Patent Application
20230339877
The present invention relates to the development of improved drugs for the treatment of infections caused by parasitic nematodes.
This patent application claims the benefit of U.S. Provisional Pat. Application No. 63/333,826, filed Apr. 22, 2022, which application is hereby incorporated, in its entirety, by reference.
Parasitic nematodes infect up to 1.5 billion people worldwide and untold billions of economically valuable plants and animals. Lymphatic filarisis (LF) is a neglected tropical disease that caused by the parasitic nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori. LF is the second leading cause of long-term disability worldwide. Because the drugs used to treat diseases caused by parasitic nematodes have been used routinely for many years and have been overused, drug resistance is a well-documented problem in some species and drives the need for new and potent drugs to kill these common pathogens. Another concern with current drugs is that they typically do not kill adult parasites. The result is that the drugs must be used repeatedly to prevent transmission of the infection. Another concern with current treatments is that caution must be exercised not to kill the parasites in an infected individual too quickly since this can cause a potentially dangerous immunological reaction. Patients with the filarial parasite Loa loa have died when the parasites were killed too quickly.
Neurolenin D mesylate, a novel compound, having the structural formula
is herein disclosed.
In some embodiments, formulations of neurolenin D mesylate are provided for the treatment of an organism suffering from a disease caused by an infection with a parasitic nematode, the organism selected from the group consisting of human, animal, and plant.
A method of synthesizing neurolenin D mesylate is disclosed, according to which neurolenin D is reacted with mesyl chloride to form Neurolenin D mesylate.
In some embodiments, methods are disclosed for treating an organism suffering from a disease caused by an infection with a parasitic nematode, the organism selected from the group consisting of human, animal, and plant, the method comprising administering to the organism a therapeutically effective dose of neurolenin D mesylate. In some embodiments, the organism is a human. In some embodiments, the organism is a companion animal. In some embodiments, the organism is livestock. In some embodiments the organism is a plant.
In some embodiments the parasitic nematode is a causative agent of lymphatic filariasis and elephantiasis in humans. In some embodiments, the parasitic nematode is a causative agent of heartworm in dogs. In some embodiments, the parasitic nematode is a causative agent of a roundworm disease in livestock.
Herein disclosed are ten neurolenin D esters:
In some embodiments, formulations of one or more of these ten neurolenin D esters are provided for the treatment of an organism suffering from a disease caused by a parasitic nematode, the organism selected from the group consisting of human, animal, and plant.
A method of synthesizing neurolenin D esters is disclosed, which includes a step of reacting neurolenin D with a compound having a formula selected from the group consisting of
under reaction conditions which include the presence of an amine base. For these compounds, R is selected from the group consisting of:
For some such embodiments the compound reacting with neurolenin D has the formula
For other embodiments the compound reacting with neurolenin D has the formula
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).
A method is herein disclosed of an organism suffering from a disease caused by an infection with a parasitic nematode, the organism selected from the group consisting of human, animal, and plant, the method comprising administering a therapeutically effective dose of one or more of the referenced ten neurolenin D esters. In some embodiments the organism is a human, a companion animal, livestock, or a plant.
In some embodiments the parasitic nematode causing the treated disease is selected from the group consisting of lymphatic filariasis, elephantiasis, and river-blindness in humans. In some embodiments the parasitic nematode is a causative agent of heartworm in dogs. In some embodiments the parasitic nematode is a causative agent of a roundworm disease in livestock.
The foregoing features of embodiments will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:
Definitions. As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires:
“Neurolenin D” is the chemical compound having the formula:
A method of purifying Neurolenin D from the plant Neurolaena lobata is described in Example 1 below.
“Neurolenin D Mesylate” (NDM) is the chemical compound having the formula:
A method of synthesizing NDM from Neurolenin D is described in Example 2 below.
As used herein, a “therapeutically effective dose” or a “therapeutically effective amount” of a compound, including a crystalline form thereof, or a pharmaceutically acceptable salt thereof, refers to an amount of the compound, or a crystalline form thereof, or a pharmaceutically acceptable salt thereof, which is effective, upon single or multiple dose administration to an organism, in treating a cell, or in curing, alleviating, relieving or improving the organism with a disorder beyond that expected in the absence of such treatment.
As used herein, a “formulation” of a compound is a composition comprising the compound together with one or more pharmaceutically acceptable excipients, suitable for delivery to an organism for the treatment of a disease.
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 widely distributed in Central and South America and is a commonly used plant in Mayan folk medicine. Traditional uses include the treatment of parasitic ailments.
The preparation of NDM from Neurolenin D involves first isolating Neurolenin D from N. lobata, and then mesylating Neurolenin D by reaction with mesyl chloride, according to the scheme:
The details of this procedure are provided in Examples 1 to 3 below.
Additional Neurolenin D esters are herein disclosed, having the formulae:
Neurolenin D was extracted from the plant Neurolaena lobata by continuous extraction with a Soxhlet extractor. Dried plant leaves of N. lobata from Belize were purchased from Grenada Market in Brooklyn, NY. A bag of N. lobata was ground to powder in a food processor and the powder was added to a cellulose extraction thimble which was filled ⅘ of the way with powder. The thimble was placed into the main chamber of a Soxhlet extractor. To a 1000-mL round bottomed flask, a stir bar and dichloromethane (600 mL) were added. The flask was connected to the main chamber of the Soxhlet extractor. A condenser cooled with a steady flow of cold water was placed atop the main chamber. The dichloromethane was heated to its reflux temperature by a heating mantle connected to a variable voltage outlet. The voltage was set to 10 V and the Soxhlet apparatus was watched carefully until the reflux ring, the highest point of condensation, reached the second bulb of the condenser. If the reflux ring did not reach the second bulb of the condenser, voltage was increased until the ring reached that point. Likewise, the voltage was decreased if the reflux ring reached above the second bulb. Once the reflux ring and temperature remained constant, the extraction was left to run for 24 h.
