Synthesis and composition of neurorenin D mesylate - a new drug for the treatment of parasitic nematode infections in humans, animals and plants

Abstract

The natural product Neurolenin D is isolated from Neurolaena lobata using successive extractions, stirring with charcoal, flash column chromatography, and recrystallization. The secondary alcohol in this molecule is converted to a methanesulfonyl ester (mesylate) to produce a new molecule, Neurolenin D mesylate. This previously unknown molecule was tested for biological activity in assays for human toxicity, mutagenicity, and nematicidal efficacy. It is not toxic or mutagenic, and has demonstrated the ability to kill the nematode responsible for the neglected tropical disease lymphatic filariasis, and may have a similar ability to kill other nematodes responsible for a wide range of diseases in humans, animals, and plants. Additional esters of Neurolenin D are claimed and procedures for their synthesis are outlined.

Description

[Technical field]

[0001] Technical Field The present invention relates to the development of improved drugs for the treatment of infections caused by parasitic nematodes.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 333,826, filed April 22, 2022, which is hereby incorporated by reference in its entirety. [Background technology]

[0003] Background technology Parasitic nematodes infect up to 1.5 billion people worldwide as well as countless other economically valuable plants and animals. Lymphatic filarisis (LF) is a neglected tropical disease caused by the parasitic nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori. LF is the second leading cause of long-term disability worldwide. Because drugs used to treat diseases caused by parasitic nematodes have been routinely used and abused for many years, drug resistance is a well-documented problem in some species, driving the need for new and more effective drugs to kill these common pathogens. Another concern with current drugs is that they typically do not kill adult parasites. As a result, drugs must be used repeatedly to prevent transmission of the infection. Another concern with current treatments is that care must be taken not to kill the parasites too quickly in infected individuals as this can trigger a potentially dangerous immune response. Patients with the parasitic worm Loa loa have died when the parasites are killed too quickly. Summary of the Invention [Means for solving the problem]

[0004] Overview of the embodiment Structural formula [ka] Disclosed herein is a novel compound, neurorenin D mesylate, having the formula:

[0005] In some embodiments, a formulation of Neurorenin D Mesylate is provided for the treatment of an organism suffering from a disease caused by infection with a parasitic nematode, wherein the organism is selected from the group consisting of humans, animals, and plants.

[0006] A method for synthesizing neurorenin D mesylate is disclosed, whereby neurorenin D is reacted with mesyl chloride to form neurorenin D mesylate.

[0007] In some embodiments, a method is disclosed for treating an organism suffering from a disease caused by infection with a parasitic nematode, the organism being selected from the group consisting of humans, animals, and plants, the method comprising administering a therapeutically effective dose of neurorenin D mesylate to the organism. In some embodiments, the organism is a human. In some embodiments, the organism is a companion animal. In some embodiments, the organism is a livestock. In some embodiments, the organism is a plant.

[0008] In some embodiments, the parasitic nematode is the causative agent of lymphatic filariasis and elephantiasis in humans. In some embodiments, the parasitic nematode is the causative agent of heartworm in dogs. In some embodiments, the parasitic nematode is the causative agent of roundworm disease in livestock.

[0009] Disclosed herein are ten Neurorenin D Esters: [ka]

[0010] In some embodiments, a formulation of one or more of these ten neurorenin D esters is provided for the treatment of an organism suffering from a disease caused by a parasitic nematode, the organism being selected from the group consisting of humans, animals, and plants.

[0011] A method for synthesizing neurorenin D esters is disclosed, which comprises the steps of: [ka] and a compound having a formula selected from the group consisting of: [ka] is selected from the group consisting of:

[0012] For some such embodiments, the compound reacted with Neurorenin D has the formula [ka] has.

[0013] In another embodiment, the compound reacted with Neurorenin D has the formula [ka] has.

[0014] For some embodiments, the amine base is selected from the group consisting of pyridine, diazabicycloundecene (DBU), imidazole, 4-dimethylaminopyridine (DMAP), and 1,4-diazabicyclo[2.2.2]octane (DABCO).

[0015] Disclosed herein is a method for treating an organism suffering from a disease caused by infection with a parasitic nematode, the organism being selected from the group consisting of humans, animals and plants, and the method comprises administering a therapeutically effective dose of one or more of the ten neurorenin D esters referred to.In some embodiments, the organism is a human, a companion animal, a livestock or a plant.

