Spleen tyrosine kinase inhibitors, compositions and methods of use

Abstract

Compounds, compositions and methods for inhibiting Syk phosphorylated red blood cell anion transporter band 3 and / or Syk tyrosine kinase. Also disclosed are methods of treating a subject having a disease involving release of microvesicles from blood cells, including alpha-thalassemia or beta-thalassemia, sickle cell disease, glucose-6-phosphate dehydrogenase (G6PD) deficiency, or glutathione reductase deficiency.

Description

priority

[0001] This patent application is related to and claims priority to U.S. Provisional Patent Application No. 63 / 319,486 filed on March 14, 2022 and U.S. Provisional Patent Application No. 63 / 398,599 filed on August 17, 2022. The contents of the foregoing applications are hereby incorporated by reference into this disclosure in their entirety. Government Support Statement

[0002] This invention was made with government support under Grant No. GM024417 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0003] The present disclosure relates to spleen tyrosine kinase (Syk) inhibitors, compositions comprising the same, and methods of use thereof in treating sickle cell disease and other diseases involving the release of microvesicles from blood cells. Background Art

[0004] Sickle cell disease is a group of inherited blood disorders caused by abnormalities in hemoglobin, specifically the structure of β-globin (glu6val), the molecule in red blood cells (RBCs) that transports oxygen to cells throughout the body. Sickle cells can be characterized by a mutation in one of the amino acids of this β chain from glutamic acid to valine, which causes deoxy-sickled hemoglobin (deoxy-HbS) to form insoluble polymers within the RBC and deform the RBC into a rigid shape or sickle (i.e., crescent-shaped) cell.

[0005] Sickle cell disease affects a significant number of people worldwide. In the United States, people with sickle cell disease die in their 40s on average, suffer from poor quality of life, and experience high medical costs. Signs and symptoms of sickle cell disease typically begin in early childhood. Characteristics of the disease can include low RBC counts (i.e., anemia), recurring infections, and periodic pain.

[0006] The signs and symptoms of sickle cell disease are caused by sickling of the RBCs. When the RBCs sickle, they break down prematurely, which can lead to anemia and other complications such as jaundice, pulmonary hypertension, and heart failure.

[0007] Currently, standard treatment for sickle cell disease patients two years of age and older is blood transfusions and treatment with the anticancer drug hydroxyurea. Not all patients respond to hydroxyurea, and hydroxyurea is poorly tolerated, causing bone marrow suppression in some patients. Treatments based on pyruvate kinase agonists (e.g., imatinib) are also available; however, such inhibitors may also interact with (i.e., inhibit) growth factor receptors involved in tissue revascularization, which is often necessary because of the frequent vascular occlusive events that occur in patients with sickle cell disease. In addition, undesired inhibition of growth factor receptors is particularly problematic in children because it can interfere with their growth and development.

[0008] In view of the above, it is an object of the present disclosure to provide such a therapeutic agent. This and other objects and advantages and inventive features will be apparent from the detailed description provided herein. Summary of the invention

[0009] A compound is provided: (i) which comprises a rigidly fused bicyclic ring substituted with at least two substituents, at least one of which is an N-linked, optionally substituted carbocyclic or heterocyclic ring, and the other of which is indole substituted with at least one substituent, at least one of which is a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring, (ii) it inhibits the phosphorylation of human erythrocyte anion transporter band 3 by spleen tyrosine kinase (Syk), and (iii) the concentration at which it inhibits Syk tyrosine kinase is at least about 20 times less than the concentration at which the compound inhibits hepatocyte growth factor receptor (HGFR), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor 3 (FGFR3), insulin-like growth factor 1 receptor (IGF1R) and / or vascular endothelial growth factor receptor (VEGFR), Wherein, in addition to or as an alternative to (ii) and (iii), the compound of (i) further has: (iv) a CLogP value of about 2 to about 5 (e.g., 2 to about 5, about 2 to 5, or 2 to 5), (v) a polar surface area of ​​less than about 250 square angstroms (e.g., less than 250 square angstroms), (vi) no more than 5 hydrogen bond donors, (vii) not more than 15 hydrogen bond acceptors, and / or (viii) not more than 20 rotatable bonds.

[0010] The compound can be of Formula X (eg, has a structure of Formula X): ABC(X) or a pharmaceutically acceptable salt thereof, wherein: A is a nitrogen-linked and optionally substituted first ring, which includes a fused bicyclic or monocyclic ring, wherein each ring of the first ring contains a 4-6 membered heterocyclic or carbocyclic ring; B is a second ring, which is optionally substituted with at least one substituent, the second ring comprising a monocyclic ring or a fused bicyclic or tricyclic ring, and wherein each ring of the second ring comprises a 5-membered or 6-membered heterocyclic or carbocyclic ring; and C is a third bicyclic ring comprising a 5- or 6-membered carbocyclic ring fused to a 5- or 6-membered carbocyclic or heterocyclic ring, wherein the third bicyclic ring is substituted with at least a first substituent comprising: or amines, or alcohols, in: X4 is CH2, NH, CO, O or S, Y5 is CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and and is the point of attachment of the first substituent to the third bicyclic ring.

[0011] In certain embodiments, the compound A is: in: Each X' is independently selected from CH and NH; Each Y2 is independently selected from CH2, NH, O and S; Z2 is CH or N; and Each R3 is independently selected from H, F, Cl, OH and -Y-(CH2) n -, where n = 1-14.

[0012] The N connected to the first ring of compound A can form a nitrogen bridge with compound B. In certain embodiments, A is a first monocyclic ring substituted with a bulky substituent located para to the nitrogen bridge. For example, the bulky substituent can be morpholine.

[0013] In certain embodiments, each ring in the first ring of A is a 6-membered, optionally substituted, open or closed heterocyclic or carbocyclic ring. A may be a first monocyclic ring substituted with two methoxy groups.

[0014] In certain embodiments, the compound of B is: in: Each X2 is independently selected from CH and N; Y3 is selected from O, NH and S; and Z3 is selected from OH, NH2 and NHCH3.

[0015] In certain embodiments, the compound B is not In other words, the compound is any compound described herein, provided that B is not B may be a monocyclic ring substituted with an amide. B may be rigid. In certain embodiments, B is a pyrazine substituted with at least one substituent (eg, an amide).

[0016] The C of the compound may be indole. The C of the compound may be: in: Each X3 is independently selected from CH and N; and Y4 is CH2, NH, O or SH.

[0017] The third bicyclic ring of C may be indole, and the first substituent of C may include a 4-6 membered, optionally substituted carbocyclic or heterocyclic substituent. In certain embodiments, the carbocyclic or heterocyclic substituent is a lipophilic ester.

[0018] The first substituent of C can be

[0019] The first substituent of X, Z, may be -OR, wherein R is And n=1-14.

[0020] In certain embodiments, the first substituent of the third bicyclic ring Z comprises a phosphate or a phosphonate. In certain embodiments, the first substituent of C comprises -OR, wherein R is a phosphate or a phosphonate. In certain embodiments, the first substituent of C is wherein Q is a phosphate or phosphonate. In certain embodiments, the first substituent of C is wherein Q is RC(=O)O-, wherein R is an alkyl group (eg, a C1-C6 alkyl group).

[0021] B of the compound can form a hydrogen bond with amino acid residue Ala451 in the catalytic domain of Syk tyrosine kinase, and the first substituent of C (eg, Z therein) can form a hydrogen bond with amino acid residue Asp512 in the catalytic domain of Syk tyrosine kinase.

[0022] One or more hydrogens of this compound may optionally be replaced by deuterium.

[0023] The compound may include the following structure: Wherein ring A is in: Each X' is independently selected from CH and NH; Each Y2 is independently selected from CH2, NH, O and S; Z2 is CH or N; and Each R3 is independently selected from H, F, Cl, OH and -Y-(CH2) n -, where n = 1-14; wherein ring B is in: Each X2 is independently selected from CH and N; Y3 is selected from O, NH and S; and Z3 is selected from OH, NH2 and NHCH3; The condition is that B is not Wherein ring C is in: Each X3 is independently selected from CH and N; and Y4 is selected from CH2, NH, O and SH; Wherein ring D is or amines, or alcohols, in: X4 is selected from CH2, NH, CO, O and S; Y5 is selected from CH and N; and Z is selected from OH, SH, NH2, NHCH3 and and One or more hydrogens may optionally be replaced by deuterium.

[0024] Ring B can form a hydrogen bond with amino acid residue Ala451 in the catalytic domain of Syk tyrosine kinase, and Z on ring D (e.g., the first substituent of C) can form a hydrogen bond with amino acid residue Asp512 in the catalytic domain of Syk tyrosine kinase. Ring A can be substituted with a bulky substituent located para to the nitrogen bridge. The bulky substituent can be a 6-membered, optionally substituted, open or closed carbocyclic or heterocyclic ring. The bulky substituent can be morpholine.

[0025] The compound has a higher docking score to Syk tyrosine kinase than the otherwise identical compound lacking the bulky substituent at the nitrogen bridge position. The compound may comprise the following structure:

[0026] The compound may include or have the following structure: in: R1 is, R2 includes or an amine, or an alcohol, wherein: X4 is CH2, NH, CO, O or S, Y5 is CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and is the point of attachment of the first substituent to the third bicyclic ring; and R4 is H, CH3, amine or methanol.

[0027] The compound may include the following structure: or a pharmaceutically acceptable salt thereof.

[0028] The compound may include the following structure: or a pharmaceutically acceptable salt thereof.

[0029] The compound may include the following structure: or a pharmaceutically acceptable salt thereof.

[0030] The compound may have or include the following structure: Or a pharmaceutically acceptable salt of any one of the above.

[0031] The compound may include the following structure: or a pharmaceutically acceptable salt thereof, wherein each X is independently selected from CH2, N, NH, O and S; Y is O, NH or SH; and Z is OH or NH2. The 4-6 membered, optionally substituted carbocyclic or heterocyclic substituents on the indole may comprise lipophilic esters. Z on ring D may be -OR, wherein R is And n=1-14.

[0032] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure:

[0033] In certain embodiments, the compound has or comprises the following structure, or is a pharmaceutically acceptable salt of the following structure: wherein the hydroxyl group (HO-) is substituted by: (a) a phosphate or phosphonate; or (b) RC(=O)O-, wherein R is an alkyl group (eg, a C1-C6 alkyl group).

[0034] The compound may be: (i) it comprises a pyrazine substituted with at least three substituents, at least one of which is a carboxamide or a lactam, another of which is an N-linked, optionally substituted carbocyclic or heterocyclic ring, another of which is a carbocyclic or heterocyclic ring having at least one substituent comprising a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring, (ii) It inhibits the phosphorylation of human erythrocyte anion transporter band 3 by Syk tyrosine kinase, (iii) the concentration at which it inhibits Syk tyrosine kinase is at least about 20 times less than the concentration at which the compound inhibits HGFR, EGFR), FGFR3, IGF1R and / or VEGFR, Wherein, in addition to or as an alternative to (ii) and (iii), the compound of (i) further has: (iv) a clogP value of about 2 to about 5 (e.g., 2 to about 5, about 2 to 5, or 2 to 5), (v) a polar surface area of ​​less than about 250 square angstroms (e.g., less than 250 square angstroms), (vi) no more than 5 hydrogen bond donors, (vii) not more than 15 hydrogen bond acceptors, and / or (viii) not more than 20 rotatable bonds.

[0035] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure: Where X is CH or N, and R is

[0036] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure: Where X is CH or N, and R is

[0037] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure: Where X is CH or N, and R is

[0038] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure: Where X is CH or N, and R is

[0039] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure: Wherein X is CH or N; Y is CH or N, and when X and Y are all CH, the compound may have a weaker binding affinity than when at least one or more X and / or Y is N.

[0040] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure: Wherein X is CH or N; Y is CH or N, and when X and Y are all CH, the compound may have a weaker binding affinity than when at least one or more X and / or Y is N.

[0041] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure: Where R is and each X is independently CH or N.

[0042] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure: wherein each X is independently CH or N; each Y is CH or N; and R is a carbocycle or a heterocycle; and wherein when X and Y are all CH, the compound may have a weaker binding affinity than when at least one or more X and / or Y is N.

[0043] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure: wherein each X is independently CH or N; Y is CH or N; and R is carbocyclic or heterocyclic; and wherein when X and Y are all CH, the compound may have a weaker binding affinity than when at least one or more X and / or Y is N.

[0044] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure: wherein each X is independently CH or N; Y is CH or N; and R is carbocyclic or heterocyclic; and wherein when X and Y are all CH, the compound may have a weaker binding affinity than when at least one or more X and / or Y is N.

[0045] The compound may have or include the following structure, or a pharmaceutically acceptable salt of the following structure: In an alternative embodiment, the hydroxyl group (HO-) of the above compounds can be substituted by phosphate or phosphonate. In another alternative embodiment, the hydroxyl group (HO-) can be substituted by RC(=O)O-, wherein R is an alkyl group (e.g., C1-C6 alkyl group).

[0046] Also provided is a pharmaceutical composition comprising a compound described herein (or a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable carrier or excipient. The composition may include, for example, Cremophor, polysorbate, nanoparticles, polymers or hydrogels.

[0047] A method for treating sickle cell disease is also provided. The method comprises administering an effective amount of: (a) the above compound; (b) fostamatinib; (c) PRT062607; (d) TAK-659; (e) TAE-684; (f) entosatinib; (g) lanraprinib; (h) sedutinib; (i) piceatannol; (j) S701; (k) Syk II; (l) Syk IV; (m) TAS05567; (n) GSK143; (o) Syk-IN-3; (p) Syk-IN-4; (q) Syk-IN-1; (r) SRX3207; (s) RO9021; (t) gusartinib; (u) R112; (v) PRT-060318; (w) OXSI-2; (x) (a) and (b); (y) (a) and (c); (z) (a) and (d); (aa) (a) and (e); (ab) (a) and (f); (ac) (a) and (g); (ad) (a) and (h); (ae) (a) and (i); (a f)(a) and (j); (ag)(a) and (k); (ah)(a) and (l); (ai)(a) and (m); (aj)(a) and (n); (ak)(a) and (o); (al)(a) and (p); (am)(a) and (q); (an)(a) and (r); (ao)(a) and (s); (ap)(a) and (t); (aq)(a) and (u); (ar)(a) and (v); (as)(a) and (w); or (at)(a) and one or more of (b)-(w); optionally as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

[0048] Also provided are methods of treating red blood cell (RBC) mediated diseases. In certain embodiments, the method of treating an RBC mediated disease comprises administering to a subject (e.g., a human patient) an effective amount of: a compound as described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein. In certain embodiments, the compound is: Compound of formula X: ABC(X) or a pharmaceutically acceptable salt thereof, wherein: A is a nitrogen-linked and optionally substituted first ring, including a fused bicyclic or monocyclic ring, wherein Each ring of the first ring contains a 4-6 membered heterocyclic or carbocyclic ring; B is a second ring, which is optionally substituted with at least one substituent, wherein the second ring comprises a monocyclic ring or a fused bicyclic or tricyclic ring, and wherein each ring of the second ring comprises a 5-membered or 6-membered heterocyclic or carbocyclic ring; and C is a third bicyclic ring comprising a 5- or 6-membered carbocyclic ring fused to a 5- or 6-membered carbocyclic or heterocyclic ring, wherein The third bicyclic ring is substituted with at least a first substituent comprising: or amines, or alcohols, in: X4 is each independently CH2, NH, CO, O or S, Y5 is each independently CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and and is the point of attachment of the first substituent to the third bicyclic ring, wherein the concentration of the compound inhibiting Syk tyrosine kinase is at least one twentieth less than the concentration of the compound inhibiting HGFR, EGFR, FGFR3, IGF1R and / or VEGFR; or The invention relates to a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt thereof.

[0049] The RBC-mediated disease may be selected from the group consisting of sickle cell disease, thalassemia, glucose-6-phosphate dehydrogenase deficiency, glutathione reductase deficiency, and a disease involving the release of microvesicles from blood cells. In certain embodiments, the RBC-mediated disease is alpha-thalassemia or beta-thalassemia alone or in combination with sickle cell disease.

[0050] Also provided are methods of preventing or inhibiting phosphorylation of human erythrocyte anion transporter band 3 in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of a compound described herein (or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition thereof.

