Bivalent Organophosphorus Compounds—Synthesis and Acetylcholinesterase Inhibitory Activity

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  • This article was downloaded by: [University of Auckland Library]On: 09 December 2014, At: 19:16Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

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    Bivalent OrganophosphorusCompoundsSynthesis andAcetylcholinesterase Inhibitory ActivityRuliang Xie a , Ting Zhang b , Qianfei Zhao a , Tao Zhang a ,Xiangdong Mei a , Huizhu Yuan a & Jun Ning aa Key Laboratory of Pesticide Chemistry and Application, Ministryof Agriculture, Institute of Plant Protection, Chinese Academy ofAgricultural Sciences , Beijing , 100193 , Chinab State Key Laboratory of Chemical Resource Engineering , BeijingUniversity of Chemical Technology , Beijing , 100029 , ChinaAccepted author version posted online: 19 Oct 2012.Publishedonline: 22 Jul 2013.

    To cite this article: Ruliang Xie , Ting Zhang , Qianfei Zhao , Tao Zhang , Xiangdong Mei , HuizhuYuan & Jun Ning (2013) Bivalent Organophosphorus CompoundsSynthesis and AcetylcholinesteraseInhibitory Activity, Phosphorus, Sulfur, and Silicon and the Related Elements, 188:8, 1095-1103, DOI:10.1080/10426507.2012.736097

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    http://www.tandfonline.com/page/terms-and-conditionshttp://www.tandfonline.com/page/terms-and-conditions

  • Phosphorus, Sulfur, and Silicon, 188:10951103, 2013Copyright C Taylor & Francis Group, LLCISSN: 1042-6507 print / 1563-5325 onlineDOI: 10.1080/10426507.2012.736097

    BIVALENT ORGANOPHOSPHORUSCOMPOUNDSSYNTHESIS ANDACETYLCHOLINESTERASE INHIBITORY ACTIVITY

    Ruliang Xie,1 Ting Zhang,2 Qianfei Zhao,1 Tao Zhang,1

    Xiangdong Mei,1 Huizhu Yuan,1 and Jun Ning11Key Laboratory of Pesticide Chemistry and Application, Ministry of Agriculture,Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing100193, China2State Key Laboratory of Chemical Resource Engineering, Beijing University ofChemical Technology, Beijing 100029, China

    GRAPHICAL ABSTRACT

    Abstract A series of novel symmetric S,S-2,2-(ethane-1,2-diylbis(azanediyl)) bis(2-oxoethane-2,1-diyl) O,O,O,O-tetraethyl diphosphorodithioate derivatives (12) was designedand synthesized based on the cluster effect and the multiple binding sites of acetylcholinesterase(AChE). The structures of all the newly synthesized title compounds were characterized by1H and 13C NMR as well as elemental analyses. Their inhibitory activities against AChEwere tested, and compound 12b exhibited the best activity (6.60-fold higher than ethion). Theresults suggested that the compound would bind to the catalytic center and the narrow gorgeof the AChE simultaneously.

    Supplementary materials are available for this article. Go to the publishers online edition ofPhosphorus, Sulfer, and Silicon and the Related Elements for the following free supplementalfiles: Additional table.

    Keywords Cluster effect; bivalent organophosphorus compound; acetylcholinesterase in-hibitory activity; dual binding

    INTRODUCTION

    Multi-valent ligand-receptor interaction, also called cluster effect, is defined as spe-cific simultaneous associations of multiple ligands presented on a molecular construct withreceptors presented on a biological entity.14 The cluster effect has been widely applied in

    Received 4 June 2012; accepted 23 September 2012.This work was supported by the National Basic Research Program of China (2012BC11410X) and the Major

    State Basic Research Development Program of China (No.2010CB126106).Ruliang Xie and Ting Zhang contributed equally to this work and should be considered as co-first authors.Address correspondence to Jun Ning, Key Laboratory of Pesticide Chemistry and Application, Ministry

    of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.E-mail: jning@ippcaas.cn

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  • 1096 R. XIE ET AL.

    drug design because of its high affinity59 and selectivity.1014 Thus, it could also be appliedin the design of agrochemicals that target proteins that have two or more active sites.

    AChE plays a vital role in the regulation of the neurotransmitter acetylcholine inmammals and insects, and it has been employed as a target for pesticides for a long time.Research results indicated that this enzyme has multiple binding sites, including a catalyticcenter that contains catalytic triad (Ser200, His440, and Glu327), a peripheral anionicsite (PAS) as well as a deep and narrow gorge which contains 14 aromatic amino acidresidues.15,16 Based on the potent binding characteristics of these active sites for AChE in-hibitors, a large number of compounds were designed, synthesized, and evaluated with theirinhibitory activities on AChE. Most of them exhibited excellent inhibitory activities andtheir IC50 values ranged from subnanomolar to picomolar.17 Generally, the primary struc-ture of the compounds with high inhibitory activities contains an aromatic pharmacophorethat is capable of binding to PAS by means of interactions and another substruc-ture that can bind to the catalytic triad, and two moieties are connected with a linker in afitted length. Therefore, the cluster effect can significantly improve the binding activity ofthe ligands to receptors, which provides a novel insight for the design of agrochemicals.

