Spleen tyrosine kinase inhibitors, compositions, and methods of use

Abstract

The present invention relates to compounds, compositions, and methods that prevent Syk from phosphorylating the erythrocyte anion transporter band 3 and / or Syk tyrosine kinase.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 319,486, filed March 14, 2022, and U.S. Provisional Patent Application No. 63 / 398,599, filed August 17, 2022. The contents of the aforementioned applications are incorporated by reference in their entirety into this disclosure.

[0002] Government support statement This invention was made with Government support under GM024417 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present disclosure relates to spleen tyrosine kinase (Syk) inhibitors, compositions comprising the inhibitors, and methods of use in the treatment of sickle cell disease, as well as other diseases involving microvesicle release from blood cells. [Background technology]

[0004] Sickle cell disease is a group of inherited blood disorders resulting from an abnormality in hemoglobin, the molecule in red blood cells (RBCs) that delivers oxygen to cells throughout the body, specifically an abnormality in the structure of β-globin (glu6val). Sickle cell can be characterized by a mutation of an amino acid in this β-chain from glutamic acid to valine, which causes deoxygenated sickle hemoglobin (deoxy-HbS) to form insoluble polymers inside the RBC, deforming the RBC into a rigid-shaped or sickle-shaped (i.e., crescent) red blood cell.

[0005] Sickle cell disease affects a significant number of individuals worldwide. In the United States, those with sickle cell disease can experience an average mortality rate in their 40s, poor quality of life, and high medical costs. Signs and symptoms of sickle cell disease usually begin in childhood. Features of the disorder can include low RBC counts (i.e., anemia), repeated infections, and periodic episodes of pain.

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

[0007] Currently, the standard of care for sickle cell patients aged 2 years and older is blood transfusions and treatment with the anticancer drug hydroxyurea. Not all patients respond to hydroxyurea, which can worsen symptoms and cause bone marrow suppression in some patients. Pyruvate kinase agonist (e.g., imatinib)-based treatments are also available, but such inhibitors can also interact with (i.e., inhibit) growth factor receptors involved in the revascularization of tissues that is often required due to the common occurrence of vaso-occlusive events in sickle cell patients. In addition, unwanted inhibition of growth factor receptors can be problematic, especially 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, as well as other objects and advantages, and features of the invention will become apparent from the detailed description provided herein. Summary of the Invention

[0009] (i) Compounds comprising a rigid, fused bicyclic ring, which is 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 an indole substituted with at least one substituent, at least one of which is a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring; (ii) a compound that prevents spleen tyrosine kinase (Syk) from phosphorylating the human erythrocyte anion transporter band 3; (iii) where the compound inhibits Syk tyrosine kinase at a concentration that is at least about 20-fold lower than the concentration that 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). and In addition to or instead of (ii) and (iii), the compound (i) is (iv) a CLogP value of between about 2 and about 5 (e.g., between 2 and about 5, between about 2 and 5, or between 2 and 5); (v) a polar surface area of ​​less than about 250 Å2 (e.g., less than 250 Å2); (vi) up to 5 H-bond donors; (vii) 15 or fewer H-bond acceptors, and / or (viii) 20 or fewer rotatable bonds A compound is provided having the following formula:

[0010] The compound has the formula X: ABC(X) or a pharma- ceutically acceptable salt thereof, wherein A is an optionally substituted first ring linked at the nitrogen, the first ring comprising a fused bicyclic ring or a monocyclic ring, each ring of each first ring comprising 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, each ring of the second ring comprising a 5- or 6-membered heterocyclic or carbocyclic ring; C is a third bicyclic ring comprising a 5- or 6-membered carbocyclic ring fused with a 5- or 6-membered carbocyclic or heterocyclic ring, said third bicyclic ring being

[0011] [ka] or an amine, or an alcohol; During the ceremony, X4 is CH2, NH, CO, O or S; Y5 is CH or N; Z is OH, SH, NH2, CO2H, NHCH3, and

[0012] [ka] is selected from

[0013] [ka] is the point of attachment of the first substituent to the third bicyclic ring.

[0014] In certain embodiments, A of the compound is

[0015] [ka] and During the ceremony, 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; Each R3 is independently H, F, Cl, OH, and -Y-(CH2) n -, wherein n=1 to 14.

[0016] The N-linkage of A to the first ring of the compound can form a nitrogen bridge with B of the compound. In certain embodiments, A is a first monocyclic ring that is substituted with a bulky substituent that is para to such nitrogen bridge. The bulky substituent can be, for example, morpholine.

[0017] In certain embodiments, each ring of the first ring of A is a six-membered, optionally substituted, open or closed heterocyclic or carbocyclic ring. A may be a first monocyclic ring substituted with two methoxy groups.

[0018] In certain embodiments, B of the compound is

[0019] [ka] and During the ceremony, Each X2 is independently selected from CH and N; Y3 is selected from O, NH and S; Z3 is selected from OH, NH2 and NHCH3.

[0020] In certain embodiments, B of the compound is

[0021] [ka] In other words, the compound is any of those described herein, except that B is not:

[0022] [ka] is not. B can be a monocyclic ring substituted with an amide. B can be rigid. In certain embodiments, B is a pyrazine substituted with at least one substituent (e.g., an amide).

[0023] C of the compound may be an indole. C of the compound may be

[0024] [ka] can be, During the ceremony, Each X3 is independently selected from CH and N; Y4 is CH2, NH, O or SH.

[0025] The third bicyclic ring at C can be indole and the first substituent at C can include a 4-6 membered, optionally substituted carbocyclic or heterocyclic substituent. In certain embodiments, the carbocyclic or heterocyclic substituent is a lipophilic ester.

[0026] The first substituent at C is

[0027] [ka] It can be.

[0028] The first substituent Z of X is -OR, where R is

[0029] [ka] where n=1 to 14).

[0030] In certain embodiments, the first substituent Z of the third bicyclic ring comprises a phosphate or a phosphonate. In certain embodiments, the first substituent of C comprises -OR, where R is a phosphate or a phosphate. In certain embodiments, the first substituent of C comprises:

[0031] [ka] wherein Q is a phosphate or phosphonate ester. In certain embodiments, the first substituent on C is

[0032] [ka] where Q is RC(=O)O-, where R is alkyl (e.g., C1-C6 alkyl).

[0033] Compound B can form a hydrogen bond with amino acid residue Ala451 in the catalytic domain of Syk tyrosine kinase, and the first substituent of C (e.g., its Z) can form a hydrogen bond with amino acid residue Asp512 in the catalytic domain of Syk tyrosine kinase.

[0034] One or more of the hydrogens of the compound can optionally be replaced with deuterium.

[0035] The compound has the structure:

[0036] [ka] may include In the formula, ring A is

[0037] [ka] and During the ceremony, 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; Each R3 is independently H, F, Cl, OH, and -Y-(CH2) n -, wherein n=1 to 14; In the formula, ring B is

[0038] [ka] and During the ceremony, Each X2 is independently selected from CH and N; Y3 is selected from O, NH and S; Z3 is selected from OH, NH2 and NHCH3; However, B is

[0039] [ka] Instead, In the formula, ring C is

[0040] [ka] and During the ceremony, Each X3 is independently selected from CH and N; Y4 is selected from CH2, NH, O and SH; In the formula, ring D is

[0041] [ka] or an amine, or an alcohol, During the ceremony, X4 is selected from CH2, NH, CO, O and S; Y5 is selected from CH and N; Z is OH, SH, NH2, NHCH3, and

[0042] [ka] is selected from One or more hydrogens can optionally be replaced with deuterium.

[0043] Ring B can form a hydrogen bond with amino acid residue Ala451 in the catalytic domain of Syk tyrosine kinase, and Z (e.g., the first substituent of C) on ring D 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 that is 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 a morpholine.

[0044] The compound may have an increased docking score for Syk tyrosine kinase compared to an otherwise identical compound lacking the bulky substituent para to the nitrogen bridge.

[0045] [ka] The structure may include:

[0046] The compound is

[0047] [ka] may include or have the structure During the ceremony, R1 is,

[0048] [ka] and R2 is

[0049] [ka] or an amine, or an alcohol, wherein X4 is CH2, NH, CO, O or S; Y5 is CH or N; Z is OH, SH, NH2, CO2H, NHCH3, and

[0050] [ka] is selected from

[0051] [ka] is the point of attachment of the first substituent to the third bicyclic ring; R4 is H, CH3, an amine, or methanol.

[0052] The compound has the structure:

[0053] [ka] or a pharma- ceutically acceptable salt thereof.

[0054] The compound has the structure:

[0055] [ka] or a pharma- ceutically acceptable salt thereof.

[0056] The compound has the structure:

[0057] [ka] or a pharma- ceutically acceptable salt thereof.

[0058] The compound has the structure:

[0059] [ka] may have or include or a pharma- ceutically acceptable salt thereof of any of the foregoing.

[0060] The compound has the structure:

[0061] [ka] or a pharma- ceutically acceptable salt thereof; wherein each X is independently selected from CH, N, NH, O, and S; Y is O, NH or SH; Z is OH or NH2. The 4-6 membered, optionally substituted carbocyclic or heterocyclic substituents on the indole can include lipophilic esters. Z on ring D can be -OR, where R is

[0062] [ka] (where N=1 to 14) It can be.

[0063] The compound has the structure:

[0064] [ka] or a pharma- ceutically acceptable salt thereof.