[0051] In each of the methods herein, administering a compound, a pharmaceutical salt thereof, or a pharmaceutical composition to a subject can stabilize the subject's red blood cell membranes and reduce the release of hemoglobin from sickle cells in the subject. In this way, for example, the risk of a subject experiencing a vaso-occlusive crisis can be reduced. In certain embodiments, the compound, a pharmaceutical salt thereof, or a pharmaceutical composition is administered at an IC of 200 nM or less. 50 Inhibition of Syk tyrosine kinase. In certain embodiments, the subject is a human child.

[0052] In certain embodiments, the method herein further comprises administering to the subject an effective amount of at least one second active agent. The at least one second active agent can be selected from the group consisting of fotatinib, PRT062607, TAK-659, TAE-684, entosatinib, lanraprinib, sedutinib, piceatannol, S701, Syk II, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, Syk-IN-1, SRX3207, RO9021, gusartinib, R112, PRT-060318, and OXSI-2. In certain embodiments, at least one second active agent is two or more of fotatinib, PRT062607, TAK-659, TAE-684, entosatinib, lanraprinib, sedutinib, piceatannol, S701, Syk II, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, Syk-IN-1, SRX3207, RO9021, gusartinib, R112, PRT-060318, and OXSI-2. At least one second active agent can be administered simultaneously with the compound (or a pharmaceutically acceptable salt thereof) or pharmaceutical composition or sequentially in any order.

[0053] The method may further comprise administering to the subject an effective amount of hydroxyurea. Hydroxyurea may be administered simultaneously with the compound (or a pharmaceutically acceptable salt thereof) or pharmaceutical composition or sequentially in any order.

[0054] In certain embodiments, the at least one second active agent comprises more than one second active agent. In certain embodiments, the more than one second active agent is all selected from the group consisting of fotatinib, PRT062607, TAK-659, TAE-684, entosatinib, lanraprinib, sedutinib, piceatannol, S701, Syk II, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusartinib, R112, PRT-060318 and OXSI-2, optionally, wherein the at least one second active agent is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

[0055] Another method of treating sickle cell disease in a subject is provided. The method comprises administering an effective amount of a compound or a pharmaceutically acceptable salt thereof: (i) which comprises a rigidly fused bicyclic ring substituted with at least two substituents, at least one of which is an N-linked, optionally substituted carbocyclic or heterocyclic ring, and the other of which is indole substituted with at least one substituent, at least one of which is a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring, (ii) It inhibits Syk phosphorylation of human erythrocyte anion transporter band 3, (iii) the concentration at which it inhibits Syk tyrosine kinase is at least about 20 times less than the concentration at which the compound inhibits HGFR, EGFR, FGFR3, IGF1R and / or VEGFR, Wherein, in addition to or as an alternative to (ii) and (iii), the compound of (i) further has: (iv) a CLogP value of about 2 to about 5 (e.g., 2 to about 5, about 2 to 5, or 2 to 5), (v) a polar surface area of ​​less than about 250 square angstroms (e.g., less than 250 square angstroms), (vi) no more than 5 hydrogen bond donors, (vii) not more than 15 hydrogen bond acceptors, and / or (viii) not more than 20 rotatable bonds. The subject is thereby treated for sickle cell disease. The compound (or a pharmaceutically acceptable salt thereof) can inhibit the phosphorylation of tyrosine 8 and tyrosine 21 in human erythrocyte anion transporter band 3 by Syk. The compound (or a pharmaceutically acceptable salt thereof) can stabilize erythrocyte membranes and reduce the release of sickle cell microvesicles. The compound (or a pharmaceutically acceptable salt thereof) can stabilize erythrocyte membranes and reduce the release of sickle cell hemoglobin. The risk of a vaso-occlusive crisis in a subject can be reduced. The compound (or a pharmaceutically acceptable salt thereof) can be used to treat sickle cell disease in a subject with an IC of 200 nM or less. 50 Inhibition of Syk tyrosine kinase. The subject may be a human child.

[0056] The method may also include administering (a) the compound, its pharmaceutically acceptable salt or composition, and (b) (e.g., an effective amount) at least one second active agent selected from the group consisting of fotatinib, PRT062607, TAK-659, TAE-684, entolinib, lanraprinib, sedutinib, piceatannol, S701, SykII, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusartinib, R112, PRT-060318 and OXSI-2 simultaneously or sequentially in any order, optionally wherein the at least one second active agent is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient. The compound, its pharmaceutically acceptable salt or pharmaceutical composition and at least one second active agent may be administered by the same route. The compound, its pharmaceutically acceptable salt or pharmaceutical composition and at least one second active agent can be administered by different routes. In certain embodiments, at least one second active agent includes hydroxyurea. In certain embodiments, the at least one second active agent comprises more than one second active agent, all of which are selected from the group consisting of fotatinib, PRT062607, TAK-659, TAE-684, entolinib, lanraprinib, sedutinib, piceatannol, S701, Syk II, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusartinib, R112, PRT-060318 and OXSI-2, optionally, wherein the at least one active agent is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

[0057] Also provided is a method for treating a subject for a disease involving the release of microvesicles from blood cells. The method comprises administering an effective amount of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a rigid fused bicyclic ring substituted with at least two substituents, wherein at least one substituent is an N-linked, optionally substituted carbocyclic or heterocyclic ring, and the other substituent is an indole substituted with at least one substituent comprising a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring. The disease may be thalassemia. The thalassemia may be alpha-thalassemia or beta-thalassemia alone or in combination with sickle cell disease. The disease may be glucose-6-phosphate dehydrogenase deficiency. The disease may be glutathione reductase deficiency.

[0058] The method may also include administering the compound or its pharmaceutically acceptable salt and (e.g., an effective amount) Src inhibitor simultaneously or in any order, and optionally wherein the Src inhibitor is formulated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient. The Src inhibitor and the compound (or its pharmaceutically acceptable salt) may be administered by the same or different routes. The Src inhibitor may be dasatinib, ibrutinib, barfitinib, PP1, PP2, PP121, or a combination of two or more of the above.

[0059] Also provided is a method of treating sickle cell disease in a subject, the method comprising administering an effective amount of the following compound or a pharmaceutically acceptable salt thereof: (i) it comprises a pyrazine substituted with at least three substituents, at least one of which is a carboxamide or a lactam, another of which is an N-linked, optionally substituted carbocyclic or heterocyclic ring, another of which is a carbocyclic or heterocyclic ring having at least one substituent comprising a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring, (ii) It inhibits the phosphorylation of human erythrocyte anion transporter band 3 by Syk tyrosine kinase, (iii) the concentration at which it inhibits Syk tyrosine kinase is at least about 20 times less than the concentration at which the compound inhibits HGFR, EGFR, FGFR3, IGF1R and / or VEGFR, Wherein, in addition to or as an alternative to (ii) and (iii), the compound of (i) further has: (iv) a CLogP value of about 2 to about 5 (e.g., 2 to about 5, about 2 to 5, or 2 to 5), (v) a polar surface area of ​​less than about 250 square angstroms (e.g., less than 250 square angstroms), (vi) no more than 5 hydrogen bond donors, (vii) not more than 15 hydrogen bond acceptors, and / or (viii) not more than 20 rotatable bonds, The subject is thereby treated for sickle cell disease. The compound (or a pharmaceutically acceptable salt thereof) can inhibit the phosphorylation of tyrosine 8 and tyrosine 21 in band 3 of human erythrocyte anion transporter by Syk tyrosine kinase. The compound (or a pharmaceutically acceptable salt thereof) can stabilize erythrocyte membranes and reduce the release of microvesicles from sickle cells. The compound (or a pharmaceutically acceptable salt thereof) can stabilize erythrocyte membranes and reduce the release of hemoglobin from sickle cells. The risk of a vaso-occlusive crisis in a subject can be reduced. The compound (or a pharmaceutically acceptable salt thereof) can be administered at an IC of 200 nM or less. 50 Inhibition of Syk tyrosine kinase. The subject may be a human child.

[0060] The method may also include administering (a) a compound (or a pharmaceutically acceptable salt thereof) and (b) one or more of fostamatinib, PRT062607, TAK-659, TAE-684, entosatinib, lanraprinib, sedutinib, piceatannol, S701, Syk II, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusartinib, R112, PRT-060318, and OXSI-2 simultaneously or sequentially in any order, optionally as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient. One or more other compounds may be administered by the same or different routes.

[0061] Also provided is a method for treating a disease involving the release of microvesicles from blood cells in a subject. The method comprises administering an effective amount of a compound (or a pharmaceutically acceptable salt thereof) comprising a pyrazine substituted with at least three substituents, at least one of the substituents being a carboxamide or a lactam, another of the substituents being an N-linked, optionally substituted carbocyclic or heterocyclic ring, another of the substituents being a carbocyclic or heterocyclic ring having at least one substituent comprising a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring, thereby treating a disease involving the release of microvesicles in the subject. The disease may be thalassemia. The thalassemia may be alpha-thalassemia or beta-thalassemia alone or in combination with sickle cell disease. The disease may be glucose-6-phosphate dehydrogenase deficiency. The disease may be glutathione reductase deficiency. The method may further comprise administering the compound (or a pharmaceutically acceptable salt thereof) and a Src inhibitor simultaneously or sequentially in any order, optionally as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient. The Src inhibitor may be administered by the same or different route as the compound (or a pharmaceutically acceptable salt thereof). The Src inhibitor can be dasatinib, ibrutinib, bafitinib, PP1, PP2, PP121, or a combination of two or more thereof. Sequence Listing

[0062] The sequence herein (SEQ ID NO: 1) is also provided in computer readable form, encoded in a file filed with this article, and incorporated herein by reference. The information recorded in computer readable form is the same as the written sequence listing provided below, in accordance with 37 CFR § 1.821 (f).

[0063] SEQ ID NO: 1 is the amino acid sequence of spleen tyrosine kinase (Syk) protein:.

[0064] The contents of the XML file for the sequence listing (named "PRF69753-03SeqListingXML.xml", 2.7 kb in size, created on March 13, 2023, and electronically submitted through Patent Center on March 14, 2023) are incorporated herein by reference in their entirety. The information recorded in a computer-readable form is the same as the written sequence listing (paper) provided herein pursuant to 37 CFR § 1.821(f). DETAILED DESCRIPTION

[0065] Although the concepts of the present disclosure are specifically illustrated and described in the specification herein, the results in the specification should be regarded as illustrative rather than restrictive; it should be understood that only exemplary embodiments have been shown and described, and all changes and modifications within the spirit of the disclosure are intended to be protected.

[0066] The present disclosure is based, at least in part, on the unprecedented discovery of a compound comprising a small molecule inhibitor that inhibits spleen tyrosine kinase (Syk) without significantly disrupting other pathways that provide regulated growth factor kinases associated with growth and development in a subject. The compound can be used to treat red blood cell (RBC) diseases, such as hemoglobinopathies (e.g., sickle cell disease (SCD) and thalassemia) and other RBC-mediated diseases, such as glucose-6-phosphate dehydrogenase (G6PD) deficiency and glutathione reductase deficiency.

[0067] Phosphorylation / dephosphorylation of protein tyrosine residues is involved in the regulation of a variety of erythrocyte functions, including cell shape. The above-mentioned RBC diseases involve a mechanism in which increased band 3 tyrosine phosphorylation triggers sequelae that lead to vascular occlusion and disease symptoms. In this mechanism, accelerated denaturation of sickle hemoglobin (HbS), heme formation, and heme release can work together to induce oxidative stress within erythrocytes. Increased oxidative stress leads to the inhibition of erythrocyte / RBC tyrosine phosphatases, which normally prevent constitutive band 3 tyrosine phosphorylation. Following inhibition of these phosphatases, hyperphosphorylation of band 3 subsequently induces global instability of the erythrocyte membrane, accelerating intravascular hemolysis and microparticle (MP) release. Increased plasma hemoglobin and heme can also "activate" the vascular endothelium, leading to the expression of adhesion receptors (e.g., p-selectin, E-selectin, and von Willebrand factor), as well as the sequestration of vasodilators (NO), while the release of MPs can trigger intravascular thrombosis by activating prothrombin.

[0068] Ultimately, these processes can promote vaso-occlusive events and lead to symptoms of SCD and other RBC diseases, such as tissue hypoxia, ischemia-reperfusion injury, organ damage and associated pathology, and debilitating pain, which can cause tremendous suffering and may require medical treatment and / or hospitalization. When occurring simultaneously with loss of RBC deformability and increased vascular adhesion, the sequelae associated with membrane weakening can exacerbate already compromised blood flow, leading to microemboli and progressive tissue damage. Clinical symptoms can be further exacerbated by sickle cell hemolysis, shortened sickle cell lifespan, and anemia.

[0069] At least one of the kinases involved in the phosphorylation of Band 3 is Syk (p72 syk) Syk is a non-receptor protein tyrosine kinase that is abundantly expressed in RBCs. For discussion of the amino acids in Syk, reference is made to the long isoform (Uniprot ID DP43405-1), which has SEQ ID NO: 1. Alternative splicing results in the deletion of amino acids 283-305, resulting in the short isoform (Uniprot ID P43405-2).

[0070] Syk binds to the cytoplasmic region of cells bearing Fcγ-activated receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs). Receptor binding results in phosphorylation, which leads to Syk activation by increasing its autophosphorylation activity. Syk activation leads to the activation of various downstream kinases, ultimately leading to the release of proinflammatory cytokines and the above-mentioned cascade reactions.

[0071] Compound

[0072] The compounds herein include small molecule inhibitors that inhibit Syk (i.e., bind to and inhibit Syk receptors), thereby inhibiting and / or preventing the cascade by inhibiting band 3 tyrosine phosphorylation in affected RBCs. In addition, these compounds can inhibit the release of MP and hemoglobin by affected RBCs.

[0073] In certain embodiments, the compound has Formula X: ABC(X) or a pharmaceutically acceptable salt thereof, wherein: A is a nitrogen (N)-linked and optionally substituted first ring, the first ring comprising a fused bicyclic or monocyclic ring, wherein each ring of the first ring comprises a 4-6 membered heterocyclic or carbocyclic ring; B is a second ring optionally substituted with at least one substituent, the second ring comprising a monocyclic ring or a fused bicyclic or tricyclic ring, and wherein each ring of the second ring comprises a 5-membered or 6-membered heterocyclic or carbocyclic ring; and C is a third bicyclic ring comprising a 5- or 6-membered carbocyclic ring fused to a 5- or 6-membered carbocyclic or heterocyclic ring, wherein the third bicyclic ring is substituted with at least the following substituents: or an amine, or a ketone, or an amide, or an alcohol (such as propanol), in: X4 is CH2, NH, CO, O or S, Y5 is CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and and is the attachment point.

[0074] A of the compound can be: Or a pharmaceutically acceptable salt of any of the foregoing structures, wherein: Each X' is independently selected from CH and NH; Each Y2 is independently selected from CH2, NH, O and S; Z2 is CH or N; and Each R3 is independently selected from H, F, Cl, OH and -Y-(CH2)n -, where n = 1-14.

[0075] The compound A can be in is the point of attachment to B, and W is O, NH, or SH.

[0076] The compound A can be in is the point of attachment to B.

[0077] The compound A can be in is the point of attachment to B.

[0078] The nitrogen attached to the first ring of A (eg, a fused bicyclic or monocyclic ring) can form a nitrogen bridge with B of the compound.

[0079] The first ring of A may include a 4-6 membered heterocyclic ring (e.g., containing one or more N, O or S). The first ring of A may include a 4-6 membered carbocyclic ring. The first ring of A may be a fused bicyclic ring including two 6 membered rings. The first ring of A may be a fused bicyclic ring including a 6 membered ring and a 5 membered ring.

[0080] In certain embodiments, A is substituted (e.g., the first ring of A is substituted). A can be a single ring substituted with a bulky substituent para to the nitrogen bridge. Compared to the compound lacking the bulky substituent para to the nitrogen bridge but otherwise identical, the compound has a higher docking score to Syk tyrosine kinase (see Table 1 below).

[0081] In certain embodiments, the bulky substituent is morpholine, for example having the structure:

[0082] The first ring of A can be or comprise a monocyclic carbocycle (eg, a 6-membered carbocycle). In certain embodiments, the first ring of A comprises benzene.

[0083] In certain embodiments, the first ring of A is benzene substituted with at least two substituents. The first ring of A can be or include a monocyclic carbocyclic ring (e.g., a 6-membered carbocyclic ring), which is optionally substituted (e.g., substituted with two methoxy groups). The first ring of A can include a closed ring. The first ring of A can include a first closed ring (e.g., a carbocyclic ring) fused with a second open ring. The first ring of A can be a bicyclic ring and include a first closed ring (e.g., a 6-membered carbocyclic ring) fused with a second closed ring (e.g., a 5-membered carbocyclic ring or a heterocyclic ring), any of which can be substituted or not substituted.

