and crystallizedfrom ethanol.

6-(2-Nitrophenyl)-4-phenyl-6H-1,3-thiazin-2-amine (4a). Yellow crys-tals; Yield: 78%; mp: 163–164 �C; Rf: 0.45; FT-IR (mmax; per cm KBr):3356 (NH),1687 (C-N), 1596 (C=C), 1656–1645 (aromatic C=N), 1522,1351 (Ar-NO2), 876, 756; 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm:5.63 (s, 1H, H5), 7.33–7.30 (m, 3H), 7.66 (d, 1H, J = 7.9 Ar-H), 8.35 (d,1H, J = 7.1, 5.43 Ar-H), 5.52 (bs, 1H); Anal. Calcd. For C16H13N3O2S:C, 61.72; H 4.21; N, 13.50. Found: C, 61.66; H, 4.23; N, 13.52.

6-(2-Nitrophenyl)-4-(4-nitrophenyl)-6H-1,3-thiazin-2-amine (4b). Browncrystals; Yield: 67%; mp: 169–171 �C; Rf: 0.65; FT-IR (mmax; per cmKBr): 3359 (NH),1683 (C-N), 1599 (C=C), 1656–1645 (aromatic C=N),1524, 1353 (Ar-NO2), 876, 756; 1H NMR (400 MHz, CDCl3-d6, TMS)d ppm: 5.68 (s, 1H, H5) 7.43–7.40 (m, 3H), 7.69 (d, 1H, J = 7.8 Ar-H), 8.06 (d, 1H, J = 7.1, 5.4 Ar-H), 5.56 (bs, 1H); Anal. Calcd. ForC16H12N4O4S: C, 53.93; H 3.39; N, 15.72. Found: C, 53.90; H, 3.39;N, 15.71.

4-(2-Amino-6-(2-nitrophenyl)-6H-1,3-thiazin-4-yl)phenol (4c). BrownYellow crystals; Yield: 69%; mp: 176–178 �C; Rf: 0.54; FT-IR (mmax;per cm KBr): 3359 (NH),1686 (C-N), 1600 (C=C), 1646–1635 (aro-matic C=N), 1521, 1353 (Ar-NO2), 889, 778; 1H NMR (400 MHz,CDCl3-d6, TMS) d ppm: 5.46 (s, 1H, H5) 7.37–7.67 (m, 3H), 7.69 (d,1H, J = 7.6 Ar-H), 8.06 (d, 1H, J = 7.1, 5.4 Ar-H), 5.45 (bs, 1H);Anal. Calcd. For C16H13N3O3S: C, 58.70; H 4.00; N, 12.84. Found: C,57.45; H, 4.12; N, 12.85.

6-(2-Chlorophenyl)-4-(4-nitrophenyl)-6H-1,3-thiazin-2-amine (4d).Light Yellow crystals; Yield: 84%; mp: 183–184 �C; Rf: 0.65; FT-IR(mmax; per cm KBr): 3359 (NH),1686 (C-N), 1634 (C=C), 1648–1630(aromatic C=N), 1520, 1353 (Ar-NO2), 890, 772; 1H NMR (400 MHz,CDCl3-d6, TMS) d ppm: 5.73 (s, 1H, H5) 7.31–7.38 (m, 3H), 7.62 (d,1H, J = 7.9 Ar-H), 8.09 (d, 1H, J = 7.2, 5.3 Ar-H), 5.45 (bs, 1H);Anal. Calcd. For C16H12ClN3O2S: C, 55.57; H 3.50; N, 12.15. Found:C, 55.51; H, 3.55; N, 12.07.

4-(4-Chlorophenyl)-6-(4-nitrophenyl)-6H-1,3-thiazin-2-amine (4e).Dark Brown crystals; Yield: 76%; mp: 189–192 �C; Rf: 0.58; FT-IR(mmax; per cm KBr): 3354 (NH),1683 (C-N), 1631 (C=C), 1648–1630(aromatic C=N), 1524, 1350 (Ar-NO2), 880, 782; 1H NMR (400 MHz,CDCl3-d6, TMS) d ppm: 5.52 (s, 1H, H5) 7.35–7.36 (m, 3H), 7.56 (d,1H, J = 7.6 Ar-H), 8.06 (d, 1H, J = 7.3, 5.3 Ar-H), 5.56 (bs, 1H);Anal. Calcd. For C16H12ClN3O2S: C, 55.57; H 3.50; N, 12.15. Found:C, 55.51; H, 3.55; N, 12.07.

4,6-Bis(4-nitrophenyl)-6H-1,3-thiazin-2-amine (4f). Orange crystals;Yield: 79%; mp: 196–198 �C; Rf: 0.57; FT-IR (mmax; per cm KBr):3359 (NH),1682 (C-N), 1635 (C=C), 1648–1630 (aromatic C=N),1524, 1353 (Ar-NO2), 890, 756; 1H NMR (400 MHz, CDCl3-d6,TMS): d 5.76 (s, 1H, H5) 7.34–7.36 (m, 3H), 7.64 (d, 1H, J = 7.6Ar-H), 8.10 (d, 1H, J = 7.3, 5.2 Ar-H), 5.52 (bs, 1H); Anal. Calcd.For C16H12N4O4S: C, 53.93; H 3.39; N, 15.72. Found: C, 53.91; H,3.40; N, 15.72.

Step 2: Procedure for synthesis of 6-chloro-N2,N4-bis(3-nitrophenyl)-1,3,5-triazine-2,4-diamine (7)3-Nitro aniline 6 (0.2 mol) was added into 100 mL of acetone attemperature 40–45 �C. The solution of 2,4,6-trichloro-1,3,5-triazine(5) (0.1 mol) in 25 mL acetone was added slowly, stirred the reac-tion for 3 h followed by drop-wise addition of NaHCO3 solution(0.1 mol) taking care that reaction mixture does not become acidic.Completion of the reaction was analyzed by TLC utilizing benzene:ethyl acetate as mobile phase (9:1). The product was filtered andwashed with cold water and re-crystallized with ethanol to affordpure compound 6-chloro-N2,N4-bis(3-nitro phenyl)-1,3,5-triazine-2,4-diamine, 7.

Brownish black crystals; Yield: 86%; mp: 143–145 �C; MW: 387.74;Rf: 0.55; FT-IR (mmax; per cm KBr): 3289.56 (N–H secondary), 3055.70(C–H broad), 1548.28–1446.06 (aromatic C=N); 1H NMR (400 MHz,CDCl3-d6, TMS): d 7.40 (t, 4H, 4 = CH-aromatic), d 7.32(t, 4H,4 = CH aromatic), 3.62 (d, 2H, 2-NH aromatic); Elemental analysisfor C15H10ClN7O4: Calculated: C, 46.46; H, 2.60; N, 25.29. Found: C,46.48; H, 2.65; N, 25.26.

