Synthesis, characterization, molecular modeling and biological activity against Artemia salina of new symmetrical bisphosphoramidates

Article abstract of DOI:10.1080/10426500490257122

A series of N,N′-bis(dialkylphosphoryl)diamines were prepared by Todd-Atherton reaction of dialkylphosphites with symmetrical diamines in a biphasic system. They were characterized by IR, ¹ H-NMR, ¹³C-NMR and mass spectroscopy. Compounds with butoxy groups, isobutoxy groups and isopropoxy groups on the phosphorus atoms showed the lowest LD₅₀ values when tested against Artemia salina. All other compounds that showed LD₅₀ values higher than 363 ppm are considered non toxic. The results of a molecular modeling study suggest that the biological activity of the compounds may be related to AChE inhibition. Contrary to classical organophophorus AChE inhibitors, the compounds synthestized in this study do not possess a good leaving group, which suggests that they may act only as reversible inhibitors.

Full text of DOI:10.1080/10426500490257122

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SYNTHESIS, CHARACTERIZATION, MOLECULAR
MODELING AND BIOLOGICAL ACTIVITY AGAINST
ARTEMIA SALINA OF NEW SYMMETRICAL
BISPHOSPHORAMIDATES
Viviane Martins Rebello dos Santos a , João Batista Neves DaCosta a , Carlos Mauricio R.
Sant'Anna a & Marcia Cristina Campos Oliveira a
a Departamento de Qu'imica, Universidade Federal Rural do Rio de Janeiro, Seropédica,
Brazil
Published online: 11 Aug 2010.
To cite this article: Viviane Martins Rebello dos Santos , João Batista Neves DaCosta , Carlos Mauricio R. Sant'Anna & Marcia
Cristina Campos Oliveira (2004): SYNTHESIS, CHARACTERIZATION, MOLECULAR MODELING AND BIOLOGICAL ACTIVITY AGAINST
ARTEMIA SALINA OF NEW SYMMETRICAL BISPHOSPHORAMIDATES, Phosphorus, Sulfur, and Silicon and the Related Elements,
179:1, 173-184
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Phosphorus, Sulfur, and Silicon, 179:173–184, 2004
ꢀC
Copyright Taylor & Francis Inc.
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500490257122
SYNTHESIS, CHARACTERIZATION, MOLECULAR
MODELING AND BIOLOGICAL ACTIVITY AGAINST
ARTEMIA SALINA OF NEW SYMMETRICAL
BISPHOSPHORAMIDATES
Viviane Martins Rebello dos Santos, Joa˜o Batista Neves
DaCosta,1 Carlos Mauricio R. Sant’Anna
and Marcia Cristina Campos de Oliveira
Departamento de Qu´ımica, Universidade Federal Rural do Rio
de Janeiro, Serope´dica, Brazil
(Received February 6, 2003; accepted July 26, 2003)
A series of N,N ꢁ-bis(dialkylphosphoryl)diamines were prepared by
Todd-Atherton reaction of dialkylphosphites with symmetrical di-
amines in a biphasic system. They were characterized by IR, 1 H-NMR,
13C-NMR and mass spectroscopy. Compounds with butoxy groups, isob-
utoxy groups and isopropoxy groups on the phosphorus atoms showed
the lowest LD50 values when tested against Artemia salina. All other
compounds that showed LD50 values higher than 363 ppm are consid-
ered non toxic. The results of a molecular modeling study suggest that
the biological activity of the compounds may be related to AChE in-
hibition. Contrary to classical organophophorus AChE inhibitors, the
compounds synthestized in this study do not possess a good leaving
group, which suggests that they may act only as reversible inhibitors.
Keywords: Artemia salina; biological activity; bisphosphoramidates;
organophosphorus compounds
Various organophosphorus compounds have been widely used as
pesticides,1 herbicides, nematicides and fungicides.2 Their mode of
action is well known and involves inhibition of acetylcholinesterase
(AChE).3–6
Organophosphorus compounds in general have low residual action,
low stability in the environment and limited accumulation in living
We thank the Brazilian agencies CAPES, Faperj, and CNPq for financial support.
