Toxiferine Synthesis Essay

Abstract

A new series of analogues of the calabash curare alkaloid toxiferine I was prepared and pharmacologically evaluated at α7 and muscle-type nAChRs and the allosteric site of muscarinic M2 receptors. The new ligands differ from toxiferine I by the absence of one (2ac) or two (3ac) hydroxy groups, saturation of the exocyclic double bonds, and various N-substituents (methyl, allyl, 4-nitrobenzyl). At the muscle-type nAChRs, most ligands showed similar binding to the muscle relaxant alcuronium, indicating neuromuscular blocking activity, with the nonhydroxylated analogues 3b (Ki = 75 nM) and 3c (Ki = 82 nM) displaying the highest affinity. At α7 nAChRs, all ligands showed a moderate to low antagonistic effect, suggesting that the alcoholic functions are not necessary for antagonistic action. Compound 3c exerted the highest preference for the muscle-type nAChRs (Ki = 82 nM) over α7 (IC50 = 21 μM). As for the allosteric site of M2 receptors, binding was found to be dependent on N-substitution rather than on the nature of the side chains. The most potent ligands were the N-allyl analogues 2b and 3b (EC0.5,diss = 12 and 36 nM) and the N-nitrobenzyl derivatives 2c and 3c (EC0.5,diss = 32 and 49 nM). The present findings should help delineate the structural requirements for activity at different types of AChRs and for the design of novel selective ligands.

Results and Discussion

The ideal starting material for the modification of the side chains of the toxiferine I scaffold is the bistertiary amine bisnortoxiferine I (Chart 1). However, because no synthesis of the latter has been reported to date and previous attempts to prepare it from its easily available cyclization product caracurine V failed,22,23 it was decided to use alcuronium chloride as a starting material. In the course of catalytic hydrogenation of alcuronium chloride using Pd/C 10% and H2 (15 bar), both N-allyl groups as well as both exocyclic double bonds were saturated. Moreover, one or two alcoholic groups were eliminated, leading after hydrogenation of the resulting double bonds to terminal ethyl substituents. The crude hydrogenation product was subjected to Hofmann elimination using KOH in DMF. In the course of the latter, both N-propyl groups were eliminated as propene, to give a mixture of the bistertiary amines 2 and 3, which could be separated by silica gel chromatography. The double quaternization of 2 and 3 using methyl iodide, allyl bromide, and p-NO2-benzyl bromide to yield 2ac and 3ac readily proceeded in a chloroform solution at room temperature (Scheme 1).

In the course of hydrogenation of bisnortoxiferine I, both exocyclic double bonds C-19–C-20 and C-19′–C-20′ were saturated, generating two novel stereogenic centers, C-20 and C-20′. 1H and 13C NMR spectra of the hydrogenation products 2 and 3 showed single sets of signals indicating a stereoselective course of the hydrogen addition that took place from the less sterically hindered side of the double bonds. The absolute configuration of C-20 and C-20′ was determined by a 600 MHz NOESY experiment for compound 3 (see Supporting Information). Strong NOEs between H-20 (and H-20′) and H-14a (H-14a′) adopting an axial orientation in the piperidine ring revealed the axial position of H-20 (and H-20′). The resulting S-configuration of C-20 (and C-20′) is in agreement with the stereochemistry of the corresponding atoms in the related tetrahydrocaracurine V ring system.24 The essential NOEs of H-20 confirming its axial orientation in the piperidine ring are displayed in Figure ​1.

Figure 1

Essential NOEs of compound 3. The 3D structure of 3 was built starting from the 3D structure of toxiferine I24 by removal of the N-methyl groups and exchange of the allyl alcohol side chains with ethyl groups. The structure was optimized using the Trident...

The antagonistic potency at the α7 nAChRs was assessed as the ability to inhibit by 50% (IC50) the effect of 20 μM ACh in ha7-GH3 cells using a Ca2+/Fluo-4 functional assay.14 The equilibrium inhibition constants Ki of ligand binding to the muscle-type nAChRs were determined according to a previously developed assay in membrane fractions of the Torpedo californica electric organ using (±)-[3H]epibatidine as radioligand.15 As a measure of test compound affinity at the allosteric binding site of the muscarinic M2 receptor, their potency to inhibit the dissociation of the orthosteric antagonist [3H]N-methylscopolamine from the receptors in homogenates of porcine heart ventricles was determined and expressed as EC0.5,diss.26 The results are presented in Table 1.

Table 1

Pharmacological Characterization of the Compounds at α7 and Muscle-Type nAChRs and the Allosteric Binding Site of Muscarinic M2 Receptors

All compounds synthesized in the present study displayed a moderate to low antagonistic effect at α7 nAChR. Most ligands were less potent than the equally substituted ring-closed caracurine V analogues.14 These findings confirmed that the caracurine V ring system is more favorable for binding at α7 nAChRs than the spatially different bisnortoxiferine I skeleton present in ligands 2ac and 3ac. The most potent α7 antagonists among the new series were found to be the N,N-dimethyl analogues 2a and 3a, bearing one or no hydroxy groups, respectively. These compounds showed similar IC50 values at the α7 nACh receptor (590 and 820 nM, respectively) and were 10–15 times more potent than toxiferine I, having two hydroxy groups. These results indicated that the alcohol functions are not necessary for antagonistic action at the α7 nAChRs and may in fact impair the binding properties of these compounds to the receptor. The latter conclusion is supported by the fact that the highly potent α7 nAChR ligands from a previously reported caracurine V series (1ac) also lack OH groups.14

At the muscle-type nAChRs, all ligands showed significantly reduced binding when compared to the strong neuromuscular blocking agent toxiferine I (Ki = 14 nM), but an affinity similar to alcuronium (Ki = 234 nM).15 However, in contrast to the action at the α7 nAChRs, the new compounds displayed considerably higher binding for the muscle-type nAChRs than the caracurine V analogues, indicating stronger neuromuscular blocking potential. The binding affinity in the monodeoxy (2ac) and dideoxy (3ac) series seems to be less dependent on the N-substituent than for the bisnortoxiferine analogues toxiferine I and alcuronium. For example, while the N-methyl-substituted toxiferine I displayed a 17-fold higher affinity than the N-allyl analogue alcuronium (Ki = 234 nM), the equivalently substituted 2a and 2b showed very similar binding constants (Ki = 455 and 250 nM, respectively). The highest affinity was observed for the nonhydroxylated analogues 3b and 3c, with Ki = 75 and 82 nM, respectively, indicating that the hydroxy groups are not essential for binding.

Since toxiferine I, alcuronium, and the caracurine derivatives bind to the allosteric site of the muscarinic M2 receptor,17 the new compounds were also tested for their ability to inhibit the dissociation of the antagonist N-methylscopolamine. With regard to the allosteric M2 receptor site, the binding is dependent on N-substitution rather than on the nature of the side chains. For example, all the N-methyl-substituted analogues, toxiferine I, 2a, and 3a, displayed similar EC0.5,diss values in the three-digit nanomolar range of concentration. In accordance with the data for the previously reported series of bisquaternary caracurine V derivatives, 1 and 1a1c,17 the highest affinity was observed for the N-allyl derivatives alcuronium (2 nM), 2b (12 nM), and 3b (36 nM). The di(4-nitrobenzyl) analogues 3c and 4c displayed slightly reduced affinities. Interestingly, the curve slopes n of 2 and 3c observed in the current study (cf. legend of Table 1) were significantly larger compared to the value reported earlier for the typical muscarinic allosteric modulator alcuronium26 and may point to an atypical allosteric binding mode.27 It should be mentioned that a direct interassay comparison of the M2 data was hampered for three reasons. First, the affinity measure, EC0.5,diss, was obtained in orthosterically (i.e., at the ACh binding site) occupied (with NMS) M2 receptors, whereas pIC50 and Ki (cf. Table 1) were obtained in orthosterically unoccupied nicotinic acetylcholine receptors. Second, the allosteric potency measure EC0.5,diss was determined in a low ionic strength, i.e., high affinity, buffer, known to facilitate a direct labeling of the allosteric site28 and an elucidation of allosteric agent binding mechanisms.27 Third, the allosteric action, i.e., EC0.5,diss, depends on the structure of the orthosteric ligand applied.29

Comparing the ring systems, the activities of the caracurine V analogues 1ac at the receptors under investigation were found to be different from those observed for the new compounds 2ac and 3ac. While the ring-closed caracurine V ligands 1ac showed high antagonistic potency at α7 nAChRs and high affinity to the allosteric site of M2 receptors, their affinity for the muscle-type nAChRs proved to be low. In contrast, the new compounds showed low antagonistic potency at α7 nAChRs, moderate affinity for the allosteric site of M2 receptors, and high binding to the muscle-type nAChRs. These differences can be explained by the diverse geometries of the ring-closed caracurine-V skeleton and the ring-opened bisnortoxiferine I ring system.24 Interestingly, the α7 nAChR is far more sensitive to these conformational changes than the other two receptors.