[0065] In certain embodiments, the compound has the structure:

[0066] [ka] or a pharma- ceutically acceptable salt thereof, wherein the hydroxyl (HO-) group is replaced with (a) a phosphate or phosphonate ester, or (b) RC(=O)O-, where R is alkyl (e.g., C1-C6 alkyl).

[0067] The compound is (i) Compounds comprising pyrazine, which is substituted with at least three substituents, at least one of which is a carboxamide or lactam and the other of which is an N-linked, optionally substituted carbocycle or heterocycle, the other of which is a 4-6 membered, optionally substituted carbocycle or heterocycle having at least one substituent, including a 4-6 membered, optionally substituted carbocycle or heterocycle. (ii) a compound that prevents Syk tyrosine kinase from phosphorylating human erythrocyte anion transporter band 3; (iii) where the compound inhibits Syk tyrosine kinase at a concentration at least about 20-fold lower than the concentration at which it inhibits HGFR, EGFR, FGFR3, IGF1R and / or VEGFR. can be, In addition to or instead of (ii) and (iii), the compound (i) is (iv) a CLogP value of between about 2 and about 5 (e.g., between 2 and about 5, between about 2 and 5, or between 2 and 5); (v) a polar surface area of ​​less than about 250 Å2 (e.g., less than 250 Å2); (vi) up to 5 H-bond donors; (vii) 15 or fewer H-bond acceptors, and / or (viii) 20 or fewer rotatable bonds has.

[0068] The compound has the structure:

[0069] [ka] or a pharma- ceutically acceptable salt thereof, wherein X is CH or N and R is

[0070] [ka] It is.

[0071] The compound has the structure:

[0072] [ka] or a pharma- ceutically acceptable salt thereof, wherein X is CH or N and R is

[0073] [ka] It is.

[0074] The compound has the structure:

[0075] [ka] or a pharma- ceutically acceptable salt thereof, wherein X is CH or N and R is

[0076] [ka] It is.

[0077] The compound has the structure:

[0078] [ka] or a pharma- ceutically acceptable salt thereof, wherein X is CH or N and R is

[0079] [ka] It is.

[0080] The compound has the structure:

[0081] [ka] or a pharma- ceutically acceptable salt thereof, wherein X is CH or N and 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 of X and / or Y are N.

[0082] The compound has the structure:

[0083] [ka] or a pharma- ceutically acceptable salt thereof, wherein X is CH or N and 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 of X and / or Y are N.

[0084] The compound has the structure:

[0085] [ka] or a pharma- ceutically acceptable salt thereof, In the formula, R is

[0086] [ka] and each X is independently CH or N.

[0087] The compound has the structure:

[0088] [ka] or a pharma- ceutically acceptable salt thereof, wherein each X is independently CH or N, each Y is CH or N, and R is a carbocycle or a heterocycle, wherein when X and Y are all CH, the compound may have a weaker binding affinity than when at least one or more of X and / or Y are N.

[0089] The compound has the structure:

[0090] [ka] or a pharma- ceutically acceptable salt thereof, wherein each X is independently CH or N, Y is CH or N, and R is a carbocycle or heterocycle, wherein when X and Y are all CH, the compound may have a weaker binding affinity than when at least one or more of X and / or Y are N.

[0091] The compound has the structure:

[0092] [ka] or a pharma- ceutically acceptable salt thereof, wherein each X is independently CH or N, Y is CH or N, and R is a carbocycle or heterocycle, wherein when X and Y are all CH, the compound may have a weaker binding affinity than when at least one or more of X and / or Y are N.

[0093] The compound has the structure:

[0094] [ka] or a pharma- ceutically acceptable salt thereof. In alternative embodiments, the hydroxyl (HO-) group of the aforementioned compounds may be replaced with a phosphate or phosphonate ester. In yet another alternative embodiment, the hydroxyl (HO-) group may be replaced with RC(=O)O-, where R is alkyl (e.g., C1-C6 alkyl).

[0095] Further provided is a pharmaceutical composition comprising a compound described herein (or a pharmaceutical salt thereof) and a pharma- ceutical acceptable carrier or excipient. The composition can include, for example, cremophor, polysorbate, nanoparticles, polymers, or hydrogels.

[0096] Still further provided is a method of treating sickle cell disease comprising administering to a subject an effective amount of (a) the compound described above, (b) fostamatinib, (c) PRT062607, (d) TAK-659, (e) TAE-684, (f) entospletinib, (g) lanlaprenib, (h) celdulatinib, (i) piceatannol, (j) S701, (k) SykII, (l) SykIV, (m) TAS05567, (n) GSK143 , (o) Syk-IN-3, (p) Syk-IN-4, (q) Syk-IN-1, (r) SRX3207, (s) RO9021, (t) gusacitinib, (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 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), optionally as a pharmaceutical composition comprising a pharma- ceutical ...

[0097] Methods of treating red blood cell (RBC) mediated diseases are also provided. In certain embodiments, the methods of treating RBC mediated diseases include administering to a subject (e.g., a human patient) an effective amount of a compound described herein, or a pharma- ceutical acceptable salt thereof, or a pharmaceutical composition described herein. In certain embodiments, the compound is Formula X: ABC(X) or a pharma- ceutically acceptable salt thereof, wherein A is an optionally substituted first ring linked at the nitrogen, the first ring comprising a fused bicyclic ring or a monocyclic ring, each ring of each first ring comprising 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, each ring of the second ring comprising a 5- or 6-membered heterocyclic or carbocyclic ring; C is a third bicyclic ring comprising a 5- or 6-membered carbocyclic ring fused with a 5- or 6-membered carbocyclic or heterocyclic ring, said third bicyclic ring being

[0098] [ka] or an amine, or an alcohol; During the ceremony, Each X4 is independently CH2, NH, CO, O or S; Each Y5 is independently CH or N; Z is OH, SH, NH2, CO2H, NHCH3, and

[0099] [ka] is selected from

[0100] [ka] is the point of attachment of the first substituent to the third bicyclic ring; the compound inhibits Syk tyrosine kinase at a concentration at least 20-fold lower than the concentration at which the compound inhibits HGFR, EGFR, FGFR3, IGF1R and / or VEGFR; or A pharmaceutical composition comprising the above compound or a pharma- ceutical acceptable salt thereof.

[0101] 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 diseases involving the release of microvesicles from blood cells. In certain embodiments, the RBC-mediated disease is α-thalassemia or β-thalassemia, alone or in combination with sickle cell disease.

[0102] Also provided are methods of preventing or inhibiting phosphorylation in a subject of human erythrocyte anion transporter band 3. In certain embodiments, the methods comprise administering to the subject an effective amount of a compound described herein (or a pharma- ceutically acceptable salt thereof), or a pharmaceutical composition herein.

[0103] In each of the methods herein, administration of a compound, pharmaceutical salt thereof, or pharmaceutical composition to a subject can stabilize red blood cell membranes in the subject and reduce the release of hemoglobin from sickle red blood cells in the subject. In this manner, for example, the risk of a vaso-occlusive crisis in the subject can be reduced. In certain embodiments, the compound, pharmaceutical salt thereof, or pharmaceutical composition has an IC of 200 nM or less. 50 In certain embodiments, the subject is a human child.

[0104] In certain embodiments, the methods herein further comprise administering to the subject an effective amount of at least one second active agent, which can be selected from the group consisting of fostamatinib, PRT062607, TAK-659, TAE-684, entospletinib, ranlaprenib, celduratinib, piceatannol, S701, SykII, SykIV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, Syk-IN-1, SRX3207, RO9021, gusacitinib, R112, PRT-060318 and OXSI-2. In certain embodiments, the at least one second active agent is two or more of ostamatib, PRT062607, TAK-659, TAE-684, entospletinib, lanlaprenib, celdulatinib, piceatannol, S701, SykII, SykIV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, Syk-IN-1, SRX3207, RO9021, gusacitinib, R112, PRT-060318, and OXSI-2. The at least one second active agent may be administered together with the compound (or a pharma- ceutical acceptable salt thereof), or the pharmaceutical composition may be administered simultaneously or sequentially, in either order.

[0105] The method can further include administering to the subject an effective amount of hydroxyurea, which may be administered simultaneously or sequentially, in either order, with the compound (or a pharma- ceutically acceptable salt thereof) or pharmaceutical composition.

[0106] 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 fostamatinib, PRT062607, TAK-659, TAE-684, entospletinib, lanlaprenib, celduratinib, piceatannol, S701, SykII, SykIV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusacitinib, R112, PRT-060318 and OXSI-2, and optionally, the at least one second active agent is formulated as a pharmaceutical composition comprising a pharma- ceutical acceptable carrier or excipient.

