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Synthesis of neolignans as microtubule stabilisers : Author Accepted Manuscript

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Synthesis of neolignans as microtubule stabilisers : Author Accepted Manuscript
  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy Synthesis of neolignans as microtubule stabilisers B. Sathish Kumar a , Aastha Singh a , Amit Kumar b , Jyotsna Singh b , Mohammad Hasanain b , Arjun Singh a ,Nusrat Masood a , Dharmendra K. Yadav a , Rituraj Konwar b , Kalyan Mitra b , Jayanta Sarkar b Suaib Luqman a , Anirban Pal a , Feroz Khan a , Debabrata Chanda a , Arvind S. Negi a, ⇑ a CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow 226015, India b CSIR-Central Drug Research Institute (CSIR-CDRI), B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India a r t i c l e i n f o  Article history: Received 31 October 2013Revised 27 December 2013Accepted 30 December 2013Available online 9 January 2014 Keywords: NeolignansLignansAnticancerMicrotubulesIn silico studiesAcute oral toxicity a b s t r a c t Tubulin is a well established target for anticancer drug development. Lignans and neolignans were syn-thesized as tubulin interacting agents. Neolignans  10  and  19  exhibited significant anticancer activityagainst MCF-7 and MDAMB-231 human breast cancer cell lines. Both the compounds effectively inducedstabilization of microtubule at 4 and 20 l M concentrations respectively. Neolignan  10  induced G2/Mphase arrest in MCF-7 cells. Docking experiments raveled that  10  and  19  occupied the same bindingpocket of paclitaxel with some difference in active site amino acids and good bioavailability of boththe compounds. In in vivo acute oral toxicity  10  was well tolerated up to 300mg/kg dose in Swiss-albinomice.   2014 Elsevier Ltd. All rights reserved. 1. Introduction Lignans and neolignans are important biodynamic agents withvariedstructuraldiversity.Inplants,theseareproducedassecond-ary metabolites derived from phenylpropanoids (C6–C3). Bothlignansandneolignansaredimericcompoundswithdifferentlink-ages. Lignans are formed by C2–C2 0 linkage through carbons of propyl chains, while other C–C linkages are known as neolignans.Both lignans and neolignans are widespread in plants. 1–4 Lignansand neolignans have exhibited a wide range of biological activitiessuch as antimalarial, 5,6 antitubercular, 7,8 anticancer, 9–14 apoptosisinducers, 15,16 antiviral 17,18 and antioxidants, 19 etc. More interest-ingly, lignans and neolignans have been good ligands for estrogenreceptors, 19 aldose reductase, 20,21 tyrosinase, 22 topoisomerase II, 23 GABA A  receptor, 24 voltage gated K + channels, 25 etc. acting asinhibitors by curtailing these enzymatic actions.Several potent leads likepodophyllotoxin(PDT) as anticancer, 26 silymarin, phyllanthin, hypophyllanthin and cleomiscosins ashepatoprotectives 27 have been obtained from this class of com-pounds. Podophyllotoxin is an aryl tetralin lignan isolated from Podophyllum  spp. 26 It acts as a mitotic inhibitor by binding revers-ibly to tubulin and inhibiting microtubule assembly. 26,28 Due totoxicity reasons podophyllotoxin could not be developed asanticancer drug. However, two of its semisynthetic derivatives,etoposideandteniposideareusedclinicallytotreatsmall-celllungcancer, testicular cancer, leukemia, lymphoma, and othercancers. 26 3,4,5-Trimethoxyphenyl unit in several antitubulins plays acrucial role in interacting with tubulin. There are antitubulinagents like podophyllotoxin, colchicine, combretastatin A4, etc.possessing this unit. 26 We designed lignans (prototype-I) andneolignans (prototype-II) as possible anticancer agents with thisfragment (Fig. 1). Several analogues were synthesized and evalu-ated against human cancer cell lines. The mode of action of activecompounds was evaluated against tubulin polymerase enzyme.The tubulin interaction was further confirmed by in silico dockingstudies. The most active compound  10  was evaluated for its effecton cell cycle phases. It was also evaluated for acute oral toxicity inSwiss-albino mice at various doses. 