Address correspondence to Joa˜o Batista Neves DaCosta, Departamento de Qu´ımica,
ICE, Universidade Federal Rural do Rio de Janeiro, Km 47, BR 465—Serope´dica - RJ
23890-000, Brazil. E-mail: dacosta@ufrrj.br
173

174
V. M. Rebello dos Santos et al.
FIGURE 1 The new synthesized bisphosphoramidates.
organisms such that 80% to 90% of these compounds are eliminated
after 48 h of contact. An increasing number of research projects have
been reported which aim at developing weaker mammal AChE in-
hibitors and such is the case of the phosphoramidates synthesized by
Mavrommatis et al.7,8
The mechanism of inhibition of AChE by organophosphorus com-
pounds generally involves the replacement of a good leaving group
bound to P atom by nucleophilic adition of a serine residue in the enzyme
active site. The aim of this work is to investigate alternative structures
which do not follow this mechanism of action.
This work describes the synthesis and characterization of twenty
phosphoramidates (Figure 1) along with the evaluation of their biologi-
cal activity employing the Artemia salina. lethality assay. The sensitiv-
ity of simple organisms such as Artemia salina Leach provides as fast
and simple way to monitor biological response.9–11 The results may be
easily analyzed statistically. This assay allows the evaluation of the gen-
eral toxicity and for this reason it is essential as a preliminary bioassay
for the investigation of compounds with potential pesticide activity.12,13
In order to investigate the possible action of the bisphosphoramidates
as AChE inhibitors, the enthalpy of interaction between compound 3
(the most active) and a model of the active site of the enzyme14–16 was
calculated employing a semiempirical molecular modeling method. The
choice of this method was based on the fact that it allows the evaluation
of systems containing a high number of atoms, in the order of 102, within
the quantum approximation.
RESULTS AND DISCUSSION
Bisphosphoramidates are compounds derived from phosphoramidic
acid ((HO)2P(O)NH2). Their preparation can be achieved in two steps.

Synthesis, Characterization, Molecular Modeling
175
The first one involves the synthesis of dialkylphosphites, which is car-
ried out by the reaction of (3 mols) of the appropriate alcohol with (1 mol)
of PCl3 to form the trialkyl phosphite which in turn suffers protonation
and is finally attacked by a chloride ion furnishing the corresponding
phosphites.17–19
The second step involves the reaction of (2 mols) of the dialkylpho-
posphites with (1 mol) of the symmetrical diamines in a biphasic system
with carbon tetrachloride and basic water/ethanol, following the Todd-
Atherton20 reaction (Scheme 1). Water and ethanol work as solvents
while carbon tetrachloride is the electrophile.21
SCHEME 1 Synthesis of N,Nꢁ-bis(dialkylphopshoryl)diamines.
Following the syntheses and characterization of the N,N ꢁ-
bis(dialkylphosphoryl)diamines, the biological activity towards
Artemia salina was evaluated and the corresponding LD50 values are
shown in (Table I).
The LD50 is lower for compounds with a butoxy group (3, 8, 13 and
18) followed by compounds with a isobutoxy group (5, 10, 15 and 20)
and finally those compounds with an isopropoxy group (4, 9 and 19).
TABLE I Values of LD50 of N,Nꢁ-Bis
(dialkylphosphoryl)diamines
Compounds
aLD50
Compounds aLD50
1
2
3
4
5
6
7
8
9
>363
>363
40
11
12
13
14
15
16
17
18
19
20
>363
>363
154
>363
160
>363
>363
50
108
55.3
>363
>363
80
113
100
111
85
10
aLD50 in ppm.

176
V. M. Rebello dos Santos et al.
GRAPH 1 Compounds with the lowest LD50 values.
The remaining compounds showed LD50 values higher than 363 ppm
and are considered non toxic. Graph 1 above shows only compounds
with the lowest LD50 values.
Molecular modeling can help better understand the higher toxicity
of compound 3. According to the mechanism described in the literature,
the H atom of Ser238 hydroxy group has been transferred to one of the N
atoms of the His480 side chain, so as to increase serine nucleophilicity.
The initial geometry of the adduct was built locating the phophoroami-
date P atom in the vicinity of the negatively charged O atom of Ser 238.
After optimization, the adduct’s structure is shown to be 20 kcal/mol
more stable than those of compound 3 and the active site separated but
also optimized (Table II), which indicates that this bisphosphoramidate
may effectively bind to the active site of the enzyme.
