Synthesis and bioactivity of new phosphorylated R,R′-substituted sulfoximines

Article abstract of DOI:10.3390/10111369

R,R′-disubstituted sulfoximines were phosphorylated with O,O-diethylchloro phosphate and phosphorothionate to obtain new organophosphorus compounds. After purification they were characterized by GC-MS and ¹H-NMR. The toxicity of the synthesized

Full text of DOI:10.3390/10111369

Molecules 2005, 10, 1369-1376
molecules
ISSN 1420-3049
http://www.mdpi.org
Synthesis and Bioactivity of New Phosphorylated
R,R’-substituted Sulfoximines
Monica Bellozas Reinhard 1 and Susana Arnstein de Licastro 2,*
1
Departamento de Química – FCEyN – Univ. Nac. La Pampa - Santa Rosa – Pcia. La Pampa –
Argentina – E-mail: bellozas@exactas.unlpam.edu.ar
2
Centro de Investigaciones de Plagas e Insecticidas (CITEFA-CONICET) – J.B.La Salle 4397 – V.
Martelli – 1603 – Buenos Aires – Argentina
* Author to whom correspondence should be addressed; E-mail: slicastro@citefa.gov.ar
Received: 20 January 2005; in revised form: 24 January 2005 / Accepted: 14 March 2005 / Published:
30 November 2005
Abstract: R,R’-disubstituted sulfoximines were phosphorylated with O,O–diethylchloro
phosphate and phosphorothionate to obtain new organophosphorus compounds. After
1
purification they were characterized by GC-MS and H-NMR. The toxicity of the
synthesized O,O-diethyl N-(R,R’-disubstituted sulfoximine) phosphoro-amidothionates
was assayed on Musca domestica. It was found that the methyl phenyl derivative was the
most toxic compound, followed by the dipropyl and dibutyl derivatives. The dihexyl
compound was the less toxic of all the assayed compounds, being one hundred times less
toxic than a paraoxon standard The anticholinesterasic activity of the corresponding
phosphoramidates was assayed on homogenates of house flies’ heads, giving values
similar to paraoxon for the methyl phenyl derivative.
Keywords: Disubstituted sulfoximines; phosphoroamidates; phosphoroamidothionates¸
inscticidal activity, toxicity; Musca domestica, house fly acetylcholinesterase
Introduction
Phosphoramidates and amidothionates, insecticidal compounds with P-N bonds, are very
interesting compounds from a toxicological point of view [1,2]. On the other hand, sulfoximines were
discovered by Bentley in 1950 [3] and are considered as the nitrogen analogues of sulfones. They

Molecules 2005, 10
1370
share some of their properties such as the acidic α-hydrogen but the nitrogen atom provides an
additional place for structural modifications by nucleophilic reactions [4].
In the literature some patents can be found for new substituted sulfoximines for use as herbicides,
detergents, additives, bacterial and antifungal compounds, but there is very little information on
phosphorylated sulfoximines to be used as insecticides.
Organophosphorus insecticides are neurotoxicants acting by inhibition of acetylcholinesterase,
phosphorylating the ester site of the enzyme. It is known that this process depends largely on the
structure of the organophosphorus compound, namely its steric or electronic configuration and its
hydrophobic properties to reach the target site [5-6]
In our search for new insecticides, a series of O,O-diethyl N-(R,R’-disubstituted sulfoximine)
phosphoroamidates and phosphoro amidothionates (R,R’ = dipropyl, dibutyl, dihexyl and methyl-
phenyl, Figure 1) were synthesized by phosphorylation on the nitrogen atom of R,R’disubstituted
sulfoximines and their anticholinesterase activity studied on house fly head acetylcholinesterase.
Toxicity of the synthesized compounds was evaluated by topical application on Musca domestica (L.).
Thus, the present study was aimed at synthesis, structural elucidation and reactivity of the novel
compounds depicted in Figure 1.
Figure 1
O
S
O(S)
C2H5O
C2H5O
R
R
P
N
R,R’: • CH (CH ) - • CH (CH ) - • CH (CH ) - • CH , Ph
2 2 2 3 2 5
3
3
3
3
Results and Discussion
Chemistry
R,R’-substituted sulfoxides were chosen as the starting material for these studies. In order to obtain
the substituted sulfoximines the corresponding sulfoxides were treated in an ice bath with sodium
azide in the presence of sulfuric acid, according to Bentley et al. [7]. Dipropyl, dibutyl, dihexyl and
methylphenyl sulfoximines were thus synthesized according to Scheme 1.
Scheme 1. Synthesis of R,R’ disubstituted sulfoximines
O
O
O
-
N2
H2SO4
CHCl3
H2N N+2
+
R2S
R S R`
NH
+
NaN3
`
R S R
- H+
1
2
R,R’: • a CH (CH ) - • b CH (CH ) - • c CH (CH ) - • d CH , Ph
2 2 2 3 2 5
3
3
3
3

