Synthesis, characterization, oxidative degradation, antibacterial activity and acetylcholinesterase/butyrylcholinesterase inhibitory effects of some new phosphorus(V) hydrazides

Article abstract of DOI:10.1016/j.ejmech.2010.08.025

Some new phosphorus(V) hydrazides 1a-12a were synthesized and characterized by ¹H, ¹³C, ³¹P NMR, IR spectroscopy and elemental analysis. Moreover, the interaction of Cu(M)₂· nH₂O with 1a, 3a and 7a ga

Full text of DOI:10.1016/j.ejmech.2010.08.025

European Journal of Medicinal Chemistry 45 (2010) 5130e5139
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization, oxidative degradation, antibacterial activity and
acetylcholinesterase/butyrylcholinesterase inhibitory effects of some new
phosphorus(V) hydrazides
,
a
a
b
Khodayar Gholivand a , Zahra Hosseini , Sedigheh Farshadian , Hossein Naderi-Manesh
*
a Department of Chemistry, Faculty of Science, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran
b Department of Biochemistry and Biophysics, Faculty of Science, Tarbiat Modares University, Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Some new phosphorus(V) hydrazides 1ae12a were synthesized and characterized by 1H, 13C, 31P NMR, IR
spectroscopy and elemental analysis. Moreover, the interaction of Cu(M)2$nH2O with 1a, 3a and 7a gave
4,40-bis(morpholine)diazene (1b). In fact, in these reactions, copper(II) ions acted as oxidizing agent. The
results supported the proposed mechanism. The structures of compounds 1a, 1b and 1c were further
determined by X-ray crystallography. Compounds 1ae12a were screened for their antibacterial activities.
Also, the acetyl- and butyrylcholinesterase inhibitory activity of 1a, 3a, 7a, 11a and 12a was measured
using Ellman’s method. It is interesting that these compounds were more potent inhibitors of BChE than
of AChE. Also, using LineweavereBurk plots, it was indicated these compounds are mixed inhibitors.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Article history:
Received 4 May 2010
Received in revised form
9 August 2010
Accepted 10 August 2010
Available online 18 August 2010
Keywords:
Phosphorus(V) hydrazide
Oxidative degradation
Antibacterial activity
Acetylcholinesterase/butyrylcholinesterase
inhibitory
1. Introduction
erythrocyte acetylcholinesterase and bovine erythrocyte butyr-
ylcholinesterase activity [18,19]. The results showed that the
Hydrazides and hydrazones based on N-aminomorpholine and
N-aminopiperidine are biologically active materials, in particular
those which decrease the growth of malignant tumors [1,2] and are
the intermediates for bulk drugs, linsidomine and molsidomine [3].
In recent years, there has been significant growth in the field of
phosphorus acid hydrazides [4e7]. The presence of two nucleo-
philic nitrogen atoms together with the phosphorus atom in
phosphorus acid hydrazides has led to extensive synthetic possi-
bilities of these compounds. Substances with properties useful in
practice have been discovered among them. Also, the hydrazides of
phosphorothioic acid esters, diarylphosphinic acid esters of meth-
ylphosphonic and methylphosphonothioic acid exhibit pesticidal,
bactericidal and fungicidal activities [8e10]. The inhibition of AChE
by organophosphorus compounds is well documented [11e15], but
investigations into the inhibition of BChE with organophosphorus
compounds have been less systematic [16,17]. In our previous
studies, we reported the inhibition potency of carbacylamido-
phosphates with general formula RC(O)NHP(O)(morpholine)2;
R ¼ p-Cl, Br, Me, HeC6H4, CCl3, CF3, CH2Cl and CHCl2 on human
differences in the inhibition potencies of organophosphorus agents
are a manifestation of differing molecular properties of the inhib-
itors involved in the interaction with the active site of the enzyme.
To our knowledge, there are no reports in the literature of the
cholinesterase inhibitory effects of hydrazinophosphorus deriva-
tives containing PeNeN and NeNePeNeN structures.
From the above consideration, and in connection with our
current works in the field of phosphoramidate chemistry and their
enzyme inhibitory effects [20,21], here we synthesized and char-
acterized 12 new phosphorus(V) hydrazides based on N-amino-
morpholine and N-aminopiperidine 1ae12a as drawn in Scheme 1
and the crystal structure of compound 1a was studied. Besides, the
reactions of 1a, 3a and 7a with CuM2$nH2O (M ¼ Cl, NO3, ClO4, OAc)
were considered and the crystal structure of the products (1b and
1c) was determined.
Compounds 1ae12a were screened for the antibacterial activity
against Bacillus subtilis, Bacillus cereus, Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia.
In addition, the inhibitory effects of 1a, 3a, 7a, 11a and 12a on
AChE and BChE activity were determined using a modified Ellman’s
method [22] and their inhibition mechanisms were evaluated by
obtaining LineweavereBurk plots [23].
* Corresponding author. Tel.: þ982182884422; fax: þ982182883455.
E-mail address: gholi_kh@modares.ac.ir (K. Gholivand).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.025

K. Gholivand et al. / European Journal of Medicinal Chemistry 45 (2010) 5130e5139
5131
Scheme 1. The synthesis pathways of 1ae12a.
2. Chemistry
3. Pharmacology
2.1. Procedure for the synthesis of compounds (1ae12a)
3.1. Antibacterial activity
N-Benzoyl phosphoramidic dichloride, 4-chloro-N-benzoyl
phosphoramidic dichloride, 4-methoxy-N-benzoyl phosphoramidic
dichloride 2,6-difluoro-N-Benzoyl phosphoramidic dichloride and
(4-tolyl) dichlorophosphate were prepared according to the litera-
ture methods [24,25].
Compounds 1ae11a were prepared by the reaction of 15 mmol
phosphoric dichloride derivatives with 30 mmol of the corre-
sponding amines in the presence of 30 mmol of triethylamine as HCl
scavenger in dry tetrahydrofurane or acetonitrile. The temperature
was controlled in the range ꢀ5 to ꢀ8 ꢁC. After stirring for 8e14 h, the
precipitate was filtered and the product was washed with cool
acetone. Colorless crystals of 1a were obtained by slow evaporation
of acetone.
The in vitro antibacterial activities of synthesized compounds
were tested against six bacteria: three Gram-positive [B. subtilis, B.
cereus and S. aureus] and three Gram-negative [E. coli, P. aeruginosa
and K. pneumonia], by using the filter paper disc method [26] in
nutrient agar medium. The bacteria were cultured in nutrient agar
medium and used as inoculums. Whatmann filter paper discs
(diameter 6.5 mm) were saturated with the solutions of test
compound (concentration: 5, 10 mg mlꢀ1
) or reference drug,
chloroamphenicol (concentration: 5 and 10 mg mlꢀ1). The discs
were incubated at 37 ꢂ 1 ꢁC for 20e24 h. The zone of inhibition of
growth was measured, which indicates the inhibitory activity of the
compounds on the growth of the bacteria. The average of three
diameters was calculated for each sample.
Compound 12a was prepared by the reaction of 15 mmol
phosphoric dichloride derivative with 60 mmol of the corre-
sponding amine in dry acetonitrile at ꢀ5 to ꢀ8 ꢁC. After stirring for
12 h, the precipitate was filtered and the solvent was evaporated to
give the product that was washed with acetone.
