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Bivalent OrganophosphorusCompounds—Synthesis 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 Compounds—Synthesis 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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Phosphorus, Sulfur, and Silicon, 188:1095–1103, 2013Copyright C© Taylor & Francis Group, LLCISSN: 1042-6507 print / 1563-5325 onlineDOI: 10.1080/10426507.2012.736097

BIVALENT ORGANOPHOSPHORUSCOMPOUNDS—SYNTHESIS ANDACETYLCHOLINESTERASE INHIBITORY ACTIVITY

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

Xiangdong Mei,1 Huizhu Yuan,1 and Jun Ning1

1Key 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 publisher’s 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.1–4 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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drug design because of its high affinity5–9 and selectivity.10–14 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 π–π 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.19–21 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 12a–h were prepared according to a general synthetic procedureshown in Scheme 4. Using liner diamines as the chain linker, it was easy to obtain theintermediates 11a–h 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 12a–h were obtained in 54–70% yields after purification bysilica gel chromatography from the intermediates 11a–h and the OP pharmacophore O,O-diethyl S-hydrogen phosphorodithioate that reacted in methanol for 24–48 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). 3a–h, 6a–c, and 9a–i, as shown inSchemes 1–3, 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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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 12a–h and fromsix or eight to seven in compounds 9b–j, 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 μmol/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:77.2–78.5 ◦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:41.2—42 ◦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:81.8–83.4 ◦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:61.0–62.2 ◦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:

55.6–56.5 ◦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) (δ) (ppm)

12a 1.32–1.39 (m, 12H, CH3), 3.42 (t, 4H, J = 5.5 Hz, NCH2), 3.56 (d, 4H, J = 19.8 Hz, SCH2),4.12–4.27 (m, 8H, OCH2), 6.91 (s, 2H, NH); 15.77 (CH3), 35.7 (SCH2), 38.8(NCH2),63.8(OCH2), 166.9(CONH).

12b 1.34–1.39 (m, 12H, CH3), 1.70 (t, 2H, J = 12.4 Hz, NCH2CH2), 3.29–3.36 (m, 4H, NCH2),3.57 (d, 4H, J = 19.5 Hz, SCH2) 4.11–4.27 (m, 8H, OCH2), 7.00 (s, 2H, NH); 15.7 (CH3),35.6 (SCH2), 38.9(NCH2), 63.82(OCH2), 167.1 (CONH).

12c 1.34–1.39 (m, 12H, CH3), 1.57–1.60 (m, 4H, NCH2CH2), 3.29 (t, 4H, J = 5.9 Hz, NCH2),3.55 (d, 4H, J = 20.3 Hz, SCH2) 4.11–4.27 (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 1.34–1.39 (m, 16H, CH3,, NCH2CH2CH2), 1.53 (t, 4H, J = 12.8 Hz, NCH2CH2CH2),3.22–3.29 (m, 4H, NCH2), 3.55 (d, 4H, J = 20.0Hz, SCH2), 4.10–4.26 (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 1.34–1.39 (m, 18H, CH3,, NCH2CH2CH2CH2), 1.52 (t, 4H, J = 13.0 Hz,NCH2CH2CH2CH2), 3.22–3.28 (m, 4H, NCH2), 3.55 (d, 4H, J = 20.1 Hz, SCH2),4.10–4.26 (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 1.31–1.39 (m, 20H, CH3,, NCH2CH2CH2CH2), 1.52 (t, 4H, J = 13.4 Hz,NCH2CH2CH2CH2), 3.22–3.28 (m, 4H NCH2), 3.54 (d, 4H, J = 19.0 Hz, SCH2),4.08–4.26 (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 1.30–1.39 (m, 22H, CH3,, NCH2CH2CH2CH2CH2), 1.49–1.54 (m, 4H,NCH2CH2CH2CH2CH2), 3.22–3.29 (m, 4H, NCH2), 3.54 (d, 4H, J = 20.4 Hz, SCH2),4.10–4.26 (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 1.26–1.39 (m, 24H, CH3, NCH2CH2CH2CH2CH2), 1.51 (t, 4H, J = 13.4 Hz,NCH2CH2CH2CH2CH2), 3.22–3.29 (m, 4H, NCH2), 3.54 (d, 4H, J = 20.4 Hz, SCH2),4.10–4.26 (m, 8H, OCH2), 6.49 (s, 2H, NH); 15.7 (CH3), 26.7 (NCH2CH2CH2CH2CH2),27.9 (NCH2CH2CH2CH2), 28.6 (NCH2CH2CH2), 29.2 (NCH2CH2) 36.2 (SCH2), 39.9(NCH2), 64.5 (OCH2), 167.2 (CONH).