After cooling the extraction, the dichloromethane was rotovapped off to leave behind a green viscous substance in the flask. The green concentrate was dissolved in ethyl acetate (500 mL) and transferred to a 1000-mL Erlenmeyer flask. Three large scoopulas of activated charcoal were added to the Erlenmeyer, and the flask was left 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 activated charcoal treatments. Once the resulting filtrate was clear in color, the ethyl acetate was rotovapped off the yield a light green viscous oil.
Thin layer chromatography (TLC) confirmed the sample contained a mixture of neurolenin D (Rf ~ 0.3), neurolenin C (Rf ~ 0.4) and neurolenin B (Rf ~ 0.5). The sample was dissolved in the least amount of eluent possible and loaded onto the column, after which flash column chromatography was performed using silica gel (1:100 sample: silica) and eluent (1:1 hexanes: ethyl acetate). The fraction number at which the sample eluted from the column varied widely. However, the pattern of elution was constant: neurolenin B was first to elute, followed by neurolenin C, and then neurolenin D. Fractions containing only neurolenin D were combined and rotovapped to prepare for recrystallization.
The least amount of ethyl acetate possible (~5 mL) was added to dissolve the impure neurolenin D obtained from the column. This solution was transferred to a small Erlenmeyer flask with a stir bar and heated carefully on a hotplate. Two Erlenmeyer flasks of heptane (20 mL) and ethyl acetate (10 mL) were also heated to boiling via the hotplate. The boiling heptane was added dropwise to the solution of neurolenin D until the solution turned cloudy, indicating that the product had precipitated out. Once cloudy, a minimal amount of refluxing ethyl acetate was added dropwise until the solution was clear again. The Erlenmeyer was removed from the hotplate, covered with aluminum foil, and left to cool in the freezer for a day or two, resulting in the formation of yellowish white crystals.
The crystals were separated from the mother liquor via suction filtration using a Buchner funnel. Small amounts of ice-cold heptane and ethyl acetate were used to rinse the crystals until there were no hints of yellow. The white crystals were left to dry open to the air.
The chemical synthesis of NDM proceeds through esterification of neurolenin D with mesyl chloride, according to:
A three-necked round bottomed flask containing neurolenin D (50 mg, 0.13 mmol, 1 equiv) was equipped with a condenser, rubber septum, ground glass stopper, and a 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 dropwise addition of mesyl chloride (15.1 µL, 0.20 mmol, 1.5 equiv). The resulting mixture was allowed to stir for one hour at 0° C., after which the flask was immersed in an oil bath at 30° C. and left to stir overnight.
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 × 10 mL), saturated NaHCO3 (2 × 5 mL), and brine (2 × 5 mL). The resulting solution was dried with MgSO4 and rotovapped to yield a yellowish oil.
The yellow oil was dissolved with the least amount of ethyl acetate (~5 mL) and transferred to a small Erlenmeyer flask with a stir bar. The solution, along with two Erlenmeyer flasks of heptane (20 mL) and ethyl acetate (10 mL), were heated to boiling via a hotplate. Boiling heptane (~6 mL) was added dropwise to the solution until it turned cloudy, after which refluxing ethyl acetate was added dropwise until the solution was clear again. The flask was removed from the hotplate, covered with aluminum foil, and allowed to cool in the freezer for a day or two, until white crystals of neurolenin D mesylate (27.0 mg, 0.06 mmol, 45% yield) had formed. If needed, the crystals were cleaned to the point of whiteness following the procedure of cleaning neurolenin D crystals.
As demonstrated in
Preliminary data on NDM indicates that the compound is not toxic or mutagenic in biological activity assays.
As shown in
This combined mode of action of killing both larval and adult forms is important since killing microfilariae is important for preventing transmission of the disease, while killing adult worms is important for eliminating the infection in the diseased individual (and ultimately preventing transmission).
When the results shown in
Moreover, because all nematodes share similar biology and biochemistry, it is very likely that this new drug will be effective in killing a wide variety of nematode parasites that infect 1.5 billion people worldwide and are important drivers of the cycle of disease and poverty in low and middle income countries. It should be noted that these diseases are not solely limited to the low and middle income countries. One of these diseases, hookworm, is still found in the United States for example. In addition to human diseases, nematode parasites are important agents of disease in companion animals and livestock. Examples include dog heartworm disease and roundworm diseases of cattle.
Parasitic nematodes are also important pathogens of economically important crops and include root knot nematode and many others nematode species. Together these diseases infect billions of humans and have an incredible economic impact in terms of infections of companion animals, livestock and crop plants. Because the drugs used to treat these diseases have been used routinely for many years and have been overused, drug resistance is a well-documented problem in some species and drives the need for new and potent drugs to kill these common pathogens. NDM is a new compound that could make an important contribution to controlling these pathogens.
Further Neurolenin D esters can be synthesized by reaction of neurolenin D with an acyl chloride or with an anhydride. For the acyl chloride reaction the scheme is:
Whereas for the anhydride reaction the scheme is:
For either reaction scheme, R is a moiety selected from the group consisting of:
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).
The embodiments of the invention described above are intended to be merely exemplary; 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 present invention as defined in any appended claims.
| Number | Date | Country | |
|---|---|---|---|
| 63333826 | Apr 2022 | US |