In summary, even though receptors of very different structures and functionalities were examined, general structure–activity relationships were derived for the different series of compounds investigated. Whereas the caracurine V skeleton is a suitable lead structure for α7 nAChR antagonists, the N-substituted bisnortoxiferine I ring system is a lead for the muscle-type nAChR ligands. Since the substituents attached to the nitrogen atoms seem to play a pivotal role, further variations with regard to substituents of different electronic, lipophilic, and hydrogen-bonding properties should be studied. As the allosteric binding site of the M2 acetylcholine receptor is located at the extracellular vestibule of the M2 receptor30 and is more flexible than orthosteric binding sites, each compound was found to exert an optimal binding mode, with the N-allyl-substituted analogues alcuronium, 1b, 2b, and 3b showing the highest affinity.

When taken together, the new compounds derived from the natural products caracurine V and toxiferine I have been shown to be excellent leads for the development of new ligands for nicotinic and muscarinic receptors.

Experimental Section

General Experimental Procedures

Melting points were determined using a capillary melting point apparatus (Gallenkamp, Sanyo) and are uncorrected. Bruker AV400 and Bruker AV600 NMR spectrometers were used to obtain 1H NMR and 13C NMR spectra, respectively. 1H NMR chemical shifts are referred to CHCl3 (7.26 ppm) and DMSO-d6 (2.50 ppm). 13C NMR chemical shifts are referred to CDCl3 (77.26 ppm) and DMSO-d6 (39.52 ppm). The NMR resonances were assigned by means of COSY and HMQC experiments. EIMS were determined on a Finnigan MAT 90. MALDIMS were run on a Bruker Daltonic MALDI-TOF spectrometer. Elemental analyses were performed by the microanalytical section of the Institute of Inorganic Chemistry, University of Würzburg. All reactions were carried out under an argon atmosphere. Column chromatography was carried out on silica gel 60 (0.063–0.200 mm) obtained from Merck. Alcuronium chloride was synthesized in pure form from Wieland-Gumlich aldehyde as previously reported.31

19,20,19′,20′-Tetrahydro-18′-deoxybisnortoxiferine I (2) and 19,20,19′,20′-Tetrahydro-18,18′-dideoxybisnortoxiferine I (3)

Pd/C 10% (500 mg) was added to a solution of alcuronium chloride (500 mg, 0.678 mmol) in EtOH (100 mL), and the reaction mixture was hydrogenated at 15 bar H2 at rt for 48 h. The catalyst was filtered over Celite and washed with EtOH, and the combined filtrates were concentrated in vacuo to give a crude product (400 mg), which was subjected to Hofmann elimination without purification. Crude products resulting from two catalytic hydrogenations were combined (800 mg), absolute DMF (50 mL) and KOH pellets (650 mg) were added, and the reaction mixture was heated under reflux for 2 h. Water (300 mL) was added, and the products were extracted with EtOAc (3 × 70 mL). The combined organic layers were washed with water (4 × 20 mL) and dried over Na2SO4, and the solvent was removed in vacuo. The residue was subjected to column chromatography on silica gel (CHCl3–MeOH–25%NH3, 100:10:1) to give 2 (120 mg, 16%) and 3 (180 mg, 25%) as colorless solids.

Compound 2

1H NMR (CDCl3, 600 MHz) δ 7.15–7.10 (2H, m, H-11, H-11′), 7.07 (2H, d, J = 7.3 Hz, H-9, H-9′), 6.82–6.76 (2H, m, H-10, H-10′), 6.43–6.38 (2H, m, H-12, H-12′), 6.10 (1H, s, H-17), 6.05 (1H, s, H-17′), 5.15 (1H, s, H-2′), 5.13 (1H, s, H-2), 3.80 (2H, t, J = 6.5 Hz, CH2-18), 3.13 (2H, ddd, J = 10.7, 9.5, 4.1 Hz, H-5a, H-5a′), 3.03 (2H, dd, J = 6.0, 3.1 Hz, H-3, H-3′), 2.98–2.93 (2H, m, H-5b, H-5b′), 2.77 (1H, dd, J = 11.6, 3.5 Hz, H-21a), 2.73 (1H, dd, J = 11.6, 3.7 Hz, H-21a′), 2.66 (1H, s, H-15′), 2.63 (1H, s, H-15), 2.39–2.31 (2H, m, H-6a, H-6a′), 2.30–2.22 (2H, m, H-6b, H-6b′), 2.15 (1H, t, J = 11.6 Hz, H-21b), 2.05 (1H, t, J = 11.6 Hz, H-21b′), 2.03–1.97 (2H, m, H-20), 1.93 (1H, m, H-14a), 1.89 (1H, m, H-14a′), 1.80 (1H, m, H-19a), 1.72–1.67 (2H, m, H-14b, H-14b′), 1.69–1.63 (1H, m, H-20′), 1.58 (1H, m, H-19b), 1.48 (1H, m, H-19a′), 1.37 (1H, m, H-19b′), 1.04 (3H, t, J = 7.4 Hz, CH3-18′); 13C NMR (CDCl3, 100 MHz) δ 146.9 (C, C-13), 146.8 (C, C-13′), 137.9 (C, C-8′), 137.7 (C, C-8), 131.0 (CH, C-17), 130.6 (CH, C-17′), 128.28 (CH, C-11), 128.25 (CH, C-11′), 122.37 (CH, C-9′), 122.34 (CH, C-9), 119.40 (CH, C-10), 119.33 (CH, C-10′), 117.9 (C, C-16), 117.6 (C, C-16′), 107.83 (CH, C-12), 107.78 (CH, C-12′), 73.22 (CH, C-2), 73.15 (CH, C-2), 67.95 (CH, C-3)′, 67.70 (CH, C-3), 60.7 (CH2, C-18), 55.20 (CH2, C-5′), 55.14 (CH2, C-5), 53.92 (C, C-7), 53.88 (C, C-7′), 51.39 (CH2, C-21), 51.32 (CH2, C-21′), 43.9 (CH, C-20′), 43.36 (CH2, C-6), 43.23 (CH2, C-6′), 38.7 (CH, C-20), 35.6 (CH2, C-19), 33.2 (CH, C-15) 32.2 (CH, C-15′), 26.52 (CH2, C-14), 26.48 (CH2, C-14′), 25.0 (CH2, C-19′), 11.9 (CH3, C-18′); EIMS m/z 573 [M]+ (45), 572 (100), 571 (43), 286 (8); anal. C 78.99, H 7.38, N 9.93%, calcd for C38H44N4O, C 79.68, H 7.74, N 9.78%.

