Ligand-based design and synthesis of novel sodium channel blockers from a combined phenytoin–lidocaine pharmacophore

Abstract

The voltage-gated sodium channel remains a rich area for the development of novel blockers. In this study we used comparative molecular field analysis (CoMFA), a ligand-based design strategy, to generate a 3D model based upon local anesthetics, hydantoins, and α-hydroxyphenylamides to elucidate a SAR for their binding site in the neuronal sodium channel. Correlation by partial least squares (PLS) analysis of in vitro sodium channel binding activity (expressed as pIC₅₀) and the CoMFA descriptor column generated a final non-cross-validated model with q² = 0.926 for the training set. The CoMFA steric and electrostatic maps described a binding site predominately hydrophobic in nature. This model was then used to design and predict a series of novel sodium channel blockers that utilized overlapping structural features of phenytoin, hydroxy amides, and the local anesthetic lidocaine. Synthesis and evaluation of these compounds for their ability to inhibit [³H]-batrachotoxin revealed that these compounds have potent sodium channel blockade. Furthermore, the CoMFA model was able to accurately predict the binding of these compounds to the neuronal sodium channel. Synthesis and subsequent sodium channel evaluation of compound 37 (predicted IC₅₀ = 7 μM, actual IC₅₀ = 6 μM), established that novel compounds based on overlapping regions of phenytoin and lidocaine are better binders to the sodium channel than phenytoin itself (IC₅₀ = 40 μM).

1. Introduction

Neuronal voltage-gated sodium channels (NVSC) play an important role in the generation and propagation of action potentials in neurons and other excitable cells. Thus, NVSC blocking agents represent a clinically important class of drugs used in the treatment of pain, seizures and arrhythmia.

Cloning, purification and sequence analysis of NVSC’s have identified at least ten highly conserved α-subunits. The NVSC is composed of six transmembrane segments (S1–S6) that form the 260 kDa α-subunit complex and at least four important accessory β-subunits.¹

Information concerning channel topography has been obtained through the use of neurotoxins. In fact, voltage-sensitivity is characterized based on the channel’s sensitivity to tetrodotoxin (TTX), a deadly neurotoxin of the puffer fish. The binding site for batrachotoxin (BTX), an alkaloid from poison dart frogs, binds to the inactivated state of the NVSC, thereby disrupting the equilibrium of protein states. Substitution with a para-[³H] benzoate ester at C₂₀ on BTX resulted in the development of a very useful radioligand [³H]-BTX-B.² The binding of [³H]-BTX-B to the NVSC protein is allosterically linked³ to and overlaps with the binding sites of both anticonvulsants (phenytoin) and local anesthetics (lidocaine). Functionally, BTX stabilizes the NVSCs in their open conformation, whereas local anesthetics block Na⁺ conductance.

The use of [³H]-BTX-B as a NVSC screening tool has indicated an important relationship between molecular structure and pharmacological effects for interactions of small molecule inhibitors. Using this information, a ligand-based molecular modeling study was successful in the design of a new small molecule which binds potently (IC₅₀ = 9 μM) to the NVSC^4–6 and a relationship between [³H]-BTX-B inhibition and anticonvulsant activity in mice has been established.⁷

Site-directed mutagenesis studies have identified clusters of common residues at D1–S6, D3–S6, and D4–S6 segments within the NVSC α-subunit that are critical for binding of hydantoins (phenytoin) and local anesthetics (lidocaine).^8,9 Mutations of amino acid residues in specific segments of the α-subunit have revealed important voltage dependence and modification of protein conformational states (open, inactivated, closed).¹⁰ Altogether, these studies suggest that phenytoin and lidocaine share a non-identical but overlapping receptor¹¹ on the extracellular side of the NVSC.¹²

Considering the accepted overlapping nature of the binding sites of phenytoin and lidocaine, we have employed the use of comparative molecular field analysis (CoMFA) to assist in identifying important overlapping structural features and to provide insight into essential SAR for the development of novel NVSC inhibitors. CoMFA, a 3D ligand-based discovery program is useful in correlating biological activity with structural features and has become a valuable tool in the design of new inhibitors of biological receptors. In this study, we have proposed a series of novel grafted phenytoin/lidocaine and hydroxyamide/lidocaine compounds (Fig. 1) that were identified and predicted by CoMFA to have potent NVSC activity. We have validated our CoMFA alignment by synthesizing these proposed compounds and evaluating them for inhibition of [³H]-BTX-B.

Figure 1.

Design of novel grafted phenytoin/lidocaine compounds.