Besides the expected participation of the residues of the oxyanion
hole, hydrophobic interactions inside the active site of the enzyme
with butyl groups of compound 3 must also be important, along with
TABLE II Calculated ꢀHf (PM3 Method)
for Bisphosphoramidate 3, for the Enzyme
Active Site and for the Interaction of
Compound 3 with the Active Site
Structure
ꢀHf (Kcal/mol)
3
−401,96
−961,06
−1383,05
DmAChE
DmAChE/3 adduct

Synthesis, Characterization, Molecular Modeling
177
FIGURE 2 Representation of the Dm AchE/3 adduct after optimization (PM3
method). H atoms attached to carbons can be omitted for clarity.
a hydrogen bond between the Gly150 N atom and the NH group of
the phosphoroamidate moiety which undergoes nucleophilic addition
of Ser238 in Figure 2.
EXPERIMENTAL
1. Syntheses of Dialkyl Phosphites
1.1 Dialkyl phophites were prepared by treating tricholride phosphorus
with three molar equivalents of the apropriate alcohol.17–19
2. Syntheses of N,Nꢀ-bis(dialkylphosphoryl)diamines
General procedure: In a 200 ml round flask equipped with an addi-
tion funnel, were placed diamine and NaOH in a 1:2 ratio dissolved

178
V. M. Rebello dos Santos et al.
TABLE III Mass (g) and Volume (ml) of Reagents
Phosphites
Diamines
Compostos
R
V (ml)
R1
R1
V (ml) NaOH (g) CCl4 (ml)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Et
Pr
Bu
2.3
2.5
3.5
-CH2CH2-
-CH2CH2-
-CH2CH2-
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.6
0.5
0.6
0.3
0.2
1.5
0.6
0.6
0.6
0.9
0.4
0.4
0.9
1.0
1.1
0.7
0.6
0.6
0.4
0.2
1.4
0.6
0.5
0.6
0.9
0.3
0.4
0.9
0.8
0.9
0.7
0.4
0.5
0.4
0.8
2.0
1.8
2.0
2.0
0.5
4.0
1.7
1.5
1.7
2.5
0.9
1.1
2.5
2.5
2.5
2.0
1.1
1.5
1.2
2.0
i-Pr 1.1∗ -CH2CH2-
i-Bu 0.9∗ -CH2CH2-
Et
Pr
Bu
4.5
2.4
2.6
-CH2CH2-CH2-
-CH2CH2-CH2-
-CH2CH2-CH2-
i-Pr 2.4∗ -CH2CH2-CH2-
i-Bu 4.6∗ -CH2CH2-CH2-
Et
Pr
Bu
1.0
1.6
4.6
-CH2CH2CH2CH2-
-CH2CH2CH2CH2-
-CH2CH2CH2CH2-
i-Pr 2.0∗ -CH2CH2CH2CH2-
i-Bu 4.1∗ -CH2CH2CH2CH2-
Et
Pr
Bu
2.2
1.6
2.5
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2- 0.7∗
-CH2CH2- 0.4∗
-CH2CH2- 0.5∗
-CH2CH2- 0.4∗
-CH2CH2- 0.9∗
i-Pr 1.1∗ -CH2CH2-
i-Bu 4.1∗ -CH2CH2-
∗Expressed in grams.
in the same volume of distilled water and distilled ethanol (5.0 ml).
The corresponding dialkylphosphite, dissolved in carbon tetrachloride,
was placed in the addition funnel which was 20% excess to ensure com-
plete chlorination of the phosphite. The flask was immersed in an ice
bath and the dropwise addition of the phosphite was carried out with
stirring. The reaction mixture was then left at room temperature for
5 h. Dichloromethane (5.0 ml) and water in equal amounts were then
added to the reaction mixture and the organic layer was separated and
the aqueous layer was washed 3 more times with dichloromethane (30
ml). Finally, the organic extract was dried with magnesium sulfate and
the solvent was evaporated under reduced pressure.