2. Results2.1. Chemistry  The synthetic strategy was as depicted in Scheme 1. Firstly,3,4,5-trimethoxycinnamic acid ( 1 ) was esterified to correspondingethyl ester ( 2 ) by treating it with thionyl chloride and ethanol atroomtemperature. The  p -methoxygroup of ester  2  was selectivelydemethylatedbytreatmentwithanhydrousaluminiumchloridein 0968-0896/$ - see front matter    2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +91 522 2718583; fax: +91 522 2342666. E-mail address: (A.S. Negi).Bioorganic & Medicinal Chemistry 22 (2014) 1342–1354 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage:  Author's personal copy dry methylenechloride to get 3,5-dimethoxy, 4-hydroxycinnamicacid ethyl ester ( 3 ). 29 The free radical coupling of ester  3  was donein potassium ferricyanide solution in aqueous alkali-benzene sys-tem.Useofphasetransfercatalyst(tetrabutylammoniumbromide,TBAB) was not beneficial inthis reaction, as reactionfailed inpres-ence of it. Incase of ester  3 , twoproducts were formed. Compound 4  was identified as neolignan and  5a  was identified as lignan. Sim-ilarly ethyl esters of   8  and  9  were processed to get neolignans  10 and  11  respectively. In case of   8  and  9 , we did not get lignans, onlyneolignans were obtained. But, the yields in these reactions werepoor(21–29%only). So, alternatively, wesynthesizedtheselignansthrough Stobbe’s condensation. Here, substituted aromatic alde-hyde ( 12  or  13 ) was condensed with diethyl succinate ( 14 ) inpresence of sodium hydride in dry dimethylformamide (DMF) un-der reflux condition in inert atmosphere (N 2 ). The yields of thelignans  15  and  16  as diacids were significantly higher (43–49%)by this method. The diacids were further converted to ethyl esters( 17  and  18 ) by using diethyl sulfate in anhydrous potassium car-bonate in dry acetone.Further these neolignans  10  and  11  were modified to varioussimple derivatives (Scheme 2) by acetylation ( 19  and  20 ), methyl-ation( 21  and 22 )andamideformation( 23  and 26 ),etc.usingstan-dard protocols. 30 Ester  22  was hydrolysed to corresponding diacid( 25 ) using 5% methanolic–KOH at 60  C. Ester  22  was treated with30% aqueous ammonia at 0  C to get corresponding diamide ( 26 ).On reduction with lithium borohydride in THF ester  22  yielded a OOOOOCH 3 OCH 3 H 3 COOH Podophyllotoxin ROROOCH 3 OCH 3 H 3 COOCH 3 H 3 COH 3 COOR 2 OHR 1 R 2 OHOR 1 Prototype-IPrototype-II OCH 3 H 3 COH 3 COOCH 3 OH Combretastatin A4 OH 3 COOCH 3 OCH 3 HNO Colchicine OCH 3 H 3 COH 3 CO 3,4,5-trimethoxyphenyl unit Figure 1.  Structures of antitubulins (podophyllotoxin, colchicine, CA4), prototypes I and II, and trimethoxyphenyl fragment. OHOOCH 3 H 3 COH 3 CO (A) OEtOOCH 3 H 3 COH 3 COOEtOOCH 3 HOH 3 CO i iiiii OEtOHOH 3 COH 3 COEtOOCOHOCH 3 OCH 3 +OEtOOCH 3 ORH 3 COOCH 3 ROH 3 COOOEt 5a : R=H; 5b : R=Ac iv 41 2 3 OHOHOROEtOHOR OEtOHOR 6:  R=H; 7:  R=OCH 3 iiii EtOOOH 8: R=H; 9: R=OCH 3  10: R=H; 11: R=OCH 3 R (B) (C)RH 3 COH 3 CO CHO+OEtOEtOOOHOOHOOCH 3 RH 3 CORH 3 COH 3 CO OEtOOEtOOCH 3 RH 3 CORH 3 COH 3 CO vvi 12:  R=H; 13:  R=OCH 3 15:  R=H; 16:  R=OCH 3 17:  R=H; 18:  R=OCH 3 14 Scheme 1.  Reagentsandconditions:(i)EtOH, SOCl 2 , RT, 2h, 89–94%;ORconcdH 2 SO 4 , EtOH, 85  C, 4h,84–91%; (ii)anhydrousAlCl 3 , DCM, 40  C,2h,84%; (iii)aqueous KOH,K 3 [Fe(CN) 6 ], benzene, RT, 30min, neolignan 21%, lignan 24–29%; (iv) dry pyridine, Ac 2 O, RT, overnight, 92%; (v) NaH, DMF, reflux, 4h, 43–49%; (vi) Et 2 SO 4 , K 2 CO 3 , acetone/DMSO=3:1, reflux, 2h, 82%. B. Sathish Kumar et al./Bioorg. Med. Chem. 22 (2014) 1342–1354  1343  Author's personal copy diol derivative  27 . Diol  27  was finally acetylated with pyridine–Ac 2 O to get diacetyl derivative  24 . All the compounds werecharacterized by spectroscopy. 