Step 3: General procedure for synthesis ofhybrid 1,3-thiazine-1,3,5-triazine analogues 8(a–f)6-Chloro-N2,N4-bis(3-nitrophenyl)-1,3,5-triazine-2,4-diamine (7) (0.1 mol)was added into 50 mL of 1,4-dioxane at temperature 40–45 �C. Asolution of substituted 1,3-thiazine 4(a–f) (0.1 mol) in 35 mL 1,4-diox-ane was added slowly to above solution and stirred for 90 min fol-lowed by drop-wise addition of K2CO3 (0.1 mol), re-fluxed the reactionmixture at 135–145 �C for 9 h. The product was filtered and washedwith cold water and re-crystallized with ethanol to afford the corre-sponding pure products 8(a–f).

N2,N4-Bis(3-nitrophenyl)-N6-(6-(2-nitrophenyl)-4-phenyl-6H-1,3-thiazin-2-yl)-1,3,5-triazine-2,4,6-triamine (8a). Black crystals; Yield: 86%;mp: 145–147 �C; Rf: 0.56; FT-IR (mmax; per cm KBr): 3278 (N-Hstretch,-NH2), 2927 (C-Hstretch, Aromatic), 1577 Aromatic (C=C ringstretch),1529 (-C=N ringstretch), 1349 (-NO2stretch), 1127 (C-Nstretch), 834 (N-Hdeformation), 1403–1092 Aromatic (-C-Hin plane deformation), 737 Aro-matic (-C-Hout of plane deformation), 1238 (C-Sstretch); 1H NMR(400 MHz, CDCl3-d6, TMS): d 3.90 (bs, 3H, NH), 6.91 (d, 1H,J = 9.8Hz, Ar-H), 6.92–6.98 (d, 1H, J = 9.9Hz, Ar-H), 7.26 (s, 1H, thi-azine), 7.42–7.44 (d, 1H, J = 8.4Hz, Ar-H), 7.63–7.99 (m, 1H, Ar-H),7.73 (d, 1H, J = 8.7Hz, Ar-H), 7.99–8.01(d, 1H, J = 4.8Hz, 1H, Ar-H),8.39–8.42 (d, 1H, J = 8.4Hz, Ar-H), 8.66 (S, 1H, Ar-H); 13C NMR(400 MHz, CDCl3): d 170.42, 133.95, 114.91, 77.65, 77.23, 76.80;ESI-MS (m ⁄ z): 525.10(M + H+); Anal. Calcd. For C31H22N10O6S: C,56.19; H, 3.35; N, 21.14. Found: C, 55.12; H, 3.41; N, 21.12.

N2,N4-Bis(3-nitrophenyl)-N6-(6-(2-nitrophenyl)-4-(4-nitrophenyl)-6H-1,3-thiazin-2-yl)-1,3,5-triazine-2,4,6-triamine (8b). Yellow crystals; Yield:78%; mp: 169–171 �C; Rf: 0.51; FT-IR (mmax; per cm KBr): 3276 (N-Hstretch, -NH2), 2366 (C-Hstretch, Aromatic), 1585–1433 Aromatic (C=Cringstretch), 1613 (-C=N ringstretch), 1348 (-NO2stretch), 1317 (C-Sstretch),1299 (C-Nstretch), 994 (N-Hdeformation), 1401-1090 Aromatic (-C-Hin plane

deformation), 700 Aromatic (-C-Hout of plane deformation), 1317 (C-Sstretch);

Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of 1,3,5-triazines

Chem Biol Drug Des 2012 3

1H NMR (400 MHz, CDCl3-d6, TMS): 4.07 (bs, 3H, NH), 7.03 (d, 1H,J = 8.1Hz, Ar-H), 7.15 (d, 1H, J = 6Hz, Ar-H), 7.27 (s, 1H, thiazine),7.73 (d, 1H, J = 7.5Hz, Ar-H), 7.95–7.97 (d, 1H, J = 8.4Hz, Ar-H), 7.79(d, 1H, J = 7.8Hz, Ar-H), 8.00 (d, 1H, J = 7.2Hz, 1H, Ar-H), 8.07–8.62(m, 1H, Ar-H), 8.62 (S, 1H, Ar-H); 13C NMR (400 MHz, CDCl3): d129.84, 126.45, 77.43, 77.00, 76.58, 29.67; ESI-MS (m ⁄ z):424.17(M + H+); Anal. Calcd. For C31H21N11O8S: C, 52.62; H, 2.99; N,21.77. Found: C, 52.60; H, 3.01; N, 21.81.

4-(2-(4,6-Bis(3-nitrophenylamino)-1,3,5-triazin-2-ylamino)-6-(2-nitro-phenyl)-6H-1,3-thiazin-4-yl)phenol (8c). Light Yellow crystals; Yield:76%; mp: 182-184 �C; Rf: 0.46; FT-IR (mmax; per cm KBr): 3383 (N-Hstretch, -NH2), 3567 (O-Sstretch) 2366 (C-Hstretch, Aromatic), 1527 Aro-matic (C=C ringstretch), 1570 (-C=N ringstretch), 1400-1093 Aromatic (-C-Hin plane deformation), 1347 (-NO2stretch), 1296 (C-Ostretch), 1211 (C-Sstretch), 1168 (C-Nstretch), 835 (N-Hdeformation), 753 Aromatic (-C-Hout

of plane deformation), 670 Aromatic (Ar-Clstretch); 1H NMR (400 MHz,CDCl3-d6, TMS): d 3.78 (bs, 3H, NH), 6.92-7.95 (d, 1H, J = 6.8Hz,Ar-H), 7.48–7.51 (d, 1H, J = 6.9Hz, Ar-H), 7.55–7.58 (d, 1H,J = 6.3Hz, Ar-H), 8.18–8.20 (d, 1H, J = 6.6Hz, 1H, Ar-H), 8.20–8.42(d, 1H, J = 9.6Hz, Ar-H), 7.32 (s, 1H, thiazine), 7.66–7.97 (m, 1H, Ar-H), 6.92-6.95 (d, 1H, J = 6Hz, Ar-H), 8.66 (S, 1H, J = 5.5Hz, Ar-H);13C NMR (400 MHz, CDCl3): d 148.44, 133.54, 130.38, 129.33,124.85, 122.25, 77.43, 77.00, 76.58, 40.49, 40.21; ESI-MS (m ⁄ z):657.19 (M + H+); Anal. Calcd. For C31H22N10O7S: C, 54.86; H, 3.27;N, 20.64. Found: C, 55.05; H, 3.27; N, 20.65.