Synthesis of N,N ꢁ-bis(diethylphosphoryl)ethylenediamine (1): (2.0 g,
67%), m.p. 80–82◦C; MS (m/z): 333 (M+1); δH (DMSO) 4.9 (N-H), 3.9 (-
CH2-O, q/d, JPOCH2 7.0 Hz), 1.2 (CH3CH2-O, t,JPOCH2CH3 7.0 Hz) and 2.8
(-HN-CH2-, m); δC (CDCl3) 61.3 (-CH2-O, d), 16.1 (CH3CH2-O, d) and
42.2 (-HN-CH2-, d); νmax/cm−1 3187 (N-H), 1243 (P O), 1037 (P-O), 971
(P-N).
Synthesis of N,N ꢁ-bis(dipropylphosphoryl)ethylenediamine (2): a yel-
lowish oil (2.0 g, 67%); MS (m/z) 389 (M+1); δH (CDCl3) 3.5 (N-H), 4.0

Synthesis, Characterization, Molecular Modeling
179
(-CH2-O, t/d, J
1.0 (CH3CH2CH2-O, t, J
6.7 Hz), 1.7(-CH2CH2-O, m, J
7.0 Hz),
POCH2
POCH2CH2
7.0 Hz) and 3.0 (-HN-CH2-, m); δC
POCH2CH2CH3
(CDCl3) 67.0 (-CH2-O, d), 23.0(-CH2CH2-O, d), 9.4 (CH3CH2CH2-O, d)
and 42.1 (-HN-CH2-, d); νmax/cm−1 3191 (N-H), 1240 (P O), 1064 (P-O),
1003 (P-N).
Synthesis of N,N ꢁ-bis(dibutylphosphoryl)ethyelenediamine (3): a col-
orless oil (1.7 g, 43%); MS (m/z) 445 (M+1); δH (CDCl3) 3.8 (N-H), 4.1
(-CH2-O, t/d,J
(-CH2CH2CH2-O, m, J
6.0 Hz), 1.5 (-CH2CH2-O, m, J
6.4 Hz), 1.3
POCH2
POCH2CH2
7.3 Hz), 0.8 (CH3CH2CH2CH2-O,
POCH2CH2CH2
t, J
7.2 Hz) and 2.8 (-HN-CH2-); δC (CDCl3) 65.4
POCH2CH2CH2CH3
(-CH2-O, d), 31.9 (-CH2CH2-O, d), 18.4 (-CH2CH2CH2-O, d), 13.2
(CH3CH2CH2CH2-O, d) and 42.3 (-HN-CH2-, d); νmax/cm−1 3243 (N-H),
1234 (P O), 1066 (P-O), 984 (P-N).
Synthesis of N,N ꢁ-bis(diisopropylphosphoryl)ethylenediamine (4): a
colorless oil (1.0 g, 79%); MS (m/z) 389 (M+1); δH (DMSO) 4.8 (N-H), 4.4
(-CH-O, m, J
6.2 Hz), 1.2 ((CH3)2CH-O, d, J
6.1 Hz) and
POCH
POCH(CH3)2
2.7 (-HN-CH2-, m); δC (CDCl3) 70.2 (-CH-O, d), 23.7 ((CH3)2CH-O, d)
and 44.4 (-HN-CH2-, d); νmax/cm−1 3242 (N-H), 1236 (P O), 1109 (P-O),
991 (P-N).
Synthesis of N,N ꢁ-bis(diisobutylphosphoryl)ethylenediamine (5): a
yellowish oil (0.9 g, 85%); MS (m/z) 445 (M+1); δH (DMSO) 4.9 (N-H),
3.6 (-CH2-O, d/d, J
6.5 Hz), 0.9 ((CH3)2CHCH2-O), d, J
6.4 Hz), 1.9 ((CH3)2CHCH2-O, m, J
POCH2CH
POCH2
6.5 Hz) and 2.8 (-HN-
POCH2CH(CH3)2
CH2-, m); δC (CDCl3) 71.3 (-CH2-O, d), 28.2 ((CH3)2CHCH2-O, d), 17.9
((CH3)2CHCH2-O, d) and 42.1 (-HN-CH2-, d); νmax/cm−1 3237 (N-H),
1232 (P O), 1020 (P-O), 961 (P-N).
