WO1989007098A1

WO1989007098A1 - Butyrl-tyrosinyl spermine, analogs thereof and methods of preparing and using same

Info

Publication number: WO1989007098A1
Application number: PCT/US1989/000482
Authority: WO
Inventors: Koji Nakanishi; Amira T. Eldefrawi; Mohyee E. Eldefrawi; Peter N. R. Usherwood
Original assignee: The Trustees Of Columbia University In The City Of
Priority date: 1988-02-08
Filing date: 1989-02-07
Publication date: 1989-08-10

Abstract

The present invention concerns a compound having structure (I), wherein R1 is a branched- or unbranched-chain alkylamine having from 2 to 20 atoms in the chain and wherein each of R2, R3 and R4 is the same or different and is hydrogen or a C1 to C20 alkyl, alkenyl, alkynyl, or alkenynyl group. The invention also concerns a method of preparing the compound from the venom, venom sacs or venom glands of the wasp Philanthus triangulum F.. Additionally, the invention provides a method of chemically synthesizing the compound. Another aspect of the invention concerns a method of treating a subject afflicted by a disorder associated with binding an etiological agent to a glutamate receptor. Lastly, the invention provides an insecticidal composition which comprises an effective amount of the compound and a method of combatting insects which comprises administering to the insects the insecticidal composition.

Description

BUTYRL-TYROSINYL SPERMINE, ANALOGS THEREOF AND METHODS OF PREPARING AND USING SAME

The invention described herein was made in the course of work under Grant No. INT-8610138 from the National Science Foundation, and Grant Nos. Al 10187 and ES 02594 from the National Institute for Health, U.S. Department of Health and Human Services. The U.S. Government has certain rights in the invention.

Background of the Invention

Throughout this application various publications are referenced and citations are provided in parentheses for them. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Glutamate receptors are believed to be the principal excitatory neurotransmitter receptors in the mammalian brain. Based on the chemicals that activate glutamate receptors, such receptors are generally divided into three major subtypes: quisqualate, N-methyl-D-aspartate (NMDA), and kainate. These receptors are involved in development, learning and neuropathology and likely mediate the neurodegenerative consequences of hypoxemia, status epilepticus and Huntington's disease (Cotman, C.W. and Iversen, L.L., Trends Neurosci. 1987, 10:263-265; Robinson, M.B. and Coyle, J.T., FASEB J., 1987, 1:446-455; Silverstein, F.S., Torke, L., Barks, J. and Johnston, M.V., Developmental Brain Res., 1987, 34: 33-39). There is considerable interest in developing agents that block glutamate receptors, particularly antagonists of the NMDA type receptor because of their anticonvulsant action and possible protection from ischemic brain damage (Wong, E.H.F., Kemp, J.A., Priestley, T., Knight, A.R., Woodruff, G.N. and Iversen, L.L., Proc. Natl. Acad. Sci. USA, 1986, 83:7104-7108). NMDA receptors are involved in a variety of neurological and psychiatric disorders, and antagonists of this receptor may be therapeutically valuable in movement disorders, such as epilepsy, and in various acute and chronic neurodegenerative disorders.

