Positive allosteric modulator (PAM) of the EAAT2 / GLT-1 glutamate transporter for use in the treatment or prevention of pruritus

Abstract

The positive allosteric modulator (PAM) of the EAAT2 / GLT-1 transporter for use in the treatment or prevention of pruritus is disclosed.

Description

[0001] Positive allosteric modulator (PAM) of the EAAT2 / GLT-1 glutamate transporter for use in the treatment or prevention of pruritus

[0002] The invention discloses chemical compounds that are structurally modified derivatives of a-D-alanine, which act effectively in murine models of both histamine-dependent and histamine-independent pruritus. These substances, in terms of their chemical structure and mechanism of action, are different compared to the drugs currently used in the treatment of pruritus. For this reason, they constitutea new chemical and pharmacological class of substances intended potentially for the treatment of pruritus of various etiologies, and the results obtained suggest their potential effectiveness in indications that are currently a therapeutic problem. These derivatives may be useful in monotherapy or in combination therapy, especially for the treatment of allergic pruritus (including those associated with insect bites) and pruritus occurring in the case of renal failure (so-called uraemic pruritus), hyperbilirubinaemia, hypercalcaemia, iron deficiency or overload, lymphomas, granulomas and solid tumors, diabetes, amyloidosis, neurological diseases (e.g. multiple sclerosis, stroke), infectious diseases (e.g. shingles, viral hepatitis, HIV), autoimmune diseases (e.g. atopic dermatitis, psoriasis), many other rare clinical cases, e.g. chikungunya fever, as well as pruritus resulting from the use of drugs such as opioid analgesics, anti-EGFR monoclonal antibodies, tyrosine kinase inhibitors, oral contraceptives, etc.

[0003] State of the art

[0004] Pruritus is an unpleasant feeling that triggers the need to scratch and is a common health problem that negatively affects the quality of life of the affected person (Yosipovitch, G. et al. N. Engl. J. Med. 2013, 368, 1625–1634). Cross-sectional studies conducted in Oslo, Norway (2000-2001) involving a total of 40,888 adults for the first time provided data on the incidence of pruritus (Dalgard, F. et al. Br. J. Dermatol. 2004, 151, 452-457), which amounted to 8.4%, and pruritus was the most frequently reported of all skin symptoms. Women had a higher incidence of pruritus than men (11.9% and 9.6%, respectively). In addition, pruritus was more prevalent among people with lower socioeconomic status and lower household incomes. In another study involving 18,137 French people with skin problems and diseases, 42% said they experienced pruritus. However, it was not determined whether the pruritus was acute or chronic (Wolkenstein, P. et al. Arch. Dermatol. 2003, 139, 1614–1619). A German pilot study of chronic pruritus in a sample of the general population (n = 200) showed a prevalence of 13.9%. In this study, 16.5% experienced chronic pruritus in the last 12 months and the lifetime incidence of chronic pruritus was 22.6% (Matterne, U. et al. Acta Derm. Venereol. 2009, 89, 250–256). In addition, pruritus is particularly prevalent in the elderly population. Research shows that chronic pruritus is present in approximately 22% of people over 65 years of age, making it a significant clinical problem in this age group. In addition, pruritus can be associated with many chronic diseases, such as chronic kidney failure, liver disease, diabetes, as well as various dermatological conditions. Due to its widespread prevalence and significant impact on patients' quality of life, pruritus is considered a significant medical problem requiring effective treatment (Weisshaar, E.; Dalgard, F. Acta Derm. Venereol. 2009, 89, 339–350). Another serious clinical problem is the occurrence of chronic pruritus of unknown origin (CPUO), which in the general pruritus population accounts for 3.6% to 44.5%, and the highest incidence is observed among the elderly. What is extremely important, the treatment of CPUO is particularly difficult and ineffective due to unknown pathophysiological mechanisms (Andrade, A. et al. Cochrane Database Syst. Rev. 2020, 25, CD013128).

