Synthesis, Nicotinic Acetylcholine Receptor Binding, Antinociceptive and Seizure Properties of Methyllycaconitine Analogs

Abstract

A series of methyllycaconitine (1a, MLA) analogs was synthesized where the (S)-2-methylsuccinimidobenzoyl group in MLA was replaced with a (R)-2-methyl, 2,2-dimethyl-, 2,3-dimethyl, 2-phenyl- and 2-cyclohexylsuccinimidobenzoyl (1b–f) group. The analogs 1b–f were evaluated for their inhibition of [¹²⁵I]iodo MLA binding at rat brain α7 nicotinic acetylcholine receptors (nAChR). In order to determine selectivity, MLA and the analogs 1b–f were evaluated for inhibition of binding to rat brain α, β nAChR using [³H]epibatidine. At the α7 nAChR, MLA showed a K_i value of 0.87 nM, analogs 1b–e possessed K_i values of 1.68–2.16 nM, and 1f showed a K_i value of 26.8 nM. Surprisingly, the analog 1e containing the large phenyl substituent (K_i = 1.68 nM) possessed the highest affinity. None of the compounds possessed appreciable affinity for α, β nAChRs. MLA antagonized nicotine-induced seizures with an AD₅₀ = 2mg/kg. None of the MLA analogs were as potent as MLA in this assay. MLA and all of the MLA analogs, with the exception of 1b, antagonized nicotine’s antinociceptive effects in the tail-flick assay. Compound 1c (K_i = 1.78 nM at α7 nAChR) with an AD₅₀ value of 1.8 mg/kg was 6.7 times more potent than MLA (AD₅₀ = 12 mg/kg) in antagonizing nicotine’s antinociceptive effects but was 5-fold less potent than MLA in blocking nicotine-induced seizures. Since MLA has been reported to show neuroprotection against β-amyloid_1–42, these new analogs which have high α7 nAChR affinity and good selectivity relative to α, β nAChRs will be useful biological tools for studying the effects of α7 nAChR antagonist and neuroprotection.

1. Introduction

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels formed by the association of five subunits, leading to heteromeric and homomeric structures. The α7 receptor is the only homomeric nAChR widely distributed in the mammalian central nervous system. Binding sites in the brain that show high affinity for [¹²⁵I] α-bungarotoxin ([¹²⁵I] α-BTX) have been correlated with the α7 nAChR.^1–4 Methyllycaconitine (1a, MLA), an alkaloid isolated from Delphinium brownii,^5,6 is reported to be the most potent non-protein competitive α7 nAChR antagonist presently available.⁷ Recent studies have suggested that α7 nAChRs may play an important role in cognitive dysfunction, neurodegenerative diseases, vestibular function, epilepsy, and possibly smoking cessation.⁸ It is particularly interesting to note that MLA (1a) protects against the toxicity produced by the Alzehimer’s disease related peptide β-amyloid_1–42(Aβ₄₂).⁹ The availability of potent and selective α7 nAChR antagonist will enhance our ability to study these processes. Although MLA binds potently to the α7 subtype, recent evidence suggests that MLA can also interact with α4β2 nAChR and presynaptic α3/α6 nAChR.¹⁰ Clearly, there is a need for additional and selective α7 nAChR antagonists to further characterize the α7 nAChR pharmacophore and to further study its biological and pharmacological roles.

In this study, we report the syntheses and inhibition of [¹²⁵I]Iodo-MLA and [³H]epibatidine binding at the α7 and α, β nAChRs for MLA (1a) and the MLA analogs 1b–f. Unfortunately, there is a lack of selective behavioral measure for α7-mediated response. Therefore, we evaluated the ability of MLA analogs to antagonize nicotine-induced seizures and antinociception in mice using both the tail-flick and hot-plate assays in mice because of MLA unique actions in these three assays. These assays were chosen because seizures are mediated at least in part through α7 receptors (sensitive to MLA), the hot-plate assay is α4β2-receptor mediated (insensitive to MLA), and the tail-flick assay is mediated through multiple nAChRs (sensitive to MLA).

1.1. Chemistry

The MLA analogs 1b–f were synthesized as outlined in Scheme 1. The succinic acids 2b–f were converted to the corresponding succinic anhydrides 3 by refluxing the appropriate acid with acetic anhydride for 12 h. Treatment of each anhydride with anthranilic acid in chloroform provided the desired substituted methyllycoctonic acids, which are most likely a mixture of 4b–f and 5b–f except for the symmetrical analog (4d or 5d).¹¹ The mixture of acids was refluxed under a Dean Stark tube in toluene containing triethylamine for 12 h to yield the appropriate 2-substituent or 2,3-disubstituted succinimidobenzoic acids 6b–f. The acids 6b–f were coupled to the primary hydroxyl group of lycoctonine (7) in the presence of p-toluenesulfonyl chloride and pyridine to give the desired MLA analogs 1b–f.¹²

Scheme 1.

