Synthesis and crystal structure of new temephos analogues as cholinesterase inhibitor: Molecular docking, QSAR study, and hydrogen bonding analysis of solid state

Article abstract of DOI:10.1021/jf5011726

A series of temephos (Tem) derivatives were synthesized and characterized by ³¹P, ¹³C, and ¹H NMR and FT-IR spectral techniques. Also, the crystal structure of compound 9 was investigated. The hydrogen bonding energies (E²) were calculated by NBO analysis of the crystal cluster. The activities and the mixed-type mechanism of Tem derivatives were evaluated using the modified Ellman's and Lineweaver-Burk's methods on cholinesterase (ChE) enzymes. The inhibitory activities of Tem derivatives with a P=S moiety were higher than those with a P=O moiety. Docking analysis disclosed that the hydrogen bonds occurred between the OR (R = CH₃ and C₂H₅) oxygen and N-H nitrogen atoms of the selected compounds and the receptor site (GLN and GLU) of ChEs. PCA-QSAR indicated that the correlation coefficients of the electronic variables were dominant compared to the structural descriptors. MLR-QSAR models clarified that the net charges of nitrogen and phosphorus atoms contribute important electronic function in the inhibition of ChEs. The validity of the QSAR model was confirmed by a LOO cross-validation method with q² = 0.965 between the training and testing sets.

Full text of DOI:10.1021/jf5011726

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Article
Synthesis and crystal structure of new Temephos analogous
as cholinesterase inhibitor: molecular docking, QSAR
study and hydrogen bonding analysis of solid state
Khodayar Gholivand, Ali Asghar Ebrahimi Valmoozi, and Mahyar Bonsaii
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5011726 • Publication Date (Web): 03 Jun 2014
Downloaded from http://pubs.acs.org on June 4, 2014
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1 Synthesis and crystal structure of new Temephos analogous as
2 cholinesterase inhibitor: molecular docking, QSAR study and
3 hydrogen bonding analysis of solid state
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Khodayar Gholivand,٭a Ali Asghar Ebrahimi Valmoozi,a Mahyar Bonsaiib
aDepartment of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran,
Iran
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bDepartment of Chemistry, Islamic Azad University, North Tehran Branch, Tehran, Iran
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ABSTRACT: A series of Temephos (Tem) derivatives were synthesized and characterized by 31P, 13C,
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1H NMR and FT–IR spectral techniques. Also, the crystal structure of compound 9 was investigated. The
hydrogen bonding energies (E2) were calculated by NBO analysis of the crystal cluster. The activities and
the mixed–type mechanism of Tem derivatives were evaluated using the modified Ellman’s and
Lineweaver–Burk’s methods on cholinesterase (ChE) enzymes. The inhibitory activities of Tem derivatives
with P=S moiety were higher than those with P=O moiety. Docking analysis disclosed that the hydrogen
bonds occurred between the OR (R = CH3 and C2H5) oxygen and N–H nitrogen atoms of the selected
compounds and the receptor site (GLN and GLU) of ChEs. PCA–QSAR indicated that the correlation
coefficients of the electronic variables were dominant comparing to the structural descriptors. MLR–QSAR
models clarified that the net charges of nitrogen and phosphorus atoms contribute as important electronic
function in the inhibition of ChEs. Validity of QSAR model was confirmed by LOO cross–validation
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method with
between the training and testing sets.
q  0.965
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KEYWORDS: Bisphosphoramidothioate, Crystal structure, Cholinesterase inhibitor, QSAR, Molecular
docking
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49 ■ INTRODUCTION
50 In recent years, organophosphorus compounds have been of great interest. A wide range
51 of application in the areas of medicinal, agricultural, and industrial chemistry have been
52 found owing to their significant biological and physical properties.1 Among many known
53 phosphorus compounds, bisphosphoramidothioates (BPAT) with the general structure of
54 R1R2P(S)NH–X–NHP(S)R1R2 are important class of compounds that exhibit the
55 insecticide properties to inhibit the cholinesterase (ChE) enzymes.2 Little attention has
56 been given on the interaction mechanism of the ChE enzymes and BPATs.3,4 Temephos
57 (Scheme 1) with the general formula is a useful insecticide to control the larvae of
58 mosquitoes, midges and moths;5,6 however, it has side effects on human as anti–
59 acetylcholinesterase (AChE) and carcinogenic potential.7 Therefore, designing and
60 producing the selective compounds of Tem category having high insecticide potential
61 with less anti–AChE and carcinogenic effects are required. To extend and to evaluate this
62 problem, two new methods have so far been introduced in order to overcome the
63 inhibition mechanism.8 An integrated molecular docking and QSAR approaches were
64 employed to explore the binding interactions between the Tem analogous and the AChE
65 and butyrylcholinesterase (BChE).9 Molecular docking was performed to define a model
66 for the comprehension of the binding interactions between ligands and receptor. QSAR
67 models elucidated the effective parameters of molecular structure computed by the
68 Density Function Theory (DFT) in the inhibition process.10 In this study, 24 novel Tem
69 analogous with the general formula of (RO)2P(X)YP(X)(OR)2, (X = O and S; Y = NH–
70 (CH2)2–NH, NH–CH(CH3)–CH2–NH, N(CH3)–(CH2)2–N(CH3), NH–(CH2)3–NH, NH–
71 CH2–C(CH3)–CH2–NH, NH–(CH2)4–NH, N–(CH2)4–N, N–(CH2)5–N and NH–(C6H10)–
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72 NH; R = OCH3 and OC2H5) (1–24) were synthesized and characterized by P, C, H
73 NMR and IR spectroscopy. The solid state structure of
74 (CH3O)2P(S)NH(C6H10)NHP(S)(OCH3)2 (9) was determined by X–ray crystallography
75 and used as reference for quantum mechanical (QM) calculations at B3LYP level. The
76 electronic aspects of two different hydrogen bonds (P=S…H–N) in the crystal structure
77 of compound 9 were studied by NBO and Atoms in Molecules (AIM) analyses. The
78 activities of Tem derivatives on AChE and BChE were determined using a modified
79 Ellman’s method.11 Also the inhibition mechanisms of the prepared compounds were
80 evaluated by Lineweaver–Burk plot.12 Docking analysis was used to find the most
81 efficient parameters to introduce a better mechanism of interaction between the selected
82 molecules and the receptor site of human AChE and BChE. The appropriate molecular
83 structural parameters were computed by DFT method and adopted to construct QSAR
84 equations.
