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Methyllycaconitine

January 01, 1970

Methyllycaconitine (MLA) is a diterpenoid alkaloid found in many species of Delphinium (larkspurs).[1][2] In common with many other diterpenoid alkaloids, it is toxic to animals, although the acute toxicity varies with species.[3][4] Early research was focused on identifying, and characterizing the properties of methyllycaconitine as one of the principal toxins in larkspurs responsible for livestock poisoning in the mountain rangelands of North America.[3][5] Methyllycaconitine has been explored as a possible therapeutic agent for the treatment of spastic paralysis,[6] and it has been shown to have insecticidal properties.[7] Most recently, it has become an important molecular probe for studying the pharmacology of the nicotinic acetylcholine receptor.[8]

Methyllycaconitine
Identifiers
CAS Number
  • 21019-30-7 Y
PubChem CID
  • 5311278
ChemSpider
  • 4470788
UNII
  • 8H7EX9Z9QE
ChEMBL
  • ChEMBL510275
CompTox Dashboard (EPA)
  • DTXSID80943320
Chemical and physical data
FormulaC37H50N2O10
Molar mass682.811 g·mol−1
3D model (JSmol)
  • Interactive image
  • CCN1CC2(CCC(C34C2C(C(C31)(C5(CC(C6CC4C5C6OC)OC)O)O)OC)OC)COC(=O)C7=CC=CC=C7N8C(=O)CC(C8=O)C

Isolation edit

The first isolation of MLA, from Delphinium brownii, Rydb., was probably made by Richard Manske at the National Research Laboratories in Ottawa, Canada, in 1938.[9] Presumably because he did not obtain the compound in sufficiently pure form, Manske declined to give it a name. The name "methyl-lycaconitine" was assigned by John Goodson, working at the Wellcome Chemical Research Laboratories in London, England, when he isolated the alkaloid, in purer form, from seeds of Delphinium elatum, L. in 1943.[10] A more modern isolation procedure is described by Pelletier and his co-workers, who used seeds of the "garden larkspur", Consolida ambigua (also referred to as Delphinium ajacis) as their plant source.[11]

Structure determination edit

The complete molecular structure for MLA, correct in all but one detail, was first published by Kuzovkov and Platonova in 1959.[12] This structure, supported in part by X-ray crystallography (considered usually to be a "definitive" analytical technique) of a chemical derivative of MLA performed by Maria Przybylska,[13] was accepted as correct until the early 1980s. At that time, the research groups of Pelletier[14] and of Edwards and Przybylska[15] independently corrected the stereochemistry of the methoxy group at C-1 from the β- to α- configuration. Thus any drawing of MLA appearing before Pelletier's 1981 paper[14] will show the structure with the incorrect stereochemistry at C-1.

Chemistry edit

Synonyms edit

[1α,4(S),6β,14α,16β]-20-Ethyl-1,6,14,16-tetramethoxy-4-[[[2-(3-methyl-2,5-dioxo-1-pyrrolidinyl)benzoyl]oxy]methyl]aconitane-7,8-diol; also referred to, incorrectly, as "N-methyl lycaconitine" in a few publications.

Physico-chemical properties edit

MLA is soluble in chloroform, but does not dissolve well in water.[10] The free base of MLA has not been obtained in crystalline form, and in its amorphous form it melts ultimately at 128 °C;[10] the hydriodide salt has a melting point of 201 °C.;[10] the perchlorate salt melts at 195 °C[16] The citrate salt is the most common form in which MLA is currently available commercially.[17]

A pKa does not seem to have been recorded for MLA, but it is considered to be a weak base because it can be readily extracted into diethyl ether from an aqueous solution at pH 7.5-8.[14]

The optical rotation of the free base, [α]D was found to be +49° in alcohol.[10]

Molecular structure edit

Although commonly referred to as a "diterpenoid" alkaloid, MLA is, strictly speaking, a nor-diterpenoid, since its carbon skeleton only contains 19 C atoms, one having been deleted somewhere during its biosynthesis.[18] Otherwise, the MLA molecule comprises a tertiary amine, two tertiary alcohols, four methyl ether groups, and a complex ester based on anthranilic acid and methylsuccinic acid. This N-(2-carboxyphenyl)-methylsuccinamido-ester is quite rare amongst natural products.

Synthesis edit

As of April, 2012 no total synthesis of MLA has been reported. A semi-synthesis of MLA, starting from its "parent" amino-alcohol, lycoctonine (obtained by simple alkaline hydrolysis of natural MLA [10]) was reported in 1994.[19]

Pharmacology edit

In many respects, the pharmacology of MLA closely resembles that of the classical neuromuscular blocker, d-tubocurarine. The "curare-like" properties of MLA seem to have been first mentioned in 1958 by Kuzovkov and Bocharnikova,[20] working at the Ordzhinikidze All-Union Institute for Scientific Research in Pharmaceutical Chemistry, in the former USSR. A detailed paper on the pharmacology of MLA (in the form of its hydriodide salt, given the drug name "mellictine") in classical animal preparations was published from the same Institute in the following year by Dozortseva.[21]

These studies, together with related others and some original observations, are summarized in the review by Benn and Jacyno.[3]

They revealed that MLA blocked neuromuscular transmission in skeletal muscle, but not smooth muscle, and had some ganglion-blocking action. Such properties are characteristic of an antagonist of acetylcholine exerting its effects at nicotinic, but not muscarinic sites.

In the rat phrenic nerve-diaphragm preparation, for example, a 2 x 10−5M concentration of MLA produced a 50% decrease in response, and total inhibition was caused by a 3 x 10−5M concentration of the drug. In this preparation, MLA-treated muscle responded normally to direct electrical stimulation, but the inhibition of contractions was only partially antagonized by physostigmine. Similar results were obtained with frog nerve-muscle preparations, in which it was shown that MLA blocked response of the gastrocnemius muscle to electrical stimulation of the sciatic nerve, inhibited post-synaptic action potentials in the sartorius muscle elicited by stimulation of the sciatic nerve, and reduced the amplitude of miniature end-plate potentials in the extensor digitus IV muscle.