12h 15.68 (CH3), 26.09(NCH2CH2CH2CH2CH2), 27.62(NCH2CH2CH2CH2),28.56(NCH2CH2CH2), 29.48(NCH2CH2) 35.65(SCH2), 39.33(NCH2), 63.65(OCH2),166.79(CONH). S,S′-2,2′-(decane-1,10-diylbis(azanediyl))bis(2-oxoethane-2,1-diyl)O,O,O′,O′-tetraethyl diphosphorodithioate15.73 (CH3), 26.70(NCH2CH2CH2CH2CH2), 27.93(NCH2CH2CH2CH2),

28.62(NCH2CH2CH2), 29.22(NCH2CH2) 36.16(SCH2), 39.92(NCH2), 64.52(OCH2),167.24(CONH).

the gorge by forming hydrogen bonds through which the amide groups interacted with theresidues in AChE along the active gorge. Some important organophosphorus insecticidessuch as prothoate, menazon, morphothion, and dimethoate belong to O,O-dialkyl-S-(N-methylcarbnoyl alkyl) phosphorodithioate derivatives, and their inhibitory activities comefrom the binding of the acetamide groups to the anionic sites of CAS. Because two moietieswere connected with a suitable linker, the bivalent compounds, like compound 12b, had astronger binding interaction with AChE and thus exhibited higher inhibitory activity. TheAChE-SERT dual inhibitors,25–28 which did not extend to the peripheral site, bound to theresidues halfway of the gorge and still exhibited potent AChE inhibitory activity. Likewise,

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the two active molecules (12b and 9f) would bind to the catalytic site and the residuesnear the catalytic site at the same time. Moreover, the specific spatial configurations of12b and 9f were also a key factor to the binding affinity to AChE. In particular, compound9b with optical configuration bound to CAS and the residues along the active gorge byhydrogen bonds. Organophosphorus insecticides with P=S structure showed lower in vitroinhibitory activity than those with P=O structure. For example, the AChE inhibitory activityof dimethoate was about 1/200-fold and 1/4-fold of that of omethoate in different testingmethods.28 A series of compounds with P=O structure were synthesized and their bioassaywere studied in our group,23 and the results showed that the activation of ethion and thecompounds synthesized in this study through oxidation was less possible, whereas theinteraction between the phosphorodithioate acetamide groups or phthalimide groups andsome specific residues in AChE may be responsible for the high inhibitory activity.

Compound 12e had the IC50 values of 121 μmol/L (0.38-fold of the AChE inhibitoryactivity of ethion), which was more active than 12d and 12f. Like bis(7)-tacrine and bis(7)-(6-chloro)tacrine,29 one organophosphorus group might extend and bind to the peripheralsite, but with low affinity. These data suggested that the peripheral site of AChE could nottolerate the simultaneous substitution at positions 6 and 8 of the bivalent organophosphorusskeleton. From the activities of 12b and 12e, it seemed that the residues near the catalyticsite instead of the peripheral site might be more interesting for the design of new bivalentinhibitors.

Compound 3j had better AChE inhibitory acitivity than compound 3c, which sug-gested 1,4-buty-2-ene substructure strengthened the interaction with AChE more than1,4-butane, and C = C bond may have π–π interaction with some aromatic residues. Withone or two oxygen atoms in the linker, the physicochemical properties of the compoundssuch as logP and pKa would change. However, compounds 6a, 6b, and 6c did not showhigher AChE inhibitory ability than other compounds, which suggested the change of logPand pKa would not increase the in vitro activity.

MATERIALS AND METHODS

Preparation of Compounds

Reagents and solvents were purchased from Beijing Chemical Reagents Company andwere used without further purification. Column chromatography was performed on silica gel200–300 mesh obtained from Qingdao Haiyang Chemical Co., Ltd. Analytical thin-layerchromatography was performed with silica gel plates, and the plots were visualized underUV light at 254 nm or iodine vapor. Elemental analyses (C, H, N, P, S) were performed atInstitute of Chemistry, Chinese Academy of Sciences. Nuclear magnetic resonance spectrawere recorded in CDCl3 solution, using Brucker DPX 300MHz spectrometer (performedby China Agricultural University).

Preparation of Products 12a–h. A series of compounds containing acetyl N-alkylamine linkers were synthesized, as shown in Scheme 4. Acetyl N-alkylamine group wasemployed because it would enhance the killing effect of O,O-dialkyl-S-(N-methylcarbnoylalkyl) phosphorodithioates which were important organophosphorus insecticides. The syn-thetic procedure for N,N′-(alkane-α,ω-diyl) bis(2-chloroacetamide) (11a–h) was describedas follow: To a solution of methyl 2-chloroacetate (13.0 g, 120.0 mmol) in methanol(20.0 mL), ethane-1,2-diamine (3.0g, 50.0 mmol) of methanol was added dropwise at

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SYNTHESIS OF ACETYLCHOLINESTERASE INHIBITORS 1101

Scheme 4

below 10 ◦C, and the mixture was stirred for 10 h. Afterwards, the product N,N′-(ethane-1,2-diyl) bis(2-chloroacetamide) (11a) was filtered, washed, dried, and collected as a whitesolid. The filtrate was condensed and subjected to recrystallization to afford additionalproduct. The same procedure was also employed in the preparation of products 11b–h byusing the corresponding alkanyl diamines.

