Patent Application
20240299380
The present invention relates to the field of medicament, specifically, relates to a use of a compound in the preparation of the medicament for treating myelofibrosis and the symptoms/signs associated with myelofibrosis, and the use thereof.
Myelofibrosis (MF) is a myeloproliferative neoplasm (MPNs) caused by the clonal proliferation of hematopoietic stem cells, including primary myelofibrosis, myelofibrosis secondary to polycythemia vera and essential thrombocythemi. Myelofibrosis (MF) is a disease characterized by the occurrence of reticular fibers and collagen fibers in the bone marrow, the clinical manifestations of MF include anemia, fatigue, early satiety, abdominal imbalance, poor mobility, difficulty concentrating, night sweats, itchy skin, diffuse bone pain, fever, weight loss, splenomegaly, hepatomegaly, which impacts quality of life seriously. The overall median survival time is 3.5 to 5.5 years, but the higher the risk level, the shorter the survival time, the median survival time of high-risk patients is about 2 years. The cause of myelofibrosis (MF) has not been well understood, most patients carry an activating mutation of the Janus kinase 2 gene (JAK2), CALR or MPL or other genes, which make the JAK-STAT pathway overreacted. About 85% of MF patients have at least one driver gene mutation among JAK, MPL and CALR (Klampfl T, et al. NEJM 2013; 369(25):2379-90; Nangalia J, et al. NEJM 2013; 369(25)2391-405). In patients with primary fibromyelopathy, mutational frequency of JAK2, CALR and MPL is ˜65%, ˜25% and ˜10% respectively. (Tefferi A. Am J Hematol. 2021 January; 96(1):145-162.).
The treatment of Myelofibrosis (MF) is to control disease symptoms and complications, enhancing quality of life in the short term, but in the long term the treatment is to extend survival, manage/reverse Myelofibrosis (MF) even cure it. Among the current treatments for MF, traditional drugs are symptomatic treatments based on clinical manifestations, and their efficacy is limited. Currently, there are few targeted therapies for myelofibrosis. Only two targeted drugs for the treatment of myelofibrosis have been developed, which are small molecule JAK1/JAK2 kinase inhibitor ruxolitinib phosphate from Novartis and highly specific JAK2 inhibitor fedratinib from Bristol-Myers Squibb's (BMS). At present, ruxolitinib is the only targeted drug approved in China for the treatment of myelofibrosis. But JAK inhibitors has shown some side effects in patients including thrombocytopenia, anemia, gastrointestinal disturbances, metabolic abnormalities, peripheral neuropathy, and hyperacute relapse of symptoms during treatment discontinuation. (Tefferi, A. JAK inhibitors for myeloproliferative neoplasms: clarifying facts from myths[J]. Blood, 2012, 119(12):2721-2730.).
For the above reasons, there is an urgent need to develop a drug that can effectively treat myelofibrosis and its related symptoms/signs.
The present invention is to provide a use of a compound in the preparation of a medicament for treating myelofibrosis and the symptoms/signs associated with myelofibrosis, the use of the compound as a new medicament for the treatment of myelofibrosis and the symptoms/signs associated with myelofibrosis.
In order to achieve the above purpose, according to one aspect of the present invention, there is provided use of a compound in the preparation of a medicament for treating myelofibrosis and the symptoms/signs associated with myelofibrosis, wherein the compound is selected from one or more of the compounds of formula I and the pharmaceutically acceptable salts, prodrugs, solvates and hydrates thereof:
Y is
wherein,
represents a single bond or a double bond, the U, W or Z is independently selected from C or N; R3 is absent or selected from H, halogen, hydroxyl, amino, C1-6 alkyl, C1-6 alkoxy, cyano, oxo, or —N(R4)—SO2—R5; R4 and R5 are each independently selected from H, C1-6 alkyl or halo C1-6 alkyl.
