Patent Application
20240415825
The present invention relates to the fields of medicine and parasitology and particularly, to compounds and compositions to benefit human health, useful as medicaments for the treatment of diseases, particularly malaria.
WO2011065980A2 describes dyes, compositions and methods useful for detection of protein aggregates. One of said dyes is compound YAT2150 which is included in the world-wide known commercial product Proteostat®. This product has been commercialized for over a decade only for the purpose of detecting protein aggregation for qualitative and quantitative assays in the field of biomedicine. To the best of inventors' knowledge, no disclosure on any medical or pharmaceutical effect of YAT2150 or any other compound disclosed in WO2011065980A2 has been made. YAT2150 corresponds to the following formula:
Malaria is a disease caused by protozoan parasites of the genus Plasmodium, which is transmitted by the bite of female Anopheles mosquitoes. Five species cause disease in humans (P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi), where they live and multiply first in hepatocytes (namely, liver stage) and later in red blood cells (namely, blood stage). P. falciparum is the deadliest and most virulent species, especially when the infection occurs in young children and pregnant women. Each cycle of blood stage parasite multiplication lasts 48-72 hours and leads to the destruction of the host erythrocyte. It is manifested by mild symptoms such as fever and headache, but if left untreated, it may quickly develop into life-threatening complications such as cerebral malaria.
There are several drugs able to kill plasmodial parasites and are used to treat malaria. Artemisinin-based combination therapy (ACT) is currently the first-line treatment for falciparum malaria, along with a non-ACT combination of amodiaquine-sulfadoxine-pyrimethamine. However, the efficacy of these treatments is declining due to the rampant evolution of resistance by Plasmodium. In fact, previous first-line anti-malarial treatments such as chloroquine and sulfadoxine-pyrimethamine were already phased out due to drug resistance and consequently treatment failure. Therefore, it appears clear that the currently available arsenal of antimalarial drugs is insufficient, and the deployment of novel compounds is an urgent need to progress towards eradication of the disease. In those lines, the fact that there is only a small collection of antimalarial drugs belonging to a handful of chemical families significantly contributes to the parasite resistance to every drug deployed against it. For those molecules belonging to chemical classes where currently used antimalarials belong, resistance might be achieved with relative rapidity through the adaptation of already existing resistance mechanisms. However, resistance would be slowed down significantly for those molecules belonging to chemical families where no antimalarials have been described so far.
In these lines, an undesirable characteristic is resistance that emerges rapidly through single gene modification. As an example of this, resistance to the aminoquinolines chloroquine and piperaquine has been associated with distinct sets of point mutations in the gene encoding for P. falciparum chloroquine resistance transporter PfCRT. PfCRT is an efflux pump evolved by the parasite to expel chloroquine and which rendered this drug virtually useless as antimalarial in many areas of the world.
Further, desirable characteristics of yet to be discovered antimalarial compounds would include targeting several parasite stages (both in the human and in the mosquito), an easy and inexpensive synthesis, low in vivo toxicity and potential for oral administration formulations.
In view of the above, it is of great interest to find new therapies and compounds for the treatment of subjects suffering from malaria, which can avoid a rapid emerge of resistance towards them while targeting several stages of the pathogen and therefore ensure long-term therapy effectiveness.
The problem to be solved by the present invention is to provide compounds useful for the treatment of diseases, particularly malaria, in a subject in need thereof.
Surprisingly, the inventors have found that a compound, herein referred as AID-X-2020, shows an outstanding antimalarial activity by inhibiting the growth of Plasmodium falciparum in critical stages of the parasite. These antimalarial effects of AID-X-2020 consequently lead to the arrest of the pathogen's life cycle at trophozoite stage and a significant reduction of parasite's growth in new invaded red blood cells (RBCs). Furthermore, the mechanism underlying such antimalarial activity has been found to be the inhibition of protein aggregates, which appears to be a highly desirable mechanism of antimalarial drugs because protein aggregation in malaria parasites is prominent in most stages of the pathogen in both the human and mosquito hosts. Additionally, impairing generalized protein aggregation targets many Plasmodium gene products, thus rapid evolution of resistance by the parasite to inhibitors of protein aggregation is unlikely to occur.
AID-X-2020 has the same organic moiety as YAT2150 previously described in WO2011065980A2. Nevertheless, the compound synthesized in the present invention differs from YAT2150 in that it includes two bromide anions, whereas YAT2150 comprises two iodide anions instead.
YAT2150 is the compound of the commercial product Proteostat®, which has been commercialized only for the purpose of performing protein aggregation monitoring assays for over a decade. In those lines, YAT2150 has been used as a dye to detect protein aggregation, therefore the skilled person would not expect this compound to have any effect on protein aggregates nor on Plasmodium's growth. Indeed, an antiaggregating effect of YAT2150 would not be expected considering that any compound used as a dye to detect protein aggregation should be neutral in terms of the activity measured by the dye. Therefore, from teachings of WO2011065980A2, the skilled person would not derive to the inhibition effect of YAT2150 on protein aggregates and the herein observed anti-malarial activity.
Working examples herein provide detailed experimental data demonstrating the antimalarial effect of AID-X-2020 through the inhibition of P. falciparum growth, which compromises the parasite's viability in early stages of the infection. Further, inventors have been able to describe the mechanism underlying such antimalarial activity to be the inhibition of protein aggregation. AID-X-2020 has also been proved herein to be safe for administration in vivo at therapeutic doses. Therefore, the compound described herein shows a clear potential to become an effective first-line treatment for malaria.
EXAMPLE 1 shows that AID-X-2020 has an outstanding antimalarial activity, which is comparable or even superior to that of other antimalarial treatments currently in use and known in the art. Other dyes for protein aggregation such as Thioflavin T (ThT) and Congo Red herein used as controls do not exhibit any antimalarial activity, therefore suggesting that this is not a common characteristic shared by all dyes detecting protein aggregation, but rather a specific and unexpected effect of AID-X-2020.
EXAMPLE 2 shows that AID-X-2020 is capable to arrest the life cycle of the parasite at trophozoite stage when added to ring stages. Thus, results evidence the potential of AID-X-2020 to exhibit its high antimalarial activity in the earliest stages of infection, which is critical for an antimalarial treatment to be highly effective. Moreover, in late Plasmodium forms there is a significant reduction of parasite's growth in new invaded RBCs. Furthermore, EXAMPLE 6 shows that AID-X-2020 is also capable of inhibiting the transition of the gametocyte to ookinete, unlike DONE3TCI, a protein antiaggregating compound which had previously shown to have high antimalarial activity.
EXAMPLES 3, 4 and 5 show that AID-X-2020 is a strong inhibitor of Aβ40 protein aggregation and consistently able to rapidly deregulate cell proteostasis. Hence, the potential mechanism underlying the antimalarial activity of AID-X-2020 relies on its protein antiaggregating capacity.
In EXAMPLE 8, eighteen compounds were synthesized, which are representative of compounds included in formula (I) other than AID-X-2020. Seventeen of these compounds (EMA357, EMA359, EMA366, EMA368, EMA377, PRC-5, PRC-8, PRC-14, PRC-15, PRC-20, PRC-25, PRC-28, PRC29, PRC-31, PRC-39, PRC-42 and PRC-50) were tested and all show a potent antimalarial activity, comparable to the antimalarial activity of AID-X-2020, and even superior in four cases (Table 4).
Therefore, evidence in EXAMPLE 8 confirms that structural analogs of AID-X-2020, featuring the core bis(aminostyrylpyridinium) scaffold, exhibit similar biological properties, particularly antimalarial activity.
EXAMPLE 9 shows that AID-X-2020 is active against chloroquine-, artemisinin- and multi-resistant P. falciparum strains, confirming that the compound belongs to a chemical family where no antimalarials have been described so far, thus being a potential new antimalarial drug with no potential resistance in comparison to the existing antimalarials.
Of note—in line with EXAMPLES 3, 4 and 5—EXAMPLE 10 shows that AID-X-2020 is capable of disassembling preformed Aβ40 fibrils, confirming not only its activity as an inhibitor of the aggregation of a model of amyloidogenic peptides, but also its disaggregating activity.
EXAMPLE 11 shows that in parasitized red blood cells (pRBCs), AID-X-2020 is mainly located in the cytosol in association with the endoplasmic reticulum regions. These findings are consistent with the role of AID-X-2020 as a protein aggregation inhibitor capable of disrupting parasite's aggresome.
EXAMPLE 12 shows that AID-X-2020 treatment causes a decrease in aggregated protein load in live parasites, which is consistent with the mechanism of action of this drug as described in previous examples.
EXAMPLE 13 shows the capacity of AID-X-2020 to block the development of gametocytes in vitro considerably more efficiently than DONE3TCI, in line with the results shown in EXAMPLE 6.
EXAMPLE 14 shows the presence of circulating ring forms of the parasite through the microscopic observation of AID-X-2020-stained clinical blood samples of a malaria-infected person.
Altogether, inventors have found different compounds to have a high antimalarial activity through the inhibition of protein aggregation in Plasmodium, which significantly reduce the parasite's viability in vitro and efficiently block the development of Plasmodium gametocytes in blood stages. These compounds belong to a novel structural class of antimalarials with mechanism of action that potentially targets many genes, therefore rapid resistance appearance to such compounds is unlikely to occur, making them good candidates to significantly contribute to malaria eradication.
Overall, the compounds of the present invention encompass the most desirable properties of future antimalarials in order to minimize the risk of resistance evolution: (i) a high therapeutic index to allow for the administration of high doses; (ii) classification into a chemical group where no antimalarials have been described so far to minimize the risk of adaptation of existing resistance mechanisms; and (iii) a target present in several stages of the pathogen and which is not a single-gene product to reduce the chances that resistance can emerge rapidly.
Accordingly, a first aspect of the invention relates to a compound of formula (I)
or a pharmaceutically acceptable salt thereof for use as a medicament, wherein:
The first aspect can alternatively be formulated as related to the compound as defined above for use as an active pharmaceutical ingredient or for treating a disease. Alternatively, the invention also relates to the use of the compounds defined above for the manufacture of a medicament or a pharmaceutical composition. Alternatively, the invention also relates to a method to prevent and/or treat a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound as defined in the first aspect of the invention.
A second aspect of the invention relates to a compound as defined in the first aspect of the invention, for use in the treatment, prevention or amelioration of malaria, or symptoms, complications and/or sequela thereof. Alternatively, the invention relates to the use of a compound as defined in the first aspect for the manufacture of a medicament for the treatment, prevention or amelioration of malaria, or symptoms, complications and/or sequela thereof. Alternatively, the invention relates to a method to treat, prevent, or ameliorate malaria, or symptoms, complications and/or sequela thereof, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound as defined in the first aspect of the invention.
A third aspect of the invention relates to a pharmaceutical composition comprising an effective amount of a compound as defined in the first aspect of the invention, together with appropriate amounts of pharmaceutically acceptable excipients or carriers.
A fourth aspect of the invention relates to a compound of formula (I)
or a pharmaceutically acceptable salt thereof, wherein:
A fifth aspect of the invention relates to a process for the preparation of the compound of formula (I) according to the fourth aspect, which comprises the following steps:
Throughout the description and claims the word “comprise” and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein. The following examples and drawings are provided herein for illustrative purposes, and without intending to be limiting to the present invention.
Excipient: The terms “excipient” and “carrier” are used interchangeably and refer to an inert substance added to a e.g., pharmaceutical composition, to further facilitate administration of a compound of the present disclosure.
Prevent: The terms “prevent,” “preventing,” “prophylaxis” and variants thereof as used herein, refer, e.g., to
Subject: The terms “subject”, “patient”, “individual”, “host” and variants thereof are used interchangeably herein and refer to any mammalian subject, particularly humans, but also including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired. The compositions and methods described herein are applicable to both human therapy and veterinary applications.
Subject in need thereof: As used herein, “subject in need thereof” includes subjects, such as mammalian subjects, that would benefit from administration of the compositions of the disclosure.
Therapeutically effective amount: As used herein the term “therapeutically effective amount” is the amount of a compound/composition of the present disclosure that is sufficient to produce a desired therapeutic effect, pharmacological and/or physiological effect on a subject in need thereof.
Treatment: The terms “treat”, “treatment”, “therapy” as used herein refer to, e.g., the reduction in severity of disease or condition disclosed herein; the amelioration or elimination of one or more symptoms, complications, or sequelae associated with a disease disclosed herein (e.g., malaria); the provision of beneficial effects to a subject with a condition/disease disclosed herein, without necessarily curing the disease or condition. The term also includes prophylaxis or prevention of a disease or condition or symptoms, complications, or sequelae thereof.
The term refers to a clinical or nutritional intervention to prevent the disease or condition; cure the disease or condition; delay onset of the disease or condition; delay onset of a symptom, complication or sequela; reduce the seriousness of the disease or condition; reduce the seriousness of a symptom, complication, or sequela; improve one or more symptoms; improve one or more complications; improve one or more sequelae; prevent one or more symptoms; prevent one or more complications; prevent one or more sequelae; delay one or more symptoms; delay one or more symptoms; delay one or more complications; delay one or more sequelae; ameliorate one or more symptoms; ameliorate one or more complications; ameliorate one or more sequelae; shorten the duration of one or more symptoms; shorten the duration of one or more complications; shorten the duration of one or more sequelae; reduce the frequency of one or more symptoms; reduce the frequency of one or more complications; reduce the frequency of one or more sequelae; reduce the severity of one or more symptoms; reduce the severity of one or more complications; reduce the severity of one or more sequelae; improve the quality of life; increase survival; prevent a recurrence of the disease or condition; delay a recurrence of the disease or condition; or any combination thereof, e.g., with respect to what is expected in the absence of the treatment with the compound/composition of the present disclosure.
Symptom: As used herein, the term “symptom” refers to subjective or physical sign, indication, or evidence of disease or physical disturbance observed by the subject. In general, the term refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. Symptoms are felt or noticed by the individual experiencing the symptom but may not easily be noticed by others. In some aspects, a symptom can be a mild symptom, a moderate symptom, or a severe symptom. As used herein, the term “mild symptom” refers to a symptom that is not life threatening and does not require, e.g., intensive care treatment (e.g., at a hospital ICU). As used herein, the term “moderate symptom” refers to a symptom that requires monitoring because it may become life threatening and may require, e.g., hospitalization. As used herein, the term “severe symptom” refers to a symptom that is life threatening and requires, e.g., intensive care treatment (e.g., at a hospital ICU).