N2-(6-(2-chlorophenyl)-4-(4-nitrophenyl)-6H-1,3-thiazin-2-yl)-N4,N6-bis(3-nitrophenyl)-1,3,5-triazine-2,4,6-triamine (8d). Yellow crystals;Yield: 86%; mp: 174–176 �C; Rf: 0.31; FT-IR (mmax; per cm KBr):3385 (N-Hstretch, -NH2), 2927 (C-Hstretch, Aromatic), 1599 (-C=N ring-stretch), 1528 Aromatic (C=C ringstretch), 1400–1091 Aromatic (-C-H in plane

deformation), 1354 (-NO2stretch), 1178 (C-Nstretch), 834 (N-Hdeformation),738 Aromatic (Ar-Clstretch), 611 Aromatic (-C-Hout of plane deformation),572.50 (C-Sstretch); 1H NMR (400 MHz, CDCl3-d6, TMS): d 3.57 (bs,3H, NH), 6.70 (s, 1H, J = 6.8Hz, Ar-H), 6.93–6.91 (d, 1H, J = 6Hz,Ar-H), 6.97 (d, 1H, J = 8.7Hz, Ar-H), 7.22 (s, 1H, thiazine), 7.38 (d,1H, J = 7.8Hz, Ar-H), 7.51 (d, 1H, J = 7.8Hz, Ar-H), 7.90-7.78 (m, 1H,Ar-H), 8.14 (d, 1H, J = 9Hz, 1H, Ar-H), 8.59 (s, 1H, J = 8.4Hz, Ar-H);13C NMR (300 MHz, CDCl3): d 148.60, 129.80, 129.16, 127.22,126.48, 123.86, 113.91, 77.43, 77.01, 76.58, 67.05, 30.29; ESI-MS(m ⁄ z): 655.10(M + H+); Anal. Calcd. For C31H21ClN10O6S: C, 53.41; H,3.04; N, 20.09. Found: C, 53.43; H, 3.03; N, 20.07.

N2-(4-(4-Chlorophenyl)-6-(4-nitrophenyl)-6H-1,3-thiazin-2-yl)-N4,N6-bis(3-nitrophenyl)-1,3,5-triazine-2,4,6-triamine (8e). Light Browncrystals; Yield: 83%; mp: 168-170 �C; Rf: 0.52; FT-IR (mmax; per cmKBr): 3385 (N-Hstretch, -NH2), 2924 (C-Hstretch, Aromatic), 1522 (-C=Nringstretch), 1431 Aromatic (C=C ringstretch), 1402–1091 Aromatic (-C-Hin plane deformation), 1351 (-NO2stretch), 1238 (C-Sstretch), 1177 (C-Nstretch), 834 (N-Hdeformation), 670 Aromatic (Ar-Clstretch), 612 Aromatic(-C-Hout of plane deformation); 1H NMR (400 MHz, CDCl3-d6, TMS): d3.98 (bs, 1H, NH), 6.93-6.91 (d, 1H, J = 6Hz, Ar-H), 7.13 (d, 1H,J = 9.3Hz, Ar-H), 7.26 (s, 1H, thiazine), 7.50–7.53 (d, 1H, J = 6.0Hz,Ar-H), 7.62–7.80 (m, 1H, Ar-H),7.87 (d, 1H, J = 8.7Hz, Ar-H), 7.99–8.02 (d, 1H, J = 9.2Hz, Ar-H), 8.06–8.09 (d, 1H, J = 7.8Hz, Ar-H),8.24–8.28 (d, 1H, J = 8.1Hz, Ar-H); 13C NMR (300 MHz, CDCl3): d77.42, 77.00, 76.58, 67.05; ESI-MS (m ⁄ z): 655.10 (M + H+); Anal.

Calcd. For C31H21ClN10O6S: C, 53.41; H, 3.04; N, 20.09. Found: C,53.45; H, 3.01; N, 20.08.

N2-(4,6-Bis(4-nitrophenyl)-6H-1,3-thiazin-2-yl)-N4,N6-bis(3-nitrophenyl)-1,3,5-triazine-2,4,6-triamine (8f). Yellow crystals; Yield: 68%; mp:154–156 �C; Rf: 0.47; FT-IR (mmax; per cm KBr): 3384 (N-H stretch, -NH2), 1522 Aromatic (C=C ringstretch), 1399–1091 Aromatic (-C-Hin

plane deformation), 1343 (-NO2stretch), 1239 (C-Sstretch), 1177 (C-Nstretch),994 (N-Hdeformation) 801 (N-Hrocking), 670 Aromatic (-C-Hout of plane

deformation); 1H NMR (400 MHz, CDCl3-d6, TMS): d 3.76 (bs, 3H, NH),7.10-7.07 (d, 1H, J = 8.8Hz, Ar-H), 7.33 (s, 1H, thiazine), 7.66–7.67(d, 1H, J = 8.8Hz, Ar-H), 7.94–7.92 (d, 1H, J = 8.4Hz, Ar-H), 8.01–7.99 (d, 1H, J = 8.8Hz, Ar-H), 8.10–8.08 (d, 1H, J = 8.4Hz,Ar-H),8.15–8.13 (d, 1H, J = 7.2Hz, Ar-H), 8.72–8.35 (d, 1H, J = 8.8Hz, Ar-H), 8.20–8.18 (d, 1H, J = 8.8Hz, Ar-H); 13C NMR (400 MHz, CDCl3):d 148.60, 129.80, 129.23, 127.22, 126.48, 123.86; ESI-MS (m ⁄ z):424.17(M + H+); Anal. Calcd. For C31H21N11O8S: C, 52.62; H, 2.99;N, 21.77. Found: C, 52.70; H, 3.02; N, 21.79.

Molecular docking studiesThe 3D X-ray crystal structure of paromomycin docked into the eu-bacterial ribosomal decoding A site (E. coli 16S rRNA A site;1j7t:pdb) was used as starting model for this study. The proteinwas prepared, docked, scored, and the molecular dynamics simula-tion carried out using standard procedures. All computational analy-sis was carried out using Discovery Studio 2.5 (Accelrys SoftwareInc., San Diego; http://www.accelrys.com).