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86 ■ MATERIALS AND METHODS
87
88 Instrument. The enzyme AChE (human erythrocyte; Sigma, Cat. No. C0663) and
89 BChE (bovine erythrocyte, Sigma, Cat. No. B4186), acetylthiocholine iodide (ATCh,
90 99%, Fluka), 5, 5’-dithiobis (2-nitrobenzoic acid)) (DTNB, 98%, Merck), Na2HPO4,
91 NaH2PO4 (99%), ethylene diamine, propylene diamine, 1, 2-propylene diamine, 2,2-
92 dimethylpropylene diamine, butylene diamine and 1,2-cyclohexylane diamine (99%,
93 Merck), N,N'-dimethylethylene diamine (95%, Alfa Aesar), piperazine (99%. Acros),
94 homopiperazine (98%, Acros), triethylamine (99.5%, Merck), CDCl3 (99%, Sigma
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95 Aldrich), (CH3O)2P(S)Cl, (CH3CH2O)2P(S)Cl and (CH3CH2O)2P(O)Cl (97%, Sigma
1
96 Aldrich) were used as supplied. H, 13C and 31P spectra were recorded on a Bruker
1
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97 Avance DRX 500 spectrometer. H and C chemical shifts were determined relative to
98 internal TMS, and 31P chemical shifts relative to 85% H3PO4 as external standard.
99 Infrared (IR) spectra were recorded on a Shimadzu spectrometer (model IR-60) using
100 KBr pellets. Melting points were obtained with an electrothermal instrument. UV
101 spectrophotometer was used using a PERKIN–ELMER Lambda 25. The insecticide and
102 anti–AChE activities of Tem derivatives were predicted by the Prediction of Activity
103 Spectrum for Substances (PASS) software (version 1.193).13 The three-dimensional X–
104 ray structures of human AChE (PDB code: 1B41) and BChE (PDB code: 1POI) were
105 chosen as the template for the modeling studies of selected compounds. The PDB files
106 about the crystal structure of the ChE enzymes domain bound to P22303 (1B41.pdb) and
107 P06276 (1POI.pdb) were obtained from the RCSB protein data bank
108 (http://www.pdb.org). Molecular docking to both ChEs was carried out by using the
109 AutoDock 4.2.3 package software.14 The correlation analysis was performed by the
110 Statistical Package for Social Scientists (SPSS), version 16.0 for Windows.15 X–ray data
111 of compound 9 were collected at 120 K on a Bruker SMART 1000 CCD area detector
112 with graphite monochromated Mo-Kα radiation (λ= 0.71073 Å) and refined by full-
113 matrix least–squares methods against F2 with SHELXL97.16 CCDC 863609 contains the
114 supplementary crystallographic data for compound 9. These data can be obtained free of
115 charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge
116 Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: (+44)
117 1223-336-033; or E-mail: deposit@ccdc.cam.ac.uk.
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119 Statistical analysis. In order to identify the effect of descriptors on the activity of
120 AChE and BChE, QSAR studies were undertaken using the model described by Hansch
121 and Fujita.17 The stepwise multiple linear regression (MLR) procedure was used for
122 model selection, which is a common method used in QSAR studies for selection
123 descriptors. MLR fits a linear model of the form (eq 1):
124 Y  b0 b X1 b2 X2 ...bn Xk e
(1)
1
125 Where, Y is dependent variable, X1, X2, …, Xk are independent variables (descriptors), e
126 is a random error, and b0, b1, b2, …, bk are regression coefficients.18 The MLR method
127 performed by the software package SPSS 16.0 was used for selection of the descriptors.