Studies of glutamate receptors, in particular studies employing biochemical techniques, have been made difficult by the relative paucity of potent antagonists for these receptor proteins. Selective, competitive and non-competitive antagonists of the NMDA receptor have become available during the past few years, but the search for antagonists of the L-quisqualate-sensitive receptor has only recently shown signs of success (Usherwood, P.N.R. (1987); Watkins, J.C. and Evans, R.H., Ann. Rev. Pharmac. Toxicol., 1981, 21:165-204; Meldrum, B., in "Neurotoxins and their pharmacological implications". Ed. P. Jenner. Raven Press. New York, 1987, pp. 133-152; Kemp, J.A., Foster, A.C. and Olverman, H.J., Trend Neurosci., 1987, 10:265-272). Quisqualate-sensitive glutamate receptors are distributed widely in excitable tissues of multicellular animals (Usherwood, P.N.R., Adv. Comp. Physiol. Biochem., 1978, 7:222-309) and studies of the effects of the venoms of certain wasps and spiders on vertebrate and invertebrate neurons and muscle fibers suggest that one source of antagonists for this class of receptor might be the venoms of some species of predaceous arthropods (Kawai, A., Miwa, T. and Abe, T., Brain Res., 1982, 247:169-171; Boden, P., Duce, I.R. and Usherwood, P.N.R., J. Britt. Pharmac, 1984, 83:221P; Bateman, A., Boden, P., Dell, A., Duce, I.R., Quicke, D.L.J. and Usherwood, P.N.R., Brain Res., 1985, 339:237-244; Grishin, L.G., Voldova, T.M., Arsoniev, A., Reshetova, A.S. Onorprienko, V.V., Magazanic, L.G., Antonov, S.M. and Fedorova, I.M., Bioorg. Khim., 1986, 12:1121-1124; Piek, T., Mantel, P. and Engels, E., Comp. Gen. Pharmacol., 1971, 2:317-331; Piek, T. and Njio, K.D., Toxicon., 1975, 13:199-201). The solitary digger wasp Philanthus triangulum F., which is a sphecid wasp that preys on honey bees, manufactures a venom which blocks glutamate receptors on locust skeletal muscle (Piek, T., et al. (1971); Piek, T., et al. (1975)). Piek and colleagues have shown that the venom of this wasp contains a component (termed δ-philanthotoxin) which exhibits a number of pharmacological properties including open channel block of junctional glutamate receptors (Piek, T., Mantel, P. and Jas, H., J. Insect Physiol., 1980, 26: 345-349) and extrajunctional glutamate D-receptors (Clark, R.B., Donaldson, P.L., Gration, K.A.F., Lambert, J.J., Piek, T., Ramsey, R.L., Spanjer, W. and Usherwood, P.N.R., Brain Res., 1982, 171:360-364) of locust leg muscle, most of which are quisqualate-sensitive (Gration, K.A.F., Clark, R.B. and Usherwood, P.N.R., Brain Res., 1979, 171:360-364). However, Piek and colleagues did not isolate or determine the active compound of the venom component.

In order to deduct the active ingredient contained in venom from the wasp Philanthus triangulum F., a series of extractions were performed to isolate an active fraction. From the chemical analysis of the fraction, a series of related compounds were snythesized and their activities and chemical properties compared to those of the venom extract fraction. This resulted in the unexpected discovery of the active compound of the venom. The present invention concerns the active ingredient contained in venom from the wasp Philanthus triangulum F., the chemical structure of this active ingredient, a method for synthesizing the ingredient. designated philanthotoxin-433 (PTX-433), and the use of PTX-433 as a potent inhibitor of the glutamate receptors. In addition, the present application describes the synthesis of pharmacologically active analogs of this ingredient, e.g. PTX-334 and PTX-343 wherein the numerals denote the number of methylenes between the amino groups of the spermine moiety.

Summary of the Invention:

The present invention concerns a compound having the structure:

wherein R₁ is a branched- or unbranched-chain alkylamine having from 2 to 20 atoms in the chain and wherein each of R ₂, R₃ and R₄ is the same or different and is hydrogen or a C₁ to C₂₀ alkyl, alkenyl, alkynyl, or alkenynyl group. Presently preferred embodiments are those wherein R₁ is:

The invention also concerns a method of preparing the compound which comprises treating venom, venom sacs or venom glands or the wasp Philanthus triangulun F. to produce a aqueous extract, and recovering the compound from the resulting aqueous extract. Additionally, the invention provides a method of preparing the compound which comprises contacting a branched- or unbranchedchain alkylamine having from two to twenty atoms in the chain and having hydrogen or a protection group attached to each nitrogen atom of the chain with a compound having the structure:

wherein R₃ and R₄ are defined previously, so as to form a product, treating the product to produce the compound and recovering the compound.