Preparation of receptorThe target protein that complexed with paramomycin (PDB ID: 1j7t)was taken, the ligand paramomycin extracted, and the bond orderwere corrected. The hydrogen atoms were added, and their posi-tions were optimized using the all-atom CHARMm (version c32b1)forcefield with Adopted Basis set Newton Raphson (ABNR) minimi-zation algorithm, until the root mean square (r.m.s) gradient forpotential energy was <0.05 kcal ⁄ mol ⁄ � (18,19). Using the 'BindingSite' tool panel available in DS 2.5, the minimized eubacterial ribo-somal decoding A site (E. coli 16S rRNA A site) was defined asreceptor, binding site was defined as volume occupied by the ligandin the receptor, and an input site sphere was defined over the bind-ing site with a radius of 5 �. The center of the sphere was takento be the center of the binding site, and side chains of the residuesin the binding site within the radius of the sphere were assumedto be flexible during refinement of postdocking poses. The receptorhaving defined binding site was used for the docking studies.

Ligand setupUsing the built-and-edit module of DS 2.5, various ligands werebuilt all-atom CHARMm forcefield parameterization was assigned,and then minimized using the ABNR method. A conformationalsearch of the ligand was carried out using a stimulated annealingmolecular dynamics (MD) approach. The ligand was heated to atemperature of 700 K and then annealed to 200 K. Thirty suchcycles were carried out. The transformation obtained at the end ofeach cycle was further subjected to local energy minimization, using

Singh et al.

4 Chem Biol Drug Des 2012

the ABNR method. The 30 energy-minimized structures were thensuperimposed and the lowest energy conformation occurring in themajor cluster was taken to be the most probable conformation.

Docking and ScoringMolecular docking is a significant computational method used toforecast the binding of the ligand to the receptor binding site byvarying position and conformation of the ligand keeping the recep-tor rigid. LigandFit (20) protocol of DS 2.5 was used for the dockingof ligands with eubacterial ribosomal decoding A site (pdb id:1j7t)a. The LigandFit docking algorithm combines a shape compari-son filter with a Monte Carlo conformational search to generatedocked poses consistent with the binding site shape. These initialposes are further refined by rigid body minimization of the ligandwith respect to the grid based calculated interaction energy usingthe Dreiding forcefield (21). The receptor protein conformation waskept fixed during docking, and the docked poses were further mini-mized using all-atom CHARMm (version c32b1) forcefield and smartminimization method (steepest descent followed by conjugate gradi-ent), until r.m.s gradient for potential energy was <0.05 kcal ⁄ mol ⁄ �.The atoms of ligand and the side chains of the residues of thereceptor within 5 � from the center of the binding site were keptflexible during minimization. The description of ligand scoring (-PLP1, -PLP2, -PMF, Lig_Internal_Energy, Binding Energy, and DockScore) were given earlier in result and discussion section. Further-more, determination of binding energy to assess the binding affinityof ligands for receptor was calculated by employing highest stableligand-receptor complex through the protocol 'Calculate BindingEnergies' within DS 2.5 using the default settings.

Antibacterial screening

Minimum inhibitory concentrationEntire hybrid compounds were screened for their minimum inhibitoryconcentration (MIC, lg ⁄ mL) against selected Gram-positive organ-isms, viz. Bacillus subtilis (NCIM-2063), Bacillus cereus (NCIM-2156)and Staphylococcus aureus (NCIM-2079) and Gram-negative organ-ism viz., E. coli (NCIM-2065) by the broth dilution method as recom-mended by the National Committee for Clinical LaboratoryStandards with minor modifications (22). Levofloxacin was used as

standard antibacterial agent. Solutions of the test compounds andreference drug were prepared in dimethyl sulfoxide (DMSO) at con-centrations of 100, 50, 25, 12.5, 6.25, 3.125 lg ⁄ mL. Eight tubeswere prepared in duplicate with the second set being used as MICreference controls (16–24 h visual). After sample preparation, thecontrols were placed in a 37 �C incubator and observed for anymacroscopic growth (clear or turbid) the next day.

Into each tube, 0.8 mL of nutrient broth was pipette (tubes 2–7),tube 1 (negative control) received 1.0 mL of nutrient broth and tube8 (positive control) received 0.9 mL of nutrient. Tube 1, the negativecontrol, did not contain bacteria or antibiotic. The positive control,tube 8 contained bacteria, but not antibiotic. The test compoundwere dissolved in DMSO (100 lg ⁄ mL), 0.1 mL of increasing concen-tration of the prepared test compounds which are serially dilutedfrom tube 2 to tube 7 from highest (100 lg ⁄ mL) to lowest(3.125 lg ⁄ mL) concentration (tube 2–7 containing 100, 50, 25, 12.5,6.25, 3.125 lg ⁄ mL). After this process, each tube was inoculatedwith 0.1 mL of the bacterial suspension whose concentration corre-sponded to 0.5 McFarland scale (9 · 108 cells ⁄ mL), and each tubewas incubated at 37 �C for 24 h at 150 rpm. The incubation cham-ber was kept humid. At the end of the incubation period, MIC val-ues were recorded as the lowest concentration of the substancethat gave no visible turbidity, i.e., no growth of inoculated bacteria.Results are presented in Table 1.

Disc diffusionThe inoculum can be prepared by making a direct broth or salinesuspension of isolated colonies of the same strain from 18 to 24 hM�eller-Hinton agar plate. The suspension is adjusted to match the0.5 McFarland turbidity standard, using saline and a vortex mixer.Optimally, within 15 min after adjusting the turbidity of the inocu-lum suspension, a sterile cotton swab is dipped into the adjustedsuspension and then the dried surface of an agar plate is inocu-lated by streaking the swab over the entire sterile agar surface.Any surface moisture to be absorbed before applying the drugimpregnated disk (23).

