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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2012 October 27.
Published in final edited form as:
PMCID: PMC3200116
NIHMSID: NIHMS326516

Deconstruction of the α4β2 Nicotinic Acetylchloine (nACh) Receptor Positive Allosteric Modulator des-Formylflustrabromine (dFBr)

Abstract

des -Formylflustrabromine (dFBr; 1), perhaps the first selective positive allosteric modulator of α4β2 neuronal nicotinic acetylcholine (nACh) receptors, was deconstructed to determine which structural features contribute to its actions on receptors expressed in Xenopus ooycytes using 2-electrode voltage clamp techniques. Although the intact structure of 1 was found optimal, several deconstructed analogs retained activity. Neither the 6-bromo substituent nor the entire 2-position chain is required for activity. In particular, reduction of the olefinic side chain of 1, as seen with 6, not only resulted in retention of activity/potency but in enhanced selectivity for α4β2 versus α7 nACh receptors. Pharmacophoric features for the allosteric modulation of α4β2 nACh receptors by 1 were identified.

Introduction

The neurotransmitter acetylcholine (ACh) has been implicated in several neurological and neuropsychiatric disorders, such as Alzheimer’s disease, autism, and schizophrenia, and might play a role in cognition, memory, and pain.13 Receptors for ACh are of two broad types: muscarinic receptors and nicotinic receptors.1,4 Muscarinic receptors (m1-m5) are G-protein coupled, whereas nicotinic acetylcholine (nACh) receptors are associated with ion channels and are composed of five membrane-spanning subunits.1,4 Neuronal nACh receptors, an object of the present study, consist of either α or α and β subunits; multiple neuronal nACh receptor types are possible because several isoforms of α and β subunits exist (α2-α10 and β2-β4).4 The two most prevalent neuronal nACh receptor subtypes in human brain are the heteromeric α4β2 receptors and the homomeric α7 receptors. A large number of nACh receptor agonists and antagonists have been identified [reviewed: 4], but a persistent problem is their low (or lack of) selectivity amongst the various nACh receptor subtypes.

Apart from direct-acting agonists and competitive antagonists that bind at the same (i.e., orthosteric) site as ACh, an alternate approach to influence or modulate the actions of ACh is via allosteric modulation of its receptors.e.g. 2,3,5 Positive allosteric modulators, for example, can enhance the actions of an endogenous ligand (e.g. a neurotransmitter such as ACh) by interaction at a binding site distinct from the orthosteric site. By definition, endogenous neurotransmitters and other orthosteric ligands normally do not interact with high affinity at an allosteric binding site(s) and, conversely, allosteric ligands do not bind with high affinity at the orthosteric site. Consequently, due to lack of suitable design templates, it is virtually impossible, in the absence of any other available information (e.g. on some other allosteric agent that might bind in a similar fashion), to design allosteric agents de novo. Yet, the availability of allosteric agents can be advantageous because they typically fail to desensitize receptors, are usually active only in the presence of the orthosteric agonist, and might achieve selectivity of effect if different subtypes of receptors possess different allosteric binding sites. 3

Several years ago, a positive allosteric modulator of α4β2 nACh receptors was serendipitously discovered: des-formylflustrabromine (dFBr; 1).6,7 Initially isolated from a North Sea marine organism,6 1 was later synthesized as a water soluble salt.8 In addition to increasing whole cell current when co-applied with effective concentrations of ACh to α4β2-containing preparations, 1 failed to potentiate the actions of ACh at α3β2, α3β4, α4β4, and α7 nACh receptors expressed in Xenopus oocytes.79 As such, 1 can now serve as a template for the development of newer, more selective agents.

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Because of its actions as a positive allosteric modulator, des-formylflustrabromine (1) represents a novel lead structure; however, nothing is known about its structure-activity relationships for producing this effect. In order to understand which structural features of des-formylflustrabromine (1) are required for, or contribute to, this action, the structure of 1 was “deconstructed”. Other goals are to identify a pharmacophore for the action of 1 as a positive allosteric modulator at α4β2 nACh receptors and, more long-term, to identify structures that retain this action but more readily lend themselves to convenient scale-up synthesis in order to have available sufficient quantities of compound for in vivo behavioral (and other) studies. We began our investigation by attempting to determine exactly what it is, structurally, that makes des-formylflustrabromine (1) a positive allosteric modulator at α4β2 nACh receptors.

Chemistry

Compound 1(as its HCl salt) was prepared exactly as previously reported.8 That is, 6-bromoindole (13; Scheme 1) was subjected to a Speeter/Anthony synthesis (i.e., reaction of an indole with oxalyl chloride, treatment of the glyoxylyl chloride product with methylamine followed by reduction of the resulting glyoxylamide with a hydride reducing agent – dimethylethylamine-alane in the case of 1) to afford tryptamine 3; protection of the basic amine of 3 with a Boc group (i.e., 14) followed by prenylation (to 16), and acidic deprotection provided 1. The primary amine counterpart of 1 (i.e., 2) was prepared in the same manner by prenylation of N-Boc-protected 6-bromotryptamine, rather than 6-bromo-N-methyltryptamine (14) used in the synthesis of 1, followed by de-protection.

Scheme 1
Synthesis of target compounds 4–6 and 10. The synthesis of 1 has been previously reported8 and is shown here only for comparison of methods. Reagents: (a) (1) CO2Cl2, Et2O; (2) MeNH2; (3) Dimethylethylamine-alane, THF; (b) (Boc)2O, Et3N, DMF; ...

Compound 14,8 however, was a useful intermediate for the synthesis of several other desired targets (Scheme 1). Treatment of 14 with tert-butyl hypochlorite followed by allyl tributyltin and boron trifluoride afforded intermediate 15. Reduction of the double bond of 15 using sodium borohydride and cobalt chloride,10 followed by de-protection, provided 4.

De-protection of 15 without the intervening reduction step should have afforded 5. It appears, however, that some double-bond migration occurred from the 2′-position to the 1′-position. That is, although elemental analysis for 5 was correct, the 1H-NMR spectrum of the product showed several minor spurious peaks, such as, for example, a small doublet of doublets at δ 1.85 (presumably a C-CH3 signal). Elemental analysis and integration of the 1H-NMR signals indicated that two different positional isomers with the same elemental composition were obtained with 86% of the product being the desired target 5 and 14% being the more conjugated 1′-alkenyl derivative. Neither isomer could be obtained in pure form. Compound 15 was also employed in the synthesis of 10 (Scheme 1); olefinic reduction of 15 and hydrogenolysis of the halogen afforded 10 following de-protection.