    One type of the classical AChE inhibitors is organophosphorus compounds (OPs).18

    OPs inhibit AChE by phosphylation (denotes phosphorylation and phosphonylation) at theactive site Serine, resulting in a deactivation of AChE and a gradual accumulation of theneurotransmitter acetylcholine, which led to an overstimulation of cholinergic receptorsand finally the paralysis of neuromuscular functions.1921 Based on the unique mechanismof organophosphorus compounds and the multibinding sites of AChE, a large numberof dual-binding compounds, including organophosphorus pharmacophore, were designed,synthesized, and tested in vitro with AChE by our group.22,23 Comparing to monomericones, many dual-binding compounds showed better inhibitory activity against AChE. TheOP pharmacophore and the length of the linker to the dual-binding compounds were thecrucial factors for their high activity. In addition to the active sites CAS and PAS, the deepand narrow gorge suggested another affinity site. As a continuous work of our previousresearch, herein we report the synthesis of the compounds 12 and the preliminary results onAChE inhibition. In the structure of the inhibitor, two ethion moieties were linked togetherwith linear amides chain in different lengths. The AChE inhibitory activities of all the newcompounds and their analogs that were reported by Yang22 were tested using the AChEisolated from heads of housefly, and their SAR rationalization was also carried out.

    RESULTS AND DISCUSION

    Synthesis

    The compounds 12ah were prepared according to a general synthetic procedureshown in Scheme 4. Using liner diamines as the chain linker, it was easy to obtain theintermediates 11ah under vigorous stir for 6 h at the temperature below 10 C. Products11 were filtered and washed with cold methanol for avoiding significant loss in the solvent.More importantly, good yield was achieved with proper adding sequence in which thediamine was slowly added to the solution of methyl chloroformate in cold methanol. It wasalso found that an optimum ratio of methyl chloroformate to diamine in 2.4 equivalentsafforded the desired product in good yield. In a word, the optimum reaction conditions forhigh yields were established as the substrates reacted in reasonable ratio and right addingsequence at suitable temperature for proper reaction time, under which up to 90% yields

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  • SYNTHESIS OF ACETYLCHOLINESTERASE INHIBITORS 1097

    Scheme 1

    were achieved and the isolated products were used directly in the next step without furtherpurification.

    The title compounds 12ah were obtained in 5470% yields after purification bysilica gel chromatography from the intermediates 11ah and the OP pharmacophore O,O-diethyl S-hydrogen phosphorodithioate that reacted in methanol for 2448 h under refluxingconditions. The overall yields of the desired products from these two steps ranged from49% to 63%. The structures of all the target compounds were well characterized by 1H and13C NMR as well as elemental analyses (Table 1 and 2). 3ah, 6ac, and 9ai, as shown inSchemes 13, were known compounds.22

    Biological Activity

    The inhibitory activity against AChE of bivalent organophosphorus compounds wassummarized in Table S1 (Supplemental materials), expressed as IC50 values with ethion

    Scheme 2

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  • 1098 R. XIE ET AL.

    Scheme 3

    as the reference standard. As shown in Table S1, most compounds demonstrated lowerinhibitory activities against AChE than ethion. Comparing with the ethylene in ethion,linkers in these compounds affected the binding activity to AChE, except 2,2-(propane-1,3-diylbis (azanediyl))bis(2-oxoethane-2,1-diyl) group in 12b and heptyl group in 9f.Ethion belongs to traditional organophosphorus pesticide which binds to the esteratic sitesand the anionic sites of AChE.24 Some of the target compounds were able to simultaneouslyinteract with aromatic amino acid residues lining the gorge and the catalytic sites of AChE.We noticed that the inhibitory activity significantly increased when the number of methyleneunits in the linker was changed from two or four to three in compounds 12ah and fromsix or eight to seven in compounds 9bj, which indicated that some specific favorableinteraction strongly affected the binding affinity of 9f and 12b to the active-gorge of AChE.

    The most potent AChE inhibitory activity to isolated enzyme was observed fromcompound 12b with the IC50 value of 6.96 mol/L (6.60-fold higher than ethion, and >400-fold higher than compound 9i in which the linker had equal unit number to that of compound9c). The result indicated that the acetamide groups of the linker assisted the molecule tobind to the gorge of AChE. These amide groups would force the tether to reorient along

    Table 1 Yields and elemental analyses of compounds

    Elemental analyses (Found)

    Compd Physical properties Yield (%) C H N P S

    12a white solid (mp:77.278.5 C)

    65 32.80 (32.9) 5.90 (5.92) 5.46 (5.31) 12.08 (12.0) 27.25 (27.1)

    12b white solid (mp:41.242 C)

    72 34.21 (33.9) 6.12 (6.22) 5.32 (5.44) 11.76 (11.9) 24.35 (24.2)

    12c white solid (mp:81.883.4 C)

    70 35.54 (35.7) 6.34 (6.26) 5.18 (5.08) 11.46 (11.3) 23.72 (23.9)

    12d white solid (mp:61.062.2 C)