Ganglion-blocking effects of MLA were observed using the cat nictitating membrane preparation: complete inhibition of the response was produced by 4 mg/kg of "mellictine" given intravenously.

No significant effects were produced by the drug in smooth muscle preparations from rabbit, guinea pig or cat, indicating the lack of activity at typically muscarinic sites. In electrically stimulated guinea pig ileum, for example, contractions were unaffected by a concentration of 5 x 10−4M of MLA.

A more detailed summary of the above data, together with much related material, may be found in a review written by Kip Panter and collaborators at USDA-ARS laboratories in Utah and California.[22]

A significant advance was made towards understanding the pharmacology of MLA when Jennings and co-workers[7] at the American Cyanamid Company reported that MLA (as its citrate salt) strongly inhibited the binding of tritiated propionyl-α-bungarotoxin to a receptor preparation from house-fly heads, with a Ki of ~ 2.5 x 10−10M. Subsequently, Macallan and his co-workers[23] showed that MLA also competed with 125I-α-bungarotoxin (Ki ~1 x 10−9M) and tritiated (−)-nicotine (Ki ~4 x 10−6M) in a receptor preparation from rat brain. These workers also reported that MLA displaced125I-α-bungarotoxin from purified Torpedo (electric ray) nicotinic acetylcholine receptors (nAChRs) with a Ki ~1 x 10−6M. Similar experiments performed later by Ward et al.[24] showed that MLA bound to nAChRs extracted from human muscle with a Ki of ~8 x 10−6M; it was also reported that MLA, at a concentration of 10−4M, had no affinity for muscarinic AChRs, as labeled by tritiated quinuclidinyl benzilate, from rat brain.

Further details about the binding of MLA to nAChRs were presented by Wonnacott and her co-workers,[8] who provided evidence that MLA bound preferentially to different sub-units, as expressed in Xenopus frog oocytes, of the nAChR cloned from avian DNA: MLA was found to have an IC50 of ~8 x 10−8M at α3β2 and ~7 x 10−7M at α4β2 receptor sub-types. Although it was also established that MLA bound strongly to α7 sub-types, experimental difficulties precluded the determination of an IC50. Subsequently, research groups from Abbott Laboratories in the USA, and the University of Geneva in Switzerland reported that MLA displaced 125I-α-bungarotoxin from α7 receptors cloned from the human K28 cell line, with a Ki of ~ 1 x 10−8.[25]

One last milestone in the ongoing saga of MLA pharmacology (there are, as of April 2012, approximately 660 references to articles in journals covered by PubMed) to be mentioned is the characterization of the receptor-interactions of tritium-labeled MLA, by researchers at the University of Bath, in the UK.[26]

One relatively recent study which sheds light on the interaction of MLA with acetylcholine-binding proteins (AChBP) at the molecular level is that of Hansen et al.,[27] who made observations on the crystal structure of a complex between MLA and an AChBP isolated from the salt-water snail, Aplysia californica.

Toxicology edit

The toxicology of MLA has been studied largely in the context of livestock poisoning by wild larkspurs. The seminal work by John Jacyno and Mike Benn at the University of Calgary in Canada showed that MLA was most likely to be the agent responsible for the toxicity of a local larkspur, D. brownii, and provided some preliminary acute toxicity data in several animal species.[3][4][5] These LD50s are as follows: mouse, 3–5 mg/kg; frog, 3–4 mg/kg; rabbit, 2–3 mg/kg (after parenteral administration). Cats appeared to have comparable susceptibility to rabbits, whereas dogs were ~ 1.5 x more sensitive.[21] These early observations have been comprehensively extended by USDA researchers,[22] who have estimated the LD50 of MLA to be ~10 mg/kg in sheep, ~ 5 mg/kg in rats, and ~2 mg/kg in cattle.

Although most LD50s are usually determined from parenteral administration of the test drug, MLA is also active when taken orally.[21]

Signs of toxicity in calves, sheep, rats and mice, at low doses, included agitation, respiratory difficulty, and loss of motor control; symptoms appeared within 2–3 minutes of injection, and disappeared within 10 minutes. Doses large enough to produce collapse also caused an increase in heart and respiration rates, as well as tremor, with significant convulsions evident in mice and rats, but not in cattle or sheep.[22] In cases where death seemed imminent, the poisoning in sheep could be counteracted by the i.v. administration of neostigmine and atropine,[22] whereas poisoning in calves was reversed by the administration of physostigmine.[4] In animals that were allowed to die, death appeared to be the result of complete motor paralysis and respiratory arrest.[21][22]

It is worth noting that although a LD50 for man is not available, the clinical studies of Kabelyanskaya showed that an oral dose of 0.02 g of MLA hydriodide ("mellictine") might be given to patients up to 5 times per day, over the course of 1 month. However, some subjects could only tolerate single doses of 0.02 g per day without experiencing side-effects.[6]

Structure-Activity relationships edit

The earliest observation on a relationship between the molecular structure of MLA and a biological activity concerned the effect of the C-18 ester group on acute toxicity. When this group was hydrolyzed, the resulting amino-alcohol (named lycoctonine as a consequence of its natural occurrence) was found to be much less poisonous to animals than was MLA.[3] A recent study comparing the LD50 of MLA and lycoctonine, given i.v. to mice, showed that lycoctonine was more than 100x less toxic than MLA.[22] In other functional pharmacological assays, lycoctonine resembled MLA qualitatively but was roughly ten times less potent.[3]

When compared in nAChR-binding studies, MLA was found to compete for 125I-α-bungarotoxin binding sites (i.e. α7 sub-types) over 1000x more strongly than did lycoctonine.[28]