The double bond of 168 was reduced using the same (i.e., sodium borohydride and cobalt chloride) reduction procedure described above (to yield 17), and de-protection of 17 afforded the desired 6 (Scheme 1).

The des-bromo analog of 1 (i.e. 7) was obtained from 18 (Scheme 2). Compound 18 was prepared in modest yield according to a reported procedure.11 However, upon changing the reaction solvent from dioxane to methylene chloride, the product could be obtained in 80% yield as a white solid (mp 86–89 °C) with a 3-h reaction time relative to that reported with dioxane (15 h). Compound 8 was prepared from the same intermediate (i.e. 19) except that the double bond was catalytically reduced prior to de-protection.

Scheme 2
Synthesis of target compounds 7 and 8. Reagents: (a) (1) t-BuOCl, THF; (2) prenyl 9-BBN, THF; (b) 10% Pd/C, H2, 50 psi; (c) TFA, CH2Cl2.

Compounds 9 and 12 were prepared in a similar fashion (shown for 12 in Scheme 3). That is, the requisite indole (e.g. 23) was treated with 1-dimethylamino-2- nitroethylene to afford nitroalkene 24 which was readily reduced to primary amine 25; acylation of 25 using ethyl chloroformate followed by reduction of the resulting carbamate 26 using lithium aluminum hydride provided the desired target 12. Compound 9 was prepared from 2-sec-butylindole (21) in like manner. The tertiary butyl derivative 11 was prepared via a classical Speeter/Anthony synthesis from 2-tert-butylindole.

Scheme 3
Synthesis of compound 12. Reagents: (a) 1-Dimethylamino-2-nitroethylene, TFA; (b) LiAlH4, THF; (c) Cl-COOC2H5, CHCl3. The exact same method was employed to synthesize 9 from 2-sec-butylindole (21).

Results and Discussion

A near-maximal effect in the present functional assays was consistently observed at an ACh concentration of 10 μM. The deconstructed analogs of 1 were examined using two-electrode voltage clamp recordings with α4β2 nACh receptors expressed in Xenopus oocytes in the presence and absence of this concentration of ACh. None of the analogs produced an effect in the absence of ACh. Several of the deconstructed analogs of 1 potentiated the effect achieved by ACh (Table 1). As with 1 itself, all deconstructed analogs that potentiated the action of ACh produced an inhibitory effect at high concentrations (representative tracings are shown in Figures 1 and and2).2). The mechanism underlying this action requires further investigation; however, data obtained with 1 suggest that the mechanism of the inhibitory component might be due to open channel blockade.9

Figure 1
Potentiation of ACh-evoked responses by representative deconstructed analogs des-formylflustrabromine (1). Responses were elicited by application of 100 μM ACh to Xenopus oocytes expressing α4β2 nicotinic receptors. Test compounds ...
Figure 2
Concentration/response curves for representative deconstructed analogs of des-formylflustrabromine (1). Deconstructed compounds 6 (A), 7 (B), and 8 (C) were co-applied with 100 μM ACh (i.e., a concentration of ACh that produced the maximal effect ...
Table 1
Effect of deconstructed analogs of des-formylflustrabromine (1) on the action (potentiation or antagonism) of the functional activity of ACh at α4β2 nACh receptors expressed in Xenopus oocytes as measured using two-electrode voltage clamp ...

At concentrations slightly higher than those required to potentiate its ACh-enhancing action in the α4β2 receptor preparation, 1 inhibited (but failed to potentiate) the action of ACh in an α7 nACh receptor preparation.8 As an initial measure of selectivity, the compounds prepared in this investigation were also examined at α7 nACh receptors in functional assays (Table 2).

Table 2
Potency of examined analogs of des-formylflustrabromine (1), relative to 1, to inhibit the effect of ACh on human α7 nACh receptors expressed in Xenopus oocytes as measured using two-electrode voltage clamp methods.

Effect of deconstructed analogs of 1 on α4β2 nACh receptor function

The pEC50 for 1 (6.48; Table 1; EC50 ca. 0.32 μM) for the potentiation of 100 QM ACh was quite comparable to what we originally reported (pEC50 ca. 6.8; EC50 = 0.12 μM);8 the small difference likely reflects experimental variation and the much greater number of determinations that now have been performed with 1. The N-des-methyl primary amine counterpart of 1 (i.e., 2; pEC50 = 6.29; Table 1) was found to be about half as potent as 1 as a positive allosteric modulator at α4β2 receptors. Hence, all subsequent compounds retained the N-methyl group associated with 1.

The next several compounds addressed the role of the indole 2-position substituent. Complete elimination of the 2-position substituent (i.e., N-methyl-6-bromotryptamine; 3) resulted in a compound that failed to enhance the action of ACh at the highest concentrations at which it was evaluated (Table 1). Similar results were obtained with the simple n-propyl analog 4 (Table 1). Either unsaturation and/or at least one of the gem dimethyl groups is important for the ability of 1 to behave as a positive allosteric modulator at α4β2 nACh receptors. The propenyl compound 5, lacking the gemdimethyl groups of 1, also lacked potentiating activity, whereas the saturated gemdimethyl analog of 1, 6 (pEC50 = 6.86), was at least as potent, if not twice as potent, as 1. Evidently, the gem-dimethyl groups, but not the unsaturation associated with the chain, is required for allosteric action.

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Before continuing to investigate 2-position substitution, the necessity of the 6-bromo group was examined. des-Bromo 1 and des-bromo 6 (i.e., 7 and 8; pEC50 = 5.14 and 5.52, respectively) (Table 1) retained the allosteric action of their parents but were substantially less potent. Apparently, the bromo group contributes to potency, but its presence is not required for the compounds to behave as positive allosteric modulators at α4β2 nACh receptors.

Further deconstruction of 7 and 8 provided analogs 9–12. Again suggesting that the gem-dimethyl groups are important, the racemic monomethyl counterpart of 8 (i.e., 9) (Table 1) was inactive (as a positive allosteric modulator). This is further supported by the inactivity of 10. Interestingly, although the inactive compound 12 possesses a gem-dimethyl group, compound 11 (pEC50 = 5.40) is active (Table 1). It would seem that it is not simply the presence of the gem-dimethyl group but, rather, the quaternary nature of this carbon atom that contributes to activity.

Effect of deconstructed analogs of 1 on blocking the functional effects of ACh at α4β2 nACh receptors

As previously shown for 1,8 high concentrations of all the deconstructed compounds blocked the action of 10 μM ACh at α4β2 nACh receptors. For those compounds acting as positive allosteric modulators, pIC50 values could not be determined with accuracy. pIC50 values for compounds not acting as positive allosteric modulators ranged from 5.45 for 5 to 4.14 for 4; additional results are shown in Table 1. The mechanism(s) whereby these analogs are able to functionally antagonize the actions of ACh at α4β2 receptors remains to be elucidated.