[0084] As described above, the first ring of A may be optionally substituted with one or more substituents, each of which is independently selected from hydrogen, deuterium, halogen, azido, cyano, nitro, hydroxyl, amino, thio, carboxyl, ester, amide and derivatives thereof, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloheteroalkyl, cycloheteroalkenyl, acyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, morpholine and nitrogen heterocycle.

[0085] In certain embodiments, the compound has the following structure: or a pharmaceutically acceptable salt thereof, wherein: R1 is R2 is or an amine, or an alcohol (such as propanol), R4 is H, CH3, amine or methanol, X4 is independently CH2, NH, CO, O or S, Y and Y5 are each independently CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and and is the attachment point.

[0086] B of the compound may be a second ring, which is optionally substituted by at least one substituent, wherein the second ring is a monocyclic ring or includes a fused bicyclic ring or a fused tricyclic ring. Each ring of the second ring may include a 5-membered or 6-membered carbon ring or heterocyclic ring.

[0087] The one or more substituents of B may each be independently selected from the group consisting of hydrogen, deuterium, amide and derivatives thereof, carbonyl and alkyl.

[0088] Compound B may be rigid.

[0089] B may be a monocyclic ring, optionally substituted with an amide. B may be pyridine. B may be pyrazine. B may be pyrazine substituted with at least one substituent. B may include imidazole substituted with at least one amide.

[0090] B can be a fused bicyclic ring, optionally substituted (eg, optionally substituted with an amide or carbonyl group). In certain embodiments, the fused bicyclic ring includes a pyrimidine fused to an optionally substituted N-containing 5-membered heterocyclic ring.

[0091] In certain embodiments, B is: in: Each X2 is independently selected from CH and N; Y3 is selected from O, NH and S; and Z3 is selected from OH, NH2 and NHCH3.

[0092] In certain embodiments, the compound B is not For example, B of the compound may be or include: in: Each X2 is independently selected from CH and N; Y3 is selected from O, NH and S; and Z3 is selected from OH, NH2 and NHCH3, provided that B is not:

[0093] When the compound (or a pharmaceutically acceptable salt thereof) is administered to a subject, B can form a hydrogen bond with amino acid residue Ala451 in the catalytic domain of Syk tyrosine kinase, and Z of the first substituent of B can form a hydrogen bond with amino acid residue Asp512 in the catalytic domain of Syk tyrosine kinase.

[0094] The C of the compound comprises a third bicyclic ring. The third ring may comprise a 5-membered or 6-membered carbocyclic ring fused to a 5-membered or 6-membered carbocyclic ring or heterocyclic ring. In certain embodiments, C comprises a third bicyclic ring having a 6-membered carbocyclic ring fused to a 5-membered heterocyclic ring (e.g., an N-containing heterocyclic ring) or a carbocyclic ring. In certain embodiments, C comprises a third bicyclic ring having a 6-membered carbocyclic ring fused to a 6-membered heterocyclic ring (e.g., an N-containing heterocyclic ring) or a carbocyclic ring. The bicyclic ring of C may be indole.

[0095] The third fused bicyclic ring is substituted by at least one substituent (eg, the first substituent). C may include the following structure: wherein each X3 is independently selected from CH and N; Y4 is CH2, NH, O or SH; and is the point of attachment of at least one substituent of the third ring (C).

[0096] In certain embodiments, the third bicyclic ring of C is substituted with at least a first substituent comprising: or an amine, or a ketone, or an amide, or an alcohol (e.g., propanol), wherein: X is each independently CH2, NH, CO, O or S, Y is each independently CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and and is the point of attachment (eg, to a 5- or 6-membered carbocyclic or heterocyclic ring of C in the compound).

[0097] In certain embodiments, Z of the first substituent is -OR, wherein R is And n=1-14.

[0098] In certain embodiments, C comprises the structure: wherein each X3 is independently selected from CH and N; Y is CH2, NH, O or SH; and is the point of attachment to the first substituent, wherein the first substituent of the third bicyclic ring of C is:

[0099] The bicyclic ring of C may be indole, and the first substituent of C may comprise a 4-6 membered, optionally substituted carbocyclic or heterocyclic substituent. In certain embodiments, the first substituent of C is a lipophilic ester. In certain embodiments, the bicyclic ring of C is indole, and the first substituent of C is a lipophilic ester.

[0100] The first substituent Z of C may include a phosphate or a phosphonate. In certain embodiments, the substituent of C is wherein: Q is a phosphate or phosphonate; or Q is RC(=O)O-, wherein R is an alkyl group.

[0101] C may further comprise at least a second substituent. In certain embodiments, C is substituted with CH3, H, CH2OH, amine and / or alkyl.

[0102] One or more hydrogen (H) of the compound may be optionally replaced by deuterium.

[0103] In certain embodiments, (e.g., when administered to a subject) B of the compound can form a hydrogen bond with amino acid residue Ala451 in the catalytic domain of Syk tyrosine kinase, and the first substituent of C can form a hydrogen bond with amino acid residue Asp512 in the catalytic domain of Syk tyrosine kinase.

[0104] The compound may have a CLogP value of about 2 to about 5 (e.g., 2 to about 5, about 2 to 5, or 2 to 5). The compound may have a polar surface area of ​​less than about 250 square angstroms (e.g., less than 250 square angstroms). The compound may have no more than 5 hydrogen bond donors. The compound may have no more than 15 hydrogen bond acceptors. The compound may have no more than 20 rotatable bonds. In certain embodiments, the compound may have a CLogP value of about 2 to about 5 (e.g., 2 to about 5, about 2 to 5, or 2 to 5); a polar surface area of ​​less than about 250 square angstroms (e.g., less than 250 square angstroms); no more than 5 hydrogen bond donors; no more than 15 hydrogen bond acceptors; and / or no more than 20 rotatable bonds.

[0105] The compound can specifically bind to Syk tyrosine kinase. When administered to a subject, in certain embodiments, the compound inhibits Syk from phosphorylating erythrocyte (eg, human erythrocyte) anion transporter band 3.

[0106] The compound may include a structure of formula (XX): or a pharmaceutically acceptable salt thereof, wherein ring A is in: Each X is independently selected from CH and NH, Each Y is independently selected from CH2, NH, O and S, Z is CH or N, and Each R is independently selected from H, F, Cl, OH and -Y-(CH2) n -, wherein n = 1-14; wherein ring B is in: Each X2 is independently selected from CH and N; Y3 is selected from O, NH and S; and Z3 is selected from OH, NH2 and NHCH3, provided that B is not Wherein ring C is in: Each X3 is independently selected from CH and N, and Y4 is selected from CH2, NH, O and SH; and Wherein ring D is in: X4 is selected from CH2, NH, CO, O and S, Y5 is selected from CH and N; Z is selected from OH, SH, NH2, NHCH3 and And one or more hydrogens may optionally be replaced by deuterium.

[0107] Rings A, B, C and D of formula (XX) may comprise any of the above embodiments of A, B and C of the compound of formula (X) above. For example, ring A of formula (XX) may be substituted by a bulky substituent para to the nitrogen bridge. The bulky substituent may be a 6-membered, optionally substituted, open or closed carbocyclic or heterocyclic ring. The bulky substituent may be morpholine. In addition, ring D of formula (XX) may comprise any embodiment of the first substituent of C in formula (X).

[0108] The compound may include the following structure: (Docking scores for Syk tyrosine kinase are shown directly below each compound) or a pharmaceutically acceptable salt of any of the foregoing.

[0109] The compound may include the following structure: or or a pharmaceutically acceptable salt of any of the foregoing.

[0110] The compound may include the following structure: or a pharmaceutically acceptable salt thereof, wherein each X is independently selected from CH2, N, NH, O and S; Y is O, NH or SH; and Z is OH or NH2. The 4-6 membered, optionally substituted carbocyclic or heterocyclic substituents on the indole may include lipophilic esters. Z on ring D may be -OR, wherein R is And n=1-14.

[0111] The compound may be a compound comprising pyrazine (or a pharmaceutically acceptable salt thereof), wherein the pyrazine is substituted with at least three substituents, wherein at least one substituent is a carboxamide or a lactam, another substituent is an N-linked, optionally substituted carbocyclic or heterocyclic ring, and another substituent is a carbocyclic or heterocyclic ring having at least one substituent, wherein at least one substituent is a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring.

[0112] The compound may have the following structure: or a pharmaceutically acceptable salt thereof, wherein: R1 is R2 is or an amine, or an alcohol (such as propanol), X is each independently CH2, NH, CO, O or S, Y is each independently CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and and is the attachment point.

[0113] In certain embodiments, the compound comprises: or a pharmaceutically acceptable salt thereof, wherein X is CH or N, and R is

[0114] In certain embodiments, the compound comprises: or a pharmaceutically acceptable salt thereof, wherein X is CH or N, and R is

[0115] In certain embodiments, the compound comprises: or a pharmaceutically acceptable salt thereof, wherein X is CH or N, and R is

[0116] In certain embodiments, the compound comprises: or a pharmaceutically acceptable salt thereof, wherein X is CH or N, and R is

[0117] In certain embodiments, the compound comprises: or a pharmaceutically acceptable salt thereof, wherein each X is independently CH or N, and Y is CH or N. In certain embodiments, when each X and Y is CH, the compound may exhibit a weaker binding affinity to Syk tyrosine kinase than when at least one X and / or Y is not CH.

[0118] In certain embodiments, the compound comprises: or a pharmaceutically acceptable salt thereof, wherein each X is independently CH or N, and Y is CH or N. In certain embodiments, when each X and Y is CH, the compound may exhibit a weaker binding affinity to Syk tyrosine kinase than when at least one X and / or Y is not CH.

[0119] The compound may include the following structure: or a pharmaceutically acceptable salt thereof, wherein: R is and X is independently CH or N.

[0120] The compound may include the following structure: or a pharmaceutically acceptable salt thereof, wherein: R is a carbocyclic or heterocyclic ring; X is independently CH or N; and Y is CH or N.

[0121] The compound may include the following structure: or a pharmaceutically acceptable salt thereof.

[0122] In certain embodiments, the hydroxyl (HO-) group of the first substituent of C of formula (X) or the first substituent of ring D of formula (XX) may be substituted with a phosphate or phosphonate. For example, the first substituent of the bicyclic ring of C may be For example, see compound S2053P. This change can improve the solubility and / or cell permeability of the compound. In an alternative embodiment, the hydroxyl (HO-) group can be replaced by RC(=O)O-, where R is an alkyl group (e.g., C1-C6 alkyl). This change can improve cell permeability.

[0123] The compound may have the following structure: Or a pharmaceutically acceptable salt of any of the foregoing structures.

[0124] The compound may also include a phosphate prodrug version. In certain embodiments, for example, the compound is: or a pharmaceutically acceptable salt thereof.

[0125] The compound may have or contain the structure Or a pharmaceutically acceptable salt of any of the foregoing structures.

[0126] In certain embodiments, the compound (or a pharmaceutically acceptable salt thereof) comprises a rigid fused bicyclic ring substituted with at least two substituents, wherein at least one substituent is an N-linked, optionally substituted carbocyclic or heterocyclic ring, and another substituent is indole substituted with at least one substituent, wherein at least one substituent is a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring.

[0127] As described above, the compound can be a Syk specific inhibitor. The compound can specifically bind to the Syk receptor, thereby reducing off-target interactions, such as interactions with growth factor receptors. When referring to small molecules / receptors or other binding pairs, "specific binding", "high affinity binding" or "specific" or "selective" binding means that under specified conditions, a specific compound or its recognition region binds to a specific receptor or site (e.g., a Syk inhibition site, such as the ATP binding catalytic site of a tyrosine kinase domain), and will not bind to other receptors present (e.g., off-target growth factor receptors) in large quantities. Specific binding or high affinity binding can also mean, for example, that the affinity of a binding compound, ligand or binding composition to its target (e.g., Syk inhibition) is generally higher than the affinity of the compound, ligand or binding composition to another receptor (e.g., growth factor receptor) by at least 20%, at least 25%, at least 50% or at least 100% (2 times).

[0128] In typical embodiments, a molecule that specifically binds to a target will have at least about 10 6 Liter / mol (K ′O =10 ～6 The binding specificity of a compound to a Syk receptor is determined using standard techniques known in the art and described herein.

[0129] When administered to a subject, the compound can inhibit Syk from phosphorylating erythrocyte anion transporter band 3 of the subject (e.g., a human). For example, the overall design of the compound can allow for inhibition of Syk without disrupting other pathways (e.g., pathways regulated by growth factor kinases). Although other Syk inhibitors are known, conventional Syk inhibitors are not specific for Syk (i.e., they inhibit several kinases in addition to inhibiting Syk, including, for example, growth factor receptors necessary for growth and development (e.g., vascular endothelial growth factor receptors (VEGFRs, such as VEGFR3) or other growth factor receptors)). Inhibition of growth factor kinases is generally undesirable because it may lead to dwarfism and other growth disorders in pediatric patients. For example, studies support that imatinib can delay vertical growth, hormone production, and vitamin D synthesis in subjects. In addition, in order to cope with situations such as vascular occlusion, it is often necessary to upregulate (rather than inhibit) these growth factors because it can promote tissue repair and regeneration (e.g., tissue angiogenesis).

[0130] Non-limiting examples of such growth factor receptors include hepatocyte growth factor receptor (HGFR), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor 3 (FGFR3), and insulin-like growth factor 1 receptor (IGF1R).

[0131] HGFR (or MET) binds to hepatocyte growth factor (HGF, also known as discrete factor (SF)) to transduce multiple biological processes such as cell motility, morphogenesis, proliferation, and survival. The HGFR precursor is proteolytically cleaved to form α and β subunits, which in turn form disulfide-linked heterodimers. HGFR is widely expressed in early development, and deletion of the gene fatally disrupts embryogenesis. After kidney, liver, or heart damage, HGFR is upregulated to promote tissue repair and regeneration.

[0132] EGFR (or ErbB-1 or HER1) can bind to members of the epidermal growth factor (EGF) protein family. EGFR is a member of the ErbB receptor family, which includes EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4). EGFR is activated by EGF and converts from an inactive monomeric form to an active homodimer. EGFR can also form heterodimers with another ErbB receptor to form active heterodimers. These receptors are involved in early development and in the renewal of stem cells in tissues such as the skin, liver, intestines and mammary ducts.

[0133] FGFR3 binds to members of the fibroblast growth factor (FGF) family of proteins. The FGF family is the largest family of growth factor receptor ligands, consisting of 22 factors. Alternative splicing of the four FGFR genes can produce more than 48 different FGFR isoforms. In addition, FGFR dimerizes into heterodimers and homodimers. FGFR3 regulates chondrocyte differentiation, proliferation and apoptosis, vitamin D metabolism, osteogenesis, and postnatal bone mineralization by osteoblasts, and is required for normal skeletal development and inner ear development.

[0134] IGF1R is activated by the hormone insulin-like growth factor 1 (IGF-1) and a related hormone called IGF-2. IGF1R regulates prenatal and postnatal development, glucose metabolism, and neutrophil physiology, and promotes protective physiological hypertrophy of the left ventricle of the heart.

[0135] VEGFR interacts with vascular endothelial growth factor (VEGF), which is a signaling protein that stimulates angiogenesis. There are three major isoforms of VEGFR - 1, 2 and 3. Depending on alternative splicing, they can be membrane-bound or soluble. There are five members of the VEGF family, namely VEGF-A, placental growth factor (PGF), VEGF-B, BEGF-C and VEGF-D. Alternative splicing can produce multiple isoforms. Not inhibiting VEGFR (such as VEGFR3) is advantageous because administration of the compound will not prevent the necessary revascularization of tissues that have been infarcted due to vaso-occlusive events in patients with sickle cell disease.

[0136] In certain embodiments, the compound includes a small molecule inhibitor that is specific for Syk and inhibits Syk at least about 20-fold better (eg, 20-fold better) than it inhibits a growth factor receptor (eg, the small molecule inhibitor is specific for Syk).