If the succinimide ring is deleted so as to leave only the -NH2 group attached to the benzene ring (as in the alkaloid anthranoyllycoctonine, which also occurs naturally), the resulting compound is intermediate between MLA and lycoctonine in potency and toxicity: it is less acutely toxic than MLA by a factor of about 4, but its affinity for 125I-α-bungarotoxin binding sites is over 200x lower than that of MLA.[29]

If the -NH2 group of anthranoyllycoctonine is removed, giving the compound lycoctonine-18-O-benzoate, the affinity for α7 receptors, as well as for α4β2 receptors is reduced by about a factor of 10 in comparison to MLA.[30] When compared with MLA in the rat phrenic nerve-diaphragm assay, lycoctonine-18-O-benzoate was also about 10x less potent, and a similar reduction in potency was observed in an electrophysiological study involving frog extensor muscle.[3]

Even the absence of the methyl group from the methylsuccinimido- ring, as in the alkaloid lycaconitine, reduces the affinity for α7 receptors by a factor of about 20,[31]> but in this case affinity for α4β2 receptors is not significantly changed in comparison with MLA.[30]

Another approach that has been explored in the attempt to elucidate structure-activity relationships in MLA has been to start with 2-(methylsuccinimido)-benzoic acid (the carboxylic acid produced when MLA is split at the C-18 ester group) and to esterify it with various alcohols and amino-alcohols that might be considered as "molecular fragments" of MLA. None of these compounds showed any significant degree of the biological actions characteristic of MLA, however, in the limited number of assays to which they were subjected.[3][22]

Therapeutic applications edit

MLA has been used for treating a variety of neurological disorders,[6][32] although there are no references to such use in the last few decades.

More recently, it has been proposed that MLA might be useful in reducing nicotine reward without precipitating symptoms of nicotine withdrawal.[33] This suggestion was made on the basis of experiments in which intraperitoneal doses of ~4 mg/kg and 8 mg/kg of MLA significantly reduced nicotine self-administration in rats.

Most recently, it has been suggested[34] that MLA had potential in the treatment of cannabis dependence. However, this suggestion was apparently based only on work by Solinas et al.[35] who showed that doses of 0.3-5.6 mg/kg, i.p., in rats, dose-dependently antagonized the discriminative-stimulus effects of 3 mg/kg THC.

Given that the early Soviet work[6] with "mellictine" indicated that as little as ~0.2-0.3 mg/kg, orally, in man (assuming a weight of 60–70 kg, for the sake of making the dose conversion) could produce symptoms of toxicity, and that oral administration of most drugs typically requires more drug than parenteral administration, it is uncertain if MLA will prove to be a practical treatment for either nicotine or cannabis addiction, based on the effective doses required in the rat experiments.

In a recent review, Wu and co-workers[36] have cited research in which α7-antagonists such as MLA show potential in cancer treatment, but this work is still in its very early stages.

Insecticidal action edit

Jennings and co-workers, in addition to making their key observations (see Pharmacology above) about the receptor-binding of MLA, found it to be toxic (50+% mortality) to the following insect species: Empoasca abrupta[37] (at 100 ppm), Heliothis virescens (at 1000 ppm), Musca domestica (at 1000 ppm) and Spodoptera eridana (at 1000 ppm). Species which were not significantly affected by MLA were: Anopheles quadrimaculatus, Aphis fabae, Diabrotica undecimpunctuata howardi and Tetranychus urticae. MLA also behaved as a feeding deterrent, with an LC50 of ~300 ppm, to Spodoptera larvae feeding on bean leaves.[7]