2. Results

Our previous sodium channel SAR studies suggested that logP was one of the parameters important for enhanced binding to the NVSC.⁵ In the training set, logP correlation to pIC₅₀ gave an R² of 0.59 (Fig. 3). This is poor correlation, in fact, the groups of compounds at logP ≈ 2.5 and logP ≈ 3.0 have IC₅₀ values that span 2 log units. It is clear that local anesthetic binding to the NVSC is dependent on other factors besides lipophilicity. Comparisons within the present model of local anesthetics and hydantoins with the same logP and different structures often show very different binding affinities to the NVSC (e.g. 3 and piperocaine). We were thus encouraged to use CoMFA to investigate the differences in structural and electrostatic features of these analogues in an effort to formulate an improved model. The sodium channel binding affinities and lipophilicities for the CoMFA training set are presented in Table 1. PLS correlation of in vitro sodium channel binding, expressed as −logIC₅₀, and CoMFA descriptors generated a CoMFA model with q² = 0.926 for the training set (Fig. 4). Further more, the CoMFA model provided better correlation than that for the logP model (R² = 0.59) from the same data set.

Figure 3.

Plot of logP versus pIC₅₀ for compounds in the training set.

Table 1.

Sodium channel binding activities for the training set used in the final non-cross-validated CoMFA model^a

Compound	R1	R2	R3	R4	n	LogP	3H-BTX IC50 (μM)	Final model, −pIC50b
								Obsd	Pred	Res
1	n-Propyl	H	H			1.96	162	−2.21	−2.10	−0.11
2	n-Butyl	H	H			2.46	103	−2.01	−1.91	−0.10
3	n-Butyl	H	H			3.01	285	−2.45	−2.16	−0.30
4	n-Pentyl	H	Methyl			2.96	39	−1.59	−1.55	−0.04
5	n-Hexyl	H	H			3.40	13	−1.11	−1.17	0.06
6	n-Heptyl	H	H			3.96	5	−0.70	−0.82	0.12
7	n-Nonyl	H	H			4.96	5	−0.70	−0.54	−0.16
8	Methyl	Ethyl	H			2.02	720	−2.86	−2.87	0.01
9					1	0.96	2112	−3.32	−2.87	−0.46
10					2	1.46	851	−2.93	−2.66	−0.27
11					3	1.96	251	−2.40	−2.52	0.12
12					4	2.46	251	−2.40	−2.43	0.03
13						1.52	250	−2.40	−2.46	0.06
14	n-Butyl	Methyl	H	H		2.96	225	−2.35	−2.03	−0.33
15	n-Butyl	H	Methyl	H		2.96	58	−1.76	−1.78	0.02
16	n-Butyl	H	H	Methyl		2.96	95	−1.98	−2.22	0.25
17	Cyclohexyl	H	H	H		2.96	58	−1.76	−1.91	0.14
18					2	2.01	700	−2.85	−3.11	0.27
19					3	2.51	427	−2.63	−2.79	0.16
20						3.70	9	−0.95	−0.96	0.01
21						1.39	910	−2.96	−3.10	0.14
22						3.86	5.4	−0.73	−0.95	0.22
23							51	−1.71	−1.65	−0.06
24						1.82	49	−1.69	−1.71	0.02
25						3.77	1.4	−0.15	0.22	−0.37
26						3.87	3.5	−0.54	−0.99	0.44
27						3.34	7.1	−0.85	−0.81	−0.04
28						2.70	240	−2.38	−1.94	−0.44
29						2.74	24	−1.38	−1.12	−0.26
30						3.01	12.9	−1.11	−0.86	−0.25
31						2.18	53.7	−1.73	−1.81	0.08
32						2.53	109.6	−2.04	−1.96	−0.08
33						2.42	2	−0.28	−0.72	0.44
34						2.42	24	−1.38	−1.50	0.12
35						3.27	3.4	−0.53	−0.73	0.20
36						2.46	40	−1.60	−1.95	0.35

^aNumber of components = 4; s = 0.245; q² = 0.926.

^b−pIC₅₀ = −log IC₅₀.

Figure 4.

Plot of observed pIC₅₀ versus predicted pIC₅₀ values for the non-cross-validated CoMFA model in Table 1.

Investigations of the CoMFA steric map revealed that a long alkyl chain at the C5 position appears to be required for tight binding to the hydantoin site. The steric contour map in Figure 5 reveals a favorable steric volume near the 5-alkyl side chain. In addition, steric bulk in the amide portion of the local anesthetics appears to also be favorable as revealed by comparison of binding by benzocaine (IC₅₀ = 910 μM) and etidocaine (IC₅₀ = 3.5 μM) to the local anesthetic site.

Figure 5.

Electrostatic and steric CoMFA fields for the model developed from compounds in Table 1. For electrostatic contours, increased binding results from placing more positive (+) charge near blue and negative (−) charge near red. For steric contours increased binding results from placing more bulk near green and less bulk near yellow. (A) Overlay of compounds used in the training set. (B) Active analogue 44. (C) Active analogue 44 with electronic contour overlay. (D) Active analogue 44 with steric contour overlay.