The synthetic procedure for title products 12a–h was described as follow: To a solu-tion of O,O-diethyl hydrogen phosphorodithioate (2.1 g, 11.0 mmol) in methanol (10.0 mL)was added 20% methanolic potassium hydroxide slowly until the pH value of the solutionwas approximately 7.0. Then, N,N′-(ethane-1,2-diyl) bis(2-chloroacetamide) (11a) (1.1 g,5.5 mmol) was added, and the mixture was refluxed for 4 h. The reaction mixture wasfiltered and the filtrate was concentrated under vacuum. The viscous residue was dissolvedin ethyl acetate (20 mL), then washed with water and the ethyl acetate layer was driedover anhydrous sodium sulfate. The dried solution was concentrated under reduced pres-sure and the crude residue was purified on silica gel chromatography to furnish S,S′-2,2′-(ethane-1,2-diylbis(azanediyl)) bis(2-oxoethane-2,1-diyl) O,O,O′,O′-tetraethyl diphospho-rodithioate (12a) as the product. The same procedure was followed to afford the products(12b–h) by using the corresponding N,N′-(alkane-α,ω-diyl)bis(2-chloroacetamide).

Characterization data for S,S′-2,2′-(ethane-1,2- diylbis(azanediyl)) bis(2-oxoethane-2,1-diyl) O,O,O′,O′-tetraethyl diphosphorodithioate (12a): white solid; yield 65.3%; mp77.2–78.5 ◦C; 1H NMR (300 MHz, DMSO-d6) (δ) (ppm): δ 6.91 (s, 2H, CONH), 4.12–4.27(m, 8H, OCH2), 3.56 (d, 4H, JHH = 19.8 Hz, SCH2CO), 3.42 (t, 4H, JHH = 5.5Hz, CH2CH2),1.32–1.39 (m, 12H, CH3); 13C NMR (DMSO-d6) (δ) (ppm) 15.8 (CH3), 35.6 (SCH2), 38.8(NCH2), 63.8 (OCH2), 166.9 (CONH). Anal. calc. for C14H30O6P2 S4 : C 32.80%, H5.90%, N 5.46%, P 12.08%, S 25.02%; found: C 32.95%, H 5.92%, N 5.31%, P 12.01%,S 24.86%.

Compounds 12b–h were characterized on the basis of elemental analyses and spectraldata (1H and 13C NMR, Table 1 and 2)

O,O,O′,O′-tetraethyl S,S′-2,2′-(propane-1,3-diyl))bis(azanediyl)bis(2-oxoethane-2,1-diyl) diphosphorodithioate (12b).

S,S′-2,2′-(butane-1,4-diylbis(azanediyl))bis(2-oxoethane-2,1-diyl) O,O,O′,O′-tetr-aethyl diphosphorodithioate (12c).

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1102 R. XIE ET AL.

O,O,O′O′-tetraethyl S,S′-2,2′-(hexane-1,6-diylbis(azanediyl))bis(2-oxoethane-2,1-diyl) diphosphorodithioate (12d).

O,O,O′,O′-tetraethyl S,S′-2,2′-(heptane-1,7-diylbis(azanediyl))bis(2-oxoethane-2,1-diyl) diphosphorodithioate (12e).

O,O,O′,O′-tetraethyl S,S′-2,2′-(octane-1,8-diylbis(azanediyl))bis(2-oxoethane-2,1-diyl) diphosphorodithioate (12f).

O,O,O′,O′-tetraethyl S,S′-2,2′-(nonane-1,9-diylbis(azanediyl))bis(2-oxoethane-2,1-diyl) diphosphorodithioate (12g).

S,S′-2,2′-(decane-1,10-diylbis(azanediyl))bis(2-oxoethane-2,1-diyl) O,O,O′,O′-tetr-aethyl diphosphorodithioate (12h).

AChE Inhibitory Bioassay

Inhibition of Isolated AChE. AChE activity was determined according to previ-ously reported method31,32 and presented in Table S1 (Supplemental materials).

CONCLUSION

In conclusion, a suitable substituent of organophosphorus compound that simulta-neously bound to the catalytic sites and the other residues lining the gorge significantlyincreased the AChE inhibitory activity. The two potent AChE inhibitors, 12b (an interestinghomovalent compound) and 9f (a heterovalent compound), belong to two different typesof dual-site inhibitor of AChE. Further evaluation of 12b and 9f is underway.

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