128 The electronic and structural descriptors (X) were obtained by both the quantum chemical
129 calculations and theoretical studies.19 The electronic descriptors include the highest
130 occupied and the lowest unoccupied molecular orbital (EHOMO and ELUMO),
131 electrophilicity (ω), polarizability (PL) and net atomic charges (Q). Dipole moment (μ)
132 and molecular volume (Mv) are the structural descriptors. EHOMO, ELUMO, ω (is expressed
133 in terms of chemical potential and hardness), PL (the charge difference between the
134 atoms in functional groups), Q, μ and Mv (the size and geometrical shape of the molecule
135 and is dependent on three-dimensional coordinate of atoms in a molecule) values were
136 obtained from the DFT results by using the Gaussian 03 program package.20 In this study,
137 only the variables containing the information required for modeling were used. The
138 principal component analysis (PCA) was utilized to find the relationship between the
139 dependent and independent variables and reducing the set of independent variables
140 (MINITAB software, version14).21 The linear combinations form a new set of variables,
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141 namely principal components (PCs), which are mutually orthogonal. The first PC
142 contains the largest variance and the second new variable contains the second largest
143 variance and so on.22 The validity of the QSAR model was evaluated by LOO cross-
144 validation method, and an external data set was tested to evaluate the model. The high
145 square value of the cross-validation coefficient (q2) in the training set shows only a good
146 internal validation; however, it does not automatically refer to its high validity for an
147 external test set, because q2 usually overestimates the validity of the model. Therefore,
148 the QSAR model should be determined with a test set to confirm its validity. The
149 performance of external validation was characterized by determination coefficient (R2),
150 standard error (Sreg) and q2.23
151
152 Synthesis. The synthesis pathway of compounds 1-24 is represented in Scheme 2. For
153 example, compound 1 was prepared by the reaction of a solution of diethylenamine (1
154 mmol) and triethylamine (2 mmol) in THF was added to a solution of (OCH3)2P(S)Cl (2
155 mmol) in THF. After 4 h stirring at 0 °C, the solvent was removed in vacuum and the
156 resulting white powder was washed with distilled solvent.
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158 Crystal structure determination. A crystal suitable for X–ray crystallography was
159 obtained from a mixture of THF at room temperature for compound 9 (Figure S1). The
160 solid state structure as starting point was fully optimized by using DFT calculations in the
161 gas phase. Whereas X–ray crystallography cannot determine accurately the position of
162 hydrogen atoms, optimization of hydrogen atoms, positions was performed to investigate
163 the hydrogen bond characters in solid state structures. The H atoms of N–H groups were
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164 objectively localized in the difference Fourier synthesis and refined in isotropic
165 approximation. To achieve this goal, the solid state structure of crystal was modeled as
166 cluster, in which the target molecule is surrounded by two neighboring molecules (Figure
167 1). Other atoms were kept frozen during the optimization. Such computational
168 justifications have also been used to describe well the geometry and electronic aspects of
169 X-ray structure.24,25 Taking into consideration the large number of atoms in the model
170 cluster, all optimizations were performed at B3LYP/6–311+G** level. The NBO26
171 analysis was performed to compare the electronic features of gas phase structures of
172 compound 9 with those of the model clusters at B3LYP/6–311+G** level. Natural
173 population analysis (NPA) was performed at the same level by using the Reed and
174 Weinhold scheme.27 As part of this study deals with investigation of the hydrogen bonds
175 between S…H atoms, AIM analysis at the B3LYP/6–311+G** has much importance.
176 Two hydrogen bonds with different lengths were observed in compound 9. The hydrogen
177 bonding energies were calculated on the basis of energy difference between the hydrogen
178 bonded trimer and its fragments, as represented in equation EHB = Etrimmer – (Edimmer
+
179 Emonomer); where, dimmer is composed of two monomers (central and neighboring
180 molecules in the right hand, in Figure 1). Then the hydrogen bonding energies were
181 corrected for basis set superposition error (BSSE) using the counterpoise method.24,28 All
182 quantum chemical calculations were carried out by using the Gaussian 03 program
183 package.29
184
185 Cholinesterase assay. Human cholinesterase activity measurements were performed
186 essentially according to the method of Ellman.11 The reaction was carried out at 37˚C in
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187 70 mM phosphate buffer (Na2HPO4/NaH2PO4, pH=7.4) containing the AChE enzyme
188 (10µl volume, diluted 100 times in phosphate buffer, pH=7.4), DTNB (5, 5’-dithiobis (2-
189 nitrobenzoic acid)) (10-4M concentration) and ATCh (1.35×10-4M concentration). Each
190 compound was dissolved in dimethyl sulfoxide (DMSO, 99%, Merck), which was then
191 added to the buffer for in vitro cholinesterase assays. The highest concentration of DMSO
192 used in the assays was 5%. In the independent experiments without the inhibitor, 5%
193 DMSO had no effect the inhibition activity of the enzyme. The absorbance change at
194 37ºC was monitored with the spectrophotometer at 412 nm for 3 min, and three replicates
195 were run in each experiment (Figure S2). In the absence of inhibitor, the absorbance
196 change was directly proportional to the enzyme level. The reaction mixtures for
197 determination of IC50 values (the median inhibitory concentration) consisted DTNB
198 solution, 100 μl; inhibitor, x μl; acetylthiocholine iodide (ATCh) solution, 40 μl;
199 phosphate buffer, (850–x) μl; and AChE (human erythrocyte; Sigma, Cat. No. C0663)
200 solution, 10 μl (26.7u). The activity of BChE (bovine erythrocyte, Sigma, Cat. No.
201 B4186) was determined the same as the AChE activity by measuring the concentration of
202 thiocholine, which reacted with DTNB after hydrolysis of BTCh. The lyophilized BChE
203 was diluted with 100 mM phosphate buffer (pH=7.4) for using in the activity assay. The
204 plot of VI/V0 (VI and V0 are the activity of the enzyme in the presence and absence of
205 inhibitors, respectively) against log[I] (where, [I] is the inhibitor’s concentration) gave
206 the IC50 values of 12 compounds 1–4, 6–9, 15, 17–18, 20 and 24 (as anti–AChE) and 11
207 compounds 1–2, 4, 6–11, 13, 15 and 24 (as anti–BChE) (Table 1; Figures 2A and 2B).