An important part of a 3D-QSAR model is the ability to accurately predict the relative binding potencies of novel analogues before synthesis. Based upon the model described here, we proposed that novel analogues containing both the phenytoin (hydantoin) pharmacophore or the hydroxyamide pharmacophore and the local anesthetic lidocaine would be potent binders to the neuronal sodium channel as they would potentially have the ability to bind to an expanded binding region encompassing both the phenytoin and local anesthetic binding sites. Using the CoMFA model described above, our newly developed grafted analogues were predicted to have IC₅₀ values of 3 μM (40) to 66μM (38) (Table 3).

Table 3.

Predicted and actual sodium channel binding activities for novel grafted analogues forming the test set

Compound	R1	R2	LogPa	[3H]-BTX Inhibition at 40 μM (%)	−pIC50
					Obsd	Predb	Res
37	n-Heptyl	Methyl	3.6	83 ± 1.53	-0.78	-0.85	-0.07
38	Phenyl	H	1.4	4 ± 4.56	nd	-1.82
39	Phenyl	Methyl	2.4	16 ± 5.23	nd	-1.74
40	n-Nonyl	Methyl	4.4	63 ± 1.01	nd	-0.51
41	Phenyl	H	1.6	50 ± 0.92	-1.60	-1.65	-0.05
42	Phenyl	Methyl	2.5	49 ± 2.72	-1.60	-1.70	-0.10
43	n-Hexyl	Methyl	3.3	68 ± 4.26	nd	-1.09
44	n-Heptyl	Methyl	3.7	71 ± 0.30	-1.13	-0.87	-0.26
45	n-Nonyl	Methyl	4.5	69 ± 0.89	nd	-0.59

^aCalculated using the chemical properties tool of ChemDraw (CambridgeSoft, Inc.).

^bData generated from the CoMFA non-cross-validated run (see QSAR methods).

After successfully completing the syntheses of analogues 37–45 (Scheme 1) we evaluated them for activity using [³H]-BTX-B binding assay with each compound at a concentration of 40 μM (IC₅₀ of phenytoin in this assay). This series of analogues encompasses a range of lipophilicities (logP = 1.4–5.3) as well as phenyl ring substitutions (R₂ = H or CH₃), pharmacophore changes (hydroxyamides vs hydantoins), and chain length (R₁ = Ph, C₆H₁₃, C₇H₁₅, and C₉H₁₉). The predictive ability of the CoMFA model was evaluated by comparing the observed versus predicted activities of the novel hybrid phenytoin/lidocaine and hydroxyamide/lidocaine analogues.

Scheme 1.

Reagents and conditions: (a) PPA, 180–200 °C; (b) bromoacetyl bromide, DMAP, THF; (c) diethylamine, NaHCO₃, KI, MeOH; (d) (1) TMSCN, KCN, 18-Crown-6, (2) 15% HCl; (e) 1,4-dioxane, HCl, HCl (g); (f) KCN, (NH)₄CO₃, EtOH, 60–65 °C.

Observed [³H]-BTX displacement revealed that compound 37 displaced 83% [³H]-BTX at 40 μM. Compounds 41 and 42, the direct grafts of phenytoin and lidocaine, displaced 50 and 49% [³H]-BTX, respectively. As a comparison between the hydroxyamides and hydantoins, compound 44 (hydantoin analogue of 37) displaced 71% [³H]-BTX at 40 μM. The latter data is shown in Table 3, and these four analogues were chosen based on their potent inhibition at 40 μM and to allow comparisons between direct phenytoin/lidocaine grafts and hydroxyamide/hydantoin pharmacophores. As previously mentioned, compounds 37 and 44 represent optimal binders based upon initial [³H]-BTX data when tested at a concentration of 40 μM (IC₅₀ of phenytoin). Compounds 41 and 42 represent grafts of phenytoin and lidocaine with and without the phenyl ring methyl groups of lidocaine. It is evident from this data that the CoMFA model has excellent predictive ability. Residual values (the difference between the observed IC₅₀ and the predicted IC₅₀) ranged from 0.05 to 0.26 for these novel compounds. As expected, compounds 37 and 44, which were designed to take advantage of the steric requirement at C5 with a long alkyl chain demonstrating the best activity with IC₅₀ values of 6.0 and 13.5 μM, respectively. Compounds 41 and 42 showed identical IC₅₀ values of about 39.8 μM (predicted = 44.7 and 50.1 μM, respectively). It should be noted that when tested at 40 μM in the preliminary [³H]-BTX assay, these two compounds showed [³H]-BTX displacement of 50% and 49%, further validating the predictive ability of the CoMFA model. Compound 42 was designed as a direct graft of the anticonvulsant phenytoin and the local anesthetic lidocaine. The sodium channel binding potency of this analogue was correctly predicted by the CoMFA model (observed IC₅₀ = 39.8 μM). Interestingly, the IC₅₀ of lidocaine in this assay is 240 μM and that of phenytoin is 40 μM, thus this analogue increases the binding potency of the lidocaine moiety 6-fold.