Synthesis of N,N ꢁ-bis(diethylphosphoryl)1,3-propylenediamine (6): a
yellowish oil (2.2 g, 36%); MS (m/z) 347 (M+1); δH (CDCl3) 4.0 (N-H), 3.3
(-CH2-O, q/d, J
7.3 Hz), 1.2 (CH3CH2-O, t, J
6.3 Hz), 2.8
POCH2
POCH2CH3
(-HN-CH2-, m) and 1.6 (-HN-CH2-CH2-, m); δC (CDCl3) 66.5 (-CH2-O,
d), 15.9 (CH3CH2-O, d), 38.3 (-HN-CH2-, d) and 32.5 (-HN-CH2-CH2-,
d); νmax/cm−1 3240 (N-H), 1232 (P O), 1020 (P-O), 966 (P-N).
Synthesis of N,N ꢁ-bis(dipropylphosphoryl)1,3-propylenediamine (7):
a colorless oil (1.8 g, 60%); MS (m/z) 402 (M+1); δH (DMSO) 4.8 (N-H),
3.8 (-CH2-O, t/d, JPOCH2 6.8 Hz), 1.6 (-CH2CH2-O, m, JPOCH2CH2 7.0 Hz),
0.9 (CH3CH2CH2-O, t, JPOCH2CH2CH3 6.7 Hz), 2.8 (-HN-CH2-, m) and 1.6
(-HN-CH2-CH2-, m); δC (CDCl3) 66.5 (-CH2-O, d), 33.2 (-CH2CH2-O, d),
9.9 (CH3CH2CH2-O, d), 38.3 (-HN-CH2-, d) and 23.1 (-HN-CH2-CH2,
d); νmax/cm−1 3239 (N-H), 1234 (P O), 1108 (P-O), 990 (P-N).
Synthesis of N,N ꢁ-bis(dibutylphosphoryl)1,3-propylenediamine (8): a
colorless oil (2.4 g, 80%); MS (m/z) 459 (M+1); δH (CDCl3) 3.5 (N-H), 3.9
(-CH2-O, t/d, JPOCH2 6.2 Hz), 1.5 (-CH2CH2-O, m, JPOCH2CH2 6.2 Hz), 1.3
(-CH2CH2CH2-O, m, J
7.5 Hz), 0.8 (CH3CH2CH2CH2-O, t,
POCH2CH2CH2

180
V. M. Rebello dos Santos et al.
JPOCH2CH2CH2CH3 7.5 Hz), 2.9 (HN-CH2-, m) and 1.5 (HN-CH2-CH2-, m);
δC (CDCl3) 65.1 (-CH2-O, d), 31.5(-CH2CH2-O, d), 17.9 (-CH2CH2CH2-
O), 12.7 (CH3CH2CH2CH2-O, d), 37.0 (-HN-CH2-, d) and 31.5 (-HN-
CH2-CH2, d); νmax/cm−1 3243 (N-H), 1233 (P O), 1067 (P-O), 984 (P-N).
Synthesis of N,N ꢁ-bis(diisopropylphosphoryl)1,3-propylenediamine
(9): a colorless oil (2.0 g, 70%); MS (m/z) 404 (M+1); δH (CDCl3) 3.2
(N-H), 4.6 (-CH-O, m, J
6.1 Hz), 1.3 ((CH3)2CH-O, d, J
POCH
POCH(CH3)2
6.3 Hz), 3.0 (-HN-CH2-, m) and 1.6 (-HN-CH2-CH2-, m); δC (CDCl3)
70.5 (-CH-O, d), 23.7 ((CH3)2CH-O, d), 32.7 (-HN-CH2-, d) and 34.5
(-HN-CH2-CH2-, d); νmax/cm−1 3238 (N-H), 1233 (P O), 1063 (P-O), 999
(P-N).
Synthesis of N,N ꢁ-bis(diisobutylphosphoryl)1,3-propylenediamine
(10): a colorless oil (4.7 g, 87%); MS (m/z) 459 (M+1); δH (DMSO)
4.8 (N-H), 3.6 (-CH2-O, d/d, J
6.4 Hz), 1.8 ((CH3)2CHCH2-O,
POCH2
m, J
6.6 Hz), 0.9 ((CH3)2CHCH2-O, d, J
POCH2CH(CH3)2
POCH2CH(CH3)2
5.5 Hz), 2.8 (-HN-CH2-, m) and 1.5 (-HN-CH2-CH2-, m); δC (CDCl3) 72.0
(-CH-O, d), 28.8 ((CH3)2CHCH2-O, d), 19.8 ((CH3)2CHCH2-O, d), 37.8
(-HN-CH2-, d) and 32.8 (-HN-CH2-CH2-,d); νmax/cm−1 3237 (N-H), 1235
(P O), 1019 (P-O), 960 (P-N).