[0016] In some embodiments, the disease caused by the parasitic nematode to be treated is selected from the group consisting of lymphatic filariasis, elephantiasis, and river blindness in humans. In some embodiments, the parasitic nematode is the causative agent of heartworm disease in dogs. In some embodiments, the parasitic nematode is the causative agent of roundworm disease in livestock.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] The foregoing features of the embodiments will be more readily understood by reference to the following detailed description, taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0019] [Figure 1] Figure 1 shows data demonstrating the ability of neurorenin D mesylate (NDM) to kill adult female Brugia pahangi parasites (BP AF) in culture.

[0020] [Diagram 2] Figure 2 shows data demonstrating the ability of NDM to kill adult male Brugia pahangi parasites (BP AM) in culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description of Specific Embodiments Definitions. As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context requires otherwise:

[0022] "Neurorenin D" has the formula: [ka] It is a chemical compound having the formula:

[0023] A method for purifying neurorenin D from the plant Neurolaena lobata is described in Example 1 below.

[0024] "Neurorenin D mesylate" (NDM) has the formula: [ka] It is a chemical compound having the formula:

[0025] A method for synthesizing NDM from Neurorenin D is described in Example 2 below.

[0026] As used herein, a "therapeutically effective dose" or "therapeutically effective amount" of a compound (including a crystalline form thereof or a pharma- ceutically acceptable salt thereof) refers to an amount of a compound, or a crystalline form thereof, or a pharma- ceutically acceptable salt thereof, which, upon administration in single or multiple doses to an organism, is more effective for treating a cell or in treating, palliating, alleviating, or improving an organism having a disorder than would be expected in the absence of such treatment.

[0027] As used herein, a "formulation" of a compound is a composition containing the compound, together with one or more pharma- ceutically acceptable excipients, suitable for delivery to an organism for the treatment of a disease.

[0028] Neurolenin D is a natural product that can be isolated from the perennial flowering plant Neurolaena lobata, commonly known as jackass bitters. N. lobata is a plant widely distributed in Central and South America and commonly used in Mayan folk medicine.

[0029] The preparation of NDM from neurorenin D involves first isolating neurorenin D from N. lobata and then analyzing the mechanism: [ka] This involves mesylation of neurorenin D by reaction with mesyl chloride according to the method described above.

[0030] Details of this procedure are provided in Examples 1 to 3 below.

[0031] formula: [ka] Disclosed herein are additional neurorenin D esters having the following structure: EXAMPLES

[0032] Example 1: Extraction of Neurorenin D from Neurolaena lobata Soxhlet Extraction Neurolenin D was extracted from the plant Neurolaena lobata by continuous extraction using a Soxhlet extractor. Dried leaves of N. lobata from Belize were purchased from the Grenada Market in Brooklyn, NY. A bag of N. lobata was ground into powder in a food processor and the powder was added to a cellulose extraction thimble that was filled 4 / 5 with powder. The thimble was placed inside the main chamber of the Soxhlet extractor. A stir bar and dichloromethane (600 mL) were added to a 1000-mL round-bottom flask. The flask was connected to the main chamber of the Soxhlet extractor. A condenser cooled by a steady stream of cold water was placed on top of the main chamber. The dichloromethane was warmed to its reflux temperature by a heating mantle connected to a variable voltage power supply. The voltage was set to 10 V and the Soxhlet apparatus was carefully observed until the reflux ring reached the second bulb of the condenser, which is the highest point of condensation. When the reflux ring did not reach the second bulb of the condenser, the voltage was increased until the ring reached that point. Similarly, when the reflux ring reached above the second bulb, the voltage was decreased. Once the reflux ring and temperature remained constant, the extraction was allowed to run for 24 hours.

[0033] Charcoal filtration After cooling the extract, the dichloromethane was removed under reduced pressure, leaving a green viscous material in the flask. The green concentrate was dissolved in ethyl acetate (500 mL) and transferred to a 1000-mL Erlenmeyer flask. Three large spoonfuls of activated charcoal were added to the Erlenmeyer and the flask was allowed to stir for at least 3 hours and up to 24 hours. After the charcoal treatment, the suspension was gravity filtered to remove the charcoal. This entire process was repeated two more times for a total of three charcoal treatments. Once the resulting filtrate was clear in color, the ethyl acetate was removed under reduced pressure, yielding a light green viscous oil.