208
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209 Inhibition mechanism study. One of the ways in which the inhibition of enzyme
210 catalyzed reactions can be discussed, is in terms of a general scheme shown below:
211
212 It is assumed that the enzyme-containing complexes are in equilibrium with each other,
213 i.e. the breakdown of ES to generate product does not significantly disturb the
214 equilibrium. In the present work, all data obtained for a particular system were analyzed
215 using the mix model of enzyme inhibition as described in the following equation:
[I0 ]
[I0 ]
Km (1
)
(1
)
Ki
KI
Vmax
1
V0
1
[S0 ]
216
(3)


Vmax
217 Where, V0 is the initial reaction rate, Vmax is the maximum reaction rate, Km is the
218 Michaelis constant for the substrate to the enzyme, and Ki and KI are the inhibition
219 constants for binding of the inhibitor to the enzyme or to the enzyme–substrate complex,
220 respectively. In this model, a mix inhibitor displays finite but unequal affinity for both the
221 free enzyme (E) and the ES complex; hence, the dissociation constants from each of these
222 enzyme forms inhibitors must be considered in their kinetic analysis.12,30 Ki and KI were
223 determined using the secondary plots as described by the following equation:
[I0]
(1
)
1
V'max
Ki
Vmax
224

(4)
[I0]
Ki
225 Slope for inhibited reaction = Slope for uninhibited reaction (1
)
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226 Hence, a secondary plot of 1/V′max against [I0] will be linear, the intercept on [I0 ] axis
227 gives –KI; a graph of the slope of primary plot against [I0] will also be linear, and the
228 intercept on [I0] axis gives –Ki. The Lineweaver–Burk plots related to the reversible
229 inhibitory effects of compounds showed a typical pattern of mixed inhibition (Figures
230 3A–3B and 3A'–3B'). The Km and Vmax values were calculated in the absence and
231 presence of inhibitor from which the secondary plots were obtained, and the equilibrium
232 dissociation constants, Ki and KI, were calculated (Table 2). A mixed inhibitor displays
233 affinity for both the free enzyme (Ki) and the enzyme-substrate complex (KI). Thus,
234 mixed–type inhibitors interfere with the substrate binding (increase Km) and hamper the
235 catalysis in the ES complex (decrease Vmax). When KI>Ki, the inhibitor preferentially
236 binds to the free enzyme, and the plots cross to the left of the 1/V0 axis but above the
237 1/[S0] axis (Figures 3A–3B and 3A'–3B'). In this situation, it is also termed competitive
238 noncompetitive inhibition.
239
240 ■ RESULTS AND DISCUSSION
241
242 Spectral Study. The 31P NMR chemical shift at room temperature in CDCl3 appears in
243 the range 68.31–78.82 ppm for P=S and 5.42–8.64 ppm for P=O derivatives. The 31P
1
244 NMR spectra of compounds 2, 9, 11 and 18 appeared as two separated signals. The H
245 NMR spectrum of compound 3 revealed that two doublets at 2.76 (3JPNH = 10.0 Hz) and
246 3.63 (3JPOH = 15.0 Hz) ppm are related to the methyl proton in the NCH3 and OCH3
1
247 groups, respectively. The H NMR spectra of compounds 9, 16 and 24 exhibited two
248 signals for the methylene protons of the six membered piperazinyl rings. Two protons of
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249 the NH group of all compounds are exhibited as a multiple peak at the range 3.48–5.37
250 ppm for P=S and 3.06–3.88 ppm for P=O derivatives. The 13C NMR spectra of
251 compounds 9, 16 and 24 indicated three separated peaks for the six carbon atoms that are
252 due to different orientations of the aliphatic six membered rings. The analysis of the IR
253 spectra indicated that the fundamental ν(P=S) stretching modes for compounds 1–16
254 appeared at the range 770.4–955.1 cm–1. The P=O stretching frequencies for compounds
255 17–24 were exhibited at the range of 1223.8–1252.1 cm–1. Moreover, the N–H stretching
256 frequencies for all compounds were observed at the range of 3189.1–3580.0 cm–1.