Effect of deconstructed analogs of 1 on α7 nACh receptor function

As an initial measure of selectivity, compounds were examined for their ability to potentiate the electrophysiological effects of ACh at α7 receptors. None proved to be active at the highest concentrations examined. However, all of the examined compounds inhibited the action of 10 μM ACh at α7 receptors (Table 2). Interestingly, whereas 1 attenuated the action of ACh at α7 receptors at a concentration of only six times greater than that required for potentiation of ACh at α4β2 receptors, its reduced counterpart 6 was 190- fold selective for the latter.

Conclusion

Deconstruction of the structure of the positive allosteric modulator 1 has shown, although several “deconstructed” analogs retained activity, that the intact structure of 1 seems optimal as a positive allosteric modulator at α4β2 nACh receptors. The reduced counterpart of 1 (i.e., 6), retains the action and potency of 1; however, 6 offers no synthetic advantage in that it was synthesized by reduction of 1. Also learned was that a) N-demethylation of 1 to its primary amine reduces potency by 50%, b) the 6-bromo group of 1 (and 6) contributes to potency, not action, c) the presence of the gem-dimethyl groups plays an important role in conferring activity, and d) it is likely that the quaternary nature of the branched carbon atom, not simply the presence of gem-dimethyl groups, defines this action. Finally, as with 1,8 analogs 2–11 were capable of attenuating the action of ACh at α7 receptors; however, compound 6 was considerably more selective than 1 as a positive allosteric modulator at α4β2 receptors. This might be an advantage for functional studies where both receptor types are present. In summary, structural features of 1 important for its action and potency as a positive allosteric modulator at α4β2 nACh receptors were determined, pharmacophoric features were identified, and 6 was found to be at least as potent as, and more selective than, 1 in this regard.

Experimental Section

Chemistry

Melting points were taken in glass capillary tubes on a Thomas Hoover melting point apparatus and are uncorrected. 1H NMR spectra were recorded either with a Varian EM-390 or Bruker 400 MHz spectrometer and peak position are given in parts per million (δ) downfield from tetramethylsilane as an internal standard. Purity of compounds (>95%) was established by elemental analysis; microanalyses were performed by Atlantic Microlab (GA) for the indicated elements, and the results are within 0.4% of theoretical values. Reactions and product mixtures were routinely monitored by thin-layer chromatography (TLC) on silica gel precoated F254 Merck plates, and chromatographic separations were performed using Aldrich Silica gel 60 columns unless otherwise stated.

6-Bromo-2-(1,1-dimethylallyl)tryptamine Hydrochloride (2)

Following the addition of tert-BuOCl (0.10 g, 0.9 mmol) to a stirred solution of tert-butyl-2-(6-bromo-1H-indol-3-yl)ethylcarbamate12 (0.25 g, 0.7 mmol) and Et3N (0.90 g, 0.9 mmol) in THF (10 mL) at −78 °C, stirring was allowed to continue for 45 min. Freshly prepared prenyl 9-BBN (1.5 mmol)13 was added in a dropwise manner over a 20-min period while maintaining temperature at −78 °C. The reaction mixture was allowed to warm to room temperature and stirring was continued for an additional 2 h. Aqueous NaOH (3M, 3 mL) and H2O2 (30%, 3 mL) were added in a dropwise fashion and stirring continued for another 1 h. The reaction mixture was diluted with Et2O (100 mL), the organic portion was washed with H2O (3 × 30 mL), brine (40 mL), and dried (Na2SO4). The solution was evaporated to dryness and the residue was purified by chromatography using hexanes/EtOAc (10:1) as eluent to afford 0.10 g (33%) of a white foam.

Gaseous HCl was bubbled into a stirred solution of the above compound (0.09 g, 0.22 mmol) in dry EtOAc (10 mL) at 0 °C; stirring was allowed to continue for 24 h and the solvent was evaporated to yield a white solid. Recrystallization from EtOAc/MeOH provided 0.03 g (33%) of 2 as white crystals: mp 256–258 °C; 1H NMR (DMSO-d6) δ 1.49 (s, 6H, 2CH3), 2.85 (t, 2H, CH2), 3.05 (t, 2H, CH2), 5.05–5.10 (m, 2H, CH), 6.10–6.17 (m, 1H, CH), 7.12 (dd, 1H, ArH), 7.48–7.51 (m, 2H, ArH), 7.98 (br s, 3H, NH3 +), 10.76 (s, 1H, NH). Anal. Calcd for (C15H20BrN2·HCl·0.25 H2O) C, H, N.

N-Methyl-6-bromo-2-n-propyltryptamine Oxalate (4)

According to the procedure described for preparation of 17, compound 15 (0.50 g, 1.27 mmol) was treated with NaBH4 (0.10 g, 2.54 mmol) and CoCl2·6H2O (0.3 g, 1.27 mmol) to give 0.40 g (78%) of the reduced compound as a white solid: mp 157–159 °C; 1H NMR (CDCl3) δ 1.01 (t, J = 7.2 Hz, 3H, CH3), 1.42 (s, 9H, t-Bu), 1.62–1.77 (m, 2H, CH2), 2.63–2.78 (m, 2H, CH2), 2.80–2.96 (m, 2H, CH2), 2.88 (s, 3H, N-CH3), 3.33–3.46 (m, 2H, CH2), 2.20 (dd, J = 1.5, 8.4 Hz, 1H, ArH), 7.35–7.47 (m, 2H, ArH), 7.86 (br s, 1H, NH).