    62 38.01 (38.3) 6.73 (6.62) 4.93 (5.01) 10.89 (10.7) 22.55 (22.7)

    12e colorless liquid 64 39.16 (39.0) 6.92 (6.83) 4.81 (4.86) 10.63 (10.8) 22.01 (22.1)12f white solid (mp:

    55.656.5 C)60 40.25 (40.3) 7.09 (7.00) 4.69 (4.63) 10.38 (10.2) 21.49 (21.6)

    12g pale yellow liquid 54 41.29 (41.4) 7.26 (7.37) 4.59 (4.50) 10.14 (10.1) 21.00 (20.9)12h colorless liquid 56 42.29 (42.2) 7.42 (7.53) 4.48 (4.40) 9.91 (10.02) 20.53 (20.4)

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  • SYNTHESIS OF ACETYLCHOLINESTERASE INHIBITORS 1099

    Table 2 Spectral data of compounds

    Compound 1H NMR (CDCl3/TMS) () (ppm)

    12a 1.321.39 (m, 12H, CH3), 3.42 (t, 4H, J = 5.5 Hz, NCH2), 3.56 (d, 4H, J = 19.8 Hz, SCH2),4.124.27 (m, 8H, OCH2), 6.91 (s, 2H, NH); 15.77 (CH3), 35.7 (SCH2), 38.8(NCH2),63.8(OCH2), 166.9(CONH).

    12b 1.341.39 (m, 12H, CH3), 1.70 (t, 2H, J = 12.4 Hz, NCH2CH2), 3.293.36 (m, 4H, NCH2),3.57 (d, 4H, J = 19.5 Hz, SCH2) 4.114.27 (m, 8H, OCH2), 7.00 (s, 2H, NH); 15.7 (CH3),35.6 (SCH2), 38.9(NCH2), 63.82(OCH2), 167.1 (CONH).

    12c 1.341.39 (m, 12H, CH3), 1.571.60 (m, 4H, NCH2CH2), 3.29 (t, 4H, J = 5.9 Hz, NCH2),3.55 (d, 4H, J = 20.3 Hz, SCH2) 4.114.27 (m, 8H, OCH2), 7.00 (s, 2H, NH); 15.7 (CH3),29.4 (NCH2CH2), 35.6 (SCH2), 38.9(NCH2), 63.9(OCH2), 167.1 (CONH).

    12d 1.341.39 (m, 16H, CH3,, NCH2CH2CH2), 1.53 (t, 4H, J = 12.8 Hz, NCH2CH2CH2),3.223.29 (m, 4H, NCH2), 3.55 (d, 4H, J = 20.0Hz, SCH2), 4.104.26 (m, 8H, OCH2),6.65 (s, 2H, NH); 15.7 (CH3), 26.1 (NCH2CH2CH2), 29.1 (NCH2CH2), 36.1 (SCH2), 39.6(NCH2), 64.5 (OCH2), 167.3 (CONH).

    12e 1.341.39 (m, 18H, CH3,, NCH2CH2CH2CH2), 1.52 (t, 4H, J = 13.0 Hz,NCH2CH2CH2CH2), 3.223.28 (m, 4H, NCH2), 3.55 (d, 4H, J = 20.1 Hz, SCH2),4.104.26 (m, 8H, OCH2), 6.62 (s, 2H, NH); 15.6 (CH3), 26.4 v(NCH2CH2CH2CH2),28.9 (NCH2CH2CH2), 29.1 (NCH2CH2) 36.10 SCH2), 41.1 (NCH2), 64.4 (OCH2), 167.4(CONH).

    12f 1.311.39 (m, 20H, CH3,, NCH2CH2CH2CH2), 1.52 (t, 4H, J = 13.4 Hz,NCH2CH2CH2CH2), 3.223.28 (m, 4H NCH2), 3.54 (d, 4H, J = 19.0 Hz, SCH2),4.084.26 (m, 8H, OCH2), 6.52 (s, 2H, NH); 15.7 (CH3), 26.6 (NCH2CH2CH2CH2), 28.9(NCH2CH2CH2), 29.2 (NCH2CH2) 36.1 (SCH2), 39.9 (NCH2), 64.5 (OCH2), 167.2(CONH).

    12g 1.301.39 (m, 22H, CH3,, NCH2CH2CH2CH2CH2), 1.491.54 (m, 4H,NCH2CH2CH2CH2CH2), 3.223.29 (m, 4H, NCH2), 3.54 (d, 4H, J = 20.4 Hz, SCH2),4.104.26 (m, 8H, OCH2), 6.51 (s, 2H, NH); 15.7 (CH3), 26.1 (NCH2CH2CH2CH2CH2),27.6 (NCH2CH2CH2CH2), 28.6 (NCH2CH2CH2), 29.5 (NCH2CH2) 35.7 (SCH2), 39.3(NCH2), 63.7 (OCH2), 166.8 (CONH).

    12h 1.261.39 (m, 24H, CH3, NCH2CH2CH2CH2CH2), 1.51 (t, 4H, J = 13.4 Hz,NCH2CH2CH2CH2CH2), 3.223.29 (m, 4H, N...

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