1.2. Biology

The K_i values for the inhibition of [¹²⁵I]iodo-MLA and [³H]epibatidine binding for MLA (1a) and the MLA analogs 1b–f were determined using previously reported procedures and are listed in the Table 1.^13,14 MLA (1a) and the MLA analogs 1b–f were evaluated for their ability to antagonize the effects of nicotine in the tail-flick, hot-plate, and seizure test using previously reported procedures and the AD₅₀ values are listed in Table 2.¹⁵

Table 1.

Radioligand binding data for MLA (1a) and the MLA analogs 1b–f^a

compd	RTI-7527-	R1	R2	R3	α7 nAChR [125I]iodo-MLA Ki, nM (-nH)	α, β nAChR [3H]epibatidine Ki, nM (-nH)
1a	MLA	CH3	H	H	0.87 ± 0.13 (1.09 ± 0.15)	739 ± 43 (0.68 ± 0.02)
1b	58	H	CH3	H	2.12 ± 0.47 (1.2 ± 0.34)	352 ± 47 (0.89 ± 0.08)
1c	60	CH3	CH3	H	1.78 ± 0.15 (0.92 ± 0.05)	602 ± 127 (0.93 ± 0.09)
1d	62	CH3	H	CH3	2.46 ± 0.87 (0.98 ± 0.18)	321 ± 33 (0.88 ± 0.08)
1e	59	C6H5	H	H	1.67 ± 0.75 (1.44 ± 0.42)	219 ± 23 (0.75 ± 0.04)
1f	61	C6H11	H	H	26.8 ± 3.62 (0.88 ± 0.21)	1103 ± 400 (1.21 ± 0.02)

^aData represent the mean ± SE from at least three independent experiments. -nH is the Hill coefficient determined from a four-parameter logistic fit to the data.

Table 2.

Pharmacological evaluation of MLA (1a) and MLA analogs 1b–f as nicotinic antagonists. Results were expressed as AD₅₀ (mg/kg) ± Confidence Limits (CL) or % effect at the highest dose tested.^a

compd	Seizures (AD50 mg/kg)	Tail-Flick (AD50 mg/kg)	Hot-Plate (AD50 mg/kg)
1a	2 (0.5–4)	12 (2–59)	15% @ 20
1b	40% @ 50	10% @ 30	0% @ 30
1c	10 (9–11)	1.8 (0.4–9)	13% @ 20
1d	0% @ 50	7.0 (3–15)	25% @ 20
1e	0% @ 50	18.3 (10–31)	7% @ 20
1f	33 (10–113)	7.0 (2.5–18)	0% @ 20

^aDose response curves were determined using a minimum of 4 different doses of test compound and at least eight mice were used per dose group.

2. Results and Discussion

The α7 nAChR subtype is the second most prevalent in the brain and has been implicated as playing a key role in conditions such as nicotine addiction, schizophrenia, Alzheimer’s disease, and epilepsy. Despite recent progress in the synthesis of competitive agonists selective for the α7 nAChR subtype, very few antagonists are known that bind with high affinity and selectivity at this receptor. These include the peptide toxins α-bungarotoxin and the norditerpenoid alkaloid methyllycaconitine (MLA, 1a).

β-Amyloid_1–42 binds to α7 nAChRs with high affinity.¹⁶ Functional studies have shown both α7 nAChR activation^17,18 and inhibition¹⁹ with this peptide. Many studies suggest that α7 nAChRs agonist, such as TC-1698 (8),²⁰ show neuroprotection against β-Amyloid_1–42 effects on α7 nAChR function. It is well known that α7 nAChRs undergo extensive desensitization^21–24 so long-term agonist treatment may not be desirable. Interestingly, Martin et al. reported that α7 nAChR antagonist MLA (1a) has neuroprotective actions against Aβ₄₂-induced neurotoxicity and suggested that α7 nAChR antagonist might be useful pharmacotherapies in treating neurodegenerative disorders such as Alzheimer’s disease.⁹ Unfortunately, lack of highly selective agonists and antagonists have complicated the elucidation of the physiological function of a7 nAChRs.

MLA (1a) is an ester composed of the alcohol lycoctonine (7) and the acid, (S)-2-methylsuccinimidobenzoic acid. A number of truncated analogs of the parent MLA (1a) have been synthesized.²⁵ None of the compounds showed appreciable activity.^25,26 Reports from the literature have shown that neither the alcohol 7 nor the acid 9 possess appreciable affinity for the α7 nAChR.^12,27 Thus, it appears that the (S)-2-methylsuccinimidobenzoyl group connected to the C18 oxygen of lycoctonine is essential for potent α7 nAChR binding. In addition, esters formed from the acid 9 and alcohols possessing parts of the lycoctonine structure as well as esters formed from lycoctonine with other acids were reported to possess low affinity for the α7 nAChR.^27,28 In addition, the lycoctonine ester (10a), a nonditerpenoid alkaloid lacking the 2-(S)-methyl group on the succinimidobenzoyl group of MLA, and the dehydro analog (10b) are reported to show 5- and 10-fold loss in affinity relative to MLA at the α7 nAChR, which suggest that the angular-2-methyl-group on the succinimidobenzoyl group is essentially for potent α7 nAChR binding.^12,29