The reduced product was deprotected as follows: TFA (0.8 mL) was added to a solution of the above compound (0.13 g, 0.33 mmol) in CH2Cl2 (5 mL) at room temperature and the reaction mixture was allowed to stir for 0.5 h. An additional 10 mL of CH2Cl2 was added; the solution was cooled to 0 °C, treated with a saturated solution of NaHCO3, and extracted with EtOAc (3 × 10 mL). The combined organic portion was dried (Na2SO4) and the solvent was removed. The crude product was purified by column chromatography (CH2Cl2/MeOH/NH4OH; 9:1:0.1) to give 0.10 g (98%) of the free base of 4 as a brown oil: 1H NMR (CDCl3) δ 0.96 (t, J = 7.2 Hz, 3H, CH3), 1.59–1.74 (m, 2H, CH2), 2.52 (s, 3H, N-CH3), 2.63–2.73 (m, 2H, CH2), 2.86–3.03 (m, 4H, 2CH2), 4.65 (br s, 1H, amine NH), 7.16 (dd, J = 1.8, 8.4 Hz, 1H, ArH), 7.35–7.43 (m, 2H, ArH), 8.25 (br s, 1H, NH). The free base in Et2O (5 mL) was treated with ethereal oxalic acid. The precipitated oxalate salt was collected by filtration, washed with anhydrous Et2O (3 × 5 mL), and recrystallized from absolute EtOH/anhydrous Et2O to afford 0.05 g (37%) of the salt as a off-white solid: mp 211–213 °C; 1H NMR (DMSO-d6) δ 0.91 (t, J = 7.2 Hz, 3H, CH3), 1.58–1.72 (m, 2H, CH2), 2.60 (s, 3H, N-CH3), 2.63–2.69 (m, 2H, CH2), 2.91–3.04 (m, 4H, 2CH2), 7.09 (dd, J = 1.5, 8.4 Hz, 1H, ArH), 7.41–7.48 (m, 2H, ArH), 11.09 (br s, 1H, NH). Anal. Calcd for (C14H19BrN2·C2H2O4) C, H, N.

N-Methyl-2-allyl-6-bromotryptamine Oxalate (5)

According to the procedure described for preparation of 4, compound 15 (0.07 g, 0.17 mmol) was treated with TFA (0.5 mL) to give 0.04 g (74%) of the free base 5 as a pale-yellow oil which was treated with ethereal oxalic acid to afford the oxalate salt. Recrystallization from absolute EtOH/anhydrous Et2O afforded 0.02 g (24%) of product as a pale-yellow solid: mp 198–203 °C (dec.); 1H NMR (DMSO-d6) δ 2.62 (s, 3H, N-CH3), 2.91–3.08 (m, 4H, 2CH2), 3.50 (d, J = 6.0 Hz, 2H, allylic CH2), 5.08–5.20 (m, 2H, vinylic H), 5.99 (m, 1H, vinylic H), 7.13 (br d, J = 8.7 Hz, 1H, ArH), 7.43–7.53 (m, 2H, ArH), 11.09 (br s, 1H, NH). Anal. Calcd for (C14H17BrN2·C2H2O4) C, H, N. Note: subsequent studies showed this product to be an isomeric mixture of positional isomers (see text).

N-Methyl-6-bromo-2-(1,1-dimethylpropyl)tryptamine Oxalate (6)

Sodium borohydride (0.02 g, 0.37 mmol) was added in portions to a solution of 168 (0.08 g, 0.18 mmol) and CoCl2·6H2O (0.04 g, 0.18 mmol) in EtOH (2 mL) at 0 °C. The reaction mixture was allowed to stir under a N2 atmosphere at room temperature for 1 h and quenched by addition of H2O (5 mL). The aqueous solution was extracted with Et2O (3 × 10 mL). The combined organic portion was dried (Na2SO4) and solvent was removed under reduced pressure. The crude product was purified by column chromatography (hexane/EtOAc; 5:1) to give 0.06 g (76%) of 17 as a white solid: mp 154–157 °C; 1H NMR (CDCl3) δ 0.79 (t, J =7.2 Hz, 3H, CH3), 1.47 (s, 6H, 2CH3), 1.52 (s, 9H, t-Bu), 1.78 (q, J = 7.2 Hz, 2H, CH2), 2.93 (s, 3H, N-CH3), 3.02–3.09 (m, 2H, CH2), 3.36–3.46 (m, 2H, CH2), 7.19 (dd, J = 1.5, 8.4 Hz, 1H, ArH), 7.41–7.48 (m, 2H, ArH), 7.92 (br s, 1H, NH).

The reduced product (0.08 g, 0.18 mmol), was treated with TFA (0.45 mL) to give the free base of 6 (0.06 g, 96%) as a brown oil which was converted to the oxalate salt: mp 192–194 °C following recrystallization from absolute EtOH/anhydrous Et2O; 1H NMR (DMSO-d6) δ 0.68 (t, J = 7.2 Hz, 3H, CH3), 1.39 (s, 6H, 2CH3), 1.70 (q, J = 7.2 Hz, 2H, CH2), 2.63 (s, 3H, N-CH3), 2.90–3.01 (m, 2H, CH2), 3.06–3.16 (m, 2H, CH2), 7.10 (dd, J = 1.5, 8.7 Hz, 1H, ArH), 7.44 (d, J = 1.5 Hz, 1H, ArH), 7.48 (d, J = 8.7 Hz, 1H, ArH), 10.70 (br s, 1H, NH). Anal. Calcd for (C16H23BrN2·C2H2O4) C, H, N.

N-Methyl-2-(1,1-dimethylallyl)tryptamine Hydrochloride (7)

tert-Butyl hypochlorite (0.19 mL, 2.15 mmol) was added in a dropwise manner to a stirred solution of 1811 (0.49 g, 1.8 mmol) and Et3N (0.30 mL, 2.2 mmol) in anhydrous THF (7.0 mL) at −78 °C. The clear solution was allowed to stir for an additional 0.5 h before freshly prepared prenyl 9-BBN13 (0.68 g, 3.59 mmol) solution in THF was added in a dropwise manner. After 30 min the reaction mixture was allowed to warm to room temperature, and stirring was continued for 1 h. The addition of 3M NaOH (1.8 mL) and 30% H2O2 (1.8 mL) was followed by stirring for 1 h. The reaction mixture was diluted with Et2O (30 mL), the organic layer was washed with 3M NaCl solution (3 × 50 mL), dried (Na2SO4), and solvent was removed under reduced pressure. The resultant residue was subjected to flash chromatography (hexanes/EtOAc; 10:1) to give 0.30 g (40%) of 19 as an oil which crystallized upon standing: mp 141–143 °C; 1H NMR (CDCl3): δ 1.51 (s, 9H, Boc), 1.58 (s, 6H, 2CH3), 2.94 (s, 3H, N-CH3), 2.93–3.10 (m, 2H, CH2), 3.38–3.52 (m, 2H, CH2), 5.18–5.22 (m, 2H, vinylic H), 6.16 (dd, J = 10.5, 17.1 Hz, 1H, vinylic H), 7.16 (m, 2H, ArH), 7.30–7.35 (m, 1 H, ArH), 7.61 (m, 1H, ArH), 7.91 (br s, 1H, NH).