[0137] First, the docking scores of several compounds were evaluated and are provided in Table 1. Determining the three-dimensional structure of the protein-receptor complex is an important component of structure-based drug design. The root mean square deviation was less than about Corresponds well to the docking scheme. Table 1. Docking scores of selected compounds with SYK enzyme

[0138] Additional unexpected and surprising properties of the compounds (e.g., S970, S970D, S1115, S1213, S1219, S1213-CH3, S2053, S2054, and S1214) compared to commercially available / previously known Syk inhibitors are shown in Tables 2 and 3. Table 2 shows the activity (IC 50 nM) results. IC 50 The value was determined as the concentration of compound (or other tested Syk inhibitor) that reduced the activity of the identified receptor by 50%. Table 3 shows the selectivity ratio of each selected compound, expressed as IC 50 The Syk / Kinase ratio is represented. Table 2: Selectivity and enzyme potency data of compounds compared with commercially available Syk inhibitors and FDA-approved Syk inhibitors Table 3: Selectivity results

[0139] As shown in Tables 2 and 3, the following FDA-approved inhibitors are known to inhibit growth factor receptor kinases (i.e., HGR, EGFR, VEGFR, IGF1R, and FGFR3): afatinib, ametinib, anlotinib, axitinib, brigatinib, bosutinib, cabozantinib, capmatinib, serotoninib, ceritinib, ceritinib, crizotinib, dacomitinib, dasatinib, erdafitinib, erlotinib, fostamatinib, fruquintinib, gefitinib, icotinib, lapatinib, lenvatinib, lestaurine, lorlatinib, midostaurin, neratinib, nintedanib, osimertinib, pemigatinib, pralsetinib, pyrotinib, regorafenib, selpatinib, sorafenib, tepotinib, and vandetanib. The following inhibitors were all no more than 20-fold more potent against Syk than against growth factor receptor kinases: alectinib, afatinib, entrectinib, fedratinib, imatinib, larotrectinib, nilotinib, pazopanib, ripretinib, ruxolitinib, selumetinib, sunitinib, tofacitinib, and tucatinib. Of the ten marketed Syk inhibitors (i.e., certinib, entrotinib, fostamatinib, lanraprinib, piceatannol, PRT062067, Syk II, Syk IV, TAE-684, and TAK-659), only entrotinib, PRT062067, and Syk II were at least 20-fold more potent against Syk than against growth factor receptor kinases.

[0140] In addition, Table 4 shows the cellular potency data of these compounds compared to selected commercially available Syk inhibitors and FDA-approved inhibitors known to have Syk inhibition. 50 ) represents the concentration of drug required to elicit half the maximal possible effect after a specific exposure. Table 4: Inhibition of band 3 tyrosine phosphorylation

[0141] The above compounds can be synthesized according to methods known in the art and exemplified herein. See, for example, Examples 1 and 3-5.

[0142] The solubility of the selected compounds was also tested using methods known in the art, and the resulting data are shown in Table 5. Table 5: CLogP data Compound LogP CLogP S970 2.29 3.97 S970D 2.28 3.46 S1115 1.42 3.15 S1213 1.04 3.20 S1219 1.54 1.18 S2053 2.63 3.69

[0143] The CLogP value of a compound may be from about 2.0 to about 5.0 (e.g., 2.0 to about 5.0, from about 2.0 to 5.0, or from 2.0 to 5.0). In certain embodiments, such as compound S1213, the CLogP value of the compound is from about 1.0 to about 2.0 (e.g., 1.0 to about 2.0, from about 1.0 to 2.0, or from 1.0 to 2.0). The term "cLogP" refers to a calculated distribution coefficient, which in physical science is the ratio of the concentration of a compound in a mixture of two immiscible phases at equilibrium. These coefficients are a measure of the difference in solubility of a compound in these two phases. In general, this is a measure of the hydrophobicity of a compound, because one phase is aqueous (traditionally water) and the other phase is hydrophobic (traditionally octanol). The higher the value, the more the compound is distributed in the hydrophobic phase, and the stronger the hydrophobicity.

[0144] In certain embodiments, the compound has a polar surface area of ​​less than about 250 square angstroms (e.g., less than 250 square angstroms). In certain embodiments, the compound has no more than 5 hydrogen bond donors. In certain embodiments, the compound has no more than 15 hydrogen bond acceptors. In certain embodiments, the compound has no more than 20 rotatable bonds. In certain embodiments, the compound may exhibit more than one of the above characteristics.

[0145] The compounds herein also exhibit adequate oral bioavailability. Table 6 shows data on the absolute oral bioavailability of selected compounds (studies conducted using methods well known in the art). Table 6: Absolute oral bioavailability data Compound F S970 NP S970D NP S1115 2％ S1213 2％ S1219 1.1％ S2053 8％

[0146] Administration of the compounds, pharmaceutically acceptable salts or compositions herein inhibits the phosphorylation of erythrocyte anion transporter band 3 by Syk. The compound or its pharmaceutically acceptable salt has an effect on Syk that is at least 20 times more potent than that on growth factor receptor kinase; in other words, the compound has a higher affinity for Syk than conventional Syk inhibitors, and this affinity is discriminatory, meaning that the compounds herein do not have such a high affinity for other receptors (e.g., growth factor receptor kinase).

[0147] Compounds (or pharmaceutically acceptable salts thereof) may contain one or more chiral centers, or may otherwise exist in multiple stereoisomeric forms, such as enantiomers, diastereomers, and mixtures enriched in enantiomers or diastereomers. Unless otherwise indicated, all stereoisomeric forms of the compound are intended to be considered. When a compound contains an olefin double bond, unless otherwise indicated, it is intended to include E and Z geometric isomers (e.g., cis or trans). Likewise, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included. It will be further understood by those of ordinary skill in the art that a compound may be "deuterated," which means that one or more hydrogen atoms may be substituted by deuterium.

[0148] The compound (or its pharmaceutically acceptable salt) can exist in unsolvated form as well as solvated form, including hydrated form. In general, the solvated form is equivalent to the unsolvated form. The compound can exist in a variety of crystalline or amorphous forms. In general, all physical forms are equivalent for the intended use. The formula includes pharmaceutically acceptable salts (e.g., acid addition salts and base salts), hydrates and / or solvates.

[0149] Pharmaceutically acceptable salts

[0150] Also contemplated are "pharmaceutically acceptable salts" of the compounds. The term "pharmaceutically acceptable salts" refers to those salts whose counterions are useful in pharmaceuticals. In various embodiments, such salts include, but are not limited to: 1) acid addition salts, which can be obtained by reacting the free base of the parent compound with an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid, etc.) or an organic acid (e.g., acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, or malonic acid, etc.); or 2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion); or salts formed when coordinated with an organic base (e.g., ethanolamine, diethanolamine, triethanolamine, trimethylamine, N-methylglucamine, etc.). Pharmaceutically acceptable salts are well known to those skilled in the art, and any such pharmaceutically acceptable salts are relevant to the embodiments described herein.

[0151] In various embodiments, suitable acid addition salts are formed from acids that form nontoxic salts. Illustrative examples include acetate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, edisylate, ethanesulfonate, formates, fumarates, glucoheptonates, gluconates, glucuronates, hexafluorophosphates, hydroxybenzoates, hydrochlorides / chlorides, hydrobromides / bromides, hydroiodides / iodides, isethionates, lactates, malates, maleates, malonates, mesylates, methylsulfates, naphthoates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, oxalates, palmitates, pamoates, phosphates / hydrogenphosphates / dihydrogenphosphates, saccharates, stearates, succinates, tartrates, mesylates, and trifluoroacetates.

[0152] In various embodiments, suitable base salts are formed from bases that form non-toxic salts. Illustrative examples include arginine salts, benzathine penicillin salts, calcium salts, choline salts, diethylamine salts, diethanolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, potassium salts, sodium salts, tromethamine salts, and zinc salts. Hemisalts of acids and bases can also be formed, such as hemisulphate salts and hemicalcium salts.

[0153] Pharmaceutical compositions, routes of administration and dosages

[0154] A pharmaceutical composition is also provided, which includes the above-mentioned compound and a pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents or encapsulated materials, which are suitable for administration to humans or other vertebrates. The term "carrier" refers to an organic or inorganic natural or synthetic component, with which the active ingredient is combined to facilitate administration. The carrier can be an excipient. The choice of carrier can depend on factors such as the specific mode of administration, the effect of the carrier on solubility and stability, and the properties of the dosage form. Pharmaceutical compositions suitable for delivering compounds described herein and methods for preparing the same can be found in, for example, Remington: The Science & Practice of Pharmacy, 21st edition (Lippincott Williams & Wilkins, 2005).

[0155] The components of the compositions may also be mixed with the compounds, or with each other, in such a manner that there is no interaction that would significantly impair the desired pharmaceutical efficacy.

[0156] For example, the composition can include Cremophor, polysorbate, nanoparticles, polymers or hydrogels. In certain embodiments, the pharmaceutical composition includes a variety of compounds and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition also includes at least one additional pharmaceutically active agent. At least one additional pharmaceutically active agent can be a reagent that can be used to treat ischemia-reperfusion injury.

[0157] Pharmaceutical compositions can be prepared by combining one or more compounds with a pharmaceutically acceptable carrier and optionally one or more additional ingredients (e.g., pharmaceutically active ingredients). The formulations can be administered in pharmaceutically acceptable solutions, which typically contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

[0158] Pharmaceutically acceptable carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, etc., and combinations thereof that are physiologically compatible. The carrier may be suitable for parenteral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injection solutions or dispersions. Examples of such carriers (or excipients) include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers (e.g., polyethylene glycol). One or more other active agents may also be incorporated into the pharmaceutical composition.

[0159] The pharmaceutical composition can be formulated as a liquid, such as a suspension or solution. The liquid formulation can include water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or a suitable oil, and one or more emulsifiers and / or suspending agents. The liquid formulation can be prepared by solid reconstitution.

[0160] Pharmaceutical preparations (e.g., for parenteral administration) include aqueous solutions of active compounds in water-soluble form. In addition, suspensions of active compounds can be prepared as suitable oily injection suspensions. Aqueous suspensions can contain compounds mixed with suitable excipients alone or in combination with one or more other active agents. Excipients include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic; dispersants or wetting agents, such as naturally occurring phospholipids, such as lecithin; condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate; condensation products of ethylene oxide with long-chain aliphatic alcohols, such as heptadecaethyleneoxyhexadecanoate; condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate; or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyoxyethylene sorbitan monooleate. Aqueous suspensions can also include one or more preservatives, such as ascorbic acid or ethyl, n-propyl or p-hydroxybenzoate, and one or more coloring agents. In certain embodiments, aqueous suspensions can also include suitable lipophilic solvents or carriers, including fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglyceride, or liposomes. Optionally, suspensions can also include suitable stabilizers or agents that increase compound solubility, to allow preparation of highly concentrated solutions.

[0161] Alternatively, the pharmaceutical composition can be in powder form and formulated with a suitable carrier (e.g., sterile pyrogen-free water) before use. Dispersible powders and granules suitable for preparing aqueous suspensions by adding water can provide the active ingredient blended with a suspending agent, a dispersant or a wetting agent and one or more preservatives. Other excipients, such as coloring agents, may also be present.

[0162] Suitable emulsifiers include naturally occurring gums, such as gum arabic or tragacanth; naturally occurring phospholipids, such as soybean lecithin; and esters, including partial esters derived from fatty acids and hexitol anhydrides, such as sorbitol monooleate, and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Isotonic agents, such as sugars, polyols (such as mannitol, sorbitol or sodium chloride) may be included in the composition. Extended absorption of the injection composition may be achieved by including one or more agents (such as monostearate and gelatin) that delay absorption in the composition.

[0163] For medical or therapeutic use, an effective amount of the compound or composition can be administered to a subject by any method of delivering the compound as needed. Administration of the composition can be accomplished by any method known to those skilled in the art. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (bladder), oral, subcutaneous, direct injection, mucosal (e.g., topical for eyes), inhalation, and topical.

[0164] Colorants and / or flavoring agents may be included. For example, the compound may be formulated (eg, by liposome or microsphere encapsulation) and then further included in an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

[0165] Exemplary forms for oral administration include, but are not limited to, tablets, capsules, elixirs, syrups, and the like.

[0166] In certain embodiments, the compound can be directly administered into the bloodstream, muscle or internal organs. Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, intranasal and subcutaneous. Suitable methods for parenteral administration include needle (including microneedle) syringe, needle-free syringe and infusion technology. When systemic delivery of compound and / or composition is required, compound and / or composition can be formulated for parenteral administration by injection, such as by push or continuous infusion. Injection preparations can exist in unit dosage form, such as in ampoules or more dosage containers, and are added with preservatives. Compositions can be in the form of suspensions, solutions or emulsions in oily or aqueous carriers, and can contain preparatons, such as suspending agents, stabilizers and / or dispersants.

[0167] Parenteral formulations are typically aqueous solutions that may contain carriers or excipients such as salts, carbohydrates and buffers (preferably pH 3-9), but for some applications they may be more suitably formulated as sterile non-aqueous solutions or in dry form to be used with a suitable carrier such as sterile pyrogen-free water.

[0168] Liquid preparations may be suitable for parenteral administration of the compound. Parenteral preparations are prepared under sterile conditions, for example, by lyophilization under sterile conditions, which can be easily accomplished using standard pharmaceutical techniques well known to those skilled in the art. The solubility of the compound can be increased by using appropriate formulation techniques (e.g., incorporation of a solubilizing agent).

[0169] Preparations for parenteral administration can be formulated to release immediately and / or slow release. The compound can be administered in the form of a slow release formulation, for example, in the form of a composition comprising a slow release polymer. The compound can be prepared with a carrier that protects it from rapid release, such as a controlled release formulation, including implants and microencapsulation delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic acid, polyglycolic acid copolymers (PGLA). The method for preparing such preparations is generally known to those skilled in the art.

[0170] Sterile injection solutions can be prepared by adding the compound alone or in combination with one or more other active agents, in the desired amount, to an appropriate solvent, adding one or more combinations of the above ingredients as needed, and then filtering and sterilizing. Typically, dispersions are prepared by adding the compound to a sterile carrier containing a dispersion medium and any additional ingredients described above. For sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze drying, which obtain a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution, or these ingredients can be sterile filtered together.

[0171] Pharmaceutical compositions can be formulated into solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium, containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating (e.g., lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant.

[0172] Where appropriate, the compound or pharmaceutical composition containing the compound may be administered continuously.

[0173] Treatment

[0174] Provided are methods for treating RBC-mediated diseases (e.g., SCD). The methods may include administering an effective amount of the above-described compound, a pharmaceutically acceptable salt thereof, or any of the above-described pharmaceutical compositions to a subject (e.g., a subject experiencing an RBC-mediated disease or at risk of experiencing an RBC-mediated disease). In certain embodiments, the compound has formula (X): ABC(X) or a pharmaceutically acceptable salt thereof, wherein: A is a nitrogen-linked and optionally substituted first ring, which includes a fused bicyclic or monocyclic ring, wherein each ring of the first ring contains a 4-6 membered heterocyclic or carbocyclic ring; B is a second ring, which is optionally substituted with at least one substituent, the second ring comprising a monocyclic ring or a fused bicyclic or tricyclic ring, and wherein each ring of the second ring comprises a 5-membered or 6-membered heterocyclic or carbocyclic ring; and C is a third bicyclic ring comprising a 5- or 6-membered carbocyclic ring fused to a 5- or 6-membered carbocyclic or heterocyclic ring, wherein the third bicyclic ring is substituted with at least a first substituent comprising: or amines, or alcohols, in: X4 is each independently CH2, NH, CO, O or S, Y5 is each independently CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and and is the point of attachment of the first substituent to the third bicyclic ring; The concentration of the compound or its pharmaceutically acceptable salt in inhibiting Syk tyrosine kinase is at least one twentieth less than the concentration of the compound (or its pharmaceutically acceptable salt) in inhibiting HGFR, EGFR, FGFR3, IGF1R and / or VEGFR.