257
258 H–bonds analysis. The crystal data and the details of the X–ray analysis of the
259 compound 9 are given in Table S1. The phosphorus centers are in typical tetrahedral
260 environments. The P=S bond lengths of 1.9288(9) Å and 1.9316(9) Å were observed for
261 P(1)–S(1) and for P(2)–S(2), respectively, in (CH3O)2P(S)NH(C6H10)NHP(S)(OCH3)2
262 (9). It seems that the difference in the bond lengths is correlated to the various
263 orientations of cyclohexyldiamine rings and OCH3 groups. These orientations lead to the
264 creation of different hydrogen bonding patterns between the P=S and N–H functional
265 groups. The one-dimensional polymeric chains form in the crystal lattice with cyclic
266
R22(8) motifs in which the monomers are connected to each other via two P=S…H–N
267 hydrogen bonds distance of 3.464(2) and 3.522(2) Å (Figure 1, Table 3). The RYX (Z)
268 graph–set notation is descriptive of a Z–membered ring produced by the X hydrogen
269 bonds between the Y donor–acceptor units.31,32 Weak interactions, particularly P(2)–
270 S(2)…H(3)–C(3) (2.970 Å) and P(2)–S(2)…H(2)–C(2) (2.902 Å) cause to create
271 different hydrogen bonding lengths of the motifs. The large atomic radius and the low
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272 electronegativity of sulfur atoms decreased the strength of the hydrogen bonding between
273 P=S and NH. The electronic parameters of the hydrogen bonded clusters of compound 9
274 were calculated by AIM and NBO methods. The results of AIM and NBO analyses for
275 the mentioned clusters are presented in Table 3 and Figure 1. As shown, the bond lengths
276 in this cluster are equal to those obtained from the X–ray structures, except for the C–H
277 and N–H bonds, since the optimizations have been performed only for the hydrogen
278 atoms’ positions. Also the donor–acceptor distances for the hydrogen bonds in the model
279 cluster are equal to the experimental values. The results of AIM analysis show that the
280 electron density (ρ) value at the bond critical point (bcp) of S(1)…H(1) (0.104 eÅ–3) bond
281 path is larger in magnitude than the that calculated for the S(2)…H(2) (0.093 eÅ–3) in the
282 model cluster. The smaller ρ value at the bcp of N–H bond confirms the presence of the
283 stronger hydrogen bonds in P(1)–S(1)…(1)H–N(1) with the linear N–H…S contact angle
284 in comparison with the values obtained for P(2)–S(2)…(2)H–N(2). The ρ value at the bcp
285 of N–H bonds is 2.24 eÅ–3 for the fully optimized structure in the gas phase, which
286 decreases to 2.211 and 2.217 eÅ–3, respectively, in N(1)–H(1) and N(2)–H(2). The mean
287 N–H distance increases from the isolated molecules from 1.012 Å to 1.017 Å in their
288 hydrogen bonded of the modeled cluster. The electronic delocalization of Lp(S)i→σ*(N–
289 H)j occurs when the hydrogen bonds are formed between the subunits i and j within a
290 cluster. Such an electronic effect leads to weakening of the N–H bond. It has been
291 previously explained that the stabilizing energy E2 increases by a decrease in the donor-
292 acceptor distance of hydrogen bond (Gholivand and Mahzouni, 2011). The stabilizing
293 energies E2 of Lp(S)i→σ*(N–H)j electron density transfer in P=S…H–N hydrogen bonds
294 in the model cluster have been calculated as 19.94 and 16.34 kJ mol–1, respectively. This
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295 is in agreement with the values of distance for these hydrogen bonds in two P(1)–
296 S(1)…(1)H–N(1) (3.464(2) Å) and P(2)–S(2)…(2)H–N(2) (3.522(2) Å) models. The
297 hydrogen bonding energy in P(1)–S(1)…(1)H–N(1) model (–33.3 kJ mol–1) is smaller
298 than the value calculated for P(2)–S(2)…(2)H–N(2) (–42.4 kJ mol–1), although the
299 stabilizing energies E2 of P=S…H–N hydrogen bonds are larger in the former (Table 3).
300 It is noteworthy that the term E2 refers to the stabilization energy of electronic
301 delocalization between the donor-acceptor orbital and differs from the hydrogen bonding
302 energy. The molecule–molecule interaction energy is the sum of the total attractive and
303 repulsive forces between two hydrogen-bonded fragments. The results of AIM analysis
304 revealed some critical points with very small ρ values for the C–H(methyl)…S(2)–P(2) and
305 C–H(cyclohexane)…S(2)–P(2) contacts. It can be said that the steric repulsion between the
306 diaminocyclohexane, OCH3 group and P=S bond in the model crystal leads to a decrease
307 in P(2)–S(2)…(2)H–N(2) hydrogen bonding energy rather than P(1)–S(1)…(1)H–N(1)
308 model.
309
310 Prediction of biological potential. PASS software predicts 900 types of biological
311 activities based on the structural formula.33 The default list of predictable biological
312 activities (Pa) includes the main and side pharmacological effects, molecular mechanisms
313 and specific toxicities. The PASS prediction results for a compound are presented as a list
314 of activity names and probability activity (Pa) values. The Pa values are interpreted as: if
315 Pa > 0.7, 0.5 < Pa < 0.7, and Pa < 0.5, then the chance of finding this activity in the
316 experiments is high, low and lower, respectively.34,35 Insecticide potential, anti–AChE
317 activity and carcinogenic effect of 24 newly designed molecules were obtained by using
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318 the PASS software (Table 1). Table 1 shows that anti–AChE and carcinogenic properties
319 of compounds decrease owing to the change of (CH3O)2P=S to (C2H5O)2P=S in
320 compounds 1–16. Furthermore, the replacement of P=S in 10–16 to P=O in 17–24
321 increased the anti–AChE property and decreased the carcinogenic activity. The
322 insecticidal properties of all compounds are predicted in the range of 0.496–0.635. The
323 comparison of experimental data and the prediction of anti–AChE activities are shown in
324 Figure S2A. As shown in Figure S2B, a linear relationship gives the plot of probable
325 insecticide potential against anti–AChE activity. To test the anti–ChE activity of the
326 synthesized compounds, we evaluated the inhibitory potential of titled compounds
327 against AChE and BChE enzymes.