2.2. Biological results All the lignans and neolignans were evaluated against MCF-7and MDAMB-231 human breast cancer cell lines (Table 1). Onlyeight of the derivatives showed some cytotoxicity against breastcancer cell lines. Rests of the derivatives were inactive at100 l M. The best compound of the series was  10  exhibiting IC 50 12 and 15 l Magainst MCF-7 and MDAMB-231, respectively. How-ever, IC 50  of   10  and  19  were much better when incubation timewas enhanced to 48 and 72h. 2.3. Effect of lead molecule on cell cycle phases of MCF-7 andMDA-MB-231 cells MCF-7 cells were treated with compound  10  for 24h and cellcycle phase distribution was recorded with PI-staining method(Fig. 2). Neolignan  10  showed significant increase in G2 phase atall concentrations evaluated suggesting G2/M arrest, along with adose dependent increase in sub-diploid population indicatingpossible apoptosis. There was also decrease in S-phase populationfor all the concentrations of compound  10  in comparison to un-treated control. However, G1 phase was not affected by any of the concentrations of compound  10 .In case of MDA-MB-231 cells, compound  10  did not affect celldivision cycle in treated cells, except dose-dependent increase of  OOEtOEtOHO OHRR 10:  R=H; 11:  R=OCH 3 OOEtOEtO AcO OHRR 19:  R=H; 20:  R=OCH 3 ONH 2 OH 2 NHO OHOCH 3 H 3 COOOEtOEtOH 3 CO OCH 3 RR 21:  R=H; 22:  R=OCH 3 ONH 2 OH 2 NH 3 CO OCH 3 OCH 3 H 3 COOOHOHOH 3 CO OCH 3 OCH 3 H 3 COOHHOH 3 CO OCH 3 OCH 3 H 3 CO 23262725 OAc AcOH 3 CO OCH 3 OCH 3 H 3 CO 24 viiviiiixxixxivii Scheme 2.  Reagents and conditions: (vii) pyridine, Ac 2 O, RT, overnight, 89–92%; (viii) Me 2 SO 4 , K 2 CO 3 , DMF, reflux, 80  C, 2h, 87–91%; (ix) 30% aqueous NH 3 , 0  C–rt, 2h,58–62%; (x) 5% methanolic–KOH, 60  C, 2h, 86%; (xi) LiBH 4 , THF, 0  C, 4h, 87%.  Table 1 In vitro cytotoxicity of lignans and neolignans by MTT assay Compound no. Mol wt. MCF-7 IC 50# ( l M) MDAMB-231 IC 50# ( l M) HEK-293 IC 50# ( l M) 4  502 74±0.012 92±0.026 Nd 5a  502 — * 82±0.037 Nd 5b  586 — — Nd 10  382 12±0.0052 (24h) 15±0.0085 (24h) 12 l M±0.129.29±0.03 (48h) 6.47±0.03 (48h)3.27±0.02 (72h) 0.45±0.02 (72h) 11  442 — — Nd 17  470 Nd Nd Nd 18  530 100±0.0013 — Nd 19  424 66±0.003 (24h) 16±0.0025 (24h) 20 l M±0.2339.65±0.02 (48h) 3.22±0.03 (48h)0.82±0.06 (72h) 0.21±0.05 (72h) 20  484 — — Nd 21  410 — — Nd 22  470 — — Nd 23  384 100±0.0019 80±0.099 Nd 24  470 — — Nd 25  414 Nd 85±0.022 Nd 26  412 100±0.018 32±0.0068 Nd 27  386 — — Nd 28  470 100±0.011 100±0.0247 NdPodophyllotoxin 414 64.99±4.38 35.7±11.80 50 l M±0.224Tamoxifen 371 9±0.003 10±0.010 26 l M±0.22 * Means not active, IC 50  >100 l M, Nd=not done. # Incubation time=24h.1344  B. Sathish Kumar et al./Bioorg. Med. Chem. 22 (2014) 1342–1354  Author's personal copy sub-diploid cells at higher concentrations (15 and 30 l M).Compound  10  might have only caused apoptosis in MDA-MB-231without causing cell cycle arrest at any phase. This also suggestedthat compound  10  affects cell cycle phases of MDA-MB-231 differ-ently than MCF-7 cells (Fig. 3). 2.4. Biochemical measure of tubulin polymerization activity of lead molecules Neolignans  10  and  19  were further evaluated for their effect ontubulin polymerisation (Fig. 4). For this experiment, we incorpo-rated tubulin destabilizing agent PDT and stabilizing agent taxolas controls to improve reliability of our assay. As can be seen inFigure 4, both the neolignans showed stabilization of tubulinassembly similar to taxol at various concentrations (10, 20 and40 l M) whereas, the standard tubulin destabilizing agent PDT,effectively inhibited tubulin polymerization in comparison to con-trolgroups. Compounds 10  showedbetterstabilizationofmicrotu-bule polymerization in comparison to compound  19 . 2.5. Effect of lead molecule on actin–tubulin cytoskeletonstructure with confocal microscopy  In order to observe the phenotypic effect of compound  10  (asshowed better activityin tubulin polymerization assay) on cellularcytoskeletal networkof actinandtubulin, MCF-7cellswereimmu-nostained and analyzed under confocal microscope. As illustratedin Figure 5, substantial stabilization of microtubules in the formof bundle like appearance was observed in paclitaxel-treated cellsthat were used as positive control in this assay. However, incompound  10  treated cells, stabilization of tubulin network wasnot apparent up to the level in comparison to positive controlgroups. No changes in actin network were evident in control aswell as in treatment groups. 