[0005] The mechanism of pruritus is multifactorial and complex. There are many exogenous and endogenous substances (pruritogens) that cause pruritus by directly affecting the peripheral sensory endings responsible for the formation of pruritic stimuli. A well-known pruritogen is histamine, which activates peripheral histamine H1receptors and thereby induces pruritus. Such a mechanism is involved in the development of pruritus accompanying contact allergy, urticaria, or insect bites (Estelle, F. et al. J. Allergy Clin. Immunol. 2011, 128, 1139–1150). It is effectively combated by the use of antihistamines, i.e. inverse agonists of the Hl receptor(Church, M. K.; Maurer, M. Exp. Dermatol. 2015, 24, 332–333). A much larger problem is pruritus, in which histamine does not play a key role, the so-called histamine-independent pruritus. It occurs in the case of renal failure (so-called uraemic pruritus), hyperbilirubinaemia, hypercalcaemia, iron deficiency or overload, lymphomas, granulomas and solid tumors, diabetes, amyloidosis, neurological diseases (e.g. multiple sclerosis, stroke), infectious diseases (e.g. shingles, viral hepatitis, HIV), autoimmune diseases (e.g. atopic dermatitis, psoriasis) and many other rare clinical cases, e.g. chikungunya fever (Cunha, B. A. et al. IDCases 2016, 6, 29–30, Lavery, MJ. et al. Ulster Med. J. 2016, 85, 164–173). It may also be the result of the use of drugs such as, among others opioid analgesics, anti-EGFR monoclonal antibodies, tyrosine kinase inhibitors, oral contraceptives (Butler, D. C. et al. JAMA 2024, 331, 2114–2124). In these cases, antihistamines show little or no efficacy (Stander, S. et al. Exp. Dermatol. 2008, 17, 161–169, Twycross, R. et al. QJM-An. Int. J. Med. 2003, 96, 7–26). Despite continuous attempts to treat histamine-independent pruritus with drugs such as pregabalin, gabapentin, aprepitant, or antidepressants, many patients are deprived of effective and safe therapy (Pereira, M. P. et al. Curr. Opin. Pharmacol. 2019, 46, 1–6, Tey, H. L.; Yosipovitch, G. Br. J. Dermatol. 2011, 165, 5–17). For example, the only effective method of permanent control of uremic pruritus is kidney transplantation (Millington, G. W. M. et al. Br. J. Dermatol. 2018, 178, 34–60, Tey, H. L.; Yosipovitch, G. Br. J. Dermatol. 2011, 165, 5–17). Therefore, new pharmacological tools are needed that are effective in combating this symptom, which significantly worsens quality of life, namely pruritus. Glutamic acid and neuropeptides such as gastrin-releasing peptide (GRP) and natriuretic peptide B (NPPB) are key neurotransmitters in the transmission of pruritus from peripheral tissues to the central nervous system where it is felt and perceived. Recent research has shown that among these neurotransmitters, it is glutamate, and not GRP, as previously thought, that is the main transmitter at the first synapse, in the ascending pruritusconducting pathways that form between the C-fibers and GRP-positive neurons of the superficial part of the dorsal horn. GRP is probably a neuromodulator for the basic neurotransmitter, i.e. glutamic acid (Chen, Q. Y., Zhuo, M. Mol. Pain 2023, 19, 17448069231152101). Limiting glutamatergic transmission in the posterior horns of the spinal cord and in the brain may hypothetically result in an antipruritic effect in all disease entities accompanied by this symptom and regardless of its cause. This is because glutamate is responsible for the transmission of pruritic impulses in the spinal cord, irrespective of the pruritogen that induces the pruritus. However, it is necessary to use a pharmacological mechanism that will not be associated with unacceptable side effects related to blocking the action of glutamate, such as memory and learning impairments or hallucinations. Such symptoms are typical, for example, for NMDA receptor antagonists (glutamic acid ionotropic receptor). Positive allosteric modulation (PAM) of the glutamate transporter (EAAT2) appears to be the optimal mechanism. Enhancement of the activity of this protein, which transports glutamic acid from the synaptic cleft into the cell (mainly astrocytes), reduces the synaptic concentration of the neurotransmitter and limits the stimulation of postsynaptic receptors, thereby inhibiting the entire neurotransmission process in the neural pathways that conduct pruritic signals. (Pajarillo, E. et. al. Neuropharmacol. 2019, 161, 107559). However, it should be emphasized that this hypothesis has not yet been confirmed experimentally for compounds with this mechanism of action, nor is it suggested by the specialized literature, therefore any positive data obtained in pruritus models are not obvious in relation to the current state of knowledge.

[0006] Technical Problem

[0007] In view of the above, the technical problem addressed by the present invention is to provide compounds that act effectively in models of both histamine-dependent and histamine-independent pruritus in mice. Substances with such a biological profile can act effectively in pruritus of varying etiology, which may determine their clinical advantage over currently used antipruritic agents.

[0008] Summary of Invention

[0009] Surprisingly, the aforementioned problem has been solved by the present invention. The invention therefore comprises a group of compounds described by the general formula (I), which are, with regard to their structure, derivatives of pyrrolidine-2, 5-dione modified with a fragment of substituted a-D-alanine, which act as positive allosteric modulators (PAM) of the glutamate transporter EAAT2 in humans and the corresponding GLT-1 transporter in rodents. These compounds, in line with the solved technical problem, exhibit activity at low doses in murine models of both histamine-dependent and histamine-independent pruritus following intraperitoneal (i.p.) administration, thereby representing potential candidates for the treatment of various forms of pruritus. It should be emphasized that this is a completely non-obvious effect, because the disclosed compounds have a different structure and have a different mechanism of action compared to the preparations currently in clinical use. In addition, it has not yet been experimentally proven, nor is it suggested in the specialized literature, that compounds or substances that are PAMs of the EAAT2 / GLT-1 transporter may be effective in relieving histamine-dependent and histamine-independent pruritus.

[0010] The subject of the invention is a positive allosteric modulator (PAM) of the EAAT2 / GLT-1 transporter for use in the treatment or prevention of pruritus.

[0011] A further object of the invention is a compound selected from pyrrolidine-2, 5-dione derivatives modified with a fragment of substituted a-D-alanine represented by the general formula (I):

[0012]

[0013] B

[0014] Formula I

[0015] wherein:

[0016] A is hydrogen or deuterium;

[0017] B is hydrogen or deuterium;

[0018] X is hydrogen or deuterium or fluorine;

[0019] Y is hydrogen or deuterium;

[0020] for use in the treatment or prevention of pruritus, especially histamine-dependent and histamine-independent pruritus.

[0021] Preferably:

[0022] A is hydrogen or deuterium, wherein at least one A is deuterium, particularly preferably each A is deuterium;

[0023] B is hydrogen;

[0024] X is hydrogen or fluorine, preferably hydrogen;

[0025] Y is hydrogen or deuterium, preferably hydrogen.

[0026] Particularly preferably, the compound for use according to the invention was selected from the group comprising / V-benzyl-2-(2,5-dioxopyrrolidin-l-yl)propanamide derivatives of the R-configuration of the stereogenic center located at C-2 in the general formula (I):

[0027] (R)-N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide (compound (R)-l, wherein A=H, B=H, X=H, Y=H),

[0028] (R)-N-benzyl-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)propanamide (compound d4-(R)-1, wherein A=D, B=H, X=H, Y=H),

[0029] (R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)-N-(phenylmethyl-d2)propanamide (compound d6-(R)-1, wherein A=D, B=H, X=H, Y=D),

[0030] (R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)-N-((phenyl-d5)methyl)propanamide (compound d9-(R)-1, wherein A=D, B=D, X=D, Y=H),

[0031] (R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)-N-((phenyl-d5)methyl-d2)propanamide (compound d11-(R)-1: A=D, B=D, X=D, Y=D), (R)-2-(2,5-dioxopyrrolidin-1-yl)-N-(2-fluorobenzyl)propanamide (compound (R)-2, wherein A=H, B=H, X=F, Y=H),