In the present study, we found that replacement of (S)-2-methylsuccinimidobenzoyl group in MLA (1a, K_i = 0.87 nM), with the enantiomeric (R)-2-methylsuccinimidobenzoyl group to give 1b, K_i = 2.12 nM, resulted in a 2.4-fold loss in affinity for the α7 nAChR. Compounds 1c, K_i = 1.78 nM, and 1d, K_i = 2.62 nM, which possess an additional methyl group at the 2- or 3-position, showed 2.1- and 3-fold loss in affinity, respectively. Surprisingly, replacement of the 2-methyl group on the 2-methylsuccinimido group of MLA with a phenyl ring to give 1e resulted in a K_i value of 1.68 nM. Thus, the MLA pharmacophore will accommodate large flat aromatic groups in this part of the structure. In contrast, replacement of the 2-methyl group with a cyclohexyl group to give 1f resulted in a K_i value of 26.8 nM and thus a 31-fold loss of affinity. This alteration suggests that even though a phenyl ring is allowed in the 2-position of the 2-methylsuccinimido group of MLA, larger more bulky groups like cyclohexyl are not allowed. The weak ability of MLA and all the analogs 1b–f to inhibit [³H]epibatidine binding indicate that these compounds have low affinity for α, β nAChRs.

MLA analogs 1b–1e showed equipotent binding affinities toward the α7 nAChR. However, their in vivo profile in the seizures and tail-flick assays was different from each other. MLA analogs 1d–f were weak antagonists in the tail-flick assay (AD₅₀ = 7–18.3 mg/kg). Analog 1b, which differs from MLA in only the stereochemistry of the methyl group [(R)-methyl] on the succinimidobenzoyl group, was inactive in this assay. The most potent compound, 1c, with an AD₅₀ value of 1.8 mg/kg, was 6.7 times more potent than MLA, which has an AD₅₀ of 12 mg/kg. The nAChR subtype or subtypes that mediate the tail-flick response are not known, but the lack of a high correlation between α7 receptor affinity and antagonistic potency in the tail-flick assay, particularly the discrepancy with enantioselectivity found in the 2-methylbenzoyl group, does not support a role for α7 receptors in mediating antinociception in this assay. It should be noted that earlier reports failed to observe a significant blockade of nicotine’s effects in the tail-flick test by MLA after s.c. injection in mice (5 mg/kg)³⁰ and i.c.v. administration in rats (10 μg).³¹ However, the doses of MLA used in the present study are much higher than the reported ones. This difference in the effect observed could mean MLA lacks nAChR selectivity at higher doses or it is operating by some other mechanism. MLA and all the analogs had poor affinity for α β nAChRs as determined in the [³H]epibatidine binding assay and they were inactive at antagonizing the nicotinic effects in the hot-plate test. This is consistent with earlier observations that α4β2 receptors are required for this effect.³²

MLA was also effective in blocking nicotine-induced seizures in an enantioselective manner. The seizures are mediated by α7 nAChRs and these results are consistent with an α7 mechanism. However, as with the tail-flick assay, there was a poor correlation between α7 receptor affinity and antagonistic potency. The results from the seizure study also support the view that nicotine-induced seizures and antinociception may be mediated by non-α4β2 and non-α7 receptor subtypes. Indeed, pharmacological and genetic approaches initially suggested the involvement of α7 nicotinic subtypes in nicotine-induced seizures. However, additional studies on different mouse inbred strains and transgenic mice have also implicated α4, α5, α3, and β4.³³ The in vivo results show also that analogs 1b and 1e are much less potent than MLA in blocking nicotinic effects despite having good affinity for the α7 nAChR. This suggests that these two analogs have a different selectivity profile than MLA. The difference seen between in vitro binding affinity and in vivo potency in the various behavioral tests suggests that these MLA analogs have lower selectivity for the α7 receptor subtype. Ultimately, in vitro functional selectivity on various nAChRs subtypes in expressed cells would confirm this in vivo selectivity. While MLA and its derivatives antagonize both nicotine-induced antinociception and seizures, they apparently do so by acting at multiple sites or different mechanisms, since there is little correlation between antagonistic potencies in these two measures. The fact that MLA does not readily cross the blood brain barrier raises the question of whether pharmacokinetic differences may explain some of these findings. However, it seems unlikely that the pharmacokinetics would be so different among these analogs to explain their pharmacological dissimilarities.