Another aspect of the invention concerns a method of treating a subject afflicted by a disorder associated with binding of an etiological agent to a glutamate receptor which comprises administering to the subject an amount of the compound effective to inhibit binding of the etiological agent to the receptor. The invention also concerns a method of treating a subject afflicted by a stroke-related disorder associated with excessive binding of glutamate to glutamate receptors which comprises administering to the subject an amount of. the compound effective to inhibit the excessive binding of the glutamate to the receptors. Lastly, the invention provides an insecticidal composition which comprises a effective amount of the compound and a suitable carrier and a method of combatting insects which comprises administering to the insects an amount of the insecticidal composition effective to produce paralysis in the insects. Brief Description of Figures

Fig 1- Venom sac (VS), gland (VG) and the sting apparatus (Stg) dissected from Philanthus triangulum.

Fig. 2- Fractionation of Philanthus venom by reverse phase high pressure liquid chromatography (HPLC). (A) Fractionation of lyophilyzed venom glands, extracted in 50% acetonitrile/water. 450 μl (representing extracts of 225 wasps) were chromatographed on a YMC-ODS 20x280 mm column and developed by a linear gradient of 5% CH₃CN/0.1% TFA-95% CH₃CN/0.1% TFA for 30 min at a flow rate of 8 ml/min. UV absorption was monitored at 215 nm. (B) Fractionation of main toxic fraction (hatched peak in Fig 2A).

Fig. 3- The chemical structures and synthesis of the natural philanthotoxin (PTX-433) and two isomers PTX-334 and PTX-343. (A) The structures of the three toxins. (B) Synthesis of intermediates of compounds 1 and 2. (C) The final steps in synthesis of the three toxins.

Fig. 4- Effects of PTX-433 (A) and PTX-334 (B) on the neurally-evoked twitch contraction, of locust metathoracic retractor urguis muscle. (A) and (B) are data from different nerve-muscle preparations dissected from the same adult, female locust (Schistocerca gregaria). The nerve-muscle preparations were superfused with standard locust saline (23) for 30 min before the toxins were applied. The retractor unguis nerve was stimulated with single, brief (0.1 s), supramaximal stimuli applied at a constant, low frequency, before and after toxin application (in locust saline), but during the period of toxin application the stimulation frequency was sometimes reversed temporarily.

Detailed Description of the Invention:

The invention concerns a compound having the structure:

wherein R₁ is a branched- or unbranched- chain alkylamine having from 2 to 20 atoms in the chain and wherein each of R₂, R₃ and R₄ is the same or different and is hydrogen or a C₁ to C₂₀ alkyl, alkenyl, alkynyl, or alkenynyl group. A branched-chain alkylamine is contemplated to be an alkylamine having lower (e.g. C₁ to C₂₀) alkyl groups attached to one or more of the atoms in the chain. Preferably, the alkylamine is unbranched and has up to three nitrogen atoms and up to fifteen carbon atoms in the chain. In certain embodiments R₂ and R₃ are the same and are hydrogen, while R₄ is a lower alkyl group, particularly -CH₂CH₂CH₃. In the presently preferred embodiments, R₁ is an alkylamine having 3 nitrogen atoms and 10 carbon atoms connected in a chain. For instance, R₁ may have the structure:

To date the most useful compounds have the structures:

The invention also provides a method of preparing or isolating the compound:

wherein R₂ and R₃ are hydrogen, R₄ is -CH₂CH₂CH₃ and R₁ is an unbranched alkylamine having up to three nitrogen atoms and up to fifteen carbon atoms in the chain, preferably having the structure:

The method comprises treating venom, venom sacs or venom glands or the wasp Philanthus triangulum F. to produce an aqueous extract, and recovering the compound from the resulting aqueous extract. The recovering may be effected by a variety of separation techniques known to those skilled in the art to which the invention pertains, such as filtration, centrifugation, and chomotography. An especially preferred recovery method is high pressure liquid chomotography. The treating of the venom, venom sacs, or venom glands may be effective by extraction with numerous organic solvents,

such as 50% CH₃CN-H₂O. Preferably, a series of extractions is performed wherein each subsequent extraction is performed on the fraction resulting from the previous extraction.