Complication: As used herein, the term “complication” refers to a pathological process or event occurring during a disease or condition that is not an essential part of the disease or condition; where it may result from the disease/condition or from independent causes. Accordingly, the term complication refers to medical/clinical problems that are observed in subjects diagnosed with a disease or condition disclosed herein, e.g., malaria. In some aspects, a complication can be temporary. In some aspects, a complication can be chronic or permanent.
Sequela: As used herein, the term “sequela” refers to a long term, chronic, or permanent complication.
Pharmaceutically acceptable salt: As used herein, the term “pharmaceutically acceptable salt” refers to that the salt derived from the corresponding compound is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.
In some embodiments of the first aspect, the invention provides a family of bis(aminostyrylpyridinium) compounds containing a tether chain to connect both aminostyrylpyridinium moieties, differently substituted by pyridinium and aminophenyl rings. In a particular embodiment, the invention provides a family of homodimeric bis(aminostyrylpyridinium) compounds, i.e., R1=R7, R2=R8, R3=R6, and R4=R5 or alternatively, when used in combination, R1—N—R2=R7—N—R8, R3=R6, and R4=R5. In another particular embodiment, the invention provides a family of heterodimeric bis(aminostyrylpyridinium) compounds.
In some embodiments, the invention relates to salts of compounds of formula (I). Examples of salts can be but are not limited to hydrochloride, hydrobromide, sulfate, nitrate, phosphates, acetate, propionate, benzoate, maleate, hemimaleate, fumarate, lactate, tartrate, citrate, succinate, hemisuccinate, glycollate, gluconate, tosylate, mesylate, esylate, napsylate, isethionate, besylate, hexanoate, octanoate, decanoate, oleate, or stearate.
It is understood that since the compounds described in the present invention comprise cationic groups, there will also be anionic counterions present. Any anion may serve this purpose if it does not interfere with the use of the compounds. Examples of anions that may serve as counterions can include but are not limited to halide anions, such as bromide (Br−), iodide (I−), or chloride (Cl−), sulfonate anions, such as mesylate, tosylate, esylate, isethionate, napsylate or besylate, or any other pharmaceutically acceptable anions, such as those derived from carboxylic acids (acetate, propionate, maleate, benzoate, fumarate), hydroxy acids (citrate, lactate, succinate, tartrate) or fatty acids (hexanoate, octanoate, decanoate, oleate, stearate). Particularly, anions are selected from the group consisting of iodide, bromide and chloride.
It should also be appreciated by those skilled in the art that the stoichiometric number of counterion or counterions which balance the charge or charges on the compound can be the same or they can be different provided that the counterions balance the charge(s) on the compound. The combination of counterions can be selected from any of the above-mentioned anions.
In some embodiments, the diradical, also referred to as diradical linker (L), that connects the two aminostyrylpyridinium moieties is an oligomethylene chain from 2 to 12 carbon atoms, in which one to three methylene groups can be optionally replaced by oxygen atoms.
In some embodiments, the diradical can be an ortho-, meta, or para-phenylene group (1,2-phenylenebis(methylene), 1,3-phenylenebis(methylene) or 1,4-phenylenebis(methylene)).
In some embodiments, the aminostyrylpyridinium moieties can be unsubstituted or optionally substituted at position 3 (R4 and R5) of the pyridinium ring with a (C1-C4)-alkyl group, a halogen atom, an amino group, an amino-(C1-C4)-alkyl group, a (C1-C2)-alkylamino-(C1-C4)-alkyl group, a (C1-C4)alkanamido group, a (C1-C4)-alkanesulfonamido group, a benzenesulfonamido group, a naphthalenesulfonamido group, a toluenesulfonamido group. More particularly, the pyridinium ring contains hydrogen at position 3 or is substituted at position 3 with chlorine, bromine, an amino group, or a methyl group.
In some embodiments, the aminostyrylpyridinium moieties can be substituted at the amino group (R1, R2, R7, and R8) with a (C1-C4)-alkyl group, a hydroxy-(C1-C4)-alkyl group, a cyano-(C1-C4)-alkyl group, a (C1-C4)-alkanoyloxy-(C1-C4)-alkyl group, a phenyl group, a (C1-C4)-alkanesulfonyl group, a benzenesulfonyl group, a naphthalenesulfonyl group or a toluenesulfonyl group. More particularly, the amino group is substituted with two methyl, ethyl, phenyl, or 2-acetyloxyethyl groups or it is substituted with a methyl and a 2-cyanoethyl group.
In some embodiments, the nitrogen atom of the amino group of the aminostyrylpyridinium moiety can be part of a heterocycle such as azetidine, pyrrolidine, piperidine, azepine, piperazine or morpholine. More particularly, the nitrogen atom of the amino group is part of a pyrrolidine, a piperidine, a piperazine or a morpholine ring.
In some embodiments, the aminostyrylpyridinium moieties can be substituted at position 2 of the phenyl ring (R3 and R6) with a (C1-C4)-alkyl group, a nitro group, an amino group, a (C1-C4)alkylamino group, a (C1-C4)-alkanamido group, a (C1-C4)-alkanesulfonamido group, a benzenesulfonamido group, a naphthalenesulfonamido group or a toluenesulfonamido group. Alternatively, the aminostyrylpyridinium moieties can be substituted at position 2 of the phenyl ring (R3 and R6) with a hydroxy group, a (C1-C4)-alkoxy group or a (C1-C4)-alkanoyloxy group. In a particular embodiment, the aminostyrylpyridinium moieties are substituted at position 2 of the phenyl ring with a hydroxy group. More particularly, the compound is 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)-2-hydroxystyryl]-3-methylpyridin-1-ium} dibromide (PRC-69).
In some embodiments, the compound for use is according to the first aspect, wherein:
In a particular embodiment, the compound for use is according to the first aspect, wherein:
In some embodiments, the compound for use according to the first aspect of the invention is selected from the group consisting of:
In a more particular embodiment, the compound for use according to the first aspect of the invention is selected from the group consisting of:
In an even more particular embodiment, the compound for use according to the first aspect of the invention is selected from the group consisting of:
In a particular embodiment, the compound for use according to the first aspect of the invention is of formula (VI) or a pharmaceutically acceptable salt thereof (also referred herein as AID-X-2020).
In a more particular embodiment, the compound for use is of formula (VI), wherein the anion (A) is bromide.
As discussed, within the compounds encompassed by formula (I), novel aminostyrylpyridinium compounds are provided (fourth aspect of the invention).
In some embodiments, the compound is according to the fourth aspect of the invention, wherein:
In a particular embodiment, the compound is according to the fourth aspect, wherein:
In some embodiments, the compound is as described in the fourth aspect of the invention, wherein R1=R7, R2=R8, R3=R6, and/or R4=R5 or alternatively, when used in combination, R1—N—R2=R7—N—R8, R3=R6, and/or R4=R5. In particular embodiments, the compound is as described in the fourth aspect of the invention, wherein R1=R7, R2=R8, R3=R6, and R4=R5 or alternatively, when used in combination, R1—N—R2=R7—N—R8, R3=R6, and R4=R5. Accordingly, in a particular embodiment, the compound as described in the fourth aspect of the invention is a homodimer.
In some embodiments, the compound is according to the fourth aspect of the invention, wherein
Alternatively, the aminostyrylpyridinium moieties can be substituted at position 2 of the phenyl ring (R3 and R6) with a hydroxy group, a (C1-C4)-alkoxy group or a (C1-C4)-alkanoyloxy group. In a particular embodiment, the aminostyrylpyridinium moieties is substituted at position 2 of the phenyl ring with a hydroxy group. More particularly, the compound is 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)-2-hydroxystyryl]-3-methylpyridin-1-ium} dibromide (PRC-69).
In particular embodiments, the compound is according to the fourth aspect of the invention, wherein R1, R7 (R1=R7) and R2, R8 (R2=R8) are radicals independently selected from the group consisting of hydrogen, methyl, ethyl, phenyl, 2-hydroxyethyl, 2-cyanoethyl, and 2-(acetyloxy)ethyl, or alternatively, when taken in combination, R1—N—R2=R7—N—R8 is selected from the group consisting of pyrrolidine, piperidine, piperazine and morpholine ring;
Particularly, the compound according to the fourth aspect of the invention is selected from the group consisting of:
In a more particular embodiment, the compound according to the fourth aspect of the invention is selected from the group consisting of:
In an even more particular embodiment, the compound according to the fourth aspect of the invention is selected from the group consisting of:
In an even more particular embodiment, the compound according to the fourth aspect of the invention is selected from the group consisting of:
Chemical structures of the above-mentioned compounds are disclosed in Table 1 presented above.
In some embodiments, the invention is also directed to combinations comprising at least one compound described herein and one or more other therapeutic agents. The compound and the other therapeutic agent can be formulated for a separate, sequential, concomitant administration or in admixture in a pharmaceutical composition.
In particular embodiments, the other therapeutic agent is an antimalarial drug. Non-limiting examples of antimalarial drugs are artemether, artesunate, dihydroartemisinin, lumefantrine, mefloquine, tafenoquine, amodiaquine, piperaquine, atovaquone, pyrimethamine, pyronaridine, artemisinin, chloroquine, doxycycline, or quinine.
In a particular embodiment, the composition comprises AID-X-2020 and one or more other therapeutic agents. In another particular embodiment, the composition comprises EMA357 and one or more other therapeutic agents. In another particular embodiment, the composition comprises EMA359 and one or more other therapeutic agents. In another particular embodiment, the composition comprises EMA366 and one or more other therapeutic agents. In another particular embodiment, the composition comprises EMA368 and one or more other therapeutic agents. In another particular embodiment, the composition comprises EMA377 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC-5 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC8 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC-14 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC-15 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC-20 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC-25 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC-28 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC-29 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC-31 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC-39 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC-42 and one or more other therapeutic agents. In another particular embodiment, the composition comprises PRC-50 and one or more other therapeutic agents. Alternatively, the composition comprises PRC-69 and one or more other therapeutic agents.
As described previously, the examples of the present invention provide evidence of the antimalarial activity of compounds described herein. Thus, the present invention described for the first time the positive effect of the compounds of the invention in the treatment, prevention and amelioration of a disease, symptoms, complications and/or sequelae thereof.
The invention also encompasses the compounds/compositions described herein for the treatment of a pathological condition or disease susceptible to amelioration by inhibition of protein aggregation comprising administering a therapeutically effective amount of at least one compound/composition described herein.
The present invention further relates to a compound/composition described herein, for use in the treatment, prevention and/or amelioration of an infectious disease, symptoms, complications and/or sequelae thereof. In a particular embodiment, the present invention relates to a compound/composition described herein, for use in the treatment of an infectious disease.
In a particular embodiment, the infectious disease can be, but it is not limited to malaria. In a particular embodiment, the present invention relates to a compound/composition described herein, for use in the treatment of malaria.
The invention also encompasses a method of treatment, prevention and/or amelioration of an infectious disease (e.g., malaria), symptoms, complications and/or sequelae thereof comprising administering a therapeutically effective amount of a compound or composition of the invention.
The invention also encompasses a method of treatment, prevention and/or amelioration of an infectious disease (e.g., malaria), symptoms, complications and/or sequelae thereof by inhibiting protein aggregation comprising administering a therapeutically effective amount of a compound or composition of the invention.
The invention also encompasses a method of treatment, prevention and/or amelioration of malaria, symptoms, complications and/or sequelae thereof comprising administering a therapeutically effective amount of a compound, salt thereof or composition of the invention. In a particular embodiment, the invention encompasses a method of treatment of malaria comprising administering a therapeutically effective amount of a compound, salt thereof or composition of the invention.
In some embodiments, the administration of the compound/composition of the invention results in the treatment, prevention or amelioration of at least one symptom of malaria selected from the group consisting of: fever, cough chills, shaking, headache, muscle aches, muscle or joint pain, general discomfort, profuse sweating, fatigue, prostration, nausea, vomiting, diarrhea, abdominal pain, rapid breathing, rapid high rate, anemia, acidosis, convulsions, coma, bloody stools, renal failure, pulmonary edema, jaundice and/or low oxygen saturation in blood, particularly an oxygen saturation SaO2 or SpO2<92%.
As known by the skilled person, SaO2 is an alternative measure of oxygen saturation in blood, that refers to oxygen saturation measured in arteries and the later to oxygen saturation measured by pulse oximetry. SaO2 measures equivalent to SpO2 measures can be used indistinctly in this description.
In some embodiments, the administration of the compound/composition of the invention results in at least one outcome (i.e., effect) selected, but not limited to, from the group consisting of:
In some embodiments, the administration of the compound/composition of the invention to the subject can avoid hospitalization or reduce hospitalization time, avoid ICU admission (i.e., eliminate the need for ICU treatment), delay the need for ICU treatment, reduce (shorten) ICU treatment time, reduce patient mortality rate, increase patient survival rate, reduce patient's chance of death, increase patient's chance of survival, decrease patient's risk of death, reduce the severity of at least one symptom, reduce the severity of at least one complication, reduce the severity of at least one sequela, reduce the duration of at least one symptom, reduce the duration of at least one complication, reduce the duration of at least one sequela, reduce disease transmission, or any combination thereof. Accordingly, the present disclosure provides methods to avoid or reduce hospitalization, reduce (shorten) ICU treatment time, delay the need for ICU treatment, avoid ICU admission (i.e., eliminate the need for ICU treatment), reduce patient mortality rate, increase patient survival rate, reduce patient's chance of death, increase patient's chance of survival, decrease patient's risk of death, reduce the severity of at least one symptom, reduce the severity of at least one complication, reduce the severity of at least one sequela, reduce the duration of at least one symptom, reduce the duration of at least one complication, reduce the duration of at least one sequela, reduce disease transmission, or any combination thereof, in a subject and comprises administering the compound/composition to the subject.
As described previously, EXAMPLES 3-5 of the present invention provide evidence of the capacity of the compounds described herein to inhibit the aggregation of proteins (e.g., β-amyloid proteins). Further, the present invention provides evidence of the capacity of the compounds to also disassemble aggregative proteins (EXAMPLE 10). Therefore, evidence herein shows the potential of compounds and compositions of the present invention to have a positive effect in the treatment, prevention and amelioration of a disease, symptoms, complications and/or sequelae thereof, wherein the disease is characterized by protein aggregation.