Synthesis of N,N ꢁ-bis(diethylphosphoryl)1,4-butylenediamine (11): a
yellowish oil (0.4 g, 28%); MS (m/z) 362 (M+2); δH (CDCl3) 2.6 (N-H), 4.0
(-CH2-O, q/d, J
7.3 Hz), 1.3 (CH3CH2-O, t, J
7.1 Hz), 2.9
POCH2
POCH2CH3
(-HN-CH2-, m) and 1.6 (-HN-CH2-CH2-, m); δC (CDCl3) 61.6 (-CH2-O,
d), 15.6 (CH3CH2-O, d), 40.4 (-HN-CH2-, d) and 28.1 (-HN-CH2-CH2-,
d); νmax/cm−1 3260 (N-H), 1228 (P O), 1032 (P-O), 967 (P-N).
Synthesis of N,N ꢁ-bis(dipropylphosphoryl)1,4-butylenediamine (12):
a yellowish oil (0.5 g, 25%); MS (m/z) 417 (M+1); δH (CDCl3) 2.4 (N-H),
4.5 (-CH2-O, t/d, JPOCH2 7.1 Hz), 1.2 (-CH2CH2-O, m, JPOCH2CH2 6.5 Hz),
0.9 (CH3CH2CH2-O, t, JPOCH2CH2CH3 6.5 Hz), 2.8 (-HN-CH2-, m) and 1.5
(-HN-CH2-CH2-, m); δC (CDCl3) 70.8 (-CH2-O, d), 30.6 (-CH2CH2-O, d),
20.4 (CH3CH2CH2-O, d), 39.6 (-HN-CH2-, d) and 34.6 (-HN-CH2-CH2,
d); νmax/cm−1 3425 (N-H), 1220 (P O), 1040 (P-O), 970 (P-N).
Synthesis of N,N ꢁ-bis(dibutylphosphoryl)1,4-butylenediamine (13): a
colorless oil (3.2 g, 57%); MS (m/z) 474 (M+2); δH (CDCl3) 3.2 (N-H), 3.9
(-CH2-O, t/d, JPOCH2 6.1 Hz), 1.5 (-CH2CH2-O, m, JPOCH2CH2 6.4 Hz), 1.4
(-CH2CH2CH2-O, m, J
6.3 Hz), 0.9 (CH3CH2CH2CH2-O, t,
POCH2CH2CH2
JPOCH2CH2CH2CH3 6.0 Hz), 2.8 (-HN-CH2-, m) and 1.4 (HN-CH2-CH2-, m);
δC (CDCl3) 66.0 (-CH2-O, d), 28.9 (-CH2CH2-O, d), 17.7 (-CH2CH2CH2-
O), 13.8 (CH3CH2CH2CH2-O, d), 41.1 (-HN-CH2-, d) and 32.5 (-HN-
CH2-CH2, d); νmax/cm−1 3342 (N-H), 1232 (P O), 1067 (P-O), 983
(P-N).
Synthesis of N,N ꢁ-bis(diisopropylphosphoryl)1,4-butylenediamine
(14): (1.4 g, 57%), m.p. 75–77◦C; MS (m/z) 417 (M+1); δH (CDCl3) 2.5

Synthesis, Characterization, Molecular Modeling
181
(N-H), 4.6 (-CH-O, m, JPOCH 6.2 Hz), 1.3 ((CH3)2CH-O, d, JPOCH(CH3)2 6.2
Hz), 2.9 (-HN-CH2-) and 1.5 (-HN-CH2-CH2-); δC (CDCl3) 70.2 (-CH-O,
d), 23.6 ((CH3)2CH-O, d), 40.9 (-HN-CH2-, d) and 28.5 (-HN-CH2-CH2-,
d); νmax/cm−1 3227 (N-H), 1234 (P O), 1107 (P-O), 1007 (P-N).
