Syntheses and spectroscopic characterization of some phosphoramidates as reversible inhibitors of human acetylcholinesterase and determination of their potency

Abstract

The ability of phosphoramidates Me₂NP(O)(Cl)(p-NHC₆H₄NO₂) 1, Me₂NP(O)(p-NHC₆H₄NO₂)₂ 2, (CH₃C₆H₄O-p)P(O)(p-NHC₆H₄NO₂)₂ 3 and (CH₃C₆H₄O-p)₂P(O)(p-NHC₆H₄NO₂) 4 to inhibit human acetylcholinesterase (hAChE) has been evaluated by a modified Ellman's method and spectrophotometric measurements. Results showed that compounds 1 and 2 do not have any inhibitory potency, whereas compounds 3 and 4 were reversible mixed inhibitors. The IC₅₀ values for inhibitors 3 and 4 were 0.143 and 0.581 mM, respectively. The previously unknown compounds 3 and 4 were synthesized and characterized by ¹H, ¹³C, ³¹P NMR and IR spectroscopy and elemental analysis.

Keywords::

• Ellman's method
• human acetylcholinesterase
• IC₅₀ values
• phosphoramidates
• reversible mixed inhibitors
• inhibition

Introduction

Many phosphoramidate compounds inhibit human acetylcholinesterase (hAChE, EC 3.1.1.7) by phosphorylation of a serine hydroxyl group in the active site of this enzyme leading to its inactivation. This inhibition increases acetylcholine's levels in cholinergic synapses of both peripheral and central nervous systems 1-8. During the inhibition, phosphorylated enzyme undergoes a postinhibitory process and its spontaneous reactivation is very slow [5,8]. The mechanism of inhibition by these compounds has been widely studied. Many papers have reported the irreversible inhibition of human acetylcholinesterase by phosphoramidates 2-479–19 and some authors have reported reversible inhibitors 156820-26. Drug design is a matter of great interest based on reactivation of hAChE with site-directed nucleophiles such as 2-pralidoxime (2-PAM) and its analogs. The nucleophile binds to the active site and reacts with the phosphorylated hydroxyl group to release free and active enzyme which in turn becomes phosphorylated [1,5,8,21,22].

Bollinger et al. studied the inhibitory effect of (Me₂N)₂P(O)(p-NHC₆H₄NO₂), 5, and Me₂P(O) (p-NHC₆H₄NO₂), 6, on acetylcholinesterase [27]. They found that only molecule 5 with a (O)PN₃ moiety has inhibitory potency and acts as reversible inhibitor. To extend investigation in this area, we designed and synthesized compounds 1–4, which have a XY(O)P(p-NHC₆H₄NO₂) skeleton and examined their inhibitory potency and inhibition mechanism with hAChE using the spectrophotometric method based on Ellman's procedure [28].

Materials and methods

All reactions for synthesis of the phosphoramidates were carried out under an argon atmosphere. Melting points were determined on a Gallenkamp apparatus. ¹H, ¹³C and ³¹P NMR spectra were recorded on a Bruker (Avance DRS) 500 spectrometer and chemical shifts were determined relative to TMS and 85% H₃PO₄, respectively, as external standards. IR spectra (KBr pellets) were obtained with a Shimadzu, IR-60 model spectrometer. Elemental analysis was performed using a Heraeus CHN-O-RAPID instrument. UV measurements were performed on a Shimadzu UV-2100 spectrophotometer. All chemicals and solvents for syntheses were from Merck. Phosphoramidodichloridicacid-4-methylphenylester was prepared by the literature method [29].

Synthesis

N,N-(dimethyl)-N′-(4-nitrophenyl)phosphoramidochloride, 1 and N,N-(dimethyl)-N′,N″-bis(4-nitrophenyl)phosphoramide, 2

were synthesized and characterized by ¹H, ¹³C, ³¹P NMR and IR spectroscopy and elemental analysis [30].