Therefore, the present invention also relates to the compounds/compositions described herein for use in the treatment, prevention and/or amelioration of disease characterized by protein aggregation, symptoms, complications and/or sequelae thereof. Alternatively, the invention relates to the use of the compounds described herein for the manufacture of a formulation or medicament for treating a disease characterized by protein aggregation. The invention also encompasses a method of treatment of a disease characterized by protein aggregation, comprising administering at least one compound or composition described herein.
Proteinaceous aggregates such as amyloids and prions were first discovered in neurodegenerative diseases and were quickly attributed to a pathological state of the otherwise properly folded protein. Many pathologies are related to a failure of protein folding and the aggregation of partially folded intermediates, including Alzheimer's, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis, transmissible spongiform encephalopathies like Creutzfeldt-Jacob disease and scrapies, and spinocerebellar ataxia, to name just a few. The cytotoxicity of protein aggregation has been also shown at the root of the evolutionary tree, where susceptibility of bacteria to protein misfolding has been proposed as the target of future antibiotics.
Consequently, in one embodiment, the disease can be, but is not limited to a neurodegenerative disease or a bacterial infection. In a particular embodiment, the disease is a neurodegenerative disease. Particularly, the neurodegenerative disease is a conformational disease.
In a particular embodiment, the neurodegenerative disease can be, but is not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), prion disease (PrD), Gehrig's disease, cystic fibrosis, Gaucher's disease, frontotemporal dementia (FTD), corticobasal degeneration, progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), multiple system atrophy (MSA), spinal muscular atrophy (SMA), dementia with Lewy bodies (DLB) and motor neurone diseases (MND).
In a particular embodiment, the prion disease can be, but is not limited to, Creutzfeldt-Jacob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), scrapie and spinocerebellar ataxia (SCA), Gerstmann-Straussler-Scheinker Syndrome, fatal familial insomnia, or Kuru.
In some embodiments, the composition described herein is in a pharmaceutical form, such as a capsule, a powder, a suspension, a tablet, a topical cream, or an ointment.
The term “pharmaceutical form” is understood in its widest meaning, including any composition that comprises an active ingredient, in this case, a compound of formula (I) or the composition described herein together with at least a pharmaceutically (also referred as nutraceutically or veterinary) acceptable excipient. The term “pharmaceutical form” is not limited to medicaments but includes e.g., pharmaceutical compositions, nutraceutical compositions or veterinary compositions. A pharmaceutical form can adopt different names depending on the product regulatory approval route and also depending on the country.
A nutraceutical composition can also be named e.g., as food supplement or dietary supplement. A nutraceutical composition is understood as a preparation or product intended to supplement the diet, made from compounds usually used in foodstuffs, which provide nutrients or beneficial ingredients that are not usually ingested in the normal diet or may not be consumed in sufficient quantities. Nutraceutical compositions are usually sold “over the counter”, i.e., without prescription.
In some embodiments, the composition described herein is formulated as pharmaceutical form in which the compounds described herein is the only active agent or is mixed with one or more other active agents and/or are mixed with pharmaceutically/nutraceutically/veterinary acceptable excipients. Particularly, the additional active agent or agents are other antimalarial agents which are not antagonistic to the compounds described herein forming the composition of the invention. Depending on the formulation, the additional antimalarial agent may be added alone or together with suitable carriers or ingredients.
The term “pharmaceutically/nutraceutical/veterinary acceptable” is art-recognized, and includes excipients, compounds, materials, compositions, carriers, vehicles and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., human or animal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical/nutraceutical/veterinary texts.
Thus, some embodiments of the invention relate to a pharmaceutical composition, a nutraceutical composition, and a veterinary composition comprising a compound of the invention described herein together with at least a pharmaceutically/nutraceutically/veterinary acceptable excipient as described above. Excipients are selected, without limitation, from the group comprising: fillers/diluents/bulking agents, binders, antiadherents, disintegrants, coatings, anti-caking agents, antioxidants, lubricants, sweeteners, flavors, colors, or tensides.
In each case, the presentation of the composition will be adapted to the type of administration used. Thus, the composition may be presented in the form of solutions or any other form of clinically permissible administration and in a therapeutically effective amount. The pharmaceutical composition can be thus formulated into solid, semisolid or liquid preparations, such as tablets, capsules, powders (such as those derived from lyophilization (freeze-drying) or air-drying), granules, solutions, suppositories, gels or microspheres. In a particular embodiment, the composition is formulated for administration in liquid form or in solid form.
In a particular embodiment, the composition is in solid form such as tablets, lozenges, sweets, chewable tablets, chewing gums, capsules, sachets, powders, granules, coated particles or coated tablets, pills, troches, gastro-resistant tablets and capsules, dispersible strips and films. More particularly, the composition is in form of a capsule, a powder, a tablet, a pill, lozenges, sachets, sticks, or granules. In a more particular embodiment, the composition is in form of a pill, a tablet or a capsule.
In another embodiment, the composition is in liquid form such as oral solutions, drops, suspensions (e.g., oil), emulsions and syrups.
In some embodiments, the composition is in the form of an oily suspension to be administered alone or mixed with a liquid. This form is particularly useful for children and babies. The oily suspension comprises at least one edible oil such as olive oil, maize oil, soybean oil, linseed oil, sunflower oil or rice oil. The oil is present in a quantity of e.g., at least 70% weight/weight. In a particular embodiment, the oily suspension also comprises at least one excipient which is an emulsifier, stabilizer or anti-caking agent, in an amount of 0.1-15% w/w. Suitable agents are silicon dioxide, silica gel, colloidal silica, precipitated silica, talc, magnesium silicate, lecithin, pectin, starch, modified starches, konjac gum, xanthan gum, gellan gum, carrageenan, sodium alginate, mono- or diglycerides of fatty acids such as glycerol monostearate or glycerol monooleate and citric acid esters of mono- or diglycerides. Particularly, compositions comprise compounds of formula (I) wherein the counteranions are fatty acids.
Oral tablets are often crushed to help make them easier for children to swallow. Consequently, in another embodiment, the composition is in the form of a syrup, granules, powders or tablets which can be dissolved in water. Particularly, the composition further comprises a flavoring agent.
In a particular embodiment, the compounds/compositions of the invention are formulated for oral administration. Oral administration is preferred for non-complicated malaria (most of the cases in residents of endemic areas) and as prevention (e.g., for travelers to endemic areas).
In other embodiments, e.g., in cases of severe malaria, the compounds/compositions of the invention can be in the form of an injectable for e.g., intravenous or intraperitoneal administration.
The composition of the invention targets also the mosquito stages of the malaria parasite and inhibits the maturation of some of them, thus it can also be directly delivered to mosquitoes to block in the insect the life cycle of Plasmodium. This strategy should bypass expensive clinical trials and would therefore lead to a much less expensive formulation.
Therefore, in another embodiment, the composition is administered directly against mosquitoes. Particularly, the composition is administered against female Anopheles mosquitoes. In another embodiment, the composition is administered in combination with mosquito attractants. More particularly, the composition is in form of dispensers or sprays, which may be delivered in surfaces such as walls or bednets.
The present invention also relates to a process for the preparation of compounds of formula (I) which comprises reacting 4-methylpyridines of formula (II) with alkylating reagents of formula (III), wherein X is a common leaving group, such as halide, acyloxy, sulfonate or sulfate, followed by reacting the resulting compound of formula (IV) with a 4-aminobenzaldehyde of formula (V), and, followed, if necessary by standard transformation of functional groups that are present in radicals R1-R8. It is to be understood by the skilled in the art that the reagents used in the process of preparation are selected according to the radicals of the compound of the fourth aspect of the invention intended to prepare.
The inventors of the present invention performed a study of the antimalarial effect of AID-X-2020, compared to compounds known to have antimalarial activity and dyes in in vitro cultures of P. falciparum.
All reagents and solvents were obtained from commercial suppliers and used without further purification. Automatic flash column chromatography was performed on a CombiFlash Rf 150 (Teledyne Isco) with prepacked RediSep Rf silica gel cartridges. Melting points were determined in open capillary tubes with a MFB 595010M Gallenkamp melting point apparatus. IR spectra were run on a Perkin Elmer Spectrum RX I spectrophotometer. Absorption values are expressed as wavenumbers (cm−1). 400 MHz 1H NMR spectra and 500 MHz 1H/125 MHz 13C NMR spectra were recorded on a Varian Mercury 400 and a Bruker Avance Neo 500 MHz spectrometers, respectively, at the Centres Cientifics i Tecnològics of the University of Barcelona (CCiTUB). The chemical shifts are reported in ppm (δ scale) relative to solvent signals (DMSO-d6 at 2.50 and 39.5 ppm in the 1H and 13C NMR spectra, respectively; CD3OD at 3.31 ppm in the 1H NMR spectra), and coupling constants are reported in Hertz (Hz). High resolution mass spectra were carried out at the CCiTUB with a LC/MSD TOF Agilent Technologies spectrometer.
1.1.2 Synthesis of 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (AID-X-2020)
A mixture of 1,10-dibromodecane (1.50 g, 5.00 mmol) and 3,4-dimethylpyridine (1.2 mL, 1.14 g, 10.7 mmol) was heated to 120° C. for 3 h. Then, isopropanol (5 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et2O (2×40 mL) and the remaining brown sticky oil was dried in vacuo, taken up in MeOH (1 mL) and treated with cold Et2O (2×40 mL), drawing off the liquids. After drying the residue in vacuo, 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (2.48 g, 96%) was obtained as a brown oil that solidified on standing; mp: 69-71° C.; IR (ATR) v: 3443, 3396, 3027, 2988, 2921, 2851, 1635, 1512, 1483, 1471, 1391, 1224, 1143, 1031, 870, 838, 710, 597, 559 cm−1; 1H NMR (500 MHz, DMSO-d6) δ:1.20-1.32 (m, 12H), 1.89 (tt, J=J′=7.5 Hz, 4H), 2.40 (s, 6H), 2.52 (s, 6H), 4.50 (t, J=7.5 Hz, 4H), 7.95 (d, J=6.0 Hz, 2H), 8.85 (dd, J=6.0 Hz, J′=1.5 Hz, 2H), 8.96 (br s, 2H); 13C NMR (125 MHz, DMSO-d6) δ: 16.3 (2 CH3), 19.6 (2 CH3), 25.4 (2 CH2), 28.3 (2 CH2), 28.7 (2 CH2), 30.5 (2 CH2), 59.7 (2 CH2), 127.9 (2 CH), 137.6 (2 C), 141.5 (2 CH), 142.8 (2 CH), 157.6 (2 C); HRMS-ESI+m/z calculated for [C24H38N2]2+/2: 177.1512, found 177.1513.
A solution of 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (514 mg, 1.00 mmol) and 4-(diethylamino)benzaldehyde (390 mg, 2.20 mmol) in n-butanol (5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 4 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/7N ammonia solution in MeOH 9:1), to provide 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (351 mg, 42%) as a red oil that solidified on standing; mp: 173-174° C.; IR (ATR) v: 3399, 2975, 2927, 2853, 1641, 1574, 1520, 1479, 1404, 1351, 1311, 1260, 1219, 1186, 1128, 1076, 1011, 958, 807, 572 cm−1; 1H NMR (500 MHz, DMSO-d6) δ: 1.13 (t, J=7.0 Hz, 12H), 1.21-1.31 (m, 12H), 1.87 (tt, J=J′=7.5 Hz, 4H), 2.48 (s, 6H), 3.43 (q, J=7.0 Hz, 8H), 4.37 (t, J=7.5 Hz, 4H), 6.74 (d, J=9.0 Hz, 4H), 7.08 (d, J=16.0 Hz, 2H), 7.64 (d, J=9.0 Hz, 4H), 7.88 (d, J=16.0 Hz, 2H), 8.26 (d, J=6.5 Hz, 2H), 8.66 (dd, J=6.5 Hz, J′=1.5 Hz, 2H), 8.73 (br s, 2H); 13C NMR (125 MHz, DMSO-d6) δ: 12.5 (4 CH3), 16.5 (2 CH3), 25.5 (2 CH2), 28.4 (2 CH2), 28.7 (2 CH2), 30.5 (2 CH2), 43.9 (4 CH2), 58.9 (2 CH2), 111.3 (4 CH), 113.4 (2 CH), 119.8 (2 CH), 122.1 (2 C), 130.8 (4 CH), 132.9 (2 C), 140.5 (2 CH), 142.4 (2 CH), 143.3 (2 CH), 149.5 (2 C), 152.4 (2 C); HRMS-ESI+m/z calculated for [C46H64N4]2+/2: 336.2560, found: 336.2550.
1.1.3 P. falciparum Growth Inhibition Assay
P. falciparum parasites of the strain 3D7 MRA-102 (BEI Resources, managed by ATCC) were 5% sorbitol-synchronized in order to obtain a culture enriched in ring stage parasites. After the synchronization process, a new culture at 1.5% parasitemia and 2% hematocrit was established and 150 μL aliquots of it were transferred to 96-well plates.
The characterization of AID-X-2020 in P. falciparum in vitro cultures was performed, and other dyes were included as controls: ThT and Congo Red. The required amounts of peptides, antimalarial compounds and dyes were added in each well at different concentrations and in triplicates. A positive growth control of untreated parasites and a negative growth control of parasites treated with a lethal dose of chloroquine (1 μM) were also included.
Parasites were grown for 48 h, a complete replication cycle, in standard culturing conditions (5% O2, 5% CO2, and 90% N2 at 37° C.). After the incubation period, 3 μL of culture from each well were mixed with 197 μL of PBS containing 0.1 μM Syto 11 (Thermo Fisher Scientific Inc), to obtain a final concentration of ca. 1-10×106 cells/ml.
Parasitemia was assessed by flow cytometry using a LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) set up with the 4 lasers, 20 parameters standard configuration. The single-cell population was selected on a forward-side scatter scattergram. Syto 11 fluorescence signal was detected by exciting samples at 488 nm and collecting the emission with a 530/30 nm bandpass filter.
Growth inhibition was calculated taking as reference values both the growth rate of the untreated culture and the growth rate of the culture treated with chloroquine. Growth inhibition data was transformed through sigmoidal fitting and used to determine the IC50 values of compounds that actually inhibited parasite growth.
The results obtained indicated that AID-X-2020 has a potent antimalarial activity in vitro, with an IC50 of 90±2 nM, comparable and even superior to other compounds previously described to have antimalarial activity (Table 2). Other protein aggregation dyes such as ThT and Congo Red did not exhibit significant antiplasmodial activity.