Further more, Y is
Further more, R1 is selected from H or C1-4 alkyl, preferably, R1 is H, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl; and/or, R2 is selected from H or C1-3 alkyl; preferably, R2 is H, methyl, ethyl, propyl or isopropyl; and/or, Q is absent or C1-3 alkylene; preferably, Q is selected from methylene or ethylene; and/or, X is selected from H, C1-3 alkyl or phenyl, alternatively, phenyl can be substituted with halogen, halo C1-3 alkyl, C1-3 alkyl, C1-3 alkoxy or C1-3 alkyl-SO2—.
Further more, the compound is selected from one or more of the compound of formula II and the pharmaceutically acceptable salts, prodrugs, solvates and hydrates thereof:
Further more, the compound is selected from one or more of:
preferably, the compound is 6-methyl-4-(2-methyl-1-(4-(trifluoromethyl)benzyl)-1H-imidazo[4,5-b]pyrazin-6-yl)-1H-py rrolo[2,3-c]pyridin-7(6H)-one.
Further more, the medicament also comprising excipients.
Further more, the medicament is administered intravenously, intramuscularly, parenterally, nasally, orally or rectally.
According to another aspect of the present invention, there is also provided a method of treating myelofibrosis and the symptoms/signs associated with myelofibrosis, wherein the method comprising: providing a medicament comprising the compound, wherein the compound is selected from one or more of the compound of formula I and the pharmaceutically acceptable salts, prodrugs, solvates and hydrates thereof; and administering therapeutically effective amount of the medicament to subject having myelofibrosis;
wherein,
represents a single bond or a double bond, the U, W or Z is independently selected from C or N; R3 is absent or selected from H, halogen, hydroxyl, amino, C1-6 alkyl, C1-6 alkoxy, cyano, oxo, or —N(R4)—SO2—R5; R4 and R5 are each independently selected from H, C1-6 alkyl or halo C1-6 alkyl.
Further more, Y is
Further more, R1 is selected from H or C1-4 alkyl, preferably, R1 is H, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl; and/or, R2 is selected from H or C1-3 alkyl; preferably, R2 is H, methyl, ethyl, propyl or isopropyl; and/or, Q is absent or C1-3 alkylene; preferably, Q is selected from methylene or ethylene; and/or, X is selected from H, C1-3 alkyl or phenyl, alternatively, phenyl can be substituted with halogen, halo C1-3 alkyl, C1-3 alkyl, C1-3 alkoxy or C1-3 alkyl-SO2—.
Further more, the compound is selected from one or more of the compound of formula II and the pharmaceutically acceptable salts, prodrugs, solvates and hydrates thereof:
Further more, the compound is selected from one or more of:
Further more, the myelofibrosis is selected from the group consisting of primary myelofibrosis, Post-Polycythemia Vera Myelofibrosis or Post-Essential Thrombocythemia Myelofibrosis.
Further more, the medicament also comprising excipients.
Further more, the medicament is administered intravenously, intramuscularly, parenterally, nasally, orally or rectally.
Further more, the compound is administered in a dosage of 1-500 mg/day.
Further more, the compound is administered orally in a dosage of about 3 mg/day, or 5 mg/day, or 10 mg/day, or 20 mg/day, or 25 mg/day, or 30 mg/day, or 35 mg/day, or 40 mg/day, or 45 mg/day, or 50 mg/day, or 55 mg/day, or 60 mg/day, or 70 mg/day, or 80 mg/day, or 90 mg/day, or 100 mg/day, or 150 mg/day, or 200 mg/day.
The present invention provides a use of the compound in the preparation of a medicament for treating myelofibrosis and the symptoms/signs associated with myelofibrosis; the compound is selected from one or more of the compound of formula I and the pharmaceutically acceptable salts, prodrugs, solvates and hydrates thereof:
The drawings that constitute a part of the present invention are used to provide a further understanding, the illustrative examples and there descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.
In the drawings:
It should be noted that, as long as there is no conflict, the examples and features in the examples of the present invention can be combined with each other. The present invention will be described in detail below with reference to the drawings and examples.
In the present invention, unless otherwise stated, the term “halogen” refers to fluorine, chlorine, bromine or iodine. Preferably, halogen refers to fluorine, chlorine and bromine.
