Bivalent organophosphorus compounds - Synthesis and acetylcholinesterase inhibitory activity

Article abstract of DOI:10.1080/10426507.2012.736097

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 designed and 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 by ¹ H and ¹³ C NMR as well as elemental analyses. Their inhibitory activities against AChE were tested, and compound 12b exhibited the best activity (6.60-fold higher than ethion). The results suggested that the compound would bind to the catalytic center and the narrow gorge of the AChE simultaneously. Supplementary materials are available for this article. Go to the publisher's online edition of Phosphorus, Sulfer, and Silicon and the Related Elements for the following free supplemental files: Additional table.

Full text of DOI:10.1080/10426507.2012.736097

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Bivalent Organophosphorus
Compounds—Synthesis and
Acetylcholinesterase Inhibitory Activity
Ruliang Xie a , Ting Zhang b , Qianfei Zhao a , Tao Zhang a
Xiangdong Mei a , Huizhu Yuan a & Jun Ning a
,
a Key Laboratory of Pesticide Chemistry and Application, Ministry
of Agriculture, Institute of Plant Protection, Chinese Academy of
Agricultural Sciences , Beijing , 100193 , China
b State Key Laboratory of Chemical Resource Engineering , Beijing
University of Chemical Technology , Beijing , 100029 , China
Accepted author version posted online: 19 Oct 2012.Published
online: 22 Jul 2013.
To cite this article: Ruliang Xie , Ting Zhang , Qianfei Zhao , Tao Zhang , Xiangdong Mei , Huizhu
Yuan & Jun Ning (2013) Bivalent Organophosphorus Compounds—Synthesis and Acetylcholinesterase
Inhibitory 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, 2013
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2012.736097
BIVALENT ORGANOPHOSPHORUS
COMPOUNDS—SYNTHESIS AND
ACETYLCHOLINESTERASE 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, Beijing
100193, China
2State Key Laboratory of Chemical Resource Engineering, Beijing University of
Chemical Technology, Beijing 100029, China
GRAPHICAL ABSTRACT
Abstract A series of novel symmetric S,Sꢁ-2,2ꢁ-(ethane-1,2-diylbis(azanediyl)) bis(2-
oxoethane-2,1-diyl) O,O,Oꢁ,Oꢁ-tetraethyl diphosphorodithioate derivatives (12) was designed
and 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 by
1H and 13C NMR as well as elemental analyses. Their inhibitory activities against AChE
were tested, and compound 12b exhibited the best activity (6.60-fold higher than ethion). The
results suggested that the compound would bind to the catalytic center and the narrow gorge
of the AChE simultaneously.
Supplementary materials are available for this article. Go to the publisher’s online edition of
Phosphorus, Sulfer, and Silicon and the Related Elements for the following free supplemental
files: 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 with
receptors 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
1095

1096
R. XIE ET AL.
drug design because of its high affinity5–9 and selectivity.10–14 Thus, it could also be applied
in 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 in
mammals 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 catalytic
center that contains catalytic triad (Ser200, His440, and Glu327), a peripheral anionic
site (PAS) as well as a deep and narrow gorge which contains 14 aromatic amino acid
residues.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 their
inhibitory activities on AChE. Most of them exhibited excellent inhibitory activities and
their IC50 values ranged from subnanomolar to picomolar.17 Generally, the primary struc-
ture of the compounds with high inhibitory activities contains an aromatic pharmacophore
that 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 a
fitted length. Therefore, the cluster effect can significantly improve the binding activity of
the 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 the
active site Serine, resulting in a deactivation of AChE and a gradual accumulation of the
neurotransmitter acetylcholine, which led to an overstimulation of cholinergic receptors
and finally the paralysis of neuromuscular functions.19–21 Based on the unique mechanism
of organophosphorus compounds and the multibinding sites of AChE, a large number
of dual-binding compounds, including organophosphorus pharmacophore, were designed,
synthesized, and tested in vitro with AChE by our group.22,23 Comparing to monomeric
ones, many dual-binding compounds showed better inhibitory activity against AChE. The
OP pharmacophore and the length of the linker to the dual-binding compounds were the
crucial factors for their high activity. In addition to the active sites CAS and PAS, the deep
and narrow gorge suggested another affinity site. As a continuous work of our previous
research, herein we report the synthesis of the compounds 12 and the preliminary results on
AChE inhibition. In the structure of the inhibitor, two ethion moieties were linked together
with linear amides chain in different lengths. The AChE inhibitory activities of all the new
compounds and their analogs that were reported by Yang22 were tested using the AChE
isolated 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 procedure
shown in Scheme 4. Using liner diamines as the chain linker, it was easy to obtain the
intermediates 11a–h under vigorous stir for 6 h at the temperature below 10 ◦C. Products
11 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 the
diamine was slowly added to the solution of methyl chloroformate in cold methanol. It was
also found that an optimum ratio of methyl chloroformate to diamine in 2.4 equivalents
afforded the desired product in good yield. In a word, the optimum reaction conditions for
high yields were established as the substrates reacted in reasonable ratio and right adding
sequence at suitable temperature for proper reaction time, under which up to 90% yields