References edit

  1. ^ Willaman JJ, Liu HJ (1970). "Missing". Lloydia. 33 (Supplement 3A): 180–181.
  2. ^ Harbourne JB, Baxter H (1993). Phytochemical Dictionary. London: Taylor & Francis. p. 153.
  3. ^ a b c d e f g h Benn MH, Jacyno JM. Pelletier SW (ed.). Chemical and Biological Perspectives. Vol. 1. New York: Wiley. pp. 153–210.
  4. ^ a b c Nation PN, Benn MH, Roth SH, Wilkens JL (September 1982). "Clinical signs and studies of the site of action of purified larkspur alkaloid, methyllycaconitine, administered parenterally to calves". The Canadian Veterinary Journal. 23 (9): 264–6. PMC 1790203. PMID 17422179.
  5. ^ a b Aiyar VN, Benn MH, Hanna T, Jacyno J, Roth SH, Wilkens JL (October 1979). "The principal toxin of Delphinium brownii Rydb., and its mode of action". Experientia. 35 (10): 1367–8. doi:10.1007/BF01964013. PMID 499426. S2CID 19818861.
  6. ^ a b c d Kabelianskaia LG (1959). "[Use of mellictin in clinical diseases of the nervous system in spastic paralysis]". Farmakologiia I Toksikologiia. 22 (1): 38–42. PMID 13653123.
  7. ^ a b c Jennings KR, Brown DG, Wright DP (1986). "Methyllycaconitine, a naturally occurring insecticide with a high affinity for the insect cholinergic receptor". Experientia. 42 (6): 611–613. doi:10.1007/BF01955557. S2CID 1460936.
  8. ^ a b Wonnacott E, Albuquerque X, Bertrand D (1993). Conn PM (ed.). Methods in Neurosciences. Vol. 12. San Diego: Academic Press. pp. 263–275.
  9. ^ Manske RH (March 1939). "Missing". Canadian Journal of Research. 16: 57–60. doi:10.1139/cjr39b-010.
  10. ^ a b c d e f Goodson JA (1943). "42. The alkaloids of the seeds of Delphinium elatum, L.". Journal of the Chemical Society (Resumed): 139–41. doi:10.1039/JR9430000139.
  11. ^ Pelletier SW, Sawhney RS, Desai HK, Mody NV (May 1980). "The diterpenoid alkaloids of Consolida ambigua". Journal of Natural Products. 43 (3): 395–406. doi:10.1021/np50009a011.
  12. ^ Kuzovkov AD, Platonova TF (1959). "Missing". J. Gen. Chem. (Eng. Trans.). 29: 2746–2749.
  13. ^ Przybylska M, Marion L (February 1956). "The Crystal Structure of Des-(oxymethylene)_lycoctonine Hydroidodide Monohydrate". Canadian Journal of Chemistry. 34 (2): 185–7. doi:10.1139/v56-026.
  14. ^ a b c Pelletier SW, Mody NV, Varughese KI, Maddry JA, Desai HK (October 1981). "Structure revision of 37 lycoctonine-related diterpenoid alkaloids". Journal of the American Chemical Society. 103 (21): 6536–8. doi:10.1021/ja00411a062.
  15. ^ Edwards OE, Przybylska M (November 1982). "The lycoctonine family of alkaloids: a stereochemical revision". Canadian Journal of Chemistry. 60 (21): 2661–7. doi:10.1139/v82-382.
  16. ^ Stern ES (1954). Manske RH, Holmes HL (eds.). The Alkaloids. Vol. 4. New York: Academic Press. pp. 275–333.
  17. ^ No citation is given here because the information is date-specific, and because it is inappropriate to endorse any particular supplier.
  18. ^ The biosynthetic pathway by which MLA is created in the plant is still not known in any great detail.
  19. ^ Blagbrough IS, Coates PA, Hardick DJ, Lewis T, Rowan MG, Wonnacott S, Potter BV (November 1994). "Acylation of lycoctonine: semi-synthesis of inuline, delsemine analogues and methyllycaconitine". Tetrahedron Letters. 35 (46): 8705–8. doi:10.1016/S0040-4039(00)78477-2.
  20. ^ Kuzovkov AD, Bocharnikova AV (1958). "Missing". J. Gen. Chem. (Eng. Trans.). 28: 546–548.
  21. ^ a b c d Dozortseva PM (1959). "The pharmacology of the alkaloid methyllycaconitine (mellictine)". Farm Toks. 22: 34–8.
  22. ^ a b c d e f g Panter KE, Manners GD, Stegelmeier BL, Lee S, Gardner DR, Ralphs MH, Pfister JA, James LF (February 2002). "Larkspur poisoning: toxicology and alkaloid structure–activity relationships". Biochemical Systematics and Ecology. 30 (2): 113–28. doi:10.1016/S0305-1978(01)00123-5.
  23. ^ Macallan DR, Lunt GG, Wonnacott S, Swanson KL, Rapoport H, Albuquerque EX (January 1988). "Methyllycaconitine and (+)-anatoxin-a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems". FEBS Letters. 226 (2): 357–63. doi:10.1016/0014-5793(88)81454-6. PMID 3338564. S2CID 23927788.
  24. ^ Ward JM, Cockcroft VB, Lunt GG, Smillie FS, Wonnacott S (September 1990). "Methyllycaconitine: a selective probe for neuronal alpha-bungarotoxin binding sites". FEBS Letters. 270 (1–2): 45–8. doi:10.1016/0014-5793(90)81231-c. PMID 2226787. S2CID 85319834.
  25. ^ Gopalakrishnan M, Buisson B, Touma E, Giordano T, Campbell JE, Hu IC, et al. (August 1995). "Stable expression and pharmacological properties of the human alpha 7 nicotinic acetylcholine receptor". European Journal of Pharmacology. 290 (3): 237–46. doi:10.1016/0922-4106(95)00083-6. PMID 7589218.
  26. ^ Davies AR, Hardick DJ, Blagbrough IS, Potter BV, Wolstenholme AJ, Wonnacott S (May 1999). "Characterisation of the binding of [3H]methyllycaconitine: a new radioligand for labelling alpha 7-type neuronal nicotinic acetylcholine receptors". Neuropharmacology. 38 (5): 679–90. doi:10.1016/s0028-3908(98)00221-4. PMID 10340305. S2CID 23349607.
  27. ^ PDB entry 2byr. Hansen SB, Sulzenbacher G, Huxford T, Marchot P, Taylor P, Bourne Y (October 2005). "Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations". The EMBO Journal. 24 (20): 3635–46. doi:10.1038/sj.emboj.7600828. PMC 1276711. PMID 16193063.
  28. ^ Coates PA, Blagbrough IS, Hardick DJ, Rowan MG, Wonnacott S, Potter BV (November 1994). "Rapid and efficient isolation of the nicotinic receptor antagonist methyllycaconitine from Delphinium: Assignment of the methylsuccinimide absolute stereochemistry as S.". Tetrahedron Letters. 35 (46): 8701–4. doi:10.1016/S0040-4039(00)78476-0.
  29. ^ Hardick DJ, Blagbrough IS, Cooper G, Potter BV, Critchley T, Wonnacott S (November 1996). "Nudicauline and elatine as potent norditerpenoid ligands at rat neuronal alpha-bungarotoxin binding sites: importance of the 2-(methylsuccinimido)benzoyl moiety for neuronal nicotinic acetylcholine receptor binding". Journal of Medicinal Chemistry. 39 (24): 4860–6. doi:10.1021/jm9604991. PMID 8941400.
  30. ^ a b Jacyno JM, et al. (1995). Gustine DL, Flores HE (eds.). Phytochemicals and Health. Current Topics in Plant Physiology. Vol. 15. Rockville: American Society of Plant Physiologists. pp. 294–295.
  31. ^ Jacyno JM, Harwood JS, Lin NH, Campbell JE, Sullivan JP, Holladay MW (July 1996). "Lycaconitine revisited: partial synthesis and neuronal nicotinic acetylcholine receptor affinities". Journal of Natural Products. 59 (7): 707–9. doi:10.1021/np960352c. PMID 8759171.
  32. ^ Gubanov IA (1965). "Missing". Planta Medica. 13: 200–205.
  33. ^ Markou A, Paterson NE (2001). "Missing". Nicotine Tob. Res. 3 (4): 361–373. doi:10.1080/14622200110073380. PMID 11694204.
  34. ^ Weinstein AM, Gorelick DA (2011). "Pharmacological treatment of cannabis dependence". Current Pharmaceutical Design. 17 (14): 1351–8. doi:10.2174/138161211796150846. PMC 3171994. PMID 21524266.
  35. ^ Solinas M, Scherma M, Fattore L, Stroik J, Wertheim C, Tanda G, et al. (May 2007). "Nicotinic alpha 7 receptors as a new target for treatment of cannabis abuse". The Journal of Neuroscience. 27 (21): 5615–20. doi:10.1523/JNEUROSCI.0027-07.2007. PMC 6672748. PMID 17522306.
  36. ^ Wu CH, Lee CH, Ho YS (June 2011). "Nicotinic acetylcholine receptor-based blockade: applications of molecular targets for cancer therapy". Clinical Cancer Research. 17 (11): 3533–41. doi:10.1158/1078-0432.CCR-10-2434. PMID 21444681.
  37. ^ The western potato leafhopper.