The objective of this experiment was the evaluation of the inhibition effect of AID-X-2020 on each stage of the parasite: rings, early trophozoites, mature trophozoites and schizonts.
For stage of growth inhibition analysis, P. falciparum cultures were synchronized at ring or trophozoite stages by repeated treatment with 5% sorbitol or 70% Percoll, respectively. Half of each culture remained untreated and the other half was treated with the IC80 of AID-X-2020. At different time points, culture samples were stained with Giemsa and the number of rings, early and mature trophozoites and schizonts was counted by microscopic examination of at least 100 pRBCs for each sample. Pictures were taken with a Nikon Eclipse 50i microscope equipped with a DS-Fi1 camera (Nikon).
When added to ring stages at its IC80, AID-X-2020 arrested the life cycle of the pathogen at trophozoite stage (
In order to determine the mechanism of action of AID-X-2020 on the inhibition of P. falciparum that was previously proved in vitro, the effect of AID-X-2020 on Aβ40 aggregation in vitro was first studied. Amyloid-β peptide fragment 1-40 (Aβ40) was used as a model of a highly aggregative peptide to evaluate the potential antiaggregatory effect of AID-X-2020. Firstly, an in vitro analysis of Aβ40 aggregation was performed by ThT fluorescence assay. Secondly, to dismiss the possibility that the decrease in ThT fluorescence was due to the presence of AID-X-2020, the Aβ40 samples treated with AID-X-2020 were examined by transmission electron microscopy (TEM).
For the in vitro analysis of As peptide fragment 1-40 (Aβ40) aggregation, 1 mg of Aβ40 (GenScript) was dissolved in 500 μL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Fluka) under vigorous stirring for 1 h and sonicated for 30 min in a sonication bath. Afterwards, the solution was stirred for 1 h and maintained at 4° C. for 30 min. Aliquots were prepared, HFIP was evaporated under a nitrogen stream for a few seconds and the peptide was stored at −20° C. Prior to use, aliquots of Aβ40 were resuspended with DMSO, sonicated for 10 min, further diluted to 25 μM in PBS containing different concentrations of test compounds (0.1 μM AID-X-2020, 1 μM AID-X-2020 and 10 μM AID-X-2020), and incubated for 24 h at 37° C. and 1400 rpm. The final sample always contained less than 5% DMSO to avoid interference of this solvent on Aβ40 amyloid fibril formation. Finally, ThT treatment was performed as described above.
25 μM ThT was added to each sample and fluorescence was measured by exciting samples at 440 nm and recording the emission from 465 nm to 600 nm. A blank measurement of each sample was done before adding ThT.
For in vitro peptide aggregation analysis, 25 μM Aβ40 solution was prepared as explained above and incubated for 48 h at 37° C. and 1400 rpm in the presence of growing concentrations of AID-X2020 (0, 0.1, 1 and 10 μM). A carbon-coated copper grid was deposited on top of a 50-μL drop of each solution for 30 min. Then, the excess liquid was removed with filter paper and the grid was placed on top of a water drop for 30 s and finally negatively stained for 2 min with 20 μL of 2% uranyl acetate. Samples were observed using a JEM 1010 transmission electron microscope (JEOL Ltd., Japan). Images were acquired using a CCD Orius camera (Gatan, Inc., USA).
According to ThT fluorescence assays (
To discard the possibility that the decrease in ThT fluorescence observed in Aβ40 aggregation assays resulted from a steric hindrance imposed to ThT binding of amyloid fibrils by the presence of AID-X-2020, TEM was used to examine Aβ40 samples treated with AID-X-2020 (
To explore if there was a correlation between the observed in vitro antimalarial activity of AID-X2020 and a disruption of protein homeostasis in the treated parasites, dot blots and Western blots of protein extracts of AID-X-2020-treated P. falciparum cultures were performed, where the presence of ubiquitinated proteins and of amyloid fibrils was examined.
Cultures of the P. falciparum 3D7 strain were sorbitol synchronized in ring stages, and after 24 h were treated for 90 min with AID-X-2020 concentrations ranging from 33 nM to 33 μM. After that time, cultures were spun down and pellets were washed once with ice-cold PBS supplemented with EDTA-free protease inhibitor cocktail (PIC, Roche; 1 PIC tablet/10 mL PBS). For anti-ubiquitin Western blots, PBS was also supplemented with 20 mM N-ethylmaleimide. Once washed, parasite pellets were treated with 0.15% saponin at 4° C. for 15 min and washed by centrifugation (10,000×g, 15 min, 4° C.) with appropriately supplemented PBS until no hemoglobin was observed in the supernatant. Protein extracts were quantified with the bicinchoninic acid assay (ThermoFisher Scientific), following the manufacturer's instructions.
For dot blots, 4-μL drops of saponin extract containing 0.5 or 1 mg/mL protein were spotted on a nitrocellulose membrane. Once protein extracts were completely absorbed by the membranes, these were incubated for 3 h in blocking solution: 5% milk powder in TBS-Tween (200 mM trisbase, 1.5 M NaCl, 0.1% Tween-20). The blocked membranes were washed 3×5 min with TBS-Tween and incubated overnight at 4° C. with rabbit polyclonal anti-amyloid fibrils OC antibody (Millipore) diluted 1:500 in blocking solution or mouse monoclonal anti-spectrin a/p (Sigma) 1:10,000 in TBS-Tween.
For Western blots, 15 μg of saponin-extracted proteins were incubated for 5 min at 95° C. diluted in Laemmli solution (0.14 M SDS, 0.125 M tris-HCl, pH 6.8, 20% glycerol, 10% 2-mercaptoethanol, 3 mM bromophenol blue) and resolved by SDS-PAGE in 12% bis-tris acrylamide (Bio-Rad) gels run at 80 V until samples entered the resolving gel and at 120 V afterwards. Proteins were transferred from the gel to polyvinylidene difluoride membranes activated with methanol 100%. After transference, membranes were blocked with blocking solution for 1 h at room temperature, washed 3×5 min with TBS-Tween and probed overnight at 4° C. with rabbit polyclonal anti-ubiquitin antibody (Cell Signaling Technology) diluted 1:1,000 in blocking solution, or mouse monoclonal anti-spectrin a/p (Sigma) 1:10,000 in TBS-Tween. Then, membranes were washed 5 times with TBS-Tween and incubated for 1 h with either goat anti-rabbit (Upstate) or goat anti-mouse (Amersham Life Science, Inc.) IgG-horseradish peroxidase conjugate antibody diluted 1:10,000 in TBS-Tween. After probing with secondary antibodies, for both dot blot and Western blot membranes, four washes with TBS-Tween and one last wash with TBS were done and peroxidase substrate (ECL Prime Western Blotting Detection Reagent, Amersham Life Science, Inc.) was poured on the membrane and chemiluminescent signal was measured in a LAS 4000 reader (ImageQuant) at different exposure times.
At the concentration of 90 nM, close to its IC50 in vitro, AID-X-2020 treatment of P. falciparum cultures led to a reduction in ubiquitinated proteins along time (
These results suggested a causal effect between decreasing protein aggregation and a deleterious effect on the parasite ultimately leading to a rapid generalized deregulation of proteostasis.
Artemisinin, one of the most potent antimalarials in use, has been described to cause protein damage/unfolding and to inhibit folding of newly synthesized proteins, likely inducing protein aggregation. As shown in the previous examples, AID-X-2020 appears to inhibit protein aggregation, the opposite action of artemisinin. Thus, this study was performed to confirm the capacity of AID-X2020 to inhibit protein aggregation.
For assays of AID-X-2020 and artemisinin, serial dilutions of both compounds were prepared at different concentration ratios (1:0, 0:1, 1:1, 1:2, 2:1, 1:5 and 5:1).
To assess the synergistic effect of AID-X-2020 and artemisinin, IC50 values for each individual compound in the mixtures were calculated as described above and plotted in an isobologram (“x” value=AID-X-2020 IC50 and “y” value=artemisinin IC50). Fractional inhibitory concentration (FIC) values were calculated by dividing the IC50 of one of the compounds in the mixture by the IC50 of the same compound in the 1:0 or 0:1 ratio mixture.
When P. falciparum cultures were treated with artemisinin and AID-X-2020 combined in different ratios, the resulting ZFIC values were always higher than 1.5 (
Because Plasmodium is known to have protein aggregates in the mosquito stages of Plasmodium, the effect of AID-X-2020 was tested on one of the key steps of the pathogen's development in the insect, namely the gametocyte to ookinete transition. Gametocytes are the sole stage of malaria parasites present in the blood circulation capable of transmitting the infection to the mosquito vector following their ingestion by a blood-feeding Anopheles female. Transmission-blocking strategies are therefore one of the main approaches being explored to disrupt the life cycle of Plasmodium, but active drugs at this critical step are scarce.
Eight days before ookinete production, 200 μL of Plasmodium berghei CTRP-GFP (which expresses GFP when reaching ookinete stage) (Vlachou, D. et al., 2004) in cryopreservation solution (RBC pellet:RPMI:30% glycerol in water, 1:1:2) was administered i.p. to a BALB/c mouse. Four days later this mouse was the donor to infect i.p. with 5×107 parasitized red blood cells in 200 μL of PBS three mice that one hour before the infection had been pretreated i.p. with phenylhydrazine (120 μL of a 10 mg/mL solution in PBS).
For ookinete production, up to 1 mL of blood carrying gametocytes was collected from each animal by intracardiac puncture and diluted in 30 mL of ookinete medium: 10.4 g/L of RPMI supplemented with 2% w/v NaHCO3, 0.05% w/v hypoxanthine, 0.02% w/v xanthurenic acid, 50 U/mL penicillin and 50 μg/mL streptomycin (Invitrogen), 20% heat-inactivated fetal bovine serum (FBS, Invitrogen), 25 mM HEPES, pH 7.4.
The effect of AID-X-2020 and DONE3TCI on gametocyte to ookinete conversion was assessed in three independent replicas by plating 120 μL of each culture mixed with growing concentrations of the compounds in 96-well plates and incubated for 24 h at 21° C. with orbital shaking at 50 rpm. 24 h later, samples were diluted 1:100 in PBS and analyzed in a LSRFortessa™ flow cytometer (BD Biosciences) set up with the 5 lasers, 20 parameters standard configuration. The GFP positive cell population was selected and counted using 488 nm laser excitation and a 525/40 nm emission collection filter. BD FACSDiva software (BD biosciences) was used in data collection, and Flowing Software 2.5.1 (Turku Centre for Biotechnology) was used for analysis.
In vitro inhibition assays of the gametocyte to ookinete transition in the murine malaria parasite Plasmodium berghei indicated that ca. 0.5 μM AID-X-2020 abolished ookinete production in this model (
BALB/c female and male mice (Janvier Laboratories) were maintained with unlimited access to food and water under standard environmental conditions (20-24° C. and 12/12 h light/dark cycle). Three 100 μL doses of an AID-X-2020 solution prepared to administer 0.0959, 0.3069 and 0.9822 mg/kg were tested. First, the lowest dose was intravenously injected to one female and one male mice. An oxygen stream of 4% isoflurane was used to anesthetize the mice, which were then maintained during the whole procedure (less than 3 minutes) with 2.5% isoflurane.
After the administration, mice were observed and different parameters related to their behavior (lethargy, motility alterations, seizures, coma, automutilation, aggressiveness, vocalizations, stereotyped movements) and physical conditions (pain, respiratory disturbances, tachycardia or bradycardia, dehydration, hair loss, body weight loss, dermatitis, bad hygiene, pruritus, tearing) were followed. If after 48 h no deleterious effects were observed, the following dose was administered to two other male and female mice. All mice were observed for 14 days after treatment in order to detect long-term side effects.
The animal care and protocol followed here adhered to the specific national and international guidelines stated in the Spanish Royal Decree 53/2013, which is based on the European regulation 2010/63/UE.
In vivo toxicity assays in mice indicated that AID-X-2020 and DONE3TCI started inducing adverse effects in female mice at 10 mg/kg. In male mice toxicity started being observed at 17 and 3 mg/kg for AID-X-2020 and DONE3TCI, respectively. 10 mg/kg then is a tentative maximal dose of reference for future preclinical assays.
In human umbilical vein endothelial cell cultures, the AID-X-2020 concentration required for the reduction of cell viability by 50% (CC50) was determined to be 3.4 μM, which resulted in a therapeutic index (CC50/IC50) of 37.8. Despite this relatively high in vitro toxicity, in vivo toxicity assays indicated that AID-X-2020 starts inducing adverse effects in female and male mice at 10 and 17 mg/kg respectively.
Eighteen compounds were synthesized, which are representative of compounds included in formula (I) other than AID-X-2020, and a study of the antimalarial effect of seventeen of these compounds was performed. The antimalarial activity of these compounds was then compared to the antimalarial activity of AID-X-2020 (IC50=0.09 μM).
Steps and conditions referring to general aspects of the synthesis procedures were performed as described in section 1.1.1 of EXAMPLE 1.
8.1.2 Synthesis of 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(dimethylamino)styryl]-3-methylpyridin-1-ium} dibromide (EMA357)
Compound EMA357 was prepared following the first steps and conditions as described for the synthesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1—1st paragraph) until 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained.
A solution of 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (257 mg, 0.50 mmol) and 4-(dimethylamino)benzaldehyde (149 mg, 1.00 mmol) in n-butanol (2.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 16 h, and then concentrated under reduced pressure. The resulting black sticky residue was purified by automatic flash column chromatography (CH2Cl2/7N ammonia solution in MeOH 9.5:0.5 to 8:2), to provide 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(dimethylamino)styryl]-3-methylpyridin-1-ium} dibromide (127 mg, 33%) as a red oil; IR (ATR) v: 3390, 2925, 2852, 1640, 1573, 1525, 1478, 1435, 1364, 1310, 1218, 1185, 1170, 1128, 944, 814 cm−1; 1H NMR (400 MHz, CD3OD) δ:1.29-1.43 (m, 12H), 1.97 (m, 4H), 2.55 (s, 6H), 3.06 (s, 12H), 4.40 (t, J=7.6 Hz, 4H), 6.79 (d, J=9.2 Hz, 4H), 7.14 (d, J=16.0 Hz, 2H), 7.64 (d, J=9.2 Hz, 4H), 7.81 (d, J=16.0 Hz, 2H), 8.18 (d, J=6.8 Hz, 2H), 8.47 (br d, J=6.8 Hz, 2H), 8.56 (br s, 2H); HRMS-ESI+m/z calculated for [C42H56N4]2+/2: 308.2247, found: 308.2241.