In the present invention, unless otherwise stated, the term “alkyl” includes a straight, branched or cyclic saturated monovalent hydrocarbon. For example, alkyl includes methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, 3-(2-methyl)butyl, 2-pentyl, 2-methylbutyl, neopentyl, cyclopentyl, n-hexyl, 2-hexyl, 2-methylpentyl and cyclohexyl. Similarly, “C1-6” in C1-6 alkyl refers to a group containing 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a linear or branched chain.
In the present invention, unless otherwise stated, the term “heteroaryl” refers to a substituted or unsubstituted stable monocyclic or bicyclic group containing at least one aromatic ring of 5 to 10 ring atoms, the aromatic ring containing one, two or three ring heteroatoms selected from N, O and S, the remaining ring atoms are C atoms. Examples of such heteroaryl groups include, but are not limited to, pyridyl, pyrimidinyl, pyrrolyl, imidazolyl, thiazolyl, thienyl, and benzimidazole.
In the present invention, the term “prodrugs” refers to functional derivatives of the compounds that are readily converted in vivo into the required compound. Thus, in the methods for the treatment of the present invention, the term “administering” shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the subject, for the treatment of myelofibrosis and the symptoms/signs associated with myelofibrosis.
When the compound of Formula I and pharmaceutically acceptable salts thereof exist in the form of solvates or polymorphic forms, the “compound” of the present invention includes any possible solvates and polymorphic forms. A type of a solvent that forms the solvate is not particularly limited so long as the solvent is pharmacologically acceptable. For example, water, ethanol, propanol, acetone and similar solvents can be used.
The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compound of the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (ic and ous), ferric, ferrous, lithium, magnesium, manganese (ic and ous), potassium, sodium, zinc and the like salts. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed include ion exchange resins such as, for example, arginine, betaine, caffeine, choline, N′,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. When the compound of the present invention is basic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic, formic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids, particularly preferred are formic and hydrochloric acid. Since the compounds of Formula I are intended for pharmaceutical use they are preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure, especially at least 98% pure (% are on a weight for weight basis).
In order to treat myelofibrosis and its associated symptoms/signs with medicament, the present invention provides a use of the compound in the preparation of a medicament for treating myelofibrosis and the symptoms/signs associated with myelofibrosis, and provides a method of treating myelofibrosis and the symptoms/signs associated with myelofibrosis. The above method comprising: providing a medicament comprising the compound, and administering therapeutically effective amount of the medicament to subject having myelofibrosis. The above compound is selected from one or more of the compound of formula I and the pharmaceutically acceptable salts, prodrugs, solvates and hydrates thereof:
wherein,
represents a single bond or a double bond, the U, W or Z is independently selected from C or N; R3 is absent or selected from H, halogen, hydroxyl, amino, C1-6 alkyl, C1-6 alkoxy, cyano, oxo, or —N(R4)—SO2—R5; R4 and R5 are each independently selected from H, C1-6alkyl or halo C1-6 alkyl.
The inventors of the present invention unexpectedly discovered that the compound of Formula I has a significant inhibitory effect on the proliferation of human myeloproliferative tumor cells HEL and SET-2 cells with JAK2V617F mutation phenotype, and also has a good inhibitory effect on the transcriptional activity of NF-κB and the secretion of the inflammatory factor IL-6, its associated symptoms/signs, and can be useful in the treatment of myelofibrotic disease and its associated symptoms/signs.
In addition, it should be noted that, the Y group of the aboved compound of formula I is required to be
compared to other 5- and/or 6-membered merged rings, such as
the compound of formula I of the present invention has significant differences and obvious effectiveness in the treatment of myelofibrosis. More preferably, Y is
In order to further improving the effectiveness of treatment of myelofibrosis and the symptoms/signs associated with myelofibrosis, in the preferred embodiment, R1 is selected from H or C1-6 alkyl, preferably, R1 is H, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl; more preferably. R1 is methyl.