3.2. AChE assay
Human acethylcholinesterase activity measurements were
performed essentially according to the method of Ellman. The
reaction mixtures for determination of IC50 values, the median
inhibitory concentration, consisted DTNB (5,50-dithiobis(2-nitro-
2.2. Procedure for the oxidative degradation with CuM2$nH2O
benzoic acid)) (10ꢀ4 M concentration) solution, 50
ml; inhibitor,
CuCl2$2H2O (30 mg, 1 mmol) was dissolved in methanol (5 ml)
and added to the solution of C6H5P(O)(NH(NC4H8O))2 (1a) (107 mg,
2 mmol) in 10 ml methanol. The mixture was stirred at 70 ꢁC for an
hour and at room temperature for 48 h. Slow evaporation of the
solution afforded crystalline material that consists of the mixture of
crystals of 4,40-bis(morpholine)diazene (1b), colorless prisms (60%),
bis(morpholinium) tetrachlorocuprate (1c), green needles (20%) and
C6H5P(O)(OCH3)2(1d) (20%). The crystals were separated manually.
The reactions of Cu(NO3)2$6H2O, Cu(ClO4)2$6H2O and Cu
(OOCCH3)2$2H2O with 1a were also carried out and produced 1b and
1d. Additionally, the interaction of the solution of CuCl2$2H2O with
the solution of 3a, 7a, and 4-aminomorpholine in methanol afforded
the same colorless prism crystals of 1b. Unfortunately, it was not
possible to mimic the above reaction with other compounds.
x
m
l
(5e250); acetylthiocholin iodide (ATCh) (1.35 ꢃ10ꢀ4 M
concentration) solution, 30 ml; 70 mM phosphate buffer (Na2HPO4/
NaH2PO4, pH ¼ 7.4) (885 ꢀ x)
m
l and hAChE (20
ml volume, diluted
100 times in phosphate buffer solution, 30
ml). The absorbance
change at 37 ꢁC was monitored with the spectrophotometer at
412 nm for 3 min and three replicates were run in each experi-
ment. In the absence of inhibitor, the absorbance change was
directly proportional to the enzyme level. The plot of VI/V0 (VI and
V0 are the activity of the enzyme in the presence and absence of
inhibitors, respectively) against log [I], where [I] is the inhibitor
concentration, gave the IC50 values of compounds 1a, 3a, 7a, 11a
and 12a (Figs. 1 and 2). Km and Vmax values were determined in
the absence and presence of inhibitor from the double reciprocal
LineweavereBurk plots (Figs. 3e5).

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K. Gholivand et al. / European Journal of Medicinal Chemistry 45 (2010) 5130e5139
similar moieties, on decreasing the NeN vibrational frequencies in
IR spectra, the 2J(PNH)amine values become greater approximately.
The 31P chemical shifts show when PheP linkage changes to
PheOeP in 1a and 2a; PheC(O)NHeP in compounds 3ae11a, C(O)
NHgroups cause moredeshieldingof thephosphorusatom. Also, the
31P chemical shifts of 2a, 6a and 11a with aminopiperidine deriva-
tives appear in upfield compared with their isostructures,1a, 3a and
7a with aminomorpholine derivatives. The carbons with one bond
distance from the N atom in all of the synthesized compounds are
equivalent and 3J(P,Caliphatic) values vary between 4.5 and 10.0 Hz
(Table 1). For ipso carbon atoms of phenyl rings, nJ(P,Caromatic) are
153.4 and 152.0 Hz (n ¼ 1) in 1a and 2a; 6.6e4.7 Hz (n ¼ 2) in 3a, 4a,
5a, 6a and 12a; 4.5 and 7.9 Hz (n ¼ 3) in 7a and 11a respectively.
The mass spectra of 1a and 9a showed the existence of frag-
mentation at m/z ¼ 101 and 86, which belong to C4H9N2Oþ and
C4H8NOþ cations with high intensity, respectively, that the latter
attributed to the break of NeN bond.
Fig. 1. The plot of VI/V0 against (log[I], mM) for inhibitors 1a, 3a, 7a, 11a and 12a. VI and
V0 are the acetylcholinesterase enzyme activity (OD minꢀ1) and [I] is the inhibitor
concentration (mM).
3.3. BChE assay
4.2. X-ray crystallography investigation
The activity of BChE was determined the same as AChE activity
by measuring thiocholine which reacted with DTNB after hydro-
lysis of BTCh. The lyophilized BChE was diluted with 100 mM
phosphate buffer (pH ¼ 8) for using in activity assay.
Single crystals were obtained from a solution of acetone (for 1a)
and methanol (for 1b and 1c) after slow evaporation at room
temperature. The crystal data and the details of the X-ray analysis
are given in Table 2, selected bond lengths and angles of 1a and 1b
are given in Tables 3 and 4 respectively. The molecular structure of
1a is shown in Fig. 6. It is interesting to note that the P]O bond
distance is considerably lengthened in comparison to the normal
3.4. Hydrophobic parameter evaluation
ꢀ
Calculated log P, hydrophobicity extent, of compounds 1a, 3a,
7a, 11a and 12a were performed using software log P, chemdraw
Ultra, 8.0.3, 2003.
P]O bond length (1.45 A) (Table 2). Also, the PeNamine bond
ꢀ
lengths are shorter than PeN single bond length (1.77 A) and longer
ꢀ
than PeN double bond length (1.57 A), in agreement with similar
compounds reported in the literature [28]. The coordination envi-
ronment around the central phosphorus atom of 1a is largely
tetrahedral. The morpholine moiety adopts a chair conformation
with tetrahedral nitrogen atoms (N2 and N4). In most phosphino-
amines, the PeN bonded nitrogen is planar, but the structural
4. Results and discussion
4.1. Spectral study
Structural assignment of 1ae12a was carried out using IR, 31P, 1H,
13C NMR spectroscopy and the results are summarized in Table 1. In
7ae11a, as weobserved earlier for phosphoramidate analogues [27],
the PeNamine stretching vibrations (1002e1017 cmꢀ1) appear at
higher frequencies than the PeNamide vibrations (902e920 cmꢀ1).
The NeN vibrational frequencies are observed in the range of
911e948 cmꢀ1. A comparison between similar compounds 1ae11a
indicate that the nP]O in compounds with X ¼ O in amino ring
moieties is stronger than compounds with X ¼CH2. 1H NMR spectra
of 1ae12a show the coupling constants 2J(PNH)amine between
feature of interest in 1a is the presence of pyramidalized nitrogen
P
atoms ( :N(1) and N(3) are 355.57 and 347.39ꢁ respectively). This
pyramidal nitrogen center was previously observed in some orga-
nophosphonic diamides [29]. The observed NeN distances (1.422
ꢀ
(2), 1.434(2) A) show slightly multiple bonding character. The data
of hydrogen bonding are presented in Table 5. The two NeH and
phosphoryl groups on each molecule are involved in intermolecular
hydrogen bonding with two neighboring molecules and one
dimensional polymeric structures shown in Fig. 7 along the a axis
are obtained.