8.1.3 Synthesis of 1,1′-(decane-1,10-diyl)bis{4-{(E)-4-[(2-cyanoethyl)(methyl)amino]styryl}-3-methylpyridin-1-ium} dibromide (EMA359)
Compound EMA359 was prepared following the first steps and conditions as described for the synthesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1—1st paragraph) until 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained.
A solution of 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (257 mg, 0.50 mmol) and 3-[(4-formylphenyl)(methyl)amino]propanenitrile (188 mg, 1.00 mmol) in n-butanol (2.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 16 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/7N ammonia solution in MeOH 9:1), to provide 1,1′-(decane-1,10-diyl)bis{4-{(E)-4-[(2-cyanoethyl)(methyl)amino]styryl}-3-methylpyridin-1-ium} dibromide (204 mg, 48%) as a red solid; mp: 130-131° C.; IR (ATR) v: 3377, 2925, 2854, 1633, 1578, 1523, 1478, 1383, 1311, 1218, 1184, 1117, 959, 808 cm−1; 1H NMR (400 MHz, CD3OD) δ: 1.31-1.43 (m, 12H), 1.97 (m, 4H), 2.56 (s, 6H), 2.76 (t, J=6.8 Hz, 4H), 3.13 (s, 6H), 3.83 (t, J=6.8 Hz, 4H), 4.41 (t, J=7.6 Hz, 4H), 6.87 (d, J=8.8 Hz, 4H), 7.20 (d, J=16.0 Hz, 2H), 7.68 (d, J=8.8 Hz, 4H), 7.82 (d, J=16.0 Hz, 2H), 8.20 (d, J=6.8 Hz, 2H), 8.50 (br d, J=6.8 Hz, 2H), 8.59 (br s, 2H); HRMS-ESI+m/z calculated for [C46H58N6]2+/2: 347.2356, found: 347.2348.
8.1.4 Synthesis of 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(diphenylamino)styryl]-3-methylpyridin-1-ium} dibromide (EMA366)
Compound EMA366 was prepared following the first steps and conditions as described for the synthesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1—1st paragraph) until 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained.
A solution of 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (257 mg, 0.50 mmol) and 4-(diphenylamino)benzaldehyde (273 mg, 1.00 mmol) in n-butanol (2.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 16 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/7N ammonia solution in MeOH 9:1 to 85:15), to provide 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(diphenylamino)styryl]-3-methylpyridin-1-ium} dibromide (236 mg, 46%) as a red solid; mp: 163-165° C.; IR (ATR) v: 3352, 3186, 2929, 2854, 1658, 1581, 1510, 1485, 1388, 1318, 1285, 1222, 1192, 1178, 1138, 973, 820, 757, 697, 571 cm−1; 1H NMR (500 MHz, CD3OD) δ: 1.30-1.43 (m, 12H), 1.98 (tt, J=J′=7.5 Hz, 4H), 2.57 (s, 6H), 4.45 (t, J=7.5 Hz, 4H), 7.00 (d, J=8.5 Hz, 4H), 7.11-7.17 (m, 12H), 7.29 (d, J=16.0 Hz, 2H), 7.34 (ddm, J=8.5 Hz, J′=7.5 Hz, 8H), 7.63 (d, J=8.5 Hz, 4H), 7.81 (d, J=16.0 Hz, 2H), 8.24 (d, J=6.5 Hz, 2H), 8.57 (dd, J=6.5 Hz, J′=1.5 Hz, 2H), 8.65 (s, 2H); HRMS-ESI+m/z calculated for [C62H64N4]2+/2: 432.2560, found: 432.2565.
8.1.5 Synthesis of 1,1′-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-(pyrrolidin-1-yl)styryl]pyridin-1-ium} dibromide (EMA368)
Compound EMA368 was prepared following the first steps and conditions as described for the synthesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1—1st paragraph) until 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained.
A solution of 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (257 mg, 0.50 mmol) and 4-(pyrrolidin-1-yl)benzaldehyde (175 mg, 1.00 mmol) in n-butanol (2.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 22 h, and then concentrated under reduced pressure. The resulting red oily residue was purified by automatic flash column chromatography (CH2Cl2/MeOH 10:0 to 9.3:0.7), to provide 1,1′-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-(pyrrolidin-1-yl)styryl]pyridin-1-ium} dibromide (233 mg, 56%) as a red solid; mp: 167-168° C.; IR (ATR) v: 3391, 2928, 2850, 1641, 1617, 1571, 1523, 1478, 1387, 1303, 1218, 1177, 1136, 961, 807 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 1.21-1.31 (m, 12H), 1.86 (m, 4H), 1.98 (t, J=5.6 Hz, 8H), 2.48 (s, 6H), 3.01 (t J=5.6 Hz, 8H), 4.36 (t, J=7.6 Hz, 4H), 6.62 (d, J=8.8 Hz, 4H), 7.09 (d, J=16.0 Hz, 2H), 7.66 (d, J=8.8 Hz, 4H), 7.89 (d, J=16.0 Hz, 2H), 8.26 (d, J=6.8 Hz, 2H), 8.65 (br d, J=6.8 Hz, 2H), 8.72 (br s, 2H); HRMS-ESI+m/z calculated for [C46H60N4]2+/2: 334.2404, found: 334.2410.
8.1.6 Synthesis of 1,1′-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-morpholinostyryl]pyridin-1-ium} dibromide (EMA377)
Compound EMA377 was prepared following the first steps and conditions as described for the synthesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1—1st paragraph) until 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained.
A solution of 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (514 mg, 1.00 mmol) and 4-morpholinobenzaldehyde (383 mg, 2.00 mmol) in n-butanol (5.0 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 4 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/MeOH 10:0 to 9:1), to provide 1,1′-(decane-1,10-diyl)bis{3-methyl-4-[(E)-4-morpholinostyryl]pyridin-1-ium} dibromide (281 mg, 33%) as a red solid; mp: 153-155° C.; IR (ATR) v: 3390, 2925, 2851, 1642, 1580, 1519, 1478, 1447, 1428, 1381, 1358, 1307, 1247, 1222, 1187, 1134, 1110, 1049, 925, 810, 635, 604, 572 cm−1; 1H NMR (400 MHz, CD3OD) δ: 1.30-1.42 (m, 12H), 1.98 (tt, J=J′=7.6 Hz, 4H), 2.57 (s, 6H), superimposed in part with the solvent signal 3.30 (t, J=4.8 Hz, 8H), 3.83 (t, J=4.8 Hz, 8H), 4.43 (t, J=7.6 Hz, 4H), 7.02 (d, J=9.2 Hz, 4H), 7.25 (d, J=16.0 Hz, 2H), 7.69 (d, J=9.2 Hz, 4H), 7.81 (d, J=16.0 Hz, 2H), 8.23 (d, J=6.8 Hz, 2H), 8.54 (dd, J=6.8 Hz, J′=1.6 Hz, 2H), 8.63 (br s, 2H); HRMS-ESI+m/z calculated for [C46H60N4O2]2+/2: 350.2353, found: 350.2361.
8.1.7 Synthesis of 1,1′-(butane-1,4-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (PRC-5)
A mixture of 1,4-dibromobutane (0.23 mL, 416 mg, 1.93 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120° C. for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature the resulting brown residue was washed with ice-cold Et2O (15 mL) and the remaining beige solid was dried in vacuo, taken up in MeOH (5 mL) and treated with cold Et2O (2×15 mL), drawing off the liquids. After drying the residue in vacuo, 1,1′-(butane-1,4-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (757 mg, 88%) was obtained as a beige solid; mp: 109-110° C.; IR (ATR) v: 3431, 3360, 3041, 1641, 1621, 1512, 1472, 1460, 1234, 1149, 1027, 847, 706 cm−1; 1H NMR (400 MHz, CD3OD) δ: 2.10 (tm, J=7.2 Hz, 4H), 2.48 (s, 6H), 2.59 (s, 6H), 4.62 (br t, J=7.2 Hz, 4H), 7.87 (d, J=6.4 Hz, 2H), 8.71 (dd, J=6.4 Hz, J′=1.6 Hz, 2H), 8.81 (br s, 2H); HRMS-ESI+ m/z calculated for [C18H26N2]2+/2: 135.1043, found: 135.1051.
A solution of 1,1′-(butane-1,4-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (669 mg, 1.55 mmol) and 4-(diethylamino)benzaldehyde (604 mg, 3.41 mmol) in n-butanol (8 mL) was treated with twelve drops of piperidine and the reaction mixture was stirred under reflux for 4 h, and then concentrated under reduced pressure. The resulting oily residue was purified twice by automatic flash column chromatography (CH2Cl2/7N ammonia solution in MeOH 95:5 to 85:15), to provide 1,1′-(butane-1,4-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (511 mg, 44%) as a red solid; mp: 275-276° C.; IR (ATR) v: 3406, 2964, 1641, 1577, 1522, 1484, 1467, 1434, 1402, 1372, 1350, 1307, 1262, 1220, 1191, 1132, 1067, 975, 810 cm−1; 1H NMR (400 MHz, CD3OD) δ:1.21 (t, J=7.2 Hz, 12H), 2.07 (m, 4H), 2.54 (s, 6H), 3.49 (q, J=7.2 Hz, 8H), 4.48 (m, 4H), 6.76 (d, J=9.2 Hz, 4H), 7.10 (d, J=16.0 Hz, 2H), 7.61 (d, J=9.2 Hz, 4H), 7.81 (d, J=16.0 Hz, 2H), 8.17 (d, J=6.8 Hz, 2H), 8.47 (dd, J=6.8 Hz, J′=1.6 Hz, 2H), 8.56 (br s, 2H); HRMS-ESI+ m/z calculated for [C40H52N4]2+/2: 294.2091, found: 294.2098.
8.1.8 Synthesis of 1,1′-(octane-1,8-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (PRC-8)
A mixture of 1,8-dibromooctane (0.37 mL, 546 mg, 2.00 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120° C. for 4 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature the resulting brown residue was washed with ice-cold Et2O (15 mL) and the remaining brown sticky oil was dried in vacuo, taken up in MeOH (5 mL) and treated with cold Et2O (2×15 mL), drawing off the liquids. After drying the residue in vacuo, 1,1′-(octane-1,8-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (510 mg, 52%) was obtained as a beige solid; mp: 183-184° C.; IR (ATR) v: 3459, 3026, 2925, 2853, 1639, 1511, 1484, 1460, 1444, 1393, 1337, 1230, 1141, 1020, 951, 839, 711 cm−1; 1H NMR (400 MHz, CD3OD) δ: 1.41 (m, 8H), 2.00 (tt, J=7.6 Hz, J′=7.2 Hz, 4H), 2.48 (s, 6H), 2.59 (s, 6H), 4.53 (t, J=7.6 Hz, 4H), 7.86 (d, J=6.4 Hz, 2H), 8.68 (dd, J=6.4 Hz, J′=1.6 Hz, 2H), 8.77 (br s, 2H); HRMS-ESI+m/z calculated for [C22H34N2]2+/2: 163.1356, found: 163.1358.
A solution of 1,1′-(octane-1,8-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (410 mg, 0.84 mmol) and 4-(diethylamino)benzaldehyde (328 mg, 1.85 mmol) in n-butanol (4.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 17 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/MeOH 95:5 to 85:15), to provide 1,1′-(octane-1,8-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (230 mg, 34%) as a red solid; mp: 235-236° C.; IR (ATR) v: 3393, 2970, 2928, 1641, 1570, 1521, 1478, 1404, 1350, 1310, 1259, 1219, 1185, 1128, 1076, 960, 807, 702 cm−1; 1H NMR (400 MHz, CD3OD) δ:1.21 (t, J=7.2 Hz, 12H), 1.41 (m, 8H), 1.87 (tt, J=J′=7.2 Hz, 4H), 2.54 (s, 6H), 3.48 (q, J=7.2 Hz, 8H), 4.40 (t, J=7.2 Hz, 4H), 6.76 (d, J=8.8 Hz, 4H), 7.10 (d, J=16.0 Hz, 2H), 7.61 (d, J=8.8 Hz, 4H), 7.79 (d, J=16.0 Hz, 2H), 8.16 (d, J=6.8 Hz, 2H), 8.46 (dd, J=6.8 Hz, J′=1.6 Hz, 2H), 8.55 (br s, 2H); HRMS-ESI+m/z calculated for [C44H60N4]2+/2: 322.2404, found: 322.2417.
8.1.9 Synthesis of 1,1′-(nonane-1,9-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (PRC-25)
A mixture of 1,9-dibromononane (0.41 mL, 577 mg, 2.02 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120° C. for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et2O (2×20 mL) and the remaining brown sticky oil was dried in vacuo, taken up in MeOH (1 mL) and treated with cold Et2O (2×20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-(nonane-1,9-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (665 mg, 66%) was obtained as a brown oil that solidified on standing; mp: 88-90° C.; IR (ATR) v: 3435, 2933, 2852, 1638, 1505, 1469, 1335, 1228, 1152, 1138, 1023, 834, 754, 729, 707 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 1.17-1.31 (m, 10H), 1.88 (tt, J=J′=7.2 Hz, 4H), 2.39 (s, 6H), 2.51 (s, 6H), 4.49 (t, J=7.2 Hz, 4H), 7.94 (d, J=6.4 Hz, 2H), 8.84 (br d, J=6.4 Hz, 2H), 8.94 (br s, 2H); HRMS-ESI+m/z calculated for [C23H36N2]2+/2: 170.1434, found 170.1435.
A solution of 1,1′-(nonane-1,9-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (250 mg, 0.50 mmol) and 4-(diethylamino)benzaldehyde (195 mg, 1.10 mmol) in n-butanol (2.5 mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux for 4 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/MeOH 95:5 to 85:15). Then fractions containing the desired product were evaporated and the residue was dissolved in hot isopropanol (15 mL) and after addition of Et2O (30 mL) and drawing off the liquids, 1,1′-(nonane-1,9-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (193 mg, 47%) was obtained as a red solid; mp: 138-142° C.; IR (ATR) v: 3381, 2920, 2852, 1642, 1567, 1519, 1482, 1402, 1347, 1308, 1258, 1217, 1185, 1126, 1070, 954, 807 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 1.13 (t, J=7.2 Hz, 12H), 1.22-1.31 (m, 10H), 1.87 (tt, J=J′=7.2 Hz, 4H), 2.48 (s, 6H), 3.43 (q, J=7.2 Hz, 8H), 4.36 (t, J=7.2 Hz, 4H), 6.74 (d, J=9.2 Hz, 4H), 7.08 (d, J=16.0 Hz, 2H), 7.64 (d, J=9.2 Hz, 4H), 7.87 (d, J=16.0 Hz, 2H), 8.26 (d, J=6.8 Hz, 2H), 8.66 (dd, J=6.8 Hz, J′=1.6 Hz, 2H), 8.73 (br s, 2H); HRMS-ESI+m/z calculated for [C45H62N4]2+/2: 329.2482, found: 329.2485.