In a preferred embodiment, R2 is selected from H or C1-3 alkyl; preferably, R2 is H, methyl, ethyl, propyl or isopropyl; more preferably, R2 is H.
In a preferred embodiment, Q is absent or C1-3 alkylene; more preferably, Q is selected from methylene or ethylene; more preferably, Q is methylene.
In a preferred embodiment, X is selected from H, C1-3 alkyl or phenyl, alternatively, phenyl can be substituted with halogen, halo C1-3 alkyl, C1-3 alkyl, C1-3 alkoxy or C1-3 alkyl-SO2—.
In a preferred embodiment, the compound is selected from one or more of the compound of formula II and the pharmaceutically acceptable salts, prodrugs, solvates and hydrates thereof:
Illustratively, the above compounds are selected from one or more of:
In one embodiment of the invention, the symptoms/signs associated with myelofibrosis is one or more of the following: anemia, splenomegaly, hepatomegaly, fatigue, early satiety, abdominal discomfort, inactivity, inability to concentrate, night sweats, itching, diffuse bone pain, fever, and weight loss.
In practical applications, the medicament also comprises excipients. Specific excipients include, but not limited to, pharmaceutically acceptable carriers, excipients or adjuvants, etc. Moreover, the medicament can be prepared into different dosage forms to adapt to diverse administration route, the specific suitable administration routes include intravenous, intramuscular, parenterally, nasally, orally or rectally. The dosage forms of the medicament include tablets, capsules, pills, powders, suppositories, solutions, suspensions, aerosols, emulsions, ointments, lotions, dusting powders or other similar dosage forms.
The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers include such as sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include such as carbon dioxide and nitrogen. In preparing the compositions for oral dosage form, any convenient pharmaceutical media may be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like may be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets may be coated by standard aqueous or nonaqueous techniques.
The above tablet may be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Each tablet preferably contains from about 0.05 mg to about 5 g of the active ingredient and each cachet or capsule preferably containing from about 0.05 mg to about 5 g of the active ingredient. For example, a formulation intended for the oral administration to humans may contain from about 0.5 mg to about 5 g of active agent, compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition. Unit dosage forms will generally contain between from about 1 mg to about 2 g of the active ingredient, typically 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 5 mg, 15 mg, 20 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg or 1000 mg.
The above mentioned medicament suitable for parenteral administration may be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.
The above mentioned medicament suitable for injectable use includes sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.
The above mentioned medicament can be in a form suitable for rectal administration with solid as a carrier. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.
In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above may include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including antioxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing a compound described by Formula I, or pharmaceutically acceptable salts thereof, may also be prepared in powder or liquid concentrate form.
The term “effective amount” is therapeutically effective amount. The “therapeutically effective amount” is an amount sufficient to provide a desired therapeutic result over the necessary dosage and specified duration. The therapeutically effective amount of the medicament can vary depending on various factors, for example, the individual's condition, age, gender, and weight, as well as the ability of drugs to elicit the desired response in the individual's body. The therapeutically effective amount is also the amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the subject. In one embodiment, in the method of treating myelofibrosis and the symptoms/signs associated with myelofibrosis provided by the present invention, the above compound is administered in dosage of 1-500 mg/day, preferably the dosage of the compound is 3˜200 mg/day, preferably the dosage of the compound is 5˜150 mg/day. Exemplarily, the above compound is administered in dosage of about 3 mg/day, or 5 mg/day, or 10 mg/day, or 20 mg/day, or 25 mg/day, or 30 mg/day, or 35 mg/day, or 40 mg/day, or 45 mg/day, or 50 mg/day, or 55 mg/day, or 60 mg/day, or 70 mg/day, or 80 mg/day, or 90 mg/day, or 100 mg/day, or 150 mg/day, or 200 mg/day for oral administration.
The present invention will be described in detail below in conjunction with examples, which are merely illustrative and not limited to the scope of application of the present invention.