[0032] (R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)-N-(2-fluorobenzyl)propanamide (compound d4-(R)-2, wherein A=D, B=H, X=F, Y=H),

[0033] (R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)-N-((2-fluorophenyl)methyl-d2)propanamide (compound d6-(R)-2, wherein A=D, B=H, X=F, Y=D),

[0034] Preferably, the compound as defined above is intended for use in the prevention or treatment of pruritus of any origin, preferably allergic pruritus (including those associated with insect bites) and pruritus occurring in the case ofrenal failure (so-called uraemic pruritus), hyperbilirubinaemia, hypercalcaemia, iron deficiency or overload, lymphomas, granulomas and solid tumors, diabetes, amyloidosis, neurological diseases (e.g. multiple sclerosis, stroke), infectious diseases (e.g. shingles, viral hepatitis, HIV), autoimmune diseases (e.g. atopic dermatitis, psoriasis), many other rare clinical cases, e.g. chikungunya fever, as well as pruritus resulting from the use of drugs such as, amongst others, opioid analgesics, anti-EGFR monoclonal antibodies, tyrosine kinase inhibitors, oral contraceptives.

[0035] In studies conducted in mice after intraperitoneal (i.p.) administration, exemplary and representative compounds for the whole series: (R)-l, (R)-2 and deuterated analog d6-(R)-2 inhibited pruritic reactions induced by subcutaneous administration of histamine (histaminedependent pruritus), as well as chloroquine or SLIGRL-NH2(histamine-independent pruritus).

[0036] For a better explain of their activity, the invention has been illustrated by figures. Figure 1 shows the antipruritic activity of compound (R)-l at 30 and 60 mg / kg, compound WAY 213613 at 6 mg / kg, and a combination of (R)-l at 60 mg / kg with WAY 213613 at 6 mg / kg in a histamine-induced pruritus model, as well as pyrilamine, a model antihistamine drug, at a dose of 10 mg / kgThe mice were observed for 45 minutes. In the control group, 1% Tween 80 solution was injected. Statistical calculations: one-way analysis of ANOVA variance: * p<0.05, ** p<0.01, **** p<0.0001 and Student's t-test for independent samples: ## p<0.01.

[0037] Figure 2 shows the antipruritic activity of compound (R)-l at 60 and 90 mg / kg, WAY 213613 compound at 6 mg / kg, and a combination of (R)-l at 60 mg / kg with WAY 213613 at 6 mg / kg and pyrilamine, a model antihistamine drug, at 10 mg / kg in a pruritus model induced by chloroquine injection. The mice were observed for 30 minutes. The table presents the average results (± SD) of the number of pruritic reactions for groups of 8-10 animals. In the control group, 1% Tween 80 solution was injected. Statistical analysis: one-way analysis of ANOVA variance: ** p<0.01, *** p<0.001 and Student's t-test for independent samples: ### p<0.001.

[0038] Figure 3 shows the antipruritic activity of compound (R)-l at 60 and 90 mg / kg, WAY 213613 compound at 6 mg / kg, and a combination of (R)-l at 60 mg / kg with WAY 213613 at 6 mg / kg and pyrilamine, a model antihistamine drug, at 10 mg / kg in a pruritus model induced by SLIGRL-NH2injection. The mice were observed for 30 minutes. The average results (± SD) of the number of pruritic reactions have been presented. In the control group, 1% Tween 80 solution was injected. Statistical analysis: one-way analysis of ANOVA variance: * p<0.05, ** p<0.01 and Student's t-test for independent samples: # p<0.05.

[0039] Figure 4 shows the antipruritic activity of compound (R)-2 at 30 and 60 mg / kg in a histamine-induced pruritus model. The mice were observed for 45 minutes. In the control group, 1% Tween 80 solution was injected. Statistical calculations: one-way analysis of ANOVA variance: * p<0.05, ** p<0.01.

[0040] Figure 5 shows the antipruritic activity of (R)-2 at 30 and 60 mg / kg in a pruritus model induced by chloroquine injection. The mice were observed for 45 minutes. In the control group, 1% Tween 80 solution was injected. Statistical calculations: one-way analysis of ANOVA variance: ** p<0.01.

[0041] Figure 6 shows the antipruritic activity of d6-(R)-2 compound at 15, 30 and 60 mg / kg in a histamine-induced pruritus model. The mice were observed for 45 minutes. In the control group, 1% Tween 80 solution was injected. Statistical calculations: one-way analysis of ANOVA variance: ** p<0.01.

[0042] Figure 7 shows the antipruritic activity of d6-(R)-2 compound at 30 and 60 mg / kg in a pruritus model induced by chloroquine injection. The mice were observed for 45 minutes. In the control group, 1% Tween 80 solution was injected. Statistical calculations: one-way analysis of ANOVA variance: * p<0.05.

[0043] Detailed description of the invention

[0044] The invention proposes the use of a positive allosteric modulator (PAM) of the EAAT2 / GLT-1 transporter in the treatment or prevention of pruritus.

[0045] In an exemplary embodiment, the present invention discloses a group of pyrrolidine-2, 5-dione derivatives modified with a / V-benzyl-substituted a-D-alanine fragment, being, in terms of the mechanism of action, the PAMs of the EAAT2 / GLT-1 transporter. In vitro studies performed using the COS-7 cell line overexpressing the EAAT2 transporter showed that the selected compounds, encompassing all structural variants according to Formula (I), markedly enhanced glutamate uptake in the examined experimental system.. The EC50values (i.e. half of the maximum effective concentration required to achieve a 50% effect after a specified exposure time) were 0.05-62.4 nM, while the glutamate uptake efficiency was in the range of 116.0-152.5%. These values for most of the compounds described by Formula (I) are shown in Table 1.

[0046] Table 1. EC50parameters and glutamate uptake efficiency for selected PAMs of the EAAT2 transporter described by Formula (I).