Synthesis of N,N ꢁ-bis(diisobutylphosphoryl)1,4-butylenediamine
(15): a colorless oil (2.9 g, 58%); MS (m/z) 471 (M+1); δH (DMSO) 4.8
(N-H), 3.6 (-CH2-O, d/d, J
6.0 Hz), 1.8 ((CH3)2CHCH2-O, m,
POCH2
J
6.5 Hz), 0.9 ((CH3)2CHCH2-O, d, J
6.5 Hz),
POCH2CH(CH3)2
POCH2CH(CH3)2
2.7 (-HN-CH2-, m) and 1.4 (-HN-CH2-CH2-,m); δC 71.7 (-CH-O, d), 29.2
((CH3)2CHCH2-O, d), 19.2 ((CH3)2CHCH2-O, d), 40.6 (-HN-CH2-, d)
and 29.2 (-HN-CH2-CH2-); νmax/cm−1 3400 (N-H), 1232 (P O), 1019
(P-O), 960 (P-N).
Synthesis of N,N ꢁ-bis(diethylphosphoryl)piperazine (16): a yellowish
oil, (2.5 g, 82%); MS (m/z) 360 (M+1); δH (CDCl3) 3.5 (-CH2-O, q/d, J
POCH2
7.4 Hz), 0.8 (CH3CH2-O, t, JPOCH2CH3 6.4 Hz) and 2.8 (-HN-CH2-, m); δC
60.2 (-CH2-O, d), 13.1 (CH3CH2-O, d) and 41.2 (-HN-CH2-, d); νmax/cm−1
1248 (P O), 1030 (P-O), 974 (P-N).
Synthesis of N,N ꢁ-bis(dipropylphosphoryl)piperazine (17): a yellow-
ish oil, (1.0 g, 50%); MS (m/z) 415 (M+1); δH (CDCl3) 3.8 (-CH2-O, q/d,
J
m, J
6.5 Hz), 0.9 (CH3CH2-O, t, J
6.9 Hz), 1.6 (-CH2CH2-O,
POCH2
POCH2CH2
6.8 Hz), 3.0 (-HN-CH2-, m); δC (CDCl3) 68.4 (-CH2-O,
POCH2CH2CH3
d), 24.1 (-CH2CH2-O, d), 10.5 (CH3CH2CH2-O, d) and 45.0 (-HN-CH2-,
d); νmax/cm−1 1250 (P O), 1062 (P-O), 989 (P-N).
Synthesis of N,N ꢁ-bis(dibutylphosphoryl)piperazine (18): a colorless
oil, (1.2 g, 40%); MS (m/z) 472 (M+2); δH (CDCl3) 3.9 (-CH2-O, t/d, J
POCH2
6.6 Hz), 1.6 (-CH2CH2-O, m, J
6.3 Hz), 1.3 (-CH2CH2CH2-O,
POCH2CH2
m, J
6.9 Hz), 0.9 (CH3CH2CH2CH2-O, t, JPOCH2CH2CH2CH3
POCH2CH2CH2
6.6 Hz) and 3.1 (-HN-CH2-, m); δC (CDCl3) 65.4 (-CH2-O, d), 31.7
(-CH2CH2-O,d), 18.2 (-CH2CH2CH2-O,d), 13.1 (CH3CH2CH2CH2-O,
d) and 45.3 (-HN-CH2-, m); νmax/cm−1 1249 (P O), 1026 (P-O), 979
(P-N).
Synthesis of N,N ꢁ-bis(diisopropylphosphoryl)piperazine (19): a col-
orless oil, (1.1 g, 78%); MS (m/z) 415 (M+1); δH (CDCl3) 4.5 (-CH-O, m,
J
POCH
6.3 Hz), 1.2 ((CH3)2CH-O, d, J
6.1 Hz) and 3.0 (-HN-
POCH(CH3)2
CH2-, m); δC (CDCl3) 71.9 (-CH-O, d), 25.1 ((CH3)2CH-O, d) and 46.1
(-HN-CH2-,d); νmax/cm−1 1250 (P O), 1106 (P-O), 977 (P-N).