The molecular structure of 1b is shown in Fig. 8. The selected
bond lengths and angles are given in Table 4. The torsion angle of
tetrazene (N1eN2eN2eN1 ¼180.0ꢁ) shows that the molecular
structure is exactly E configuration about the azo (N2]N2A) bond,
however in 2,5,20-triazido-1,10-azo-1,3,4-triazole [30] and 1,4-bis-
[1-methyl-tetrazol-5-yl]-1,4-dimethyl-2-tetrazene [31] this angle
was 177.8(2)ꢁ. The sum of N(2)eN(1)eC(1), N(2)eN(1)eC(2), C(1)e
N(1)eC(4) bond angle is 339.21ꢁ, therefore, the atoms N(1) and N
(1A) are sp3 hybridized. The bond length of the N1eN2 is 1.3950
phosphorus atom and the
range of 25.2e35.0 Hz. As shown in Table 1, in compounds with
a proton of the hydrazino group in the
ꢀ
(11) A in 1b, which is shorter than the accepted NeN single bond
ꢀ
length (1.44 A). Also, the bond length between the N atoms of the
ꢀ
azo group (N2]N2A) is 1.2556(17) A, which is the same for the
ꢀ
accepted NeN double bond length (1.25 A). In fact, there is a local-
ization of the azo
p electrons along the N2 moiety within 1b.
The crystal structure of bis(morpholinium) tetrachlorocuperate
(1c) was previously determined [32] at 22 ꢁC. We report here
a redetermination of 1c, at low temperature (120(2) K), in good
agreement with previous study and its packing diagram is pre-
sented in Fig. 9.
Fig. 2. The plot of VI/V0 against (log[I],
V0 are the butyrylcholinesterase enzyme activity (OD minꢀ1) and [I] is the inhibitor
concentration ( m).
mm) for inhibitors 1a, 3a, 7a, 11a and 12a. VI and
m

K. Gholivand et al. / European Journal of Medicinal Chemistry 45 (2010) 5130e5139
5133
Fig. 3. The plot of 1/[V] against 1/[S] for inhibitors 3a, 7a, 11a and acetylcholinesterase activation without inhibitor. [V] is the enzyme activity (OD minꢀ1) and [S] is the ATCh
concentration (mM).
4.3. Oxidative degradation results
depicts our proposed mechanism for the oxidative degradation
of candidate phosphorus(V) hydrazides to tetrazene by use of Cu
(II) under methanol solvent. The first step was the nucleophilic
substituent reaction of phosphorus(V) hydrazide (I) to phos-
phorus acid ester (II). The second step involved the removal of
hydrogen atoms from 4-aminomorpholine by CuCl2$2H2O giving
rise the aminonitrene(III), which finally the formation of the tet-
razene is explained in terms of the dimerization of the amino-
nitrene. It seems that the formation of 1c takes place in
a competitive mechanism with above proposed mechanism.
Phosphoryl-containing compounds have been used as effective
complexation agents in coordination chemistry [33e36]. In this
paper, our aim was the coordination of synthesized phosphorus(V)
hydrazides to Cu(II) ion due to the biological properties of Cu(II)
complexes [37]. Unexpectedly, the reaction of 2 equiv PhP(O)(NH
(NC4H8O))2 (1a) with 1 equiv of CuCl2$2H2O afforded 4,40-bis
(morpholine)diazene (1b), bis(morpholinium) tetrachlorocuprate
(1c) and dimethyl phenyl phosphonic acid (1d).
N-Unsubstituted hydrazides of phosphorus diesters were
previously oxidized by iodine in aqueous solutions in the presence
of tertiary amines with liberation of nitrogen and the formation
of phosphoric acid diesters [38]. In addition, the oxidation
of phosphorodi(N,N-alkylarylamidic) 2-methoxy(ethoxy)carbon-
ylhydrazides with lead tetraacetate led to the formation of the
corresponding azo-derivative [39]. Also, the oxidation of N-ami-
nomorpholine by use of benzeneseleninic acid [40], manganese
dioxide [41] and hexamethyldisilane [42] as oxidizing agents
afforded 1b in the moderate yield, whereas in this work, 1b was
prepared by copper ion as oxidizing agent with high yield.
2-Tetrazenes are composed of a four nitrogen chain with a central
double bond and they are widely applicable. For example, they
serve as chain-transfer agents in vinyl polymerization [43], source
of free amine radicals [44] and anticancer agents [45]. Scheme 2
4.4. Antibacterial activity results
The antibacterial results are given in Table 6. The antibacterial
activity is directly proportional to the concentration of the tested
compounds. It may be concluded that as the concentration is
increased, these compounds inhibit the growth of bacteria to
a greater extent. The most sensitive bacterial species on compounds
tested is Gram-positive bacteria, S. aureus and E. coli, while Gram-
negative bacteria P. aeruginosa is the most resistant species and only
12a exhibits antibacterial activity toward it. The compounds con-
taining aminopiperidine substituents have more activity toward
tested bacteria than their isostructural compounds containing
aminomorpholine substituents. Comparing the obtained results to
the standard antibiotic, chloramphenicol, 12a has stronger anti-
bacterial effect against P. aeruginosa.
Fig. 4. The plot of 1/[V] against 1/[S] for inhibitors 1a, 7a, 11a and butyr-
Fig. 5. The plot of 1/[V] against 1/[S] for inhibitors 3a, 12a and butyrylcholinesterase
activation without inhibitor. [V] is the enzyme activity (OD minꢀ1) and [S] is the BTCh
concentration (mM).
ylcholinesterase activation without inhibitor. [V] is the enzyme activity (OD minꢀ1
and [S] is the BTCh concentration (mM).
)

5134
K. Gholivand et al. / European Journal of Medicinal Chemistry 45 (2010) 5130e5139
Table 1
IR vibration frequencies (cmꢀ1), 31P chemical shifts (ppm) and coupling constants (Hz), of the synthesized compounds.
PeNa
PeNb
NeHa
NeHb
NeN
d
31P
d
NHb
2J(PNH)
d
NHa
2J(PNH)
3J(PNC)
nJ(P,Cipso)
b
a
Comp.
P]O
C]O
1a
2a
3a
4a
5a
6a
7a
8a
1183
1185
1222
1193
1197
1222
1212
1207
1219
1211
1211
1171
e
e
948
910
929
914
917
e
3435
3430
3445
3420
3455
3430
3355
3445
3265
3420
3265
3150
990
958
902
945
956
920
970
925
970
936
926
910
15.77
15.12
6.78
7.16
6.87
6.95
4.72
0.25
2.31
2.88
2.49
ꢀ4.49
5.81
e
25.2
br
e
e
10.0
9.9
5.2
6.6
5.1
5.1
4.8
4.5
4.6
4.7
4.4
4.8
153.4
152.0
6.6
5.0
4.8
6.6
0.0
0.0
8.6
e
e
e
e
e
e
6.32
6.51
6.27
4.77
4.74
5.99
4.74
5.99
5.63
4.11
35.0
31.4
31.3
32.6
27.0
30.5
27.0
30.5
30.0
br
e
e
e
e
e
e
e
e
e
e
e
e
e
e
920
e
e
e
e
920
912
912
910
902
e
1009
1010
1011
1002
1017
876
3159
3260
2960
3260
3260
e
8.86
9.62
9.20
9.02
9.08
e
5.2
6.6
5.0
0.0
0.0
e
1670
1651
1647
1713
e
9a
10a
11a
12a
0.0
7.9
4.8
a
Amide.