[0034] Column chromatography Thin layer chromatography (TLC) showed that the sample was f Approximately 0.3), Neurorenin C (R f Approximately 0.4) and neurorenin B (R fIt was confirmed that the fraction contained a mixture of neurorenin B and neurorenin C (approximately 0.5%). The sample was dissolved in the smallest possible volume of eluent, loaded onto the column, and then flash column chromatography was performed using silica gel (1:100 sample:silica) and eluent (1:1 hexane:ethyl acetate). The fraction numbers of the samples eluted from the column varied greatly; however, the elution pattern was consistent: neurorenin B eluted first, followed by neurorenin C, and then neurorenin D. The fractions containing only neurorenin D were combined, concentrated under reduced pressure, and prepared for recrystallization.

[0035] Recrystallization The minimum amount of ethyl acetate possible (approximately 5 mL) was added to dissolve the impure Neurorenin D from the column. This solution was transferred to a small Erlenmeyer flask equipped with a stir bar and carefully heated on a hot plate. Two Erlenmeyer flasks containing heptane (20 mL) and ethyl acetate (10 mL) were similarly heated to boiling by a hot plate. Boiling heptane was added dropwise to the solution of Neurorenin D until the solution became cloudy, indicating that the product had precipitated. Once cloudy, a minimum amount of refluxing ethyl acetate was added dropwise until the solution was clear again. The Erlenmeyer was removed from the hot plate, covered with aluminum foil, and allowed to cool in the freezer for 1 or 2 days, resulting in the formation of yellow-white crystals.

[0036] Washing of crystals The crystals were separated from the mother liquor by suction filtration using a Buchner funnel. The crystals were rinsed with small amounts of ice-cold heptane and ethyl acetate until no signs of yellow color remained. The white crystals were allowed to dry in the air.

[0037] Example 2: Chemical synthesis of neurorenin D mesylate [ka] According to this, the chemical synthesis of NDM proceeds through the esterification of neurorenin D with mesyl chloride.

[0038] procedure A three-neck round-bottom flask containing neurorenin D (50 mg, 0.13 mmol, 1 equiv.) was fitted with a condenser, rubber septum, ground glass stopper, and stir bar. The flask was flushed with nitrogen and anhydrous dichloromethane (5 mL) was added. The flask was immersed in a dry ice bath at 0° C. Anhydrous triethylamine (40.4 μL, 0.29 mmol, 2.2 equiv.) was added, followed by the dropwise addition of mesyl chloride (15.1 μL, 0.20 mmol, 1.5 equiv.). The resulting mixture was allowed to stir at 0° C. for 1 h, after which the flask was immersed in a 30° C. oil bath and left to stir overnight.

[0039] Workup The reaction was diluted with dichloromethane, quenched with saturated NaHCO3 (5 mL) and transferred to a separatory funnel. The aqueous layer was extracted with dichloromethane. The combined organic layers were washed with HCl (1M, 3 x 10 mL), saturated NaHCO3 (2 x 5 mL), and brine (2 x 5 mL). The resulting solution was dried over MgSO4 and concentrated under reduced pressure to give a yellowish oil.

[0040] Recrystallization The yellow oil was dissolved in a minimal amount of ethyl acetate (approximately 5 mL) and transferred to a small Erlenmeyer flask equipped with a stir bar. The solution was heated to boiling using a hot plate along with two Erlenmeyer flasks of heptane (20 mL) and ethyl acetate (10 mL). Boiling heptane (approximately 6 mL) was added dropwise to the solution until it became cloudy, and then refluxing ethyl acetate was added dropwise until the solution became clear again. The flask was removed from the hot plate, covered with aluminum foil, and chilled in a freezer for 1 or 2 days until white crystals of neurorenin D mesylate (27.0 mg, 0.06 mmol, 45% yield) formed. If necessary, the crystals were washed until white, following the procedure for washing neurorenin D crystals. 1H NMR (500 MHz, CDCl3) δ 6.60 (d, 1H), 6.37 (s, 1H), 6.05 (t, 1H), 5.85 (s, 1H), 5.65 (d, 2H), 5.19 (d, 1H), 4.52 (dd, 1H), 4.14 (s, 1H), 3.11 (s, 1H), 3.11 (m, 3H), 2.58 (s, 1H), 2.19 (qd, 2H), 2.00 (m, 1H), 1.85 (td, 1H), 1.57 (d, 8H), 1.45 (td, 1H), 1.16 (d, 3H), 0.90 (d, 7H) ppm 13 C NMR (125 MHz, CDCl3) δ 204.0, 172.0, 168.9, 149.0, 135.0, 127.0, 125.2, 81.7, 79.9, 77.3, 73.2, 43.2, 41.3, 40.3, 38.8, 28.5, 25.3, 22.5, 19.9, 0.3ppm HRMS(ESI):C 21 H 30 The calculated value of O9S[M+Na] is 481.5130, and the measured value is 481.1491