Molecules 2005, 10
1371
The oily products thus obtained were purified by column chromatography with 7:3 (v/v) hexane -
acetone as the elution solvent. The products were pure by TLC. The structures of all synthesized
1
compounds were confirmed by spectral data: H-NMR (complete data to be published elsewhere [8])
and GC-MS spectroscopy.
Results of the fragmentation analysis indicate that the parent peaks of all sulfoximines studied are
observed clearly at the corresponding expected m/e values The fragments obtained for aliphatic
sulfoximines depend on the length of the chain and are markedly different from the ones obtained for
aryl methyl sulfoximine, which is in accordance with the fragmentation observed by Oae et al. [9].
1
By H-NMR the chemical shifts measured showed the different -CH3 and -CH2 protons clearly
identified. The protons adjacent to the sulfoximine group appeared to shift towards a lower magnetic
field compared to a sulfone group [9] The imino proton moves towards higher fields as the length of
the chain increases. For the methyl phenyl sulfoximine the imino proton is found at lower field
probably due to the strong influence of the aromatic ring.
The synthesized sulfoximines were converted into the corresponding phosphoramidates or
phosphoroamidothionates by reaction with O,O-diethylchlorophosphate or O,O-diethylchlorothio-
phosphate according to Wieczorkowski’s method [10], as seen in Scheme 2.
Scheme 2. Phosphorylation reaction
O(S)
O(S)
O
S
(C2H5)3N
NH
O
C2H5O
C2H5O
C2H5O
C2H5O
R
R
R
P
Cl
P
N
S
2
+
+
HCl
R
3
4/5
They were obtained by a nucleophilic substitution of the O,O-diethylchlorophosphate or
O,O-diethylchlorophosphorothionate on the nitrogen atom of the R,R’-substituted sulfoximines.
Triethylamine was used as a catalyst. The reaction was performed in chloroform solution and in an ice
bath, since it is exothermic. The side products increase and the yield decreases if the temperature
increases. The obtained oily products were purified by column chromatography. The structures of all
synthesized compounds were confirmed by spectral data: 1H-NMR [8] and GC-MS.
1H-NMR for the phosphorylated compounds synthesized gave chemical shifts similar to those of
the corresponding sulfoximines. The peak corresponding to the proton attached to the nitrogen atom
disappeared, while the peaks corresponding to phosphoramidates and phosphoroamidothionates
appeared at similar values.
Mass spectral fragmentation showed the parent peaks of all compounds studied at the
corresponding expected m/e. A difference between phosphoramidates and amidothionates can be
observed in the M+ peak. The fragmentation involving P-N bond depends strongly on the substrate’s
structure, particularly on the nature of the substituents of the sulfoximine moiety. Obtained spectra
showed no P-N bond cleavage in the primary fragmentation reactions, in agreement with the results
found by Wieczorkowski’s group for thionylamidates [11]. The bond P-N in the P(O)-N=S=O system
is unusually strong, despite the electron-withdrawing effect of the N-thionyl group. Mastrantonio [12],