Amine.
b
4.5. AChE and BChE activity results
electronic effects. It is well known that the hydrophobic character
of organophosphorus compounds as operationally defined by
octanol/water partition coefficient (log P) plays an important role in
toxicity of them [46e48]. It is now quite clear that small difference
in hydrophobic character can be important. The replacement of
eCH2-moiety by eO-moiety in amine substituents, increases the
hydrophobicity. As we expected there is a linear relationship
between IC50 values and hydrophobicity (log P) in isostructure
compounds 1a, 3a, 7a and 11a with NeNePeNeN structures, that
is, as the IC50 values decrease, log P increase. It is revealed that
hydrophobic substituents enhance the association of organophos-
phorus with AChE and BChE. The electropositivity of the phos-
phorus atom in the Coulombic association of the inhibitors with the
active site serine of AChE is important. The 31P chemical shift is one
Enzymatic experiments on synthesized compounds were per-
formed to illustrate the inhibition behavior and different biological
activity characteristics of new derivatives of phosphoramidates.
Values for the inhibitory activity of the compounds 1a, 3a, 7a, 11a
and 12a against AChE and BChE were expressed as IC50 and log P
values are reported in Tables 7 and 8. The inhibition potency of the
compounds indicates an increasing inhibitory effect on AChE:
11a > 12a > 3a > 7a and on BChE: 11a > 12a > 3a > 7a > 1a as
obtained by IC50 values comparison. The data demonstrate that 1a
gives no significant change in the activity of the AChE, but inhibits
the activity of BChE. The inhibitory process of candidate
compounds was compared with respect to hydrophobicity and
Table 2
Crystal data collection and structure refinement parameters for compounds 1a, 1b and 1c.
Compound
1a
1b
1c
Formula
Formula weight
Temperature (K)
C14H23N4O3P
326.33
120(2)
C8H16N4O2
200.25
120(2)
C8H20Cl4CuN2O2
381.60
120(2)
ꢀ
Wavelength (A)
0.71073
0.71073
0.71073
Crystal system
Space group
Orthorhombic
Pna21
7.7634(12)
11.5332(15)
18.477(2)
90
90
90
1654.4(4)
Monoclinic
P2(1)/c
7.8668(13)
6.0462(10)
10.7655(18)
90
109.940(3)
90
481.36(14)
2
Orthorhombic
Pnma
7.6201(4)
20.8219(11)
9.1887(5)
90
ꢀ
a (A)
ꢀ
b (A)
ꢀ
c (A)
a
b
g
(deg)
(deg)
(deg)
90
90
3
ꢀ
V (A )
1457.92(13)
4
1.739
2.223
780
Z
4
Dcalc (Mg mꢀ3
)
1.310
0.184
696
1.382
0.102
216
Absorption coefficient (mmꢀ1
F(000)
)
Crystal size (mm)
0.21 ꢃ 0.17 ꢃ 0.15
2.08e29.49
ꢀ6 ꢄ h ꢄ 10
ꢀ13 ꢄ k ꢄ 15
ꢀ21 ꢄ l ꢄ 25
8893/4287(0.0357)
99.2
Semi-empirical from equivalents
0.972 and 0.959
Full-matrix least-squares on F2
4287/1/200
1.002
0.17 ꢃ 0.12 ꢃ 0.08
2.75e28.99
ꢀ10 ꢄ h ꢄ 10
ꢀ8 ꢄ k ꢄ 8
ꢀ14 ꢄ l ꢄ 14
5058/1282 (0.0230)
100.0
Semi-empirical from equivalents
0.989 and 0.980
Full-matrix least-squares on F2
7872/1/334
0.997
0.451 ꢃ 0.354 ꢃ 0.128
1.96e26.97
ꢀ9 ꢄ h ꢄ 9
ꢀ26 ꢄ k ꢄ 26
ꢀ11 ꢄ l ꢄ 11
13068/1443(0.0324)
100.0
Semi-empirical from equivalents
0.748 and 0.397
Full-matrix least-squares on F2
1636/0/82
1.011
Theta range for data collection (ꢁ)
Index ranges
Reflections collected/unique (Rint
Completeness to (%)
Absorption correction
)
q
Maximum and minimum transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices
R1 ¼ 0.0442
wR2 ¼ 0.0936
R1 ¼ 0.0574
wR2 ¼ 0.0989
0.493 and ꢀ0.269
R1 ¼ 0.0436
wR2 ¼ 0.1154
R1 ¼ 0.0488
wR2 ¼ 0.1230
0.268 and ꢀ0.282
R1 ¼ 0.0401
wR2 ¼ 0.0940
R1 ¼ 0.0450
wR2 ¼ 0.0982
1.078 and ꢀ1.117
R indices (all data)
ꢀꢀ3
Largest difference in peak and hole (e A
)

K. Gholivand et al. / European Journal of Medicinal Chemistry 45 (2010) 5130e5139
5135
Table 3
Selected bond lengths and bond angels for 1a.
ꢀ
ꢀ
Bond
Distance (A)
Bond
Distance (A)
P(1)eO(1)
P(1)eN(1)
P(1)eN(3)
P(1)eC(9)
1.4975(14)
1.6402(17)
1.6449(19)
1.809(2)
N(1)eN(2)
N(1)eH(1N)
N(3)eN(4)
N(3)eH(3N)
1.422(2)
0.9000
1.434(2)
0.9000
Angle
Amplitude (ꢁ)
Angle
Amplitude (ꢁ)
O(1)eP(1)eN(1)
O(1)eP(1)eN(3)
N(1)eP(1)eN(3)
O(1)eP(1)eC(9)
N(1)eP(1)eC(9)
N(3)eP(1)eC(9)
N(2)eN(1)eP(1)
N(2)eN(1)eH(1N)
P(1)eN(1)eH(1N)
109.02(8)
118.19(10)
106.71(10)
110.65(10)
111.56(10)
100.45(11)
117.87(13)
121.2
N(1)eN(2)eC(4)
N(1)eN(2)eC(1)
C(4)eN(2)eC(1)
N(4)eN(3)eP(1)
N(4)eN(3)eH(3N)
P(1)eN(3)eH(3N)
N(3)eN(4)eC(5)
N(3)eN(4)eC(8)
C(5)eN(4)eC(8)
111.69(16)
109.42(17)
109.76 (17)
116.89(15)
116.8
113.7
108.01(17)
110.70(18)
109.17(17)
116.5
of the factors that can indicate electron density around phosphorus
nuclei. It might be expected that the inhibitory of 1a, with the least
electron density around phosphorus nuclei (
d
31P ¼ 15.77 ppm),
Fig. 6. Molecular structure of 1a with numbering (50% probability ellipsoids).
should be larger than the others. However, as shown in Tables 7 and
8, this compound has no activity against AChE and less inhibition
potency on BChE. Also, 12a with the high electronegativity of
5. Conclusion
phosphorus atom (
d
31P ¼ ꢀ4.49 ppm) has more activity against
In summary, some new phosphorus(V) hydrazides 1ae12a
based on N-aminomorpholine and N-aminopiperidine were
reported. Also, the interaction of methanol solution of Cu
(M)2$nH2O (M ¼ Cl, NO3, ClO4, OAc) with 1a, 3a and 7a gave 4,40-bis
(morpholine)diazene (1b). The structures of compounds 1a, 1b and
1c were further determined by X-ray crystallography. Compounds
1ae12a were screened for their in vitro antibacterial activities
against six bacteria. From the activity studies, it was concluded that
S. aureus and E. coli were the most sensitive species. Also, the
compounds bearing aminopiperidine moiety, exhibited better
activities toward tested bacteria than those bearing amino-
morpholine moiety. The inhibitory potencies of 1a, 3a, 7a, 11a and
12a against human acetyl- and butyrylcholinesterase were studied.