8.1.10 Synthesis of 1,1′-(undecane-1,11-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (PRC-14)
A mixture of 1,11-dibromoundecane (0.47 mL, 627 mg, 2.00 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120° C. for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et2O (2×20 mL) and the remaining brown sticky oil was dried in vacuum, taken up in CH2Cl2 (1 mL) and treated with cold Et2O (2×20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-(undecane-1,11-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (0.98 g, 93%) was obtained as a brown oil; IR (ATR) v: 3403, 3020, 2924, 2853, 1638, 1510, 1481, 1466, 1389, 1229, 1146, 1026, 843, 707 cm−1; 1H NMR (400 MHz, CD3OD) δ: 1.29-1.42 (m, 14H), 1.99 (tt, J=J′=7.6 Hz, 4H), 2.48 (s, 6H), 2.59 (s, 6H), 4.52 (t, J=7.6 Hz, 4H), 7.86 (d, J=6.4 Hz, 2H), 8.67 (dd, J=6.4 Hz, J′=1.6 Hz, 2H), 8.76 (br s, 2H); HRMS-ESI+m/z calculated for [C25H40N2]2+/2: 184.1590, found: 184.1598.
A solution of 1,1′-(undecane-1,11-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (396 mg, 0.75 mmol) and 4-(diethylamino)benzaldehyde (293 mg, 1.65 mmol) in n-butanol (3.75 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 4 h, and then concentrated under reduced pressure, to afford 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (180 mg, 28%) as a red solid, which was used in the following step without further purification; mp: 81-82° C.; IR (ATR) v: 3389, 2924, 2847, 1641, 1572, 1522, 1478, 1405, 1351, 1310, 1259, 1217, 1186, 1157, 1133, 1075, 1008, 959, 808, 783, 701 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 1.12 (t, J=6.8 Hz, 12H), 1.20-1.31 (m, 14H), 1.86 (tt, J=J′=7.2 Hz, 4H), 2.48 (s, 6H), 3.43 (q, J=6.8 Hz, 8H), 4.36 (t, J=7.2 Hz, 4H), 6.74 (d, J=9.2 Hz, 4H), 7.08 (d, J=16.0 Hz, 2H), 7.64 (d, J=9.2 Hz, 4H), 7.88 (d, J=16.0 Hz, 2H), 8.26 (d, J=6.8 Hz, 2H), 8.65 (br d, J=6.8 Hz, 2H), 8.73 (br s, 2H); HRMS-ESI+m/z calculated for [C47H66N4]2+/2: 343.2638, found: 343.2640.
8.1.11 Synthesis of 1,1′-(dodecane-1,12-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (PRC-15)
A mixture of 1,12-dibromododecane (656 mg, 2.00 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120° C. for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et2O (2×20 mL) and the remaining brown sticky oil was dried in vacuum, taken up in CH2Cl2 (1 mL) and treated with cold Et2O (2×20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-(dodecane-1,12-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (850 mg, 78%) was obtained as a brown oil that solidified on standing; mp: 82-83° C.; IR (ATR) v: 3438, 3373, 2994, 2923, 2853, 1636, 1516, 1466, 1387, 1332, 1228, 1147, 1027, 856, 837, 721, 709, 619, 592 cm−1; 1H NMR (400 MHz, CD3OD) δ:1.28-1.41 (m, 16H), 1.99 (tt, J=J′=7.6 Hz, 4H), 2.48 (s, 6H), 2.59 (s, 6H), 4.52 (t, J=7.6 Hz, 4H), 7.86 (d, J=6.4 Hz, 2H), 8.68 (dd, J=6.4 Hz, J′=1.6 Hz, 2H), 8.77 (br s, 2H); HRMS-ESI+m/z calculated for [C26H42N2]2+/2: 191.1669, found: 191.1675.
A solution of 1,1′-(dodecane-1,12-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (379 mg, 0.70 mmol) and 4-(diethylamino)benzaldehyde (273 mg, 1.54 mmol) in n-butanol (3.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 4.5 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/MeOH 95:5), to provide 1,1′-(dodecane-1,12-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (116 mg, 19%) as a red solid; mp: 112-114° C.; IR (ATR) v: 3400, 2971, 2927, 2852, 1641, 1574, 1519, 1477, 1435, 1403, 1348, 1312, 1259, 1219, 1188, 1157, 1126, 1077, 961, 824, 789, 571 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 1.13 (t, J=7.2 Hz, 12H), 1.19-1.32 (m, 16H), 1.88 (tt, J=J′=7.2 Hz, 4H), 2.48 (s, 6H), 3.43 (q, J=7.2 Hz, 8H), 4.36 (t, J=7.2 Hz, 4H), 6.74 (d, J=8.8 Hz, 4H), 7.08 (d, J=16.0 Hz, 2H), 7.64 (d, J=8.8 Hz, 4H), 7.88 (d, J=16.0 Hz, 2H), 8.26 (d, J=6.8 Hz, 2H), 8.66 (br d, J=6.8 Hz, 2H), 8.73 (br s, 2H); HRMS-ESI+m/z calculated for [C48H68N4]2+/2: 350.2717, found: 350.2721.
8.1.12 Synthesis of 1,1′-(3,6-dioxaoctane-1,8-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} diiodide (PRC-20)
A mixture of 1,2-bis(2-iodoethoxy)ethane (0.27 mL, 548 mg, 1.48 mmol) and 3,4-dimethylpyridine (0.37 mL, 353 mg, 3.29 mmol) was heated to 120° C. for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et2O (2×20 mL) and the remaining brown sticky oil was dried in vacuum, taken up in MeOH (1 mL) and treated with cold Et2O (2×20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-(3,6-dioxaoctane-1,8-diyl)bis(3,4-dimethylpyridin-1-ium) diiodide (620 mg, 72%) was obtained as a brown oil that solidified on standing; mp: 135-136° C.; IR (ATR) v: 3426, 3029, 2983, 2937, 2878, 1636, 1510, 1476, 1450, 1369, 1353, 1327, 1297, 1247, 1230, 1217, 1139, 1102, 1091, 1026, 992, 977, 912, 886, 813, 713, 704, 609, 558 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 2.39 (s, 6H), 2.53 (s, 6H), 3.48 (s, 4H), 3.84 (t, J=5.2 Hz, 4H), 4.64 (t, J=5.2 Hz, 4H), 7.95 (d, J=6.0 Hz, 2H), 8.71 (dd, J=6.0 Hz, J′=1.6 Hz, 2H), 8.81 (br s, 2H); HRMS-ESI+m/z calculated for [C20H30N2O2]2+/2: 165.1148, found 165.1147.
A solution of 1,1′-(3,6-dioxaoctane-1,8-diyl)bis(3,4-dimethylpyridin-1-ium) diiodide (409 mg, 0.70 mmol) and 4-(diethylamino)benzaldehyde (273 mg, 1.54 mmol) in n-butanol (3.5 mL) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 4.5 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/MeOH 95:5), to provide 1,1′-(3,6-dioxaoctane-1,8-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} diiodide (200 mg, 32%) as a red solid; mp: 71-72° C.; IR (ATR) v: 3417, 2967, 2920, 2859, 1640, 1567, 1519, 1477, 1435, 1404, 1350, 1307, 1258, 1218, 1184, 1154, 1128, 1072, 1009, 812, 700, 603 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 1.12 (t, J=7.2 Hz, 12H), 2.44 (s, 6H), 3.43 (q, J=7.2 Hz, 8H), 3.54 (s, 4H), 3.85 (t, J=5.2 Hz, 4H), 4.53 (t, J=5.2 Hz, 4H), 6.72 (d, J=8.8 Hz, 4H), 7.04 (d, J=16.0 Hz, 2H), 7.61 (d, J=8.8 Hz, 4H), 7.83 (d, J=16.0 Hz, 2H), 8.22 (d, J=6.8 Hz, 2H), 8.54 (br d, J=6.8 Hz, 2H), 8.61 (br s, 2H); HRMS-ESI+m/z calculated for [C42H56N4O2]2+/2: 324.2196, found: 324.2197.
8.1.13 Synthesis of 1,1′-(3,6,9-trioxaundecane-1,11-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dichloride (PRC-28)
A mixture of bis[2-(2-chloroethoxy)ethyl] ether (0.26 mL, 307 mg, 1.33 mmol), 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol), and NaI (15 mg, 0.10 mmol) was heated to 120° C. for 4 h. Then, isopropanol (1.5 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et2O (2×20 mL) and the remaining brown sticky oil was dried in vacuum, taken up in MeOH (1 mL) and treated with cold Et2O (2×20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-(3,6,9-trioxaundecane-1,11-diyl)bis(3,4-dimethylpyridin-1-ium) dichloride (467 mg, 79%) was obtained as a brown oil; IR (ATR) v: 3371, 3043, 2923, 2871, 1639, 1510, 1480, 1449, 1233, 1086, 1026, 931, 836, 706 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 2.38 (s, 6H), 2.52 (s, 6H), 3.38 (tm, J=4.8 Hz, 4H), 3.49 (tm, J=4.8 Hz, 4H), 3.87 (t, J=4.8 Hz, 4H), 4.67 (t, J=4.8 Hz, 4H), 7.93 (d, J=6.4 Hz, 2H), 8.75 (dd, J=6.4 Hz, J′=1.6 Hz, 2H), 8.85 (br s, 2H); HRMS-ESI+ m/z calculated for [C22H34N2O3]2+/2: 187.1279, found 187.1292.
A solution of 1,1′-(3,6,9-trioxaundecane-1,11-diyl)bis(3,4-dimethylpyridin-1-ium) dichloride (190 mg, 0.43 mmol) and 4-(diethylamino)benzaldehyde (164 mg, 0.93 mmol) in n-butanol (2.2 mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/MeOH 95:5 to 85:15), to provide 1,1′-(3,6,9-trioxaundecane-1,11-diyl)bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dichloride (55 mg, 17%) as a red oil; IR (ATR) v: 3364, 2970, 2925, 2859, 1640, 1573, 1522, 1479, 1436, 1406, 1352, 1311, 1260, 1223, 1187, 1156, 1132, 1075, 1011, 814, 700, 607 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 1.12 (t, J=7.2 Hz, 12H), 2.44 (s, 6H), superimposed in part 3.42 (tm, J=4.8 Hz, 4H), 3.43 (q, J=7.2 Hz, 8H), 3.52 (tm, J=4.8 Hz, 4H), 3.86 (t, J=4.8 Hz, 4H), 4.54 (t, J=4.8 Hz, 4H), 6.73 (d, J=8.8 Hz, 4H), 7.05 (d, J=16.0 Hz, 2H), 7.62 (d, J=8.8 Hz, 4H), 7.85 (d, J=16.0 Hz, 2H), 8.22 (d, J=6.8 Hz, 2H), 8.57 (br d, J=6.8 Hz, 2H), 8.63 (br s, 2H); HRMS-ESI+m/z calculated for [C44H60N4O3]2+/2: 346.2327, found: 346.2328.
8.1.14 Synthesis of 1,1′-[1,4-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (PRC-29)
A mixture of 1,4-bis(bromomethyl)benzene (527 mg, 2.00 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120° C. for 2 h. Then, isopropanol (4 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et2O (2×20 mL) and the remaining brown sticky oil was dried in vacuum, taken up in MeOH (1 mL) and treated with cold Et2O (2×20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-[1,4-phenylenebis(methylene)]bis(3,4-dimethylpyridin-1-ium) dibromide (813 mg, 85%) was obtained as a white solid; mp: >300° C.; IR (ATR) v: 3008, 2984, 2920, 1634, 1507, 1484, 1448, 1364, 1339, 1297, 1248, 1223, 1142, 1039, 1027, 961, 931, 887, 866, 770, 744, 608, 557 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 2.37 (s, 6H), 2.50 (s, 6H), 5.74 (s, 4H), 7.59 (s, 4H), 7.96 (d, J=6.4 Hz, 2H), 8.94 (dd, J=6.4 Hz, J′=1.6 Hz, 2H), 9.03 (br s, 2H); HRMS-ESI+m/z calculated for [C22H26N2]2+/2: 159.1043, found 159.1032.
A solution of 1,1′-[1,4-phenylenebis(methylene)]bis(3,4-dimethylpyridin-1-ium) dibromide (231 mg, 0.48 mmol) and 4-(diethylamino)benzaldehyde (156 mg, 0.88 mmol) in n-butanol (2 mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/MeOH 95:5). The fractions containing the desired product were evaporated and the resulting oily residue was dissolved in hot isopropanol (15 mL) and treated with Et2O (40 mL), drawing off the liquid. After drying the residue in vacuum, 1,1′-[1,4-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (320 mg, 84%) was obtained as a red solid; mp: 246-248° C.; IR (ATR) v: 3380, 2969, 2910, 1635, 1567, 1520, 1478, 1446, 1356, 1312, 1260, 1211, 1185, 1151, 1128, 1074, 1013, 970, 813, 701, 645 cm1; 1H NMR (500 MHz, DMSO-d6) δ: 1.12 (t, J=7.0 Hz, 12H), 2.46 (s, 6H), 3.43 (q, J=7.0 Hz, 8H), 5.62 (s, 4H), 6.73 (d, J=9.0 Hz, 4H), 7.06 (d, J=16.0 Hz, 2H), 7.58 (s, 4H), 7.63 (d, J=9.0 Hz, 4H), 7.88 (d, J=16.0 Hz, 2H), 8.27 (d, J=6.5 Hz, 2H), 8.76 (dd, J=6.5 Hz, J′=1.5 Hz, 2H), 8.83 (br s, 2H); HRMS-ESI+m/z calculated for [C44H52N4]2+/2: 318.2091, found: 318.2090.