[0047] Compound Glutamate uptake parameters mediated by EAAT2 StructureaNumber EC50[nM]bEfficiency [%]c

[0048] (R)-1 0.28 ± 0.1 140 ± 8.0

[0049] d4-(R)-1 0.82 ± 0.8 116 ± 3.4

[0050] O D D

[0051] 1.0 ± 2.8 116 ± 4.4

[0052] <^O

[0053] O D

[0054] d9-(R)-1 4.5 ± 4.2 134.3 ± 1.6

[0055] HVr

[0056] 0 1 11 A0

[0057] D'^ Y^'D

[0058] D O D D D

[0059] d11-(R)-1 0.1 ± 0.04 137.0 ± 4.8

[0060] O 1H11 J

[0061] D'' YXO

[0062] D

[0063] (R)-2 4.5 ± 2.1 149.7 ± 7.1

[0064] ZY-o

[0065] d4-(R)-2 0.05 ± 0.03 118.0 ± 5.0

[0066] D

[0067] 0 1 H

[0068]

[0069]

[0070] aThe studies were conducted on the COS-7 cell line transfected with EAAT2. The results were normalized and described as % control (EAAT2). The data show the mean ± S. E. M. from 3-6 independent experiments performed in triplicate;bHalf of the maximum effective concentration required to achieve a 50% effect;cGlutamate uptake efficacy expressed as % control (EAAT2). Unidirectional analysis of variance (ANOVA) with Dunnett's post-hoc multiple comparison test was used to analyze differences in glutamate uptake at different concentrations of test compounds compared to control.

[0071] Studies conducted in mice after i.p. administration showed that an exemplary, representative compound (R)-l inhibited pruritic reactions induced by subcutaneous administration of histamine (histamine-dependent pruritus) and administration of chloroquine or SLIGRL-NH2(histamine-independent pruritus). The compounds (R)-2 (a fluorinated analogue) and its deuterated derivative d6-(R)-2 also exhibited pronounced antipruritic activity, as they statistically significantly inhibited the pruritic response induced in mice by administration of histamine or chloroquine.. It is worth emphasizing that the described therapeutic effect was not accompanied by undesirable side effects such as the occurrence of sedation or motor coordination disorders (these results for compound (R)-l are described in Abram, M. et al. J. Med. Chem. 2022, 65, 11703-11725 and Kaminski, K. et al. Ann. Neurol. 2025, 97, 344-357). This confirms that the compound of the invention may be a preferred alternative to current therapies or may complement them, particularly in clinical cases where the efficacy of currently available drugs is insufficient.

[0072] In the conducted research, the antipruritic effect of compounds (R)-l, (R)-2 and d6-(R)-2 was determined. In addition, the activity of WAY 213613, which is an EAAT2 / GLT-1 inhibitor, and thus works in the opposite way to (R)-l, (R)-2 and d6-(R)-2, was determined. The effect of simultaneous administration of the model compound (R)-l and WAY 213613 was also tested to clearly confirm that the previously demonstrated mechanism (in vitro), i.e. positive allosteric modulation of the glutamate transporter ( enhancement of glutamate uptake), is responsible for the observed antipruritic effect in vivo. Such a relationship has not yet been demonstrated for any chemical compound or antipruritic drug. In addition, the activity of (R)-1 compound was compared with the reference compound, i.e. pyrilamine, which is a model representative of the entire group of antihistamines, i.e. drugs currently most often used in the symptomatic treatment of pruritus. First, compound (R)-l was studied in a mouse model of histamine-induced pruritus. A model of pruritus induced by subcutaneous injection of histamine in the amount of 800 μg / 20 μL (single dose) near the neck of the mouse was used.

[0073] For the control group, the number of pruritic reactions was 118.30 ± 58.99 (Fig. 1).

[0074] Administration of (R)-l at a dose of 30 mg / kg resulted in a decrease in the response to 56.53 ± 40.23, which indicates a statistically significant inhibition of the pruritic reaction (p<0.05) by 52.13%. (R)-l at a dose of 60 mg / kg also resulted in a statistically significant inhibition of the pruritic reaction (p<0.01) by 64.71%, the response was 41.75 ± 32.17. After administration of WAY 213613 at a dose of 6 mg / kg, the tested parameter was 111.20 ± 38.15%, the pruritic reaction was inhibited by 6.0%, however, this effect was not statistically significant.. Administration of the tested compound at a dose of 60 mg / kg together with the WAY 213613 at a dose of 6 mg / kg resulted in a statistically significant (p<0.01) attenuation of the antipruritic effect of (R)-l. The pruritic reaction was 94.80 ± 39.72, which, compared to the control group, indicates an inhibition of the pruritic reaction by only 19.86% (vs. 64.71% for (R)-l alone). Such results clearly indicate that (R)-l has an antipruritic effect in the histamine pruritus model (an experimental model of pruritus accompanying e.g. allergies or insect bites), and this effect results from the enhancement of the EAAT2 / GLT-1 transporter function. In this model, a statistically significant (p<0.0001) activity was also demonstrated by pyrilamine, the administration of which at a dose of 10 mg / kg resulted in a reduction of the pruritic reaction to 22.56 ± 9.15. This is equivalent to an 80.92% reduction in pruritic reaction.