The data demonstrated these compounds were shown to be potent
inhibitors of BChE versus AChE, respectively and 11a and 12a
having aminopiperidine substituents have a stronger inhibitory
effect on BChE than AChE. Also, these compounds were found to be
mixed type of inhibitors.
AChE and BChE. Therefore 31P NMR spectra are inadequate to
explain the relationship between IC50 and the 31P chemical shifts of
these compounds. By comparing the IC50 values of Tables 7 and 8, it
is clear that the inhibitory effects of these compounds are nearly 4-
to 9-fold higher on BChE than on AChE.
Early hypotheses and modeling experiments indicated that the
main functional difference between the AChE and BChE active sites
is related to the structure of the acyl pocket [49]. Due to a larger
substrate-binding pocket that can be accommodate bulky
substrates [50], the specificity of BChE is much larger than that of
AChE. It is interesting that compounds 11a and 12a with more
hydrophobicity due to bulky aminopiperidine substituent have
a stronger inhibitory effect on BChE than AChE.
The dependence of inhibitory potency and substrate concen-
tration was studied initially in a classical way in order to determine
the mechanism of inhibition. In Figs. 3e5, double reciprocal plots
for candidate compounds yielded increasing slopes and intercepts,
indicating the inhibitors are mixed ones. In these plots, y-inter-
cept ¼ 1/Vmax, x-intercept ¼ ꢀ1/Km and regression coefficients are
between 0.91 and 0.99. The Km and Vmax values of these compounds
and AChE and BChE are obtained under experimental conditions
(Tables 7 and 8). The Km’s for ATCh and BTCh are 0.083 mM and
0.11 mM, respectively. The patterns of lines reveal that the plots for
6. Experimental
6.1. Spectroscopic measurements
varying inhibitor concentrations depend on the value of
a. The
All reactions were performed under an argon atmosphere and
in dry solvents. 1H, 13C and 31P NMR spectra were recorded
on a Bruker Avance DRS 500 spectrometer. 1H and 13C chemical
shifts were determined relative to internal TMS and 31P chemical
shifts relative to 85% H3PO4 as an external standard. Infrared (IR)
spectra were recorded on a Shimadzu model IR-60 spectrometer.
Elemental analysis was performed using a Heraeus CHN-O-RAPID
apparatus. Mass spectra were obtained on a MS Model 5973
Network apparatus at ionization potential of 20 eV. Melting points
double reciprocal plots for compounds 3a, 7a, 11a and 12a with
AChE show that in these compounds, the lines are intersect at
a value of 1/[S] less than zero on the x-axis and a value of 1/[V] of
greater than zero (Fig. 3) and
a >1. In compounds 11a and 12a with
BChE, the lines intersect below the x and y axes, at negative values
of 1/[S] and 1/[V], where
a <1 (Figs. 4 and 5).
Table 4
Selected bond lengths and bond angels for 1b.
Table 5
ꢀ
ꢀ
Bond
Distance (A)
Bond
Distance (A)
Hydrogen bonds for compound 1a (A, ꢁ).
ꢀ
N(1)eN(2)
N(1)eC(1)
1.3950(11)
1.4637(13)
N(1)eC(4)
N(2)eN(2)#1
1.4671(12)
1.2556(17)
Complex
DeHeA
d(DeH)
d(H/A)
d(D/A)
<DHA
1a
N(1)eH(1N)/O(1)
[x þ 1/2, ꢀy þ 1/2, z]
N(3)eH(3N)/O(1)
[x ꢀ 1/2,ꢀy þ 1/2,z]
0.90
2.12
3.002(2)
168
Angle
Amplitude (ꢁ)
Angle
Amplitude (ꢁ)
N(2)eN(1)eC(1)
N(2)eN(1)eC(4)
118.05(8)
109.06(8)
C(1)eN(1)eC(4)
N(2)#1eN(2)eN(1)
112.10(8)
112.99(10)
0.90
2.04
2.900(2)
159

5136
K. Gholivand et al. / European Journal of Medicinal Chemistry 45 (2010) 5130e5139
Fig. 9. A view of the unit cell packing of 1c.
31P NMR (202.46 MHz, d6-DMSO, ppm):
(500.13 MHz, d6-DMSO, ppm):
d
¼ 15.77 (m). 1H NMR
3
d
¼ 7.82 (d, JP,H ¼ 11.2 Hz, 2H;
3
4
AreH), 7.48 (t, JH,H ¼ 7.3 Hz, 1H; AreH), 7.42 (t, JP,H ¼ 3.1 Hz, 2H;
AreH), 5.81 (d, 2JPNH ¼ 25.2 Hz; NHamine), 3.77e2.98 (t,
3
3JH,H ¼ 4.5 Hz; 8H), 2.64e2.54 (t, JH,H ¼ 4.2 Hz; 8H). 13C NMR
(125.77 MHz, d6-DMSO, ppm):
d
¼ 57.7 (d, 3JP,C ¼ 10.0 Hz), 63.24 (s),
2
Fig. 7. A view of the unit cell packing of 1a.
65.42 (s), 66.1 (s), 127.52 (d, JP,C ¼ 12.6 Hz), 130.81, 131.85 (d,
1
3JP,C ¼ 7.6 Hz), 132.87 (d, JP,C ¼ 153.4 Hz; Cipso). IR (KBr,
n
, cmꢀ1):
were obtained with an Electrothermal instrument. UV measure-
ments were recorded on a Shimadzu UV-2100 spectrometer.
3435(w, br, NeH), 3055 (m, CeHaromatic), 1435 (m, PePh),
1318e1358 (w, Ph), 1183 (s, P]O), 990 (w, NeN), 948 (s, PeNamine).
6.4. N,N0-Bis(4-aminopiperidine) phenylphosponic diamide (2a)
6.2. X-ray measurements
Yield: 43%. M.p. ¼ 232 ꢁC. Anal. Calc. for C16H27N4OP (322.43): C,
X-ray data of compounds 1a, 1b and 1c were collected on
59.55; H, 8.37; N, 17.36; Found: C, 59.50; H, 8.31; N, 17.50%. 31P NMR
a Bruker SMART 1000CCD [51] with graphite monochromated Mo
ꢀ
¼ 15.29 (m). 1H NMR (500.13 MHz,
Ka
radiation (
l
¼ 0.71073 A). The structures were solved by the
(202.46 MHz, d6-DMSO, ppm):
d
¼ 7.80 (d, 3JP,H ¼ 10.0 Hz, 2H; AreH), 7.68 (m, 1H;
direct method using the SHELX-97 program package [52]. The non-
hydrogen atoms were refined anisotropically and all located from
subsequent difference Fourier maps and refer to final R values of
0.0442, 0.0436 and 0.0401 for 1a, 1b and 1c, respectively.