N,N′-bis(4-nitrophenyl)phosphoramidicacid-4-methylphenylester, 3

To a solution of phosphoramidodichloridicacid-4-methylphenylester (2.25 g, 10 mmol) in dry benzene (20 ml), the sodium salt of 4-nitroaniline (2.76 g, 20 mmol) was added under an argon atmosphere. After 4 h stirring, the precipitate was removed and washed with dry chloroform (20 ml). The purity of the product was up to 98%. (yield 65%), m. p. = 174–176°C. Anal. Calc. for C₁₉H₁₇N₄O₆P: C, 53.27; H, 3.97; N, 13.08. Found: C, 53.20; H, 3.93; N, 12.99%. ¹H NMR (DMSO-d₆), δ (ppm): 2.24 (s, 3H, p-CH₃), 6.63 (d, ³J_H–H = 8.28 Hz, 4H_ortho),7.0 (d, ³J_H–H = 7.97 Hz, 2H_meta), 7.1 (d, ³J_H–H = 7.93 Hz, 2H_ortho), 7.86 (s, 2 N–H), 7.92 (d, ³J_H–H = 8.28 Hz, 4H_meta); ¹³C NMR (DMSO-d₆), δ (ppm): 20.28 (s, p-CH₃), 113.02 (s), 119.94 (s), 126.32 (s), 129.86 (s), 133.57 (s), 136.07 (s), 148.76 (s), 155.03 (s); ³¹P NMR (DMSO-d₆), δ (ppm): − 19.09 (s); IR (KBr), ν (cm^{− 1}): 2855 (w, N-H), 1962 (w), 1589 (s), 1514 (vs, NO₂), 1342 (s, NO₂), 1309 (m), 1198 (m, P = O), 1121 (m), 990 (m, P-N), 919 (m), 855 (m), 824 (m), 735 (m), 672 (m), 499 (m), 493 (s).

N-(4-nitrophenyl)phosphoramidicacid-bis(4-methylphenyl)ester, 4

The sodium salt of 4-nitroaniline (1.6 g, 10 mmol) was added to a solution of phosphoramidodichloridicacid-4-methylphenylester (2.25 g, 10 mmol) in dry benzene (20 ml) under an argon atmosphere and the mixture was stirred at room temperature for 5 h. The yellow precipitate obtained was washed with dry chloroform (20 ml). The purity of the product was up to 98%. (yield 58%), m.p. = 165–168°C. Anal. Calc. for C₂₀H₁₉N₂O₅P: C, 60.3; H, 4.77; N, 7.03. Found: C, 60.1; H, 4.72; N, 6.988%. ¹H NMR (DMSO-d₆), δ (ppm): 2.24 (s, 6H, p-CH₃), 6.61 (d, ³J_H–H = 7.58 Hz, 2H_meta), 6.75 (s, NH), 7.01 (d, ³J_H–H = 7.3 Hz, 4H_meta), 7.07 (d, ³J_H–H = 7.36 Hz, 4H_ortho), 7.91 (d, ³J_H–H = 7.47 Hz, 2H_ortho); ¹³C NMR (DMSO-d₆), δ (ppm): 20.74 (s, p-CH₃), 112.93 (s), 120.44 (s), 126.82 (s), 130.12 (s), 133.37 (s), 136.06 (s), 149.82 (s), 156.25 (s); ³¹P NMR (DMSO-d₆), δ (ppm): − 17.64 (s); IR (KBr), ν (cm^{− 1}): 3400 (w, N–H), 2920 (m), 1598 (w), 1499 (s, NO₂), 1337 (m, NO₂), 1255 (s), 1203 (vs, P = O), 1163 (m), 1080 (m), 1031 (s), 990 (m), 949 (s, P–N), 920 (s), 820 (m), 705 (w), 548 (w), 499 (m), 475 (m).

Kinetic experiments

All reagents for enzymatic experiments were from Fluka. Human acetylcholinesterase (hAChE) from SIGMA (50 units/785 μl) was diluted 25-fold in phosphate buffer (Na₂HPO₄/NaH₂PO₄, 70 mM, pH = 7.8).

The activity of the enzyme was measured at 25°C by a modified Ellman's method [28]. The reaction mixture for determination of IC₅₀ values consisted of: DTNB solution, 50 μl; Inhibitor, x μl (5 < x < 400); acetylthiocholine (ASCh) solution, 15 μl; phosphate buffer, (835-x) μl; AChE solution, 100 μl. The concentrations of substrate (s₀), DTNB and inhibitors 1–4 were 1.35 × 10^{− 4}, 10^{− 4} and 0.019, 0.014, 0.012 and 0.013 M, respectively, and the enzyme concentration under the assay conditions was 33.4 × 10^{− 9} M. K_m and V_max were obtained in the absence and presence of inhibitor from double-reciprocal Linweaver-Burk plots [31]. A control solution containing all of above materials except inhibitor was used to determine the activity of the enzyme.