2.6. Molecular docking for binding studies The modulation of anticancer activity of   10  and  19  was eluci-datedthroughinteractionwithtubulinpolymeraseandidentifyingbinding site pocket. The molecular docking results also confirmedthat both  10  and  19  stabilize the polymerisation of tubulin. Theorientations and binding affinities (in terms of total score) of   10 and  19  were established towards tubulin (PDB ID: 1TUB). Thedocking reliability was validated by using the known X-ray crystalstructure of tubulin complexed with taxol and docked conforma-tionwiththehighesttotalscoreof6.3796wasselectedasthemostprobable binding conformation. The low root mean-square devia-tion (RMSD) of 0.6014Å between the docked and the crystal con-formations indicates the high reliability of Surflex-dock softwarein reproducing the experimentally observed binding mode fortaxol. As shown in Figure 6A, redocked molecules were almost inthesamepositionwithco-crystallizedattheactivesite.Crystallog-raphydatashowedthat the aminoacidThreonine-276is the‘gate-keeper’ residue, animportant determinant of stabilizingspecificityin the tubulin binding pocket.The docking results of   19  against target protein showed highbinding affinity docking score indicated by total score of 8.6059and formation of two hydrogen bonds of length 2.0 and 1.8Åthrough hydrophobic residues THR-276 and HIS-229. In dockingpose, theconservedbindingsitepocketaminoacidresidueswithina selection radius of 4Å from bound ligand were hydrophobic res-idue Val-23 (Valine) PHE-272; nucleophilic (polar, hydrophobic),for example, THR-276 (Threonine), SER-232, SER-236, SER-277(Serine);basicLYS-218(Lysine),ARG-320(Arginine)CYS-213(Cys-teine), GLN-281(Glutamine), HIS-299 (Histidine); acidic (polar,negative charged), for example, ASP-26 (Aspartic acid), Hydropho-bic, for example, ALA-233, ARG-278 (Alanine), LEU-217, LEU-219,LEU-230, LEU-275, LEU-371 (Leucine), and imino acids PRO-274,PRO-360 (Proline), as a result, the bound compound  19  showedstronghydrophobicinteractionswithtubulin,thusleadingtomorestability and activity in this compound (Fig. 6B).The binding affinity obtained in the docking study allowed acomparisonbetweentheactivitiesofthe 10  tobecomparedtothatof the standard anticancer drug taxol. Compound  10  showed ahigher binding affinity against tubulin, the target protein. Duringthe comparison of the nature of interaction between the bindingpocket amino acid residues of target protein and compound  10 , itwas found that the compound  10  showed molecular interactionswith conserved hydrophobic amino acid residues, thus leading tomore stability and potency (Table 2). The docking results for  10 showed that the compound docked on tubulin with a high bindingaffinitydockingscoreindicatedbyitstotalscoreof6.3796andalsoshowed the formation of a two H-bond of length 1.9 and 1.8Å tothe acidic residues, Glu-22 and basic (hydrophobic) residues, Lys-19. The  10  tubulin-docked complex also showed a similar type of binding site residues within a radius of 4Å of bound ligand suchas nucleophilic (polar, hydrophobic), for example, SER-232, SER-236 (Serine); basic LYS-19 (Lysine), ARG-320, HIS-299 (Histidine);acidic (polar, negative charged), for example, ASP-26 (Asparticacid); Hydrophobic, for example, ALA-233 (Alanine), ARG-278,ARG-369 (Arginine) LEU-219, LEU-230, LEU-275, LEU-371(Leucine), Val-23 (Valine), PHE-272 (Phenylalanine); imino acidsPRO-274, PRO-360 (Proline); and acidic (polar, negative charged)residues, for example, GLU-22 (Glutamic acid), therefore, thedocked molecule also showed a strong hydrophobic interactionwith tubulin, thus leading to more stability (Fig. 6C).The docking results for the negative control compoundpodophyllotoxin (tubulin inhibitor) with tubulin showed a low Figure 2.  Effect of neolignan  10  in cell cycle phases of MCF-7 cells. Figure 3.  Effect of compound  10  in cell cycle phases of MDA-MB-231 cells. B. Sathish Kumar et al./Bioorg. Med. Chem. 22 (2014) 1342–1354  1345
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