328
329 Bioassay. The inhibition constant (IC50) values of AChE against compounds 1–4, 6–9,
330 15, 17–18, 20 were in the range of 5.01–11402.50 μM (Table 1). Compound 3 displayed
331 the most potent inhibitory activity (IC50 = 5.01 μM). The inhibitory potential of
332 compound 15 with the OC2H5 was more than 7 with OCH3 substituent (Table 1). The
333 review of literature demonstrates that the inhibitory potential of monophosphoramides
334 (P=O moiety) is higher than the monophosphoramidothiates (P=S moiety),36 while
335 comparison of bisphosphoramidothioate 2 (IC50 = 674.53 μM) and bisphosphoramide 18
336 (IC50 = 11402.50 μM) reveals the inhibitory activity of P=S > P=O in contrast with the
337 AChE. In the present work, the synthesized compounds 1–2, 4, 6–11, 13, 15 showed
338 inhibition of BChE with the IC50 values between 149.62 and 7481.69 μM. In general, the
339 inhibitory activities of Tem derivatives with the P=S moiety were better than the P=O
340 moiety. The mixed–type and reversible mechanisms of these compounds were evaluated
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341 by Lineweaver–Burk plots (Table 2). To gain a better understanding of the inhibitory
342 potential of the synthesized compounds and to study on the reversible mechanism in
343 more detail, it was necessary to examine the interaction of the Tem derivatives with the
344 ChE structures by molecular docking method.
345
346 Molecular docking study. The interactions between Tem derivatives and AChE
347 receptor were achieved by molecular docking, which can facilitate the selection of
348 appropriate molecular parameters in the subsequent QSAR studies. The binding models
349 of titled compounds against AChE and BChE are depicted in Figs. 2A–2D. They are
350 located in the active site gorge of both AChE and BChE so as to maximize the favorable
351 contacts. The hydrogen bonds and van der Waals forces are the main features of the
352 interactions of compounds 2 (Figure 4A) and 18 (Figure 4B)with the polar charged site of
353 AChE, as well as the interactions of 2 (Figure 4C) and 11 (Figure 4D) with the esterastic
354 site of BChE. H–bond formation in the polar charged site was found to occur between the
355 P–OCH3 and P–OC2H5 oxygen of compounds 2 (P=S) and 18 (P=O) with the hydrogen
356 of H–N of GLN181 (d = 3.131 Å) and GLN291 (d = 2.100 Å), respectively. Figures 4C
357 and 4D show the 2D representation of the interaction mode of compounds 2 and 11 at the
358 active site of BChE. It can be clearly seen from Figures 4C and 4D that the hydrogen
359 atoms of the N–H group of compounds 2 and 11 forms an H–bond with the C=O group
360 containing GLU197 (d = 2.760 Å) and (d = 2.822 Å) moieties. The docking data
361 including inhibition constant (Ki), electrostatic energy (Eelect), and the rest of H–bonds
362 distance are given for the ChEs in Table S3. Figs. 3A and 3A' indicate that the increase of
363 the H–bond distances of RO…H–N leads to enhance their inhibitory potential, while
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364 these correlations are reversed in the interaction between BChE and ligands (Figures 5B
365 and 5B'). To continue work, QSAR technique was used to find the effective electronic
366 and structural parameters.
367
368 QSAR analysis. QSAR studies were done in order to recognize the effect of
369 descriptors on the activity of AChE and BChE enzymes. The stepwise MLR procedure,
370 which is a common method used in QSAR studies was used for model selection. The
371 electronic and structural descriptors were obtained by quantum chemical calculations
372 (Table S3). The number of independent variables is equal to the training set compounds,
373 as shown in Tables 1 and S3. Therefore, PCA method was used to reduce the independent
374 variables. The variable selection in PCA was performed by using the Fisher’s weights
375 approach37 and the results are summarized as the following equations (4a and 4b):
PC1  0.411QP(1)  0.411QP(2) 0.400QX (S,O) 0.080QN  0.410PLPX
376
(4a)
0.407EHOMO 0.052ELUMO  0.316  0.142  0.035Mv
PC2  0.064QP(1) 0.053QP(2) 0.042QX (S,O) 0.547QN 0.121PLPX
0.028EHOMO 0.500ELUMO 0.334 0.343 0.437Mv
377
(4b)
378 The results showed the total variance of the first and second factor PC was 51.4% and
379 19.0%, respectively. Figure S3 shows the score and the loading plot of PC1×PC2. The
380 score plot shows that separation of the compounds with P=O (right side) and P=S (left
381 side) groups has been provided by PC1, which contains the most part of the variance.
382 From the above equations, it is deduced that the electronic parameters (QP, QX, QN,
ELUMO, EHOMO and
383 PLP=X, ) are the largest comparing to the structural parameters (


384 and Mv). Consequently, these nine descriptors with higher correlation coefficient were
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385 selected to carry out the stepwise multiple linear regression (MLR) analysis, which led to
386 an optimal QSAR equation based on the anti–AChE potency (eq 5):
log(1/ IC50)  0.017 64.805QP 1.690QX (S,O) 8.967QN 31.967PLPX
387
25.193EHOMO 217.942ELUMO 243.834 0.016Mv33.690
n 13;R2  0.721;Sreg  0.966;Fstatistic  0.862
(5)
388 Where, n is the number of compounds,
389
R
2 is the determination coefficient of regression,
Sreg is the standard deviation of regression and Fstatistic is the Fisher’s statistic.38 The
390 variables with a high value of Variance Inflation Factor (VIF >10) are candidates for
391 exclusion from the model.39 The low determination coefficient value (R2 = 0.721) and
392 high residual value (Sreg = 0.966) with high VIF value (see Table 4) are associated with
393 the multicollinearity problem. The improvement in eq 3 was carried out by omitting of
394 compounds 7, 8, 24 from the training set compounds and replacing ELUMO with

and
395
EHOMO, as well as QP with PLP=X and QX. The linear regression was performed using the
396 remaining five parameters that yielded the following model with increasing of R2 = 0.922
397 and decreasing of Sreg = 0.438 (eq 6):
log(1/IC )  0.175 0.845QP 29.699QN 129.903ELUMO 0.019Mv 27.921
n 10;R2  0.922;Sreg  0.438;Fstatistic  9.473
50
398
(6)