Results

Synthesis and spectral data

The interesting point in compounds 1 and 2 is their ³J_PNCH = 0. Usually, in compounds with the general formula Me₂NP(O)XY (X = Y = halide or amine or X = halide, Y = amine) a doublet with ³J_PNCH of about 10–14 Hz is observed 2732-34.

The ¹H NMR spectra of compounds 3 and 4 showed that ⁷J_P–H is zero. This phenomena was has also been observed in some phosphoramidates [32,33]. The reason for the vanishing ⁷J_P–H coupling was described as the formation of partial multiple bonds between phosphorus and nitrogen in phosphoramidates [33].

It has been demonstrated that the crystalline state of compound (MeO)₂P(O)(p-NHC₆H₄NO₂) exists as a network of linear hydrogen bonds [35]. In compounds 3 and 4, we expected the formation of intermolecular hydrogen bonding. The existence of 4-nitroaniline as an electron withdrawing group in these molecules increased the acidity of the amine hydrogen. The highly acidity of these protons caused the exchange with the moisture of DMSO-d₆ and appeared in the ¹H NMR spectra as a singlet downfield peak. The ¹³C NMR spectra of molecules 3 and 4 did not show any coupling of carbons with phosphorus (ⁿJ_P–C = 0). ³¹P NMR indicated that the phosphorus atom in compound 4 was more deshielded than in compound 3. This may be attributed to greater electronegativity of oxygen compared to nitrogen. The IR spectra showed a stronger ν_{P = O} and a weaker ν_PN in compound 4 than in compound 3.

Human acetylcholinesterase inhibition

Compounds 1 and 2: In experiments with these compounds, the activity of the enzyme showed negligible changes (Figure 1) and their inhibitory potency was negligible.

Figure 1 The plot of V_I/V₀ against log ([I] × 10⁶) for inhibitors (Me₂N)P(O)(Cl)(p-NHC₆H₄NO₂), 1, and (Me₂N)P(O)(p-NHC₆H₄NO₂)₂, 2. V_I and V₀ are the enzyme activity (OD min^{− 1}) and [I] is the inhibitor concentration (μM).

Compounds 3 and 4: By plotting the V_I/V₀ (V_I 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, the IC₅₀ values of compounds 3 and 4 were obtained as 0.143 and 0.581 mM, respectively (Figure 2). Usually, a plot of remaining activity of the enzyme versus time for irreversible inhibiting phosphoramidates shows a linearly decrease from which the rate constant for inhibition of the enzyme may be obtained [16,36]. These plots for the inhibitors 3 and 4 indicated that the activity of the enzyme remains constant with time and demonstrating that the inhibition process is reversible.

Figure 2 The plot of V_I/V₀ against log ([I] × 10⁶) for inhibitors: 1-(p-CH₃C₆H₄O)P(O)(p-NHC₆H₄NO₂)₂, 3, 2-(p-CH₃C₆H₄O)₂P(O)(p-NHC₆H₄NO₂), 4. V_I and V₀ are the enzyme activity (OD min^{− 1}) and [I] is the inhibitor concentration (μM).

To further characterize the reversible process and to obtain the K_m and V_max values in the absence and in the presence of inhibitor, 1/V was plotted against 1/[S], where the V and [S] are the enzyme activity and substrate concentration, respectively. These Lineweaver-Burk plots [31] indicated that compounds 3 and 4 were mixed inhibitors (Scheme 1) and their values were 23.19 × 10^{− 5} and 10.79 × 10^{− 5} mol L^{− 1} and values were 9.16 × 10^{− 6} and 9.97 × 10^{− 6} mol L^{− 1} min^{− 1}, respectively. Also, the K_m and V_max values for enzyme were 108.20 × 10^{− 5} mol L^{− 1} and 10.03 × 10^{− 6} mol L^{− 1}min^{− 1}, respectively.

Scheme 1 The mechanism of human acetylcholinesterase inhibition by compounds 3 and 4.

Discussion

Usually, the inhibition of acetylcholinesterase by phosphoramidates has an irreversible mechanism 2-479–19 and only a few show a reversible inhibitory effect such as (Me₂N)₂P(O)(p-NHC₆H₄NO₂), 5 [27]. Compounds 1–4 were designed and synthesized to further investigate the influence of these phosphoramidates on human acetylcholinesterase.