External links edit

  • Plant extract may block cannabis addiction

January 01, 1970
methyllycaconitine, diterpenoid, alkaloid, found, many, species, delphinium, larkspurs, common, with, many, other, diterpenoid, alkaloids, toxic, animals, although, acute, toxicity, varies, with, species, early, research, focused, identifying, characterizing, . Methyllycaconitine MLA is a diterpenoid alkaloid found in many species of Delphinium larkspurs 1 2 In common with many other diterpenoid alkaloids it is toxic to animals although the acute toxicity varies with species 3 4 Early research was focused on identifying and characterizing the properties of methyllycaconitine as one of the principal toxins in larkspurs responsible for livestock poisoning in the mountain rangelands of North America 3 5 Methyllycaconitine has been explored as a possible therapeutic agent for the treatment of spastic paralysis 6 and it has been shown to have insecticidal properties 7 Most recently it has become an important molecular probe for studying the pharmacology of the nicotinic acetylcholine receptor 8 MethyllycaconitineIdentifiersCAS Number21019 30 7 YPubChem CID5311278ChemSpider4470788UNII8H7EX9Z9QEChEMBLChEMBL510275CompTox Dashboard EPA DTXSID80943320Chemical and physical dataFormulaC 37H 50N 2O 10Molar mass682 811 g mol 13D model JSmol Interactive imageSMILES CCN1CC2 CCC C34C2C C C31 C5 CC C6CC4C5C6OC OC O O OC OC COC O C7 CC CC C7N8C O CC C8 O C Contents 1 Isolation 2 Structure determination 3 Chemistry 3 1 Synonyms 3 2 Physico chemical properties 3 3 Molecular structure 3 4 Synthesis 4 Pharmacology 5 Toxicology 6 Structure Activity relationships 7 Therapeutic applications 8 Insecticidal action 9 References 10 External linksIsolation editThe first isolation of MLA from Delphinium brownii Rydb was probably made by Richard Manske at the National Research Laboratories in Ottawa Canada in 1938 9 Presumably because he did not obtain the compound in sufficiently pure form Manske declined to give it a name The name methyl lycaconitine was assigned by John Goodson working at the Wellcome Chemical Research Laboratories in London England when he isolated the alkaloid in purer form from seeds of Delphinium elatum L in 1943 10 A more modern isolation procedure is described by Pelletier and his co workers who used seeds of the garden larkspur Consolida ambigua also referred to as Delphinium ajacis as their plant source 11 Structure determination editThe complete molecular structure for MLA correct in all but one detail was first published by Kuzovkov and Platonova in 1959 12 This structure supported in part by X ray crystallography considered usually to be a definitive analytical technique of a chemical derivative of MLA performed by Maria Przybylska 13 was accepted as correct until the early 1980s At that time the research groups of Pelletier 14 and of Edwards and Przybylska 15 independently corrected the stereochemistry of the methoxy group at C 1 from the b to a configuration Thus any drawing of MLA appearing before Pelletier s 1981 paper 14 will show the structure with the incorrect stereochemistry at C 1 Chemistry editSynonyms edit 1a 4 S 6b 14a 16b 20 Ethyl 1 6 14 16 tetramethoxy 4 2 3 methyl 2 5 dioxo 1 pyrrolidinyl benzoyl oxy methyl aconitane 7 8 diol also referred to incorrectly as N methyl lycaconitine in a few publications Physico chemical properties edit MLA is soluble in chloroform but does not dissolve well in water 10 The free base of MLA has not been obtained in crystalline form and in its amorphous form it melts ultimately at 128 C 10 the hydriodide salt has a melting point of 201 C 10 the perchlorate salt melts at 195 C 16 The citrate salt is the most common form in which MLA is currently available commercially 17 A pKa does not seem to have been recorded for MLA but it is considered to be a weak base because it can be readily extracted into diethyl ether from an aqueous solution at pH 7 5 8 14 The optical rotation of the free base a D was found to be 49 in alcohol 10 Molecular structure edit Although commonly referred to as a diterpenoid alkaloid MLA is strictly speaking a nor diterpenoid since its carbon skeleton only contains 19 C atoms one having been deleted somewhere during its biosynthesis 18 Otherwise the MLA molecule comprises a tertiary amine two tertiary alcohols four methyl ether groups and a complex ester based on anthranilic acid and methylsuccinic acid This N 2 carboxyphenyl methylsuccinamido ester is quite rare amongst natural products Synthesis edit As of April 2012 no total synthesis of MLA has been reported A semi synthesis of MLA starting from its parent amino alcohol lycoctonine obtained by simple alkaline hydrolysis of natural MLA 10 was reported in 1994 19 Pharmacology editIn many respects the pharmacology of MLA closely resembles that of the classical neuromuscular blocker d tubocurarine The curare like properties of MLA seem to have been first mentioned in 1958 by Kuzovkov and Bocharnikova 20 working at the Ordzhinikidze All Union Institute for Scientific Research in Pharmaceutical Chemistry in the former USSR A detailed paper on the pharmacology of MLA in the form of its hydriodide salt given the drug name mellictine in classical animal preparations was published from the same Institute in the following year by Dozortseva 21 These studies together with related others and some original observations are summarized in the review by Benn and Jacyno 3 They revealed that MLA blocked neuromuscular transmission in skeletal muscle but not smooth muscle and had some ganglion blocking action Such properties are characteristic of an antagonist of acetylcholine exerting its effects at nicotinic but not muscarinic