[0107] Also provided is another method of treating sickle cell disease in a subject, comprising administering an effective amount of: (i) A compound comprising a rigid, fused bicyclic ring, or a pharma- ceutically acceptable salt thereof, which is 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 an indole substituted with at least one substituent, at least one of which is a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring. (ii) a compound or a pharma- ceutical acceptable salt thereof that prevents Syk from phosphorylating the human erythrocyte anion transporter band 3; (iii) wherein the compound inhibits Syk tyrosine kinase at a concentration at least about 20-fold lower than the concentration at which the compound inhibits HGFR, EGFR, FGFR3, IGF1R and / or VEGFR, or a pharma- ceutically acceptable salt thereof. administering In addition to or instead of (ii) and (iii), the compound (i) is (iv) a CLogP value of between about 2 and about 5 (e.g., between 2 and about 5, between about 2 and 5, or between 2 and 5); (v) a polar surface area of ​​less than about 250 Å2 (e.g., less than 250 Å2); (vi) up to 5 H-bond donors; (vii) 15 or fewer H-bond acceptors, and / or (viii) 20 or fewer rotatable bonds having The subject is treated for sickle cell disease. The compound (or a pharma- ceutically acceptable salt thereof) can prevent Syk from phosphorylating tyrosine 8 and tyrosine 21 in human erythrocyte anion transporter band 3. The compound (or a pharma- ceutically acceptable salt thereof) can stabilize erythrocyte cell membranes and reduce the release of microvesicles from sickle erythrocytes. The compound (or a pharma- ceutically acceptable salt thereof) can stabilize erythrocyte cell membranes and reduce the release of hemoglobin from sickle erythrocytes. The risk of vaso-occlusive crisis in the subject can be reduced. The compound (or a pharma- ceutically acceptable salt thereof) can be administered at an IC of 200 nM or less. 50 The subject can be a human child.

[0108] The method may further comprise administering, in any order, simultaneously or sequentially, (a) the compound, a pharma- ceutically acceptable salt thereof, or the composition, and (b) at least one second active agent (e.g., an effective amount) selected from the group consisting of fostamatinib, PRT062607, TAK-659, TAE-684, entospletinib, lanlaprenib, celdulatinib, piceatannol, S701, SykII, SykIV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusacitinib, R112, PRT-060318, and OXSI-2, optionally, such at least one second active agent formulated as a pharmaceutical composition comprising a pharma- ceutically acceptable carrier or excipient. The compound, its pharmaceutically acceptable salt, or pharmaceutical composition, and at least one second active agent can 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 comprises hydroxyurea. In certain embodiments, the at least one second active agent comprises more than one second active agent all selected from the group consisting of fostamatinib, PRT062607, TAK-659, TAE-684, entospletinib, lanlaprenib, celdulatinib, piceatannol, S701, SykII, SykIV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusacitinib, R112, PRT-060318 and OXSI-2, optionally wherein the at least one active agent is formulated as a pharmaceutical composition comprising a pharma- ceutically acceptable carrier or excipient.

[0109] Still further provided is a method of 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 medicamentously acceptable salt thereof, the compound comprising a rigid, 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 an indole substituted with at least one substituent comprising a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring. The disease can be thalassemia. The thalassemia can be α-thalassemia or β-thalassemia, alone or in combination with sickle cell disease. The disease can be glucose-6-phosphate dehydrogenase deficiency. The disease can be glutathione reductase deficiency.

[0110] The method can further comprise administering the compound or a pharmaceutically acceptable salt thereof and a Src inhibitor (e.g., an effective amount) in any order, simultaneously or sequentially, and optionally the Src inhibitor is formulated as a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient. The Src inhibitor and the compound (or a pharmaceutically acceptable salt thereof) can be administered by the same or different routes. The Src inhibitor can be dasatinib, ibrutinib, bafetinib, PP1, PP2, PP121, or a combination of two or more of the above.

[0111] Still yet further provided is a method of treating sickle cell disease in a subject, the method comprising administering to a subject an effective amount of: (i) a compound comprising a pyrazine, or a pharma- ceutically acceptable salt thereof, which is substituted with at least three substituents, at least one of which is a carboxamide or lactam and the other of which is an N-linked, optionally substituted carbocycle or heterocycle, the other of which is a carbocycle or heterocycle having at least one substituent, and at least one of which is a 4-6 membered, optionally substituted carbocycle or heterocycle; (ii) a compound or a pharma- ceutical acceptable salt thereof that prevents Syk tyrosine kinase from phosphorylating human erythrocyte anion transporter band 3; (iii) wherein the compound inhibits Syk tyrosine kinase at a concentration at least about 20-fold lower than the concentration at which the compound inhibits HGFR, EGFR, FGFR3, IGF1R and / or VEGFR, or a pharma- ceutically acceptable salt thereof. administering In addition to or instead of (ii) and (iii), the compound (i) is (iv) a CLogP value of between about 2 and about 5 (e.g., between 2 and about 5, between about 2 and 5, or between 2 and 5); (v) a polar surface area of ​​less than about 250 Å2 (e.g., less than 250 Å2); (vi) up to 5 H-bond donors; (vii) 15 or fewer H-bond acceptors, and / or (viii) 20 or fewer rotatable bonds having The subject is treated for sickle cell disease. The compound (or a pharma- ceutically acceptable salt thereof) can prevent Syk tyrosine kinase from phosphorylating tyrosine 8 and tyrosine 21 in human erythrocyte anion transporter band 3. The compound (or a pharma- ceutically acceptable salt thereof) can stabilize erythrocyte cell membranes and reduce the release of microvesicles from sickle erythrocytes. The compound (or a pharma- ceutically acceptable salt thereof) can stabilize erythrocyte cell membranes and reduce the release of hemoglobin from sickle erythrocytes. The risk of vaso-occlusive crisis in the subject can be reduced. The compound (or a pharma- ceutically acceptable salt thereof) can be administered at an IC of 200 nM or less. 50 The subject can be a human child.

[0112] The method may further comprise administering (a) the compound (or a pharma- ceutically acceptable salt thereof) and (b) one or more of fostamatinib, PRT062607, TAK-659, TAE-684, entospletinib, lanlaprenib, celduratinib, piceatannol, S701, SykII, SykIV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusacitinib, R112, PRT-060318, and OXSI-2, either simultaneously or sequentially, in any order, optionally as a pharmaceutical composition comprising a pharma- ceutically acceptable carrier or excipient. The one or more other compounds may be administered by the same or a different route.

[0113] Also provided is a method of 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 pharma- ceutically acceptable salt thereof) comprising a pyrazine substituted with at least three substituents, at least one of which is a carboxamide or lactam, and the other of which is an N-linked, optionally substituted carbocycle or heterocycle, and the other of which is a carbocycle or heterocycle having at least one substituent, and at least one of which is a 4-6 membered, optionally substituted carbocycle or heterocycle, wherein the subject is treated for a disease involving the release of microvesicles. The disease can be thalassemia. The thalassemia can be α-thalassemia or β-thalassemia, alone or in combination with sickle cell disease. The disease can be glucose-6-phosphate dehydrogenase deficiency. The disease can be glutathione reductase deficiency. The method can further include administering the compound (or a pharma- ceutical acceptable salt thereof) and the Src inhibitor simultaneously or sequentially, in any order, as a pharmaceutical composition, optionally including a pharma- ceutical acceptable carrier or excipient. The Src inhibitor can be administered by the same or different route as the compound (or a pharma- ceutical acceptable salt thereof). The Src inhibitor can be dasatinib, ibrutinib, bafetinib, PP1, PP2, PP121, or a combination of two or more of the foregoing.

[0114] Sequence Listing The sequence herein (SEQ ID NO:1) is also provided in computer readable form encoded in a file with the present application and incorporated herein by reference. The information recorded in computer readable form is identical to the written sequence listing provided below in accordance with 37 C.FR § 1.821(f).

[0115] SEQ ID NO:1 is the amino acid sequence of the Spleen tyrosine kinase (Syk) protein:

[0116] [ka]

[0117] The contents of the Sequence Listing XML file (named "PRF69753-03SeqListingXML.xml", which is 2.7kb in size, was created on March 13, 2023, and submitted electronically via the Patent Center on March 14, 2023) are incorporated herein by reference in their entirety. The information recorded in computer readable form is identical to the written Sequence Listing provided herein (on paper) in accordance with 37 C.FR § 1.821(f). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0118] While the concepts of the present disclosure have been illustrated and described in detail in the description herein, it is understood that the results described are considered exemplary and are not to be limited to the letter, that only exemplary embodiments have been shown and described, and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0119] The present disclosure is based, at least in part, on the unprecedented discovery of compounds, including small molecule inhibitors, that inhibit spleen tyrosine kinase (Syk) without substantially interfering with other pathways that confer regulated growth factor kinases relevant to the growth and development of a subject. The compounds can be used to treat diseases of red blood cells (RBCs), such as hemoglobinopathies (e.g., sickle cell disease (SCD) and thalassemia), as well as other RBC-mediated diseases, such as glucose-6-phosphate dehydrogenase (G6PD) deficiency and glutathione reductase deficiency.

[0120] Phosphorylation / dephosphorylation of protein tyrosine residues has been implicated in the regulation of several red blood cell functions, including cell shape. The aforementioned diseases of RBCs involve a mechanism in which elevated tyrosine phosphorylation of band 3 initiates sequelae that lead to vaso-occlusive disease and disease symptoms. In this mechanism, accelerated denaturation of sickle hemoglobin (HbS), hemichrome formation, and heme release can collectively induce oxidative stress within RBCs. Increasing oxidative stress can then cause inhibition of red blood cell / RBC tyrosine phosphatases, which normally block constitutive band 3 tyrosine phosphorylation. Upon inhibition of these phosphatases, excessive phosphorylation of band 3 then induces globular destabilization of the red blood cell membrane, promoting intravascular hemolysis and microparticle (MP) release. Increased plasma hemoglobin and heme can also "activate" the vascular endothelium, leading to expression of adhesion receptors (e.g., p-selectin, E-selectin, and von Willebrand factor) and sequestration of vasodilators (NO), whose release can then trigger intravascular coagulation via activation of prothrombin.