Gaseous HCl was bubbled through a solution of 19 (0.10 g, 0.29 mmol) in anhydrous EtOAc (10 mL). The salt was recrystallized from MeOH/anhydrous Et2O to afford 0.04 g (50%) of 7 as an off-white solid: mp 223–225 °C; 1H NMR (DMSO-d6) δ 1.56 (s, 6H, 2CH3), 2.61 (s, 3H, N-CH3), 2.90–3.00 (m, 2H, CH2), 3.02–3.12 (m, 2H, CH2), 5.12 (dd, J = 1.2, 17.0 Hz, 2H, vinylic H), 6.16 (dd, J = 10.5, 17.0 Hz, 1H, vinylic H), 6.88–7.08 (m, 2H, ArH), 7.30 (d, J = 8.0 Hz, 1H, ArH), 7.59 (d, J = 8.0 Hz, 1H, ArH), 10.61 (br s, 1H, NH). Anal. Calcd for (C16H22N2·HCl) C, H, N.

N-Methyl-2-(1,1-dimethylpropyl)tryptamine Hydrochloride (8)

A catalytic amount of 10% Pd/C (0.02 g) was added to 19 (0.09 g, 0.26 mmol) in MeOH (15 mL) and hydrogenated at ca. 50 psi for 3 h. The catalyst was removed by filtration, and the solvent was removed under reduced pressure. Compound 20 (0.09 g, 92%; mp 172–174 °C) was obtained as an off-white solid and used without further purification.

Gaseous HCl was bubbled through a solution of 20 (0.09 g, 0.25 mmol) in dry EtOAc (10 mL). The salt was recrystallized from MeOH/anhydrous Et2O to afford 0.04 g (50%) of 8 as an off-white solid: mp 234–235 °C; 1H NMR (DMSO-d6) δ 0.69 (t, J = 7.5 Hz, 3H, CH3), 1.41 (t, J = 12 Hz, 6H, 2CH3), 1.73 (q, J = 7.5 Hz, 2H, CH2), 2.58 (s, 3H, N-CH3), 2.87–3.00 (m, 2H, CH2), 3.05–3.23 (m, 2H, CH2), 6.88–7.08 (m, 2H, ArH), 7.30 (d, J = 7.5 Hz, 1H, ArH), 7.59 (d, J = 7.5 Hz, 1H, ArH), 10.51 (br s, 1H, NH). Anal. Calcd for (C16H24N2·HCl·0.25 H2O) C, H, N.

N-Methyl-2-sec-butyltryptamine Oxalate (9)

Ethyl chloroformate (0.06 mL, 0.65 mmol) was added in a dropwise manner to a solution of 22 (0.14 g, 0.65 mmol) in CHCl3 (5 mL) at 0 °C, followed by addition of aqueous 4M NaOH (0.17 mL, 0.65 mmol). The reaction mixture was allowed to stir at room temperature for 2 h and diluted with CHCl3 (20 mL). The organic portion was separated, washed with H2O (20 mL), dried (Na2SO4), and solvent was removed under reduced pressure to afford 0.14 g (75%) of N-Boc-22 as a brown oil: 1H NMR (CDCl3) δ 0.95 (t, J = 6 Hz, 3H, CH3), 1.21–1.61 (m, 6H, 2CH3), 1.62–2.12 (m, 2H, CH2), 3.03 (m, 3H, CH2, CH), 3.32–3.70 (m, 2H, CH2), 3.95–4.30 (m, 2H, CH2), 7.13–7.21 (m, 2H, ArH), 7.38 (d, J = 9 Hz, 1H, ArH), 7.62 (d, J = 9 Hz, 1H, ArH), 8.33 (br s,1H, NH). The crude reaction product was directly used in the next step.

A solution of the above product (0.14 g, 0.48 mmol) in anhydrous THF (5 mL) was added in a dropwise manner to a stirred suspension of LiAlH4 (0.11 g, 3 mmol) in dry THF (10 mL) at 0 °C. The stirred mixture was heated at reflux under a N2 atmosphere for 2 h, cooled to 0 °C, and successively quenched with MeOH (1 mL), H2O (1.5 mL), NaOH (3M, 1 mL) and diluted with CH2Cl2 (10 mL). The organic portion was dried (Na2SO4) and concentrated under reduced pressure. The crude residue was purified on a silica gel column using CH2Cl2/MeOH (9:1) → CH2Cl2/MeOH/Et3N (9:1:0.1) as eluent to afford the amine as a brown oil. The oxalate salt was prepared and recrystallized from MeOH/anhydrous Et2O to give 0.08 g (48%) of 9 as a beige solid: mp 180–181 °C; 1H NMR (DMSO–d6) δ 0.79 (t, J = 6.9 Hz, 3H, CH3), 1.29 (d, J = 6.9 Hz, 3H, CH3), 1.57–1.72 (m, 2H, CH2), 2.57–2.67 (m, 4H, 2CH2), 2.62 (s, 3H, CH3), 2.93–3.00 (m, 1H, CH), 6.95–7.08 (m, 2H, ArH), 7.29 (d, J = 7.5 Hz, ArH), 7.50 (d, J = 7.5 Hz, 1H, ArH), 10.79 (s, 1H, NH3+). Anal. Calc for (C15H22N2·C2H4O4·0.25 H2O) C, H, N.

N-Methyl-2-(n-propyl)tryptamine Oxalate (10)

A suspension of 4 (0.13 g, 0.33 mmol) in absolute EtOH (10 mL) was hydrogenated in the presence of 10% Pd/C (0.03 g) under a H2 atmosphere (45 psi) at room temperature for 1 h. The catalyst was removed by filtration and the filtrate was evaporated to give 0.10 g (100%) of a cream-colored solid: mp 142–144 °C; 1H NMR (CDCl3) δ 1.01 (t, J = 7.5 Hz, 3H, CH3), 1.44 (s, 9H, 3CH3), 1.64–1.80 (m, 2H, CH2), 2.74 (t, J = 7.8 Hz, 2H, CH2), 2.82–3.00 (m, 2H, CH2), 2.90 (s, 3H, N-CH3), 3.43 (t, J = 7.8 Hz, 2H, CH2), 7.10 (dt, J = 1.5, 6.9 Hz, 1H, ArH), 7.14 (dt, J = 1.5, 6.9 Hz, 1H, ArH), 7.30 (dd, J = 1.5, 6.9 Hz, 1H, ArH), 7.56 (m, 1H, ArH), 7.83 (br s, 1H, NH).