The invention also provides a method of synthesizing the compound described hereinabove which comprises contacting a branched- or unbranched-chain alkylamine, having from two to twenty atoms in the chain and having hydrogen or a protection group attached to each nitrogen atom of the chain, with a compound having the structure:

wherein R₃ and R₄ are previously defined, so as to form a product, treating the product to produce the compound and recovering the compound. The treating of the product may comprise deprotection with trifluoroacetic acid or hydrogen. Presently, the component having the structure:

is obtained by the treatment of N-tert-butoxycarbonyl-O-Benzyl-L-tyrosin p-nitrophenylester (preferably with trifluoroacetic acid) to remove the tertbuutoxycarbonyl group followed by acylation (preferably with butyryl chloride). In certain embodiments, the alkylamine has the formula:

wherein each of x, y, z is the same or different and is an integer from 1 to 6 and each of R₅, R₆ and R₇ is the same or different and is hydrogen or a protection group. Several types of protection groups may be used in the practice of the present invention and these protection groups are well-known to those skilled in art to which the invention pertains. Examples of useful protection groups include tert-butoxycarbonyl and carbobenzoxy groups and derivatives thereof. In the presently preferred embodiments, the alkylamine has the structure:

wherein Boc is a tert-butoxycarbonyl group and Cbz is a carbobenzoxy group. Such an alkylamine may be obtained by contacting acylonitrile with a spermidine derivative having the structure:

so as to produce a nitrile, and reducing the nitrile and treating it with (Boc)₂O or carbobenzoxy chloride. In another preferred embodiment, the alkylamine has the structure:

It is also contemplated that the compound of the present invention may also be radioactively labeled or be formulated into a pharmaceutical composition or an insecticidal composition comprising an effective amount of the compound and a suitable carrier. The compound may also be mixed with glutamate to form an admixture which in turn may be mixed with a carrier to provide a pharmaceutical composition. The compound may also be useful as an anticonvulsant.

Another aspect of the invention concerns a method of inhibiting binding to a glutamate receptor which comprises contacting the receptor with a binding-inhibiting amount of the compound described hereinabove or the admixture of the compound with glutamate. Such methods of inhibiting binding to glutamate receptors may prove useful in medical applications, agricultural applications or as research tools for the study of humans and animals. In one embodiment, the invention provides a method of treating a subject afflicted by a disorder associated with binding of an etiological agent to a glutamate receptor which comprises administering to the subject an amount of the compound or the admixture effective to inhibit binding of the etiological agent to the receptor. The method is particularly useful where the receptor is a quisqualate or NMDA receptor. In medical applications, the present invention may have therapeutic value in epilepsy, in movement disorders, in protection from ischemic brain damage and in various neurodegenerative disorders. The method may be useful where the neurodegenerative disorder is Huntington's disease, Parkinson's disease or Alzheimer's disease. Another embodiment provides a method of treating a subject afflicted by a strokerelated disorder associated with excessive binding of glutamate to glutamate receptors which comprises administering to the subject an amount of the compound or admixture effective to inhibit the excessive binding of the glutamate to the receptors.

As previously noted, the compound may be mixed with a suitable carrier to form an insecticidal composition and the insecticidal composition may be used in a method of combating insects which comprising administering to the insects an amount of the insecticidal composition effective to induce paralysis in the insects.

This invention is illustrated in the Experimental Details section which follow. This section is set forth to aid in an understanding of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.

Experimental Details

Collection of Wasp Venom and Bioassay: Female Philanthus triangulum F. were collected from the Dakhla oasis in the great Sahara desert in Egypt in the late summer when the population of this wasp is very high. The wasps were restrained by chilling at 4ºC and their venom sacs and glands, with the sting apparati attached (Fig. 1), were removed and placed in liquid nitrogen, before being lyophilized and stored at -20ºC. To test the biological activity of the crude venom preparation (water extract of the lyophilized venom glands), it was injected into honey bees. Honey bee workers (1-3 weeks old) were restrained by chilling at 4ºC then placed on their backs in a leucite holder (16 bees to a holder) and injected in the ventral thorax behind the first pair of legs with 1 μl of water extract of the venom glands and immediately transferred to holding cages supplied with 40% sucrose solution. Controls received phosphate buffered Ringer.