In comparing the inhibition of BTX binding to Na⁺ channels between the hydroxyamides (37–40) and hydantoins (41–45), it is apparent that when R₁ is a phenyl group, hydantoins are much more active than the corresponding hydroxyamides. This difference cannot be solely attributed to lipophilicity as the logP values for these lidocaine analogues are all within one log unit of one another (1.4–2.5).

Substitution of a heptyl group for the phenyl group at R₁ in the lidocaine/hydroxyamide analogues (37 vs 39) resulted in a greater than 5-fold increase in inhibition (83% vs 16%). Similarly, the lidocaine/hydantoin analogue showed an increase in inhibition (49% for R₁ = Ph vs 71% for R₁ = heptyl) but it was not nearly as dramatic as that seen in the other analogues. The substitution of a heptyl group for a phenyl group corresponds to both an increase in size as well as lipophilicity (~1.5-fold increase in logP). The heptyl substitution appears to remain the optimal substitution in this region, as increasing the chain length to 9 carbons (37 vs 40) actually results in a less potent compound. Interestingly, in the lidocaine/hydantoin analogue, the series of 6, 7, to 9 carbon chains at R₁ showed no difference in potency. This result appears to support our previous findings that hydantoins within the same class having similar logP values exhibit similar binding potencies.⁷

We determined the effects of compound 37 on sodium currents recorded from HEK cells stably expressing human Na_v1.2. At a concentration of 100 μM compound 37 exhibited a 67.7% ± 5.6 (n = 4) block of sodium channel current (Fig. 6) and were fully reversible on washout. These results demonstrate that the newly designed compound 37 has significant functional effects on inhibiting sodium channel currents.

Figure 6.

Sample current traces shows block by compound 37 of the human Na_v1.2 sodium channel isoform stably expressed in HEK 293 cells. Currents were elicited by a step depolarization to +10 mV for 12 ms from a holding potential of −100 mV. Compound 37 blocked 67.7 ± 5.6% (n = 4) of the recorded current which was partially reversible upon washout.

3. Discussion

Voltage-gated sodium channels are important in many physiological processes and their dysregulation are known to play a key role in several pathological human diseases.¹³ In view of this, voltage-gated sodium channels remain an attractive target for medicinal chemistry. Since the X-ray structure of the voltage-gated sodium channel remains unsolved, the development of computer based strategies that contribute to the design of new sodium channel ligands are critical in our pursuit of targeted inhibitors. The CoMFA analysis presented in this study provides such a model and allowed us to encompass a range of new structures. The predictive sodium channel binding affinities for four novel compounds not present in the training set and not previously reported provided a focused rationale for targeted synthesis. The novel synthetic compounds were evaluated for direct displacement of a sodium channel specific radioligand and function block of cellular sodium channel currents was confirmed for one lead. Both the ³H-BTX and the electrophysiology confirms our hypothesis for targeting site 2 of the sodium channel and our study provides validation of this technique with functional sodium channel block. Finally, this study expands our understanding concerning the binding region encompassing both the local anesthetic and anticonvulsant binding sites within site 2 of the sodium channel.

4. Conclusion

One of the most challenging facets of ion channel drug discovery is the design of new classes of small molecule leads with appropriate potency without the direct assistance of 3D protein X-ray structures. In this study we have used ligand-based drug design through CoMFA and validated the model by synthesis and evaluation of human sodium channel interactions. This finding continues to support the use of CoMFA in transmembrane drug discovery paradigms where little or no target structural data is known.

5. Experimental

All reaction requiring anhydrous conditions were carried out in flame-dried glassware under nitrogen atmosphere. Solvents were purified by pressure filtration through activated alumina. Melting points were determined in open capillary tubes with an electrothermal Mel-temp melting point apparatus and are uncorrected. ¹H and ¹³C NMR spectra were measured on a General Electric 300 MHz spectrometer. Chemical shifts are reported in ppm relative to resonances of the solvent CDCl₃ (unless specified otherwise): 7.25 ppm in the ¹H spectra and 77.08 ppm in the ¹³C spectra.

Thin-layer chromatography was performed on silica gel 60 F 254 aluminum sheets with UV detection. Chromatography separations were carried out on silica gel columns (silica gel 60, 40–63 μm).

Mass spectra were obtained on Thermofinnigan LCQ iontrap (Classic) instrument. High-resolution mass spectrometry was performed at the University of Illinois at Urbana-Champaign, School of Chemical Science.

5.1. General procedure for the preparation of 4-aminobenzophenones

A mixture of 2,6-dimethylaniline (1.0 equiv), RCOOH (2.0 equiv) in excess PPA was heated with vigorous stirring at 180–200 °C. for 1 h. Then, 2N hydrochloric acid and water was added and stirred for an additional hour. The reaction was cooled to 25 °C and extracted with CH₂Cl₂, dried over MgSO₄, filtered, and concentrated. The residue was purified by silica gel column chromatography, eluting with EtOAc/hexane.