Using the procedure described for 4, the amine (0.1 g, 0.31 mmol) was treated with TFA (0.8 mL) to give 0.06 g (93%) of a pale-yellow oil: 1H NMR (CDCl3) δ 1.01 (t, J = 7.5 Hz, 3H, CH3), 1.64–1.78 (m, 2H, CH2), 2.47 (s, 3H, N-CH3), 2.75 (t, J = 7.5 Hz, 2H, CH2), 2.83–3.00 (m, 4H, 2CH2), 7.10 (dt, J = 1.5, 6.9 Hz, 1H, ArH), 7.15 (dt, J = 1.5, 6.9 Hz, 1H, ArH), 7.30 (m, 1H, ArH), 7.58 (dd, J = 1.5, 6.9 Hz, 1H, ArH), 8.00 (br s, 1H, NH). The free base in Et2O (5 mL) was treated with an ethereal solution of oxalic acid to afford 0.07 g (69%) of 10: mp 177–179 °C after recrystallization from absolute EtOH/anhydrous Et2O; 1H NMR (DMSO-d6) δ 0.92 (t, J = 7.2 Hz, 3H, CH3), 1.56–1.74 (m, 2H, CH2), 2.60 (s, 3H, N-CH3), 2.67 (t, J = 7.5 Hz, 2H, CH2), 2.88–3.06 (m, 4H, 2CH2), 6.91–7.04 (m, 2H, 2ArH), 7.25 (br d, J = 7.8 Hz, 1H, ArH), 7.48 (br d, J = 7.8 Hz, 1H, ArH), 10.85 (br s, 1H, NH). Anal. Calcd for (C14H20N2·C2H2O4) C, H, N.

N-Methyl-2-(tert-butyl)tryptamine Hydrogen Oxalate (11)

Oxalyl chloride (0.2 mL, 2.308 mmol) was added in a dropwise manner at −5 °C to a stirred solution of 2-tert-butyl-1H-indole14 (0.02 g, 1.154 mmol) in anhydrous Et2O (10 mL). The reaction mixture was allowed to stir for 6 h at 0 °C; after evaporation of solvent under reduced pressure, methylamine (40% aqueous solution, 5 mL) was added at room temperature, and the reaction mixture was allowed to stir overnight. The precipitate was collected by filtration and recrystallized from MeOH to afford 0.17 g (56%) of the corresponding glyoxylamide as brown solid: mp 190–191 °C; 1H NMR (CDCl3) δ 1.46 (s, 9H, 3CH3), 3.1 (s, 3H, N-CH3), 6.77 (s, 1H, NH), 7.07–7.45 (m, 4H, ArH), 7.76 (br s, 1H, NH).

A solution of the glyoxylamide (0.17 g, 0.65 mmol) in dioxane (5 mL) was added to a stirred suspension of LiAlH4 (0.25 g, 6.5 mmol) in dioxane (10 mL) at 60 °C, and then heated at reflux overnight. The reaction mixture was cooled to room temperature and diluted with THF (10 mL), MeOH (3 mL) and NaOH (3N, 3 mL). The mixture was filtered and the filtrate was heated at reflux with THF (15 mL). The combined organic portion was evaporated to dryness under reduced pressure, and the residue was purified by column chromatography using CH2Cl2/MeOH (10:1) as eluent to afford the desired amine. The oxalate salt was prepared to give 0.03 g (56%) of 11 as white flakes after recrystallization from 2-PrOH: mp 194–195 °C; 1H NMR (DMSO-d6) δ 1.43 (s, 9H, 3CH3), 2.64 (s, 3H, N-CH3), 2.94 (d, 2H, CH2), 3.15 (d, 2H, CH2), 6.96–7.51 (m, 4H, ArH), 10.55 (s, 1H, R2NH2+COO). Anal. Calcd for (C18H30N2O·(C2H2O4) ·2-PrOH) C, H, N.

N-Methyl-2-isopropyltryptamine Hydrochloride (12)

A solution of 26 (0.17 g, 0.61 mmol) in dry THF (5 mL) was added to a stirred suspension of LiAlH4 (0.14 g, 3.7 mmol) in anhydrous THF (10 mL) at 0 °C, and the reaction mixture was heated at reflux for 2 h, cooled in an ice bath and quenched with MeOH (1 mL), NaOH (15%, 1.5 mL) and H2O (1 mL). The organic portion was separated, dried (Na2SO4), and solvent was evaporated under reduced pressure to yield an oily residue. Purification by column chromatography using CH2Cl2/MeOH (9:1) → CH2Cl2/MeOH/Et3N (9:1:0.1) afforded 0.07 g of the amine as a light-yellow oil: 1H NMR (DMSO-d6) δ 1.28 (d, J = 9 Hz, 6H, 2CH3), 2.51 (s, 3H, CH3), 2.60–2.70 (m, 2H, CH2), 2.74–2.81 (m, 2H, CH2), 3.22 (m, 1H, CH), 6.90–7.00 (m, 2H, ArH), 7.25 (d, J = 9 Hz, 1H, ArH), 7.48 (d, J = 9 Hz, 1H, ArH).

Gaseous HCl was bubbled through a solution of the amine in dry EtOAc (10 mL) to obtain 0.04 g (50%) of 12 as white crystals after recrystallization from MeOH/anhydrous Et2O: mp 212–213 °C; 1H NMR (DMSO-d6) δ 1.30 (d, J = 6.9 Hz, 6H, 2 × CH3), 2.59 (s, 3H, CH3), 2.92–3.05 (m, 4H, 2CH2), 3.24 (m, 1H, CH), 6.95–7.08 (m, 2H, ArH), 7.29 (d, J = 7.5 Hz, 1H, ArH), 7.53 (d, J = 7.5 Hz, 1H, ArH), 9.00 (s, 1H, NH), 10.87 (br br s, 1H, NH3+). Anal. Calcd for (C14H20N2·HCl) C, H, N.

N-[2-(2-Allyl-6-bromo-1H-indol-3-yl)ethyl]-N-methylcarbamic acid tert-butyl ester (15)

tert-Butyl hypochlorite (0.58 mL, 5.10 mmol) was added in a dropwise manner to a stirred solution of 148 (1.50 g, 4.25 mmol) and Et3N (0.71 mL, 5.10 mmol) in THF (25 mL) at −78 °C under a N2 atmosphere. After 1.5 h at −78 °C, allyltributyltin (2.61 mL, 8.49 mmol) was added followed by addition of BF3·Et2O (1.1 mL, 8.49 mmol). The reaction mixture was allowed to stir for 0.5 h at the same temperature, and then quenched by addition of saturated aqueous solution of NaHCO3 (10 mL). Ethyl acetate (100 mL) and H2O (100 mL) were added, and the aqueous portion was extracted with EtOAc (3 × 50 mL). The combined organic portion was dried (Na2SO4) and solvent was removed under reduced pressure. The residue was purified by column chromatography (hexane/EtOAc; 7:1) to give 0.74 g (44%) of a white solid: mp 138–140 °C. The product was immediately used in the synthesis of 4.