Crystallographic data for the structures reported in this paper
have been deposited with the Cambridge crystallographic data center
as supplementary publication No. CCDC-727884 (C14H23N4O3P),
CCDC-742026 (C8H16N4O2) and CCDC-742027 (C8H20Cl4Cu1N2O2).
Copies of the data can be obtained free of charge on application to
CCDC,12 Union Road, Cambridge CB21EZ UK (fax: þ441223 336033;
e-mail: deposit@ccdc.cam.ac.uk).
d6-DMSO, ppm):
d
AreH), 7.37 (m, 2H; AreH), 5.85 (br, NHamine), 3.1 (m, 8H), 1.6 (m,
8H), 1.5 (m, 2H), 1.4 (m, 2H). 13C NMR (125.77 MHz, d6-DMSO,
ppm):
d
¼ 23.6, 24.9, 53.91 (d, 3JP,C ¼ 9.9 Hz), 127.91 (d,
2JP,C ¼ 12.2 Hz), 129.11, 132.61 (d, JP,C ¼ 6.5 Hz), 133.39 (d,
3
1JP,C ¼ 152.3 Hz; Cipso). IR (KBr,
n
, cmꢀ1): 3430 (w, br, NeH), 3140 (m,
CeHaromatic), 1450 (m, PePh), 1185 (s, P]O), 958 (w, NeN), 910 (m,
PeNamine).
6.5. N,N0-Bis (4-aminomorpholine) phosphoramidic acid phenyl
ester (3a)
6.3. N,N0-Bis(4-aminomorpholine) phenylphosponic diamide (1a)
Yield: 60%. M.p. ¼160 ꢁC. Anal. Calc. for C14H23N4O4P (342.33):
C, 49.08; H, 6.70; N, 16.36; Found: C, 49.10; H, 6.70; N,16.40%. 31P
Yield: 73%. M.p. ¼196 ꢁC. Anal. Calc. for C14H23N4O3P (326.33):
NMR (202.46 MHz, d6-DMSO, ppm):
d
¼ 6.78 (m). 1H NMR
(500.13 MHz, d6-DMSO, ppm):
d
¼ 7.32 (t, 3JH,H ¼ 8.8 Hz, 2H; AreH),
C, 51.48; H, 7.04; N, 17.16; Found: C, 51.50; H, 7.00; N, 17.50%.
7.21 (d, 3JH,H ¼ 8.8 Hz, 2H; AreH), 7.1 (t, JH,H ¼ 7.2 Hz, 1H), 6.32 (d,
3
2JPNH ¼ 35.0 Hz, 2H; NHamine), 3.51 (m, 8H), 2.60 (t, JH,H ¼ 4.7 Hz,
3
8H). 13C NMR (125.77 MHz, d6-DMSO, ppm):
d
¼ 57.80 (d,
3JP,C ¼ 5.2 Hz; 4C), 66.1 (s), 120.2 (d, JP,C ¼ 4.8 Hz), 123.55, 129.23,
3
151.55 (d, 2JP,C ¼ 6.6 Hz; Cipso). IR (KBr,
n
, cmꢀ1): 3445 (w, br, NeH),
3285 (m, CeHaromatic), 1453 (m, PePh), 1222 (s, P]O), 929 (s,
PeNamine), 902 (w, NeN).
6.6. 2-Chloro-N,N0-bis (4-aminomorpholine) phosphoramidic acid
phenyl ester (4a)
Yield: 40%. M.p. ¼185 ꢁC. Anal. Calc. for C14H22ClN4O4P (376.77):
C, 44.58; H, 5.83; N, 14.86; Found: C, 44.10; H, 5.75; N, 14.80%. 31P
Fig. 8. Molecular structure of 1b with numbering (50% probability ellipsoids).

K. Gholivand et al. / European Journal of Medicinal Chemistry 45 (2010) 5130e5139
5137
Scheme 2. The oxidative degradation process of 1a, 3a and 7a.
NMR (202.46 MHz, d6-DMSO, ppm):
(500.13 MHz, d6-DMSO, ppm):
d
¼ 7.16 (m). 1H NMR
13C NMR (125.77 MHz, d6-DMSO, ppm):
d
¼ 57.76 (d, 3JP,C ¼ 5.1 Hz),
3
d
¼ 7.59 (d, JH,H ¼ 8.2 Hz, 1H;
66.0, 119.9 (d, 3JP,C ¼ 4.8 Hz), 129.55, 129.91, 149.31 (d, 2JP,C ¼ 6.3 Hz;
3
3
AreH), 7.41 (d, JH,H ¼ 8.0 Hz; 1H; AreH), 7.23 (t, JH,H ¼ 7.55 Hz;
Cipso). IR (KBr, n
, cmꢀ1): 3455 (w, br, NeH), 3185 (m, CeHaromatic),
1303e1381 (w, Ph), 1197 (s, P]O), 956 (w, NeN), 917 (s, PeNamine).
3
1H; AreH), 7.01 (t, JH,H ¼ 7.6 Hz; 1H; AreH), 6.86 (d,
2JPNH ¼ 31.4 Hz, 2H; NHamine), 3.54 (m, 8H), 2.49 (m, 4H), 2.24 (m,
4H). 13C NMR (125.77 MHz, d6-DMSO, ppm):
d
¼ 20.20, 57.70 (d,
6.8. N,N0-Bis (4-aminopiperidine) phosphoramidic acid phenyl ester
(6a)
2
3JP,C ¼ 6.63 Hz), 66.1, 120.2 (d, JP,C ¼ 5.0 Hz; Cipso), 123.55, 129.23,
151.55. IR (KBr, n
, cmꢀ1): 3420 (w, br, NeH), 3165 (m, CeHaromatic),
Yield: 75%. M.p. ¼138 ꢁC. Anal. Calc. for C16H27N2O2P (310.12): C,
1477 (m, PePh), 1350e1390 (w, Ph), 1193 (m, P]O), 940 (w, NeN),
914 (m, PeNamine), 768 (m, CeCl).
61.91; H, 8.71; N, 9.03; Found: C, 61.89; H, 8.70; N,9.01%. 31P NMR
(202.46 MHz, d6-DMSO, ppm):
d
¼ 6.95 (m). 1H NMR (500.13 MHz,
3
6.7. N,N0-Bis (4-aminomorpholine) phosphoramidic acid (4-
methyl-phenyl) ester (5a)
d6-DMSO, ppm):
d
¼ 7.32 (t, JH,H ¼ 10.7 Hz, 2H; AreH), 7.18 (d,
3JH,H ¼ 10.0 Hz, 2H; AreH), 7.08 (m, 1H), 6.00 (d, JPNH ¼ 31.9 Hz;
2
NHamine), 3.36 (m, 2H), 2.65 (t, 3JH,H ¼ 5.2 Hz; 6H); 1.25 (m, 4H). 13
C
Yield: 60%. M.p. ¼182 ꢁC. Anal. Calc. for C15H25N4O4P (356.36):
NMR (125.77 MHz, d6-DMSO, ppm):
d
¼23.01, 25.50, 58.50 (d,
C, 50.51; H, 7.01; N, 15.71; Found: C, 50.50; H, 7.00; N, 15.49%. 31P
3JP,C ¼ 5.1 Hz), 120.24 (d, JP,C ¼ 4.9 Hz), 123.31, 129.11, 151.74 (d,
3
NMR (202.46 MHz, d6-DMSO, ppm):
(500.13 MHz, d6-DMSO, ppm):
d
¼ 6.86 (m). 1H NMR
2JP,C ¼ 6.6 Hz; Cipso). IR (KBr,
n
, cmꢀ1): 3300 (w, br, NH), 3290 (m,
d
¼ 7.06e7.16 (m, 4H; AreH), 6.28
CeHaromatic), 1425 (s, PePh), 1337e1378 (w, Ph), 1222 (s, P]O), 920
(s, PeNamine), 958 (w, NeN).