[0175] The method may also include administering to the subject an effective amount of at least one additional / other active agent (each a "second active agent"). In certain embodiments, the second active agent is selected from the group consisting of fotatinib, PRT062607, TAK-659, TAE-684, entosatinib, lanraprinib, sedutinib, piceatannol, S701, Syk II, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, Syk-IN-1, SRX3207, RO9021, gusartinib, R112, PRT-060318, and OXSI-2. Additionally or alternatively, the method may include administering an effective amount of: (a) the above compound, a pharmaceutically acceptable salt thereof, or any of the above pharmaceutical compositions; (b) fostamatinib; (c) PRT062607; (d) TAK-659; (e) TAE-684; (f) entosanitib; (g) lanlaprinib; (h) sedutinib; (i) piceatannol; (j) S701; (k) Syk II; (l) Syk IV; (m) TAS05567; (n) GSK143; (o) Syk-IN-3; (p) Syk-IN-4; (q) Syk-IN-1; (r) SRX3207; (s) RO9021; (t) gusartinib; (u) R112; (v) PRT-060318; (w) OXSI-2; (x) (a) and (b); (y) (a) and (c); (z) (a) and (d); (aa) (a) and (e); (ab) (a) and (f); (ac) (a) and (g); (ad) (a) and (h); ( ae)(a) and (i); (af)(a) and (j); (ag)(a) and (k); (ah)(a) and (l); (ai)(a) and (m); (aj)(a) and (n); (ak)(a) and (o); (al)(a) and (p); (am)(a) and (q); (an)(a) and (r); (ao)(a) and (s); (ap)(a) and (t); (aq)(a) and (u); (ar)(a) and (v); (as)(a) and (w); or (at)(a) and (b)-(w) One or more. The foregoing may optionally be administered to a subject as a single pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient, or in multiple doses using, for example, multiple pharmaceutical compositions.

[0176] The at least one second active agent may include a combination of two or more of fotatinib, PRT062607, TAK-659, TAE-684, entolinib, lanlaprinib, sedutinib, piceatannol, S701, Syk II, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, Syk-IN-1, SRX3207, RO9021, gusartinib, R112, PRT-060318 and OXSI-2. Each second active agent may be administered simultaneously with a compound, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, or sequentially and in any order (where applicable). In certain embodiments, a compound, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition and each second active agent are administered by the same or different routes.

[0177] The method may also include administering to the subject an effective amount of hydroxyurea (e.g., which may include a second active agent). Hydroxyurea may be administered simultaneously with the compound, its pharmaceutically acceptable salt, or pharmaceutical composition, or sequentially and in any order (where applicable). In certain embodiments, the compound, its pharmaceutically acceptable salt, or pharmaceutical composition, and hydroxyurea are administered by the same route. In certain embodiments, the compound, its pharmaceutically acceptable salt, or pharmaceutical composition, and hydroxyurea are administered by different routes.

[0178] Another method of treating a disease mediated by RBCs is also provided. In certain embodiments, the method comprises administering an effective amount of a compound (or a pharmaceutically acceptable salt): (i) which comprises a rigidly fused bicyclic ring substituted with at least two substituents, at least one of which is an N-linked, optionally substituted carbocyclic or heterocyclic ring, and the other of which is indole substituted with at least one substituent, at least one of which is a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring, (ii) it inhibits Syk phosphorylation of human erythrocyte anion transporter band 3, and (iii) The concentration at which the compound inhibits Syk tyrosine kinase is The concentration of IGF1R and / or VEGFR is at least about 20 times lower than that of Wherein, in addition to (ii) and (iii) or as an alternative to (ii) and (iii), the compound of (i) or the pharmaceutically Accepted salts also have: (iv) a CLogP value of about 2 to about 5 (e.g., 2 to about 5, about 2 to 5, or 2 to 5), (v) a polar surface area of ​​less than about 250 square angstroms (e.g., less than 250 square angstroms), (vi) no more than 5 hydrogen bond donors, (vii) not more than 15 hydrogen bond acceptors, and / or (viii) not more than 20 rotatable bonds, The subject's sickle cell disease is thereby treated. The compound can inhibit Syk phosphorylation of tyrosine 8 and tyrosine 21 in human erythrocyte anion transporter band 3. The compound and / or a pharmaceutical composition comprising the compound can stabilize erythrocyte membranes and reduce the release of microvesicles in sickle cells.

[0179] The RBC-mediated disease can be selected from SCD, thalassemia (e.g., α-thalassemia or β-thalassemia), G6PD, glutathione reductase deficiency, and diseases involving the release of microvesicles from blood cells. Each of these diseases is associated with phosphorylation of erythrocyte anion transporter band 3, and inhibiting such phosphorylation can prevent, slow down, and / or treat these diseases, thereby not initiating or stopping the sequelae that cause vaso-occlusion and disease symptoms. In fact, administration of an effective amount of a compound or a pharmaceutical composition thereof can stabilize the red blood cell membranes of a subject and reduce the release of hemoglobin in sickle cells in a subject. In this way, the risk of vaso-occlusive crises in a subject can be reduced.

[0180] In certain embodiments of the method, the compound, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition is administered at an IC of 200 nM or less. 50 Inhibition of Syk tyrosine kinase. The subject can be a human child (ie, a growing human or other human in which off-target inhibition of growth hormone may have significant deleterious effects).

[0181] The method may also include administering one or more other compounds selected from the group consisting of fostamatinib, PRT062607, TAK-659, TAE-684, entosatinib, lanraprinib, sedutinib, piceatannol, S701, Syk II, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusartinib, R112, PRT-060318 and OXSI-2 simultaneously or sequentially in any order, optionally as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient. One or more other compounds may be administered by the same or different routes (compared to the administration of the compound or pharmaceutical composition).

[0182] A method for preventing or inhibiting phosphorylation of human erythrocyte anion transporter band 3 in a subject comprises administering to the subject an effective amount of the above compound, a pharmaceutically acceptable salt thereof, or any of the above pharmaceutical compositions.

[0183] A method for treating a disease in which a subject is involved in releasing microvesicles from blood cells is also provided. The method includes administering an effective amount of a compound or a pharmaceutically acceptable salt thereof, wherein the compound includes a rigidly fused bicyclic ring, which is substituted by at least two substituents, wherein at least one substituent is an N-connected, optionally substituted carbocyclic or heterocyclic ring, and another substituent is an indole substituted by at least one substituent, which is a 4-6-membered, optionally substituted carbocyclic or heterocyclic ring. After administration to the subject, the subject may be treated for a disease in which microvesicles are released. The disease may be thalassemia. Thalassemia may be α-thalassemia or β-thalassemia, alone or in combination with SCD. The disease may be G6PD deficiency. The disease may be glutathione reductase deficiency.

[0184] The method may also include administering a Src inhibitor simultaneously or sequentially in any order (compared to the compounds or pharmaceutical compositions herein), optionally as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient. Compared to the compounds or pharmaceutical compositions herein, the Src inhibitor may be administered by the same or different routes. The Src inhibitor may be dasatinib, ibrutinib, barfitinib, PP1, PP2, PP121, or a combination of two or more thereof.

[0185] Also provided is a method of treating SCD in a subject, the method comprising administering an effective amount of a compound: (i) it comprises a pyrazine substituted with at least three substituents, at least one of which is a carboxamide or a lactam, another of which is an N-linked, optionally substituted carbocyclic or heterocyclic ring, another of which is a carbocyclic or heterocyclic ring having at least one substituent comprising a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring, (ii) It inhibits the phosphorylation of human erythrocyte anion transporter band 3 by Syk tyrosine kinase, (iii) The concentration at which the compound inhibits Syk tyrosine kinase is The concentration of IGF1R and / or VEGFR is at least about 20 times lower than that of Wherein, in addition to or as an alternative to (ii) and (iii), the compound of (i) further has: (iv) a CLogP value of about 2 to about 5 (e.g., 2 to about 5, about 2 to 5, or 2 to 5), (v) a polar surface area of ​​less than about 250 square angstroms (e.g., less than 250 square angstroms), (vi) no more than 5 hydrogen bond donors, (vii) not more than 15 hydrogen bond acceptors, and / or (viii) not more than 20 rotatable bonds, The subject's SCD is thereby treated. The compound or a pharmaceutically acceptable salt thereof can inhibit the phosphorylation of tyrosine 8 and tyrosine 21 in human erythrocyte anion transporter band 3 by Syk tyrosine kinase. The compound or a pharmaceutically acceptable salt thereof can stabilize erythrocyte membranes and reduce the release of sickle cell microvesicles. The compound or a pharmaceutically acceptable salt thereof can stabilize erythrocyte membranes and reduce the release of sickle cell hemoglobin. The risk of a subject developing a vaso-occlusive crisis can be reduced. The compound or a pharmaceutically acceptable salt thereof can be used to treat SCD at an IC of 200 nM or less. 50 Inhibit Syk tyrosine kinase. The subject may be a human child. The method may also include administering one or more of fotatinib, PRT062607, TAK-659, TAE-684, entosatinib, lanraprinib, sedutinib, piceatannol, S701, Syk II, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusartinib, R112, PRT-060318 and OXSI-2 simultaneously or in any order, optionally as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient. One or more other compounds may be administered by the same or different routes.

[0186] Also provided is a method for treating a subject for a disease involving the release of microvesicles from blood cells. The method comprises administering an effective amount of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a pyrazine substituted with at least three substituents, at least one of which is a carboxamide or a lactam, another is an N-linked, optionally substituted carbocyclic or heterocyclic ring, and another is a carbocyclic or heterocyclic ring having at least one substituent, wherein at least one substituent is a 4-6-membered, optionally substituted carbocyclic or heterocyclic ring, thereby treating the subject for a disease involving the release of microvesicles. The disease may be thalassemia. The thalassemia may be α-thalassemia or β-thalassemia alone or in combination with sickle cell disease. The disease may be G6PD deficiency. The disease may be glutathione reductase deficiency. The method may also comprise administering a Src inhibitor simultaneously or sequentially in any order, optionally as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient. The Src inhibitor may be administered by the same or different route as compared to the compound or a pharmaceutically acceptable salt thereof. The Src inhibitor can be dasatinib, ibrutinib, bafitinib, PP1, PP2, PP121, or a combination of two or more thereof.

[0187] The terms "treat", "treating", "treated", and "treatment" (for a disease or condition) are an approach to obtaining beneficial or desired results, including and preferably clinical results, and include, but are not limited to, one or more of the following: improving disease-related symptoms, curing the disease, lessening the severity of the disease, delaying the progression of the disease, alleviating one or more symptoms associated with the disease, improving the quality of life of patients with the disease, prolonging survival, and / or preventive or prophylactic treatment.

[0188] As used herein, "effective amount" refers to any amount of a compound for treatment, and refers to the amount of the compound in a formulation that, when administered as part of a desired dosage regimen (to a mammal, such as a human), is sufficient to achieve the desired biological effect (e.g., relief of symptoms, amelioration of symptoms, or reduction of the onset of a disease condition, according to clinically acceptable standards for the disease or condition being treated or for cosmetic purposes, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment).

[0189] In conjunction with the teachings provided herein, by selecting among various active compounds and weighing factors such as efficacy, relative bioavailability, patient weight, severity of adverse side effects and mode of administration, an effective prevention or treatment regimen can be formulated that does not cause significant undesirable toxicity but is effective for treating a particular subject. The effective amount of any particular application may vary depending on factors such as the disease or condition being treated, the specific compound being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound and / or other therapeutic agent without having to perform excessive experiments.

[0190] For example, for any compound, the effective amount can be first determined from an animal model. For compounds tested in humans and compounds known to exhibit similar pharmacological activity (e.g., other related active agents), the effective dose can also be determined from human data. Higher doses may be required for parenteral administration. The applied dose can be adjusted according to the relative bioavailability and efficacy of the administered compound. It is entirely within the capabilities of ordinary technicians to adjust the dose to achieve maximum efficacy according to the above methods and other methods well known in the art.

[0191] A maximum dose, i.e., the highest safe dose based on some medical judgment, may be used. Multiple doses may be used daily to achieve appropriate systemic levels of the compound. Appropriate systemic levels may be determined, for example, by measuring peak or sustained plasma levels of the drug in the patient.

[0192] "Dose" and "amount" are used interchangeably herein.

[0193] In general, for human subjects, the daily oral dose of the compound is about 0.01 mg / kg / day to 1,000 mg / kg / day. Oral doses of 0.5 mg / kg to 50 mg / kg administered once or more daily can produce therapeutic effects. The dose can be appropriately adjusted according to the mode of administration to achieve the desired local or systemic drug level. For example, the daily dose for intravenous administration can be one order of magnitude lower to several orders of magnitude. If the subject does not respond adequately at such a dose, a higher dose (or an effective higher dose through a different, more localized delivery route) can be used within the patient's tolerance range. Consider multiple daily dosing to achieve appropriate systemic levels of the compound.

[0194] For clinical use, any compound can be administered in an amount equal to or equivalent to 0.2-2,000 milligrams (mg) of compound per kilogram of subject body weight per day. The compound can be administered in an amount equal to or equivalent to 2-2,000 mg of compound per kilogram of subject body weight per day. The compound can be administered in an amount equal to or equivalent to 20-2,000 mg of compound per kilogram of subject body weight per day. The compound can be administered in an amount equal to or equivalent to 50-2,000 mg of compound per kilogram of subject body weight per day. The compound can be administered in an amount equal to or equivalent to 100-2,000 mg of compound per kilogram of subject body weight per day. The compound can be administered in an amount equal to or equivalent to 200-2,000 mg of compound per kilogram of subject body weight per day. When a precursor or prodrug of a compound is to be administered, it is administered in an amount equal to (i.e., sufficient to deliver) the above-mentioned compound.

[0195] The preparation of the compound or its pharmaceutically acceptable salt can be administered to a human subject in an effective amount. Typical dosage ranges are from about 0.01 micrograms / kg to about 2 mg / kg body weight per day. The dosage of the drug to be administered may depend on variables such as the type and extent of the disease, the overall health of the particular subject, the particular compound administered, the excipient used to prepare the compound, and its route of administration. Routine experiments can be used to optimize the dosage and frequency of administration of any particular compound or its pharmaceutically acceptable salt.

[0196] The compound or a pharmaceutically acceptable salt thereof can be administered at a concentration ranging from about 0.001 μg / kg to greater than about 500 mg / kg. For example, the concentration can be 0.001 μg / kg, 0.01 μg / kg, 0.05 μg / kg, 0.1 μg / kg, 0.5 μg / kg, 1.0 μg / kg, 10.0 μg / kg, 50.0 μg / kg, 100.0 μg / kg, 500 μg / kg, 1.0 mg / kg, 5.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, 20.0 mg / kg, 25.0 mg / kg, 30.0 mg / kg, 35.0 mg / kg, In some embodiments, the present invention relates to an aqueous solution of at least about 100 mg / kg, about 200 mg / kg, about 400 mg / kg, about 500 mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, about 1000 mg / kg, about 1500 mg / kg, about 2000 mg / kg, about 2500 mg / kg, about 3000 mg / kg, about 3500 mg / kg, about 4000 mg / kg, about 4500 mg / kg, to greater than about 500.0 mg / kg or any incremental value thereof. It should be understood that all values ​​and ranges between these values ​​and ranges should be covered.

[0197] The compound or a pharmaceutically acceptable salt thereof can be administered in a dosage range of about 0.2 mg / kg / day to greater than about 100 mg / kg / day. For example, the dosage can be 0.2 mg / kg / day to 100 mg / kg / day, 0.2 mg / kg / day to 50 mg / kg / day, 0.2 mg / kg / day to 25 mg / kg / day, 0.2 mg / kg / day to 10 mg / kg / day, 0.2 mg / kg / day to 7.5 mg / kg / day, 0.2 mg / kg / day to 5 mg / kg / day, 0.25 mg / kg / day to 100 mg / kg / day, 0.25 mg / kg / day to 50 mg / kg / day, 0.25 mg / kg / day to 25 mg / kg / day, 0.25 mg / kg / day to 100 mg / kg / day, 0.25 mg / kg / day to 50 mg / kg / day, 0.25 mg / kg / day to 25 mg / kg / day, 0.25 mg / kg / day to 100 mg / kg / day. / day to 10mg / kg / day, 0.25mg / kg / day to 7.5mg / kg / day, 0.25mg / kg / day to 5mg / kg / day, 0.5mg / kg / day to 50mg / kg / day, 0.5mg / kg / day to 25mg / kg / day, 0.5mg / kg / day to 20mg / kg / day, 0.5mg / kg / day to 15mg / kg / day, 0.5mg / kg / day to 10mg / kg / day, 0.5mg / kg / day to 7.5mg / kg / day, 0.5mg / kg / day to 5mg / kg / day, 0.75mg / kg / day to 50mg / kg / day, 0.75mg / kg / day to 25mg / kg / day, 0.75mg / kg / day to 20mg / kg / day, 0.75mg / kg / day to 15mg / kg / day, 0.75mg / kg / day to 10mg / kg / day, 0.75mg / kg / day to 7.5mg / kg / day, 0.75mg / kg / day to 5mg / kg / day, 1.0mg / kg / day to 50mg / kg / day, 1.0mg / kg / day to 25mg / kg / day, 1.0mg / kg / day to 20mg / kg / day, 1.0mg / kg / day to 15mg / kg / day, 1.0mg / kg / day to 10mg / kg / day, 1.0mg / kg / day to 7.5mg / kg / day, 1.0mg / kg / day to 5mg / kg / day, 2mg / kg / day to 50mg / kg / day, 2mg / kg / day to 25mg / kg / day, 2mg / kg / day to 20mg / kg / day, 2mg / kg / day to 15mg / kg / day, 2mg / kg / day to 10mg / kg / day, 2mg / kg / day to 7.5mg / kg / day, or 2mg / kg / day to 5mg / kg / day.