Compound 3

1H NMR (CDCl3, 400 MHz) δ 7.12 (2H, m, H-11, H-11′), 7.08 (2H, dd, J = 7.3, 1.0 Hz, H-9, H-9′), 6.78 (2H, m, H-10, H-10′), 6.39 (2H, d, J = 7.3 Hz, H-12, H-12′), 6.05 (2H, s, H-17, H-17′), 5.16 (2H, s, H-2, H-2′), 3.12 (2H, m, H-5a, H-5a′), 3.00 (2H, dd, J = 3.8, 2.3 Hz, H-3, H-3′), 2.97 (2H, m, H-5b, H-5b′), 2.71 (2H, dd, J = 10.6, 3.5 Hz, H-21a, H-21a′), 2.67 (2H, m, H-15, H-15′), 2.33 (2H, m, H-6a, H-6a′), 2.25 (2H, m, H-6b, H-6b′), 2.25 (2H, m, H-6b, H-6b′), 2.06 (2H, t, J = 11.3 Hz, H-21b, H-21b′), 1.89 (2H, m, H-14a, H-14a′), 1.70 (2H, m, H-14b, H-14b′), 1.64 (2H, m, H-20, H-20′), 1.50 (2H, m, H-19a, H-19a′), 1.38 (2H, m, H-19b, H-19b′), 1.05 (6H, t, J = 7.3 Hz, H-18, H-18′); 13C NMR (CDCl3, 100 MHz) δ 146.8 (C, C-13,C-13′), 137.9 (C, C-8, C-8′), 130.5 (CH, C-17, C-17′), 128.2 (CH, C-11, C-11′), 122.4 (CH, C-9, C-9′), 119.2 (CH, C-10, C-10′), 117.7 (C, C-16, C-16′), 107.6 (CH, C-12, C-12′), 73.1 (CH, C-2, C-2′), 68.1 (CH, C-3, C-3′), 55.2 (CH2, C-5, C-5′), 53.8 (C, C-7, C-7′), 51.2 (CH2, C-21, C-21′), 43.9 (CH, C-20, C-20′), 43.1 (CH2, C-6, C-6′), 32.0 (CH, C-15, C-15′), 26.4 (CH2, C-14, C-14′), 25.0 (CH2, C-19, C-19′), 11.9 (CH3, C-18, C-18′); EIMS m/z 557 [M]+ (38), 556 (100), 555 (32), 456 (14), 278 (18); anal. C 81.74, H 7.39, N 10.06%, calcd for C38H44N4, C 81.97, H 7.96, N 9.56%.

General Double Quaternization Procedure of the Tertiary Bases 2 and 3

The respective halide was added dropwise to a solution of 2 or 3 in CHCl3 (10 mL). After being stirred at room temperature for 2 h, the crystallized ammonium salt was isolated by filtration. If no crystallization occurred, the product was precipitated by adding Et2O. The collected ammonium salt was washed with CHCl3 or with a CHCl3–Et2O mixture (1:1) and dried in vacuo (0.001 mbar) at 50 °C. No further purification was necessary, as indicated by the 1H NMR spectra measured.

4,4′-Dimethyl-19,20,19′,20′-tetrahydro-18′-deoxybisnortoxiferinium I Diiodide (2a)

Compound 2a (110 mg, 81%) was obtained from 2 (90 mg, 0.157 mmol) and methyl iodide (0.2 mL) as a white solid: 1H NMR (DMSO-d6, 400 MHz) δ 7.48 (2H, d, J = 7.3 Hz, H-9, H-9′), 7.22 (2H, m, H-11, H-11′), 6.95 (2H, m, H-10, H-10′), 6.69–6.65 (2H, m, H-12, H-12′), 6.22 (1H, s, H-17), 6.20 (1H, s, H-17′), 5.23 (2H, s, H-2, H-2′), 4.57 (1H, t, J = 4.7 Hz, OH), 4.22 (2H, br s, H-3, H-3′), 3.90–3.82 (4H, m, CH2-5, CH2-5′), 3.53–3.42 (4H, m, CH2-18, H-21a, H-21a′), 3.41 (6H, s, N+-CH3, N′+-CH3), 3.40–3.30 (2H, m, H-21b, H-21b′), 2.54–2.45 (4H, m, H-15, H-15′, H-6a, H-6a′), 2.36–2.22 (5H, m, H-6b, H-6b′, H-14a, H-14a′, H-20), 2.10 (1H, m, H-20′), 1.60 (2H, m, H-14b, H-14b′), 1.48 (1H, m, H-19a), 1.38–1.26 (2H, m, H-19b, H-19a′), 1.21 (1H, m, H-19b′), 0.89 (3H, t, J = 7.3 Hz, H-18′); 13C NMR (DMSO-d6, 100 MHz) δ 147.1 (C, C-13,C-13′), 132.9 (CH, C-17), 132.8 (CH,C-17′), 132.6 (C, C-8, C-8′), 129.6 (CH, C-11, C-11′), 122.9 (CH, C-9, C-9′), 121.1 (CH, C-10, C-10′), 112.9 (C, C-16), 112.8 (C, C-16′), 110.4 (CH; C-12), 110.3 (CH, C-12′), 72.0 (CH, C-3′), 71.9 (CH, C-3), 69.9 (CH, C-2, C-2′), 64.7 (CH2, C-5, C-5′), 59.5 (CH2, C-21), 59.4 (CH2, C-21′), 58.0 (CH2, C-18), 52.99 (C, C-7′), 52.97 (C, C-7), 47.8 (CH3, N+-CH3, N′+-CH3), 39.3 (CH2, C-6, C-6′), 37.4 (CH, C-20′), 34.4 (CH, C-20), 33.8 (CH, C-15′), 33.4 (CH2, C-19), 32.9 (CH, C-15), 23.0 (CH2, C-19′), 22.6 (CH2, C-14, C-14′), 11.0 (CH3, C-18′); MALDIMS (matrix, 2,5-dihydroxybenzoic acid in MeOH–H2O, 1:3) m/z 739.5 [M – I + Na + 3H]+, 713.4 [M – I]+ (100%); anal. C 54.51, H 5.95, N 6.12%, calcd for C40H50I2N4O·H2O, C 54.93, H 5.99, N 6.41%.

4,4′-Diallyl-19,20,19′,20′-tetrahydro-18′-deoxybisnortoxiferinium I Dibromide (2b)

Compound 2b (80 mg, 56%) was obtained from 2 (100 mg, 0.175 mmol) and allyl bromide (0.5 mL) as a white solid: 1H NMR (DMSO-d6, 400 MHz) δ 7.49 (2H, d, J = 7.3 Hz, H-9, H-9′), 7.22 (2H, m, H-11, H-11′), 6.94 (2H, m, H-10, H-10′), 6.70–6.65 (2H, m, H-12, H-12′), 6.30–6.16 (2H, m, 2 × −CH2–CH=CH2), 6.24 (1H, s, H-17), 6.21 (1H, s, H-17′), 5.81–5.68 (4H, m, 2 × −CH2–CH=CH2), 5.53 (2H, s, H-2, H-2′), 4.58–4.50 (3H, m, OH, 2 × −CHH–CH=CH2), 4.30–4.20 (2H, m, 2 × −CHH–CH=CH2), 4.23 (2H, br s, H-3, H-3′), 4.00–3.90 (2H, m, H-5a, H-5a′), 3.66–3.26 (8H, m, H-5b, H-5b′, CH2-18, H-21a, H-21a′, H-21b, H-21b′), 2.60 (2H, m, H-6a, H-6a′), 2.47 (2H, br s, H-15, H-15′), 2.44–2.22 (5H, m, H-20, H-6b, H-6b′, H-14a, H-14a′), 2.12 (1H, m, H-20′), 1.59 (2H, m, H-14b, H-14b′), 1.49 (1H, m, H-19a), 1.41 (1H, m, H-19b), 1.37–1.21 (2H, m, H-19a′, H-19b′), 0.85 (3H, t, J = 7.3 Hz, H-18′); 13C NMR (DMSO-d6, 100 MHz) δ 147.2 (C, C-13,C-13′), 132.8 (C, C-8, C-8′), 132.7 (CH, C-17), 132.5 (CH,C-17′), 129.6 (CH, C-11, C-11′), 127.7 (CH2, −CH2–CH=CH2), 127.4 (CH2, −CH2′–CH′=CH2′), 126.8 (CH, −CH2–CH=CH2), 126.6 −CH2′–CH′=CH2′), 122.8 (CH, C-9, C-9′), 121.0 (CH, C-10, C-10′), 112.8 (C, C-16′), 112.6 (C, C-16), 110.22 (CH, C-12), 110.16 (CH, C-12′), 71.68 (CH, C-3), 71.62 (CH, C-3′), 69.3 (CH, C-2′), 69.2 (CH, C-2), 61.5 (CH2, C-5, C-5′), 60.56 (CH2, −CH2′–CH′=CH2′), 60.49 (CH2, −CH2–CH=CH2), 58.2 (CH2, C-18), 55.6 (CH2, C-21, C-21′), 52.7 (C, C-7, C-7′), 38.9 (CH2, C-6, C-6′), 36.9 (CH, C-20′), 34.5 (CH, C-15′), 33.9 (CH, C-15), 33.2 (CH2, C-19), 32.6 (CH, C-20), 22.9 (CH2, C-19′), 22.6 (CH2, C-14, C-14′), 10.9 (CH3, C-18′); MALDIMS (matrix, α-cyano-4-hydroxycinnamic acid in MeOH–MeCN–H2O, 2:1:1) m/z 734.5 [M – Br]+, 692.39 [M – Br – allyl]+ (100%); anal. C 62.08, H 6.50, N 6.34%, calcd for C44H54Br2N4O·2H2O C 62.12, H 6.87, N 6.59%.