3. Conclusions

Replacement of the (S)-2-methylsuccinimidobenzoyl group in MLA (1a) by an (R)-2-methylsuccinimidobenzoyl to give 1b caused only a small reduction in binding at the α7 nAChR, however, 1b showed no ability to antagonize seizures and antinociception induced by nicotine. Surprisingly, compound 1c which has methyl groups present in both the (2S) and (2R)-position of the succinimido group is 6.7 times more potent than MLA in antagonizing the antinociception induced by nicotine in the tail-flick test. However, 1c has essentially the same affinity for the α7 nAChR as 1b. These analogs have a pharmacological profile distinct from that of MLA despite relatively modest changes in structure. It would seem that they are acting at sites other than α7 receptors. Regardless of their mechanism of action, the (S)-2-methylsuccinimidobenzoyl group is a critical for receptor. Further development of MLA analogs may lead to even better probes for characterizing these receptor sites and to biological tools for studying the effects of α7 nAChR antagonist in several neurological disorders.

4. Experimental

Melting points were determined on a Thomas-Hoover capillary tube apparatus and are not corrected. Elemental analyses were obtained by Atlantic Microlabs, Inc. and are within ± 0.4% of the calculated values. All optical rotations were determined at the sodium D-line using a Rudolph Research Autopol III polarimeter (1-dm cell).¹H NMR were determined on a Bruker Avance DPX-300 or Bruker AMX-500 spectrometer using tetramethylsilane as an internal standard. Silica gel 60 (230–400 mesh) was used for all column chromatography. All reactions were followed by thin-layer chromatrography using Whatman silica gel 60 TLC plates and were visualized by UV or by charring using 5% phosphomolybdic acid in ethanol. All solvents were reagent grade.

MLA was isolated from delphenium elatum (pacific giant) seeds (purchased from Flowers of Tomorrow, Inc., Parma, Idaho 83660) and hydrolyzed to lycoctonine (7).^34,35 [³H]epibatidine (s.a. = 66.6 ci/mmol) was obtained from DuPont New England Nuclear (Boston, MA).

(R)-Methylsuccinamic Acids (4b and 5b)

A suspension of (R)-methylsuccinic acid (2b) (0.89 g, 6.75 mmol) in acetic anhydride (10 mL) was heated to reflux overnight. After removal of the excess of acetic anhydride by distillation, the residue was triturated and dried under reduced pressure to give the anhydride 3b that was dissolved in 10 mL of CHCl₃ and added to a suspension of anthranilic acid (0.925 g, 6.75 mmol) in 10 mL of CHCl₃. The reaction mixture was heated on a steam bath for 30 min, and the CHCl₃ removed under reduced pressure. The product was purified on silica gel using hexane:ethyl acetate:methanol:acetic acid (125:45:5:4) as eluent to give 1.56 g (92%) of succinamic acids (4b and 5b): mp 165–167 °C;¹H NMR (CD₃OD) δ 1.25–1.36 (2 d, 3H), 2.50–2.60 (m, 1H), 2.76–2.99 (m, 2H), 7.12–7.18 (t, 1H, aromatic), 7.53–7.56 (t, 1H, aromatic), 8.08–8.11 (d, 1H, aromatic), 8.54–8.57 (d, 1H, aromatic).

(R)-Methyllycactonic Acid (6b)

A solution of the above (R)-methylsuccinamic acids (4b and 5b) (1.5 g, 6 mmol) and triethylamine (3.05 g, 30.1 mmol) in 200 mL of toluene was heated to reflux overnight. The toluene was removed by simple distillation, and the residue was purified on silica gel using hexane:ethyl acetate:methanol:acetic acid (125:45:5:4) as eluent to obtain 0.9 g (64%) of pure (R)-methyllycactonic acid (6b): mp 117–119 °C;¹H NMR (CD₃OD) δ 1.38–1.40 (d, 3H), 2.41–2.61 (m, 1H), 3.04–3.15 (m, 2H), 7.28–7.31 (d, 1H, aromatic), 7.56–7.58 (t, 1H, aromatic), 7.65–7.68 (t, 1H, aromatic), 8.11–8.14 (d, 1H, aromatic). Anal. (C₁₂H₁₁NO₄•0.5H₂O) C, H, N.

(R)-Methyllycaconitine (1b) Citrate

To a stirred solution of imido acid 6b (49.9 mg, 0.214 mmol) in 1 mL of dry pyridine was added 76.4 mg (0.428 mmol) of p-toluenesulfonyl chloride. The mixture was cooled to 0 °C, 100 mg (0.214 mmol) of lycoctonine was added, and the solution was stirred for additional 1 h at 0 °C and then placed in the refrigerator for 24 h. The yellow solution was dissolved in 10 mL of NH₄OH (aq, pH 9) and extracted with 3 × 10 mL of CHCl₃. Organic phases were combined and washed with 10 mL of NH₄OH (aq, pH 9) and dried over anhydrous Na₂SO₄. After removal of the solvent, the residue was purified on silica gel using cyclohexane:CHCl₃:(Et)₃N (6:4:1) as eluent to give 66 mg (45%) of pure (R)-methyllycaconitine (1b). An analytical sample was prepared as the citrate salt: mp 99 °C (dec.); [α]²⁰_D +48.3° (c, 0.64, EtOH) (free base). Anal. (C₄₃H₅₈N₂O₁₇•1.5H₂O) C, H, N.