[0075] Subsequently, (R)-l was studied in models of histamine-independent pruritus, which may be regarded as models of pruritus resistant to the most commonly used treatment, i.e., antihistamines. The first of these models was a model of pruritus induced by subcutaneous injection of chloroquine at a dose of 200 μg / 20 μL. For the control group, the number of pruritic reactions was 83.40 ± 29.83 (Fig. 2). Administration of (R)-l at a dose of 60 mg / kg resulted in a statistically significant inhibition of the pruritic reaction (p<0.01) by 58.03% relative to baseline, the response was reduced to 35.00 ± 21.09. (R)-l at a dose of 90 mg / kg also caused a statistically significant inhibition of the pruritic reaction (p<0.001) by 68.15%, the response was 26.56 ± 22.83. After administration of WAY 213613 at a dose of 6 mg / kg, the tested parameter was 81.13 ± 21.72%, the pruritic reaction was inhibited by 2.72%, however, this effect was not statistically significant.. Administration of pyrilamine at a dose of 10 mg / kg resulted in a reduction of the pruritic response to a value of 52.38 ± 33.76, corresponding to a 37.20% antipruritic effect. However, this effect was not statistically significant (p>0.5).. This indicates that classic antihistamines, unlike (R)-l, are not effective or have little efficacy in relieving chloroquine-induced pruritus. Another model of histamine-independent pruritus used was SLIGRL-NHz-induced pruritus. In this model, pruritus was induced by subcutaneous injection of SLIGRL-NH2 solution at a dose of 100 pg / 20 pL. For the control group, the number of pruritic reactions was 80.63 ± 29.19 (Fig. 3). Administration of (R)-l at a dose of 60 mg / kg resulted in a statistically significant (p<0.05) decrease in the pruritic response to 37.13 ± 29.24, the pruritic reaction was inhibited by 53.90% in a statistically significant manner. (R)-l at a dose of 90 mg / kg also caused a statistically significant inhibition of the pruritic reaction (p<0.01) by 72.40%, the response was 22.25 ± 13.49. After administration of WAY 213613 at a dose of 6 mg / kg, the measured parameter was 74.50 ± 39.84%, and the pruritic reaction was inhibited by 7.60%; however, this effect was not statistically significant (p>0.05). Administration of (R)-l at a dose of 60 mg / kg together with WAY 213613 at a dose of 6 mg / kg, compared to the control group, in a statistically insignificant manner, reduced the pruritic reaction by 4.03%, the response was 77.38 ± 43.36 (vs. 53.90% for (R)-l alone). Administration of pyrilamine at a dose of 10 mg / kg resulted in a reduction of the pruritic reaction to 65.25 ± 23.24. This is equivalent to 19.10% of the antipruritic effect, however this effect was not statistically significant (p>0.05). Again, the results obtained indicate that classical antihistamines, unlike (R)-l, are not effective in relieving SLIGRL-NH2-induced pruritus.

[0076] In order to confirm the antipruritic activity, and at the same time to demonstrate the consistency of the biological effect for positive allosteric modulators of the EAAT2 / GLT-1 transporter, representing the remaining chemotypes (variants) described by the general formula (I), tests were performed for (R)-2 (where X = F) and its deuterated analogue - d6-(R)-2. These substances were tested in a representative model of histamine-induced pruritus and in a model of chloroquine-induced pruritus. Intradermal injection of histamine at a dose of 800 pg / 20pL to mice previously administered with a vehicle (1% Tween 80 solution), i.e. the control group, resulted in pruritic reactions amounting to 143.80 ± 17.06 (100% of the response) (100% of the responses) (Fig.4). In the groups of mice administered with compound (R)-2 at doses of 30 mg / kg and 60 mg / kg, the observed number of pruritic reactions was reduced to 80.63 ± 13.55 (p<0.05) and 71.63 ± 11.63 (p<0.01), respectively, which was 56.07% and 49.81% of the value observed for the control group. Administration of d6-(R)-2 at doses of 15 mg / kg, 30 mg / kg or 60 mg / kg resulted in a reduction of the pruritic reaction to 82.62 ± 8.89 (p<0.01), 81.25 ± 7.18 (p<0.01) and 124.60 ± 12.60, corresponding to57.46%, 56.50% and 86.64% of the control value, respectively (Fig. 6).

[0077] Intradermal injection of chloroquine at a dose of 200 μg / 20 μLto mice from the control group resulted in pruritic reactions in the amount of 143.80 ± 17.06. In the groups of mice administered with (R)-2 at doses of 30 mg / kg and 60 mg / kg, the observed number of pruritic reactions was reduced to 47.50 ± 6.73 (p<0.01) and 41.75 ± 9.26 (p<0.01), respectively, corresponding to54.44% and 47.85% of the value observed for the control group (Fig. 5).

[0078] Administration of compound d&-(R)-2 at a dose of 30 mg / kg resulted in a reduction of the pruritic reaction to 57.50 ± 7.94 (p<0.05), corresponding to 65.90% of the control valuee (Fig.

[0079] 7). Administration of d6-(R)-2 at a dose of 60 mg / kg resulted in a statistically insignificant increase in pruritic reaction to 104.0 ± 8.49, representing 119.19% of the control value.. For the deuterated derivative,, i.e. d6-(R)-2, it was observed in both pruritus models used that low doses are active (15 mg / kg and 30 mg / kg). However, antipruritic activity is not observed at a higher dose, i.e. 60 mg / kg.

[0080] The conducted studies clearly demonstratedthat (R)-l, (R)-2 and d6-(R)-2 compounds have a wide antipruritic activity. An extremely important observation is that, unlike the model drugpyrilamine, compounds (R)-l, (R)-2 and d6-(R)-2 act both in histamine-dependent and histamine-independent pruritus. This indicates their potentially very wide use in the treatment of pruritus, especially pruritus, which is resistant to commonly used antipruritic drugs. In addition, previous in vitro studies on (R)-l interactions with WAY 213613 (Kaminski, K. et. al. Ann. Neurol. 2025, 97, 344-357), as well as the data disclosed in the present application, confirm that the antipruritic effect of (R)-l depends on the enhancement of EAAT2 / GLT-1 activity (positive allosteric modulation). Such a mechanism of action has not yet been demonstrated for any antipruritic substance.