4,4′-Di(4-Nitrobenzyl)-19,20,19′,20′-tetrahydro-18′-deoxybisnortoxiferinium I Dibromide (2c)

Compound 2c (95 mg, 54%) was obtained from 2 (100 mg, 0.175 mmol) and 4-nitrobenzyl bromide (200 mg, 0.92 mmol) as a yellow solid; 1H NMR (DMSO-d6, 400 MHz) δ 8.44–8.38 (4H, m), 7.90–7.96 (4H, m), 7.51 (1H, d, J = 7.3 Hz, H-9,) 7.48 (2H, d, J = 7.3 Hz, H-9′), 7.22–7.28 (2H, m, H-11, H-11′), 6.92–6.98 (2H, m, H-10, H-10′), 6.64 (1H, d, J = 7.5 Hz, H-12′), 6.61 (1H, d, J = 7.5 Hz, H-12), 6.33 (1H, s, H-17), 6.27 (1H, s, H-17′), 5.67 (2H, s, H-2, H-2′), 5.31–5.22 (2H, m, 2 × N+–CHH–C6H4–NO2), 4.93–4.84 (2H, m, 2 × N+–CHH–C6H4–NO2), 4.75 (1H, t, J = 4.7 Hz, OH), 4.32–4.22 (2H, m, H-5a, H-5a′), 4.18 (2H, br s, H-3, H-3′), 3.59–3.45 (2H, m, CH2-18), 3.38–3.17 (4H, m, H-5b, H-5b′, H-21a, H-21a′), 3.06 (1H, m, H-21b′), 2.89 (1H, m, H-21b), 2.72–2.58 (2H, m, H-6a, H-6a′), 2.48–2.31 (7H, m, H-15, H-15′, H-20, H-6b, H-6b′, H-14a, H-14a′), 1.70 (2H, m, H-14b, H-14b′), 1.63 (1H, m, H-20′), 1.51–1.40 (2H, H-19a, H-19a′), 1.34–1.21 (2H, H-19b, H-19b′), 0.93 (3H, t, J = 7.3 Hz, H-18′); 13C NMR (DMSO-d6, 100 MHz) δ 148.76 (C, C-NO2), 148.69 (C, C-NO2), 146.6 (C, C-13, C-13′), 135.7 (C, 2 × C1 benzyl), 134.1 (CH, C2 benzyl), 133.8 (CH, C2 benzyl), 133.3 (CH, C-17, C-17′), 132.7 (C, C-8, C-8′), 129.6 (CH, C-11, C-11′), 124.2 (CH, C3 benzyl), 124.0 (CH, C3 benzyl), 123.0 (CH, C-9, C-9′), 120.5 (CH, C-10, C-10′), 112.3 (C, C-16′), 112.0 (C, C-16), 109.5 (CH; C-12, C-12′), 74.1 (CH, C-3), 73.9 (CH, C-3′), 68.7 (CH, C-2), 68.6 (C-2′), 61.5 (CH2, C-5, C-5′), 59.6 (CH2, 2 × CH2 benzyl), 58.1 (CH2, C-18), 54.3 (CH2, C-21′), 53.4 (CH2, C-21), 52.31 (C, C-7′), 52.24 (C, C-7), 38.4 (CH2, C-6, C-6′), 36.7 (CH, C-20′), 35.6 (CH, C-15′), 33.6 (CH, C-15), 33.1 (CH2, C-19), 32.8 (CH, C-20), 23.0 (CH2, C-19′), 22.5 (CH2, C-14, C-14′),11.0 (CH3, C-18′); MALDIMS (matrix, α-cyano-4-hydroxycinnamic acid in MeOH–MeCN–H2O, 2:1:1) m/z 842.45 [M – 2Br + 2H]+ (100%); anal. C 59.62, H 5.86, N 7.65%, calcd for C52H56Br2N6O5·2H2O C 60.00, H 5.81, N 8.07%.

4,4′-Dimethyl-19,20,19′,20′-tetrahydro-18,18′-dideoxybisnortoxiferinium I Diiodide (3a)

Compound 3a (55 mg, 91%) was obtained from 3 (40 mg, 72 mmol) and MeI (0.2 mL) as a white solid: 1H NMR (DMSO-d6, 400 MHz) δ 7.47 (2H, d, J = 7.3 Hz, H-9, H-9′), 7.22 (2H, m, H-11, H-11′), 6.95 (2H, m, H-10, H-10′), 6.66 (2H, d, J = 7.5 Hz, H-12, H-12′), 6.20 (2H, s, H-17, H-17′), 5.22 (2H, s, H-2, H-2′), 4.20 (2H, br s, H-3, H-3′), 3.91–3.79 (4H, m, CH2-5, CH2-5′), 3.50 (2H, m, H-21a, H-21a′), 3.40 (6H, s, N+-CH3, N′+-CH3), 3.32–3.26 (2H, m, H-21b, H-21b′), 2.52–2.39 (4H, m, H-15, H-15′, H-6a, H-6a′), 2.34–2.23 (4H, m, H-6b, H-6b′, H-14a, H-14a′), 2.10 (2H, m, H-20, H-20′), 1.60 (2H, m, H-14b, H-14b′), 1.31 (2H, m, H-19a, H-19a′), 1.21 (2H, m, H-19b, H-19b′), 0.89 (6H, t, J = 7.3 Hz, H-18, H-18′); 13C NMR (DMSO-d6, 100 MHz) δ 147.0 (C, C-13, C-13′), 132.8 (CH, C-17, C-17′), 132.6 (C, C-8, C-8′), 129.6 (CH, C-11, C-11′), 122.9 (CH, C-9, C-9′), 121.1 (CH, C-10, C-10′), 112.8 (C, C-16, C-16′), 110.3 (CH, C-12, C-12′), 72.0 (CH, C-3, C-3′), 69.9 (CH, C-2, C-2′), 64.7 (CH2, C-5, C-5′), 59.2 (CH2, C-21, C-21′), 53.0 (C, C-7, C-7′), 47.7 (CH3, N+-CH3, N′+-CH3), 39.3 (CH2, C-6, C-6′), 37.4 (CH, C-20, C-20′), 33.6 (CH, C-15, C-15′), 23.0 (CH2, C-19, C-19′), 22.5 (CH2, C-14, C-14′), 10.9 (CH3, C-18, C-18′); MALDIMS (matrix, 2,5-dihydroxybenzoic acid in MeOH–H2O, 1:3) m/z 739.5 [M – I + Na + 3H]+, 713.4 [M – I]+ (100%); anal. C 54.60, H 5.90, N 6.11%, calcd for C40H50N4I2·2H2O C 54.80, H 6.21, N 6.39%.