The plates containing bacterial inoculums are received a disc of lev-ofloxacin (20 lg) and synthesized compound (20 lg), whereas thecontrol plate was inoculated with DMSO which shows no inhibition

Table 1: Antibacterial activity of hybrid derivatives

Compound

Minimum Inhibitory Concentration (MIC, inlg ⁄ mL)

Inhibition halo (in mm) Percentage inhibition

Gram positive Gram negative Gram positive Gram negative Gram positive Gram negative

B. subtilis B. cereus S. aureus E. coli B. subtilis B. cereus S. aureus E. coli B. subtilis B. cereus S. aureus E. coli

8a 25 100 6.25 25 24 09 36 24 54.5 22.5 85.7 52.28b 12.5 100 25 25 32 18 29 27 72.7 45 69.0 58.68c 6.25 25 25 12.5 38 28 27 32 86.3 70 64.2 69.58d 25 100 3.125 25 29 14 38 26 65.9 35 90.4 568e 25 12.5 6.25 50 27 34 35 22 61.3 85 83 47.88f 6.25 12.5 12.5 12.5 35 30 31 32 79.5 75 73.8 69.5Levofloxacin 3.125 3.125 3.125 3.125 44 40 42 46 100 100 100 100

Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of 1,3,5-triazines

Chem Biol Drug Des 2012 5

of bacterial growth. Each disc must be pressed down to ensurecomplete contact with the agar surface. Then plates are invertedand placed in an incubator set to 35 �C within 15 min after thediscs are applied. They were then incubated at 37 �C for 24 h,after which the inhibition halo was measured with a milimetricruler. This qualitative screening was performed to verify positiveantimicrobial activity of the synthesized compound. Each test wascarried out in triplicate. These results are further quantified interms of percentage of inhibition in reference to levofloxacin asstandard. And results were shown in Table 1.

Results and Discussion

ChemistryThe synthetic route to obtain the necessary key intermediates fromcommercially available reagents is briefly outlined in Scheme 1.

The title hybrid analogues of 1,3-thiazine-1,3,5-triazine tethered via–NH– linkage 8(a–f) were accomplished by clubbing of 4,6-substiu-tuted di-phenyl-6H-1,3-thiazin-2-amine (4) fragment with 6-chloro-N2,N4-bis(3-nitrophenyl)-1,3,5-triazine-2,4-diamine (5) through anaromatic nucleophilic reaction in the presence of NaOH as baseunder vigorous condition. The mechanism of synthesis of substituteddi-phenyl thiazine-2-amine fragment 4(a–f), achieved throughsubstituted chalcone intermediates 3(a–f) prepared by crossed aldolcondensation reaction between substituted aldehydes (2) and eth-anolic acetophenone (1) (enolizable) in presence of NaOH as base,

is presented in Figure 1. The resulting substituted chalcones 3(a–f)were again allowed to undergo cyclo-condesation reaction to afford4,6-substiututed di-phenyl-6H-1,3-thiazin-2-amine amine fragment4(a–f) by refluxing for 6 h in the presence of thiourea. On theother hand, flanked ring substituents on either side of 1,3,5-triazine,namely di-m-nitro phenyl amine were generated from the aromaticnucleophilic reaction between 2,4,6-trichloro-1,3,5-triazine (5) withtwo equivalents of m-nitro phenyl amine (6) to yield 6-chloro-N2,N4-bis(3-nitrophenyl)-1,3,5-triazine-2,4-diamine (7), Scheme 1. Thestructures of all synthesized compounds were confirmed by FT-IR,1H NMR, 13C NMR, mass spectral, and elemental analysis tech-niques. Herewith, this procedure acclaims an efficient and promis-ing synthetic strategy with good to excellent yields for productionof the titled skeleton. FTIR spectrum was recorded in the range4000–650 per cm to ensure presence of various functional groups.In this context, the characteristic group stretching frequencies ofaromatic secondary amines (-NH) tend to appear at 3276–3385 percm whereas N-H deformation at 834–994 per cm. Aromatic C-H inplane deformation was observed in range 1092–1403 per cm andaromatic C=C ring stretching is easily recognized at 1431–1522 percm. Moreover, our investigations in the NMR spectrum states thatamino linker between 1,3-thiazines and 1,3,5-triazine appear at3.57–4.07 ppm as a singlet. A singlet nearer at 7.26 ppm wasattributed to aromatic protons of thiazine. Other protons corre-sponded to phenyl ring were observed in the spectrum at 6.91–8.62ppm. 13C NMR technique reveals the characteristic carbon shiftranges from 170.42 to 29.67 ppm. The highest carbon shifts areassigned to triazine carbon in case of 8a. At the end, the identified

Scheme 1: Synthesis of target hybrid 1,3-thiazine-1,3,5-triazine derivatives 8(a–f). Reagents and condition: (a) NaOH, stirring for 24 h. (b)ethanol, aq. KOH, reflux for 6 h. (c) NaHCO3, 40–45 �C. (d) reflux, 120–135 �C, K2CO3.

Singh et al.

6 Chem Biol Drug Des 2012

proton multiplet was presented in the 1H NMR spectrum with theirJ-couplings 4.8–8.4Hz. The mass spectrum of compounds is charac-terized by their M + 1 peak. Elemental analysis was within €0.4%of the theoretical values in agreement with the proposed struc-tures.

Antibacterial activityThe title hybrid derivatives 8(a–f) showed moderate to significantsusceptibilities towards Gram-negative and Gram-positive bacteria,as shown in Table 1. In the case of compound 8a, no activity wasobserved against B. cereus at highest test concentration of100 lg ⁄ mL, but at the same time, a significant inhibition patternwas observed in the case of S. aureus (6.25 lg ⁄ mL) and mildagainst B. subtilis and E. coli. In the next instance, introduction ofnitro at para position on intact phenyl ring connected to 1,3-thiazine(8b), showed no significant alteration in activity, except in the caseof B. subtilis (12.5 lg ⁄ mL). Furthermore, replacement of p-nitro withp-hydroxy, compound 8c, caused markedly increase in activityagainst B. subtilis (6.25 lg ⁄ mL), B. cereus (25 lg ⁄ mL), and E. coli(12.5 lg ⁄ mL) and no change against S. aureus (25 lg ⁄ mL). Surpris-ingly, twofold reduction in activity was observed in the case ofB. subtilis and B. cereus, onefold in the case of E. coli, but furtherthreefold increase in activity for S. aureus (3.125 lg ⁄ mL), when m-chloro and p-nitro substitution were present on both the ring atfourth and fifth position, respectively, compound 8d, of 1,3-thiazinecore. No considerable change in antibacterial activity was observedin the case of compound 8e (m-chloro to p-chloro), e.g. B. subtilis(25 lg ⁄ mL), however, drastic decline in activity was reportedagainst rest of the strains. Notably, twofold amplified antibacterialactivity was observed in the case of derivative 8f, against B. subtil-is (6.25 lg ⁄ mL) and E. coli (12.5 lg ⁄ mL) with structural variation ofp-nitro in place of p-chloro, by keeping all fragment rigid. The mini-mum inhibitory concentration of all title analogues were supportedby their respective inhibition halo (percentage inhibition), to confirmpositive antimicrobial action.

It was surprising to see that minor structural variations in hybridcompounds induced marked influence on antibacterial activity. Thus,it was summarized that position of nitro atom was the only maindeterminant for generation and escalation of bio-activity withregard to structure-activity relationships. The presence of chloro

and hydroxy too have their own contribution to structure-activityrelationships.