sites In the rat phrenic nerve diaphragm preparation for example a 2 x 10 5M concentration of MLA produced a 50 decrease in response and total inhibition was caused by a 3 x 10 5M concentration of the drug In this preparation MLA treated muscle responded normally to direct electrical stimulation but the inhibition of contractions was only partially antagonized by physostigmine Similar results were obtained with frog nerve muscle preparations in which it was shown that MLA blocked response of the gastrocnemius muscle to electrical stimulation of the sciatic nerve inhibited post synaptic action potentials in the sartorius muscle elicited by stimulation of the sciatic nerve and reduced the amplitude of miniature end plate potentials in the extensor digitus IV muscle Ganglion blocking effects of MLA were observed using the cat nictitating membrane preparation complete inhibition of the response was produced by 4 mg kg of mellictine given intravenously No significant effects were produced by the drug in smooth muscle preparations from rabbit guinea pig or cat indicating the lack of activity at typically muscarinic sites In electrically stimulated guinea pig ileum for example contractions were unaffected by a concentration of 5 x 10 4M of MLA A more detailed summary of the above data together with much related material may be found in a review written by Kip Panter and collaborators at USDA ARS laboratories in Utah and California 22 A significant advance was made towards understanding the pharmacology of MLA when Jennings and co workers 7 at the American Cyanamid Company reported that MLA as its citrate salt strongly inhibited the binding of tritiated propionyl a bungarotoxin to a receptor preparation from house fly heads with a Ki of 2 5 x 10 10M Subsequently Macallan and his co workers 23 showed that MLA also competed with 125I a bungarotoxin Ki 1 x 10 9M and tritiated nicotine Ki 4 x 10 6M in a receptor preparation from rat brain These workers also reported that MLA displaced125I a bungarotoxin from purified Torpedo electric ray nicotinic acetylcholine receptors nAChRs with a Ki 1 x 10 6M Similar experiments performed later by Ward et al 24 showed that MLA bound to nAChRs extracted from human muscle with a Ki of 8 x 10 6M it was also reported that MLA at a concentration of 10 4M had no affinity for muscarinic AChRs as labeled by tritiated quinuclidinyl benzilate from rat brain Further details about the binding of MLA to nAChRs were presented by Wonnacott and her co workers 8 who provided evidence that MLA bound preferentially to different sub units as expressed in Xenopus frog oocytes of the nAChR cloned from avian DNA MLA was found to have an IC50 of 8 x 10 8M at a3b2 and 7 x 10 7M at a4b2 receptor sub types Although it was also established that MLA bound strongly to a7 sub types experimental difficulties precluded the determination of an IC50 Subsequently research groups from Abbott Laboratories in the USA and the University of Geneva in Switzerland reported that MLA displaced 125I a bungarotoxin from a7 receptors cloned from the human K28 cell line with a Ki of 1 x 10 8 25 One last milestone in the ongoing saga of MLA pharmacology there are as of April 2012 approximately 660 references to articles in journals covered by PubMed to be mentioned is the characterization of the receptor interactions of tritium labeled MLA by researchers at the University of Bath in the UK 26 One relatively recent study which sheds light on the interaction of MLA with acetylcholine binding proteins AChBP at the molecular level is that of Hansen et al 27 who made observations on the crystal structure of a complex between MLA and an AChBP isolated from the salt water snail Aplysia californica Toxicology editThe toxicology of MLA has been studied largely in the context of livestock poisoning by wild larkspurs The seminal work by John Jacyno and Mike Benn at the University of Calgary in Canada showed that MLA was most likely to be the agent responsible for the toxicity of a local larkspur D brownii and provided some preliminary acute toxicity data in several animal species 3 4 5 These LD50s are as follows mouse 3 5 mg kg frog 3 4 mg kg rabbit 2 3 mg kg after parenteral administration Cats appeared to have comparable susceptibility to rabbits whereas dogs were 1 5 x more sensitive 21 These early observations have been comprehensively extended by USDA researchers 22 who have estimated the LD50 of MLA to be 10 mg kg in sheep 5 mg kg in rats and 2 mg kg in cattle Although most LD50s are usually determined from parenteral administration of the test drug MLA is also active when taken orally 21 Signs of toxicity in calves sheep rats and mice at low doses included agitation respiratory difficulty and loss of motor control symptoms appeared within 2 3 minutes of injection and disappeared within 10 minutes Doses large enough to produce collapse also caused an increase in heart and respiration rates as well as tremor with significant convulsions evident in mice and rats but not in cattle or sheep 22 In cases where death seemed imminent the poisoning in sheep could be counteracted by the i v administration of neostigmine and atropine 22 whereas poisoning in calves was reversed by the administration of physostigmine 4 In animals that were allowed to die death appeared to be the result of complete motor paralysis and respiratory arrest 21 22 It is worth noting that although a LD50 for man is not available the clinical studies of Kabelyanskaya showed that an oral dose of 0 02 g of MLA hydriodide mellictine might be given to patients up to 5 times per day over the course of 1 month However some subjects could only tolerate single doses of 0 02 g per day