Molecules 2005, 10
1372
when performing molecular modeling of the synthesized compounds, found also that the P-N bond has
a considerable rotation restriction, with π electrons delocalized to the P=O (S) bond.
Toxicity and anticholinesterasic assays
Toxicity of the synthesized O,O-diethyl-N-(R,R’disubstituted sulfoximine) phosphoroamido-
thionates was assayed by topical application on Musca domestica. LD50 values were calculated using a
Micro Probit program (Table 1). The most toxic compound was the methylphenyl derivative with LD50
= 0.5 (0.3 -0.7) µg/insect, followed by the dipropyl and dibutyl ones with similar LD50 values of 1.4
(1.3 – 1.5) µg/insect, while the dihexyl derivative was the less toxic compound (LD50 = 4.9 (4.5-5.4)
µg/insect. It appears that the length of the chain or the spatial distribution of the substituents could
modulate the toxicity of the compounds. It is known that acetylcholinesterase is the target for
organophosphorus compounds [5], thus steric efects could affect the formation of the enzyme-inhibitor
complex. The high toxicity of the methyl-phenyl derivative suggests that some other factor could be
involved, perhaps a better accomodation of the compound in the active site of the acetylcholiesterase.
Table 1. Toxicity of O,O-diethyl N-(R,R’-disubstituted sulfoximine)phosphoroamido-
thionates
LD50
µg/ insect
1.4
Confidence interval
95%
COMPOUND
Dipropyl
Dibutyl
1.3-1.5
1.4
1.2-1.5
Dihexyl
4.9
4.5-5.4
Methyl phenyl
0.5
0.3-0.7
In vitro, the inhibition constants (ki) on house fly acetylholinesterase (HFAChE) were measured
with the corresponding O,O diethyl-N-(R,R’substituted sulfoximine) phosphoramidates. Crude fly
head homogenates were used as the enzyme source. Ellman’s kinetic method [13] was used to measure
inhibition constants (ki) using acetylthiocholine (ATC) as the enzyme substrate and 5,5’-dithiobis 2-
nitrobenzoic acid (DTNB) as an indicator since its anion has a strong absortion at 412 nm. In Table 2,
ki values were given for the four compounds synthesized. Paraoxon was used a reference compound. It
can be seen that phosphoroamidates of R,R’-substituted sulfoximines are as potent inhibitors as
paraoxon with values ranging from 1.3 x 104 to 7.4 x 106 mol-1min-1. Again, the methylphenyl
derivative was the most potent inhibitor for HFAChE with a value similar to paraoxon, a known strong
inhibitor [14], followed by the dipropyl and dibutyl derivatives. For the dihexyl derivative, the less
active compound, a difference of two orders of magnitude was observed.

Molecules 2005, 10
1373
Table 2. Anticholinesterase activity of O,O-diethyl N -(R R’-disubstituted sulfoximine)
phosphoroamidates
Ki
COMPOUND
mol-1min-1
2.9 x 106
1.4 x 106
1.3 x 104
7.4 x 106
1.17 x 106
Dipropyl
Dibutyl
Dihexyl
Methyl phenyl
Paraoxon
Structure-activity studies (QSAR) are being conducted in order to determine the physicochemical
parameters required to explain the relationship between the chemical structure of the compounds and
its anticholiesterasic activity or toxicity.
Conclusions
Biological biassays of O,O-diethyl N- (R,R’ disubstituted sulfoximine) phosphoroamidothionates
performed on Musca domestica revealed a high toxicity of the synthesized compounds, with the
methylphenyl derivative being the most active compound. These results are in agreement with the
results obtained for the anticholiesterasic activity of the corresponding phosphoramidates. The limited
series presented in this work indicate that relatively small substituents such as methylphenyl or
dipropyl are more effective than substituents bearing a long chain such as dihexyl, probably due to a
steric effect of the substituent on the AchE of house flies.
Acknowledgements
We thank the Chemical Department of the National University of La Pampa (Argentine) and the
Centro de Investigaciones de Plagas e Insecticidas (CIPEIN) for the financial support of technical
work. Also we want to thank Dra. P. Gonzalez Audino (CIPEIN) for the support on the realization of
GC-MS spectra
Experimental
General
Some of the sulfoxides used (methylphenyl and dibutyl) were purchased from Aldrich Chemical Co.
(USA) as well as NNa3, O,O-diethyl chlorophosphate and O,O-diethyl chlorophosphorothionate. In
other cases (dipropyl and dihexyl) sulfoxides were synthesised from the corresponding sulfides by
oxidation with sodium periodate [15]. Solvents were analytical grade. GC-MS spectrometry was
performed on a Shimadzu GCMS-QP5050A on which the inlet system was heated to 250ºC,
temperature programmed to 15ºC/min up to 270ºC or 230ºC, column stationary phase was either a HP-
1, 100% dimethylpolysiloxane (50m long x 0.32mm i.d. and film thickness 0.52µm) or a CP-Wax 52