5.2. General procedure for the preparation of lidocaineketones

To a solution of bromoacetyl bromide (1.5 equiv) in THF at 0 °C was added dropwise, under stirring and nitrogen atmosphere, a solution of R–NH₂ (1.0 equiv) and 4-(dimethylamino)pyridine (0.5 equiv) in THF. The reaction mixture was stirred at 0 °C for 1.5 h, then at room temperature for 1 h. The reaction was quenched with water and extracted with CH₂Cl₂, dried over MgSO₄, filtered, and concentrated by rotary evaporator. The crude product was purified by silica gel column chromatography, eluting with EtOAc/hexane.

A solution of above product (1.0 equiv), diethylamine (1.5 equiv), sodium bicarbonate (1.5 equiv), and potassium iodide (catalytic amount) in methanol was stirred at 50 °C for 4 h. The reaction mixture was cooled to room temperature and diluted with ethyl acetate and water, then the mixture was extracted with CH₂Cl₂, dried over MgSO₄, filtered, and concentrated. The residue was purified by silica gel column chromatography, eluting with EtOAc/hexane.

5.3. General procedure for the preparation of α-hydroxyamides (37–40)

To a solution of the ketone (1.0 equiv) in CH₂Cl₂ were added KCN and 18-Crown-6 (10 mg per mol of ketone) and trimethylsilylcyanide (2.5 equiv) by syringe under nitrogen atmosphere. The reaction mixture was stirred for 24 h at 25 °C. After 24 h. the solvent was removed in vacuo.

To the residue was added 1,4-dioxane (5 ml per mmol of ketone) and cooled to 0 °C. Previously cooled concentrated HCl (1 ml per 1 mmol of ketone) was added and HCl gas (from H₂SO₄ and NH₄Cl) was then passed through the reaction mixture for 1 h at 0 °C. The mixture was allowed to stand at room temperature overnight then extracted with CH₂Cl₂. The extracts were dried over MgSO₄, filtered, concentrated, and purified by silica gel column chromatography, eluting with EtOAc/hexane.

5.3.1. 2-[4-(2-Diethylamino-acetylamino)-3,5-dimethylphenyl]-2-hydroxy-nonanoic acid amide (37)

Isolated as a light brown oil. ¹H NMR: δ 9.20–8.39 (s, 1H), 7.78–7.23 (s, 1H), 7.04–6.94 (s, 1H), 6.74–6.64 (s, 1H), 6.30–6.20 (s, 1H), 2.65–2.48 (s, 2H), 2.49–2.33 (m, 4H), 1.65–1.49 (m, 2H), 1.91–1.75 (s, 6H), 1.02–0.84 (m, 14H), 0.84–0.71 (t, J = 7.5 Hz, 6H), 0.58–0.46 (t, J = 6 Hz, 3H); ¹³C NMR: δ 177.97, 162.83, 142.56, 134.60, 133.18. 125.66, 78.42, 49.14, 36.59, 32.07, 31.45, 30.08, 29.44, 22.84, 18.93, 14.32, 12.30; HRMS (EI): calcd for C₂₃H₃₉N₃O₃ 405.5809; found, 405.5811.

5.3.2. 2-[4-(2-Diethylamino-acetylamino)-phenyl]-2-hydroxy-2-phenyl-acetamide (38)

Isolated as a light brown oil (40%). ¹H NMR: δ 9.69–8.91 (s, 1H), 7.52–6.47 (m, 9H), 3.03–2.82 (s, 2H), 2.62–2.41 (m, 4H), 1.06–0.86 (t, J = 6 Hz, 6H); ¹³C NMR: δ 176.93, 170.96, 143.78, 139.45, 137.43, 128.87, 128.43, 128.13, 119.34, 70.52, 58.32, 49.31, 12.86; HRMS (EI): calcd for C₂₀H₂₅N₃O₃ 355.4358: found 355.4357.

5.3.3. 2-[4-(2-Diethylamino-acetylamono)-3,5-dimethylphenyl]-2-hydroxy-2-phenylacetamide (39)

Isolated as a light brown oil (61%). ¹H NMR: δ 9.42–8.20 (s, 1H), 7.53–7.39 (m, 2H), 7.39–7.34 (m, 3H), 7.20–7.07 (s, 2H), 6.63–6.35 (s, 1H), 6.08–5.80 (s, 1H), 3.29–2.97 (s, 2H), 2.80–2.54 (m, 4H), 2.23–2.06 (s, 6H), 1.25–1.00 (t, J = 6 Hz, 6H); ¹³C NMR: δ 176.8, 171.18, 143.15, 142.14, 135.45, 134.19, 128.61, 128.36, 128.03, 127.99, 81.47, 57.79, 49.44, 19.22, 13.11; HRMS (EI): calcd for C₂₂H₂₉N₃O₃ 383.4906; found 383.4905.