4,4′-Diallyl-19,20,19′,20′-tetrahydro-18,18′-dideoxybisnortoxiferinium I Dibromide (3b)

Compound 3b (81 mg, 80%) was obtained from 3 (70 mg, 0.126 mmol) and allyl bromide (0.2 mL) as a white solid; 1H NMR (DMSO-d6, 400 MHz) δ 7.47 (2H, d, J = 7.5 Hz, H-9, H-9′), 7.22 (2H, m, H-11, H-11′), 6.95 (2H, m, H-10, H-10′), 6.66 (2H, d, J = 7.9 Hz, H-12, H-12′), 6.31–6.16 (2H, m, 2 x N+–CH2–CH=CH2), 6.21 (2H, s, H-17, H-17′), 5.81–5.86 (4H, m, 2 × N+–CH2–CH=CH2), 5.50 (2H, s, H-2, H-2′), 4.54 (2H, dd, J = 12.6, 7.9 Hz, 2 × N+–CHH–CH=CH2), 4.22 (2H, dd, J = 12.6, 5.5 Hz, 2 × N+-CHH-CH=CH2), 4.18 (2H, br s, H-3, H-3′), 3.93 (2H, dd, J = 19.0, 10.1 Hz, H-5a, H-5a′), 3.67–3.59 (2H, m, J = 9.2 Hz, H-5b, H-5b′), 3.47 (2H, t, J = 13.6 Hz, H-21a, H-21a′), 3.32–3.26 (2H, m, H-21b, H-21b′), 2.62–2.50 (4H, m, H-15, H-15′, H-6a, H-6a′), 2.34–2.25 (4H, m, H-6b, H-6b′, H-14a, H-14a′), 2.16–2.07 (2H, m, H-20, H-20′), 1.60 (2H, d, J = 12.3 Hz, H-14b, H-14b′), 1.40–1.20 (4H, m, H-19a, H-19a′, H-19b, H-19b′), 0.87 (6H, t, J = 7.3 Hz, H-18, H-18′); 13C NMR (DMSO-d6, 100 MHz) δ 147.1 (C, C-13, C-13′), 132.8 (CH, C-17, C-17′), 132.4 (C, C-8, C-8′), 129.6 (CH, C-11, C-11′), 127.5 (CH2, 2 × N+–CH2–CH=CH2), 126.8 (CH, 2 × N+–CH2–CH=CH2), 122.8 (CH, C-9, C-9′), 121.0 (CH, C-10, C-10′), 112.6 (C, C-16, C-16′), 110.1 (CH, C-12, C-12′), 71.8 (CH, C-3, C-3′), 69.3 (CH, C-2, C-2′), 61.5 (CH2, C-5, C-5′), 60.6 (CH2, 2 × N+–CH2–CH=CH2), 55.4 (CH2, C-21, C-21′), 52.7 (C, C-7, C-7′), 39.8 (CH2, C-6, C-6′), 36.9 (CH, C-20, C-20′), 33.7 (CH, C-15, C-15′), 22.9 (CH2, C-19, C-19′), 22.5 (CH2, C-14, C-14′), 10.9 (CH3, C-18, C-18′); MALDIMS (matrix, 2,5-dihydroxybenzoic acid in MeOH–H2O, 1:3) m/z 791.56 [M – 5H]+ 719.46 [M – Br + 2H]+ (100%); anal. C 61.37, H 6.64, N6.21%, calcd for C44H54N4Br2·3H2O C 61.97, H 7.09, N 6.57%.

4,4′-Di(4-nitrobenzyl)-19,20,19′,20′-tetrahydro-18,18′-dideoxybisnortoxiferinium I Dibromide (3c)

Compound 3c (150 mg, 84%) was obtained from 2 (100 mg, 0.180 mmol) and 4-nitrobenzyl bromide (200 mg, 0.92 mmol) as a yellow solid: 1H NMR (DMSO-d6, 400 MHz) δ 8.42 (4H, d, J = 8.5 Hz, 4 × O2N–C–CH), 7.92 (4H, d, J = 8.5 Hz, 4 × O2N–C–CH=CH), 7.51 (2H, d, J = 7.1 Hz, H-9, H-9′), 7.24 (2H, m, H-11, H-11′), 6.95 (2H, m, H-10, H-10′), 6.63 (2H, d, J = 7.5 Hz, H-12, H-12′), 6.26 (2H, s, H-17, H-17′), 5.70 (2H, s, H-2, H-2′), 5.26 (2H, d, J = 12.1 Hz, 2 × N+–CHH–C6H4–NO2), 4.95 (2H, d, J = 12.1 Hz, 2 × N+–CHH–C6H4–NO2), 4.31 (2H, dd, J = 18.8, 9.5 Hz, H-5a, H-5a′), 4.25 (2H, br s, H-3, H-3′), 3.45 (2H, t, J = 12.5 Hz, H-21a, H-21a′), 3.37–3.27 (2H, m, J = 9.2 Hz, H-5b, H-5b′), 2.88 (2H, d, J = 12.5 Hz, H-21b, H-21b′), 2.70–2.63 (4H, m, H-15, H-15′, H-6a, H-6a′), 2.54–2.31 (6H, m, H-14a, H-14a′, H-6b, H-6b′, H-20, H-20′), 1.70 (2H, d, J = 11.5 Hz, H-14b, H-14b′), 1.49–1.38 (2H, m, H-19a, H-19a′), 1.34–1.24 (2H, m, H-19b, H-19b′), 0.91 (6H, t, J = 7.3 Hz, H-18, H-18′); 13C NMR (DMSO-d6, 100 MHz) δ 148.8 (C, 2 × C–NO2), 146.8 (C, C-13, C-13′), 135.8 (C, 2 × C1 benzyl), 133.9 (CH, 2 × C2 benzyl), 133.2 (CH, C-17, C-17′), 132.2 (C, C-8, C-8′), 129.7 (CH, C-11, C-11′), 124.2 (CH, 2 × C3 benzyl), 123.0 (CH, C-9, C-9′), 120.7 (CH, C-10, C-10′), 112.1 (C, C-16, C-16′), 109.6 (CH, C-12, C-12′), 73.8 (CH, C-3, C-3′), 68.6 (CH, C-2, C-2′), 61.6 (CH2, C-5, C-5′), 59.6 (CH2, 2 × CH2 benzyl), 54.0 (CH2, C-21, C-21′), 52.4 (C, C-7, C-7′), 38.3 (CH2, C-6, C-6′), 36.7 (CH, C-20, C-20′), 33.3 (CH, C-15, C-15′), 23.0 (CH2, C-19, C-19′), 22.5 (CH2, C-14, C-14′), 11.0 (CH3, C-18, C-18′); MALDIMS (matrix, 2,5-dihydroxybenzoic acid in MeOH–H2O, 1:3) m/z 909.60 [M – Br + 2H]+, 719.46 [M – Br +2H]+, 692.58 [M – Br – CH3C6H4NO2 – Br + H]+ (100%); anal. C 59.58, H 5.85, N 7.72, calcd for C52H56N6O4Br2 × 3H2O C 59.89, H 5.99, N 8.06%.