Table I summarizes the spectral and enzymatic data for compounds 1–6. Compound 1 with a chlorine atom as a suitable leaving group was expected to be an irreversible inhibitor, but to our surprise this compound gave no significant change in the activity of the enzyme hAChE. The ³¹P NMR spectra of this compound in D₂O indicated two peak at − 10.59 and 0.29 ppm with relative ratio 1:1. The P–Cl bond in molecule 1 is labile and hydrolysis in phosphate buffer perhaps produces a P–OH bond. This may be the reason for the negligible inhibitory potency of molecule 1.

Table I.  The spectral and enzymatic data for compounds 1–6.

Compound	δ31P (ppm)	^3JPNCH (Hz)	^7JP–H (Hz)	^nJP–C (Hz), (n=2–5)	νP = O (cm^− 1)	νPN (cm^− 1)	IC50 (mM)	Km × 10^5 (mol L^− 1)	Vmax × 10^6 (mol L^− 1min^− 1)	Ref.
1	− 0.9	s, 0	–	s, 0	1221	907 (ar) 724 (al)	^aNI	101.12	9.98	^b
2	2.17	s, 0	–	s, 0	1300	902 (ar) 746 (al)	^aNI	103.08	10.01	^b
3	− 19.09	–	s, 0	s, 0	1198	990	0.143	23.19	9.16	^b
4	− 17.64	–	s, 0	s, 0	1203	949	0.581	10.79	9.97	^b
5	–	d., 10	–	–	1250	980	–	–	–	27
6	–	–	–	–	1255	980	–	–	–	27

^a Non-Inhibitor.

^b This work.

The behavior of compound 2 with hAChE was similar to that of compound 1. Although the structure of compound 2 is similar to that of molecule 5 of Bollinger et al. [27] and contains a (O)PN₃ moeity, their inhibition powers are different against hAChE.

Debord et al. [37] showed that the (O)PN₃ moeity in aliphatic phosphoramides interacts with the hydrophilic zone of the catalytic site of butyrylcholinesterase. Jarv et al. indicated [38] that in acetylcholinesterase three hydrophobic regions bind to hydrocarbon substituents and surround this zone. In phosphoramide 2, the existence of two nitroaniline groups decrease the lipophilicity of the aromatic ring. The hydrophobic constants obtained by Rekker et al. [39] are the measure of lipophilicity of a molecular fragment which is 1.69 for C₆H₄ and − 0.059 for an aromatic nitro group. Therefore, it is likely that two nitroaniline groups will decrease the lipophilicity of the molecule to a great extent, and this probably leads to the negligible inhibitory effect of this compound.

Surprisingly, compounds 3 and 4 revealed different results. Although the only structural difference between molecules 2 and 3 is the replacement of a Me₂N group by p-cresol, the interaction of molecule 3 with hAChE indicated that it was a reversible mixed inhibitor.

To further investigate the relationship between the inhibitory effects of this molecule with a p-cresol substituent, we used molecule 4 which has two p-cresol groups. The inhibitory potency of this molecule is drastically decreased in comparison to molecule 3. The IC₅₀ values for compounds 3 and 4 are 0.143 and 0.581 mM, respectively. These values show that the inhibitory potency of compound 3 is greater than that of compound 4.

The noticeable point in molecules 3 and 4 with phenolic substituents is that the P–O–ph groups are stable against hydrolysis and show only a singlet peak in ³¹P NMR spectra in D₂O.

Finally, it is concluded that changing the substituents X and Y in phosphoramidates (X)(Y)P(O) (p-NHC₆H₄NO₂) leads to compounds with different inhibitory potency. The effect of 4-nitroaniline within this research area is interesting. Compounds 1 and 2 showed no inhibitory effect while compounds 3 and 4 possesing (O)PN₂O and (O)PNO₂ moieties, respectively, are reversible mixed inhibitors. Spectral data showed that in compounds with a (O)P(p-NHC₆H₄NO₂) moiety all phosphorus-hydrogen and phosphorus-carbon couplings disappeared.

Acknowledgements

We would like to express our thanks to Dr. M. Naderimanesh in the Genetics department of Tarbiat Modares University and for his helpful discussions. Also, we thank Miss. Z. Zarandi and Mrs. Kh. Khorramishad for their laboratory assistance.