without experiencing side effects 6 Structure Activity relationships editThe earliest observation on a relationship between the molecular structure of MLA and a biological activity concerned the effect of the C 18 ester group on acute toxicity When this group was hydrolyzed the resulting amino alcohol named lycoctonine as a consequence of its natural occurrence was found to be much less poisonous to animals than was MLA 3 A recent study comparing the LD50 of MLA and lycoctonine given i v to mice showed that lycoctonine was more than 100x less toxic than MLA 22 In other functional pharmacological assays lycoctonine resembled MLA qualitatively but was roughly ten times less potent 3 When compared in nAChR binding studies MLA was found to compete for 125I a bungarotoxin binding sites i e a7 sub types over 1000x more strongly than did lycoctonine 28 If the succinimide ring is deleted so as to leave only the NH2 group attached to the benzene ring as in the alkaloid anthranoyllycoctonine which also occurs naturally the resulting compound is intermediate between MLA and lycoctonine in potency and toxicity it is less acutely toxic than MLA by a factor of about 4 but its affinity for 125I a bungarotoxin binding sites is over 200x lower than that of MLA 29 If the NH2 group of anthranoyllycoctonine is removed giving the compound lycoctonine 18 O benzoate the affinity for a7 receptors as well as for a4b2 receptors is reduced by about a factor of 10 in comparison to MLA 30 When compared with MLA in the rat phrenic nerve diaphragm assay lycoctonine 18 O benzoate was also about 10x less potent and a similar reduction in potency was observed in an electrophysiological study involving frog extensor muscle 3 Even the absence of the methyl group from the methylsuccinimido ring as in the alkaloid lycaconitine reduces the affinity for a7 receptors by a factor of about 20 31 gt but in this case affinity for a4b2 receptors is not significantly changed in comparison with MLA 30 Another approach that has been explored in the attempt to elucidate structure activity relationships in MLA has been to start with 2 methylsuccinimido benzoic acid the carboxylic acid produced when MLA is split at the C 18 ester group and to esterify it with various alcohols and amino alcohols that might be considered as molecular fragments of MLA None of these compounds showed any significant degree of the biological actions characteristic of MLA however in the limited number of assays to which they were subjected 3 22 Therapeutic applications editMLA has been used for treating a variety of neurological disorders 6 32 although there are no references to such use in the last few decades More recently it has been proposed that MLA might be useful in reducing nicotine reward without precipitating symptoms of nicotine withdrawal 33 This suggestion was made on the basis of experiments in which intraperitoneal doses of 4 mg kg and 8 mg kg of MLA significantly reduced nicotine self administration in rats Most recently it has been suggested 34 that MLA had potential in the treatment of cannabis dependence However this suggestion was apparently based only on work by Solinas et al 35 who showed that doses of 0 3 5 6 mg kg i p in rats dose dependently antagonized the discriminative stimulus effects of 3 mg kg THC Given that the early Soviet work 6 with mellictine indicated that as little as 0 2 0 3 mg kg orally in man assuming a weight of 60 70 kg for the sake of making the dose conversion could produce symptoms of toxicity and that oral administration of most drugs typically requires more drug than parenteral administration it is uncertain if MLA will prove to be a practical treatment for either nicotine or cannabis addiction based on the effective doses required in the rat experiments In a recent review Wu and co workers 36 have cited research in which a7 antagonists such as MLA show potential in cancer treatment but this work is still in its very early stages Insecticidal action editJennings and co workers in addition to making their key observations see Pharmacology above about the receptor binding of MLA found it to be toxic 50 mortality to the following insect species Empoasca abrupta 37 at 100 ppm Heliothis virescens at 1000 ppm Musca domestica at 1000 ppm and Spodoptera eridana at 1000 ppm Species which were not significantly affected by MLA were Anopheles quadrimaculatus Aphis fabae Diabrotica undecimpunctuata howardi and Tetranychus urticae MLA also behaved as a feeding deterrent with an LC50 of 300 ppm to Spodoptera larvae feeding on bean leaves 7 References edit Willaman JJ Liu HJ 1970 Missing Lloydia 33 Supplement 3A 180 181 Harbourne JB Baxter H 1993 Phytochemical Dictionary London Taylor amp Francis p 153 a b c d e f g h Benn MH Jacyno JM Pelletier SW ed Chemical and Biological Perspectives Vol 1 New York Wiley pp 153 210 a b c Nation PN Benn MH Roth SH Wilkens JL September 1982 Clinical signs and studies of the site of action of purified larkspur alkaloid methyllycaconitine administered parenterally to calves The Canadian Veterinary Journal 23 9 264 6 PMC 1790203 PMID 17422179 a b Aiyar VN Benn MH Hanna T Jacyno J Roth SH Wilkens JL October 1979 The principal toxin of Delphinium brownii Rydb and its mode of action Experientia 35 10 1367 8 doi 10 1007 BF01964013 PMID 499426 S2CID 19818861 a b c d Kabelianskaia LG 1959 Use of mellictin in clinical diseases of the nervous system in spastic paralysis Farmakologiia I Toksikologiia 22 1 38 42 PMID 13653123 a b c Jennings KR Brown DG Wright DP 1986 Methyllycaconitine a naturally occurring insecticide with a high affinity for the insect cholinergic receptor Experientia 42 6 611 613 doi 10 1007 BF01955557 S2CID 1460936 