5.3.4. 2-[4-(2-Diethylamono-acetylamono)-3,5-dimethylphenyl]-2-hydroxy-undecanoic acid amide (40)

Isolated as a light yellow oil 71%). ¹H NMR: δ 9.21–8.41 (s, 1H), 7.55–6.92 (s, 2H), 6.99–6.41 (s, 1H), 6.32–5.89 (s, 1H), 3.56–3.30 (s, 2H), 3.16–2.90 (t, J = 9 Hz, 2H), 2.68–2.42 (m, 4H), 2.19–1.99 (s, 6H), 1.35–1.10 (m, 14H), 1.09–1.049 (t, J = 6 Hz, 6H), 0.89–0.63 (t, J = 6 Hz, 3H); ¹³C NMR: δ 178.01, 170.93, 142.48, 134.92, 133.42, 125.94, 78.61, 57.83, 49.38, 39.58, 32.31, 32.20, 30.36, 30.07, 29.97, 29.74, 23.07, 19.16, 14.52, 13.04. HRMS (EI): calcd for C₂₅H₄₃N₃O₃ 433.6352; found, 433.6353.

5.4. General procedure for the preparation of hydantoins (41–45)

To a stirring solution of the ketone (1.0 equiv) in 50% ethanol were added KCN (2.0 equiv) and ammonium carbonate (4.0 equiv). The reaction mixture was stirred at 60–65 °C for more than 24 h (using TLC to monitor reaction progress). When the reaction was completed, the reaction mixture was cooled to room temperature and extracted with CH₂Cl₂. The organic extracts were dried over MgSO₄, filtered, concentrated, and purified by silica gel column chromatography, eluting with EtOAc/hexane.

5.4.1. 2-Diethylamino-N-[4-(2,5-dioxo-4-phenyl-imidazolidin-4-yl)-phenyl]-acetamide (41)

Isolated as a white solid. mp 253–244 °C; ¹H NMR: δ 9.74–9.17 (s, 1H), 8.62–8.47 (s, 1H), 8.10–7.96 (s, 1H), 7.56–7.17 (m, 9H), 3.29–2.93 (s, 1H), 2.81–2.40 (m, 4H), 1.22–0.81 (t, J = 6 Hz, 6H); ¹³C NMR (CDCl₃): δ 175.47, 171.34, 164.91, 139.63, 138.02, 135.32, 129.17, 128.86, 128.22, 127.34, 120.11, 71.72, 58.37, 49.35, 12.86; HRMS (EI): calcd for C₂₁H₂₄N₄O₃ 380.4466; found, 380.4465.

5.4.2. 2-Diethylamino-N-[4-(2,5-dioxo-4-phenyl-imidazolidin-4-yl)-2,6-dimethyl-phenyl]-acetamide (42)

Isolated as a white solid, (61%); mp 164–167 °C; ¹H NMR: δ 10.46–9.82 (s, 1H), 9.56–8.76 (s, 2H), 7.70–6.47 (m, 7H) 3.42–3.04 (s, 2H), 2.90–2.52 (m, 4H), 2.53–2.16 (s, 6H), 1.29–0.92 (t, J = 7.5 Hz, 6H); ¹³C NMR (CDCl₃): δ 174.39, 173.13, 157.58, 141.43, 140.1, 128.55, 128.45, 128.04, 126.73, 126.63,70.86, 57.44, 49.60, 19.35, 13.28; HRMS (EI): calcd for C₂₃H₂₈N₄O₃ 408.5003: found 408.5003.

5.4.3. 2-Diethylamino-N-[4-(4-hexyl-2,5-dioxo-imidazolidin-4-yl)-2,6-dimethyl-phenyl]-acetamide (43)

Isolated as a white solid (57%); mp 107–110 °C; ¹H NMR: δ 9.85–10.00 (b, 1H), 9.24–8.63 (s, 1H), 7.90–8.00 (b, 1H), 7.54–6.86 (s, 2H), 3.35–3.13 (s, 2H), 2.78–2.53 (m, 4H), 2.31–2.11 (s, 6H), 1.37–1.29 (m, 10H), 1.28–1.14 (t, J = 9.6 Hz), 0.94–0.67 (t, J = 6 Hz, 3H); ¹³C NMR: δ 176.48, 171.74, 158.08, 135.91, 133.99, 125.96, 125.42, 68.79, 57.67, 49.47, 31.95, 29.49, 23.09, 23.00, 19.20, 19.07, 14.62, 14.20; HRMS (EI): calcd for C₂₃H₃₆N₄O₃ 416.5636; found, 416.5633.