2,2-Dimethylsuccinamic Acids (4c and 5c)

2,2-Dimethylsuccinic anhydride (3c) (2.0 g, 15.6 mmol) was dissolved in 15 mL of CHCl₃ and added to a suspension of anthranilic acid (2.14 g, 15.6 mmol) in 15 mL of CHCl₃. The reaction mixture was heated on a steam bath for 30 min and CHCl₃ removed under reduced pressure. The product was purified on silica gel using hexane:ethyl acetate:methanol:acetic acid (125:45:5:4) as eluent to obtain 3.5 g (85%) of 2,2-dimethylsuccinamic acids (4c and 5c): mp 138–140 °C;¹H NMR (CDCl₃) δ 1.41 (s, 6H, 2CH₃), 2.78 (s, 2H, -CH₂), 7.07–7.20 (t, 1H, aromatic), 7.56–7.61 (t, 1H, aromatic), 8.05–8.08 (d, 1H, aromatic), 8.77–8.80 (d, 1H, aromatic). Anal. (C₁₃H₁₅NO₅•0.5H₂O) C, H, N.

2,2-Dimethyllycactonic Acid (6c)

A solution of the above 2,2-dimethylsuccinamic acids (4c and 5c) (3.5 g, 13.2 mmol) and triethylamine (6.6 g, 66 mmol) in 300 mL of toluene was heated to reflux overnight. The toluene was removed by simple distillation, and the residue was purified on silica gel using hexane:ethyl acetate:methanol:acetic acid (125:45:5:4) as eluent to obtain 1.3 g (40%) of pure 2,2-dimethyllycactonic acid (6c): mp 134–136 °C;¹H NMR (CDCl₃) δ 1.39 (s, 6H, 2CH₃), 2.70 (s, 2H, -CH₂), 7.23–7.26 (d, 1H, aromatic), 7.47–7.52 (t, 1H, aromatic), 7.63–7.68 (t, 1H, aromatic), 8.11–8.14 (d, 1H, aromatic). Anal. (C₁₃H₁₃NO₄•0.25H₂O) C, H, N.

2,2-Dimethyllycaconitine (1c) Citrate

To a stirred solution of imido acid 6c (105.8 mg, 0.428 mmol) in 2 mL of dry pyridine was added 152.8 mg (0.856 mmol) of p-toluenesulfonyl chloride. The mixture was cooled to 0 °C, 100 mg (0.214 mmol) of lycactonine was added, and the solution was stirred for an additional 1 h at 0 °C and then placed in the refrigerator for 15 h. The deep-red solution was dissolved in 15 mL of NH₄OH (aq, pH 9) and extracted with 3 × 10 mL of CHCl₃. Organic phases were combined and washed with 10 mL of NH₄OH (aq, pH 9) and dried over anhydrous Na₂SO₄. After the solvent was removed, the residue was purified on silica gel using cyclohexane:CHCl₃:(Et)₃N (6:4:1) as eluent to obtain 117.5 mg (79%) of the pure 2,2-dimethyllycaconitine (1c). An analytical sample was prepared as the citrate salt: mp 104 °C (dec.). Anal. (C₄₄H₆₀N₂O₁₇•H₂O) C, H, N.

2,3-Dimethylsuccinamic Acids (4d and 5d)

A suspension of 2,3-dimethylsuccinic acid (2d) (2.0 g, 13.7 mmol) in acetic anhydride (15 mL) was heated to reflux overnight. After removal of the excess of acetic anhydride by distillation, the residue was triturated and dried under reduced pressure to give the anhydride 3d which was dissolved in 15 mL of CHCl₃ and added to a suspension of anthranilic acid (1.88 g, 13.7 mmol) in 15 mL of CHCl₃. The reaction mixture was heated on a steam bath for 30 min and the CHCl₃ removed under reduced pressure. The product was purified on silica gel using hexane:ethyl acetate:methanol:acetic acid (125:45:5:4) as eluent to obtain 3 g (83%) of 2,3-dimethylsuccinamic acids 4d and 5d:¹H NMR (CDCl₃) δ 130–138 (m, 6H), 2.75–2.85 (m, 2H), 7.07–7.12 (t, 1H, aromatic), 7.54–7.59 (t, 1H, aromatic), 8.01–8.04 (d, 1H, aromatic), 8.64–8.67 (d, 1H, aromatic).