[0041] Example 3: Biological properties of NDM-NDM (NDM) As shown in Figures 1 and 2, Neurorenin D Mesylate shows remarkable efficacy in killing both male and female adult parasitic nematodes Brugia pahangi (BP) in culture. BP infects a variety of animals and occasionally humans and is very closely related to the important human parasite Brugia malayi (BM), one of the major causative agents of lymphatic filariasis and elephantiasis in humans. Biologically and biochemically, BP is very similar to the human parasite BM, so it is expected that NDM will show similar efficacy in killing this deadly human parasite. Furthermore, since previous drugs developed to treat nematode parasitic infections acted in killing a wide variety of parasite species, Neurorenin D Mesylate is further expected to have a similar broad spectrum efficacy against nematode parasites of humans, animals, and plants.

[0042] Preliminary data on NDM indicates that the compound is not toxic or mutagenic in bioactivity assays.

[0043] NDM kills adult female BP parasites in culture. Figure 1 shows data demonstrating the ability of neurorenin D mesylate (NDM) to kill adult female BP parasites (BP AF) in culture. The drug was applied at 1, 2, and 3 parts per million (ppm) and was 100% effective in killing the parasites at all three concentrations. Even 1 ppm killed all the parasites within 90 hours after treatment. The control treatment was feeding 1, 2, and 3 ppm ethanol to the parasites in culture under the same conditions. One adult female parasite treated with 1 ppm ethanol died in about 80 hours. This death is not experimentally significant since parasites in culture occasionally die without any drug treatment.

[0044] NDM kills adult male BP parasites in culture. Figure 2 shows data demonstrating the ability of neurorenin D mesylate (NDM) to kill adult male BP parasites (BP AM) in culture. The drug was applied at 1, 2, and 3 parts per million (ppm) and was 100% effective in killing the parasites at all three concentrations. Even 1 ppm killed all the parasites within 120 hours after treatment. The control treatment was feeding 1, 2, and 3 ppm of ethanol to the parasites in culture under the same conditions. One adult male parasite treated with 1 ppm ethanol died in about 40 hours. This death is not experimentally significant since parasites in culture occasionally die without any drug treatment.

[0045] NDM kills the parasitic nematode in both adult and juvenile forms. As shown in Figures 1 and 2, NDM is highly effective at killing adult male and adult female BP parasites. These data have been repeated in many isolated trials with the same results. 100% of all adult male and female parasites were routinely killed by treatment with NDM. In repeated experiments, the inventors have also demonstrated that NDM kills 100% of the microfilariae (larvae) produced following mating of adult male and female parasites.

[0046] This multimodal action of killing both juvenile and adult forms is important because killing the microfilariae is important for preventing transmission of the disease, while killing the adult worms is important for eliminating the infection in diseased individuals (and ultimately preventing transmission).

[0047] When comparing the results seen in Figures 1 and 2 with those of other neurorenin derivatives, the parasites are killed slower by NDM than by other neurorenin derivatives, even though NDM kills 100% of the worms at the endpoint. This surprising result is encouraging, because killing the parasites too quickly can result in a severe immune reaction, causing the death of the patient in some cases (Loa loa).

[0048] Furthermore, because all nematodes share similar biology and biochemistry, it is highly likely that the new drugs will be effective in killing a wide range of nematode parasites that infect 1.5 billion people worldwide and are important drivers of disease and the cycle of poverty in low- and middle-income countries. Of note, these diseases are not simply limited to low- and middle-income countries. For example, one of these diseases, hookworm disease, is still found in the United States. In addition to human disease, nematode parasites are important agents of disease in companion animals and livestock. Examples include heartworm disease in dogs and roundworm disease in cattle.