[0121] Ultimately, these processes may promote vaso-occlusive events leading to symptoms of SCD and other RBC diseases, such as tissue hypoxia, ischemia-reperfusion injury, organ damage, and associated morbidity and debilitating pain resulting in significant suffering that may require medical treatment and / or hospitalization. When initiated in conjunction with loss of RBC deformability and enhanced vascular adhesion, sequelae associated with membrane weakening may exacerbate already compromised blood flow, leading to pulmonary microemboli and progressive tissue damage. Sickle cell hemolysis shortens the life span of sickle red blood cells, and anemia may further exacerbate clinical symptoms.

[0122] At least one of the kinases involved in phosphorylation of band 3 is Syk (p72 syk). Syk is a non-receptor protein tyrosine kinase that is expressed in significant amounts in RBCs. For the purposes of discussing the amino acids in Syk, reference is made to the long isoform (Uniprot ID P43405-1) having SEQ ID NO:1. Alternative splicing results in a deletion of amino acids 283-305, conferring the short isoform (Uniprot ID P43405-2).

[0123] Syk binds to the cytoplasmic domain of cells bearing Fcγ activating receptors containing immunoreceptor tyrosine-based activation motifs (ITAMs). Receptor binding leads to phosphorylation, which increases its autophosphorylation activity, leading to Syk activation. Syk activity activates a variety of downstream kinases, which ultimately leads to the release of proinflammatory cytokines and the above cascade.

[0124] compound The compounds herein include small molecule inhibitors that inhibit Syk (i.e., bind to and inhibit the Syk receptor), thereby inhibiting and / or blocking this cascade by reducing band 3 tyrosine phosphorylation in affected RBCs. In addition, the compounds can inhibit MPs and hemoglobin release from affected RBCs.

[0125] In certain embodiments, the compound has formula X: ABC(X) or a pharma- ceutically acceptable salt thereof, wherein A is a nitrogen (N)-linked, optionally substituted first ring, which first ring comprises a fused bicyclic ring or a monocyclic ring, each ring of the first ring comprising 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 fused bicyclic or tricyclic ring, or a monocyclic ring, each ring of the second ring comprising a 5- or 6-membered heterocyclic or carbocyclic ring; C is a third bicyclic ring comprising a 5- or 6-membered carbocyclic or heterocyclic ring fused to a 5- or 6-membered carbocyclic or heterocyclic ring, the third bicyclic ring comprising at least

[0126] [ka] or substituted with an amine, or a ketone, or a carboxamide, or an alcohol (e.g., propanol); During the ceremony, X4 is CH2, NH, CO, O or S; Y5 is CH or N; Z is OH, SH, NH2, CO2H, NHCH3, and

[0127] [ka] is selected from

[0128] [ka] is the attachment point.

[0129] A of the compound is

[0130] [ka] or a pharma- ceutically acceptable salt of any of the preceding 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; Each R3 is independently H, F, Cl, OH, and -Y-(CH2) n -, wherein n=1 to 14.

[0131] A of the compound is

[0132] [ka] wherein

[0133] [ka] is the point of attachment to B, and W is O, NH, or SH.

[0134] A of the compound is

[0135] [ka] wherein

[0136] [ka] is the attachment point to B.

[0137] A of the compound is

[0138] [ka] wherein

[0139] [ka] is the attachment point to B.

[0140] The N-linkage of A to the first ring (eg, a fused bicyclic ring or a monocyclic ring) can form a nitrogen bridge with B of the compound.

[0141] The first ring of A can include a 4-6 membered heterocyclic ring (e.g., containing one or more of N, O, or S). The first ring of A can include a 4-6 membered carbocyclic ring. The first ring of A can be a fused bicyclic ring including two 6 membered rings. The first ring of A can be a fused bicyclic ring including a 6 membered ring and a 5 membered ring.

[0142] In certain embodiments, A is substituted (e.g., the first ring of A is substituted). A can be a monocyclic ring substituted with a bulky substituent that is para to the nitrogen bridge. The compound can have an increased docking score for Syk tyrosine kinase compared to an otherwise identical compound lacking the bulky substituent that is para to the nitrogen bridge (see Equation 1 below).

[0143] In certain embodiments, the bulky substituent can be, for example, the structure:

[0144] [ka] is morpholine having the formula:

[0145] The first ring of A can be or can include a monocyclic carbocyclic ring (e.g., one that is six-membered). In certain embodiments, the first ring of A includes benzene.

[0146] 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., six-membered) that 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 bicyclic and can include a first closed ring (e.g., a six-membered carbocyclic ring) fused with a second closed ring (e.g., a five-membered carbocyclic or heterocyclic ring), either of which may be substituted or neither of which may be substituted.

[0147] As noted above, the first ring of A can be optionally substituted with one or more substituents, each independently selected from the group consisting of hydrogen, deuterium, halo, azido, cyano, nitro, hydroxy, amino, thio, carboxy, ester, amide and derivatives thereof, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloheteroalkyl, cycloheteroalkenyl, acyl, aryl, heteroaryl, arylalkyl, arylalkenyl, arylalkynyl, morpholine, and nitrogen heterocycle.

[0148] In certain embodiments, the compound is:

[0149] [ka] or a pharma- ceutically acceptable salt thereof, wherein R1 is,

[0150] [ka] and R2 is

[0151] [ka] or an amine, or an alcohol (e.g., propanol); R4 is H, CH3, an amine or methanol; X4 is independently CH2, NH, CO, O or S; each Y and Y5 is independently CH or N; Z is OH, SH, NH2, CO2H, NHCH3, and

[0152] [ka] is selected from

[0153] [ka] is the attachment point.

[0154] B of the compound can be a second ring optionally substituted with at least one substituent, where the second ring is monocyclic or includes a fused bicyclic or fused tricyclic ring. Each ring of the second ring can include a 5- or 6-membered carbocyclic or heterocyclic ring.

[0155] The one or more substituents of B can each be independently selected from the group consisting of hydrogen, deuterium, amide and derivatives thereof, carbonyl, and alkyl.

[0156] The B of the compound can be rigid.

[0157] B can be a monocyclic ring optionally substituted with an amide. B can be a pyridine. B can be a pyrazine. B can be a pyrazine substituted with at least one substituent. B can include an imidazole substituted with at least one amide.

[0158] B can be an optionally substituted (e.g., optionally substituted with an amide or carbonyl group) fused bicyclic ring. In certain embodiments, the fused bicyclic ring comprises a pyrimidine fused with an optionally substituted N-containing 5-membered heterocycle.

[0159] In certain embodiments, B is

[0160] [ka] and During the ceremony, Each X2 is independently selected from CH and N; Y3 is selected from O, NH and S; Z3 is selected from OH, NH2 and NHCH3.

[0161] In certain embodiments, B of the compound is

[0162] [ka] For example, compound B is

[0163] [ka] may be or may include During the ceremony, Each X2 is independently selected from CH and N; Y3 is selected from O, NH and S; Z3 is selected from OH, NH2 and NHCH3, with the proviso that B is

[0164] [ka] isn't it.

[0165] When the compound (or a pharma- ceutically 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. In addition, 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.

[0166] C of the compound comprises a third bicyclic ring. This third ring can comprise a 5- or 6-membered carbocyclic ring fused to a 5- or 6-membered carbocyclic 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., N-containing heterocyclic ring) or 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., N-containing heterocyclic ring) or carbocyclic ring. The bicyclic ring of C can be indole.

[0167] The third fused bicyclic ring is substituted with at least one substituent (e.g., the first substituent). C has the structure:

[0168] [ka] may include wherein each X3 is independently selected from CH and N, and Y4 is CH2, NH, O, or SH;

[0169] [ka] is the point of attachment of at least one substituent on the third ring (C).

[0170] In certain embodiments, the third bicyclic ring of C is

[0171] [ka] or amine, or ketone, or carboxamide, or alcohol (e.g., propanol), wherein X is, independently, CH, NH, CO, O, or S; Each Y is independently CH or N; Z is OH, SH, NH2, CO2H, NHCH3, and

[0172] [ka] is selected from

[0173] [ka] is the point of attachment (e.g., to a 5- or 6-membered carbocyclic or heterocyclic ring at C of a compound).

[0174] In certain embodiments, Z of the first substituent is -OR, where R is

[0175] [ka] (where n = 1 to 14).

[0176] In certain embodiments, C has the structure:

[0177] [ka] Including, wherein each X3 is independently selected from CH and N; and Y is CH2, NH, O, or SH;

[0178] [ka] is the point of attachment to the first substituent, where the first substituent on the third bicyclic ring at C is

[0179] [ka] It is.

[0180] The bicyclic ring of C can be indole and the first substituent of C can include 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.

[0181] The first substituent Z of C can comprise a phosphate or phosphonate ester. In certain embodiments, the substituent of C is

[0182] [ka] where Q is a phosphate or phosphonate ester, or Q is RC(=O)O-, where R is alkyl.

[0183] C can also include at least a second substituent. In certain embodiments, C is substituted with CH, H, CHOH, amine, and / or alkyl.

[0184] One or more of the hydrogens (H) of the compound can optionally be replaced with deuterium.

[0185] In certain embodiments, (e.g., when administered to a subject), compound B can form a hydrogen bond with amino acid residue Ala451 in the catalytic domain of Syk tyrosine kinase, and a first substituent of C can form a hydrogen bond with amino acid residue Asp512 in the catalytic domain of Syk tyrosine kinase.