Ca2+/Fluo-4 Assay

The α7-GH3 cell line32 was cultured in Dulbecco’s modified Eagle medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 10% fetal bovine serum, and 0.1 mg/mL G-418. The Ca2+/Fluo-4 assay was performed essentially as previously described.33 The cells were split into poly-d-lysine-coated, black, 96-well plates with a clear bottom (BD Biosciences, Bedford, MA, USA), and the assay was performed 64–72 h later. The culture medium was aspirated, and the cells were incubated in 50 μL of loading buffer [Hank’s buffered saline solution (HBSS) containing 20 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, and 2.5 mM probenecid, pH 7.4], supplemented with 6 mM Fluo-4/AM (Molecular Probes, Eugene, OR, USA) at 37 °C for 1 h. The loading buffer was removed, the cells were washed once with 100 μL of assay buffer [HBSS containing 20 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, and 2.5 mM probenecid, pH 7.4], and then 100 μL of assay buffer containing 100 μM genistein and various concentrations of the test compounds was added to the wells. Following a 30 min incubation at 37 °C in a humidified 5% CO2 incubator, the 96-well plate was assayed in a NOVOstar microplate reader (BMG Labtechnologies, Offenburg, Germany), measuring emission (in fluorescence units) at 520 nm caused by excitation at 485 nm before and up to 60 s after addition of 33 μL of agonist solution (the agonists were dissolved in assay buffer). The compounds were characterized in duplicate at least three times using EC80–EC90 concentrations of ACh as agonist.

Radioligand Binding Studies at the Muscle-Type nAChRs (Competition Assays)

(±)-[3H]Epibatidine (56.3 Ci/mmol) was obtained from PerkinElmer. All other chemicals were obtained from Sigma-Aldrich. Frozen samples of the T. californica electric organ were purchased from Dr. Charles Winkler, San Pedro, CA, USA. Membrane preparation and radioligand binding assays were performed according to a previously published procedure.15

Membrane Preparation

Briefly, frozen samples of the T. californica electric organ were placed on ice and allowed to thaw slowly before the membrane preparation. The tissue was homogenized (Polytron) in ice-cold Hepes solid solution (HSS) and centrifuged (15000g, 10 min, 4 °C). The pellets were collected, washed four times with HSS buffer through rehomogenization and centrifugation at the same settings, resuspended in HSS buffer, and stored as aliquots at −80 °C.

Competition Assay

Each assay sample, with a total volume of 500 μL, contained 200 μL of the test compound, 100 μL of (±)-[3H]epibatidine, 100 μL of T. californica electroplax (60–70 μg), and 100 μL of HSS buffer. Nonspecific binding was determined in the presence of (−)-nicotine. The samples were incubated for 90 min at 22 °C. The incubation was terminated by vacuum filtration (Brandel harvester) through glass fiber filters (GF/B) presoaked in 1% PEI solution. The filters were rinsed three times with TRIS buffer. Radioactivity was measured using a liquid scintillation counter (PerkinElmer TriCarb 2910 TR).

Competition binding data were analyzed using nonlinear regression methods. Ki values were calculated by the Cheng–Prusoff equation (Ki = IC50/(1 + L/KD), where L is the used radioligand concentration) based on the measured IC50 values and KD = 2 nM for binding of (±)-[3H]epibatidine. The KD values were obtained from five independent experiments performed on the same membrane preparations that were used for the competition assays.

M2 Receptor Binding Assays

Preparation and storage of cardiac porcine membranes was carried out as described elsewhere.27 Protein content was determined by the Lowry method and amounted to 3.3 mg/mL. The [3H]NMS filtration binding assay was carried out as described earlier.27 The buffer contained 4 mM Na2HPO4 and 1 mM KH2PO4, pH 7.4 at 23 °C. [3H]NMS equilibrium binding assays applied 0.2 nM [3H]NMS. Nonspecific [3H]NMS binding was assessed in the presence of 1 μM atropine and did not exceed 5% of total binding. Homologous competition equilibrium binding experiments were carried out for 2 h in a 1.5 mL volume to determine the M2 equilibrium binding characteristics of the radioligand [3H]NMS. Specific binding of [3H]NMS under control conditions was characterized by the negative log equilibrium dissociation constant, pKD = 10.41 ± 0.32, n = 3.

In dissociation experiments, membranes were incubated with the respective radioligand for 30 min at 23 °C. Thereafter, aliquots of the mixture were added to excess unlabeled ligand in buffer over a total period of 120 min followed by filtration of the samples. To determine the effect of the test compounds on the dissociation of [3H]NMS (t1/2,control: 5.27 ± 0.03, n = 70), dissociation was measured by addition of 1 μM atropine in combination with the respective test compounds. Three-point kinetic experiments were performed in analogy to two-point kinetic experiments with measurements of specific [3H]NMS binding at t = 0, t = 10 min, and t = 30 min, respectively.34 Receptor-bound radioactivity was separated by filtration and measured as described earlier.35 The binding data from individual experiments were analyzed by computer-aided, nonlinear regression analysis using Prism 5.03 (GraphPad Software, San Diego, CA, USA).

[3H]NMS dissociation data were analyzed assuming a monoexponential decay as described previously.26 The slowing actions of the allosteric agents on [3H]NMS dissociation were analyzed as described elsewhere.36 Homologous competition data obtained with [3H]NMS were analyzed using a four-parameter logistic function to yield estimates of the bottom and top plateaux, the inflection point (IC50), and the slope factor, n, of the curve. If the observed slope factors did not differ significantly from unity (F-test, p > 0.05), the IC50 values were estimated with n constrained to −1. The pKD value of [3H]NMS equilibrium binding to the M2 receptor was calculated according to ref (37).

[3H]NMS (specific activity 82 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Homburg, Germany). Atropine sulfate and all laboratory reagents were >99% pure and purchased from Sigma Chemicals (Taufkirchen, Germany).

Acknowledgments

We thank M. Kepe, Institute of Pharmacy, University of Bonn, for her skillful technical assistance as well as Dr. M. Grüne and E. Ruckdeschel, Institute of Organic Chemistry, University of Würzburg, for recording 600 MHz NMR spectra. Dr. D. Feuerbach (Novartis Institutes for Biomedical Research, Basel, Switzerland) is thanked for the generous gift of the α7-GH3 cell line. A.A.J. thanks the Novo Nordisk Foundation for financial support. This work was financially supported in part by the National Institutes of Health P20RR016467 (D.G.)

Funding Statement

National Institutes of Health, United States

Notes

This paper was published ASAP on September 5, 2014, with errors to Table 1 and the Results and Discussion Section. The corrected version reposted on September 9, 2014.

Supporting Information Available

1H and 13C NMR spectra of compounds 2, 2ac, 3, 3ac. NOESY spectrum of compound 3. This material is available free of charge via the Internet at http://pubs.acs.org.

Notes

The authors declare no competing financial interest.

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This article is about the plant toxins. For the DC Comics character, see Curaré (Batman Beyond).

Not to be confused with Curara.

Curare[1] or [2] is a common name for various plant extract alkaloidarrow poisons originating from Central and South America. These poisons function by competitively and reversibly inhibiting the nicotinic acetylcholine receptor (nAChR), which is a subtype of acetylcholine receptor found at the neuromuscular junction. This causes weakness of the skeletal muscles and, when administered in a sufficient dose, eventual death by asphyxiation due to paralysis of the diaphragm.

According to pharmacologist Rudolf Boehm's 1895 classification scheme, the three main types of curare are:[3]

  • tube or bamboo curare (so named because of its packing into hollow bamboo tubes; main toxin is D-tubocurarine).
  • pot curare (originally packed in terra cotta pots; main alkaloid components are protocurarine, protocurine, and protocuridine). Protocurarine is the active ingredient; protocurine is only weakly toxic, and protocuridine is not toxic.
  • calabash or gourd curare (originally packed into hollow gourds; main toxin is C-curarine I).