Synthesis of N,N ꢁ-bis(diisobutylphosphoryl)piperazine (20): (2.9 g,
58%), mp 45◦C; MS (m/z) 471 (M+1); δH (CDCl3) 3.7 (-CH2-O, d/d,
J
6.4 Hz), 1.9 ((CH3)2CHCH2-O, m, J
6.4 Hz), 0.9
POCH2
POCH2CH(CH3)2
((CH3)2CHCH2-O, d, J
6.4 Hz) and 3.1 (-HN-CH2-, m); δC
POCH2CH(CH3)2
(CDCl3) 71.6 (-CH-O, d), 28.8 ((CH3)2CH-O, d) 18.7 ((CH3)2CHCH2-
O, d) and 44.4 (-HN-CH2-, d); νmax/cm−1 1252 (P O), 1052 (P-O), 980
(P-N).

182
V. M. Rebello dos Santos et al.
Lethality Assay with Larvae of Artemia salina Leach22–25
General Procedure: The Artemia salina eggs were left in artificial sea
water for 48 h in order for them to eclode.26–29 The larvae were collected
with a Pasteur pipette in such a way that 10 larvae were present in
test tubes already containing saline solution (5 ml). A stock solution
was prepared from the various N,Nꢁ-bis(dialkylphosphoryl)diamines
(20 mg) dissolved in DMSO (2 ml) and the volume was made up to 10 ml
with water. Each assay was carried out in quadruplicate by adding vol-
umes of the stock solution (50, 100, 200, 300 and 500 µl) to the test
tubes.
Blank assays were carried out as controls. The tubes were kept under
light and the live and dead organisms counted after 24 h. Employing
the Origin 6.0 software, the LD50 of the different phosphoroamidates
was determined by plotting the percentage of live animals against log
dose.
Compounds with butyl, isopropyl and isobutyl groups have LD50 val-
ues in the 40–160 ppm range while the others showed LD50 values
higher than 360 ppm. A new assay with the most active compounds
was carried out with volumes of the stock solution (10, 20, 50, 100 and
200 µl), in order to calculate the more accurate LD50 values. Compounds
with LD50 values equal or above 360 ppm were considered non toxic to
Artemia salina.
Molecular Modelling30,31
The model used in this study (DmAChE) was built by selecting
16 aminoacid residues (together with 5 molecules of water) from the
active site of the only AChE crystalline structure available in the Pro-
tein Data Bank (PDB) from the fruit fly, Drosophila melanogaster.
Details on the building of this model will be published elsewhere.
All H and side chain atoms were kept free during the structural
optimization, while the peptide bond atoms were fixed in space to
avoid great movements during the optimization process. We assumed
that only one of the phosphoroamidate groups of 3 interacts with
Ser238 of the active site (which corresponds to Ser200 of Torpedo
californica AChE). Thus, the butyl groups of the other phospho-
roamidate moiety were substituted for methyls to simplify calcula-
tions. These were made by employing the PM3 Hamiltonian from
the Mopac 6.0 software on either a Pentium II 333 MHz or a Pen-
tium III 1.1 GHz. There are some recent 16 examples of the usage of
PM3 in the calculation of reaction mechanisms involving hypervalent
phosphorus.32,33

Synthesis, Characterization, Molecular Modeling
183
CONCLUSION
The phosphorylation of diamines with alkyl phosphites is a simple and
efficient reaction which affords products in moderate yields. The use of
the Artemia salina Leach bioassay as a preliminary test for the general
toxicity of these compounds showed interesting results and warrants
further toxicity assays against Musca domestica and Stomoxys calci-
trans, especially with those compounds with lower LD50 values. The
results of the molecular modelling experiments suggest that the tox-
icity of compound 3 may be related to AChE inhibition. Contrary to
organophosphorus compounds which act as classical AChE inhibitors,
the structures in this study do not possess a good leaving group associ-
ated with the P atom, which in turn suggests that they may act only as
reversible inhibitors of the enzyme. However, the LD50 for compound
3, in the nanomolar range, suggests that it may bind to the active site
for a longer period, acting as an inhibitor which slowly dissociates from
AChE. The modeling results indicate that a hydrogen bond between
the N atom of AChE Gly150 and the NH group of the fosforamidate
moiety, together with hydrophobic interactions with the butyl groups
of compound 3 may contribute to the adduct stability.
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