399 The correlating parameters have VIF<10; thus, there is no colinearity problem (Table 4).
400 In this equation, the inhibitory potency of AChE is influenced mainly by the electronic
401 parameters. ELUMO with the coefficient value of –129.903 has the highest contribution to
402 log(1/ IC50) rather than the structural parameters. The negative sings of ELUMO in
403 log(1/ IC50) disclose that the compound with lower ELUMO is indicative of higher toxicity
404 against the AChE enzyme. The net charge of N–H nitrogen (QN) with the coefficient
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405 value of +29.699 reveals the highest effect of function on the inhibitory potential rather
406 than the net charge of P=O phosphorus (+0.845). The correlation matrix was used to
407 determine the interrelationship between the independent variables. A high
408 interrelationship was observed between ELUMO and QN (r = +0.456) (Table 5). Therefore,
409 compound 3 with QN = –0.893 and compound 18 with QN = –1.031 showed the highest
410 and the lowest inhibitory potential, respectively. Consequently, the net charge of nitrogen
411 atom is able to control the influence of molecular orbital energy in inhibition of human
412 AChE. Also the interaction of BChE as a secondary target was investigated against the
413 tested compounds (1–24) and the same procedures were carried out. The QSAR
414 modification model based on anti–BChE potency produced the following equation:
log(1/ IC50)  0.148 7.065QP 1.720QX (S,O) 0.430QN 0.469PLPX
415
100.702EHOMO 100.525ELUMO 104.965 0.002Mv 22.751
n 12;R2  0.566;Sreg  0.862;Fstatistic  0.290
(7)
416 Table 4 shows that the regression equation is not favorable with VIF>10. The
417 improvement in eq 7 was performed by excluding compounds 1, 24 from the selected
418 compounds and replacing

with ELUMO and EHOMO, as well as QP with PLP=X and QX.
419 Consequently, a new multiple regression was resulted without colinearity (eq 8):
log(1/ IC50)  0.116 3.768QP 0.32QN 106.212 0.004Mv 3.397
n 10;R2  0.848;Sreg  0.352;Fstatistic  4.479
420
(8)
421 The model described by eq 8, similar to eq 6, depicts the share of molecular orbital energy in
422 the inhibition of BChE. The most effective variable in the interaction of BChE and Tem

423 derivatives was
with the coefficient value of –106.212. The interrelationship between
424 the variables and the correlation matrix results are presented in Table 5. The high
425 interrelationship between ω and QP (r = +0.680) shows that QP controls the affect of
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426 molecular orbital energy in the inhibition of human BChE. Compounds 15 (QP = +2.027)
427 and 2 (QP = +1.997) are in order of the highest and the lowest inhibitory potential. DFT–
428 QSAR models of AChE and BChE revealed that changing in the net charge of nitrogen
429 and phosphorus atoms contributes an important function in the inhibition mechanism of
430 AChE and BChE, respectively. The above results are relatively good, but the validity of
431 QSAR models and docking output must be examined by NBO and LOO cross validation
432 methods.
433
434 Validation of MLR-QSAR Model. The LOO cross–validation method was used for
435 the training sets to select the optimum values of parameters. This procedure consists three
436 stage; i) removing one sample from the training set, ii) constructing the equation on the
437 basis of only the remaining training data, and iii) testing of the model on the removed and
438 innovative samples.40 The total of 10 samples were used as a training set, and the
439 remaining compounds (7, 8 and 24) for AChE–QSAR (eq 6) were adopted as a test set
440 for validating the models. A new equation is proposed to determine the outliers using
441 LOO cross–validation coefficient qn2i , which is equal to the q2 of compound i computed
442 by the new cross-validation procedure after leaving this datum from n compounds. The
443 compound with unduly high qn2i value can be considered as an outlier, and the
444 compound with low value can be taken as an influential point. Compound 4 has too large
445
qn2i value in the training set, so this compound can be confirmed to be an outlier from the
446 AChE–QSAR model. After omitting compound 4, two optimal models are obtained (eq
447 9):
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log(1/ IC50)  0.254 0.063QP 30.879QN 191.801ELUMO 0.018Mv 29.838
n  9;RA2dj  0.907;Sreg  0.335;r  0.10;
448
(9)
F
statistic 16.59;q2  0.965;P  0.0214
q2
449 Where,
is the square of LOO cross–validation coefficient. A good QSAR model has
S R
450 characters of large F, small r and reg , low P–value, and
2 and q2 values close to 1.41
451 So the above established eq 9 shows appropriate statistical quality. To check the validity
452 of eq 9, we selected our previous omitted compound (4) as the test set; the dependent and
453 independent variables with their residuals of test set compounds are shown in Table 1.
454 Table 1 and particularly residuals data show that the inhibition results are the same in the
455 empirical and prediction techniques. Figure 6 indicates that the predicted values of
456 log(1/ IC50) are in good agreement with the experimental ones. The integrity was
457 validated by the determination coefficient of R2 = 0.851 with the residuals between the
458 training and testing sets.