HPLC fractionation of venom extracts: Venom glands were extracted with 50% CH₃CN/H₂O and the extracts passed through a reverse-phase HPLC, YMC-ODS column 20×280 mm. A 5 to 95 linear gradient of CH₃CN/H₂O containing 0.1% TFA was used for 30 min at a flow rate of 8 ml/min. The fraction of highest pharmacological activity was further purified on a reverse-phase YMC-ODS column 4×280 mm, developed by 15% CH₃CN/H₂O containing 0.1% TFA for 15 min at a flow rate of 1 ml/min.

Electrophysiological studies: The metathoracic retractor unguis nerve-muscle preparation of the locust Schistocerca gregaria was dissected and mounted in a small Preapex bath as described previously (Usherwood, P.N.R. and Machili, P., J. Exp. Biol., 1968, 49:341-361). The muscle apodeme was attached to a Grass FT 10 strain gauge with a short strand of terylene and the muscle stretched to maximal body length. The total volume of the bath, including inlet and outlet reservoirs, was about 2.2 ml and the contents could be replaced within 1 sec. The dissection and setting up procedure were performed in continuously flowing saline. The muscle was stimulated indirectly through fine (40-80 μ) platinum wire electrode, insulated to its tip and placed on the retractor unguis nerve. The venom fractions were dissolved in locust saline of the following composition: NaCl, 140 mM; KCl, 10 mM; CaCl₂, 2mM; NaH₂PO₄, 4mM; Na₂HPO₄, 6 mM, and buffered at pH 6.8. The nerve muscle preparation was perfused with this saline at a flow rate of 5-10 ml/min at 19ºC.

Experimental Results: Honey bees injected with water extracts of the venom glands were paralyzed in a dosedependent manner. Time for recovery from paralysis was 15 ± 3 min and 55 ± 8 min for bees injected with 0.2 and 1.2 venom units (a unit is the extract from 1 wasp gland), respectively. Although all bees recovered within 1 hour, a dose-dependent mortality was evident after 24 hours (30, 80 and 100% mortality for bees injected with 0.4, 0.8 and 1.2 venom units). Polyacrylamide disc gel electrophoresis showed that the water extract of the venom glands contained a large number of proteins, all of which were precipitated by heating the extract at 100ºC for 10 min. The boiled extracts retained full activity when assayed on the locust nerve-muscle preparation or on honeybees.

The extract of each batch of 1000 venom glands was fractionated by reverse-phase HPLC and 30 fractions collected. Each of the 30 fractions was tested for pharmacological activity on the locust nerve muscle preparation, using reduction in neurally evoked twitch applitude as the measure of activity. Ten fractions were pharmacologically active. The most active fraction was the one collected at retention time 13 min (hatched peak in Fig. 2A). Further purification of this fraction by reverse-phase HPLC gave four peaks (Fig. 2B), the most pharmacologically active was in the major peak (hatched Fig. 2B). This fraction gave 1.1 mg of toxin as amorphus powder.

The UV spectrum of 1 has a maximum at 274 nirt, which shifts to 290 nm at pH 12, suggesting the presence of a tyrosine residue. This was supported by H-NMR (250 MHZ in D₂O), δ 3.00 (2H, d, J=7.8 Hz), 4.43 (1H, t, J=7.8 Hz), 6.88 (2H, d, J=8.7 Hz), 7.18 (2H, d, J=8.7 Hz). The presence of a butyryl group was also clear from ¹H-NMR, δ 0.83 (3H, t, J=7.2 Hz), 1.57 (2H, quin, J=7.2 Hz), 2.26 (2H, t, J=7.2 Hz). The ¹H-NMR signals corresponding to six methylenes a to nitrogen at δ 3.0-3.3 (12H, m) and four methylenes ß to nitrogen at δ 1.4-1.6 (4H, m) and 2.1-2.2 (4H, m) (Ohshima, T., J. Biol. Chem., 1979, 254:8720-8722), together with the FAB-MS (M+H)⁺ peak at m/z 436, showed the remainder of the molecule to be a polyamine of the spermine type. ¹H-NMR measured in DMSO-d₆ (500 MHz) clarified the connectivity of the butyryl, tyrosyl and polyamine moieties; namely, two amide protons were observed at δ 7.82 and 7.86 as a doublet and triplet, respectively, indicating that the former is due to tyrosine and the latter to polyamine. This leads to a butyryl/tyrosyl/polyamine sequence as shown in 1, 2 and 3 of Figure 3A, but since spectroscopic evidence was ambiguous to differentiate the three, all isomers were synthesized.