[0081] Examples

[0082] Chemical research

[0083] The synthesis of the compounds described by the general formula (I) is disclosed in the earlier Polish patent applications P.429656 and P.433567 and later publications by the inventors (Abram, M. et al. J. Med. Chem. 2022, 65, 11703-11725). Their physicochemical and spectral data are as follows: Compound (R)-l (wherein A=H, B=H, X=H, Y=H); (R)-N-benzyl-2-(2,5-dioxopyrrolidin-l-yl)propanamide

[0084] White solid. Melting point: 138.1-138.7°C; TLC: Rf= 0.39 (DCM: MeOH (9: 0.3; v / v)); C14H16N2O3 (260.29), Monoisotopic mass: 260.12. UPLC (100% purity): tR= 3.94 min. (M+H)+261.1. Chiral HPLC > 99% ee (tR= 22.649 min).XH NMR (500 MHz, CDCI3) 6 1.56 (d, J=7.5 Hz, 3H), 2.66 (s, 4H), 4.39 (d, J=5.7 Hz, 2H), 4.76 (q, J=7.3 Hz, 1H), 6.45 (br s, 1H), 7.22-7.26 (m, 3H), 7.30-7.32 (m, 2H).13C NMR (126 MHz, CDCI3) 6 14.5, 24.9, 25.6, 28.3, 33.7, 43.8, 49.8, 127.6, 127.7, 128.8, 137.9, 168.6, 177.0.

[0085] Compound dn-(R)-l (wherein A=D, B=H, X=H, Y=H); (R)-N-benzyl-2-(2,5-dioxopyrrolidin-l-yl-3,3,4,4-d4)propanamide

[0086] White solid. Melting point: 138.2-138.9°C; TLC: Rf= 0.39 (DCM: MeOH (9: 0.3; v / v)); C14H12D4N2O3 (264.32), Monoisotopic mass: 265.14. UPLC (100% purity): tR= 3.79 min. (M+H)+265.2. Chiral HPLC > 99% ee (tR= 24.566 min).TH NMR (500 MHz, CDCI3) 6 1.57 (d, J=7.2 Hz, 3H), 4.39 (d, J=5.7 Hz, 2H), 4.77 (q, J=7.2 Hz, 1H), 6.40 (br s, 1H), 7.22-7.26 (m, 3H), 7.29-7.32 (m, 2H).13C NMR (126 MHz, CDCI3) 614.5, 28.3, 33.7, 49.8, 127.7, 128.8, 137.9, 168.6, 177.0.

[0087] Compound de-(R)-l (wherein A=D, B=H, X=H, Y=D); (R)-2-(2,5-Dioxopyrrolidin-l-yl-3,3,4,4-d4)-N-(phenylmethyl-d2)propanamide

[0088] White solid. Melting point: 138.3-139.1°C; TLC: Rf= 0.39 (DCM: MeOH (9: 0.3; v / v)); C14H10D6N2O3 (266.33), Monoisotopic mass: 267.15. UPLC (100% purity): tR= 3.78 min (M+H)+267.2. Chiral HPLC > 99% ee (tR= 23.945 min).XH NMR (500 MHz, CDCI3) 6 1.58 (d, J=7.5 Hz, 3H), 4.78 (q, J=7.5 Hz, 1H), 6.35 (br s, 1H), 7.22-7.28 (m, 3H), 7.30-7.33 (m, 2H).13C NMR (126 MHz, CDCI3) 6 14.6, 25.7, 34.0, 49.8, 127.8, 128.8, 137.8, 168.6, 177.0.

[0089] Compound dg-f / ? -l (wherein A=D, B=D, X=D, Y=H); (2R)-2-(2,5-Dioxopyrrolidin-l-yl-3, 3,4,4-d4)-N-((phenyl-ds)methyl)propanamide

[0090] White solid. Melting point: 139.3-140.7°C; TLC: Rf= 0.44 (DCM: MeOH (9: 0.3; v / v)); C14H7D9N2O3 (269.35), Monoisotopic mass: 270.17. UPLC (100% purity): tR= 3.82 min (M+H)+270.1. Chiral HPLC > 99% ee (tR= 24.539 min).TH NMR (500 MHz, CDCI3) 6 1.58 (d, J=7.2 Hz, 3H), 4.43 (d, J=5.7 Hz, 2H), 4.79 (q, J=7.2 Hz, 1H), 6.31 (br s, 1H).13C NMR (126 MHz, CDCI3) 6 14.6, 34.0, 43.8, 49.9, 127.1, 127.3, 128.3, 137.7, 168.6, 176.9.

[0091] Compound dn- / / ? / -l (wherein A=D, B=D, X=D, Y=D); (R)-2-(2,5-Dioxopyrrolidin-l-yl-3, 3,4,4-d4)-N-((phenyl-d5)methyl-d2)propanamide White solid. Melting point: 138.1-139.0°C; TLC: Rf= 0.39 (DCM: MeOH (9: 0.3; v / v)); C14H5D11N2O3 (271.36), Monoisotopic mass: 271.19. UPLC (100% purity): tR= 3.80 min (M+H)+272.2. Chiral HPLC > 99% ee (tR= 24.017 min).XH NMR (500 MHz, CDCh) 6 1.58 (d, 7=7.5 Hz, 3H), 4.79 (q, 7=7.5 Hz, 1H), 6.32 (br s, 1H).TH NMR (500 MHz, CDCI3) 6 1.58 (d, 7=7.5 Hz, 3H), 4.79 (q, 7=7.5 Hz, 1H), 6.32 (br s, 1H).13C NMR (126 MHz, CDCI3) 614.5, 25.7, 34.0, 49.8, 127.5, 128.5, 137.7, 168.6, 177.0.

[0092] Compound (R)-2 (wherein A=H, B=H, X=F, Y=H); (R)-2-(2,5-dioxopyrrolidin-l-yl)-N-(2-fluorobenzyl)propanamide

[0093] White solid. Melting point: 115.1-115.8°C; TLC: Rf= 0.43 (DCM: MeOH (9: 0.3; v / v)); C14H15FN2O3 (278.28), Monoisotopic mass: 278.11. UPLC (100% purity): tR= 4.08 min (M+H)+279.2. Chiral HPLC > 99% ee (tR= 24.859 min).XH NMR (500 MHz, CDCh) 6 1.56 (d, 7=7.5 Hz, 3H), 2.68 (s, 4H), 4.43 (t, 7=6.0 Hz, 2H), 4.73-4.76 (m, 1H), 6.50 (br s, 1H), 7.00 (t, 7=9.1 Hz, 1H), 7.08 (t, 7=7.5 Hz, 1H), 7.21-7.28 (m, 1H), 7.29-7.31 (m, 1H)13C NMR (126 MHz, CDCh) 6 14.5, 28.2. 37.9, 37.9, 49.8, 115.3, 115.5, 124.5, 124.5, 124.8, 124.9, 129.4, 129.4, 130.2, 130.2, 160.0, 161.9, 168.8, 176.9.