Molecular docking studiesMolecular docking is a powerful tool in drug design, which couldpredict the best mode by which a given compound fits well into abinding site of a macromolecular target (12). With in vitro antimi-crobial results in hand, we thought it worthwhile to perform in sil-ico studies to support the results. The in silico molecular dockingstudy was carried out using LigandFit module within software pack-age in Discovery studio 2.5 on X-ray structure of eubacterial ribo-somal decoding A site (E. coli 16S rRNA A site) receptor domainwithout co-crystallized ligand paromomycin (1j7t.pdb) as outlined inexperimental section. Docking results were discussed on the param-eters, such as hydrogen bond because they help to stabilize andstrengthen a bound receptor-ligand complex and non-bonded p–pand p–+ interactions. The p–p and p–+ interactions are non-cova-lent interactions, which have pivotal role on protein-ligand recogni-tion and are termed as major forces that stabilize the association.The p–p stacking interaction results from frequent overlapping ofnon-polar ring-to-ring in parallel pose to generate hydrophobic con-tacts, whereas p–+ interaction also known as Doughtry-effect andtermed as a non-covalent molecular interaction results from interac-tion between the face of an electron-rich p system (e.g. benzenering) and an adjacent cation (+). This unusual interaction is anexample of non-covalent bonding between a monopole (cation) anda quadrupole (p system). According to Gallivan and Dougherty, p–+interaction energies should be considered of the same order ofmagnitude as hydrogen bonds or salt bridges and play an importantrole in molecular recognition and interaction with ligand. Only thosep–+ interaction of the title compounds have been discussed in thepresent case, where the interatomic distances were <6 �, the dis-tance imperative for effective p–+ interactions necessary for recep-tor-ligand recognition as suggested by Gallivan and Dougherty (13).

Molecular docking analysisFrom Table 2, it was clear that, compound 8a, formed two close in-teratomic hydrogen (H…O) bonds resulting from the interactionbetween hydrogen of amine linkages, connecting m-nitro phenylamine and 1,3,5-triazine. The oxygen of Gua40 (1.86 �) and Ade39

Figure 1: Mechanism of substituted chalcone intermediates synthesis 3(a–f).

Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of 1,3,5-triazines

Chem Biol Drug Des 2012 7

(2.10 �) present on B-chain of RNA of eubacterial ribosomal decod-ing site acted as a acceptor. Similarly, m-nitro phenyl amine con-nected to 1,3,5-triazine in compound 8a was shown to form twop–p interactions with purine ring of Gua37 on B-chain. Introductionof nitro group in fourth position of non-substituted phenyl con-nected to 1,3-thiazine, compound (8b) caused increase in numberof interatomic hydrogen bonds to three. The first hydrogen bondcorresponded to interaction involving hydrogen of amine linkageconnecting m-nitro phenyl amine to 1,3,5-triazine core (donor) andoxygen of Gua37 on B-chain as acceptor with distance of 2.03 �(H…O), whereas, the other two hydrogen bonds resulted from theinteraction of amine linkage, connecting 1,3,5-triazine to 1,3-thia-zine, with oxygen of Gua37 (1.79 �) (H….O) on B-chain and hydro-gen of Ade7 (A-chain) with sulfur atom of 1,3-thiazine (2.12 �)(H….S). These two hydrogen bond interactions are close enough tosupport di-substituted 1,3-thiazine fragment to inner grove of eubac-terial ribosomal site. Furthermore, stability of this fragment into aninner groove was also increased by the formation of one p–p inter-action because of phenyl of o-nitro phenyl on 1,3-thiazine and twop–+ interaction between nitro of above mentioned phenyl ring withthe purine ring of Gua37 (B-chain). Furthermore, increase in H-bondsto five was observed in the case of compound 8c, having hydroxyin place of p-nitro and all fragment kept rigid. Of which, two hydro-

gen bonds were formed between hydrogen of Cyt6 of A-chain andoxygen of nitro group present on meta position of phenyl ring on1,3-thiazine (H….O) with distance of 1.98 � and 2.49 �. Anothertwo intermolecular hydrogen bond resulted from the involvement ofhydrogen of amine linkage connecting m-nitro phenyl amine to1,3,5-triazine core on either side as donor and nitrogen of purinering (Gua37) and oxygen of Ade38 with interatomic distance 2.50and 2.08 �, respectively, as acceptor. The fifth hydrogen bond wasformed through the hydrogen of hydroxy group on phenyl at paraposition attached to 1,3-thiazine (donor) with oxygen (acceptor) ofGua40 (Chain B) with distance of 1.91 � (H….O). Moreover, oneadditional p–p and p–+ interaction was also observed, in which,Gua37 of B-chain was responsible for p–p stacking with m-nitrophenyl connected to 1,3,5-triazine and nitro group of o-nitro phenylon 1,3-thiazine for p–+ interaction with Gua4 of A-chain. Slightdecrease in number of hydrogen bonds to four was achieved onintroduction of Cl atom on second position in place of nitro on phe-nyl connected to 1,3-thiazine and further substitution of p-nitro inplace of p-hydroxy (8d). Of these, one hydrogen bond resultedthrough the interaction of hydrogen of Cyt6 (A-chain) with oxygenatom of m-nitro group present on phenyl amine connected to 1,3,5-triazine (H….O) with a distance of 2.38 �. Additional hydrogenbond formed through the involvement of hydrogen of amine of

Table 2: Molecular docking interaction of ligand with receptor in ligand-receptor docked complex

Compound

H-bonding p–p monitor p–+ monitor

Bond Distance(�)

DonorAtom

AcceptorAtom Bond

Distance(�) End1 End2 Bond

Distance(�)

End1 End2

8a 8a:H49–B:Gua40:O6 1.86 H49 O6 8a–B:Gua37 5.84 8a B:Gua37 A:Cyt3–8a:N40 6.09 A:Cyt3 8a:N408a:H50–B:Ade39:OP2 2.10 H50 OP2 8a–B:Gua37 4.41 8a B:Gua37

8b A:Ade7:H62–8b:S17 2.12 H62 S17 8b–B:Gua37 5.68 8b B:Gua37 B:Gua37–8b:N46 5.16 B:Gua37 8b:N468b:H52–B:Gua37:OP2 2.03 H52 OP2 B:Gua37–8b:N46 4.34 B:Gua37 8b:N468b:H54–B:Gua37:O6 1.79 H54 O6

