Synthesis, Identification of Some P-Chiral Organic Phosphoramidates and Their Inhibitory Action towards Enzymes
School of Studies in Chemistry, Jiwaji University, Gwalior-474011
*Corresponding Author E-mail: ibomsoram@yahoo.co.uk
ABSTRACT
Phosphoramidates (N-P) are biologically active organic compounds because they are capable of inhibiting enzyme action. A few members of this class were synthesized and checked for their anticholinesterase properties and they were found to be a good reactive phosphorylating agent to block AChE rather than BuChE in the poisoning process. The presence of chloro-substituent (either ortho- or para-) in the aryl moiety of these phosphoramidates does not show any significant contribution during toxicological study made in vitro. These phosphoramidates were characterized by FT-IR, 31P NMR and GC-MS spectral studies.
KEYWORDS: Phosphorylation, phosphoramidates, anticholinesterase, 31P NMR, toxicity
INTRODUCTION:
Unlike phosphorus compounds containing the C-O-P linkages, the organic phosphoramidates containing at least one C-N-P linkage are of great importance because of their numerous applications. Several researchers have reported the enzymatic assay of phosphorus pesticides. Organophosphorus (OP) nerve agents such as sarin, tabun, soman, etc. are highly toxic1 compounds, which have been utilized as chemical warfare agents. The primary target of nerve agents is acetylcholinesterase (AChE) the enzyme that terminates neuro transmission (at the post synapses of cholinergic neuron) by catalyzing the hydrolysis of the neurotransmitter acetylcholine. OP nerve agents irreversibly inhibit2,3 AChE by forming a covalent adduct with the hydroxyl group of the active site, resulting in successive accumulation of acetylcholine, overstimulation of cholinergic receptors, causing paralysis of neuromuscular functions and finally resulting in death of the victim. AChE and butyryl cholinesterase (BuChE) are potentially and stereoselectively inhibited2 by a particular enantiomer/isomer of an OP compound. Organophosphates are biologically active because they inhibit enzyme action through formation of inactive phosphoryl esterase. The stability of the phosphoryl esterase is very important, if this complex is unstable, the enzyme may sever on detoxification, whereas if it is stable in vivo, a definite chronic toxicity problem may result. The antiesterase action of organophosphates has been primarily investigated with cholinesterase, chymotrypsin, trypsin, etc.
Two general types of cholinesterases have been differentiated; the true or acetylcholinesterase hydrolyses acetylcholine selectively and possess both anionic and esteratic sites4 while the pseudocholinesterase attacks a variety of choline esters and the anionic type site is of lesser significance in its action.
The toxicity of OP esters has been recognized since 1932 and their myotic and anticholinesterase5 properties since 1939. Recently, the reactivation of nerve agent- inhibited AChE by oxime has been reported6 as an important step in the treatment of nerve agent poisoning. In this paper, an experimental study of the preparation and reactivity of the new synthetic compounds is described with information on their action towards AChE and BuChE.
Material and Methods:
Melting points were determined on an electric melting point apparatus and are uncorrected. Infra red spectra were determined on a FT-IR spectrophotometer (SHIMADZU PRESTIGE 21). 31P NMR was recorded on a 400 MHz spectrophotometer and GC-MS was performed on Agilent Technologies Model 6890N and 5973. The synthetic approach comprised of the conventional7-9 synthetic techniques of phosphorylation via the formation of methoxy phosphorodichloridate.
Preparation of methoxy phosphorodichloridate as a precursor (Zero): The preparation was carried out taking triethylamine (8.70 mL), phosphorus oxychloride (5.80 mL) and methanol (2.50 mL) in dry benzene. A yellow coloured layer separated out on stirring for 1.30 hours as the methoxy phosphorodichloridate. GC-MS showed the presence of 62% of the precursor.
Table 1: Preparation of methoxy, subsd. phenoxy, N-subsd. Phenyl phosphoramidates
|
Cpd. No. |
X |
Y |
Z |
m.p.(0C) |
IR10, KBr (cm-1) |
31P NMR11 |
|
1 |
H |
H |
H |
182-184 |
1548 (P-N-H), 1274 (P=O), 1082 (P-N-C), 1236 (P-O-Carom.), 1024 (P-O-C aliph.) |
-18.152 ppm (DMSO, 161MHz) |
|
2 |
H |
H |
Cl |
252-254 |
1529 (P-N-H), 1274 (P=O), 1109(P-N-C), 1240 (P-O-Carom.), 904 (P-O-C aliph.) |
- |
|
3 |
H |
Cl |
H |
188-190 |
1587 (P-N-H), 1284 (P=O), 974 (P-N-C), 1234 (P-O-Carom.), 1010 (P-O-C aliph.) |
- |
|
4 |
Cl |
H |
H |
200-201 |
1558 (P-N-H), 1274 (P=O), 1083 (P-N-C), 1234 (P-O-Carom.), 1018 (P-O-C aliph.) |
-18.343 ppm (DMSO, 161MHz) |
Preparation of methoxy, phenoxy, N-phenyl phosphoramidate (1): To the above dichloridate (Zero) phenol (4.4 mL) and aniline (4.6 mL) were added one after the other and subjected to refluxing for nearly 23 hours. A creamish-white substance was separated out and on recrystallisation with CHCl3 gave a pure solid (Table 1).