SYNTHESIS OF ACETYLCHOLINESTERASE INHIBITORS
1097
Scheme 1
were achieved and the isolated products were used directly in the next step without further
purification.
The title compounds 12a–h were obtained in 54–70% yields after purification by
silica 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 refluxing
conditions. The overall yields of the desired products from these two steps ranged from
49% to 63%. The structures of all the target compounds were well characterized by 1H and
13C NMR as well as elemental analyses (Table 1 and 2). 3a–h, 6a–c, and 9a–i, as shown in
Schemes 1–3, were known compounds.22
Biological Activity
The inhibitory activity against AChE of bivalent organophosphorus compounds was
summarized in Table S1 (Supplemental materials), expressed as IC50 values with ethion
Scheme 2

1098
R. XIE ET AL.
Scheme 3
as the reference standard. As shown in Table S1, most compounds demonstrated lower
inhibitory activities against AChE than ethion. Comparing with the ethylene in ethion,
linkers in these compounds affected the binding activity to AChE, except 2,2ꢁ-(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 sites
and the anionic sites of AChE.24 Some of the target compounds were able to simultaneously
interact 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 methylene
units in the linker was changed from two or four to three in compounds 12a–h and from
six or eight to seven in compounds 9b–j, which indicated that some specific favorable
interaction 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 from
compound 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 compound
9c). The result indicated that the acetamide groups of the linker assisted the molecule to
bind 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
12a
Physical properties
Yield (%)
C
H
N
P
S
white solid (mp:
77.2–78.5 ◦C)
white solid (mp:
41.2—42 ◦C)
white solid (mp:
81.8–83.4 ◦C)
white solid (mp:
61.0–62.2 ◦C)
colorless liquid
white solid (mp:
55.6–56.5 ◦C)
pale yellow liquid
colorless liquid
65
72
70
62
32.80 (32.9) 5.90 (5.92) 5.46 (5.31) 12.08 (12.0) 27.25 (27.1)
34.21 (33.9) 6.12 (6.22) 5.32 (5.44) 11.76 (11.9) 24.35 (24.2)
35.54 (35.7) 6.34 (6.26) 5.18 (5.08) 11.46 (11.3) 23.72 (23.9)
38.01 (38.3) 6.73 (6.62) 4.93 (5.01) 10.89 (10.7) 22.55 (22.7)
12b
12c
12d
12e
12f
64
60
39.16 (39.0) 6.92 (6.83) 4.81 (4.86) 10.63 (10.8) 22.01 (22.1)
40.25 (40.3) 7.09 (7.00) 4.69 (4.63) 10.38 (10.2) 21.49 (21.6)
12g
12h
54
56
41.29 (41.4) 7.26 (7.37) 4.59 (4.50) 10.14 (10.1) 21.00 (20.9)
42.29 (42.2) 7.42 (7.53) 4.48 (4.40) 9.91 (10.02) 20.53 (20.4)

SYNTHESIS OF ACETYLCHOLINESTERASE INHIBITORS
1099
Table 2 Spectral data of compounds
1H NMR (CDCl3/TMS) (δ) (ppm)
Compound
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
12c
12d
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).
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).
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
12f
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).
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
12h
12h
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).
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).
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,Sꢁ-2,2ꢁ-(decane-1,10-diylbis(azanediyl))bis(2-oxoethane-2,1-diyl)
O,O,Oꢁ,Oꢁ-tetraethyl diphosphorodithioate
15.73 (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 the
residues in AChE along the active gorge. Some important organophosphorus insecticides
such as prothoate, menazon, morphothion, and dimethoate belong to O,O-dialkyl-S-(N-
methylcarbnoyl alkyl) phosphorodithioate derivatives, and their inhibitory activities come
from the binding of the acetamide groups to the anionic sites of CAS. Because two moieties
were connected with a suitable linker, the bivalent compounds, like compound 12b, had a
stronger binding interaction with AChE and thus exhibited higher inhibitory activity. The
AChE-SERT dual inhibitors,25–28 which did not extend to the peripheral site, bound to the
residues halfway of the gorge and still exhibited potent AChE inhibitory activity. Likewise,