[0186] The compound can have a CLogP value between about 2 and about 5 (e.g., between 2 and about 5, between about 2 and 5, or between 2 and 5). The compound can have a polar surface area of ​​less than about 250 Å 2 (e.g., less than 250 Å 2 ). The compound can have 5 or less H-bond donors. The compound can have 15 or less H-bond acceptors. The compound can have a number of rotatable bonds of 20 or less. In certain embodiments, the compound can have a CLogP value between about 2 and about 5 (e.g., between 2 and about 5, between about 2 and 5, or between 2 and 5), a polar surface area of ​​less than about 250 Å 2 (e.g., less than 250 Å 2 ), 5 or less H-bond donors, 15 or less H-bond acceptors, and / or 20 or less rotatable bonds.

[0187] The compounds can bind with specificity to Syk tyrosine kinase, and when administered to a subject, in certain embodiments, the compounds prevent Syk from phosphorylating the erythrocyte (e.g., human erythrocyte) anion transporter band 3.

[0188] The compound has the formula (XX):

[0189] [ka] or a pharma- ceutically acceptable salt thereof, wherein ring A is

[0190] [ka] and During the ceremony, each X is independently selected from CH and NH; each Y is independently selected from CH, NH, O, and S; Z is CH or N; Each R is independently H, F, Cl, OH, and -Y-(CH2). n -, wherein n=1 to 14; In the formula, ring B is

[0191] [ka] and During the ceremony, Each X2 is independently selected from CH and N; Y3 is selected from O, NH and S; Z3 is selected from OH, NH2 and NHCH3, with the proviso that B is

[0192] [ka] Instead, In the formula, ring C is

[0193] [ka] and During the ceremony, Each X3 is independently selected from CH and N; Y4 is selected from CH2, NH, O and SH; In the formula, ring D is

[0194] [ka] and During the ceremony, X4 is selected from CH2, NH, CO, O and S; Y5 is selected from CH and N; Z is OH, SH, NH2, NHCH3, and

[0195] [ka] is selected from In the formula, one or more hydrogens can be optionally replaced with deuterium.

[0196] Ring A, ring B, ring C and ring D of formula (XX) can include any of the aforementioned embodiments of A, B and C of the compound of formula (X) described above. For example, ring A of formula (XX) can be substituted with a bulky substituent that is 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. In addition, ring D of formula (XX) can include any of the embodiments of the first substituent of C of formula (X).

[0197] The compound is

[0198] [ka] The structure may include Or a pharma- ceutically acceptable salt of any of the foregoing.

[0199] The compound has the structure:

[0200] [ka] may include Or it can be a pharma- ceutically acceptable salt of any of the foregoing.

[0201] The compound has the structure:

[0202] [ka] or a pharma- ceutically acceptable salt thereof, where 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 can include lipophilic esters. Z on ring D can be -OR, where R is

[0203] [ka] where n=1 to 14).

[0204] The compound can be a compound (or a pharma- ceutically acceptable salt thereof) that comprises a pyrazine substituted with at least three substituents, at least one of which is a carboxamide or lactam and the other of which is an N-linked, optionally substituted carbocycle or heterocycle, the other of which is a carbocycle or heterocycle having at least one substituent, and at least one of which is a 4-6 membered, optionally substituted carbocycle or heterocycle.

[0205] The compound is

[0206] [ka] or a pharma- ceutically acceptable salt thereof, wherein R1 is,

[0207] [ka] and R2 is

[0208] [ka] or an amine, or an alcohol (e.g., propanol); X is, independently, CH, NH, CO, O, or S; Each Y is independently CH or N; Z is OH, SH, NH2, CO2H, NHCH3, and

[0209] [ka] is selected from

[0210] [ka] is the attachment point.

[0211] In certain embodiments, the compound is

[0212] [ka] or a pharma- ceutically acceptable salt thereof, wherein X is CH or N and R is

[0213] [ka] It is.

[0214] In certain embodiments, the compound is

[0215] [ka] or a pharma- ceutically acceptable salt thereof, wherein X is CH or N and R is

[0216] [ka] It is.

[0217] In certain embodiments, the compound is

[0218] [ka] or a pharma- ceutically acceptable salt thereof, wherein X is CH or N and R is

[0219] [ka] It is.

[0220] In certain embodiments, the compound is

[0221] [ka] or a pharma- ceutically acceptable salt thereof, wherein X is CH or N and R is

[0222] [ka] It is.

[0223] In certain embodiments, the compound is

[0224] [ka] or a pharma- ceutically 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 are all CH, the compound may exhibit a weaker binding affinity for Syk tyrosine kinase compared to when at least one X and / or Y is not CH.

[0225] In certain embodiments, the compound is

[0226] [ka] or a pharma- ceutically 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 are all CH, the compound may exhibit a weaker binding affinity for Syk tyrosine kinase compared to when at least one X and / or Y is not CH.

[0227] The compound has the structure:

[0228] [ka] or a pharma- ceutically acceptable salt thereof, wherein R is

[0229] [ka] and X is independently CH or N.

[0230] The compound has the structure:

[0231] [ka] or a pharma- ceutically acceptable salt thereof, wherein R is a carbocycle or a heterocycle; X is independently CH or N; Y is CH or N.

[0232] The compound has the structure:

[0233] [ka] or a pharma- ceutically acceptable salt thereof.

[0234] In certain embodiments, the hydroxyl (HO-) group of the first substituent of C of formula (X) or the hydroxyl (HO-) group of ring D of formula (XX) can be replaced with a phosphate or phosphonate ester. For example, the first substituent of the bicyclic ring of C can be:

[0235] [ka] For example, see compound S2053P. Such modifications can improve compound solubility and / or cell permeability. In alternative embodiments, the hydroxyl (HO-) group can be replaced with RC(=O)O-, where R is alkyl (e.g., C1-C6 alkyl). Such modifications can improve cell permeability.

[0236] The compound has the structure:

[0237] [ka] and or a pharma- ceutically acceptable salt of any of the preceding structures.

[0238] The compound may further comprise a prodrug version of the phosphate. In certain embodiments, for example, the compound is

[0239] [ka] or a pharma- ceutically acceptable salt thereof.

[0240] The compound has the structure:

[0241] [ka] may have or include or a pharma- ceutically acceptable salt of any of the preceding structures.

[0242] In certain embodiments, the compound (or a pharma- ceutically acceptable salt thereof) comprises a rigid, fused bicyclic ring substituted with at least two substituents, at least one of which is an N-linked, optionally substituted carbocycle or heterocycle, and the other of the substituents is an indole substituted with at least one substituent, and at least one of the substituents is a 4-6 membered, optionally substituted carbocycle or heterocycle.

[0243] As noted above, the compound can be a specific inhibitor of Syk. The compound can bind to the Syk receptor with specificity, thus reducing off-target interactions, for example, with growth factor receptors. "Bind with specificity," "bind with high affinity," or "specifically" or "selectively," when referring to a small molecule / receptor or other binding pair, indicates that under specified conditions, the specified compound, or its recognition region, binds to a particular receptor or site (e.g., a Syk inhibitory site, e.g., an ATP-binding catalytic site of a tyrosine kinase domain), and does not bind in significant amounts to other receptors present (e.g., off-target growth factor receptors). Specific binding, or binding with high affinity, can also mean, for example, a binding composition that binds to its target (e.g., inhibits Syk) with an affinity that is often at least 20% greater, at least 25% greater, at least 50% greater, or at least 100% (2-fold) greater than the affinity of the binding compound, ligand, or binding composition that binds to another receptor (e.g., a growth factor receptor).

[0244] In an exemplary embodiment, a molecule that specifically binds to a target has a specific binding affinity of at least about 10, as determined, for example, by Scatchard analysis. 6 Liter / mol (K0=10 ~6M), preferably an affinity of at least about 10 liters / mol. The binding specificity of a compound to a Syk receptor is determined using standard techniques known in the art and described herein.

[0245] When administered to a subject, the compound can prevent Syk from phosphorylating the erythrocyte anion transporter band 3 of the subject (e.g., a human). For example, the overall design of the compound can allow for the inhibition of Syk without interfering with other pathways (e.g., those regulated by growth factor kinases). Although other Syk inhibitors are known, conventional Syk inhibitors are not specific to Syk (i.e., they inhibit several kinases in addition to Syk, including, for example, growth factor receptors that are necessary for growth and development (e.g., vascular endothelial growth factor receptors (VEGFRs, e.g., VEGFR3), or other growth factor receptors). Inhibition of growth factor kinases is typically undesirable because it can lead to dwarfism and other growth disorders in pediatric patients. For example, studies support that imatinib can slow vertical growth, hormone production, and vitamin D synthesis in subjects. Furthermore, upregulation (but not inhibition) of these growth factors is often desirable in response to vascular occlusions and the like, because it can promote tissue repair and regeneration (e.g., revascularization of tissues).

[0246] 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).

[0247] HGFR (or MET) binds to hepatocyte growth factor (HGF, also called scatter factor (SF)), which transduces numerous biological processes such as cell motility, morphogenesis, proliferation and survival. The HGFR precursor is proteolytically cleaved to form α and β subunits that form disulfide-linked heterodimers. HGFR is widely expressed in early development, and genetic defects lethally disrupt embryogenesis. Following kidney, liver or heart injury, HGFR is upregulated to promote tissue repair and regeneration.

[0248] 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 family of receptors, which includes EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4). Upon activation by EGF, EGFR undergoes a transition from an inactive monomeric form to an active homodimer. EGFR can also form a heterodimer with another ErbB receptor to form an active heterodimer. These receptors are involved in early development and participate in stem cell renewal in tissues such as the skin, liver, intestine and breast ducts.