2
(d, JPNH ¼ 31.3 Hz, 2H; NHamine), 3.51 (m, 8H), 2.60 (m, 8H).
Table 6
Inhibition zone (mean diameter of inhibition in millimetres) as a criterion of antibacterial activities of the synthesized compounds.
Compound
Concentration (mg/ml)
P. aeruginosa
E. coli
K. pneumonia
B. cereus
B. subtillus
S. aureus
1a
5
10
5
10
5
10
5
10
5
10
5
10
5
10
5
10
5
10
5
10
5
10
5
10
5
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
5
e
e
e
e
e
e
7
10
6
11
7
15
6
11
e
e
6
13
e
7
e
e
7
e
e
e
e
e
e
e
e
7
e
e
e
e
e
e
7
10
e
e
5
14
e
e
e
e
e
8
e
e
e
e
e
2a
e
e
3a
e
e
e
e
4a
e
e
e
e
5a
8
18
6
13
7
13
e
10
13
11
17
e
14
5
6a
15
e
e
e
e
e
e
e
e
e
e
e
e
11
15
7a
8
e
8a
e
e
9a
5
12
7
13
9
19
7
15
14
17
e
e
10a
e
e
7
e
11a
e
e
e
e
12
15
e
7
e
12a
10
e
7
11
13
17
8
17
22
Chloramphenicol
10

5138
K. Gholivand et al. / European Journal of Medicinal Chemistry 45 (2010) 5130e5139
Table 7
1219 (s, P]O), 1011 (m, PeNamine), 970 (w, NeN), 912 (s, PeNamide),
715 (CeCl).
The enzymatic data for hAChE and candidate compounds
Compound
IC50 (mM)
log P
Km (mM)
Vm (mM/min)
Inhibition
mechanism
6.12. 4-Methoxy-N-benzoyl-N0,N00-bis (4-aminomorpholine)
Enzyme
1a
3a
7a
11a
12a
e
e
0.083
e
34.48
e
e
phosphoric triamide (10a)
e
ꢀ1.12
ꢀ0.54
ꢀ0.78
1.35
1.29
e
9.55
18.62
1.95
5.49
0.047
0.049
0.045
0.051
37.04
55.55
40.01
11.11
Mixed
Mixed
Mixed
Mixed
Yield: 68%. M.p. ¼188 ꢁC. Anal. Calc. for C16H26N5O5P (399.36):
C, 48.08; H, 6.51; N, 17.53; Found: C, 48.10; H, 6.50; N, 17.60%. 31P
NMR (202.46 MHz, d6-DMSO, ppm):
d
¼ 2.88 (m). 1H NMR
2
(500.13 MHz, d6-DMSO, ppm):
d
¼ 9.02 (s, JPNH ¼ 0 Hz; 1Hamide),
7.92 (d, 3JH,H ¼ 8.8 Hz, 2H; AreH), 6.97 (d, 3JH,H ¼ 8.8 Hz, 2H; AreH),
6.9. N-Benzoyl-N0,N00-bis (4-aminomorpholine) phosphoric
triamide (7a)
2
5.94 (d, JPNH ¼ 29.6 Hz; NHamide), 3.80 (s, 3H), 3.52 (m, 8H), 2.68
(m, 8H). 13C NMR (125.77 MHz, d6-DMSO, ppm):
d
¼ 27.00, 57.81 (d,
3JP,C ¼ 4.7 Hz), 66.00, 113.5, 125.91, 129.91, 135.90, 162.31, 167.20. IR
Yield: 88%. M.p. ¼177e179 ꢁC. Anal. Calc. for C15H24N5O4P
(KBr, n
, cmꢀ1): 3420 (w, br, NeH), 3260 (m, CeHaromatic), 1647 (vs,
(396.36): C, 45.41; H, 6.05; N, 17.66; Found: C, 45.10; H, 6.10; N,
C]O), 1448 (m, C]C), 1407 (m, CeNamide) 1211 (s, P]O), 1173 (m,
CeO), 1002 (m, PeNamine), 936 (w, NeN), 910 (m, PeNamide).
17.60%. 31P NMR (202.46 MHz, d6-DMSO, ppm):
d
¼ 4.72 (m). 1H
2
NMR (500.13 MHz, d6-DMSO, ppm):
d
¼ 8.86 (d, JPNH ¼ 5.2 Hz;
1Hamide), 7.98 (d, 3JH,H ¼ 7.8 Hz,1H; AreH), 7.58 (t, 3JH,H ¼ 7.3 Hz,1H;
6.13. N-Benzoyl-N0,N00-bis (4-aminopiperidine) phosphoric triamide
(11a)
3
2
AreH), 7.29 (t, JH,H ¼ 7.5 Hz, 2H; AreH), 4.74 (d, JPNH ¼ 27.0 Hz;
NHamide), 3.67 (m, 8H), 2.81 (m, 8H). 13C NMR (125.77 MHz, d6-
3
Yield: 75%. M.p. ¼ 204 ꢁC. Anal. Calc. for C17H28N5O2P (365.36): C,
DMSO, ppm):
133.43,136.57,169.63. IR (KBr,
CeHaromatic), 1643 (vs, C]O), 1447 (m, C]C), 1421 (m, CeNamide
d
¼ 58.97 (d, JP,C ¼ 4.8 Hz), 66.43, 131.97, 132.93,
55.83; H, 7.66; N, 19.16; Found: C, 55.80; H, 7.65; N, 19.20%. 31P NMR
n
, cmꢀ1): 3400 (w, br, NeH), 3235 (m,
¼ 2.49 (m). 1H NMR (500.13 MHz,
)
(202.46 MHz, d6-DMSO, ppm):
d
2
1212 (s, P]O), 1009 (m, PeNamine), 920 (s, PeNamide), 970 (w, NeN).
d6-DMSO, ppm):
d
¼ 9.08 (s, JPNH ¼ 0 Hz; 1Hamide), 7.88 (d,
3JH,H ¼ 7.8 Hz, 2H; AreH), 7.48e7.69 (m, 3H; AreH), 5.69 (d,
2JPNH ¼ 30.0 Hz; NHamide), 3.40 (m, 8H), 3.04 (m, 8H),1.45 (m, 4H).13
C
6.10. 2,6-Difluoro-N-benzoyl-N0,N00-bis (4-aminomorpholine)
phosphoric triamide (8a)
NMR (125.77 MHz, d6-DMSO, ppm):
d
¼ 23.4, 25.51, 58.70 (d,
3JP,C ¼ 4.4 Hz), 127.43, 128.11, 132.00,134.00 (d, 4JP,C ¼ 7.9 Hz),167.81.