5.4.4. 2-Diethylamino-N-[4-(4-heptyl-2,5-dioxo-imidazolidin-4-yl)-2,6-dimethyl-phenyl]-acetamide (44)

Isolated as a light yellow solid (48%); mp 97–100 °C; ¹H NMR: δ 9.36–8.57 (s, 1H), 7.60–6.80 (s, 2H), 3.35–3.07 (s, 2H), 2.82–2.54 (m, 4H), 2.35–2.07 (s, 6H), 2.03–1.75 (m, 2H), 1.37.1.14 (m, 10H), 1.13–0.97 (t, J = 7.5 Hz, 6H), 0.95–0.67 (t, J = 6 Hz, 3H); ¹³C NMR: δ 176.44, 164.51, 157.93, 136.04, 136.01, 125.88, 125.77, 68.86, 57.72, 49.51, 32.23, 29.84, 29.48, 24.17, 23.06, 19.24, 14.54, 13.15; HRMS (EI): calcd for C₂₄H₃₈N₄O₃ 430.5907; found, 430.5905.

5.4.5. 2-Diethylamino-N-[2.6-dimethyl–4-(4-nonyl-2,5-dioxoimidazolidin-4-yl)-phenyl]-acetamide (45)

Isolated as a white solid (78%); mp 95–98 °C; ¹H NMR: δ 9.23–8.79 (s, 1H), 8.11–7.86 (s, 1H), 7.81–7.47 (s, 1H), 7.31–6.98 (s, 2H), 3.33–3.02 (t, J = 9 Hz, 2H), 2.80–2.48 (m, 4H), 2.33–2.09 (s, 6H), 1.87–1.83 (m, 2H), 1.36–1.13 (m, 14H), 1.19–1.10 (t, J = 7.5 Hz, 6H), 0.92–0.68 (t, J = 6 Hz, 3H); ¹³C NMR: δ 176.58, 171.57, 164.61, 137.39, 135.83, 134.27, 125.66, 68.88, 60.78, 57.50, 53.90, 50.47, 49.30, 32.24, 31.97, 29.90, 29.74, 23.04, 19.10, 14.52, 12.76; HRMS (EI): calcd for C₂₆H₄₂N₄O₃ 458.6445; found, 458.6444.

5.5. Molecular modeling conformational analysis

The X-ray coordinates for phenytoin (DPH) were utilized in this study.¹⁴ The conformations of training set compounds 1–36 (Fig. 2) were determined as previously described.⁶ An additional 15 common local anesthetics were added to this model and modified from a representative X-ray structure. These modified structures were energy-minimized with the Tripos force field,¹⁵ without solvent, using default bond distances and angles and neglecting electrostatics. The minimization was completed by aggregating using the SYBYL/AGGREGATE module for only the X-ray structure atoms and allowing the modified portion to minimize. For internal consistency, we used only the R-configuration for all chiral compounds. The test set analogues 37–45 (Table 3) were modified in SYBYL using the X-ray conformation of lidocaine and representative low-energy conformations were obtained using the Tripos force field. To determine the low-energy conformations for 37–45, we utilized GRIDSEARCH on rotatable bonds over 360° in 1° increments. The atomic charges for all analogues were calculated using AM1 (MOPAC).

Figure 2.

Test set compounds used in developing the CoMFA model.

5.6. Molecular alignment

Benzocaine, lidocaine, butacaine, cocaine, hexylcaine, piperocaine, parethoxycaine, and piperocaine utilized in the training set were fit to overlap the C1 and C4 carbons of the phenyl ring and the adjacent carbon or atom to C1. All hydantoins were fit as previously reported.⁶ Similarly, for analogues 41–45 in the test set, we aligned the C1 and C4 carbons of the phenyl ring and the adjacent nitrogen and the hydantoin ring was fit as previously described.⁶ The hydroxyamides 37–40 were aligned such that the OH group was superimposed with N1 and the carboxyamide was aligned with C4 and N3. The n-alkyl groups at R₁ in analogues 37, 40, and 43–45 in the test set approximated a fully extended conformation following energy minimization, which was arbitrarily selected for this study.

5.7. CoMFA calculations

CoMFA, using default parameters except where noted, was calculated in the QSAR option of SYBYL 6.8 on a Silicon Graphics with an Octane II R12000 dual processor. The CoMFA grid spacing was 2.0 Å in the x, y, and z directions, and the grid region was automatically generated by the CoMFA routine to encompass all molecules with an extension of 4.0 Å in each direction. An sp³ carbon and a charge of +1.0 were used as probes to generate the interaction energies at each lattice point. The default value of 30 kcal/mol was used as the maximum electrostatic and steric energy cutoff.

5.8. Partial least squares (PLS) regression analysis

A single conformer for each compound was selected by the smallest cross-validated residual value. Single conformers of each training set compound were used in both the non-cross-validated model (Table 1) and the cross-validated model (Table 2). We also used the final non-cross-validated CoMFA model to predict the sodium channel binding activities for all low-energy conformations of the test set compounds 37–45 (Table 3).