8.1.15 Synthesis of 1,1′-[1,3-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (PRC-31)
A mixture of 1,3-bis(bromomethyl)benzene (527 mg, 2.00 mmol) and 3,4-dimethylpyridine (0.49 mL, 467 mg, 4.36 mmol) was heated to 120° C. for 2 h. Then, isopropanol (4 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et2O (2×20 mL) and the remaining brown sticky oil was dried in vacuum, taken up in MeOH (1 mL) and treated with cold Et2O (2×20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-[1,3-phenylenebis(methylene)]bis(3,4-dimethylpyridin-1-ium) dibromide (785 mg, 82%) was obtained as a brown solid; mp: 152-153° C.; IR (ATR) v: 3460, 3414, 3006, 2920, 1636, 1509, 1480, 1450, 1387, 1362, 1340, 1225, 1163, 1139, 1020, 856, 842, 740, 705, 692, 652, 613 cm−1; 1H NMR (500 MHz, DMSO-d6) δ: 2.40 (s, 6H), 2.52 (s, 6H), 5.77 (s, 4H), 7.47-7.55 (m, 3H), 7.80 (t, J=2.0 Hz, 1H), 7.98 (d, J=6.0 Hz, 2H), 8.94 (dd, J=6.0 Hz, J′=1.5 Hz, 2H), 9.10 (br s, 2H); HRMS-ESI+m/z calculated for [C22H26N2]2+/2: 159.1043, found 159.1042.
A solution of 1,1′-[1,3-phenylenebis(methylene)]bis(3,4-dimethylpyridin-1-ium) dibromide (231 mg, 0.48 mmol) and 4-(diethylamino)benzaldehyde (156 mg, 0.88 mmol) in n-butanol (2 mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/MeOH 100:0 to 95:5). The fractions containing the desired product were evaporated and the resulting oily residue was dissolved in hot isopropanol (15 mL) and treated with Et2O (40 mL), drawing off the liquid. After drying the residue in vacuum, 1,1′-[1,3-phenylenebis(methylene)]bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide (20 mg, 5%) was obtained as a red solid; mp: 66-67° C.; IR (ATR) v: 3347, 2969, 2920, 1639, 1572, 1523, 1479, 1438, 1406, 1350, 1310, 1261, 1213, 1187, 1151, 1128, 1076, 1013, 971, 808, 785, 740, 700 cm−1; 1H NMR (500 MHz, DMSO-d6) δ: 1.13 (t, J=7.0 Hz, 12H), 2.48 (s, 6H), 3.43 (q, J=7.0 Hz, 8H), 5.64 (s, 4H), 6.74 (d, J=9.0 Hz, 4H), 7.08 (d, J=16.0 Hz, 2H), 7.49-7.51 (m, 3H), 7.60 (br s, 1H), 7.64 (d, J=9.0 Hz, 4H), 7.90 (d, J=16.0 Hz, 2H), 8.29 (d, J=7.0 Hz, 2H), 8.74 (dd, J=7.0 Hz, J′=1.5 Hz, 2H), 8.82 (br s, 2H); HRMS-ESI+m/z calculated for [C44H52N4]2+/2: 318.2091, found: 318.2090.
8.1.16 Synthesis of 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide (PRC-39)
A mixture of 1,10-dibromodecane (0.44 mL, 587 mg, 1.96 mmol) and 4-methylpyridine (0.43 mL, 412 mg, 4.42 mmol) was heated to 120° C. for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et2O (2×20 mL) and the remaining brown sticky oil was dried in vacuo, taken up in MeOH (1 mL) and treated with cold Et2O (2×20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-(decane-1,10-diyl)bis(4-methylpyridin-1-ium) dibromide (751 mg, 79%) was obtained as a orange oil that solidified on standing; mp: 56-57° C.; IR (ATR) v: 3350, 3013, 2920, 2852, 1638, 1516, 1469, 1379, 1219, 1171, 1030, 828, 730, 710 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 1.17-1.31 (m, 12H), 1.87 (tt, J=J′=7.2 Hz, 4H), 2.61 (s, 6H), 4.53 (t, J=7.2 Hz, 4H), 7.99 (d, J=6.4 Hz, 4H), 8.96 (d, J=6.4 Hz, 4H); HRMS-ESI+m/z calculated for [C22H34N2]2+/2: 163.1356, found 163.1360.
A solution of 1,1′-(decane-1,10-diyl)bis(4-methylpyridin-1-ium) dibromide (243 mg, 0.50 mmol) and 4-(diethylamino)benzaldehyde (156 mg, 0.88 mmol) in n-butanol (2. mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/MeOH 100:0 to 95:5). The fractions containing the desired product were evaporated and the resulting oily residue was dissolved in hot isopropanol (15 mL) and treated with Et2O (2×40 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide (132 mg, 33%) was obtained as a red solid; mp: 157-159° C.; IR (ATR) v: 3404, 2969, 2920, 1646, 1572, 1521, 1471, 1434, 1403, 1348, 1325, 1272, 1192, 1169, 1150, 1011, 827, 704, 572, 561 cm−1; 1H NMR (400 MHz, CD3OD) δ: 1.21 (t, J=7.2 Hz, 12H), 1.31-1.42 (m, 12H), 1.96 (tt, J=J′=7.2 Hz, 4H), 3.48 (q, J=7.2 Hz, 8H), 4.40 (t, J=7.2 Hz, 4H), 6.76 (d, J=8.8 Hz, 4H), 7.05 (d, J=16.0 Hz, 2H), 7.59 (d, J=8.8 Hz, 4H), 7.83 (d, J=16.0 Hz, 2H), 7.95 (d, J=6.8 Hz, 4H), 8.55 (d, J=6.8 Hz, 4H); HRMS-ESI+m/z calculated for [C44H60N4]2+/2: 322.2404, found: 322.2395.
8.1.17 Synthesis of 1,1′-(decane-1,10-diyl)bis{3-bromo-4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide (PRC-42)
A mixture of 1,10-dibromodecane (0.44 mL, 587 mg, 1.96 mmol) and 3-bromo-4-methylpyridine (0.49 mL, 759 mg, 4.41 mmol) was heated to 120° C. for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et2O (2×20 mL) and the remaining bluish sticky oil was dried in vacuo, taken up in MeOH (1 mL) and treated with cold Et2O (2×20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-(decane-1,10-diyl)bis(3-bromo-4-methylpyridin-1-ium) dibromide (605 mg, 48%) was obtained as a brown oil that solidified on standing; mp: 63-64° C.; IR (ATR) v: 3454, 3387, 2988, 2921, 2851, 1632, 1497, 1465, 1375, 1317, 1186, 1174, 1084, 1031, 864, 719, 697, 579 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 1.19-1.32 (m, 12H), 1.90 (tt, J=J′=7.2 Hz, 4H), 2.61 (s, 6H), 4.54 (t, J=7.2 Hz, 4H), 8.16 (d, J=6.4 Hz, 2H), 9.04 (dd, J=6.4 Hz, J′=1.6 Hz, 2H), 9.49 (d, J=1.6 Hz, 2H); HRMS-ESI+m/z calculated for [C22H32Br2N2]2+/2: 241.0461, found 241.0453.
A solution of 1,1′-(decane-1,10-diyl)bis(3-bromo-4-methylpyridin-1-ium) dibromide (322 mg, 0.50 mmol) and 4-(diethylamino)benzaldehyde (195 mg, 1.10 mmol) in n-butanol (2 mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/MeOH 100:0 to 90:10), to provide 1,1′-(decane-1,10-diyl)bis{3-bromo-4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide (45 mg, 9%) as a purple solid; mp: 136-138° C.; IR (ATR) v: 3396, 2969, 2924, 2852, 1634, 1567, 1520, 1454, 1406, 1350, 1293, 1269, 1171, 1149, 1075, 1011, 806, 689, 567 cm−1; 1H NMR (400 MHz, CD3OD) δ: 1.22 (t, J=7.2 Hz, 12H), 1.31-1.43 (m, 12H), 1.97 (tt, J=J′=7.6 Hz, 4H), 3.51 (q, J=7.2 Hz, 8H), 4.39 (t, J=7.6 Hz, 4H), 6.79 (d, J=9.2 Hz, 4H), 7.24 (d, J=16.0 Hz, 2H), 7.64 (d, J=9.2 Hz, 4H), 7.94 (d, J=16.0 Hz, 2H), 8.23 (d, J=6.8 Hz, 2H), 8.52 (br d, J=6.8 Hz, 2H), 9.02 (d, J=1.2 Hz, 2H); HRMS-ESI+m/z calculated for [C44H58Br2N4]2+/2: 400.1509, found: 400.1512.
8.1.18 Synthesis of 1,1′-(decane-1,10-diyl)bis{3-chloro-4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide (PRC-50)
A mixture of 1,10-dibromodecane (0.44 mL, 587 mg, 1.96 mmol) and 3-chloro-4-methylpyridine (484 mg, 3.79 mmol) was heated to 120° C. for 3 h. Then, isopropanol (2 mL) was added and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to room temperature, the resulting brown residue was washed with ice-cold Et2O (2×20 mL) and the remaining bluish sticky oil was dried in vacuo, taken up in MeOH (1 mL) and treated with cold Et2O (2×20 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-(decane-1,10-diyl)bis(3-chloro-4-methylpyridin-1-ium) dibromide (614 mg, 58%) was obtained as a bluish oil that solidified on standing to a light pink solid; mp: 167-168° C.; IR (ATR) v: 3411, 2945, 2924, 2896, 2859, 1638, 1504, 1468, 1449, 1366, 1311, 1250, 1219, 1200, 1169, 1099, 1025, 1007, 955, 865, 769, 726, 703, 596 cm−1; 1H NMR (400 MHz, DMSO-d6) δ: 1.21-1.31 (m, 12H), 1.90 (tt, J=J′=7.6 Hz, 4H), 2.61 (s, 6H), 4.54 (t, J=7.6 Hz, 4H), 8.18 (d, J=6.4 Hz, 2H), 9.01 (dd, J=6.4 Hz, J′=1.6 Hz, 2H), 9.43 (d, J=1.6 Hz, 2H); HRMS-ESI+m/z calculated for [C22H32Cl2N2]2+/2: 197.0966, found 197.0967.
A solution of 1,1′-(decane-1,10-diyl)bis(3-chloro-4-methylpyridin-1-ium) dibromide (222 mg, 0.40 mmol) and 4-(diethylamino)benzaldehyde (156 mg, 0.88 mmol) in n-butanol (1,6. mL) was treated with four drops of piperidine and the reaction mixture was stirred under reflux overnight, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/7N ammonia solution in MeOH 100:0 to 90:10), to provide 1,1′-(decane-1,10-diyl)bis{3-chloro-4-[(E)-4-(diethylamino)styryl]pyridin-1-ium} dibromide (54 mg, 15%) as a purple solid; mp: 124-126° C.; IR (ATR) v: 3396, 2967, 2924, 2852, 1633, 1572, 1520, 1455, 1405, 1349, 1294, 1269, 1176, 1150, 1074, 1037, 1012, 972, 917, 808, 699, 612, 570 cm−1; 1H NMR (500 MHz, CD3OD) δ: 1.22 (t, J=7.0 Hz, 12H), 1.32-1.42 (m, 12H), 1.97 (tt, J=J′=7.5 Hz, 4H), 3.51 (q, J=7.0 Hz, 8H), 4.39 (t, J=7.5 Hz, 4H), 6.79 (d, J=9.0 Hz, 4H), 7.25 (d, J=16.0 Hz, 2H), 7.64 (d, J=9.0 Hz, 4H), 7.96 (d, J=16.0 Hz, 2H), 8.26 (d, J=6.5 Hz, 2H), 8.49 (br d, J=6.5 Hz, 2H), 8.91 (d, J=1.5 Hz, 2H); HRMS-ESI+m/z calculated for [C44H58Cl2N4]2+/2: 356.2014, found: 356.2020.
8.1.19 Synthesis of 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)-2-hydroxystyryl]-3-methylpyridin-1-ium} dibromide (PRC-69)
Compound PRC-69 was prepared following the first steps and conditions as described for the synthesis of compound AID-X-2020 (section 1.1.2 of EXAMPLE 1—1st paragraph) until 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide was obtained.
A solution of 1,1′-(decane-1,10-diyl)bis(3,4-dimethylpyridin-1-ium) dibromide (307 mg, 0.60 mmol) and 5-(diethylamino)-2-formylphenyl acetate (309 mg, 1.31 mmol) in n-butanol (3 mL) was treated with nine drops of DBU and the reaction mixture was stirred under microwave irradiation at 140° C., 1 bar, 80 W for 15 min. Then, it was concentrated under reduced pressure and the resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2/isopropanol 90:10 to 75:25). The fractions containing the desired product were treated with Cs2CO3 in MeOH, until the colour changed from red to blue. The solution was evaporated under reduced pressure and the solid was suspended in CH2Cl2. The suspension was filtered, the filtrate was evaporated, and the resulting solid was washed with EtOAc (5×10 mL), drawing off the liquids. After drying the residue in vacuum, 1,1′-(decane-1,10-diyl)bis{4-[(E)-4-(diethylamino)-2-hydroxystyryl]-3-methylpyridin-1-ium} dibromide (12 mg, 2%) was obtained as a blue solid; mp: 169-171° C.; IR (ATR) v: 3277, 2924, 2853, 1556, 1512, 1395, 1373, 1350, 1243, 1186, 1104, 1075, 1011, 962, 771, 682 cm−1; 1H NMR (400 MHz, CD3OD) δ: 1.18 (t, J=7.2 Hz, 12H), 1.23-1.40 (m, 12H), 1.89 (m, 4H), 2.37 (s, 6H), 3.41 (q, J=7.2 Hz, 8H), 4.18 (t, J=7.2 Hz, 4H), 6.01 (d, J=2.0 Hz, 2H), 6.16 (dd, J=9.2 Hz, J′=2.0 Hz, 2H), 6.89 (d, J=15.2 Hz, 2H), 7.45 (dm, J=9.2 Hz, 2H), 7.89 (d, J=7.2 Hz, 2H), 8.05 (d, J=7.2 Hz, 2H), 8.11 (br s, 2H), 8.30 (d, J=15.2 Hz, 2H); HRMS-ESI+m/z calculated for [C46H64N4O2]2+/2: 352.2509, found: 352.2514.
8.1.20 P. falciparum Growth Inhibition Assay
The assay on P. falciparum growth inhibition for compounds EMA357, EMA359, EMA366, EMA368, EMA377, PRC-5, PRC-8, PRC-14, PRC-15, PRC-20, PRC-25, PRC-28, PRC-29, PRC31, PRC-39, PRC-42, and PRC-50 was performed following the same steps and conditions used in section 1.1.2 of EXAMPLE 1. The results obtained were then compared to the values previously obtained for AID-X-2020 (IC50=0.09 μM).