[0249] FGFR3 binds to members of the fibroblast growth factor (FGF) protein family. The FGF family is the largest family of growth factor receptor ligands, containing 22 factors. Alternative splicing of the four FGFR genes can result in the generation of more than 48 different isoforms of FGFR. In addition, FGFRs dimerize as heterodimers and homodimers. FGFR3 regulates chondrocyte differentiation, proliferation and apoptosis, vitamin D metabolism, bone formation, and postnatal mineralization of bone by osteoblasts, and is required for normal skeletal development and inner ear development.

[0250] IGF1R is activated by the hormone insulin-like growth factor 1 (IGF-1) and by a related hormone called IGF-2. IGF1R regulates pre- and postnatal development, glucose metabolism and neutrophil physiology, and promotes protective physiological cardiac hypertrophy in the left ventricle of the heart.

[0251] VEGFR interacts with vascular endothelial growth factor (VEGF), a signal protein that stimulates the formation of blood vessels. There are three main subtypes of VEGFR: type 1, type 2 and type 3. Depending on alternative splicing, they can be membrane-bound or soluble. The VEGF family has five members: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C and VEGF-D. Alternative splicing leads to multiple isoforms. The lack of inhibition of VEGFR such as VEGFR3 is advantageous because the administration of the compound does not prevent the essential revascularization of tissue that will become infarcted in patients with sickle cell disease due to the occurrence of vaso-occlusive events.

[0252] In certain embodiments, the compound includes a small molecule inhibitor that has specificity for Syk and inhibits Syk at least about 20 times better (e.g., 20 times better) than it inhibits the growth factor receptor (e.g., the small molecule inhibitor is Syk specific).

[0253] Mainly, the docking scores for some of the compounds were analyzed and are given in Table 1. Determination of the three-dimensional structure of a protein-receptor complex is an important part of structure-based drug design. A root mean square deviation of less than about 2 Å corresponds to a good docking solution.

[0254] [Table 1]

[0255] Further surprising and unexpected properties of the compounds (e.g., S970, S970D, S1115, S1213, S1219, S1213-CH3, S2053, S2054 and S1214) in the context of commercially available / previously known Syk inhibitors are shown in Tables 2 and 3. Activity (IC 50 The results are shown in Table 2. IC 50 IC values ​​were determined as the concentration of compound (or other tested Syk inhibitor) that reduced the determined receptor activity by 50%. Table 3 shows the IC 50 The selectivity ratios of each of the selected compounds are shown as Syk / kinase ratios.

[0256] [Table 2-1]

[0257] [Table 2-2]

[0258] [Table 3]

[0259] 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, almonertinib, anlotinib, axitinib, brigatinib, bosutinib, cabozantinib, capmatinib, cerduratinib, ceritinib, crizaotinib, dacobalaminib, and dacobalaminib. Mitinib, dasatinib, erdafitinib, erlotinib, fostamitinib, fruquintinib, gefitinib, icotinib, lapatinib, lenvatinib, lestaurtinib, lorlatinib, midostaurin, neratinib, nintedanib, osimertinib, pemigatinib, pralsetinib, pyrotinib, regorafenib, selpercatinib, sorafenib, tepotinib and vandetanib. None of the following inhibitors were >20-fold more potent for Syk versus growth factor receptor kinases: alectinib, avapritinib, entrectinib, fedratinib, imatinib, larotrectinib, nilotinib, pazopanib, ripretinib, ruxolitinib, selumetinib, sunitinib, tofacitinib, and tucatinib. Of the 10 commercially available Syk inhibitors, celduratinib, entospletinib, fostamtinib, ranlaprenib, piceatannal, PRT062067, SykII, SykIV, TAE-684, and TAK-659, only entospletinib, PRT062067, and SykII were at least 20-fold more potent for Syk than for growth factor receptor kinases.

[0260] Additionally, cellular potency data for the compounds compared to selected commercially available and FDA-approved Syk inhibitors for which Syk inhibition is known is shown in Table 4. Median maximal effective concentrations (EC 50 ) represents the concentration of a drug required to produce half of the maximum possible effect after a specified exposure.

[0261] [Table 4]

[0262] The above compounds can be synthesized according to methods known in the art and exemplified herein, see, e.g., Examples 1 and 3-5.

[0263] The solubility of selected compounds was also tested using methods known in the art, and the resulting data is presented in Table 5.

[0264] [Table 5]

[0265] The compound may have a CLogP value between about 2.0 and about 5.0 (e.g., between 2.0 and about 5.0, between about 2.0 and 5.0, or between 2.0 and 5.0). In certain embodiments, such as compound S1213, the compound has a CLogP value between about 1.0 and about 2.0 (e.g., between 1.0 and about 2.0, between about 1.0 and 2.0, or between 1.0 and 2.0. The term "cLogP" refers to the calculated partition coefficient, which in physical science is the ratio of the concentrations of a compound in a mixture of two immiscible phases at equilibrium. These coefficients are a measure of the difference in the solubility of a compound in these two phases. In general, this is a measure of the hydrophobicity of a compound, where one phase is aqueous (traditionally water) and the other phase is hydrophobic (traditionally octanol). A higher value indicates a greater partitioning of the compound into the hydrophobic phase, supporting increased hydrophobicity.

[0266] In certain embodiments, the compound has a polar surface area of ​​less than about 250 Å 2 (e.g., less than 250 Å 2 ). In certain embodiments, the compound has 5 or fewer H-bond donors. In certain embodiments, the compound has 15 or fewer H-bond acceptors. In certain embodiments, the compound has 20 or fewer rotatable bonds. In certain embodiments, the compound may exhibit more than one of the above properties.

[0267] The compounds herein also exhibit sufficient oral bioavailability. Table 6 shows the data of selected compounds regarding their absolute oral bioavailability (studies were carried out using methods generally known in the art).

[0268] [Table 6]

[0269] Administration of the compounds, pharma- ceutically acceptable salts, or compositions herein can prevent Syk from phosphorylating the erythrocyte anion transporter band 3. The compounds, or pharma- ceutically acceptable salts thereof, are at least 20 times more potent for Syk than for growth factor receptor kinases, in other words, the compounds have a higher affinity for Syk than conventional Syk inhibitors, and such affinity is differential, meaning that the compounds herein do not share this high affinity with other receptors, such as growth factor receptor kinases.

[0270] A compound (or a pharma- ceutically acceptable salt thereof) may contain one or more chiral centers or may otherwise exist as multiple stereoisomers, such as enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures.Unless otherwise stated, all stereoisomeric forms of a compound are intended to be contemplated.When a compound contains an alkene double bond, it is intended to include both E and Z geometric isomers (e.g., cis or trans) unless otherwise specified.Similarly, all possible isomers are intended to be included, as well as their racemic and optically pure forms, and all tautomeric forms.Those skilled in the art will further recognize that a compound may be "deuterated", meaning that one or more hydrogen atoms may be replaced with deuterium.

[0271] The compound (or its pharmaceutically acceptable salt) can exist in unsolvated form and solvated form, including hydrated form. In general, solvated form is equivalent to unsolvated form. The compound can exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the contemplated use. The preparation includes pharmaceutically acceptable salts (e.g., acid addition salts and base salts), hydrates and / or solvates.

[0272] Pharmaceutically acceptable salts The "pharmaceutical acceptable salts" of the compounds are contemplated. The term "pharmaceutical acceptable salts" refers to salts whose counterions can be used in pharmaceutical preparations. In various embodiments, such salts include, but are not limited to, 1) acid addition salts that can be obtained by reacting the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as 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, or 2) salts that are formed when the acidic protons present in the parent compound are either replaced by metal ions, such as alkali metal ions, alkaline earth ions, or aluminum ions, or are matched with organic bases, such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucamine, etc. Pharmaceutically acceptable salts are well known to those of skill in the art, and any such pharma- ceutically acceptable salts are contemplated in connection with the embodiments described herein.

[0273] In various embodiments, suitable acid addition salts are formed from acids which form non-toxic salts. Illustrative examples include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, tosylate, and trifluoroacetate.

[0274] In various embodiments, suitable base salts are formed from bases that form non-toxic salts.Illustrative examples include arginine salt, benzathine salt, calcium salt, choline salt, diethylamine salt, diolamine salt, glycine salt, lysine salt, magnesium salt, meglumine salt, olamine salt, potassium salt, sodium salt, tromethamine salt and zinc salt.Hemi-salts of acids and bases can also be formed, such as hemisulfate salt and hemicalcium salt.

[0275] Pharmaceutical Compositions, Routes of Administration, and Dosing Further provided is a pharmaceutical composition comprising the compound and a pharma- ceutically acceptable carrier or excipient. The term "pharma- ceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances 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. A carrier may be an excipient. The choice of carrier may depend on factors such as the particular method of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form. Pharmaceutical compositions suitable for delivery of the compounds described herein and methods for their preparation can be found, for example, in Remington: The Science & Practice of Pharmacy, 21st edition (Lippincott Williams & Wilkins, 2005).

[0276] The components of the compositions also are capable of being commingled with the compounds, and with each other, in a manner such that there is no interaction that would substantially impair the desired pharmaceutical efficacy.

[0277] The composition may include, for example, cremophor, polysorbate, nanoparticles, polymers, or hydrogels. In certain embodiments, the pharmaceutical composition includes a plurality of compounds and a pharma- ceutical acceptable carrier. In certain embodiments, the pharmaceutical composition further includes at least one additional pharma- ceutical active agent. The at least one additional pharma- ceutical active agent may be an agent useful for treating ischemia-reperfusion injury.