[0198] The compound can be administered at a dosage ranging from about 0.25 mg / kg / day to about 25 mg / kg / day. For example, the dosage can be 0.25 mg / kg / day, 0.5 mg / kg / day, 0.75 mg / kg / day, 1.0 mg / kg / day, 1.25 mg / kg / day, 1.5 mg / kg / day, 1.75 mg / kg / day, 2.0 mg / kg / day, 2.25 mg / kg / day, 2.5 mg / kg / day, 2.75 mg / kg / day, 3.0 mg / kg / day, 3.25 mg / kg / day, 3.5 mg / kg / day, 3.75 mg / kg / day, 4.0 mg / kg / day, 4.25mg / kg / day, 4.5mg / kg / day, 4.75mg / kg / day, 5mg / kg / day, 5.5mg / kg / day, 6.0mg / kg / day, 6.5mg / kg / day, 7.0mg / kg / day, 7.5mg / kg / day, 8.0mg / kg / day, 8.5mg / kg / day, 9.0mg / kg / day, 9.5mg / kg / day, 10mg / kg / day, 11mg / kg / day, 12mg / kg / day, 13mg / kg g / day, 14mg / kg / day, 15mg / kg / day, 16mg / kg / day, 17mg / kg / day, 18mg / kg / day, 19mg / kg / day, 20mg / kg / day, 21mg / kg / day, 22mg / kg / day, 23mg / kg / day, 24mg / kg / day, 25mg / kg / day, 26mg / kg / day, 27mg / kg / day, 28mg / kg / day, 29mg / kg / day, 30mg / kg / day, 31mg / kg / day, 32mg / kg / day 50 mg / kg / day, 51 mg / kg / day, 52 mg / kg / day, 53 mg / kg / day, 54 mg / kg / day, 55 mg / kg / day, 56 mg / kg / day, 57 mg / kg / day, 58 mg / kg / day, 59 mg / kg / day, 60 mg / kg / day, 61 mg / kg / day, 62 mg / kg / day, 63 mg / kg / day, 64 mg / kg / day, 65 mg / kg / day, 66 mg / kg / day, 67 mg / kg / day, 68 mg / kg / day, 69 mg / kg / day or 70 mg / kg / day.

[0199] The compound, its pharmaceutically acceptable salt or its precursor can be administered at a concentration ranging from 0.01 micromolar to greater than or equal to 500 micromolar. For example, the dosage can be 0.01 micromolar, 0.02 micromolar, 0.05 micromolar, 0.1 micromolar, 0.15 micromolar, 0.2 micromolar, 0.5 micromolar, 0.7 micromolar, 1.0 micromolar, 3.0 micromolar, 5.0 micromolar, 7.0 micromolar, 10.0 micromolar, 15.0 micromolar, 20.0 micromolar, 25.0 micromolar, 30.0 micromolar, 35.0 micromolar, 40.0 micromolar, 45.0 micromolar, 46.0 micromolar, 47.0 micromolar, 48.0 micromolar, 49.0 micromolar, 50.0 micromolar, 51.0 micromolar, 52.0 micromolar, 53.0 micromolar, 54.0 micromolar, 56.0 micromolar, 57.0 micromolar, 58.0 micromolar, 59.0 micromolar, 60.0 micromolar, 61.0 micromolar, 62.0 micromolar, 63.0 micromolar, 64.0 micromolar, 65.0 micromolar, 66.0 micromolar, 67.0 micromolar, 68.0 micromolar, 69.0 micromolar, 70.0 micromolar, 71.0 micromolar, 72.0 micromolar, 73.0 micromolar, 74.0 micromolar, 75.0 micromolar, 76.0 micromolar, 77.0 micromolar, 78.0 micromolar, 79 In some embodiments, the present invention relates to an aqueous solution of at least about 500 micromolar, about 200 micromolar, about 400 micromolar, about 500 micromolar, about 600 micromolar, about 700 micromolar, about 800 micromolar, about 900 micromolar, about 1000 micromolar, about 1500 micromolar, about 2000 micromolar, about 2500 micromolar, about 3000 micromolar, about 3500 micromolar, about 4000 micromolar, about 4500 micromolar, to greater than about 5000 micromolar or any incremental value thereof. It should be understood that all values ​​and ranges between these values ​​and ranges should be covered.

[0200] The compound or a pharmaceutically acceptable salt thereof or a precursor thereof may be administered at a concentration ranging from 0.10 micrograms / mL to 500.0 micrograms / mL. For example, the concentration can be from 0.10 micrograms / mL, 0.50 micrograms / mL, 1 microgram / mL, 2.0 micrograms / mL, 5.0 micrograms / mL, 10.0 micrograms / mL, 20 micrograms / mL, 25 micrograms / mL, 30 micrograms / mL, 35 micrograms / mL, 40 micrograms / mL, 45 micrograms / mL, 50 micrograms / mL, 60.0 micrograms / mL, 70.0 micrograms / mL, 80.0 micrograms / mL, 90.0 micrograms / mL, 100.0 micrograms / mL, 150.0 micrograms / mL, 200.0 micrograms / mL, 250.0 micrograms / mL, 250.0 micrograms / mL, 300.0 micrograms / mL, 350.0 micrograms / mL, 400.0 micrograms / mL, 450.0 micrograms / mL, to greater than about 500.0 micrograms / mL or any incremental value thereof. It should be understood that all values ​​and ranges between these values ​​and ranges should be included.

[0201] Preparations can be used in the form of pharmaceutically acceptable solutions, which solutions can generally contain pharmaceutically acceptable concentrations of salt, buffers, preservatives, compatible carriers, adjuvants and optional other therapeutic ingredients. For therapeutic use, the compound of an effective amount can be administered to a subject by any means by which the compound is delivered to the desired surface. Pharmaceutical compositions can be administered by any means known to those skilled in the art. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (bladder), oral, subcutaneous, direct injection (e.g., injection into a tumor or abscess), mucosal (e.g., local for eyes), suction and topical.

[0202] For intravenous and other parenteral administration routes, the compound can be formulated as a lyophilized preparation, a lyophilized preparation of the active compound inserted or encapsulated by liposomes, a lipid complex or a salt complex in an aqueous suspension. The lyophilized preparation is usually reconstituted in a suitable aqueous solution (e.g., sterile water or saline) shortly before administration.

[0203] For oral administration, the compound can be easily formulated by combining the active compound with a pharmaceutically acceptable carrier known in the art. Such a carrier enables the compound to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipients, optionally grinding the resulting mixture, and processing the granular mixture after adding suitable adjuvants (if necessary) to obtain tablets or dragee cores. Suitable excipients are particularly fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If necessary, a disintegrant can be added, such as cross-linked PVP, agar or alginic acid or a salt thereof, such as sodium alginate. Optionally, oral formulations may also be formulated in saline or a buffer (eg, ethylenediaminetetraacetic acid (EDTA)) for neutralizing internal acidic conditions, or may be administered without any carrier.

[0204] Oral dosage forms of the compound are also contemplated. The compound may be chemically modified so that oral delivery of the derivative is effective. Typically, the chemical modification contemplated is to attach at least one moiety to the compound itself, wherein the moiety allows (a) inhibition of acid hydrolysis; and (b) absorption from the stomach or intestine into the bloodstream. It is also desirable to improve the overall stability of the compound and increase circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, PVP, and polyproline. Abuchowski and Davis, "Soluble Polymer-Enzyme Adducts," In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383 (1981); Newmark et al., J Appl Biochem 4: 185-189 (1982). Other polymers that may be used are poly-1,3-dioxolane and poly-1,3,6-thioxoctane.For pharmaceutical uses, as described above, polyethylene glycol moieties are suitable.

[0205] The release site of the compound herein or its pharmaceutically acceptable salt may be the stomach, small intestine (e.g., duodenum, jejunum, or ileum), or large intestine. Those skilled in the art have available formulations that will not dissolve in the stomach but will release the substance in the duodenum or elsewhere in the intestinal tract. Release may avoid the harmful effects of the gastric environment by protecting the compound or by releasing the compound or its pharmaceutically acceptable salt outside the gastric environment, for example in the intestinal tract.

[0206] To ensure complete gastric tolerance, a coating that is impermeable at least at pH 5.0 may be essential. Examples of more common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S and shellac. These coatings can be applied as mixed films.

[0207] Tablets may also be coated with coatings or coating mixtures that are not intended for gastric protection. This may include sugar coatings or coatings that make the tablet easier to swallow. Capsules may consist of a hard shell such as gelatin for delivery of dry therapeutic agents (e.g., powders); for liquid forms, a soft gelatin shell may be used. The outer shell material for cachets may be thick starch or other edible paper. For pills, lozenges, molded tablets, or tablet triturates, moist massing techniques may be used.

[0208] As mentioned above, the compound or its pharmaceutically acceptable salt may be included in the formulation as fine multi-particulates in the form of granules or pellets having a particle size of about 1 mm. The formulation of the material for capsule administration may also be a powder, lightly compressed plugs or even tablets.

[0209] Pharmaceutical formulations that can be used orally include push-in capsules made of gelatin, and soft sealed capsules made of gelatin and a plasticizer (e.g., glycerol or sorbitol). Push-in capsules may contain the active ingredient blended with a filler (e.g., lactose), a binder (e.g., starch), and / or a lubricant (e.g., talc or magnesium stearate) and an optional stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres are well defined in the art. All formulations for oral administration should have a dosage suitable for such administration.

[0210] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0211] For topical administration, the compounds can be formulated into solutions, gels, ointments, creams, suspensions, etc., as is well known in the art. Systemic formulations include those designed for administration by injection, such as subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral, or pulmonary administration.

[0212] For administration by inhalation, the compound is conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer, using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example, of gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base, for example lactose or starch.

[0213] Pulmonary delivery of the compound (or its salt) is also contemplated. The compound is delivered to the lungs of a mammal upon inhalation and crosses the lung epithelial lining into the bloodstream. Other reports of inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., Int J Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., J Cardiovasc Pharmacol 13(suppl. 5):143-146 (1989) (endothelin-1); Hubbard et al., Annal Int Med 3:206-212 (1989) (a1-antitrypsin); Smith et al., 1989, J Clin Invest 84:1145-1146 (a-1-proteinase); Oswein et al., 1990, "Aerosolization of Proteins," Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant hepatocyte growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor; incorporated herein by reference). U.S. Pat. No. 5,451,569, issued Sept. 19, 1995 to Wong et al. (the disclosure of which is specifically incorporated herein by reference) describes a method and composition for pulmonary delivery of drugs for systemic effect.

[0214] A variety of mechanical devices designed for pulmonary delivery of therapeutic products are contemplated for use, including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

[0215] Nasal delivery of pharmaceutical compositions is also contemplated. Nasal delivery allows the pharmaceutical composition to enter the bloodstream directly after the therapeutic product is applied to the nasal cavity without depositing the product in the lungs. Formulations for nasal delivery include formulations with dextran or cyclodextran.

[0216] When systemic delivery is required, the compound can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection can be in unit dose form, for example in ampoules or multidose containers, with the addition of preservatives. The composition can be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain preparatants, such as suspending agents, stabilizers and / or dispersants.

[0217] Pharmaceutical preparations for parenteral administration include aqueous solutions of the active compound in water-soluble form. In addition, the suspension of the active compound can be prepared into suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of high concentration solutions.

[0218] Alternatively, the active compound may be in powder form for constitution with a suitable vehicle (eg, sterile pyrogen-free water) before use.

[0219] The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0220] In addition to the above-mentioned preparations, the compound can also be formulated into a long-acting preparation. Such a long-acting preparation can be formulated with a suitable polymer or hydrophobic material (e.g., formulated as an emulsion in an acceptable oil) or an ion exchange resin, or formulated as a poorly soluble derivative, e.g., a poorly soluble salt.

[0221] The pharmaceutical composition may also contain suitable solid or gel phase carriers or excipients. Examples of these carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0222] Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous solutions or saline solutions for suction, microcapsules, endocytosis agents, dosage forms coated on microscopic gold particles, dosage forms contained in liposomes, atomizers, aerosols, pills for implanting skin, or dosage forms dried on sharp objects to scrape into the skin. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with sustained release of active compounds, in which excipients and additives and / or adjuvants as described above are generally used, such as disintegrants, adhesives, coating agents, swelling agents, lubricants, flavoring agents, sweeteners or solubilizing agents. Pharmaceutical compositions are applicable to various drug delivery systems. For a brief review of drug delivery methods, see Langer R, Science 249:1527-1533 (1990).

[0223] Disintegrants can be included in the solid dosage form formulation of the therapeutic agent. Materials used as disintegrants include, but are not limited to, starch, including the commercial disintegrant Explotab based on starch. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, super branched starch, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge and bentonite can all be used. Another form of disintegrant is an insoluble cation exchange resin. Powdered gums can be used as disintegrants and binders, and these can include powdered gums, such as agar, carrageenan or tragacanth. Alginic acid and its sodium salt can also be used as disintegrants.

[0224] Binders can be used to hold the compound together to form a hard tablet, including materials from natural products, such as gum arabic, gum tragacanth, starch and gelatin. Other binders include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). PVP and hydroxypropylmethylcellulose (HPMC) can both be used in alcohol solutions to form particles of the therapeutic agent.

[0225] Friction reducers may be added to the formulation of the therapeutic agent to prevent sticking during formulation. Lubricants may be used as a layer between the therapeutic agent and the mold wall and may include, but are not limited to, stearic acid (including its magnesium and calcium salts), polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, and waxes. Soluble lubricants may also be used, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, Carbowax 4000 and 6000.

[0226] Glidants may be added to improve the flow properties of the drug during formulation and to aid rearrangement during compression. Glidants may include starch, talc, fumed silica, and hydrated aluminosilicates.

[0227] In order to help the therapeutic agent dissolve in the water environment, a surfactant can be added as a wetting agent. The surfactant can include anionic detergents, such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents that can be used include benzalkonium chloride and benzethonium chloride. Possible nonionic detergents that can be included in the preparation as a surfactant include lauryl alcohol 400, polyethylene glycol 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. These surfactants can be present in the preparation of the compound or its derivatives alone or in a mixture of different proportions.

[0228] Compound and optional one or more other therapeutic agents can be used in the form of itself (pure) or in a pharmaceutically acceptable salt. When used in medicine, salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare its pharmaceutically acceptable salt. Such salts include, but are not limited to, salts prepared by the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. In addition, such salts can be prepared as alkali metal or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts of carboxylic acid groups.

[0229] Inert substances may be used to dilute or increase the volume of the compound. These diluents may include carbohydrates, particularly mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextran, and starch. Certain inorganic salts may also be used as fillers, including tricalcium phosphate, magnesium carbonate, and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx1500, Emcompress, and Avicell.

[0230] Suitable buffers include acetic acid and its salts (1-2% w / v); citric acid and its salts (1-3% w / v); boric acid and its salts (0.5-2.5% w / v); phosphoric acid and its salts (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25% w / v) and thimerosal (0.004-0.02% w / v).

[0231] The pharmaceutical composition contains an effective amount of the compound described herein and optionally one or more other therapeutic agents, contained in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to humans or other vertebrates. The term "carrier" refers to a natural or synthetic organic or inorganic ingredient with which the active ingredient is combined to facilitate application. The components of the pharmaceutical composition can also be mixed with the compound, and with each other, in a manner such that there is no interaction that would significantly impair the desired drug efficacy.

[0232] Therapeutic agents, specifically including but not limited to compounds, can be provided in the form of particles. "Particles" as used herein refer to nanoparticles or microparticles (or larger particles in some cases), which can be composed of compounds described herein or other therapeutic agents in whole or in part. The particles can contain therapeutic agents in a core surrounded by a coating, including but not limited to an enteric coating. The therapeutic agent can also be dispersed throughout the particles. The therapeutic agent can also be adsorbed into the particles. The particles can have release kinetics in any order, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof. In addition to the therapeutic agent, the particles can also include any material conventionally used in the pharmaceutical and medical fields, including but not limited to erodible, non-erodible, biodegradable or non-biodegradable materials or combinations thereof. The particles can be microcapsules, which contain compounds in a solution or semi-solid state. The particles can have almost any shape.