Molecules 2005, 10
1374
CB, 5% diphenyl and 95% dimethylpolysiloxane (30m long x 0.25mm i.d. and 0.25µm film
thickness). The ionization source was electron impact with the detector voltage maintained at 1.25 kV.
1H-NMR was performed at the Chemistry Department (CEQUINOR), La Plata University [8] with a
Bruker AC250-E spectrometer, solvent CCl3D (Aldrich, USA) and TMS 0.03% v/v. Temperature
21ºC. A Shimadzu UV-visible spectrophotometer model UV-160 was used for the anticholinesterase
activity determinations.
Synthesis of R,R’-substituted sulfoximines 2a-d:
Using Whitehead and Bentley’s method [16], Na3N (30 mmol) in CHCl3 (60 ml) was added to
dipropyl, dibutyl, dihexyl or methylphenyl sulfoxide (1a-d, 75 mmol), followed finally by addition
with shaking of H2SO4 (15 mL). Shaking was continued during 5 hrs, keeping the reaction in an ice
bath. The temperature was allowed to rise to 45ºC and stored until the next day. The reaction mixture
was cooled with ice water, neutralized with NaOH and extracted with Cl3CH. The organic layer was
dried over anhydrous Na2SO4 and solvent evaporated under vacuum to obtain compounds 2a-d. The
products were characterized by 1H-NMR [8] and GC-MS
Dipropyl sulfoximine (2a): 1H-NMR: 1.08 (t, 3H, -CH3 ), 1.87-1.90 (m, 2H, -CH2 ), 2.95-2.99 (m, 2H, -
CH2-S), 2.41 (s, 1H, -NH); MS: 150 (M+), 134, 107, 91, 65, 64.
1
Dibutyl sulfoximine (2b): H-NMR: 0.98 (t, 3H, -CH3 ), 1.46-1.54 (m, 2H, -CH2 ), 1.72-1.90 (m, 2H, -
CH2 ), 2.65-2.69 (m, 2H, -CH2-S), 2.53 (s, 1H, -NH); MS: 178 (M+), 148, 120, 104, 65, 64.
1
Dihexyl sulfoximine (2c): H-NMR: 0.89 (t, 3H, -CH3 ), 1.2-1.5 (m, 6H, -(CH2 )3), 1.70-1.90 (m, 2H, -
CH2 ), 3.01-3.20 (m, 2H, -CH2-S), 3.72 (s, 1H, -NH); MS: 233 (M+), 176, 150, 132, 65.
1
Methyl phenyl sulfoximine (2d): H-NMR: 3.15 (s, 3H, -CH3 S), 7.99-8.04 (m, 2H, Ph-Ho), 7.56-7.64
(m, 3H, Ph-Hm,p); MS: 155 (M+), 140, 92, 78, 77, 65
Synthesis of O,O-diethyl-N-(R,R’-disubstituted sulfoximine) phosphoroamidates 4a-d or amido-
thionates 5a-d:
To the appropriate R,R’-substituted sulfoximine 2a-d (0.01 mol) and triethylamine (0.01 mol) in
CHCl3 (6 mL), O,O-diethylchlorphosphate or O,O-diethylchlorthiophosphate (3, 0.01 mol ) in CHCl3
(6 mL) was added dropwise with shaking, maintaining the temperature at 0ºC. After finishing the
addition, the temperature was raised to 64ºC during 15 min, and then left at 40ºC during 20 hs. The
solution was washed with Na2CO3 (5% w/v) and then with water to pH=6-7. The chloroform solution
was dried over Na2SO4 and solvent evaporated under vacuum. The remaining oily product was
purified on a column (50cm x 2 cm) of Silica gel 200 mesh (Merck, Germany) and eluted with
acetone-hexane (7:3, v/v). 10mL fractions were separated, checked by TLC and only the pure fractions
taken for characterization. They were mixed and the solvent evaporated under vacuum. Product
characterization was performed using 1H-NMR [8] and GC-MS.