[0094] Compound d4-f / ? -2 (wherein A=D, B=H, X=F, Y=H); (R)-N-(2-fluorobenzyl)-2-(2,5-dioxopyrrolidin-l-yl-3,3,4,4-d4)propanamide

[0095] White solid. Melting point: 157.2-157.3°C; TLC: Rf= 0.44 (DCM: MeOH (9: 0.3; v / v)); C14H11D4FN2O3 (282.31), Monoisotopic mass: 283.13. UPLC (100% purity): tR= 4.01 min (M+H)+283.2. Chiral HPLC > 99% ee (tR= 18.863 min).TH NMR (500 MHz, CDCh) 6 1.57 (d, 7=7.3 Hz, 3 H), 4.38-4.52 (m, 2 H), 4.76 (q, 7=7.3 Hz, 1 H), 6.48 (br s, 1 H), 6.98-7.04 (m, 1 H), 7.08-7.11 (m, 1 H), 7.20-7.24 (1 H), 7.30-7.31 (m, 1 H).13C NMR (126 MHz, CDCh) 6 14.5, 37.9, 38.0, 49.8 115.4, 124.5 (7=3.62 Hz), 124.8, 124.9, 129.4 (7=8.45 Hz), 130.2 (7=4.23 Hz), 168.7, 177.0. Compound d -(R)-2 (wherein A=D, B=H, X=F, Y=D); (R)-2-(2,5-Dioxopyrrolidin-l-yl-3,3,4,4-d4)-N-((2-fluorophenyl)methyl-d2)propanamide

[0096] White solid. Melting point: 157.2-157.7°C; TLC: Rf= 0.44 (DCM: MeOH (9: 0.3; v / v)); C14H9D6FN2O3 (284.32), Monoisotopic mass: 284.14. UPLC (100% purity): tR= 3.98 min (M+H)+285.2. Chiral HPLC > 99% ee (tR= 18.011 min).XH NMR (500 MHz, CDCh) 6 1.57 (d, J=7.2 Hz, 3H), 4.76 (q, 7=7.3 Hz, 1H), 6.44 (br s, 1H), 7.01 (ddd, J=10.2, 8.2, 1.0 Hz, 1H), 7.09 (td, 7=7.6, 1.2 Hz, 1H), 7.21-7.26 (m, 1H), 7.31 (td, 7=7.6, 1.7 Hz, 1H).13C NMR (126 MHz, CDCI3) 6 14.5, 25.7, 34.0, 49.8, 115.5, 124.5 (d, 7=3.6 Hz), 129.4 (d, 7=8.5 Hz), 130.2(d, 7=4.2 Hz), 168.8, 177.0.

[0097] Glutamate uptake study on COS-7 cells with EAAT2 overexpression A detailed procedure is described in the literature (Kaminski, K. et. al. Ann. Neurol. 2025, 97, 344-357), and a summary thereof is provided below.

[0098] Transfection of COS-7 cells with EAAT2 plasmid

[0099] COS-7 cells (Kidney Fibroblast; African Green Monkey, ATCC-CRL-1651, ATCC, Manassas, VA, USA) were seeded at 50,000 cells per well (24-well plate) and then transiently transfected with CMV vectors not comprising EAAT2 (background control) or comprising EAAT2 (CMV-hEAAT2) at 100 ng cDNA per well at a final volume of 510 pL: DMEM (ATCC, Manassas, VA, USA) supplemented with 10% bovine fetal serum (ATCC, Manassas, VA, USA), 5% 100x penicillin / streptomycin (Thermo Fisher Scientific, Waltham, MA USA) and 100 pL Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA). The transfection was performed using TurboFect transfection reagent according to the manufacturer's protocol (Thermo Fisher Scientific, Waltham, MA, USA). After transfection, the cells were incubated 48 hours at 37°C under 5% CO2.

[0100] Glutamate uptake measurement

[0101] Glutamate uptake tests were performed 48 hours after transfection using glutamic acid [3H] (Revvity, Boston, USA, specific activity 47 Ci / mmol) with a final concentration = 50 nM, according to the procedure described by Fontana (Fontana, A. C. K. Curr. Protoc. Pharmacol.

[0102] 2018, 82, e45), as amended. The transfected cells, after a single wash with phosphate buffer with calcium and magnesium (PBS-CM, pH=7.4), were incubated with 200 pL of test compound at concentrations from 10“4M to 1O“12M for 10 minutes at 37 °C. The compounds were dissolved in 100% DMSO, with a maximum DMSO content of 0.01%. After incubation, 100 pL of a radioligand was added and incubated for 5 minutes at 37°C.

[0103] Radioactivity measurement

[0104] After incubation with radioligand, after washing twice with PBS-CM buffer, the cells were lysed in lytic buffer (1% SDS / 0.1 N NaOH; 20 min / room temp.). Radioactivity as the number of decays per minute (DPM) was measured using a Beckman LS 650 scintillation counter (Beckman, USA).

[0105] Statistical analysis

[0106] The results were analyzed with GraphPad Prism 10.2.3. To analyze differences in glutamate uptake at various concentrations of the tested compounds in comparison with the control group, one-way analysis of variance (ANOVA) followed by Dunnett's post hoc multiple comparison test was used. Evaluation of antipruritic activity in models of histamine-dependent and histamine-independent pruritus

[0107] General information

[0108] The study was conducted on male Swiss white mice (CD-I) weighing 22-26 g. All procedures were performed in accordance with the applicable Polish and international guidelines on animal research ethics, after obtaining appropriate institutional consent. Substances were administered intraperitoneally (i.p.) after suspending in a 1% aqueous Tween solution, as single injections with a volume of 10 mL / kg, 30 minutes before a given test.