8c A:Cyt6:H42–8c:O48 1.98 H42 O48 8c–B:Gua37 4.89 8c B:Gua37 B:Gua40–8c:N44 6.97 B:Gua40 8c:N44A:Cyt6:H42–8c:O49 2.49 H42 O49 A:Gua4–8c:N47 5.96 A:Gua4 8c:N478c:H50–B:Ade38:OP2 2.08 H50 OP2 A:Gua4–8c:N47 6.36 A:Gua4 8c:N478c:H51–B:Gua37:N3 2.50 H51 N3 B:Ade38–8c:N44 6.55 B:Ade38 8c:N448c:H71–B:Gua40:OP2 1.91 H71 OP2

8d A:Cyt6:H42–8d:O42 2.38 H42 O42 NO B:Ade38–8d:N44 5.74 B:Ade38 8d:N44B:Ade39:H61–8d:O46 2.33 H61 O468d:H50–B:Gua40:N7 2.47 H50 N78d:H52–B:Gua37:O6 1.97 H52 O6

8e 8e:H52–B:Gua37:N7 2.06 H52 N7 NO B:Gua40–8e:N47 6.55 B:Gua40 8e:N47A:Gua4–8e:N44 5.13 A:Gua4 8e:N44A:Gua4–8e:N44 5.86 A:Gua4 8e:N44

8f A:Ade7:H61–8f:O42 2.26 H61 O42 NO B:Gua40–8f:N49 5.50 B:Gua40 8f:N49A:Cyt8:H42–8f:O41 2.42 H42 O418f:H54–B:Gua37:N7 2.08 H54 N7

Paromomycin

(reference ligand)A:Cyt6:H42–par:O25 1.77 H42 O25 NO NOA:Ade7:H62–par:O10 2.37 H62 O10par:H48–B:Ade38:OP2 1.93 H48 OP2par:H50–B:Ade39:OP2 1.78 H50 OP2par:H54–A:Ade7:N1 2.03 H54 N1par:H56–B:Urd41:O4 1.82 H56 O4par:H61–B:Gua40:OP2 1.95 H61 OP2par:H62–B:Gua40:N7 1.90 H62 N7par:H77–A:Gua4:OP2 1.85 H77 OP2par:H80–A:Urd5:OP2 1.83 H80 OP2

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Ade39 (donor) with oxygen atom of other m-nitro present on phenylamine connected to 1,3,5-triazine (H….O) with a distance 2.33 �.The other two hydrogen bonds formed through the interactionbetween nitrogen of Gua40 and oxygen of Gua37 (acceptor) of B-chain with hydrogen of amine linkages connecting m-nitro phenylamine and 1,3-thiazine to 1,3,5-triazine (donor) with interatomic dis-tance of 2.47 and 1.97 �, respectively. The compound 8d did notshow any p–p interaction, but had only one p–+ interactionbetween Ade39 and nitro at meta to phenyl amine connected to1,3,5-triazine ring. However, replacement of 2-chloro with 4-chloroon the phenyl ring of 1,3-thiazine fragment (compound 8e), causeddrastic reduction in number of hydrogen bond to only one. And thissingle H-bond formed through hydrogen of amine linkage connecting1,3-thiazine fragment of 1,3,5-triazine ring (donor) with nitrogen ofGua37 as acceptor atom with interatomic distance of 2.06 �(H….O). Compound 8e showed no p–p interaction, but for twoeffective p–+ interactions resulting from involvement of nitro atomof m-nitro phenyl amine attached to 1,3,5-triazine with purine ringof Gua4 (A-chain). Presence of nitro group on para to phenyl ringattached to either side of 1,3-thiazine nucleus and keeping all frag-ment rigid led to generation of compound 8f, which showedincrease in number of hydrogen bonds to three, of which twohydrogen bonds resulted from the interaction between oxygen ofnitro group with (acceptor) hydrogen of Ade7 and Cyt8 (donor) withinteratomic distance of 2.26 and 2.42 �, respectively. The hydrogenof amine linkage connecting 1,3-thiazine to 1,3,5-triazine is respon-sible for third hydrogen bond formation with nitrogen of Gua37 asacceptor with distance of 2.08 �. It was also shown to have onep–+ interaction with nitro of p-nitro phenyl on second position of1,3-thiazine with purine ring of Gua40, but not reported to haveany p–p interaction.

It can be inferred from the orientation of H-bonds that, hydrogenpresent on amine linkages, connecting 1,3-thiazine fragment and m-nitro phenyl to 1,3,5-triazine and nucleotides present on B-chainbackbone of eubacterial ribosomal site namely Ade38, Gua37,Ade39, and Gua40; and only Cyt6, Cyt8, and Ade7 present on A-chain were highly conserved and enhanced the binding affinity andstability of compounds into the inner groove of active site. Nucleo-tides present on B-chain act as acceptor atoms to form H-bond andA-chain nucleotides behave as donor atoms. In addition, presenceof p–p and p–+ interactions in some instances provides an addi-tional factor for stability of ligands into the receptor site for effec-tive ligand-receptor interaction. Therefore, it is worthwhile tomention here that 1,3-thiazine fragment is well tolerated on thebasis of H-bond formation as pendant substituent on 1,3,5-triazine,owing to the intriguing interaction of hydrogen of amine linkageconnecting it with nucleotides and involvement of substitutedgroups on phenyl to form hydrogen bond, p–p and p–+ interac-tions. To compare the binding mode with known inhibitor, a refer-ence ligand paromomycin (a bacterial translation inhibitor) was alsodocked at same site. This reference ligand formed ten hydrogenbonds and the binding mode was the same as that of the title com-pound (A-chain: Cyt6 and Ade7; B-chain: Ade38, Ade39, Gua37,Gua40), except additional H-bond formed with Uri5 and Gua4 of A-chain and Uri41 of B-chain, no p–p and p–+ interactions wereobserved. It further confirmed a similar mechanism of action of titleanalogues as that of reference, as a bacterial translation inhibitor.

The docked orientation of ligands as well as paromomycin in eubac-terial ribosomal decoding A site (E. coli 16S rRNA A site) receptordomain are presented in Figure 2.

ScoringLigand scoring is a method to rapidly estimate the binding affinityof a ligand, based on a candidate ligand pose geometry docked intoa target receptor structure. To define binding affinity of ligands withreceptor in a better way, hybrid title analogous and reference pa-romomycin (for comparison) were rigorously analyzed through scor-ing function, such as -PLP1, -PLP2, Jain, -PMF, Lig_Internal_Energy,binding energy, and ultimately dock score.