2-sec-Butyltryptamine (22)

A solution of 2-sec-butylindole (21)14 (0.35 g, 2 mmol) in CH2Cl2 (2 mL) was added to a stirred solution of 1-dimethylamino-2-nitroethylene (0.23 g, 2 mmol) in TFA (1.2 mL) at 0 °C. The reaction mixture was allowed to stir for 2 h, poured into ice-cold H2O (50 mL) and extracted with EtOAc (2 × 20 mL). The combined organic portion was washed with brine (20 mL), dried (Na2SO4) and solvent was removed under reduced pressure. The residue was recrystallized from EtOAc to yield the corresponding nitroalkene (0.27 g, 75% based on recovered starting material) as orange crystals: mp 141 °C; 1H NMR (CDCl3) δ 0.94 (t, J = 7.5 Hz, 3H, CH3), 1.43 (d, J = 7.0 Hz, 3H, CH3), 1.72–1.84 (m, 2H, CH2), 3.28 (m, 1H, CH), 7.30–7.34 (m, 2H, ArH), 7.42–7.48 (m, 1H, CH), 7.72–7.78 (m, 1H, CH), 7.87 (d, J = 13 Hz, 1H, ArH), 8.40 (d, J = 13 Hz, 1H, ArH), 8.74 (br s, 1H, NH).

A solution of the alkene (0.27 g, 1.1 mmol) in dry THF (5 mL) was added in a dropwise manner at 0 °C to a stirred suspension of LiAlH4 (0.26 g, 6.9 mmol) in anhydrous THF (10 mL). The reaction mixture was heated overnight at reflux under a N2 atmosphere, cooled to 0 °C, quenched with MeOH (1 mL), H2O (1.5 mL), NaOH (1 mL) and diluted with CH2Cl2 (30 mL). After acid (2N HCl, 30 mL)/base (3N NaOH, 30 mL) extraction, the organic portion was separated, dried (Na2SO4) and solvent was removed under reduced pressure to give 0.14 g (62%) of 22 as a light-brown oil: 1H NMR (CDCl3) δ 0.91 (t, J = 7.5 Hz, 3H, CH3), 1.33 (d, J = 6.0 Hz, 3H, CH3), 1.62–1.79 (m, 2H, CH2), 2.84–2.97 (m, 2H, CH2), 2.98–3.12 (m, 3H, CH, CH2), 7.03–7.25 (m, 2H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.82 (br s, 1H, NH).

2-Isopropyl-1H-indole (23)

A solution of nBuLi (1.6 M in hexane; 14 mL, 22.4 mmol) was added to a stirred solution of N-(o-tolyl)isobutyramide15 (2.0 g, 11 mmol) in anhydrous THF (20 mL) at 0°C. After stirring for 1 h, the reaction mixture was allowed to warm gradually to room temperature. After an additional 8 h, EtOAc (15 mL) and then saturated aqueous NH4Cl (5 mL) were added. The organic portion was separated, dried (Na2SO4) and solvent was removed under reduced pressure. The crude oily residue was purified by column chromatography using hexanes/EtOAc (20:1) as eluent to afford 0.70 g (66 % based on recovered material) of 23 as an off-white solid: mp 75–76 °C (lit.16 73–74°C); 1H NMR (CDCl3) δ 1.41 (d, J = 6 Hz, 6H, 2 × CH3), 3.09–3.16 (m, 1H, CH), 6.31 (s, 1H, CH), 7.10–7.16 (m, 2H, ArH), 7.35 (d, J = 9 Hz, 1H, ArH), 7.60 (d, J = 9 Hz, 1H, ArH), 7.85 (br s, 1H, NH).

2-Isopropyltryptamine (25)

A solution of 23 (0.30 g, 1.9 mmol) in CH2Cl2 (2 mL) was added to a stirred solution of 1-dimethylamino-2-nitroethylene (0.22 g, 1.9 mmol) in TFA (1.2 mL) at 0 °C. The reaction mixture was allowed to stir at room temperature for 2 h, poured into icecold water (20 mL) and extracted with EtOAc (20 mL). The organic portion was washed with brine (15 mL), dried (Na2SO4) and the solvent was removed under reduced pressure. The crude product was purified recrystallized from EtOAc to afford 0.18 g (60%) of 24 as orange crystals: mp 159 °C; 1H NMR (CDCl3) δ 1.44 (d, J = 6.0 Hz, 6H, 2 × CH3), 3.51–3.62 (m, 1H, CH), 7.28–7.35 (m, 2H, ArH), 7.42–7.46 (m, 1H, CH), 7.72 – 7.75 (m, 1H, CH), 7.84 (d, J = 13 Hz, 1H, ArH), 8.42 (d, J = 13 Hz, 1H, ArH), 8.74 (br s, 1H, NH).

A solution of 24 (0.26 g, 1.1 mmol) in anhydrous THF (5 mL) was added in a dropwise manner to a stirred suspension of LiAlH4 (0.26 g, 6.9 mmol) in THF (10 mL) at 0 °C. The reaction mixture was heated at reflux overnight, cooled in an ice bath, and quenched with MeOH (1 mL), NaOH (15%, 1.5 mL) and H2O (1 mL). After acid (3N HCl, 10 mL)/base (3N NaOH, 15 mL) extraction, the organic portion was dried (Na2SO4) and solvent was removed under reduced pressure to afford 0.14 g (61%) of 25 as a light-yellow oil: 1H NMR (DMSO-d6) δ 1.28 (d, J = 7.5 Hz, 6H, 2CH3), 1.73 (br s, 2H, NH2), 2.73–2.84 (m, 4H, 2 × CH2), 3.21 (m, 1H, CH), 6.89–7.01 (m, 2H, ArH), 7.27 (d, J = 7.5 Hz, 1H, ArH), 7.44 (d, J = 7.5 Hz, 1H, ArH), 8.78 (br s, 1H, NH). Compound 25 was used immediately in the synthesis of 26.

Ethyl [2-(2-isopropyl-1H-indol-3-yl)ethyl]carbamate (26)

Ethyl chloroformate (0.07 mL, 0.70 mmol) was added in a dropwise manner at 0 °C to a stirred solution of 25 (0.14 g, 0.7 mmol) in CHCl3 (5 mL), followed by addition of a 4N aqueous solution of NaOH (0.20 mL, 0.70 mmol). The reaction mixture was allowed to stir for 2 h at room temperature and diluted with CHCl3 (15 mL). The organic portion was dried (Na2SO4), and solvent was evaporated under reduced pressure to afford 0.17 g (88%) of 26 as an oily residue which was used in the preparation of 12 without further purification.