Of these three types, some formulas belonging to the tube curare are the most toxic, relative to their LD50 values.[3]

History[edit]

This article is missing information about use of curare by Central American people. Please expand the article to include this information. Further details may exist on the talk page.(March 2014)

Curare was used as a paralyzing poison by South American indigenous people. The prey was shot by arrows or blowgun darts dipped in curare, leading to asphyxiation owing to the inability of the victim's respiratory muscles to contract. The word 'curare' is derived from wurari, from the Carib language of the Macusi Indians of Guyana.[4] Curare is also known among indigenous peoples as Ampi, Woorari, Woorara, Woorali, Wourali, Wouralia, Ourare, Ourari, Urare, Urari, and Uirary.

In 1596, Sir Walter Raleigh mentioned the arrow poison in his book Discovery of the Large, Rich, and Beautiful Empire of Guiana (which relates to his travels in Trinidad and Guayana), though the poison he described possibly was not curare.[5] In 1780, Abbe Felix Fontana discovered that it acted on the voluntary muscles rather than the nerves and the heart.[6] In 1832, Alexander von Humboldt gave the first western account of how the toxin was prepared from plants by Orinoco River natives.[7]

During 1811–1812 Sir Benjamin Collins Brody (1783–1862) experimented with curare.[8] He was the first to show that curare does not kill the animal and the recovery is complete if the animal's respiration is maintained artificially. In 1825, Charles Waterton described a classical experiment in which he kept a curarized femaledonkey alive by artificial respiration with a bellows through a tracheostomy.[9] Waterton is also credited with bringing curare to Europe.[10]Robert Hermann Schomburgk, who was a trained botanist, identified the vine as one of the Strychnos genus and gave it the now accepted name Strychnos toxifera.[11]

George Harley (1829–1896) showed in 1850 that curare (wourali) was effective for the treatment of tetanus and strychnine poisoning.[12][13] In 1857, Claude Bernard (1813–1878) published the results of his experiments in which he demonstrated that the mechanism of action of curare was a result of interference in the conduction of nerve impulses from the motor nerve to the skeletal muscle, and that this interference occurred at the neuromuscular junction.[3][14] From 1887, the Burroughs Wellcome catalogue listed under its 'Tabloids' brand name, tablets of curare at ​112 grain (price 8 shillings) for use in preparing a solution for hypodermic injection. In 1914, Henry Hallett Dale (1875–1968) described the physiological actions of acetylcholine.[15] After 25 years, he showed that acetylcholine is responsible for neuromuscular transmission, which can be blocked by curare.[16]

The best known and historically most important (because of its medical applications) toxin is d-tubocurarine. It was isolated from the crude drug — from a museum sample of curare — in 1935 by Harold King (1887–1956) of London, working in Sir Henry Dale's laboratory. He also established its chemical structure.[17] It was introduced into anesthesia in the early 1940s as a muscle relaxant for surgery. Curare is active — toxic or muscle-relaxing, depending on the intended use — only by an injection or a direct wound contamination by poisoned dart or arrow. It is harmless if taken orally[9][18] because curare compounds are too large and highly charged to pass through the lining of the digestive tract to be absorbed into the blood. For this reason, people can eat curare-poisoned prey safely. In medicine, curare has been superseded by a number of curare-like agents, such as pancuronium, which have a similar pharmacodynamic profile, but fewer side effects.

Classification and chemical structure[edit]

This section needs expansion. You can help by adding to it.(March 2014)

The various components of curare are organic compounds classified as either isoquinoline or indole alkaloids. Tubocurarine is the major active component in the South American dart poison.[19] As an alkaloid, tubocurarine is a naturally occurring compound that consists of nitrogenous bases—though the chemical structure of alkaloids is highly variable.

Like most alkaloids, tubocurarine consists of a cyclic system with a nitrogen atom in an amine group.[20] Because of this structure, tubocurarine can bind readily to the receptors for acetylcholine (ACh) at the neuromuscular junction, which blocks nerve impulses from being sent to the skeletal muscles, effectively paralyzing the muscles of the body. Since tubocurarine binds reversibly to the ACh receptors, treatment for curare poisoning involves adding an acetylcholinesterase (AChE) inhibitor, which will stop the destruction of acetylcholine so that it can compete with curare.[21]

Pharmacological properties[edit]

Curare is an example of a non-depolarizing muscle relaxant that blocks the nicotinic acetylcholine receptor (nAChR), one of the two types of acetylcholine (ACh) receptors, at the neuromuscular junction. The main toxin of curare, d-tubocurarine, occupies the same position on the receptor as ACh with an equal or greater affinity, and elicits no response, making it a competitive antagonist. The antidote for curare poisoning is an acetylcholinesterase (AChE) inhibitor (anti-cholinesterase), such as physostigmine or neostigmine. By blocking ACh degradation, AChE inhibitors raise the amount of ACh in the neuromuscular junction; the accumulated ACh will then correct for the effect of the curare by activating the receptors not blocked by toxin at a higher rate.

The time of onset varies from within one minute (for tubocurarine in intravenous administration, penetrating a larger vein), to between 15 and 25 minutes (for intramuscular administration, where the substance is applied in muscle tissue).[22]

Curare has no effect if ingested so the meat of an animal killed by curare does not become poisonous, and it has no effect on its flavor.[23]

Anesthesia[edit]

Isolated attempts to use curare during anesthesia date back to 1912 by Arthur Lawen of Leipzig,[24] but curare came to anesthesia via psychiatry (electroplexy). In 1939 Abram Elting Bennett used it to modify metrazol induced convulsive therapy.[25]Muscle relaxants are used in modern anesthesia for many reasons, such as providing optimal operating conditions and facilitating intubation of the trachea. Before muscle relaxants, anesthesiologists needed to use larger doses of the anesthetic agent, such as ether, chloroform or cyclopropane to achieve these aims. Such deep anesthesia risked killing patients that were elderly or had heart conditions.

The source of curare in the Amazon was first researched by Richard Evans Schultes in 1941. Since the 1930s, it was being used in hospitals as a muscle relaxant. He discovered that different types of curare called for as many as 15 ingredients, and in time helped to identify more than 70 species that produced the drug.

In the 1940s, it was used on a few occasions during surgery as it was mistakenly thought to be an analgesic or anesthetic. The patients reported feeling the full intensity of the pain though they were not able to do anything about it since they were essentially paralyzed.[26]

On January 23, 1942, Harold Griffith and Enid Johnson gave a synthetic preparation of curare (Intercostrin/Intocostrin) to a patient undergoing an appendectomy (to supplement conventional anesthesia). Safer curare derivatives, such as rocuronium and pancuronium, have superseded d-tubocurarine for anesthesia during surgery. When used with halothane d-tubocurarine can cause a profound fall in blood pressure in some patients as both the drugs are ganglion blockers.[27] However, it is safer to use d-tubocurarine with ether.

In 1954, an article was published by Beecher and Todd suggesting that the use of muscle relaxants (drugs similar to curare) increased death due to anesthesia nearly sixfold.[28] This was refuted in 1956.[29]

Modern anesthetists have at their disposal a variety of muscle relaxants for use in anesthesia. The ability to produce muscle relaxation irrespective of sedation has permitted anesthetists to adjust the two effects independently and on the fly to ensure that their patients are safely unconscious and sufficiently relaxed to permit surgery. The use of neuromuscular blocking drugs carries with it a very small risk of anesthesia awareness.

Plant sources[edit]

There are dozens of plants from which isoquinoline and indole alkaloids with curarizing effects can be isolated, and which were utilized by indigenous tribes of Central and South America for the production of arrow poisons. Among them are:

In family Menispermaceae:

Other families:

Some plants in the Aristolochiaceae family have also been reported as sources.

Toxicity[edit]

The toxicity of curare alkaloids in humans hasn't been established. Administration must be parenterally, as gastro-intestinal absorption is ineffective.

LD50 (mg/kg)

human: 0.735 est. (form and method of administration not indicated)

mouse: pot:0.8-25; tubo: 5-10; calabash: 2-15.