The results obtained indicated that all the compounds exhibit good antimalarial activity, especially EMA357, EMA359, EMA377, PRC-8, PRC-14, PRC-15, PRC-25, PRC-39, PRC-42, and PRC-50, which have a potent antimalarial activity, similar to AID-X-2020. Particularly, PRC-25 shows the highest antimalarial activity among the tested compounds, with an IC50 of 47 nM (Table 4).
The aim of this experiment was to assess whether AID-X-2020 is active against chloroquine- and artemisinin-resistant P. falciparum strains, and thus to evaluate the potential of a new antimalarial resistant drug compared to existing antimalarials.
9.1.1 P. falciparum Growth Inhibition Assays
P. falciparum parasites of chloroquine-sensitive (3D7 strain) and chloroquine- and artemisinin-resistant strains were processed as described in section 1.1.3 of EXAMPLE 1.
In particular, the P. falciparum resistant strains were the following: (1) strain W2 MRA-157 (BEI Resources, managed by ATCC) which is chloroquine-resistant; (2) strains 3D7 harboring the K13 mutations associated to artemisinin resistance, either M5791 or R561H (both provided by Dr. David A. Fidock and referenced in Stokes, B. H. et al., 2021); (3) Cam 3.11 strain which is chloroquine and sulfadoxine/pyrimethamine-resistant; and (4) Cam 3.11 strain modified to carry K13 mutations associated to artemisinin-resistant strains, either R561H or R539T, therefore being multiresistant strains. All Cam 3.11 strains (3) and (4) were provided by Dr. David A. Fidock and are referenced in Straimer, J. et al., 2015 and Stokes, B. H. et al., 2021.
Then, the required amount of AID-X-2020 was added to each well at different concentrations (0.0001, 0.001, 0.01, 1 and 10 μM) and triplicates.
The effect of AID-X-2020 of chloroquine- and artemisinin-resistant strains was compared with the activity of AID-X-2020 of P. falciparum parasites of chloroquine-sensitive 3D7 strain.
Results confirm that AID-X-2020 was also strongly active against the chloroquine-resistant W2 strain (IC50 of 90±1 nM), and several artemisinin-resistant strains with IC50 values ranging from 90 to 160 nM, in comparison to parental 3D7 and Cam3.II strains (
Results suggest that resistance is slowed down significantly for AID-X-2020, a molecule belonging to a chemical family where no antimalarials have been described so far. Thus, the sensitivity to AID-X-2020 of the chloroquine-resistant W2 strain indicates that the antimalarial mode of action of AID-X-2020 is not related to that of chloroquine. Similarly, data showing a sensitivity of artemisinin-resistant strains to AID-X-2020 similar to that of the corresponding parental non-resistant lines, strongly suggest that the antimalarial modes of action of both drugs are not related. Indeed, an antagonistic action of artemisinin and AID-X-2020 was observed in P. falciparum cultures, indicating that their main effects on the pathogen are opposed (EXAMPLE 5). Therefore, in this scenario, it can be concluded that AID-X-2020 is a potential new antimalarial resistant drug compared to the existing antimalarials.
EXAMPLE 3 confirmed that AID-X-2020 is a strong inhibitor of the aggregation of Aβ40 in formation, suggesting that inhibition of protein aggregation was the responsible mechanism for the antimalarial activity of this compound. In the present example, inventors further tested the possibility of disaggregation of already formed aggregative peptides.
To test the effect of AID-X-2020 on already formed amyloid fibrils, Aβ40 DMSO solutions were diluted to 25 μM in PBS and incubated as above in order to allow fibril formation. Then, AID-X-2020 was added at different concentrations (0.1 μM AID-X-2020, 1 μM AID-X-2020 and 10 μM AID-X2020) and the mixture was incubated in the same conditions for another 24 h. The final samples always contained less than 5% DMSO to avoid interference of this solvent on Aβ40 amyloid fibril formation. Finally, ThT treatment and TEM analysis was performed as described in section 3.1 of EXAMPLE 3. The analysis of aggregation inhibition and disaggregation performed with aggregative peptides present in P. falciparum proteins were conducted in the same way.
Aggregation inhibition and disaggregation activities were also tested with six aggregative peptides present in P. falciparum proteins with SEQ ID NO: 1-6 (NVNIYN, LYWIYY, NFNNIYH, NNFYYNN, LISFIL, LQSNIG) in order to determine the effects on AID-X-2020 directly on P. falciparum peptides. AID-X-2020 was added in this case at 0.1 μM and 1 μM.
AID-X-2020 at concentrations >90 nM was also found to disassemble preformed Aβ40 fibrils (
Results confirm the in vitro activity of AID-X-2020 as inhibitor of the aggregation of a model amyloidogenic peptide like Aβ40 and of aggregative peptides present in P. falciparum proteins, as already demonstrated in EXAMPLES 3, 4 and 5. The present example further demonstrates the disaggregating activity of AID-X-2020, beyond its capacity to inhibit protein aggregation.
For AID-X-2020 staining, a P. falciparum 3D7 culture was incubated in RPMIc for 30 min at 37° C. with 4.5 μM of the compound and 4 μg/ml of Hoechst 33342.
For colocalization studies, 0.5 μM of ER Tracker™ Green (BODIPY™ FL Glibenclamide, Thermo Fisher Scientific) was included in the solution. Cells were placed in an 8-well LabTek™ II chamber slide system (Thermo Fisher Scientific), rinsed with warm PBS and diluted 1:20 for their observation in a Leica TCS SP5 confocal microscope (Leica Camera, Mannheim, Germany) equipped with a 63× objective of 1.4 NA. Hoechst 33342 was excited with a diode laser at 405 nm, ER Tracker Green with the 488 nm line of an argon laser, and AID-X-2020 with a diode-pumped solid-state laser at 561 nm. The corresponding fluorescence emissions were collected in the ranges of, respectively, 415-460, 490-590, and 600-700 nm.
To avoid crosstalk between the different fluorescence signals, sequential line scanning was performed. To quantify Manders' overlap coefficient, images were analyzed using the Just Another Colocalization Plugin (JACoP) in the Fiji software. To avoid fixation artifacts, all the fluorescence microscopy data presented in this work were obtained with live cells.
A 0.5% parasitemia RBC culture was prepared for CLEM by allowing its binding to concanavalin. Briefly, a μ-Dish 35 mm, High, Grid-500 (ibidi GmbH, Gräfelfing, Germany) was coated for 20 min at 37° C. with a 50 mg/ml concanavalin A solution in ddH2O and wells were rinsed with pre-warmed PBS before parasite seeding. P. falciparum-infected RBCs washed twice with PBS were deposited into the dish and incubated for 10 min at 37° C.; afterwards, unbound RBCs were washed away with three PBS rinses. Seeded RBCs were then incubated with 3 μM AID-X-2020, and nuclei were counterstained with 2 μg/ml Hoechst 33342. The preparation was observed with a Zeiss LSM880 confocal microscope (Carl Zeiss, Jena, Germany), with respective λex/em for AID-X-2020 and Hoechst 33342 of 405/415-520 nm and 561/565-600 nm. Images were obtained from areas corresponding to a specific coordinate of the dish-grid by tile scans that were stitched into larger mosaics. A bright field image facilitated the recognition of the grid coordinates from the plate where the cells selected for CLEM were located.
After confocal image acquisition, cells were washed three times with TEM fixation buffer (2% paraformaldehyde and 2.5% glutaraldehyde in PBS) for 5 min each. Then, the fixation buffer was changed to 1% osmium tetroxide and 0.8% potassium ferricyanide in fixation buffer and incubated at 4° C. for 45 min, followed by three 5-min washes with ddH2O. Then, a dehydration procedure was performed by gradually increasing ethanol concentration: 50% (10 min), 70% (10 min), 80% (10 min), 90% (5 min, 3×), 96% (5 min, 3×), and 100% (5 min, 3×). At this point, the plastic part of the dish was carefully separated from the crystal part containing the samples, which was embedded in Spurr resin by successive incubations with different proportions of resin/ethanol, starting with 1/3 for 1 h, 1/1 for 1 h, 3/1 for 1 h and 1/0 overnight. After the embedding procedure, a BEEM® capsule containing polymerized Spurr resin was filled with a small volume of liquid resin in order to obtain an interphase in which the dish was placed. The BEEM® capsule was incubated at 70° C. for 72 h, and the crystal part of the dish was removed by alternatively immersing samples in liquid nitrogen and boiling water. When the crystal was broken, cells remained attached to the resin, which was further cut in a microtome with a diamond blazer in order to obtain 100 nm-thick resin slides, which were mounted on a carbon-coated copper grid and negatively stained with 2% uranyl acetate for 2 min and washed with ddH2O for 1 min. Samples were observed in a JEM 1010 transmission electron microscope. Images were processed for CLEM analysis using the CORRELIA plug-in in the Fiji software (version 2.0.0-pre-8).
Confocal fluorescence microscopy colocalization analysis (
The cytosolic localization of the protein aggregation inhibitor AID-X-2020 in Plasmodium rough ER regions, where proteins are being synthesized, is consistent with the likely role of this drug in disrupting a yet to be described parasite's aggresome.
The aim of this experiment was to directly prove the level of protein aggregation in live Plasmodium cells following AID-X-2020 treatment.
12.1.1 Quantitative Analysis of Protein Aggregation in Live P. falciparum Cultures
To directly probe the level of protein aggregation in live Plasmodium cells following AID-X-2020 treatment, a ThT-based method to measure protein aggregation in parasite cultures was developed.
P. falciparum cultures enriched in early stages were treated with the IC10 (27 nM) and IC50 (90 nM) of AID-X-2020 or left untreated. After 90 min, 4 h and 30 h, a Percoll purification was done in order to isolate parasitized cells from uninfected RBCs. After Percoll purification, the pellets of late stage parasites and a control non-infected RBC suspension containing the same proportion of cells than the purified cultures were resuspended in 50 μl of lysis buffer (4.5 mg/ml NaCl in water supplemented with EDTA-free protease inhibitor cocktail, PIC, Hoffman-La Roche, Basel, Switzerland; 1 PIC tablet/10 ml water) and incubated overnight, at 4° C. under stirring, with the objective of releasing their inner content. After this time, lysed samples were spun down and the protein content in the supernatant was quantified with the bicinchoninic acid assay (Thermo Fisher Scientific), following the manufacturer's instructions. 30 μg of protein from each supernatant were further diluted with PBS to a final volume of 70 μl and plated on a 96-well black plate in triplicates. ThT fluorescence was measured as described above.
ThT fluorescence of culture extracts, normalized to have equal protein content, exhibited a reduced emission spectrum in samples that had been treated for only 90 min with 90 nM AID-X-2020, the compound's in vitro IC50 (
These results indicating a relevant decrease in aggregated protein load in live parasites following AID-X-2020 treatment at physiologically relevant concentrations are supportive of a mode of action of this drug consisting in the inhibition of protein aggregation in the pathogen.
EXAMPLE 6 studied the inhibition of the gametocyte to ookinete transition in order to determine whether AID-X-2020 was able to arrest the life cycle of malaria parasites in the mosquito host.
Contrarily, the objective of the present study was to determine if AID-X-2020 is capable of targeting gametocytes, the sexual phase of the malaria parasites' life cycle and the sole stage of malaria parasites present in the blood circulation capable of transmitting the infection to the mosquito vector, and where drugs active at this critical step are scarce.
Cultures of the P. falciparum NF54-gexp02-Tom strain (developed and authenticated by Portugaliza, H. P. et al., 2019 and kindly provided by Prof. Alfred Cortes), were maintained in standard conditions in RPMI medium supplemented with 0.5% Albumax II and 2 mM choline, synchronized in ring stages with sorbitol lysis, and diluted to 2% parasitemia. To trigger sexual conversion, choline was removed from the medium and cultures were maintained in the same conditions for 48 h after synchronization (cycle 0). In the next cycle (cycle 1), parasites were treated with 50 mM N-acetylglucosamine (GlcNac) in order to kill asexual parasites, and maintained in RPMI supplemented with 10% human serum. Medium was refreshed daily and GlcNac was kept during 4 days.
To determine the effect of AID-X-2020 and DONE3TCI in early gametocytes, the culture was distributed in triplicates (200 μl/well, 96-well plates) and drugs were added in cycle 1 and maintained for 48 h in the culture. Controls of untreated parasites as well as of parasites treated with a lethal dose of chloroquine were prepared. Gametocytemia was monitored daily by light microscopy until the majority of parasites (˜90%) could be identified as stage V gametocytes. At that point, Giemsa smears of each well were prepared and mature gametocytes were manually counted (10,000 cells were counted for each replica by two investigators blinded to group assignment). To test the effect of AID-X-2020 and DONE3TCI on mature gametocytes, cultures were grown for 14 days, when the majority of the parasites could be identified as stage V gametocytes. Afterwards, the culture was treated for 48 h with the drugs and the gametocytemia determined as above.
AID-X-2020 efficiently blocked the development of P. falciparum early and mature stage V gametocytes in vitro with respective IC50 of 95±3 nM and 103±3 nM (
Therefore, besides its activity against Plasmodium asexual blood stages, AID-X-2020 displays marked activity against the sexual phase of the malaria parasites' life cycle, paving the way for the exploration of this drug as a potential multi-stage antiplasmodial therapy.
Clinical malaria samples were provided by the Centre de Salut Internacional i Malalties Transmissibles Drassanes-Vall d'Hebron. Five μL of blood from a malaria-infected person were diluted in 100 μL of RPMIc. The solution was stained with 2 μg/mL Hoechst 33342 and 18 μM AID-X-2020 and incubated for 30 min before centrifuging at 5000× g for 30 s and discarding the supernatant. The pellet was then resuspended in 100 μL of PBS and 5 μL of the suspension were placed on a 8 well slide (Ibidi) with 200 μL of PBS. The sample was observed in a IX-51 Olympus fluorescence microscope with a 100× objective and a SPRED filter (λex: 586/20 nm, λem: 628/32 nm).
Microscopic observation of AID-X-2020-stained clinical blood samples of a P. falciparum infection allowed the identification of the circulating ring forms of the parasite that are detected in malaria diagnosis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21382949.2 | Oct 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/079438 | 10/21/2022 | WO |