Chemical synthesis of the three isomers is illustrated in Figs. 3B and C. The protected polyamine 6 was obtained from spermidine derivative 4 (Humora, M. and Quick, J., Org. Chem., 1970, 44: 1166-1168) through Fig. 3B: (1) Michael addition to acrylonitrile (76%); (2) Boc-protection (81%); and (3) reduction of the nitrile (70%). Further Cbz-protection and Boc-deprotection of 6 yielded partially protected polyamine 7 (Boc represents tert-butoxycarbonyl and Cbz represents carbobenzoxy). Deprotection of N-Boc-O-benzyl-L- tyrosine p-nitrophenylester 8 (Figure 3C) with trifluoroacetic acid (TFA) followed by acylation with butyryl chloride gave key intermediate 9 in 85% yield. Coupling of 9 with protected polyamines 7, 6 and commercial spermine (1, 12-diamino-4,9-diazadodecane), (Hashimoto, Y., Skudo, K., Aramaki, Y., Kawai, N. and Nakajima, T., Tetrahedron Lett., 1987, 28:3511-3514), ca. 65% yield, followed by deprotection gave PTX 1 and analogs, 2 and 3 (ca. 80% yield). Synthetic material derived from 7 was found to be identical with the natural product in all respects (¹H-NMR, MS, CD, HPLC and biological activities). Thus the chemical structure of the major naturally-occuring philanthotoxin is 1, which is designated PTX-433, the numerals denoting the number of methylene groups between the amino groups of the spermine moiety. All three of the synthetic end-products were biologically active, PTX-334 having a higher potency than the natural PTX-433 toxin, while PTX-343 being somewhat less active.

Preliminary pharmacological studies with PTX-433 suggested that its action on a locust nerve-muscle preparation was both time- and concentration-dependent. The effects of this toxin on the neurally-evoked twitch contraction of the locust retractor unguis muscle (Usherwood, P.N.R., et al. (1968)) were investigated using toxin concentrations of 1-10 μM. It was clear from the data presented in Fig. 4A that PTX-433 exerted a number of actions on the locust nerve-muscle system. There was an initial reduction in twitch amplitude, which was stimulus frequency independent. This was followed by a further reduction in the twitch height, the extent of this charge being directly proportional to the frequency at which the retractor unguis nerve was stimulated. Prolonged applications of PTX-433 abolished the twitch. Immediately following removal of the toxin there was a brief period of operated and prolonged contractions in response to a single stimulus applied to the retractor unguis nerve before the twitch slowly returned to normal. PTX-433 also reduced the response of the retractor unguis muscle to L-glutamate (0.1 μM; bath applied), which suggests that at least part of the reduction in twitch amplitude was due to the antagonism of postjunctional, quisqualate-sensitive glutamate receptors. PTX-334 (Fig. 4B) and PTX-343 influenced the twitch contraction in the same qualitative fashion as PTX-433. In these preliminary studies PTX-334 seemed to be more potent than PTX-433 but PTX-343 was apparently less potent.