Preparation[edit]

This section needs expansion. You can help by adding to it.(March 2014)

Traditionally prepared curare is a dark, heavy, viscid paste with a very bitter taste.[30]

Adjuvants[edit]

It is known[citation needed] that the final preparation is often more potent than the concentrated principal active ingredient. Various irritating herbs, stinging insects, poisonous worms, and various parts of amphibians and reptiles are added to the preparation. Some of these accelerate the onset of action or increase the toxicity; others prevent the wound from healing or blood from coagulating.

Diagnosis and management of curare poisoning[edit]

Curare poisoning can be indicated by typical signs of neuromuscular-blocking drugs such as paralysis including respiration but not directly affecting the heart.

Curare poisoning can be managed by artificial respiration such as mouth-to-mouth resuscitation. In a study of 29 army volunteers that were paralyzed with curare, artificial respiration managed to keep an oxygen saturation of always above 85%,[31] a level at which there is no evidence of altered state of consciousness.[32] Yet, curare poisoning mimics the total locked-in syndrome in that there is paralysis of every voluntarily controlled muscle in the body (including the eyes), making it practically impossible for the victim to confirm consciousness while paralyzed.[33]

Spontaneous breathing is resumed after the end of the duration of action of curare, which is generally between 30 minutes[34] to 8 hours,[35] depending on the variant of the toxin and dosage. Cardiac muscle is not directly affected by curare, but if more than four to six minutes has passed since respiratory cessation the cardiac muscle may stop functioning by oxygen-deprivation, making cardiopulmonary resuscitation including chest compressions necessary.[36]

Antidote[edit]

Muscle paralysis can be reversed by administration of a cholinesterase inhibitor such as pyridostigmine.[37] Other drugs have anticurare properties.

Citations[edit]

  1. ^Company, Houghton Mifflin Harcourt Publishing. "The American Heritage Dictionary entry: curare". www.ahdictionary.com. 
  2. ^curare in the Oxford Online Dictionaries
  3. ^ abcGray, TC (1947). "The Use of D-Tubocurarine Chloride in Anæsthesia"(PDF). Ann R Coll Surg Engl. 1 (4): 191–203. PMC 1940167. PMID 19309828. 
  4. ^"Online Etymology Dictionary". www.etymonline.com. 
  5. ^Carman J. A. Anaesthesia 1968, 23, 706.
  6. ^The Gale Encyclopedia of Science. Third Edition.
  7. ^[1]Personal Narrative of Travels to the Equinoctial Regions of America, During the Year 1799–1804 — Volume 2, Humboldt, Alexander von
  8. ^Phil. Trans. 1811, 101, 194; 1812, 102, 205.
  9. ^ ab"Loading..."www.yeoldelog.com. 
  10. ^Reprinted in "Classical File", Survey of Anesthesiology 1978, 22, 98.
  11. ^Waterton and Wouralia. British Journal of Pharmacology (1999) 126, 1685–1689
  12. ^Paton A. Practitioner 1979, 223, 849
  13. ^"Whonamedit – dictionary of medical eponyms". www.whonamedit.com. 
  14. ^Bernard, C (1857). "Leçons sur les effets des substances toxiques et médicamenteuses" (in French). Paris: J.B. Baillière: 369–80. 
  15. ^Dale H. H. J. Pharmac. Exp. Ther. 1914, 6, 147.
  16. ^Dale H. H. Br. Med. J. 1934, 1, 835
  17. ^King H. J. Chem. Soc. 1935, 57, 1381; Nature, Lond. 1935, 135, 469.
  18. ^"Curare – Chondrodendron tomentosum". www.blueplanetbiomes.org. 
  19. ^http://www.britannica.com/science/curare Encyclopædia Britannica 01 Dec. 2015
  20. ^Ethnopharmacology and systems biology: A perfect holistic match, Verpoorte, R. 2005
  21. ^Saladin, Kenneth S. Anatomy and Physiology The Unity of Form and Function. 7th ed. New York: McGraw Hill Education, 2015. Print.
  22. ^Curare in Drugs.com. Revised: 11/08/2001
  23. ^From the Rainforests of South America to The Operating Room: A History of Curare, By Daniel Milner, BA, CD, Summer 2009. University of Ottawa Faculty of Medicine.
  24. ^Lawen A. Beitr. klin. Chir. 1912, 80, 168.
  25. ^Bennett A. E. J. Am. Med. Ass. 1940, 114, 322
  26. ^Dennett, Daniel C. Brainstorms: Philosophical Essays on Mind and Psychology (1978), Cambridge, MA : MIT Press, p209
  27. ^Mashraqui S. Hypotension induced with d-tubocurarine and halothane for surgery of patent ductus arteriosus. Indian Journal of Anesthesia. 1994 Oct; 42(5): 346-50
  28. ^Beecher H. K.; Todd D. P. (1954). "A Study of the Deaths Associated with Anesthesia and Surgery : Based on a Study of 599,548 Anesthesias in Ten Institutions 1948–1952, Inclusive". Annals of Surgery. 140 (2): 2–35. doi:10.1097/00000658-195407000-00001. PMC 1609600. PMID 13159140.  
  29. ^Albertson HA, Trout HH, Morfin E (June 1956). "The Safety of Curare in Anesthesia'". Annals of Surgery. 143 (6): 833–837. doi:10.1097/00000658-195606000-00012. PMC 1465152. PMID 13327828. 
  30. ^Curare, a South American Arrow PoisonArchived 2012-07-28 at the Wayback Machine., from "Plants and Civilization" by Professor Arthur C. Gibson, at UCLA Mildred E. Mathias Botanical Garden.
  31. ^Page 520 in: Paradis, Norman A. (2007). Cardiac arrest: the science and practice of resuscitation medicine. Cambridge, UK: Cambridge University Press. ISBN 0-521-84700-1. 
  32. ^Oxymoron: Our Love-Hate Relationship with Oxygen, By Mike McEvoy at Albany Medical College, New York. 10/12/2010
  33. ^Page 357 in: Damasio, Antonio R. (1999). The feeling of what happens: body and emotion in the making of consciousness. San Diego: Harcourt Brace. ISBN 0-15-601075-5. 
  34. ^For therapeutic dose of tubocurarine by shorter limit as given at page 151 in: Rang, H. P. (2003). Pharmacology. Edinburgh: Churchill Livingstone. ISBN 0-443-07145-4. OCLC 51622037. 
  35. ^For 20-fold paralytic dose of toxiferine ("calabash curare"), according to: Page 330 in: The Alkaloids: v. 1: A Review of Chemical Literature (Specialist Periodical Reports). Cambridge, Eng: Royal Society of Chemistry. 1971. ISBN 0-85186-257-8. 
  36. ^"four to six minutes" given from: Cardiopulmonary Resuscitation (CPR) in Farlex medical dictionary, in turn citing Gale Encyclopedia of Medicine. Copyright 2008.
  37. ^Page 153 in: Thomas Morgan III; Bernadette Kalman (2007). Neuroimmunology in Clinical Practice. Wiley-Blackwell. ISBN 1-4051-5840-9. 

See also[edit]

References[edit]

  • Foldes, F.F. "Anesthesia before and after curare", Anasthesieabteilung des Albert-Einstein-College of Medicine. Anaesthesiol Reanim, 1993, 18(5):128-31. (retrieved June 20, 2005)
  • James, Mel. "Harold Griffith",Heirloom Series, Volume 6. (retrieved June 20, 2005)
  • "Curare", Blue Planet Biomes, 2000. (retrieved September 27, 2005)
  • Smith, Roger. "Cholernergic Transmission", (retrieved March 13, 2007)
  • Strecker G J et al. "Curare binding and the curare-induced subconductance state of the acetylcholine receptor channel.", Biophysical Journal 56: 795-806 (1989). (retrieved May 12, 2007)

External links[edit]

Nicotinic acetylcholine receptormodulators

nAChRs
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Antagonists
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Strychnos toxifera by Koehler 1887

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