[0278] Pharmaceutical compositions can be prepared by combining one or more compounds with a pharma- ceutically acceptable carrier and, optionally, one or more additional ingredients (e.g., active pharmaceutical ingredients). The formulations can be administered in pharma- ceutically acceptable solutions, which may routinely contain pharma- ceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and, optionally, other therapeutic ingredients.

[0279] Pharmaceutically acceptable carriers can include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, and combinations thereof. Carriers can be suitable for parenteral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable 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 such as polyethylene glycols. One or more other active agents can also be incorporated into the pharmaceutical composition.

[0280] Pharmaceutical compositions can be formulated as liquid, for example suspension or solution.Liquid formulations can contain water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or suitable oil, and one or more emulsifiers and / or suspending agents.Liquid formulations can be prepared by reconstitution of solid.

[0281] Pharmaceutical preparations (e.g. for parenteral administration) include aqueous solutions of the active compound in water-soluble form. In addition, suspensions of the active compound can be prepared as suitable oily injection suspensions. Aqueous suspensions can contain the compound alone or in further combination with one or more other active agents in admixture with suitable excipients. Excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as naturally occurring phospholipids, e.g. lecithin; condensation products of alkylene oxides with fatty acids, e.g. polyoxyethylene stearate; condensation products of ethylene oxide with long-chain aliphatic alcohols, e.g. heptadecaethyleneoxycetanol; condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols, e.g. polyoxyethylene sorbitol monooleate; or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, e.g. polyoxyethylene sorbitan monooleate. Aqueous suspension can also contain 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 suspension can further contain suitable lipophilic solvent or vehicle, including fatty oil such as sesame oil, or synthetic fatty acid ester, such as ethyl oleate or triglyceride, or liposome.Optionally, suspension can also contain suitable stabilizers, or agents that increase the solubility of compounds, allowing the preparation of highly concentrated solutions.

[0282] Alternatively, the pharmaceutical composition can be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use. Dispersible powders and granules suitable for preparation of an aqueous suspension by addition of water can provide the active ingredient in admixture with a suspending agent, dispersing agent or wetting agent, and one or more preservatives. Additional excipients, such as coloring agents, can also be present.

[0283] Suitable emulsifiers include naturally occurring gums, such as gum acacia or gum tragacanth; naturally occurring phospholipids, such as soybean lecithin; and esters, including partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and partial esters, condensation products with ethylene oxide, such as polyoxyethylene sorbitan monooleate.Isotonicity agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride, can be included in the composition.Prolonged absorption of injection compositions can be achieved by including one or more absorption retardants, such as monostearate salts and gelatin in the composition.

[0284] For use in therapy or treatment, an effective amount of compound or composition can be administered to a subject by any method that delivers the compound as desired. Administering the composition can be accomplished 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, mucosal (e.g. topical administration to the eye), inhalation, and topical administration.

[0285] Colorants and / or flavoring agents may be included. For example, the compounds may be formulated (such as by encapsulation in liposomes or microspheres) and then contained within an edible product, such as a refrigerated beverage, which further contains colorants and flavoring agents.

[0286] Exemplary formats for oral administration include, but are not limited to, tablets, capsules, elixirs, syrups, and the like.

[0287] In certain embodiments, the compound may be administered directly into the bloodstream, into muscle, or into an internal organ.Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, subarachnoid, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, intranasal, and subcutaneous.Suitable means for parenteral administration include needle injectors (including microneedles), needle-free injectors, and infusion techniques.Where it is desired to deliver the compound and / or composition to the whole body, the compound and / or composition can be formulated for parenteral administration by injection, for example, bolus injection or continuous infusion.The preparation for injection can be provided in unit dosage form, for example, in ampoules, or in multi-dose containers with added preservatives.The composition can take the form of a suspension, solution, or emulsion in oily or aqueous vehicles, and can contain formulating agents such as suspending, stabilizing, and / or dispersing agents.

[0288] Parenteral formulations are typically aqueous solutions which may contain carriers or excipients such as salts, carbohydrates and buffers (preferably at a pH of 3 to 9), but more preferably for some applications they are formulated as sterile non-aqueous solutions or as a dry form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.

[0289] Liquid formulations can be adapted for parenteral administration of compound.Preparation of parenteral formulations under sterile conditions, for example by lyophilization under sterile conditions, can be readily achieved by using standard pharmaceutical techniques well known to those skilled in the art.The solubility of compound can be increased by using suitable formulation techniques, such as incorporating solubility enhancers.

[0290] Preparations for parenteral administration can be formulated for immediate and / or modified release. The compound can be administered in a time-release formulation, for example in a composition that includes a slow-release polymer. The compound can be prepared with a carrier that will protect it against rapid release, for example, a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactides, polyglycol copolymers (PGLA). Methods for preparing such formulations are generally known to those skilled in the art.

[0291] Sterile injectable solution can be prepared by incorporating the compound alone or in combination with one or more other active agents, optionally in the required amount in a suitable solvent with one or combination of the above-listed components, followed by sterile filtration.Typically, dispersion is prepared by incorporating the compound into a sterile vehicle that contains dispersion medium and any additional components of the above-listed components.In the case of sterile powder for preparing sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying, which produces a powder of active ingredient plus any additional desired components from its solution that has been previously sterile filtered, or this component may be sterile filtered together.

[0292] The pharmaceutical composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration.The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, or by the maintenance of the required particle size in the case of dispersion, by the use of surfactants.

[0293] The compounds, or pharmaceutical compositions containing the compounds, may be administered continuously, where appropriate.

[0294] Treatment Method Methods for treating RBC-mediated diseases (e.g., SCD) are provided. The methods can include administering to a subject (e.g., a subject experiencing or at risk of experiencing an RBC-mediated disease) an effective amount of any of the above compounds, pharma- ceutically acceptable salts thereof, or the above pharmaceutical compositions. In certain embodiments, the compound has the formula (X): ABC(X) or a pharma- ceutically acceptable salt thereof, wherein A is an optionally substituted first ring linked at the nitrogen, the first ring comprising a fused bicyclic ring or a monocyclic ring, each ring of each first ring comprising 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, each ring of the second ring comprising a 5- or 6-membered heterocyclic or carbocyclic ring; C is a third bicyclic ring comprising a 5- or 6-membered carbocyclic ring fused with a 5- or 6-membered carbocyclic or heterocyclic ring, said third bicyclic ring being

[0295] [ka] or an amine, or an alcohol; During the ceremony, Each X4 is independently CH2, NH, CO, O or S; Each Y5 is independently CH or N; Z is OH, SH, NH2, CO2H, NHCH3, and

[0296] [ka] is selected from

[0297] [ka] is the point of attachment of the first substituent to the third bicyclic ring; The compound, or a pharma- ceutically acceptable salt thereof, inhibits Syk tyrosine kinase at a concentration that is at least 20-fold lower than the concentration at which the compound (or a pharma- ceutically acceptable salt thereof) inhibits HGFR, EGFR, FGFR3, IGF1R and / or VEGFR.

[0298] The method may further comprise 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 fostamatinib, PRT062607, TAK-659, TAE-684, entospletinib, ranlaprenib, celdulatinib, piceatannol, S701, SykII, SykIV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, Syk-IN-1, SRX3207, RO9021, gusacitinib, R112, PRT-060318 and OXSI-2. Additionally or alternatively, the method can include administering an effective amount of: (a) any of the above compounds, pharma- ceutically acceptable salts thereof, or the above pharmaceutical compositions; (b) fostamatinib; (c) PRT062607; (d) TAK-659; (e) TAE-684; (f) entospletinib; (g) lanraprenib; (h) cerdulatinib; (i) Piceatannol, (j) S701, (k) SykII, (l) SykIV, (m) TAS05567, (n) GSK143, (o) Syk-IN-3, (p) Syk-IN-4, (q) Syk-IN-1, (r) SRX3207, (s) RO9021, (t) Gusacitinib, (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).The foregoing may optionally be administered to a subject in a single pharmaceutical composition containing a pharma- ceutically acceptable carrier or excipient, or in multiple doses, e.g., using multiple pharmaceutical compositions.

[0299] The at least one second active agent may include a combination of two or more of fostamatinib, PRT062607, TAK-659, TAE-684, entospletinib, lanlaprenib, celduratinib, piceatannol, S701, SykII, SykIV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, Syk-IN-1, SRX3207, RO9021, gusacitinib, R112, PRT-060318 and OXSI-2. Each second active agent may be administered simultaneously or sequentially with the compound, a pharma- ceutical acceptable salt thereof, or a pharmaceutical composition, in any order (where applicable). In certain embodiments, the compound, a pharma- ceutical acceptable salt thereof, or a pharmaceutical composition, and each second active agent are administered by the same or different routes.

[0300] The method can further include administering to the subject an effective amount of hydroxyurea (which can, for example, include a second active agent). Hydroxyurea can be administered simultaneously or sequentially with the compound, a pharma- ceutical acceptable salt thereof, or a pharmaceutical composition, in either order (where applicable). In certain embodiments, the compound, a pharma- ceutical acceptable salt thereof, or a pharmaceutical composition, and hydroxyurea are administered by the same route. In certain embodiments, the compound, a pharma- ceutical acceptable salt thereof, or a pharmaceutical composition, and hydroxyurea are administered by different routes.