Preparation of 6-methyl-4-(2-methyl-1-(4-(trifluoromethyl)benzyl)-1H-imidazo[4,5-b]pyrazin-6-yl)-1H-py rrolo[2,3-c]pyridin-7(6H)-one
2-amino-3,5-dibromopyrazine (10.00 g), 4-trifluoromethylbenzylamine (20.98 g) and DIEA (25.54 g) was dissolved in DMSO (60 mL), heated and stirred at 120° C. for 4 h, LCMS confirmed the end of the reaction. Cooled, cold water (250 mL) was added, extracted 3 times with EA, combined organic phases, washed with saturated brine 3 times, dried with anhydrous sodium sulfate, concentrated, and purified the crude product by column chromatography (the mobile phase is PE:EA=100:15-100:30) to obtain compound 1-1(15.01 g).
LCMS: [M+1]+=347.0
Compound 1-1 (15.43 g), triethyl orthoacetate (35.96 g) and glacial acetic acid (200 mL) were mixed and reacted at 100° C. overnight. Cooled, concentrated, diluted the crude product with EA, wash 3 times with saturated Na2CO3 solution, 3 times with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography (the mobile phase is PE:EA=100:20-100:50) to obtain compound 1-2 (13.27 g).
LCMS: [M+1]+=371.0
Compound 1-2 (1.10 g), 2M−1(1.27 g), Pd(dppf)Cl2.DCM(0.25 g) were dissolved in dioxane (20 mL), K2CO3 (0.61 g) and water (4 mL) were added, protected by nitrogen, heated and stirred at 100° C. overnight. Cooled, the reaction solution was poured into a mixed solvent of EA (50 mL) and saturated Na2CO3 (50 mL), separated the liquid, aqueous phase was extracted 3 times with EA, organic phases were combined, washed with saturated brine 3 times, dried over anhydrous sodium sulfate, and concentrated, the crude product was purified by column chromatography (the mobile phase is DCM:MeOH=100:2) to obtain compound 1-4 (1 g).
LCMS: [M+1]+=593.1
Compound 1-4 (20.00 g) was dissolved in EtOH (80 mL) and DCM (250 mL), NaOH (2.70 g) was added, stirred at 60° C. overnight, concentrated. The crude product was purified by column chromatography (the mobile phase is DCM:MeOH=100:2) to obtain a brownish-yellow crude product, the brownish-yellow crude product was slurried with DCM (40 mL), the solid was collected by filtration, and dried under reduced pressure to give compound 1 as a off-white solid (6.53 g).
LCMS: [M+1]+=439.1.
1H NMR (400 MHz, DMSO) δ 12.13 (s, 1H), 8.92 (s, 1H), 7.99 (d, J=35.1 Hz, 1H), 7.76 (t, J=17.0 Hz, 2H), 7.49 (d, J=8.0 Hz, 2H), 7.28 (s, 1H), 6.75 (s, 1H), 5.68 (s, 2H), 3.63 (s, 3H), 2.63 (s, 3H).
Using a method basically similar to that of Example 1, the corresponding trifluoromethylbenzylamine derivative is used to replace
(trifluoromethylbenzylamine) to prepare the example in Table 1 below. The corresponding trifluoromethylbenzylamine derivative, such as
or
etc, can all be purchased through commercially available channels.
The effects of compound 1 on proliferation of human myeloproliferative tumor cells HEL and SET-2 cells with JAK2V617F activation mutation were tested using CTG assay in this example to evaluate half inhibitory rate (IC50) of compound 1 on HEL and SET-2 cell proliferation.
The compounds were diluted with DMSO 6.67 fold from 20 mM to 3 mM, then 3-fold serial dilutions were conducted to make 10 concentrations for each compound. Staurosporine was diluted with DMSO 6.67 fold from 20 mM to 3 mM, followed by 3-fold serial dilutions to make 10 concentrations. The vehicle control consisted of 100% DMSO, and the positive control was 3 mM Staurosporine.
IC50 (the half maximal inhibitory concentration) was calculated with GraphPad Prism 8 software and following nonlinear fitting formula:
LUMHigh is the average value of 0.33% DMSO wells, LUMempd is the value of each well of the compound, LUMLow is the average value of 10 μM Staurosporine wells.