[0233] Non-biodegradable and biodegradable polymer materials can be used to manufacture particles for delivering therapeutic agents. Such polymers can be natural or synthetic polymers. Polymers are selected based on the time period of required release. Particularly interesting bioadhesive polymers are included in Sawhney et al., Macromolecules 26: The bioerodible hydrogel described in 581-587 (1993), the teaching of which is hereby incorporated by reference into this paper. These include polyhyaluronic acid, casein, gelatin, gelatin, polyanhydride, polyacrylic acid, alginate, chitosan, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (dodecyl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate) and poly (octadecyl acrylate).

[0234] The therapeutic agent can be included in a controlled release system. The term "controlled release" is intended to refer to any drug-containing formulation, wherein the mode and profile of the drug release from the formulation is controlled. This refers to immediate release formulations and non-immediate release formulations, which include but are not limited to sustained release and delayed release formulations. The term "slow release" (also referred to as "extended release") refers to a pharmaceutical formulation that gradually releases the drug over a long period of time in its conventional sense, and can cause the blood level of the drug to be substantially constant over a long period of time. The term "delayed release" refers to a pharmaceutical formulation that has a time delay between the administration of the formulation and the release of the drug from the formulation in its conventional sense. "Delayed release" may or may not involve gradually releasing the drug over a long period of time, and may or may not be "slow release".

[0235] Long-term sustained-release implants are particularly suitable for the treatment of chronic diseases. "Long-term" release as used herein refers to that the implant is constructed and arranged to deliver the active ingredient at the therapeutic level for at least 7 days, and up to 30-60 days. Long-term sustained-release implants are well known to those of ordinary skill in the art, including some of the above-mentioned release systems.

[0236] Depending on the route of administration, various permissible doses are contemplated herein, including doses falling within about 10 6 Up to 10 11 The dosage can be a single dose or divided dose, and can be administered according to various schedules, including qd (once a day), bid (twice a day), tid (three times a day), or even once every other day, once a week, once a month, once a quarter, etc. In each case, it should be understood that the effective amount described herein corresponds to the administration situation, or corresponds to the total daily, weekly, monthly or quarterly dose determined by the dosing schedule.

[0237] In addition to the exemplary dosages and dosing regimens described herein, the attending diagnostician or physician can also determine the effective amount of any one of the compounds or mixtures of compounds described herein by using known techniques and / or by observing the results obtained under similar circumstances. In determining the effective amount or dosage, the attending diagnostician or physician will consider many factors, including but not limited to the species of mammal (including humans), its size, age and general health, the specific disease or condition involved, the degree of involvement or severity of the disease or condition, the response of the individual patient, the specific compound administered, the mode of administration, the bioavailability characteristics of the administered formulation, the selected dosage regimen, the use of concurrent medications, and other relevant circumstances.

[0238] Those skilled in the art will recognize that various modifications may be made to the above specific embodiments. The embodiments should not be limited to the specific limitations described. Other embodiments are also possible.

[0239] While compounds and pharmaceutical compositions have been illustrated and described in detail in the foregoing description, it should be considered illustrative rather than restrictive, and it should be understood that only certain embodiments have been shown and described and that all changes and modifications that fall within the spirit of the invention are desired to be protected.

[0240] It is intended that the scope of the compounds, compositions and methods of the present invention be defined by the following claims. However, the disclosure may be implemented in a manner other than that specifically explained and illustrated without departing from its spirit or scope. It will be appreciated by those skilled in the art that various alternatives to the embodiments described herein may be adopted when implementing the claims without departing from the spirit and scope defined in the following claims.

[0241] The use of any section headings is intended to aid in reading the document and should not be construed as limiting. In addition, the information associated with a section heading may appear within or outside of that particular section.

[0242] All publications, patents, patent application publications, journal articles, textbooks, and other publications mentioned in this document are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. In the event of inconsistent usage between this document and those documents incorporated by reference, the usage in the incorporated references shall be deemed supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document shall prevail.

[0243] Various techniques and mechanisms sometimes describe a connection or link between two components. Unless otherwise specified or the context clearly indicates the difference, words such as attached, linked, coupled, connected, and similar terms with their inflected morphemes are used interchangeably. These words and expressions do not necessarily indicate direct connection, but include connection through intermediate components. It should be noted that connection between two components does not necessarily mean a direct, unimpeded connection, as various other components may exist between the two components. Therefore, connection does not necessarily mean a direct, unimpeded connection unless otherwise specified. Some definitions

[0244] As used herein, the following terms and phrases shall have the following meanings. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0245] The term "about" when referring to a number or value or range (including, for example, integers, fractions, and percentages) means that the number or range referred to is an approximate value within experimental variability (or statistical experimental error), so the number or range may vary between 1% and 15% of the number or range (for example, + / - 5% to 15% of the value), provided that a person of ordinary skill in the art would consider it equivalent to the value (for example, having the same function or result). The term "substantially" allows for a certain degree of variability in the value or range, for example, within 90%, 95%, or 99% of the value or range limit.

[0246] Unless the context clearly dictates otherwise, the terms "a", "an" or "the" are used to include one or more. Thus, for example, reference to "the method" includes one or more methods and / or steps described herein and / or that will be apparent to one of ordinary skill in the art upon reading this disclosure.

[0247] Unless otherwise specified, the term "or" is used to mean a non-exclusive "or." Further, it is to be understood that the phraseology or terminology employed herein (not otherwise defined) is for the purpose of description only and not for the purpose of limitation.

[0248] "Alkyl" generally refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, such as a radical having from one to fifteen carbon atoms (e.g., C1-C 15 Unless otherwise indicated, disclosures of "alkyl" provided herein are intended to include independent references to saturated "alkyl". Alkyl groups can contain from one to thirteen carbon atoms (e.g., C1-C 13 An alkyl group may contain one to eight carbon atoms (e.g., C1-C8 alkyl). An alkyl group may contain one to five carbon atoms (e.g., C1-C5 alkyl). An alkyl group may contain one to four carbon atoms (e.g., C1-C4 alkyl). An alkyl group may contain one to three carbon atoms (e.g., C1-C3 alkyl). An alkyl group may contain one to two carbon atoms (e.g., C1-C2 alkyl). An alkyl group may contain one carbon atom (e.g., C1 alkyl). An alkyl group may contain five to fifteen carbon atoms (e.g., C5-C 15 Alkyl). The alkyl group may contain five to eight carbon atoms (e.g., C5-C8 alkyl). The alkyl group may contain two to five carbon atoms (e.g., C2-C5 alkyl). The alkyl group may contain three to five carbon atoms (e.g., C3-C5 alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (isopropyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl group is connected to the rest of the molecule by a single bond.

[0249] "Alkoxy" refers to a group of the formula -O-alkyl bonded through an oxygen atom wherein alkyl is an alkyl chain as defined above.

[0250] "Alkylene" or "alkylene chain" generally refers to a straight or branched divalent alkyl group, such as an alkyl group having one to twelve carbon atoms, such as methylene, ethylene, propylene, isopropylene, n-butylene, and the like, which attaches the rest of the molecule to the radical.

[0251] "Aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon, has 5 to 18 carbon atoms, and wherein at least one of the rings in the ring system is fully unsaturated, i.e., contains a cyclic, delocalized (4n+2) π electron system according to the Huckel theory. Ring systems derived from aryl include, but are not limited to, radicals such as benzene, fluorene, indane, indene, tetralin and naphthalene.

[0252] "Arylalkyl" refers to a group having the formula -R c -aryl radical, wherein R c is an alkylene chain as defined above, for example methylene, ethylene, etc. The alkylene chain part of the aralkyl group is optionally substituted in the manner described above for the alkylene chain.

[0253] The term "comprising" (and related terms such as "including," "having," or "containing") is not intended to exclude embodiments of any compounds, compositions, methods, processes, etc. that may "consist of" or "consist essentially of" the recited features. The invention illustratively described herein may suitably be practiced in the absence of any element or elements or limitations not specifically disclosed herein.

[0254] The term "heteroalkyl" refers to an alkyl group as defined above, wherein one or more of the skeletal carbon atoms of the alkyl group is replaced by a heteroatom (with an appropriate number of substituents or valences - for example, -CH2- can be replaced by -NH- or -O-). For example, each substituted carbon atom is independently substituted by a heteroatom, such as where the carbon is substituted by nitrogen, oxygen, selenium or other suitable heteroatoms. In some cases, each substituted carbon atom is independently substituted by oxygen, nitrogen (e.g., -NH-, -N(alkyl)- or -N(aryl)- or with another substituent contemplated herein) or sulfur (e.g., -S-, -S(=O)- or -S(=O)2-). The heteroalkyl group is attached to the rest of the molecule at a carbon atom of the heteroalkyl group. The heteroalkyl group is attached to the rest of the molecule at a heteroatom of the heteroalkyl group. A heteroalkyl group is C1-C 18 Heteroalkyl. Heteroalkyl is C1-C 12Heteroalkyl. Heteroalkyl is C1-C6 heteroalkyl. Heteroalkyl is C1-C4 heteroalkyl. Heteroalkyl can include alkoxy, alkoxyalkyl, alkylamino, alkylaminoalkyl, aminoalkyl, heterocycloalkyl, heterocycloalkyl and heterocycloalkylalkyl, as defined herein.

[0255] The terms "patient" and "subject" are used interchangeably and include human patients, laboratory animals, such as rodents (e.g., mice, rats, or hamsters), rabbits, monkeys, chimpanzees, domestic animals such as dogs, cats, or rabbits, farm animals such as cattle, horses, pigs, sheep, or goats, or captive wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, or whales. The patient to be treated is preferably a mammal, particularly a human.

[0256] The terms "protein", "polypeptide" and "peptide" refer to compounds composed of amino acids linked by peptide bonds and are used interchangeably. Example

[0257] The following examples serve to illustrate the present disclosure. The examples are not intended to limit the scope of the claims in any way. Example 1 Synthesis of S970

[0258] Tert-butyl (6-bromoimidazo [1,2-a] pyrazine-8-yl) (3,4-dimethoxyphenyl) carbamate (Compound S970-A). N, N-diisopropylethylamine (DIPEA) (2.5 mL, 5.0 equiv) was added to a stirred solution of 6,8-dibromoimidazo [1,2-a] pyrazine (1.0 g, 3.6 mmol) and 3,4-dimethoxyaniline (426 mg, 2.5 mmol) in dimethylformamide (DMF) (20.0 mL). The reaction mixture was heated to 120 ° C and stirred at the same temperature for 24 hours. After the reaction was completed (the progress of the reaction was monitored by thin layer chromatography (TLC)), it was cooled to room temperature and then added to 250 ml of ice water. The resulting precipitate was stirred at room temperature for 30-45 minutes and then filtered through a Buchner funnel. The resulting wet material was washed with water (100 mL), then with diethyl ether (100 mL), and dried under vacuum at 50° C. for 5 hours to give 6-bromo-N-(3,4-dimethoxyphenyl)imidazo[1,2-a]pyrazin-8-amine as a solid.

[0259] The crude intermediate was added to a 100 ml single-necked RBF, followed by 25 ml of dichloromethane (DCM), (Boc)2O anhydride (0.875 g, 4.0 mmol) and a catalytic amount of 4-dimethylaminopyridine (DMAP) (25 mg, 0.025 equivalent). The resulting solution was heated to 50 ° C and stirred for 3 hours. After the reaction was completed (the reaction progress was monitored by TLC), the resulting solution was concentrated to dryness in vacuo.

[0260] The resulting crude material was purified by normal phase silica gel chromatography (hexanes:EtOAc) to afford an off-white solid (1.00 g, 65% overall yield).

[0261] 1 H NMR (500 MHz, dimethyl sulfoxide (DMSO)-d6): δ8.89 (s, 1H), 8.13 (s, 1H), 7.86 (s, 1H), 6.99-6.82 (m, 2H), 6.74 (d, J=8.5 Hz, 1H), 3.71 (s, 3H), 3.68 (s, 3H), 1.29 (s, 9H) ppm; 13 CNMR (125 MHz, DMSO-d6): δ 152.9, 149.0, 148.1, 145.4, 136.9, 136.6, 133.6, 120.7, 119.7, 119.0, 116.8, 111.9, 111.8, 81.9, 56.1, 56.0, 28.0 ppm. LCMS (ESI) m / z calculated value C 19 H 22 BrN4O4:449.0819 Measured value:449.0817(M+1) + .

[0262] Tert-butyl(6-(1H-indol-6-yl)imidazo[1,2-a]pyrazin-8-yl)(3,4-dimethoxyphenyl)carbamate (compound S970-B).

[0263] Compound S970-A (500 mg, 1.11 mmol) and (1H-indol-5-yl)boronic acid (220 mg, 1.35 mmol) were dissolved in 1,4-dioxane (8 mL), and H2O (4 mL) was added to a vial equipped with a screw cap with a septum. The reaction mixture was purged with a gentle stream of N2 under stirring. After purging for 5 minutes, the outlet needle was removed and Pd(PPh3)4 (40 mg, 0.025 mmol) was added, followed by rapid addition of Na2CO3 (356 mg, 3.35 mmol). The reaction mixture was heated to 120 ° C and stirred at the same temperature for 6 hours. The reaction mixture was then concentrated to dryness, and the desired product was separated by normal phase silica gel chromatography (hexane: EtOAc) to give an off-white solid (302 mg, 56%).

[0264] 1 H NMR (500MHz, DMSO-d6): δ11.25(S,1H),9.11(s,1H),8.13(s,1H),8.00(s,1H),7.80(s,1H),7.68-7.50(m ,2H),7.42-7.30(m,1H),7.14-7.03(m,1H),6.96-6.77(m,2H),6.44(s,1H),3.72(s,3H),3.70(s,3H)ppm; 13 C NMR (125 MHz, DMSO-d6): δ 153.3, 148.9, 147.9, 145.5, 137.9, 136.9, 136.7, 135.6, 134.0, 129.0, 128.4, 127.2, 120.8, 119.6, 117.2, 116.5, 115.5, 111.95, 111.92, 109.5, 101.6, 81.3, 56.1, 56.0, 28.1 ppm. LCMS (ESI) m / z calculated value C 27 H 28 N5O4:486.2136 Measured value:486.2133(M+1) + .

[0265] 1-((6-(8-((3,4-dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-6-yl)-1H-indol-3-yl)methyl)azetidin-3-ol S970.

[0266] Compound B (250 mg, 0.56 mmol) was dissolved in DCM (2.5 mL) and trifluoroacetic acid (TFA) (2.5 mL) was added within 10 minutes at 0° C. The resulting solution was stirred at room temperature for 10 hours under N 2 atmosphere.

[0267] After the reaction was completed, the reaction mixture was concentrated to dryness under reduced pressure, and the crude material was used directly in the next step of N-(3,4-dimethoxyphenyl)-6-(1H-indol-6-yl)imidazo[1,2-a]pyrazin-8-amine without purification.

[0268] A solution of azetidine-3-ol (45 mg, 0.65 mmol) and 1,4-dioxane (1 mL) was added dropwise to a stirred solution of formaldehyde (30% aqueous solution, 83 μL) and acetic acid (1 mL) in 1,4-dioxane (1 mL) at 0°C over 10 minutes. The resulting solution was stirred at the same temperature for 30 minutes. A solution of crude N-(3,4-dimethoxyphenyl)-6-(1H-indol-6-yl)imidazo[1,2-a]pyrazine-8-amine in 1,4-dioxane (1 mL) was added dropwise to the reaction mixture at 0°C over 10 minutes. The reaction mixture was warmed to room temperature and stirred at the same temperature for 24 hours. The resulting suspension was concentrated under reduced pressure and the title product was separated by normal phase silica gel chromatography (dichloromethane: methanol) to give an off-white solid (20 mg, 10%).