Preparation of methoxy, o-chloro-phenoxy, N-phenyl phosphoramidate (2): Similarly, o-chlorophenol (4.8 mL) and aniline (4.5 mL) were added to zero compound and the reaction mixture was refluxed on an oil-bath for nearly 21 hours. The lower white mass on recrystallisation with chloroform gave a white solid (Table 1).
Preparation of methoxy, p-chloro-phenoxy, N-phenyl phosphoramidate (3): Here, 2.80 mL of aniline and 3.0 mL of p-chlorophenol were added to methoxy phosphorodichloridate (Zero) prepared as above and then refluxed for 7 hours. The pale- yellow sticky mass, was extracted with CHCl3-H2O mixture, gave a brown mass on recrystallisation with carbon tetrachloride gave a brown solid (Table 1).
Preparation of methoxy, phenoxy, N- p-chloro-phenyl phosphoramidate (4): To the methoxy phosphorodichloridate (Zero) phenol (4.4 mL) and p-chloroaniline (6.4 mL) were added in dry benzene and subjected to refluxing for 15 hours. The grey residue which on recrystallisation with dichloromethane gave a grey solid (Table 1).
The spectral (IR and 31P NMR) data of above synthesized compounds are tabulated and interpreted in Table 1.
RESULTS AND DISCUSSION:
After synthesis and characterization of the above four compounds their reactivity towards the enzymes acetyl cholinesterase (AChE) and butyryl cholinesterase (BuChE) have been checked by performing Ellman’s method12. The study was done on a Visible spectrophotometer i.e. Spectronic-20 at 412 nm. From this study, it is observed that the simplest (N-P ester) methoxy, phenoxy, N-phenyl phosphoramidate (1) inhibits the enzyme AChE when the substrate (1) was taken as 50 µL but it was found inactive towards BuChE. Compound (2) showed its reactivity towards BuChE but only at higher concentration of the substrate. The compound (3) is also reactive towards AChE but not sensitive against BuChE in very low concentration (10 µL). Interestingly, compound (4) i.e. methoxy, phenoxy, N- p-chloro-phenyl phosphoramidate, was examined to be unreactive to both the enzymes.
A comparative data had been achieved from the above study and is given in table 2.
From the above study, it is observed that these compounds follow the following order of inhibition as: 1 > 3 > 4 (AChE). With BuChE, only compound (2) shows <30% inhibition at a high concentration of even 100 μL.
It may be seen that inspite of the absence of any activating group in the aryl moieties but still compound (1) showed inhibition towards AChE which may be due to its basic chemical structure as the phosphoramidate. Compound (3) on the other hand, even though it has a p-chloro group in the phenoxy moiety showed lesser reactivity towards AChE.
Table 2: A comparative study of inhibitory action against AChE and BuChE.
|
Compounds’ No. |
1 |
2 |
3 |
4 |
|
Concentration. %age Inhibition (AChE) |
50 µL 37.0 |
- - |
50 µL 22.0 |
50 µL 0.0 |
|
Concentration. %age Inhibition (BuChE) |
10 µL 0.0 |
100 µL 29.0 |
10 µL 0.0 |
100 µL 0.0 |
It is interesting to note that compound (4) has a p-chloro group in the aniline moiety but it neither showed any activity to both the enzymes under examination which clearly states that the presence of the substituent in an aniline moiety does not contribute at all to the chemical nature of the compound. On the other hand, it is also surprising to learn that when chloro- group was present at the ortho position of the phenoxy moiety it has a drastic reactivity towards BuChE. This clearly indicates the participation of the substituent in the rate of hydrolysis as well as the extent of inhibition, although only to BuChE.
CONCLUSION:
It is generally agreed that the toxicity of OP compounds to mammals13 is associated with the inhibition of the cholinesterase enzymes, although other enzymes such as liver esterase, chymotrypsin, and trypsin are also inhibited. The most susceptible enzyme by OP compounds is AChE and phosphorylation takes place of the esteratic4 site via an electrophilic mechanism. In the present study also, it is seen that phosphoramidates (N-P) preferably act on AChE rather than BuChE which clearly indicates their specific dominating toxic property, towards the former enzyme.
ACKNOWLEDGMENT:
The financial support of MPCST (Grant No. 3287/CST/RandD/2007), Bhopal is gratefully acknowledged by the author of this manuscript.
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Received on 17.08.2009 Modified on 08.10.2009
Accepted on 30.10.2009 © AJRC All right reserved
Asian J. Research Chem. 2(4):Oct.-Dec. 2009 page 523-525