[0049] Parasitic nematodes are also important pathogens of economically important crops, including root-knot nematodes and many other nematode species. Together, these diseases infect billions of humans, with staggering economic impacts in terms of infection of companion animals, livestock and crops. Because the drugs used to treat these diseases have been routinely used and abused for many years, drug resistance is a well-documented problem in some species, driving the need for new and more potent drugs to kill these common pathogens. NDM is a new compound that can make an important contribution to controlling these pathogens.

[0050] Example 4: Chemical synthesis of additional neurorenin D esters Additional Neurorenin D esters can be synthesized by reaction of Neurorenin D with acyl chlorides or anhydrides. The mechanism for the acyl chloride reaction is: [ka] It is.

[0051] On the other hand, the mechanism for the anhydride reaction is: [ka] It is.

[0052] For both reaction mechanisms, R is [ka] is a moiety selected from the group consisting of:

[0053] In some embodiments, the amine base is trimethylamine (NEt3). In some embodiments, the amine base is selected from the group consisting of pyridine, diazabicycloundecene (DBU), imidazole, 4-dimethylaminopyridine (DMAP), and 1,4-diazabicyclo[2.2.2]octane (DABCO).

[0054] The embodiments of the invention described above are intended to be merely illustrative; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the invention as defined in any appended claims.

Claims

1. formula: 【Chemistry 15】 A compound having the formula:

2. A formulation comprising Neurorenin D Mesylate for the treatment of an organism suffering from a disease caused by infection with a parasitic nematode, said organism being selected from the group consisting of humans, animals, and plants.

3. 1. A method for synthesizing neurorenin D mesylate, comprising the steps of: A method comprising reacting neurorenin D with mesyl chloride to form neurorenin D mesylate.

4. A method for treating an organism suffering from a disease caused by infection with a parasitic nematode, said organism being selected from the group consisting of humans, animals, and plants, said method comprising administering to said organism a therapeutically effective dose of neurorenin D mesylate.

5. The method of claim 4 , wherein the organism is a human.

6. The method of claim 4, wherein the organism is a companion animal.

7. The method of claim 4, wherein the organism is a livestock animal.

8. 5. The method of claim 4, wherein the organism is a plant.

9. 5. The method of treatment of claim 4, wherein the parasitic nematode is the causative agent of a disease selected from the group consisting of lymphatic filariasis, elephantiasis, and river blindness in humans.

10. 5. The method of claim 4, wherein the parasitic nematode is the causative agent of Dirofilaria immitis in dogs.

11. 5. The method of claim 4, wherein the parasitic nematode is the causative agent of roundworm disease in livestock.

12.

16. A compound selected from the group consisting of:

13. 13. A formulation comprising one or more of the compounds according to claim 12 for the treatment of an organism suffering from a disease caused by a parasitic nematode, said organism being selected from the group consisting of humans, animals and plants.

14. 1. A method for synthesizing a neurorenin D ester, comprising the steps of: Neurorenin D and 【Chemistry 17】 with a compound having a formula selected from the group consisting of The reaction conditions include the presence of an amine base, and R is 【Chemistry 18】 The method of claim 1, wherein the

15. The compound that reacts with neurorenin D is represented by the formula 【Chemistry 19】 15. The method of claim 14, comprising:

16. The compound that reacts with neurorenin D is represented by the formula 【Chemistry 20】 15. The method of claim 14, comprising:

17. 15. The method of claim 14, wherein the amine base is selected from the group consisting of pyridine, diazabicycloundecene (DBU), imidazole, 4-dimethylaminopyridine (DMAP), and 1,4-diazabicyclo[2.2.2]octane (DABCO).

18. A method of treating an organism suffering from a disease caused by infection with a parasitic nematode, said organism being selected from the group consisting of humans, animals, and plants, said method comprising administering a therapeutically effective dose of one or more of the compounds described in claim 12.

19. 20. The method of claim 18, wherein the organism is a human.

20. 20. The method of claim 18, wherein the organism is a companion animal.

21. 20. The method of claim 18, wherein the organism is a livestock animal.

22. The method of claim 18 , wherein the organism is a plant.

23. 20. The method of claim 18, wherein the parasitic nematode is the causative agent of a disease selected from the group consisting of lymphatic filariasis, elephantiasis, and river blindness in humans.

24. 19. The method of claim 18, wherein the parasitic nematode is the causative agent of Dirofilaria immitis in dogs.

25. 20. The method of claim 18, wherein the parasitic nematode is the causative agent of roundworm disease in livestock.

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