[0269] 1H NMR (500MHz, DMSO) δ11.11(s,1H),9.47(s,1H),8.56(s,1H),8.12(d,J=2.4Hz,1H),8. 04(s,1H),8.00(dd,J=10.1,1.0Hz,1H),7.67(m,1H),7.63(dd,J=5.6,0.9Hz,3H),7.27 (d,J=1.8Hz,1H),6.98(d,J=8.8Hz,1H),4.18(p,J=6.2Hz,1H),3.83(s,3H),3.77(s,3 H),3.74–3.69(m,2H),3.48(td,J=6.1,1.9Hz,2H),2.80(td,J=6.3,1.7Hz,2H)ppm; 13C NMR (126 MHz, DMSO) δ 173.1, 148.9, 145.1, 144.4, 138.1, 137.1, 134.5, 132.5, 132.4, 130.8, 127.7, 125.5, 119.3, 117.0, 116.7, 112.6, 111.8, 109.4, 107.9, 105.4, 63.9, 61.1, 56.3, 55.8, 54.4, 22.7; LCMS (ESI) m / z calculated value C 26 H 27 N6O3:471.2139 Measured value:471.2139(M+1)+. Example 2 In vitro analysis of Syk inhibitors

[0270] Syk kinase inhibition assays were performed in 96-well plates using the ADP-Glo ​​assay kit according to the manufacturer's instructions. All kinase reactions were performed using 1x kinase reaction buffer, which consisted of 40mM Tris pH 7.5, 20mM MgCl2, 0.1mg / mL bovine serum albumin, and 0.1mM dithiothreitol (DTT) added as needed for the target kinase (each kinase listed in Table 2 above) to generate the kinase reaction buffer. The DMSO concentrated solution of the kinase inhibitor was diluted into the 1x kinase reaction buffer, and the final concentration of DMSO in each kinase reaction was kept below 0.5%. The total volume of each kinase reaction was 25μL, which was distributed as follows: 8.5μL 1x kinase reaction buffer, 5μL diluted kinase inhibitor, 5μL 1mg / mL poly-(4:1-Glu, Tyr) kinase substrate peptide, 4μL 1x kinase reaction buffer in the kinase, and 2.5μL diluted to 100μM ATP in the kinase reaction buffer. Kinase inhibitors were incubated with kinase and substrate at room temperature for 30 min before the addition of ATP.

[0271] After ATP was added to the wells, the plates were incubated at 37 °C for 30 minutes. After incubation, 25 μL of ADP-Glo ​​reagent was immediately added and incubated at ambient temperature for 30 minutes. After 30 minutes, 50 μL of kinase detection reagent was added, and the plates were incubated at ambient temperature for 45 minutes and protected from light. After 45 minutes, luminescence was recorded using a Neo2 plate reader and a 1-second signal integration was performed. The background signal was subtracted from all readings, and the signal was normalized to the maximum signal obtained in the absence of inhibitors. The data were plotted and the inhibition curves were fitted using Graphpad software, and the results are shown in Tables 2 and 3 above. Example 3 Synthesis of compound S1115A

[0272] A solution of dichloropyrazine-2-carboxamide (1.0 equiv) was added to a stirred solution of 3,4-dimethoxyaniline (1.0 equiv) in THF (20 V) and lithium bis(trimethylsilyl)amide (LHMDS) (3.0 equiv) at -78°C. The resulting reaction mixture was stirred at the same temperature for 2 hours, then warmed to room temperature and stirred for 2 hours. After completion of the reaction (progress monitored by TLC), the reaction mixture was poured into ice-cold 2N hydrochloric acid (HCl), extracted with DCM, and dried over sodium sulfate.

[0273] The solvent was then removed under vacuum. The crude intermediate obtained was triturated with hexanes to give compound S1115A (5-chloro-3-((3,4-dimethoxyphenyl)amino)pyrazine-2-carboxamide) as a dark yellow solid (yield 70%). Example 4 Synthesis of compound S1115B

[0274] Compound S1115-A (1.0 equiv) and (1H-indol-5-yl)boronic acid (1.2 equiv) were dissolved in 1,4-dioxane (20 V) and then H2O (20 V) was added in a vial equipped with a screw cap with a septum. The reaction mixture was purged with a gentle stream of N2 while stirring. After 5 min of purging, the outlet needle was removed and Pd(PPh3)4 (0.05 equiv) was added, followed by the rapid addition of Na2CO3 (3.0 equiv).

[0275] The reaction mixture was heated to 120°C and stirred at the same temperature for 6 hours. The reaction mixture was then concentrated to dryness and the desired product was isolated by normal phase silica gel chromatography (DCM:MeOH) to give compound S1115B (3-((3,4-methoxyphenyl)amino)-5-(1H-indol-6-yl)pyrazine-2-carboxamide) as a yellow solid. (Yield 58%). Example 5 Synthesis of S1115

[0276] A solution of azetidin-3-ol (3.0 eq.) and 1,4-dioxane (20 V) was added dropwise over 10 min to a stirred solution of formaldehyde (30% in H2O, 3.0 eq.) and acetic acid (10 eq.) in 1,4-dioxane (10 V) at 0°C.

[0277] The resulting solution was stirred at the same temperature for 30 minutes, and a solution of compound S1115B in 1,4-dioxane (20V) was added dropwise to the reaction mixture at 0°C within 10 minutes. The reaction mixture was allowed to reach room temperature and stirred at the same temperature for 24 hours. The resulting suspension was concentrated under reduced pressure, and compound S1115 (3-((3,4,-methoxyphenyl)amino)-5-(3-((3-hydroxyazetidine-1-yl)methyl)-1H-indol-6-yl)pyrazine-2-carboxamide) was separated by reverse phase silica gel chromatography (pH 7.4, ammonium acetate: CAN) to obtain yellow solid compound S1115 (yield of 10-15%).

Claims

1. Compounds of formula X: ABC(X) or a pharmaceutically acceptable salt thereof, wherein: A is a nitrogen-linked and optionally substituted first ring, the first ring comprising a fused bicyclic or monocyclic ring, wherein each ring of the first ring comprises a 4-6 membered heterocyclic or carbocyclic ring; B is a second ring, which is optionally substituted with at least one substituent, wherein the second ring comprises a monocyclic ring or a fused bicyclic or tricyclic ring, and wherein each ring of the second ring comprises a 5-membered or 6-membered heterocyclic or carbocyclic ring; and C is a third bicyclic ring comprising a 5- or 6-membered carbocyclic ring fused to a 5- or 6-membered carbocyclic or heterocyclic ring, wherein the third bicyclic ring is substituted with at least a first substituent comprising: or amine, or ketone, or amide, or alcohol, in: X4 is CH2, NH, CO, O or S, Y5 is CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and and is the point of attachment of the first substituent to the third bicyclic ring.

2. The compound according to claim 1, wherein A is: in: Each X' is independently selected from CH and NH; Each Y2 is independently selected from CH2, NH, O and S; Z2 is CH or N; and Each R3 is independently selected from H, F, Cl, OH and -Y-(CH2) n -, where n = 1-14.

3. The compound according to claim 1, wherein B is in: Each X2 is independently selected from CH and N; Y3 is selected from O, NH and S; and Z3 is selected from OH, NH2 and NHCH3.

4. The compound according to any one of claims 1 to 3, wherein B is not 5. The compound according to any one of claims 1 to 3, wherein one or more hydrogens are optionally replaced by deuterium. The compound according to claim 1 , wherein the N connected to the first ring of A forms a nitrogen bridge with B. .

7. The compound of claim 6, wherein A is a first monocyclic ring substituted with a bulky substituent located para to the nitrogen bridge.

8. The compound of claim 7, wherein the bulky substituent is morpholine.

9. The compound according to any one of claims 1-3 or 6-8, wherein each ring of the first ring of A is a 6-membered, optionally substituted, open or closed heterocyclic or carbocyclic ring.

10. The compound according to any one of claims 1-3 or 6-8, wherein A is a first monocyclic ring substituted with two methoxy groups.

11. The compound according to any one of claims 1-3 or 6-8, wherein B is a monocyclic ring substituted with an amide.

12. The compound of any one of claims 1-3 or 6-8, wherein B is rigid.

13. The compound according to claim 1, wherein B is pyrazine substituted with at least one substituent. The compound according to claim 13 , wherein at least one substituent of B is an amide.

15. The compound according to claim 1, wherein C is in: Each X3 is independently selected from CH and N; and Y4 is CH2, NH, O or SH.

16. The compound according to claim 15, wherein the first substituent of C is 17. The compound according to claim 1, wherein the first substituent Z is -OR, wherein R is And n=1-14.

18. The compound of any one of claims 1-3, 6-8 or 13-17, wherein the third bicyclic ring of C is indole.

19. The compound of claim 1, wherein the third bicyclic ring of C is indole and the first substituent of C comprises a 4-6 membered, optionally substituted carbocyclic or heterocyclic substituent.

20. The compound of claim 19, wherein the carbocyclic or heterocyclic substituent comprises a lipophilic ester.

21. A compound according to any one of claims 1-3, 6-8 or 13-17, wherein Z of the first substituent of C comprises a phosphonate or a phosphate.

22. according to the compound described in any one of claim 1-3,6-8 or 13-17, wherein the first substituent of C is wherein Q is a phosphate or a phosphonate.

23. according to the compound described in any one of claim 1-3,6-8 or 13-17, wherein the first substituent of C is wherein Q is RC(=O)O-, wherein R is an alkyl group.

24. A compound according to any one of claims 1-3, 6-8 or 13-17, wherein B is capable of forming a hydrogen bond with amino acid residue Ala451 in the catalytic domain of Syk tyrosine kinase, and the first substituent of C is capable of forming a hydrogen bond with amino acid residue Asp512 in the catalytic domain of Syk tyrosine kinase.

25. The compound according to claim 1, having the following structure: in: R1 is Y is each independently CH or N, R2 is or an amine, or an alcohol, wherein: X4 is CH2, NH, CO, O or S, Y5 is CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and and is the point of attachment; and R4 is H, CH3, amine or methanol.

26. The compound according to claim 1, having the following structure: in: R1 is R2 is or amine, or propanol; X is each independently CH2, NH, CO, O or S, Y is each independently CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and and is the attachment point.

27. A compound according to any one of claims 1-3, 6-8, 13-17, 19, 25 or 26, wherein the compound has: a CLogP value of about 2 to about 5 (e.g., 2 to about 5, about 2 to 5, or 2 to 5); A polar surface area of ​​less than about 250 square angstroms (e.g., less than 250 square angstroms); No more than 5 hydrogen bond donors; No more than 15 hydrogen bond acceptors; and / or No more than 20 rotatable bonds.

28. The compound of any one of claims 1-3, 6-8, 13-17, 19, 25 or 26, wherein the compound specifically binds to Syk tyrosine kinase.

29. The compound of any one of claims 1-3, 6-8, 13-17, 19, 25 or 26, wherein when administered to a subject, the compound inhibits Syk from phosphorylating erythrocyte anion transporter band 3.

30. The compound according to claim 1, having the following structure: Or a pharmaceutically acceptable salt of any of the foregoing structures.

31. The compound according to claim 1, having the following structure: Or a pharmaceutically acceptable salt of any of the foregoing structures.

32. The compound according to claim 1, having the following structure: Or a pharmaceutically acceptable salt of any of the foregoing structures.

33. The compound according to claim 31, wherein the hydroxyl group (HO-) of the compound is substituted with a phosphate or phosphonate.

34. The compound of claim 31, wherein the hydroxyl group (HO-) of the compound is substituted with RC(=O)O-, wherein R is an alkyl group.

35. The compound of claim 33, wherein the alkyl group is a C1-C6 alkyl group.

36. A pharmaceutical composition comprising a compound according to any one of claims 1 to 34 and a pharmaceutically acceptable carrier or excipient.

37. The pharmaceutical composition of claim 35, further comprising one or more of Cremophor, polysorbate, and nanoparticles.

38. The pharmaceutical composition of claim 35, further comprising a polymer or a hydrogel.

39. A method of treating a red blood cell (RBC)-mediated disease, the method comprising administering to a subject an effective amount of: A compound or a pharmaceutically acceptable salt according to any one of claims 1 to 34, or A pharmaceutical composition according to any one of claims 35-37.

40. The method of claim 38, further comprising administering to the subject an effective amount of at least one second active agent.

41. A method of treating an RBC-mediated disease, the method comprising administering to a subject an effective amount of: Compound of formula X: ABC(X) or a pharmaceutically acceptable salt thereof, wherein: A is a nitrogen-linked and optionally substituted first ring, the first ring comprising a fused bicyclic or monocyclic ring, wherein each ring of the first ring comprises a 4-6 membered heterocyclic or carbocyclic ring; B is a second ring, which is optionally substituted with at least one substituent, wherein the second ring comprises a monocyclic ring or a fused bicyclic or tricyclic ring, and wherein each ring of the second ring comprises a 5-membered or 6-membered heterocyclic or carbocyclic ring; and C is a third bicyclic ring comprising a 5- or 6-membered carbocyclic ring fused to a 5- or 6-membered carbocyclic or heterocyclic ring, wherein the third bicyclic ring is substituted with at least a first substituent comprising: or amine, or ketone, or amide, or alcohol, in: X4 is each independently CH2, NH, CO, O or S, Y5 is each independently CH or N, Z is selected from OH, SH, NH2, CO2H, NHCH3 and and is the point of attachment of the first substituent to the third bicyclic ring, wherein the concentration of the compound when inhibiting Syk tyrosine kinase is at least one 20 times less than the concentration of the compound when inhibiting HGFR, EGFR, FGFR3, IGF1R and / or VEGFR; or A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof.

42. The method of any one of claims 35-40, wherein the RBC-mediated disease is selected from the group consisting of sickle cell disease, thalassemia, glucose-6-phosphate dehydrogenase deficiency, glutathione reductase deficiency, and a disease involving the release of microvesicles from blood cells.

43. The method of any one of claims 38-40, wherein the RBC-mediated disease is alpha-thalassemia or beta-thalassemia, alone or in combination with sickle cell disease.

44. A method for preventing or inhibiting phosphorylation of human erythrocyte anion transporter band 3 in a subject, comprising administering to the subject an effective amount of: A compound or a pharmaceutically acceptable salt according to any one of claims 1 to 36, or A pharmaceutical composition according to any one of claims 35-37.

45. The method of any one of claims 38-43, wherein the compound or pharmaceutical composition stabilizes the subject's red blood cell membranes and reduces the release of hemoglobin from the subject's sickle cells.

46. ​​The method of any one of claims 38-44, wherein the subject has a reduced risk of vaso-occlusive crisis.

47. The method of any one of claims 38-45, wherein the compound, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition is administered at an IC of 200 nM or less. 50 Inhibits Syk tyrosine kinase.

48. The method of any one of claims 38-45, wherein the subject is a human child.

49. The method of any one of claims 38-45, further comprising administering an effective amount of at least one second active agent simultaneously or sequentially in any order.

50. The method of claim 48, wherein the at least one second active agent is selected from the group consisting of fostamatinib, PRT062607, TAK-659, TAE-684, entosatinib, lanraprinib, selutinib, piceatannol, S701, Syk II, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusartinib, R112, PRT-060318, and OXSI-2, optionally wherein the at least one active agent is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

51. The method of claim 48, wherein the compound, pharmaceutically acceptable salt thereof, or pharmaceutical composition and at least one second agent are administered by the same route.

52. The method of claim 48, wherein the compound, pharmaceutically acceptable salt thereof, or pharmaceutical composition and at least one second agent are administered by different routes.

53. The method of claim 48, wherein the at least one second active agent comprises hydroxyurea.

54. The method of claim 49, wherein the at least one second active agent comprises more than one second active agent, all of which are selected from the group consisting of fostamatinib, PRT062607, TAK-659, TAE-684, entosatinib, lanraprinib, selutinib, piceatannol, S701, Syk II, Syk IV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusartinib, R112, PRT-060318, and OXSI-2, optionally wherein the at least one active agent is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

55. A method for treating a disease involving the release of microvesicles from blood cells in a subject, the method comprising administering to the subject an effective amount of a compound or a pharmaceutically acceptable salt thereof, the compound comprising a rigidly fused bicyclic ring substituted with at least two substituents, wherein at least one substituent is an N-linked, optionally substituted carbocyclic or heterocyclic ring, and the other substituent is an indole substituted with at least one substituent comprising a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring, thereby treating the disease involving the release of microvesicles in the subject.

56. The method of claim 54, wherein the disease is alpha-thalassemia or beta-thalassemia alone or in combination with sickle cell disease.

57. The method of claim 54, wherein the disease is glucose-6-phosphate dehydrogenase (G6PD) deficiency or glutathione reductase deficiency.

58. The method according to any one of claims 38-47, further comprising administering a Src inhibitor simultaneously or sequentially in any order, optionally as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

59. The method of claim 57, wherein the compound, pharmaceutically acceptable salt or pharmaceutical composition thereof and the Src inhibitor are administered by the same or different routes.

60. The method of claim 57 or 58, wherein the Src inhibitor is dasatinib, ibrutinib, bafitinib, PP1, PP2, PP121, or a combination of two or more of the foregoing.

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