a b Wonnacott E Albuquerque X Bertrand D 1993 Conn PM ed Methods in Neurosciences Vol 12 San Diego Academic Press pp 263 275 Manske RH March 1939 Missing Canadian Journal of Research 16 57 60 doi 10 1139 cjr39b 010 a b c d e f Goodson JA 1943 42 The alkaloids of the seeds of Delphinium elatum L Journal of the Chemical Society Resumed 139 41 doi 10 1039 JR9430000139 Pelletier SW Sawhney RS Desai HK Mody NV May 1980 The diterpenoid alkaloids of Consolida ambigua Journal of Natural Products 43 3 395 406 doi 10 1021 np50009a011 Kuzovkov AD Platonova TF 1959 Missing J Gen Chem Eng Trans 29 2746 2749 Przybylska M Marion L February 1956 The Crystal Structure of Des oxymethylene lycoctonine Hydroidodide Monohydrate Canadian Journal of Chemistry 34 2 185 7 doi 10 1139 v56 026 a b c Pelletier SW Mody NV Varughese KI Maddry JA Desai HK October 1981 Structure revision of 37 lycoctonine related diterpenoid alkaloids Journal of the American Chemical Society 103 21 6536 8 doi 10 1021 ja00411a062 Edwards OE Przybylska M November 1982 The lycoctonine family of alkaloids a stereochemical revision Canadian Journal of Chemistry 60 21 2661 7 doi 10 1139 v82 382 Stern ES 1954 Manske RH Holmes HL eds The Alkaloids Vol 4 New York Academic Press pp 275 333 No citation is given here because the information is date specific and because it is inappropriate to endorse any particular supplier The biosynthetic pathway by which MLA is created in the plant is still not known in any great detail Blagbrough IS Coates PA Hardick DJ Lewis T Rowan MG Wonnacott S Potter BV November 1994 Acylation of lycoctonine semi synthesis of inuline delsemine analogues and methyllycaconitine Tetrahedron Letters 35 46 8705 8 doi 10 1016 S0040 4039 00 78477 2 Kuzovkov AD Bocharnikova AV 1958 Missing J Gen Chem Eng Trans 28 546 548 a b c d Dozortseva PM 1959 The pharmacology of the alkaloid methyllycaconitine mellictine Farm Toks 22 34 8 a b c d e f g Panter KE Manners GD Stegelmeier BL Lee S Gardner DR Ralphs MH Pfister JA James LF February 2002 Larkspur poisoning toxicology and alkaloid structure activity relationships Biochemical Systematics and Ecology 30 2 113 28 doi 10 1016 S0305 1978 01 00123 5 Macallan DR Lunt GG Wonnacott S Swanson KL Rapoport H Albuquerque EX January 1988 Methyllycaconitine and anatoxin a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems FEBS Letters 226 2 357 63 doi 10 1016 0014 5793 88 81454 6 PMID 3338564 S2CID 23927788 Ward JM Cockcroft VB Lunt GG Smillie FS Wonnacott S September 1990 Methyllycaconitine a selective probe for neuronal alpha bungarotoxin binding sites FEBS Letters 270 1 2 45 8 doi 10 1016 0014 5793 90 81231 c PMID 2226787 S2CID 85319834 Gopalakrishnan M Buisson B Touma E Giordano T Campbell JE Hu IC et al August 1995 Stable expression and pharmacological properties of the human alpha 7 nicotinic acetylcholine receptor European Journal of Pharmacology 290 3 237 46 doi 10 1016 0922 4106 95 00083 6 PMID 7589218 Davies AR Hardick DJ Blagbrough IS Potter BV Wolstenholme AJ Wonnacott S May 1999 Characterisation of the binding of 3H methyllycaconitine a new radioligand for labelling alpha 7 type neuronal nicotinic acetylcholine receptors Neuropharmacology 38 5 679 90 doi 10 1016 s0028 3908 98 00221 4 PMID 10340305 S2CID 23349607 PDB entry 2byr Hansen SB Sulzenbacher G Huxford T Marchot P Taylor P Bourne Y October 2005 Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations The EMBO Journal 24 20 3635 46 doi 10 1038 sj emboj 7600828 PMC 1276711 PMID 16193063 Coates PA Blagbrough IS Hardick DJ Rowan MG Wonnacott S Potter BV November 1994 Rapid and efficient isolation of the nicotinic receptor antagonist methyllycaconitine from Delphinium Assignment of the methylsuccinimide absolute stereochemistry as S Tetrahedron Letters 35 46 8701 4 doi 10 1016 S0040 4039 00 78476 0 Hardick DJ Blagbrough IS Cooper G Potter BV Critchley T Wonnacott S November 1996 Nudicauline and elatine as potent norditerpenoid ligands at rat neuronal alpha bungarotoxin binding sites importance of the 2 methylsuccinimido benzoyl moiety for neuronal nicotinic acetylcholine receptor binding Journal of Medicinal Chemistry 39 24 4860 6 doi 10 1021 jm9604991 PMID 8941400 a b Jacyno JM et al 1995 Gustine DL Flores HE eds Phytochemicals and Health Current Topics in Plant Physiology Vol 15 Rockville American Society of Plant Physiologists pp 294 295 Jacyno JM Harwood JS Lin NH Campbell JE Sullivan JP Holladay MW July 1996 Lycaconitine revisited partial synthesis and neuronal nicotinic acetylcholine receptor affinities Journal of Natural Products 59 7 707 9 doi 10 1021 np960352c PMID 8759171 Gubanov IA 1965 Missing Planta Medica 13 200 205 Markou A Paterson NE 2001 Missing Nicotine Tob Res 3 4 361 373 doi 10 1080 14622200110073380 PMID 11694204 Weinstein AM Gorelick DA 2011 Pharmacological treatment of cannabis dependence Current Pharmaceutical Design 17 14 1351 8 doi 10 2174 138161211796150846 PMC 3171994 PMID 21524266 Solinas M Scherma M Fattore L Stroik J Wertheim C Tanda G et al May 2007 Nicotinic alpha 7 receptors as a new target for treatment of cannabis abuse The Journal of Neuroscience 27 21 5615 20 doi 10 1523 JNEUROSCI 0027 07 2007 PMC 6672748 PMID 17522306 Wu CH Lee CH Ho YS June 2011 Nicotinic acetylcholine receptor based blockade applications of molecular targets for cancer therapy Clinical Cancer Research 17 11 3533 41 doi 10 1158 1078 0432 CCR 10 2434 PMID 21444681 The western potato leafhopper External links editPlant extract may block cannabis addiction Retrieved from https en wikipedia org w index php title Methyllycaconitine amp oldid 1170218804, wikipedia, wiki, book, books, library,

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