The natural philanthotoxin and its synthetic counterpart PTX-433 and analogs PTX-334 and PTX-343 (Fig. 3A) represent a new class of chemicals that are active biologically and inhibit allosterically the quisqualate-sensitive glutamate receptor in insect skeletal muscle (Fig. 4). They are smaller in molecular weight (435 daltons) than the toxins isolated from orb web spider venoms, the argiotoxins (>600 daltons) and easier to synthesize. (Bateman, A., et al. (1985); Grishin, L.G., et al. (1986); Piek, T. et al. (1975); Budd, T., Clinton, P., Dell, A., Duce, I.R., Johnson, S.J., Quicke, D.L.J., Taylor, G.M., Usherwood, P.N.R. and Usoh, G., Brain Res. (In Press); Adams, M.E., Candy, R.L., Enderlin, F.E., Fu, T.E., Jarema, M.A., Li, J.P., Miller, C.A., Schooley, D.A., Shapiro, M.J. and Venema, V.J., Biochem. Biophys. Res. Comm., 1987, 348 : 678-683; Clark, R.B., et al. (1982)). Binding PTX-433 to NMDA Receptor: The NMDA receptor is identified by its high affinity for the compound (+)-5- methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10 imine maleate (MK-801). This compound is an anticonvulsant introduced by Merck Sharp & Dohme Co. and is a potent non-competitive antagonist of the NMDA receptor. Binding of [³H]MK-801 to synaptic membranes from rat brain after thorough washing is extremely poor. However, the binding (measured by a filtration assay) is potentiated by glutamate in a dose-dependent manner and reaches maximal potentiation at 10 μM glutamate (Foster and Wong, Brit. J. Pharmacol. 91, 403 (1987)). The increase m binding of [³H]MK-801 resulting from addition of glutamate has been used as an index of NMDA receptor binding. Philanthotoxin (PTX433) inhibited the binding of [³H]MK-801 to NMDA receptors (Fig. 1) with an EC₅₀ (the concentration that inhibits 50% of binding) of 25 μM. Because of the difference in maximal glutamate-induced [³H]MK-801 binding in absence and presence of PTX-433 (Fig. 2), it is suggested that MK-801 and PTX-433 may affect the NMDA receptor by binding to distinct allosteric sites on the receptor protein.

Claims

What is claimed is:

1. A compound having the structure:

wherein R₁ is a branched- or unbranched-chain alkylamine having from two to twenty atoms in the chain and wherein each of R₂, R₃, and R₄ is the same or different and is hydrogen or a C₁ to C₂₀ alkyl, alkenyl, alkynyl, or alkenynyl group.

2. A compound of claim 1, wherein R₁ is an unbranched alkylamine having up to three nitrogen atoms and up to fifteen carbon atoms and each of R₂, R₃ and R₄ is the same or different and is hydrogen or a lower alkyl group.

3. A compound of claim 2, wherein R₂ and R₃ are hydrogen and R₄ is: CH₃-CH₂-CH₂-

4. A compound of claim 2, wherein R₁ is an unbranched alkylamine having three nitrogen atoms and ten carbon atoms.

5. A compound of claim 4 wherein R₁ is:

6. A compound of claim 5 having the structure:

7. A compound of claim 5 having the structure:

8. A compound of claim 5 having the structure:

9. A method of preparing the compound of claim 3 which comprises treating venom, venom sacs or venom glands of the wasp Philanthus triangulum F. to produce an aqueous extract, and recovering the compound from the resulting aqueous extract.

10. A method of claim 9, wherein the recovering comprises liquid chromatography.

11. A method of preparing the compound of claim 1 which comprises (a) contacting a branched- or unbranched-chain alkylamine having from two to twenty atoms in the chain and having hydrogen or a protection group attached to each nitrogen atoms of the chain with a compound having the structure:

wherein each of R₃ and R₄ are the same or different and is hydrogen or a lower alkyl group, so as to form a product, (b) treating the product to produce the compound and (c) recovering the compound.

12. A method of claim 11, wherein the treating comprises deprotection with trifluoroacetic acid or hydrogen.

13. A method of claim 11, wherein prior to step (a) N-tert-butoxycarbonyl-O-benzyl-L-tyrosine p-nitrophenylester is treated to remove tert-butoxycarbonyl and then acylated to produce the compound:

wherein each of R₃ and R₄ are the same or different and is hydrogen or a lower alkyl group.