Molecules 2005, 10
1375
O,O-diethyl-N-(dipropylsulfoximine) phosphoramidate (4a): 1H-NMR: 1.09 (t, 3H, -CH3 sulfoximine),
1.34 (t, 3H, -CH3 ethyl), 3.11-3.22 (m, 2H, -CH2 ethyl), 1.83-1.98 (m, 2H, -CH2 sulfoximine), 4.0-
4.06 (m, 2H, S-CH2); MS: 286 (M+), 258, 242, 230, 214, 200, 150, 144.
1
O,O-diethyl-N-(dibutylsulfoximine) phosphoramidate (4b): H-NMR: 0.99 (t, 3H, -CH3 sulfoximine),
1.30 (t, 3H, -CH3 ethyl), 3.17-3.30 (m, 2H, -CH2 ethyl), 1.44-1.57 (m, 2H, -CH2-), 1.78-1.90 (m, 2H, -
CH2-), 3.98-4.12 (m, 2H, S-CH2); MS: 312 (M+), 284, 268, 258, 256, 229, 200, 172, 144.
1
O,O-diethyl-N-(dihexylsulfoximine) phosphoramidate (4c): H-NMR: 0.89 (t, 3H, -CH3 sulfoximine),
1.32 (t, 3H, -CH3 ethyl), 3.1-3.3 (m, 2H, -CH2 ethyl), 1.35-1.47 (m, 6H, -CH2-)3, 1.78-1.91 (m, 2H, -
CH2), 4.01-4.12 (m, 2H, S-CH2); MS: 369 (M+), 340, 324, 312, 295, 284, 200, 144.
1
O,O-diethyl-N-(methyl phenylsulfoximine) phosphoramidate (4d): H NMR: 3.35 (s, 3H, S-CH3), 1.3
(t, 3H, -CH3 ethyl), 3.9- 4.4 (m, 2H, -CH2 ethyl), 7.5-7.7 (m, 3H, PhHm,p), 7.9-8.15 (m, 2H, PhHo );
MS: 292 (M+), 276, 248, 220, 155, 144.
1
O,O-diethyl-N-(dipropylsulfoximine) phosphoramidothionate (5a): H-NMR: 1.10 (t, 3H, -CH3
sulfoximine), 1.32 (t, 3H, -CH3 ethyl), 3.20-3.40 (m, 2H, -CH2 ethyl), 1.86-1.96 (m, 2H, -CH2
sulfoximine), 4.09-4.13 (m, 2H, S-CH2); MS: 302 (M+), 257, 216, 188.
O,O-diethyl-N-(dibutylsulfoximine) phosphoramidothionate (5b): 1H-NMR: 0.97 (t, 3H, -CH3
sulfoximine), 1.35 (t, 3H, -CH3 ethyl), 3.17-3.25 (m, 2H, -CH2 ethyl), 1.44-1.53 (m, 2H, -CH2-),
1.80-1.88 (m, 2H, -CH2-), 3.9-4.07 (m, 2H, S-CH2); MS: 327 (M+), 284, 216, 188.
O,O-diethyl-N-(dihexylsulfoximine) phosphoramidothionate (5c): 1H-NMR: 0.87 (t, 3H, -CH3
sulfoximine), 1.34 (t, 3H, -CH3 ethyl), 3.2-3.4 (m, 2H, -CH2 ethyl), 1.35-1.47 (m, 6H, -CH2-)3, 1.76-
1.88 (m, 2H, -CH2), 3.9-4.1 (m, 2H, S-CH2); MS: 386 (M+), 340, 216, 188, 160.
O,O-diethyl-N-(methyl phenylsulfoximine) phosphoramidothionate (5d): 1H-NMR: 3.4 (s, 3H, S-CH3),
1.4 (t, 3H, -CH3 ethyl), 4.05- 4.08 (m, 2H, -CH2 ethyl), 7.5-7.8 (m, 3H, PhHm,p), 7.9-8.1 (m, 2H,
PhHo); MS: 309 (M+), 294, 264, 236
Insecticidal activity (LD50)
Musca domestica: a susceptible RAC strain, reared at CIPEIN laboratory since 1980 and
maintained at constant temperature and humidity of 24-26ºC, 50-60% RH and photoperiod 12:12 hs
was used. Solutions in acetone (0.5 µL) of the synthesized compounds were topically applied to the
ventral abdomen of 10 immobilized flies (3 days old females) according to Picollo et al. [17] Three
replicates were done for each dose and four different doses were applied. Control assay was performed
by appling only acetone to female flies. Mortalities were assessed after 24 h and data analyzed using a
probit analysis programme (Micro Probit 3.0) based on Lichfield and Wilcoxon probit method [18] for
calculating LD50 values for each compound.