2,3-Dimethyllycactonic Acid (6d)

A solution of the above 2,3-dimethylsuccinamic acids 4d and 5d (3 g, 11.3 mmol) and triethylamine (5.65 g, 56.5 mmol) in 300 mL of toluene was heated to reflux overnight. The toluene was removed by simple distillation, and the residue was purified on silica gel using hexane:ethyl acetate:methanol:acetic acid (125:45:5:4) as eluent to obtain 0.8 g (29%) of pure 2,3-dimethyllycactonic acid (6d): mp 163–165 °C;¹H NMR (CDCl₃) δ 1.32–1.44 (2 d, 6H), 2.62–3.16 (2 m, 2H), 7.26–7.29 (d, 1H, aromatic), 7.51–7.56 (t, 1H, aromatic), 7.67–7.70 (t, 1H, aromatic), 8.17–8.20 (d, 1H, aromatic). Anal. (C₁₃H₁₃NO₄.0.25H₂O) C, H, N.

2,3-Dimethyllycaconitine (1d) Citrate

To a stirred solution of imido acid 6d (105.8 mg, 0.428 mmol) in 2 mL of dry pyridine was added 152.8 mg (0.856 mmol) of p-toluenesulfonyl chloride. The mixture was cooled to 0 °C and 100 mg (0.214 mmol) of lycactonine was added, and the solution was stirred for additional 1 h at 0 °C and was refrigerated for 18 h. The deep-red solution was taken up in 15 mL of NH₄OH (aq, pH 9) and extracted with 3 × 10 mL of CHCl₃. Organic phases were combined and washed with 10 mL of NH₄OH (aq, pH 9) and dried over anhydrous Na₂SO₄. After removal of the solvent, the residue was purified on silica gel using cyclohexane:CHCl₃:(Et)₃N (6:4:1) as eluent to obtain 74.6 mg (50%) of pure 2,3-dimethyllycaconitine (1d). An analytical sample was prepared as citrate salt: mp 84 °C (dec.). Anal. (C₄₄H₆₀N₂O₁₇•0.5H₂O) C, H, N.

Phenylsuccinamic Acids (4e and 5e)

Phenylsuccinic anhydride (3e) (2.0 g, 11.36 mmol) was dissovled in 15 mL of CHCl₃ and was added to a suspension of anthranilic acid (1.55 g, 11.36 mmol) in 15 mL of CHCl₃. The reaction mixture was heated on a steam bath for 30 min, and then the CHCl₃ solvent was evaporated to dryness under reduced pressure. The product was purified on silica gel, eluting with hexane:ethyl acetate:methanol :acetic acid (125:45:5:4) to obtain 3 g (84%) phenylsuccinamic acids (4e and 5e): mp 169–171 °C;¹H NMR (DMSO) δ 2.75–2.82 (dd, 1H), 3.11–3.20 (dd, 1H), 4.02–4.10 (dd, 1H), 7.12–7.17 (m, 1H, aromatic), 7.26–7.38 (m, 5H, -C₆H₅), 7.55–7.60 (m, 1H, aromatic), 7.93–7.99 (m, 1H, aromatic), 8.44–8.50 (m, 1H, aromatic).

Phenyllycactonic Acid (6e)

A solution of the above phenylsuccinamic acids (4e and 5e) (3 g, 9.58 mmol) and triethylamine (4.79 g, 47.9 mmol) in 250 mL of toluene was heated to reflux overnight. The toluene solvent was removed by simple distillation, and the residue was purified on silica gel, eluting with hexane:ethyl acetate:methanol:acetic acid (125:45:5:4) to obtain 1.1 g (39%) of pure phenyllycactonic acid (6e): mp 80–82 °C;¹H NMR (CDCl₃) δ 2.98–3.11 (dd, 1H), 3.36–3.45 (dd, 1H), 4.21–4.26 (dd, 1H), 7.33–7.40 (m, 6H, aromatic), 7.56–7.61 (m, 1H, aromatic), 7.72–7.76 (m, 1H, aromatic), 8.22–8.25 (m, 1H, aromatic). Anal. (C₁₇H₁₃NO₄•0.6H₂O) C, H, N.

Phenyllycaconitine (1e) Citrate

To a stirred solution of imido acid 6e (126.3 mg, 0.428 mmol) in 2 mL of dry pyridine was added 152.8 mg (0.856 mmol) of p-toluenesulfonyl chloride. The mixture was cooled to 0 °C, 100 mg (0.214 mmol) of lycactonine was added, and the solution was stirred for additional 1 h at 0 °C and was then placed in the refrigerator for 18 h. The deep-red solution was taken up in 15 mL of NH₄OH aqueous solution (pH ~9) and extracted with 3 × 10 mL of CHCl₃. Organic phases were combined and washed with 10 mL of NH₄OH aqueous solution (pH ~9) and dried over anhydrous Na₂SO₄. After the solvent was removed, the residue was purified on silica gel, eluted with cyclohexane:CHCl₃:(Et)₃N (6:4:1) to obtain 100 mg (63%) of the pure phenyllycaconitine (1e). An analytical sample was prepared as the citrate salt: mp 105 °C (dec.). Anal. (C₄₈H₆₀N₂O₁₇•0.5H₂O) C, H, N.