Electrophysiology

The cDNA sequences for human α4 (NCBI Reference Sequence: NM_000744.5), β2 (NCBI Reference Sequence: NM_000748.2) and α7 (NCBI Reference Sequence: NM_000746.3) nACh receptor subunits were used to synthesize a full length cDNA for each subunit. cDNA synthesis was conducted by GeneArt Inc. (Burlingame, CA). The synthetic β2 cDNA was inserted into the pcDNA3.1/Zeo(+) expression vector and the α4 cDNA was inserted into the pcDNA3.1/hygromyocin expression (vectors procured from Invitrogen, Carlsbad, CA).

Ovarian lobes were surgically removed from Xenopus laevis frogs and washed twice in Ca+2-free Barth’s buffer (82.5 mM NaCl/2.5 mM KCl/1mM MgCl2/5 mM HEPES, pH 7.4) then gently shaken with 1.5 mg/mL collagenase (Sigma type II, Sigma-Aldrich) for 1 h at 20–25 °C. Stage IV oocytes were selected for microinjection. Synthetic cRNA transcripts for human α7 and α4β2 were prepared using the mMESSAGE mMACHINE™ High Yield Capped RNA Transcription Kit (Ambion, TX). Oocytes were injected with a total of 50 nL cRNA at a concentration of 0.2 ng/nL in appropriate subunit ratios then incubated at 19 °C for 24 to 72 h prior to their use in voltage clamp experiments. Recordings were made using an automated two-electrode voltage-clamp system incorporating an OC-725C oocyte clamp amplifier (Warner Instruments, CT) coupled to a computerized data acquisition (Datapac 2000, RUN technologies) and autoinjection system (Gilson). Recording and current electrodes with resistance 1–4 M] were filled with 3M KCl. Details of the chambers and methodology employed for electrophysiological recordings have been described earlier.17 Oocytes were held in a vertical flow chamber of 200-μL volume and perfused with ND-96 recording buffer (96 mM NaCl/2 mM KCl/1.8 mM CaCl2/1 mM MgCl2/5 mM HEPES; pH 7.4) at a rate of 20 mL/min. Test compounds were dissolved in ND-96 buffer and injected into the chamber at a rate of 20 mL/min using the Gilson auto-sampler injection system. Compounds were co-applied with the EC75 concentration (100 μM) of ACh. Data Analysis: Concentration/response curves were fit by non-linear curve fitting and GraphPad Prism Software (San Diego, CA) using standard built-in algorithms. For IC50 determinations, data were fit to a single site competition model. For the potentiation/inhibition curves obtained for α4β2 modulation, the data were fit to a bell shaped dose-response equation as previously described.9

Acknowledgments

This study was funded, in part, by the National Institutes of Health, National Institute of Neurological Disorders and Stroke [Grant 1R01NS066059] and by a Virginia Center on Aging. NG was partially supported by TA grant DA 007027.

Abbreviations

ACh
acetylcholine
dFBr
des-formylflustrabromine
nACh
nicotinic acetylcholine
TFA
trifluoroacetic acid

References

1. Dani JA, Bertrand D. Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. Ann Rev Pharmacol. 2007;47:699–729. [PubMed]
2. Arias HR, Bhumireddy P, Bouzat C. Molecular mechanisms and binding site locations for noncompetitive antagonists of nicotinic acetylcholine receptors. Int J Biochem Cell Biol. 2006;38:1254–1276. [PubMed]
3. Arias HA. Positive and negative modulation of nicotinic receptors. Adv Protein Chem Struct Biol. 2010;80:153–205. [PubMed]
4. Glennon RA. Progress in Medicinal Chemistry. Vol. 42. Elsevier Science; Amsterdam: 2004. Medicinal Chemistry of α4β2 Nicotinic Cholinergic Receptor Ligands; p. 55. [PubMed]
5. Bertrand D, Gopalakrishnan M. Allosteric modulation of nicotinic acetylcholine receptors. Biochem Pharmacol. 2007;74:1155–1193. [PubMed]
6. Peters L, Wright AD, Kehraus S, Gundisch D, Tilotta MC, Konig GM. Prenylated indole alkaloids from Flustra foliacea with subtype specific binding on NAChRs. Planta Med. 2004;70:883–886. [PubMed]
7. Sala F, Mulet J, Reddy KP, Bernal JA, Wikman P, Valor LM, Peters L, Konig GM, Criado M, Sala S. Potentiation of human α4β2 neuronal nicotinic receptors by a Flustra foliacea metabolite. Neurosci Lett. 2005;373:144–149. [PubMed]
8. Kim JS, Pandya A, Weltzin M, Edmonds BW, Schulte MK, Glennon RA. Synthesis of desformylflustrabromine and its evaluation as an α4β2 and α7 nACh receptor modulator. Bioorg Med Chem Lett. 2007;17:4855–4860. [PMC free article] [PubMed]
9. Weltzin MM, Schulte MK. Pharmacological characterization of the allosteric modulator desformylflustrabromine and its interaction with α4β2 neuronal nicotinic acetylcholine receptor orthosteric ligands. J Pharmacol Exp Ther. 2010;334:917–926. [PubMed]
10. Chung SK. Selective reduction of mono- and disubstituted olefins by sodium borohydride and cobalt (II) J Org Chem. 1979;44:1014–1016.
11. Benson SC, Lee L, Shyder JK. Intramolecular inverse electron Demand Diels-Alder reactions of tryptamine with tethered heteroaromatic azadienes. Tetrahedron. 2000;56:1165–1180.
12. Burm BEA, Meijler MM, Korver J, Wanner MJ, Koomen GJ. Synthesis of the brominated marine alkaloids (±)-arborescidine A, B and C. Tetrahedron. 1998;54:6135–6146.
13. Kramer GW, Brown HC. The preparation and some unusual chemistry of B-allyl derivatives of 9-borabicyclo[3.3.1]nonane. J Organomet Chem. 1977;132:9–27.
14. Smith K, El-Hiti GA, Pritchard GJ, Hamilton A. Carbonylation of various organolithium reagents. A novel approach to heterocycles via intramolecular trapping of aromatic acyllithiums. J Chem Soc Perkin Trans. 1999;1:2299–2303.
15. Boido C, Boido V, Novelli F, Sparatore F. Formation of basic compounds during the indole cyclization of ketone phenylhydrazone. J Heterocycl Chem. 1998;35:853–858.
16. Illy H, Funderburk L. Fischer indole synthesis. Direction of cyclization of isopropylmethyl ketone phenylhydrazone. J Org Chem. 1968;33:4283–4285.
17. Joshi PR, Suryanarayanan A, Schulte MK. A vertical flow chamber for Xenopus oocyte electrophysiology and automated drug screening. J Neurosci Methods. 2004;132:69–79. [PubMed]