[0109] Routes and time schedules for administration of test substances

[0110] Compounds (R)-l, (R)-2 and d6-(R)-2 were suspended in a 1.0% Tween 80 solution and administered intraperitoneally (i.p.) on the left side of the abdominal cavity. Control groups were administered intraperitoneally (i.p.) with 1.0% Tween 80 solution. In interaction studies WAY213613, was administered (i.p.) on the right side of the abdominal cavity 10 minutes before (R)-l administration. Pyrilamine suspended in a 1.0% Tween 80 solution was administered intraperitoneally (i.p.). In pruritus models, histamine, chloroquine, or SLIGRL-NH2 were administered subcutaneously 30 minutes after the administration of the tested compounds or vehicle, and measurements were immediately initiated.

[0111] Determination of antipruritic activity in models of pruritus induced by histamine, chloroquine or SLIGRL-NH2

[0112] 24 hours before the start of the main test, the mice were placed in an experimental room and they had a piece of skin shaved off, approximately 2 cm2in size, in the neck area. For adaptation, one hour before the actual test, the animals were placed in experimental cages with dimensions of 35 x 35 x 35 cm. Then, after this time, the mice were administered intradermally with a pruritic agent in the neck area. In order to induce a pruritic reaction, the following were used: histamine at a dose of 800 pg / dose, chloroquine at a dose of 200 pg / dose or SLIGRL-NH2 at a dose of 100 pg / dose. All pruritic agents used were administered intradermally in a constant volume of 20 pL. After injection of pruritic compounds, the animals were immediately caged and observed. In the case of pruritus induced by histamine, the full observation time was 45 minutes, and in the case of pruritus induced by chloroquine or SLIGRL-NH2, it was 30 minutes. After the end of the study, the mice were removed from the cages, which were washed with ethanol before their next use. The parameter measured was the number of pruritic reactions (scratching). A single reaction was considered to be at least three times scratching with the hind paw in the area of the neck skin, to which the pruritic compound was administered (Mogilski, Sz. et al. Pharmaceuticals 2023, 16, 1481, Obara, I. et al. Pain 2015, 156, 1519-1529).

[0113] Statistical analysis

[0114] The obtained results were presented as mean measurements ± standard deviation (± SD). When comparing mean values for two different groups, the test of differences in pairs for dependent samples and the Student's t-test for independent samples and one-way analysis of variance (one-way ANOVA) with Dunnett's post hoc test were used. The difference in means was considered statistically significant at the significance level of p<0.05. The statistical analysis was performed using the GraphPadPrism 5.0 computer program (GraphPad Software Inc., San Diego, CA, USA).

Claims

Claims1. A positive allosteric modulator (PAM) of the EAAT2 / GLT-1 transporter for use in the treatment or prevention of pruritus, which preferably is a compound of formula (I):B N O H B Y BBwherein:A is hydrogen or deuterium;B is hydrogen or deuterium;X is hydrogen or deuterium or fluorine;Y is hydrogen or deuterium;for use in the treatment or prevention of pruritus, especially histamine-dependent and histamine-independent pruritus.

2. The compound for use according to claim 1 characterized in that:A is hydrogen or deuterium, wherein at least one A is deuterium, particularly preferably each A is deuterium;B is hydrogen;X is hydrogen or fluorine, preferably hydrogen;Y is hydrogen or deuterium, preferably hydrogen.

3. The compound for use according to claim 1 characterized in that it has been selected from the group comprising N-benzyl-2- (2,5-dioxopyrrolidin-l-yl)propanamide derivatives of the R configuration of the stereogenic center located at C-2 in the general formula (I), preferably from the group comprising:(?.)-N-benzyl-2-(2,5-dioxopyrrolidin-l-yl)propanamide (compound (R)-l, wherein A=H, B=H, X=H, Y=H),(7?.)-N-benzyl-2-(2,5-dioxopyrrolidin-l-yl-3,3,4,4-d4)propanamide (compound d4-(R)-1, wherein A=D, B=H, X=H, Y=H), / ?^-2-(2,5-dioxopyrrolidin-l-yl-3,3,4,4-d4)-N-(phenylmethyl-d2)propanamide (compound d6-(R)-1, wherein A=D, B=H, X=H, Y=D),(7 -2-(2,5-dioxopyrrolidin-l-yl-3,3,4,4-d4)-N-((phenyl-d5)methyl)propanamide (compound d9-(R)-1, wherein A=D, B=D, X=D, Y=H),(R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)-N-((phenyl-d5)methyl-d2)propanamide (compound d11-(R)-1: A=D, B=D, X=D, Y=D),(7 -2-(2,5-dioxopyrrolidin-l-yl)-N-(2-fluorobenzyl)propanamide (compound (R)-2, wherein A=H, B=H, X=F, Y=H),(7 -2-(2,5-dioxopyrrolidin-l-yl-3,3,4,4-d4)-N-(2-fluorobenzyl)propanamide (compound d4-( / - 2, wherein A=D, B=H, X=F, Y=H),(R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)-N-((2-fluorophenyl)methyl-d2)propanamide (compound d6-(R)-2, wherein A=D, B=H, X=F, Y=D).

4. The compound for use according to any of the above claims, characterized in that the pruritus accompanies an allergic reaction, in particular one caused by an insect bite, renal failure (uremic pruritus), hyperbilirubinemia, hypercalcemia, iron deficiency or overload, lymphoma, granuloma or solid tumor, diabetes, amyloidosis, neurological disease, especially multiple sclerosis or stroke, infectious disease, especially chickenpox, shingles, viral hepatitis, HIV, autoimmune disease, especially atopic dermatitis or psoriasis, other clinical cases such as chikungunya fever, or is the consequence of the use of drugs, preferably selected from the group comprisingopioid analgesics, anti-EGFR monoclonal antibodies, tyrosine kinase inhibitors or oral contraceptives.

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