Cyclohexylsuccinamic Acids (4f and 5f)

A suspension of cyclohexylsuccinic acid (2f) (2.0 g, 10 mmol) in acetic anhydride (15 mL) was heated to reflux overnight. The excess of acetic anhydride was removed by sample distillation, the residue (3f) was triturated and dried under reduced pressure. The anhydride (3f) (1.78 g, 9.78 mmol) was dissolved in 15 mL of CHCl₃ and was added to a suspension of anthranilic acid (1.34 g, 9.78 mmol) in 15 mL of CHCl₃. The reaction mixture was heated on a steam bath for 30 min, and the CHCl₃ solvent was evaporated to dryness under reduced pressure. The product was purified on silica gel, eluting with hexane:ethyl acetate:methanol :acetic acid (125:45:5:4) to obtain a 2.9 g (92%) of cyclohexylsuccinamic acids (4f and 5f): mp 171–173 °C,¹H NMR (CDCl₃) δ 1.05–2.04 (m, 11H, -C₆H₁₁), 2.61–2.68 (m, 1H), 2.76–2.88 (m, 2H), 7.08–7.13 (t, 1H, aromatic), 7.56–7.61 (t, 1H, aromatic), 8.06–8.08 (d, 1H, aromatic), 8.70–8.73 (d, 1H, aromatic).

Cyclohexyllycactonic Acid (6f)

A solution of the above cyclohexylsuccinamic acids (4f and 5f) (2.77 g, 8.68 mmol) and triethylamine (4.34 g, 43.4 mmol) in 300 mL of toluene was heated to reflux overnight. The toluene solvent was removed by simple distillation. The residue was purified on silica gel, eluting with hexane:ethyl acetate:methanol:acetic acid (125:45:5:4) to obtain 1.1 g (42%) of pure cyclohexyllycactonic acid (6f): mp 68–70 °C;¹H NMR (CDCl₃) δ 1.12–1.35 (m, 5H), 1.69–1.81 (m, 5H), 2.06–2.11 (m, 1H), 2.71–3.10 (m, 3H), 7.22–7.26 (d, 1H, aromatic), 7.51–7.56 (t, 1H, aromatic), 7.67–7.72 (t, 1H, aromatic), 8.16–8.19 (d, 1H, aromatic). Anal. (C₁₇H₁₉NO₄•0.33H₂O) C, H, N.

Cyclohexyllycaconitine (1f) Citrate

To a stirred solution of the imido acid (6f) (128.98 mg, 0.428 mmol) in 2 mL of dry pyridine was added 152.8 mg (0.856 mmol) of p-toluenesulfonyl chloride. The mixture was cooled to 0 °C, and 100 mg (0.214 mmol) of lycactonine was added. The solution was stirred for additional 1 h at 0 °C and was then placed in the refrigerator for 18 h. The deep-red solution was taken up in 15 mL of NH₄OH aqueous solution (pH ~9) and extracted with 3 × 10 mL of CHCl₃. Organic phases were combined and washed with 10 mL of NH₄OH aqueous solution (pH ~9) and dried over anhydrous Na₂SO₄. After the solvent was removed, the residue was purified on silica gel, eluting with cyclohexane:CHCl₃:(Et)₃N (6:4:1) to obtain 105.7 mg (66%) of the pure cyclohexyllycaconitine (1f). An analytical sample was prepared as citrate salt: mp 72 °C (dec.). Anal. (C₄₈H₆₆N₂O₁₇•1.5H₂O) C, H, N.

[¹²⁵I]Iodo-MLA Binding Assay

Frozen male rat cerebral cortex (includes hippocampus; Pel-Freez Biologicals, Rogers, AK) was homogenized (polytron) in 39 volumes of ice-cold 50 mM Tris buffer (assay buffer; pH 7.4 @ 4 °C) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, and 1 mM MgCl₂. The homogenate was centrifuged at 35,000 × g for 10 min at 4 °C and the supernatant discarded. The pellet was washed twice more with the original volume of buffer. After the last centrifugation, the pellet was resuspended in 1/10 the original volume of buffer and stored at −80 °C until needed. The competition binding experiments were carried out in 1.4 mL polypropylene tubes (Matrix Technologies Corporation, Hudson, NH) in a 96-well array. In a final volume of 0.5 mL triplicate samples contained approximately 3 mg of tissue (wet weight; added last), 40–50 pM [¹²⁵I]iodo-MLA and 10–12 different concentrations of the test compounds. The dilutions of the test compounds were made using assay buffer containing 10% DMSO (1% final concentration). The total binding and nonspecific binding (300 uM nicotine) samples also contained a final concentration of 1% DMSO. The test compounds were pipetted using a MultiProbe II_EX (Packard Instruments, Meriden, CT) robotic liquid handling system. The samples were incubated on ice for 2 h. A MultiMate harvester (Packard) was used to separate bound radioligand from free by rapid vacuum filtration onto GF/B filters presoaked for at least 30 min in assay buffer containing 0.15% bovine serum albumin. The filters were washed with approximately 4 mL of ice cold 10 mM Tris buffer (pH 7.4 @ 4 °C) without salts and dried prior to the addition of 35 μL of Microscint 20 scintillant (Packard). The amount of radioligand remaining on each filter was determined using a Packard TopCount microplate scintillation counter (70% efficiency).