The proliferation inhibition curve of compound 1 against HEL cells at different concentrations is shown in
The IC50 values of compound 1 against HEL cells and SET-2 cells are 154.1 nM and 103.6 nM, respectively. These results indicate that compound 1 exhibits a good inhibitory effect on the proliferation of both HEL and SET-2 cells.
In present example, reporter gene assay was used to determine the inhibitory effect of compound 1 on NF-κB transcriptional activity in HEK293 cells, to evaluate the effect of the compound on NF-κB transcriptional activity.
The following nonlinear fitting formula was used to calculate the IC50 of the compound with Graphpad8.0.
The inhibition curve of compound 1 on NF-κB transcriptional activity at different concentrations was shown in
Under the conditions of this experiment, compound 1 significantly inhibited the transcriptional activity of NF-κB in HEK293 cells stably transfected with the NF-κB reporter gene.
The Human IL-6 Valukine ELISA kit (purchased from R&D) was used to measure the IL-6 content in the cell supernatants by ELISA method in this example. The experimental procedures shown below were conducted to determine the effect of compound 1 on IL-6 secretion in THP-1 cell supernatants following LPS stimulation.
THP-1 cells were cultured in RPMI 1640 medium containing 1% PS and 10% FBS and 2-Mercaptoethanol (IX). The cells were incubated in a humidified incubator with 5% carbon dioxide (CO2) at 37° C.
IC50 (The half maximal inhibitory concentration) was calculated using GraphPad Prism 8 software with the following nonlinear fitting formula:
Note: When the cell viability inhibition rate is greater than 30%, IL-6 at this concentration dose not participate in the calculation of IC50.
The inhibitory curve of compound 1 on IL-6 in THP-1 cells at different concentrations was shown in
As compound concentration increased, the concentration of IL-6 in the supernatant of THP-1 cells decreased, indicating that compound 1 significantly inhibited the IL-6 secretion in THP1 monocytes.
The above data demonstrated that the compound of the present invention displayed a significant inhibitory effect on the proliferation of human myeloproliferative tumor cell lines HEL and SET-2 harboring the JAK2V617F mutantion, which is directly associated with myelofibrosis. Furthermore, the compound exhibited a substantial inhibitory effect on the transcriptional activity of NF-κB and the secretion of the inflammatory factor IL-6.
A Phase I study to evaluate safety, tolerability, pharmacokinetics and efficacy of compound 1 and determine MTD and RP2D
Target: Subjects with primary myelofibrosis, post-polycythemia vera myelofibrosis, post-essential thrombocythemia myelofibrosis.
This study used a “3+3” design and designed 3 dose levels initially, that is, after determining the initial dose of 60 mg, the test doses were increased by 33% and 25%, which were 60 mg/d, 80 mg/d and 100 mg/d respectively. “3+3” design escalation rule: {circle around (1)} If no DLTs are observed of 3 DLT-evaluable patients, the trial proceeds to next higher dose cohort; {circle around (2)}If one subject develops a DLT of 3 DLT-evaluable patients, an additional three subjects are enrolled into that same dose cohort, once 31 DLTs are observed further (total number of DLT patients≥2), the maximum tolerated dose (MTD) will be declared as the previous dose level; {circle around (3)} If 2 or more patients in a cohort experience DLTs, then stop enrollment at the specific dose and higher dose levels, the MTD will be declared as the previous dose level.
Twice daily (BID, Q12H was used in this study, two doses per day every 12 hours), then the dose level and dosing regimens (including, but not limited to, starting dose, intended escalating maximum dose, intermediate dose, dosing frequency and intervals) can be adjusted by the sponsor and researcher based on the result of pharmacokinetics, safety, tolerability and efficacy. 21 days each treatment cycle.
The 2013 ELN and IWG-MRT consensus standards were adopted.
The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/101322 | Jun 2021 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/100134 | 6/21/2022 | WO |