1100
R. XIE ET AL.
the two active molecules (12b and 9f) would bind to the catalytic site and the residues
near the catalytic site at the same time. Moreover, the specific spatial configurations of
12b and 9f were also a key factor to the binding affinity to AChE. In particular, compound
9b with optical configuration bound to CAS and the residues along the active gorge by
hydrogen bonds. Organophosphorus insecticides with P=S structure showed lower in vitro
inhibitory activity than those with P=O structure. For example, the AChE inhibitory activity
of dimethoate was about 1/200-fold and 1/4-fold of that of omethoate in different testing
methods.28 A series of compounds with P=O structure were synthesized and their bioassay
were studied in our group,23 and the results showed that the activation of ethion and the
compounds synthesized in this study through oxidation was less possible, whereas the
interaction between the phosphorodithioate acetamide groups or phthalimide groups and
some 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 inhibitory
activity 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 peripheral
site, but with low affinity. These data suggested that the peripheral site of AChE could not
tolerate the simultaneous substitution at positions 6 and 8 of the bivalent organophosphorus
skeleton. From the activities of 12b and 12e, it seemed that the residues near the catalytic
site instead of the peripheral site might be more interesting for the design of new bivalent
inhibitors.
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 than
1,4-butane, and C = C bond may have π–π interaction with some aromatic residues. With
one or two oxygen atoms in the linker, the physicochemical properties of the compounds
such as logP and pKa would change. However, compounds 6a, 6b, and 6c did not show
higher AChE inhibitory ability than other compounds, which suggested the change of logP
and pKa would not increase the in vitro activity.
MATERIALS AND METHODS
Preparation of Compounds
Reagents and solvents were purchased from Beijing Chemical Reagents Company and
were used without further purification. Column chromatography was performed on silica gel
200–300 mesh obtained from Qingdao Haiyang Chemical Co., Ltd. Analytical thin-layer
chromatography was performed with silica gel plates, and the plots were visualized under
UV light at 254 nm or iodine vapor. Elemental analyses (C, H, N, P, S) were performed at
Institute of Chemistry, Chinese Academy of Sciences. Nuclear magnetic resonance spectra
were recorded in CDCl3 solution, using Brucker DPX 300MHz spectrometer (performed
by 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 was
employed because it would enhance the killing effect of O,O-dialkyl-S-(N-methylcarbnoyl
alkyl) phosphorodithioates which were important organophosphorus insecticides. The syn-
thetic procedure for N,Nꢁ-(alkane-α,ω-diyl) bis(2-chloroacetamide) (11a–h) was described
as 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

SYNTHESIS OF ACETYLCHOLINESTERASE INHIBITORS
1101
Scheme 4
below 10 ◦C, and the mixture was stirred for 10 h. Afterwards, the product N,Nꢁ-(ethane-
1,2-diyl) bis(2-chloroacetamide) (11a) was filtered, washed, dried, and collected as a white
solid. The filtrate was condensed and subjected to recrystallization to afford additional
product. The same procedure was also employed in the preparation of products 11b–h by
using 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 solution
was approximately 7.0. Then, N,Nꢁ-(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 was
filtered and the filtrate was concentrated under vacuum. The viscous residue was dissolved
in ethyl acetate (20 mL), then washed with water and the ethyl acetate layer was dried
over 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,Sꢁ-2,2ꢁ-
(ethane-1,2-diylbis(azanediyl)) bis(2-oxoethane-2,1-diyl) O,O,Oꢁ,Oꢁ-tetraethyl diphospho-
rodithioate (12a) as the product. The same procedure was followed to afford the products
(12b–h) by using the corresponding N,Nꢁ-(alkane-α,ω-diyl)bis(2-chloroacetamide).
Characterization data for S,Sꢁ-2,2ꢁ-(ethane-1,2- diylbis(azanediyl)) bis(2-oxoethane-
2,1-diyl) O,O,Oꢁ,Oꢁ-tetraethyl diphosphorodithioate (12a): white solid; yield 65.3%; mp
77.2–78.5 ◦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%, H
5.90%, 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 spectral
data (1H and 13C NMR, Table 1 and 2)
O,O,Oꢁ,Oꢁ-tetraethyl
S,Sꢁ-2,2ꢁ-(propane-1,3-diyl))bis(azanediyl)bis(2-oxoethane-
2,1-diyl) diphosphorodithioate (12b).
S,Sꢁ-2,2ꢁ-(butane-1,4-diylbis(azanediyl))bis(2-oxoethane-2,1-diyl) O,O,Oꢁ,Oꢁ-tetr-
aethyl diphosphorodithioate (12c).

1102
R. XIE ET AL.
O,O,OꢁOꢁ-tetraethyl S,Sꢁ-2,2ꢁ-(hexane-1,6-diylbis(azanediyl))bis(2-oxoethane-2,1-
diyl) diphosphorodithioate (12d).
O,O,Oꢁ,Oꢁ-tetraethyl S,Sꢁ-2,2ꢁ-(heptane-1,7-diylbis(azanediyl))bis(2-oxoethane-2,1-
diyl) diphosphorodithioate (12e).
O,O,Oꢁ,Oꢁ-tetraethyl S,Sꢁ-2,2ꢁ-(octane-1,8-diylbis(azanediyl))bis(2-oxoethane-2,1-
diyl) diphosphorodithioate (12f).
O,O,Oꢁ,Oꢁ-tetraethyl S,Sꢁ-2,2ꢁ-(nonane-1,9-diylbis(azanediyl))bis(2-oxoethane-2,1-
diyl) diphosphorodithioate (12g).
S,Sꢁ-2,2ꢁ-(decane-1,10-diylbis(azanediyl))bis(2-oxoethane-2,1-diyl) O,O,Oꢁ,Oꢁ-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 significantly
increased the AChE inhibitory activity. The two potent AChE inhibitors, 12b (an interesting
homovalent compound) and 9f (a heterovalent compound), belong to two different types
of dual-site inhibitor of AChE. Further evaluation of 12b and 9f is underway.
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