Piecewise Linear Potential is a fast, simple, docking function thathas been shown to explain well the protein ligand binding affini-ties. PLP scores are measured in arbitrary units, with negative PLPscores reported to make them suitable for subsequent use in con-sensus score calculations. Higher PLP scores indicate strongerreceptor-ligand binding. Two versions of the PLP function are avail-able: PLP1 (15) and PLP2 (16). In the PLP1 function, each non-hydro-gen ligand or non-hydrogen receptor atom is assigned a PLP atomtype. Hydrogen atoms are excluded from consideration. In PLP2function, PLP atom typing remains the same as in PLP1. In addition,an atomic radius is assigned to each atom except for hydrogen. Itis worthwhile to mention that majority of the title compoundsshowed comparable PLP scores to the reference ligands, howevercompounds 8a (100.7), 8d (100.39), and 8f (101.1) exhibited PLP1score very close to the reference (105.6).

The PMF scoring functions were developed on the basis of statisticalanalysis of the 3D structures of protein-ligand complexes. These werefound to correlate well with protein-ligand binding free energies. Thescores are calculated by summing pairwise interaction terms over allinteratomic pairs of the receptor-ligand complex (17). In a comparisontest, it was found that all title compounds showed excellent to signif-icant PMF scores with reference to standard ligand.

The internal non-bonded ligand energy is calculated for each newconformation that is generated. Bad conformations with high inter-nal ligand energies (typically resulting from internal close contacts)are discarded. The internal ligand energy consists of a van der Wa-als (vdW) term and an optional electrostatic term. The non-bondedvdW energy is computed using a standard 9-6 (unsoftened) poten-tial using forcefield parameters consistent with the forcefieldemployed. Efficient ligand is classified with high negative ligandinternal energy. All title hybrid analogues demonstrated significantlyhigh negative values as that compared to that of the reference andcompound 8d ()11.412) and 8e ()10.068) showed higher stabiliza-tion energy than the reference.

Binding energy of the title molecules as well as of paromomycin(reference) was analyzed from their corresponding docked conforma-tion in receptor. Results illustrated that binding energy of com-pounds 8a ()151.89), 8b ()97.86), and 8c ()119.85) was higherthan that of reference, whereas in case of compounds 8d ()83.42),8e ()79.37), and 8f ()81.36), it was lower than that of reference()90.72).

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Chem Biol Drug Des 2012 9

Candidate ligand poses were evaluated and prioritized according tothe DockScore function. All the scoring data for the title hybridcompounds along with paromomycin were presented in Table 3.

It was clear from Table 3 that the reference ligand paromomycinexhibited dock score of 88.35 and none of compounds presentedthe same or greater score than reference. The entire hybrid com-pounds 8(a–f) showed significant dock score ranging from 76.94 to87.41, which suggested that all molecules have similar mode ofbinding and significant binding affinity towards RNA receptordomain as that of the reference ligand.

Validation of the accuracy and performance ofLigandFitA scoring function is deemed to be successful, if the best-docked conformation of a ligand bears a resemblance with the

bound native ligand in the experimental crystal structure. Accord-ing to Wang et al., (14) the successful scoring function is theone in which the RMSD (root mean square deviation) of thebest docked conformation is £2.0 � from the experimental one.As a consequence, the validation of function achieved in Ligand-Fit ⁄ Discovery Studio 2.5 was prepared by docking of the nativeligand into its binding site. Then the docked result was com-pared with the crystal structure of the bound ligand–proteincomplex. In the present study, paromomycin ligand was dockedinto its 16S-rRNA A-site receptor (PDB code: 1j7t), as referenceligand for the comparative study of binding action, and the newsynthesized compounds, i.e., hybrid 1,3-thiazine-1,3,5-triazinederivatives as ligands. It was revealed that the title ligandsbound in similar fashion with paromomycin with the RMSD of1.25 �. It has been confirmed that the accuracy and perfor-mance of the LigandFit ⁄ Discovery Studio 2.5 was highly satisfac-tory.

Figure 2: Binding modeof hybrid 1,3-thiazine-1,3,5-triazine derivatives 8(a–f)and paromomycin (standard)inside the eubacterial RNAactive site. (Green: H-bonds,Black: +–p and p–p stack-ing).

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Conclusion

The present study provided a novel insight into the potential che-motherapeutic value of hybrid molecules comprising 1,3-thiazineand 1,3,5-triazine as core bioactive heterocyclic scaffolds on thebasis of molecular docking analysis and in vitro antibacterial activ-ity. Furthermore, docking results suggested that the binding modeof the synthetic molecules was similar to the reference ligand andthus the synthetic molecules were supposed to work as bacterialtranslation inhibitors.

It is also proved that, expansion in pendant substituent in earlierdeveloped lead fragment (phenyl thiazole) changing to di-phenyl1,3-thiazine was well tolerated as confirmed by antibacterial activityand docking runs.

A close inspection of in vitro antibacterial activity supported by dock-ing study clearly suggested that the hybrid molecules 8c, owing tothe higher number of H-bonds, p–+ interactions, significant dock scor-ing can act as a potential bacterial translation inhibitor in an ongoingchemotherapeutic drug discovery regime. However, optimizationshould be carried out to transform these hybrid molecules to increaseits efficacy and potency. Furthermore, the structure-based optimiza-tion studies are in progress and will subsequently be reported.

Acknowledgments

Authors are grateful to SAIF, Central Drug Research Institute, Luc-know, India for providing spectral data of compounds synthesizedand SHIATS for providing basic facilities to carry out the project.One of the author, UPS is also pleased to acknowledge Prof. Ray C.F. Jones, Professor of Organic and Biological Chemistry, Loughbor-ough University, UK for his critical discussion and suggestions onthe chemistry of 1,3,5-triazines.

Conflict of Interest

None.

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Table 3: Scoring of ligands in ligand-receptor docked complex

Name -PLP1 -PLP2 -PMF Lig-Internal_Energy Binding energy (kcal ⁄ mol) Dock Score

8a 100.7 63.58 101.93 )8.531 )151.89 80.708b 82.77 46.05 69.15 )8.718 )97.86 87.418c 95.03 74.01 67.81 )8.459 )119.85 83.458d 100.39 52.31 91.25 )11.412 )83.42 76.948e 94.14 64.64 83.79 )10.068 )79.37 84.018f 101.1 56.62 90.39 )9.345 )81.36 83.03Reference ligand

(Paromomycin)105.6 104.06 88.8 )6.422 )90.72 88.35

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Note

ahttp://www.rcsb.org/pdb/explore/explore.do?structureId=1J7T

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