Table 2.

Cross-validated PLS analysis

Components	s	q2
1	0.746	0.242
2	0.663	0.418
3	0.654	0.452
4	0.644	0.485
5	0.691	0.426
6	0.674	0.473

s, standard error for the estimate of log IC₅₀; q², correlation coefficient or press value = 1.0 − {Σ(Y predicted − Y actual)² ÷ (Y actual − Y mean)²}. Optimum number of components is 4 with q² = 0.485.

Cross-validated (preliminary and final models) and non-cross-validated PLS analyses (final model) were performed within the SYBYL/QSAR routine. Cross-validation of the dependent column (pIC₅₀) and the CoMFA column was performed with 2.0 kcal/mol column filtering. Scaled by the CoMFA standard deviation, the final cross-validated analysis generated an optimum number of components equal to 4 and q² = 0.485 (Table 2). PLS analysis with non-cross-validation, performed with 4 components, gave a standard error of estimate of 0.245, a probability of R² = 0 (n₁ = 4, n₂ = 31) equal to 0.000, an F value (n₁ = 4, n₂ = 31) of 96, and a final q² = 0.926. The relative steric (0.624) and electrostatic (0.376) contributions to the final model were contoured as the standard deviation multiplied by the coefficient at 80% for favored steric (contoured in green) and favored positive electrostatic (contoured in blue) effects and at 20% for disfavored steric (contoured in yellow) and favored negative electrostatic (contoured in red) effects, as shown in Figure 5. On the basis of this analysis, the sodium channel binding activities for low-energy conformers of analogues 37–45, from the test set (Table 3), were predicted.

5.9. Chemistry

The synthesis of compounds 37–45 is shown in Scheme 1 and detailed in the experimental section. Briefly, the aminobenzophenones were generated by reaction of substituted anilines and acids in polyphosphoric acid (PPA). Subsequent reaction with diethylamine, NaHCO₃, and KI in MeOH generated the lidocaine-ketones which were carried on to final product amides and hydantoins.

5.10. Biological data

The structures and literature reference [³H]-BTX displacement data for compounds 1–20, the 15 local anesthetics (21–35), and phenytoin (36) that form the training set, are listed in Figure 2 and Table 1. Table 3 describes the structures and sodium channel binding activities of compounds 37–45, which form the test set.

5.11. [³H]-BTX-B experiment

In the sodium channel evaluation, the IC₅₀, which represents the micromolar concentration of compound required to displace 50% of specifically bound [³H]-BTX) was determined using rat cerebral cortex synaptoneurosomes. To calculate the IC₅₀ value, the percent inhibition at 5–7 concentrations, which spanned the IC₅₀ value, was determined in triplicate and the IC₅₀ value was obtained by a Probit analysis of the concentration versus percent binding curve. Briefly, rat forebrain membranes were incubated with [³H]-Batrachotoxin (30–60 Ci/mmol). Reactions are carried out in 50 mM HEPES (pH 7.4) containing 130 mM choline chloride at 37 °C for 60 min. The reaction was terminated by rapid vacuum filtration of the reaction contents onto glass fiber filters. Radioactivity trapped onto the filters was determined and compared to control values in order to ascertain any interactions of the test compound with the sodium channel site 2 binding site. Aconitine [1 μM] was used as a positive control.

5.12. Cell culture and electrophysiology

Human embryonic cells (HEK) cells stably expressing human Na_v1.2 were grown in DMEM/F12 media (Invitrogen, Corp., CA, USA) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 μg/ml) and G418 (500 μg/ml; Sigma, MO, USA). Cells were grown in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C.

Sodium currents were recorded using the whole-cell configuration of the patch clamp recording technique with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). All voltage protocols were applied using pCLAMP 8 software (Axon, USA) and a Digidata 1322A (Axon, USA). Currents were amplified and low pass filtered (2 kHz) and sampled at 33 kHz. Borosilicate glass pipettes were pulled using a Brown–Flaming puller (model P87, Sutter Instruments Co., Novato, CA) and heat polished to produce electrode resistances of 0.5–1.5MΩ when filled with the following electrode solution (in mM); CsCl 130, MgCl₂ 1, MgATP 5, BAPTA 10, HEPES 5 (pH adjusted to 7.4 with CsOH). Cells were plated on glass coverslips and superfused with solution containing the following composition; (in mM) NaCl 130, KCl 4, CaCl₂ 1, MgCl₂ 5, HEPES 5, and glucose 5 (pH adjusted to 7.4 with NaOH).

Compounds were prepared as 100 mM stock solutions in Dimethly sulfoxide (DMSO) and diluted to desired concentration in perfusion solution. The maximum DMSO concentration used was 0.3% and had no effect on current amplitude. All experiments were performed at room temperature (20–22 °C). After establishing whole-cell, a minimum series resistance compensation of 75% was applied.