IR (KBr, n
, cmꢀ1): 3455 (w, br, NeH), 3280 (m, CeHaromatic), 1648 (vs,
Yield: 83%. M.p. ¼ 212 ꢁC. Anal. Calc. for C15H22F2N5O4P
C]O), 1451 (m, C]C), 1428 (m, CeNamide) 1211 (s, P]O), 1017 (m,
PeNamine), 902 (m, PeNamide), 926 (w, NeN).
(432.36): C, 41.63; H, 5.09; N, 16.19; Found: C, 41.60; H, 6.00; N,
16.10%. 31P NMR (202.46 MHz, d6-DMSO, ppm):
d
¼ 0.25 (m). 1H
2
NMR (500.13 MHz, d6-DMSO, ppm):
d
¼ 9.62 (d, JPNH ¼ 6.6 Hz;
3
6.14. Phenyl-N-(4-aminopiperidine) phosphate (12a)
1Hamide), 7.49 (m, 2H), 7.14 (t, JH,H ¼ 5.1 Hz, 1H; AreH), 5.99 (d,
2JPNH ¼ 30.5 Hz; 2Hamide), 3.56 (t, JH,H ¼ 4.2 Hz; 8H), 2.71 (m, 8H).
3
Yield: 88%. M.p. ¼128 ꢁC. Anal. Calc. for C11H18N2O3P (257.12): C,
13C NMR (125.77 MHz, d6-DMSO, ppm):
d
¼ 57.81 (d, 3JP,C ¼ 4.5 Hz),
51.34; H, 7.00; N, 10.89; Found: C, 51.39; H, 7.10; N, 10.91%. 31P NMR
66.13, 111.9, 111.7. IR (KBr, n
, cmꢀ1): 3445 (w, br, NeH), 3260 (m,
(202.46 MHz, d6-DMSO, ppm):
d
¼ ꢀ4.49 (s). 1H NMR (500.13 MHz,
CeHaromatic), 1671 (vs, C]O), 1463 (m, C]C), 1383 (m, CeNamide
1207 (s, P]O), 1010 (m, PeNamine), 925 (w, NeN), 912 (m,
PeNamide).
)
3
d6-DMSO, ppm):
d
¼ 7.27 (t, JH,H ¼ 7.9 Hz; 2H; AreH), 7.18 (d,
3JH,H ¼ 7.8 Hz, 2H; AreH), 6.98 (t, 3JH,H ¼ 7.3 Hz, 1H; AreH), 6.11 (br,
1H; OH), 2.99 (m, 4H), 1.66 (m, 4H), 1.34 (t, 2H). 13C NMR
6.11. 4-Chloro-N-benzoyl-N0,N00-bis (4-aminomorpholine)
phosphoric triamide (9a)
(125.77 MHz, d6-DMSO, ppm):
d
¼ 19.71, 21.90, 23.41, 25.60, 57.81,
128.91, 122.31, 120.10 (d, 3JP,C ¼ 4.8 Hz; Cipso). IR (KBr,
n
, cmꢀ1): 3220
(w, br, NeH), 3150 (m, CeHaromatic), 1452 (PePh), 1356e1392 (w,
Ph), 1171 (m, P]O), 950 (w, NeN), 876 (m, PeNamine).
Yield: 80%. M.p. ¼ 201e205 ꢁC. Anal. Calc. for C15H23ClN5O4P
(404.86): C, 44.45; H, 5.68; N, 17.29; Found: C, 44.10; H, 5.60; N,
6.15. 4,40-Bis(morpholine)diazene (1b)
17.30%. 31P NMR (202.46 MHz, d6-DMSO, ppm):
d
¼ 2.31 (m). 1H
2
NMR (500.13 MHz, d6-DMSO, ppm):
d
¼ 9.08 (s, JPNH ¼ 0.0 Hz;
Yield: 60%. M.p. ¼156 ꢁC. Anal. Calc. for C8H16N4O2 (200.12): C,
1Hamide), 7.91 (d, 3JH,H ¼ 8.5 Hz, 2H; AreH), 7.54 (d, 3JH,H ¼ 8.1 Hz, 2H;
AreH), 5.63 (d, 2JPNH ¼ 30.0 Hz; 2Hamine), 3.53 (m, 8H), 2.67 (m, 8H).
55.83; H, 7.66; N, 19.16; Found: C, 55.80; H, 7.65; N, 19.20%. 1H NMR
(500.13 MHz, d6-DMSO, ppm):
d
¼ 3.71 (m, 8H), 3.05 (m, 8H). 13C
13C NMR (125.77 MHz, d6-DMSO, ppm):
66.13 (s), 128.40, 129.81, 133.43, 132.61 (d, JP,C ¼ 8.6 Hz; Cipso),
d
¼ 57.97 (d, 3JP,C ¼ 4.6 Hz),
4
NMR (125.77 MHz, d6-DMSO, ppm):
d
¼ 49.87, 65.31. IR (KBr,
n,
cmꢀ1): 2960, 1448, 1264, 1108, 1085, 985, 857.
136.91, 167.00. IR (KBr, n
, cmꢀ1): 3331 (w, br, NeH), 3265 (m,
CeHaromatic), 1651 (vs, C]O), 1432 (m, C]C), 1396 (m, CeNamide),
6.16. Dimethyl phenyl phosphonic acid (1d)
Table 8
Yield: 68%. Anal. Calc. for C8H11O3P (186.23): C, 51.55; H, 5.91;
Found: C, 51.50; H, 5.95%. 31P NMR (202.46 MHz, d6-DMSO, ppm):
The enzymatic data for hBChE and candidate compounds.
Compound
IC50 (mM)
log P
Km (mM)
Vm (mM/min)
Inhibition
mechanism
d
¼ 22.29 (s). 1H NMR (500.13 MHz, d6-DMSO, ppm):
d
¼ 7.75 (d,
3JP,H ¼ 6.5 Hz, 2H; AreH), 7.51 (m, 1H; AreH), 7.43 (m, 2H), 3.71 (d,
3JP,H ¼ 10.85 Hz, 6H; OCH3). 13C NMR (125.77 MHz, d6-DMSO, ppm):
Enzyme
1a
3a
7a
11a
12a
e
e
0.11
40.32
71.43
66.38
23.49
55.56
27.78
e
2.45
1.62
2.14
0.43
1.58
ꢀ1.12
ꢀ0.54
ꢀ0.78
1.35
1.29
0.067
0.085
0.050
0.075
0.067
Mixed
Mixed
Mixed
Mixed
Mixed
d
¼ 52.48 (d, 2JP,C ¼ 5.0 Hz), 127.83 (d, 1JP,C ¼ 189.0 Hz; Cipso), 128.39
(d, 2JP,C ¼ 15.1 Hz), 131.70 (d, 3JP,C ¼ 8.8 Hz), 132.48. IR (KBr,
n
, cmꢀ1):
3365 (m, CeHaromatic), 1436 (m, PePh), 1178 (s, P]O), 1136 (s, CeO),
791 (s, PeO).

K. Gholivand et al. / European Journal of Medicinal Chemistry 45 (2010) 5130e5139
5139
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Acknowledgement
We wish to thank the Research Council of Tarbiat Modares
University for the financial support of this work.
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