Molecules 2005, 10
1376
Anticholinesterase activity
Homogenates containing HFAChE were prepared from the heads of 3-5 day old houseflies (Musca
domestica RAC susceptible strain) in 0.1M buffer phosphate pH=7.2 (50 heads·mL-1) using a
homogenized Sorvall Omni Mixer (USA) in an ice bath. The crude homogenate was filtered through
glass wool and used without further purification. Bimolecular rate constants ki, for the inhibition of
HFAChE were determined at 412 nm by Ellman’s kinetic UV method [13] as previously described
[19].
References
1- Eto M. In Organophosphorus Pesticides: Organic and Biological Chemistry; CRC Press:
Cleveland, Ohio, USA 1974.
2- Quistad G.B.; Fukuto T.R.; Metcalf R.L. J. Agr. Food Chem. 1970, 18, 189-193
3- Bentley H.R.; Mc Dermott, E.E.; Moran T; Pace J and Whitehead, J.K. Proc. Roy. Soc. 1950,
B137, 402-417
4- Johnson, C.R. “Sulphoximides”, In Comprehensive Organic Chemistry: Synthesis and Reactions
of Organic Compounds; Barton, D.; Ollis, W.D. Eds., Pergamon Press Oxford-New York, 1979;
Vol 3, chapter 11.11, pp. 223-232 (Edited by Jones N., Univ of Sheffield)
5- O`Brien, R.D. In Drug Design; Ariens, E.J., Ed.; Academic Press: New York, 1971; Vol II, chap.
3.
6- Hollingworth, R.M.; Fukuto, T.R.; Metcalf, R.L. J. Agric. Food Chem. 1967, 15, 235-241.
7- Bentley H.R.; Mc Dermott, E.E.; Whitehead, J.K. Proc. Roy. Soc. 1951, B 138, 265-272
8- Bellozas Reinhard, M.; Mastrantonio G.; Jios J.; Della Vedova, C.O. – to be published – 2005
9- Oae, S.; Harada, K.; Tsujihara, K.; Furukawa, N. J. Sulfur Chem. 1972 A, 49-61
10- Wieczorkowski, J.; Jakobsen, P.; Treppendahl, S. Acta Chem. Scand. 1983, B 37, 27-30
11- Modro, T.A.; Wieczorkowski, J.; Org. Mass Spectrom. 1989, 24, 839-840
12- Mastantonio Garrido, G. Estudio Conformacional de Compuestos Organofosforados y sus
Mecanismos de Acción Tóxica; Ph.D. Thesis, La Plata Nacional University, Argentina, 2003 (in
Spanish): www.sedici.edu.ar/search/request.php?id_document=ARG-UNLP-TPG-0000000038
13- Ellman, G.L.; Courtney, K.D.; Andres, V.; Featherstone, R.H. Biochem. Pharmacol. 1961, 7, 88-
95 .
14- O`Brien, R. D. Insecticides, Action and Metabolism; Academic Press: New York, 1967.
15- Leonard, N.J.; Johnnson, C.R. J.Org.Chem., 1961, 27, 282- 284
16- Whitehead, J.K.; Bentley, H.R. J. Chem. Soc. 1952, 1572-74
17- Picollo, M.I.; Word, E.J.; Zerba, E.N.; Licastro, S.A.; Ruveda, M.A. Acta Bioquim. Clin.
Latinoam. 1976, 10, 67-70
18- Lichfield, J.T.; Wilcoxon, F.J. J. Exp. Therap. 1949, 96, 99-100
19- Fukuto, T.R.; Mercalf, R. L. J. Agric. Food. Chem. 1956, 4, 930-935.
Sample Availability: Contact the authors.
© 2005 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.