[³H]Epibatidine Binding Assay

Adult male rat cerebral cortices (Pelfreeze Biological, Rogers, AK) were homogenized in 39 volumes of ice-cold 50 mM Tris buffer (pH 7.4 at 4 °C) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, and 1 mM MgCl₂ and centrifuged at 37,000 g for 10 min at 4 °C. The supernatant was discarded, the pellet resuspended in the original volume of buffer, and the wash procedure repeated twice more. After the last centrifugation, the pellet was resuspended in 1/10 its original homogenization volume and stored at −80 °C until needed. In a final volume of 0.5 mL, each assay tube contained 3 mg wet weight of male rat cerebral cortex homogenate (added last), 0.5 nM [³H]epibatidine (NEN Life Science Products, Wilmington, DE) and one of 10–12 different concentrations of test compound dissolved in buffer (pH 7.4 at room temperature) containing 10% DMSO resulting in a final DMSO concentration of 1%. Total and nonspecific bindings were determined in the presence of vehicle and 300 uM (–)-nicotine, respectively. After 4-h incubation at room temperature, the samples were vacuum-filtered over GF/B filter papers presoaked in 0.03% polyethylenimine using a Brandel 48-well harvester and washed with 6 mL of ice-cold buffer. The amount of radioactivity trapped on the filter was determined by standard liquid scintillation techniques in a TriCarb 2200 scintillation counter (Packard Instruments, Meriden, CT) at approximately 50% efficiency.

Data Handling

The specific binding data were fit using the nonlinear regression equations in Prism (GraphPad Prism v. 3.0; GraphPad Software, San Diego, CA). The Cheng-Prusoff equation³⁶ [K_i = IC₅₀/(1+([L]/K_d)] was used to calculate the K_i from the IC₅₀. The data are reported as the mean ± S.E.M. from at least three independent experiments. K_d values for [¹²⁵I]iodo-MLA and [³H]epibatidine were 1.8 and 0.02 nM, respectively. These K_d values were determined under conditions identical to their respective assays.

Tail-Flick Test

Antinociception was assessed by the tail-flick method of D’Amour and Smith.³⁷ A control response (2–4 sec) was determined for each mouse before treatment, and a test latency was determined after drug administration. In order to minimize tissue damage, a maximum latency of 10 sec was imposed. Antinociceptive response was calculated as percent maximum possible effect (% MPE), where %MPE = [(test-control)/(10-control)] × 100. Groups of 8 to 12 animals were used for each dose and for each treatment. The mice were tested 5 min after i.t. injections of epibatidine analogs for the dose-response evaluation. Eight to 12 mice were treated per dose, and a minimum of 4 doses were performed for dose-response curve determination. Antagonism studies were carried out by pretreating the mice s.c. with either saline MLA (1a) or MLA analogs 1b–f at different times before nicotine. The animals were tested 5 min after administration of nicotine.

Hot-Plate Test

Mice were placed into a 10 cm wide glass cylinder on a hot plate (Thermojust Apparatus) maintained at 55.0 C. Two control latencies at least 10 min apart were determined for each mouse. The normal latency (reaction time) was 8 to 12 sec. Antinociceptive response was calculated as percent maximum possible effect (% MPE), where %MPE = [(test-control)/40-control) × 100]. The reaction time was scored when the animal jumped or licked its paws. Eight mice per dose were injected s.c. with epibatidine analogs and tested 5 min thereafter in order to establish a dose-response curve.

Nicotine-Induced Seizures

Male ICR (20–25 g) obtained from Harlan Laboratories (Indianapolis, IN) were used throughout the study. Following injection of nicotine, each animal was placed in a 30 × 30 cm² Plexiglas cage and observed for 3 min. Whether a clonic seizure occurred within a 3-min time period was noted for each animal after s.c. administration of nicotine. This time was chosen because seizures occur very quickly after nicotine administration. Antagonism studies were carried out by pretreating the mice s.c. with either saline or different MLA analogs 10 min before nicotine. The percentage of animals exhibiting a seizure was calculated and dose-response curves were constructed and AD₅₀ value determined for each of the MLA analogs.

Figure 1.

Figure 2.

Figure 3.