459
460 Natural Bond Orbital (NBO) analysis. The stabilization energies (E2) data clarify
461 that electron transfers lead to change in the net charge of phosphorus and nitrogen atoms,
462 and accordingly, alter the inhibitory properties of compounds against enzymes. The NBO
463 analysis reveals an electronic delocalization between the lone pair of X atom in the
464 P=X(O, S) group, Lp(XP), and the vacant (PO2) orbital. Stabilization energies of
465 9.211 kJ/mol for compound 2 with the P=S moiety, and 103.706 kJ/mol for compound 18
466 with the P=O moiety were obtained for the Lp(XP ) (PO2) interaction (Table 6).
467 The this interaction increases the electron density of OR (R = CH3 and C2H5) oxygen
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468 atom and rises the hydrogen bond energy of (R–O)ligand…(H–N)enzyme type (Figure 4).
469 Comparing to 18, compound 2 has higher inhibitory potential in contrast to AChE. The
470 H–bond energy of CH3–O…H–N (GLN181) for compound 2 is lower than the energy of
471 C2H5–O…H–N (GLN291) for compound 18. Hence, there is not a direct correlation
472 between the E2 of
interaction and the hydrogen bond energy
Lp(XP ) (PO2)
473 against the AChE activity. In other words, compound 2 with E2 = 18.601 kJ/mol and
474 compound 18 with E2 = 16.218 kJ/mol for the Lp(XP ) (P N1) interaction (Table
475 6) prove that the negative charge of nitrogen atom (QN) versus the hydrogen bond energy
476 of (R–O)ligand…(H–N)protein affects on the log(1/IC50) factor. For instance, compound 3
477 with the highest E2 and QN behaves as a strong inhibitor in the process of the inhibition of
478 AChE. In addition, the high E2 = 16.678 kJ/mol of Lp(N1) (PO2) interaction for
479 compound 18 comparing to compound 2 (E2 = 12.582 kJ/mol) leads to the increase of the
480 electron density of oxygen atom on the alcohol substituent and consequently to the rise of
481 the energy of the hydrogen bonding and to the decline of the inhibition potential. By
482 investigating the interaction mechanism of ligand and BChE enzyme, it can be said that
483 the Lp(O2) (P N1) interaction of selected compounds (2 and 11) increases the
484 electron density of N–H nitrogen and rises of the hydrogen bond energy of (N–
485 H)ligand…(O=C)enzyme type (Figure 4). The H–bond energy of N–H…O=C (GLU197) for
486 compound 2 is higher than that of 11, while compound 2 has lower inhibitory potential
487 comparing to compound 11 in contrast to BChE. It means that the net charge of
488 phosphorus atom’s (QP) effect inverses the H–bond energy of (N–H)ligand…(O=C)enzyme
.
489 Figures 5A'' and 5B'' demonstrate that the H–bonds energy of the docking analysis of
490 AChE and BChE has negative proportional to the QN and QP parameters.
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491
492 ■ CONCLUDING REMARKS
493 Twenty four bisphosphoramidothioate with the general formula of (RO)2P(S)XP(S)(OR)2
494 (1–24) were synthesized and characterized by spectroscopy methods. The cyclic motif of
495 (CH3O)2P(S)NH(C6H10)NHP(S)(OCH3)2 (9) was determined by X–ray crystallography.
496 The results of NBO analysis showed that the H–bond energy in P(1)–S(1)…(1)H–N(1)
497 and P(2)–S(2)…(2)H–N(2) model was –33.3 and –42.4 kJ mol–1, respectively. Docking
498 analysis disclosed the reversible noncovalent interactions, especially hydrogen bonds
499 occurred between the OR (R = CH3 and C2H5) oxygen and N–H nitrogen atoms of
500 selected compounds, and the H–N atoms of GLN with O=C of GLU. The MLR–QSAR
501 models (to R2=0.922 and 3.422 < VIF < 12.411 for AChE and to R2=0.845 and 1.281 <
502 VIF < 3.105 for BChE) and correlation matrix clarified that the net charge of N–H
503 nitrogen and R2NP=X phosphorus atoms contribute as important electronic function in
504 the inhibition of ChEs. The interrelationship shows that the net charge of nitrogen and
505 phosphorus atoms control the modification of the molecular orbital energy of Tem
506 analogues. The predicted values of log(1/ IC50) are in good agreement with the
507 experimental ones; the validity of QSAR model was confirmed by LOO cross–validation
q2  0.965
508 method with
between the training and testing sets. Increasing of E2 for the
interactions and the electron density of the
Lp(XP ) (P N) and
Lp(N) (PO)
509
510 N–H nitrogen with the OR oxygen of Tem analogous verifies the integrity of QSAR
511 equation and docking results in the inhibition process of ChE enzymes.
512
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513 ■ AUTHOR INFORMATION
514 Corresponding Author
515 ٭Phone: +98 21 82883443; fax: +98 218006544; e-mail gholi_kh@modares.ac.ir
516
517 ■ SUPPORTING INFORMATION
518 Spectral data as indicated in the text relative to Tem analogous (1–24). This information
519 is available free of charge via the Internet at http: //pubs.acs.org.
520
521 ■ ABBREVIATIONS USED
522 AChE, Acetylcholinesterase; BPAT, Bisphosphoramidothioate; ChE, Cholinesterase;
523 DFT, Density function theory; IC50, Half maximal inhibitory concentration; PASS,
524 Prediction of activity spectrum for substances; PCA, principal component analysis; PDB,
525 Protein data bonk; QSAR, Quantitative structure–activity relationships; SPSS, Statistical
526 package for social scientists; Tem, Temephos; VIF, Variance inflation factor;
527
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