14. A method of claim 13, wherein R₃ is hydrogen, R₄ is CH₃CH₂CH₂- and N-tert-butoxycarbonyl-O-benzyl-L-tyrosine p-nitrophenylester is treated with trifluoroacetic acid and acylated with butyryl chloride.

15. A method of claim 11, wherein the alkylamine has the formula:

wherein each of x, y and z is the same or different and is an integer from 1 to 6 and each of R₅, R₆ and R₇ is the same or different and is hydrogen or a protection group.

16. A method of claim 15, wherein the alkylamine has the structure:

or

wherein Boc represents tert-butoxycarbonyl and Cbz represents carbobenzoxy.

17. A method of claim 16, wherein prior to step (a) acrylonitrile is contacted with a spermidine derivative having the structure:

so as to produce a nitrile, and the nitrile is then reduced and treated with (Boc)₂O or carbobenzoxy chloride to produce the alkylamine having the structure:

wherein Boc represents tert-butoxycarbonyl.

18. A radioactively labeled compound of claim 1, 2, 6,

7 or 8.

19. A pharmaceutical composition which comprises an effective amount of the compound of claim 1, 2, 6, 7 or

8 and a pharmaceutically acceptable carrier.

20. A composition comprising the compound of claim 1, 2, 6, 7 or 8 in admixture with glutamate.

21. A method of inhibiting binding to a glutamate receptor which comprises contacting the receptor with an effective binding-inhibiting amount of the compound of claim 1, 2, 6, 7, or 8.

22. A method of claim 21, wherein the glutamate receptor is a quisqualate receptor.

23. A method of claim 21, wherein the glutamate receptor is a N-methyl-D-aspartate (NMDA) receptor.

24. A method of inhibiting binding to a glutamate receptor which comprises contacting the receptor with an effective binding-inhibiting amount of the composition of claim 20.

25. A method of claim 24, wherein the glutamate receptor is a quisqualate receptor.

26. A method of claim 24, wherein the glutamate receptor is a N-methyl-D-aspartate (NMDA) receptor.

27. A method of treating a subject afflicted by a disorder associated with binding of an etiological agent to a glutamate receptor which comprises administering to the subject an amount of the compound of claim 1, 2, 6, 7 or 8 effective to inhibit binding of the etiological agent to the receptor.

28. A method of .treating a subject afflicted by a disorder associated with binding of an etiological agent to a glutamate receptor which comprises administering to the subject an amount of the composition of claim 20 effective to inhibit binding of the etiological agent to the receptor.

29. A method of claim 27, wherein the disorder is a neurodegenerative disease or movement disorder.

30. A method of claim 28, wherein the disorder is a neurodegenerative disease or movement disorder.

31. A method of claim 29, wherein the neurodegenerative disease is Huntington's Disease, Parkinson's Disease, or Alzheimer's Disease.

32. A method of claim 30, wherein the neurodegenerative disease is Huntington's Disease, Parkinson's Disease, or Alzheimer's Disease.

33. A method of claim 29, wherein the movement disorder is epilepsy.

34. A method of claim 30, wherein the movement disorder is epilepsy.

35. A method of treating a subject afflicted by a stroke-related disorder associated with excessive binding of glutamate to glutamate receptors which comprises administering to the subject an amount of the compound of claim 1, 2, 6, 7 or 8 effective to inhibit the excessive binding of the glutamate to the receptors.

36. A method of treating a subject afflicted by a stroke-related disorder associated with excessive binding of glutamate to glutamate receptors which comprises administering to the subject an amount of the composition of claim 20 effective to inhibit the excessive binding of the glutamate to the receptors.

37. An insecticidal composition which comprises an effective amount of the compound of claim 1, 2, 6, 7 or 8 and a suitable carrier.

38. A method of combatting insects which comprises administering to the insects an amount of the composition of claim 37 effective to induce paralysis in the insects.