[0301] Another method of treating an RBC-mediated disease is also provided. In certain embodiments, the method comprises administering an effective amount of: (i) A compound (or a pharma- ceutically acceptable salt) comprising a rigid, fused bicyclic ring, which is 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 an indole substituted with at least one substituent, at least one of which is a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring. (ii) a compound (or a pharma- ceutical acceptable salt) that prevents Syk from phosphorylating the human erythrocyte anion transporter band 3; (iii) where the compound inhibits Syk tyrosine kinase at a concentration at least about 20-fold lower than the concentration at which it inhibits HGFR, EGFR, FGFR3, IGF1R and / or VEGFR (or a pharma- ceutical acceptable salt thereof). administering In addition to or instead of (ii) and (iii), the compound or pharma- ceutically acceptable salt of (i) is (iv) a CLogP value of between about 2 and about 5 (e.g., between 2 and about 5, between about 2 and 5, or between 2 and 5); (v) a polar surface area of ​​less than about 250 Å2 (e.g., less than 250 Å2); (vi) up to 5 H-bond donors; (vii) 15 or fewer H-bond acceptors, and / or (viii) 20 or fewer rotatable bonds having wherein the subject is treated for sickle cell disease. The compound can prevent Syk from phosphorylating tyrosine 8 and tyrosine 21 in human erythrocyte anion transporter band 3. The compounds, and / or pharmaceutical compositions comprising the compounds, include compounds that can stabilize red blood cell membranes and reduce the release of microvesicles from sickle red blood cells.

[0302] The RBC-mediated disease can be selected from the group consisting of 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 involves phosphorylation of the erythrocyte anion transporter band 3, and inhibition of such phosphorylation can prevent, delay, and / or treat the sequelae and disease symptoms that cause vaso-occlusive disease, so that they are either not initiated or are stopped. In fact, administration of an effective amount of the compound or a pharmaceutical composition thereof can stabilize the erythrocyte cell membrane in a subject and reduce the release of hemoglobin from sickle erythrocytes in the subject. In this way, the risk of vaso-occlusive crisis in a subject can be reduced.

[0303] In certain embodiments of the method, administration of the compound, a pharma- ceutical acceptable salt thereof, or pharmaceutical composition provides an IC 50 The subject may be a human child (i.e., a growing human or other human in whom off-target inhibition of growth hormone is believed to have significant adverse effects).

[0304] The method may further comprise administering one or more other compounds selected from the group consisting of fostamatinib, PRT062607, TAK-659, TAE-684, entospletinib, lanlaprenib, celduratinib, piceatannol, S701, SykII, SykIV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusacitinib, R112, PRT-060318 and OXSI-2, either simultaneously or sequentially, in any order, optionally as a pharmaceutical composition comprising a pharma- ceutically acceptable carrier or excipient. The one or more other compounds may be administered by the same or different route (relative to the administration of the compound or pharmaceutical composition).

[0305] A method for blocking or inhibiting phosphorylation of human erythrocyte anion transporter band 3 in a subject comprises administering to the subject an effective amount of any of the above-described compounds, pharma- ceutically acceptable salts thereof, or the above-described pharmaceutical compositions.

[0306] Still further provided is a method of 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 medicamentously acceptable salt thereof, the compound comprising a rigid, 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 an indole substituted with at least one substituent that is a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring. Upon administration to the subject, the subject can be treated for a disease involving the release of microvesicles. The disease can be thalassemia. The thalassemia can be α-thalassemia or β-thalassemia, alone or in combination with SCD. The disease can be G6PD deficiency. The disease can be glutathione reductase deficiency.

[0307] The method can further comprise administering a Src inhibitor simultaneously or sequentially (in accordance with the compound or its pharmaceutical composition) in any order, optionally as a pharmaceutical composition containing a pharma- ceutical acceptable carrier or excipient.The Src inhibitor can be administered by the same or different route in accordance with the compound or its pharmaceutical composition.The Src inhibitor can be dasatinib, ibrutinib, bafetinib, PP1, PP2, PP121, or a combination of two or more of the above.

[0308] Still yet further provided is a method of treating SCD in a subject, the method comprising administering an effective amount of (i) A compound comprising a pyrazine, which is substituted with at least three substituents, at least one of which is a carboxamide or lactam and the other of which is an N-linked, optionally substituted carbocycle or heterocycle, the other of which is a carbocycle or heterocycle having at least one substituent, and at least one of which is a 4-6 membered, optionally substituted carbocycle or heterocycle. (ii) a compound that prevents Syk tyrosine kinase from phosphorylating human erythrocyte anion transporter band 3; (iii) where the compound inhibits Syk tyrosine kinase at a concentration at least about 20-fold lower than the concentration at which it inhibits HGFR, EGFR, FGFR3, IGF1R and / or VEGFR. administering In addition to or instead of (ii) and (iii), the compound (i) is (iv) a CLogP value of between about 2 and about 5 (e.g., between 2 and about 5, between about 2 and 5, or between 2 and 5); (v) a polar surface area of ​​less than about 250 Å2 (e.g., less than 250 Å2); (vi) up to 5 H-bond donors; (vii) 15 or fewer H-bond acceptors, and / or (viii) 20 or fewer rotatable bonds having wherein the subject is treated for SCD. The compound or a pharma- ceutically acceptable salt thereof can prevent Syk tyrosine kinase from phosphorylating tyrosine 8 and tyrosine 21 in human erythrocyte anion transporter band 3. The compound or a pharma- ceutically acceptable salt thereof can stabilize erythrocyte cell membranes to reduce the release of microvesicles from sickle erythrocytes. The compound or a pharma- ceutically acceptable salt thereof can stabilize erythrocyte cell membranes to reduce the release of hemoglobin from sickle erythrocytes. The risk of vaso-occlusive crisis in the subject can be reduced. The compound or a pharma- ceutical acceptable salt thereof can inhibit erythrocyte cell membranes with an IC of 200 nM or less. 50The Syk tyrosine kinase can be inhibited by. The subject can be a human child. The method can further include administering one or more of fostamatinib, PRT062607, TAK-659, TAE-684, entospletinib, lanlaprenib, celduratinib, piceatannol, S701, SykII, SykIV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusacitinib, R112, PRT-060318 and OXSI-2, in any order, simultaneously or sequentially, optionally as a pharmaceutical composition including a pharma- ceutical acceptable carrier or excipient. The one or more other compounds can be administered by the same or different routes.

[0309] Also provided is a method of 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 pharma- ceutically acceptable salt thereof, the compound comprising a pyrazine substituted with at least three substituents, at least one of which is a carboxamide or lactam, and the other of which is an N-linked, optionally substituted carbocyclic or heterocyclic ring, and the other of which is a carbocyclic or heterocyclic ring with at least one substituent, and at least one of which is a 4-6 membered, optionally substituted carbocyclic or heterocyclic ring, wherein the subject is treated for a disease involving the release of microvesicles. The disease can be thalassemia. The thalassemia can be α-thalassemia or β-thalassemia, alone or in combination with sickle cell disease. The disease can be G6PD deficiency. The disease can be glutathione reductase deficiency. The method can further comprise administering Src inhibitors simultaneously or sequentially in any order, optionally as pharmaceutical compositions containing pharma- ceutical acceptable carriers or excipients.The Src inhibitors can be administered by the same or different routes according to the compound or its pharma- ceutical acceptable salt.The Src inhibitors can be dasatinib, ibrutinib, bafetinib, PP1, PP2, PP121, or a combination of two or more of the above.

[0310] The terms "treat," "treating," "treated," or "treatment" (in the context of a disease or condition) refer to an approach for obtaining beneficial or desired results, including, and preferably clinical results, including, but not limited to, one or more of the following: ameliorating the conditions associated with the disease, curing the disease, reducing the severity of the disease, slowing the progression of the disease, alleviating one or more symptoms associated with the disease, improving the quality of life of a person suffering from the disease, prolonging survival, and / or prophylactic or preventative treatment.

[0311] As used herein, "effective amount" refers to any amount of a compound in terms of its use in treatment, and refers to a quantity of a compound in a preparation that, when administered as part of a desired (to a mammal, e.g., a human) dosage regimen, is sufficient to achieve a desired biological effect (e.g., alleviating symptoms, ameliorating a condition, or slowing the onset of a disease state for the disorder or condition being treated or for cosmetic purposes, in accordance with clinically accepted standards, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment).

[0312] By choosing among a variety of active compounds and comparing factors such as potency, relative bioavailability, patient weight, severity of side effects and method of administration, in combination with the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be designed that is effective in treating a particular subject without causing substantial undesired toxicity. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the particular compound being administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art can determine the effective amount of a particular compound and / or other therapeutic agent on an empirical basis without necessitating undue experimentation.

[0313] For example, for any compound, the effective amount can be determined first from an animal model. Effective doses can also be determined from human data for compounds tested in humans and for compounds known to exhibit similar pharmacological activity, such as other related active agents. Higher doses may be required for parenteral administration. The applied dose can be adapted based on the relative bioavailability and efficacy of the administered compound. It is well within the capabilities of a person skilled in the art to adapt the dose to achieve maximum efficacy based on the methods described above and other methods known in the art.

[0314] The maximum dose, i.e., the safest dose according to some medical judgment, can be used. Multiple doses per day can be used to achieve an appropriate systemic level of the compound. The appropriate systemic level can be determined, for example, by measuring the patient's peak or sustained plasma level of the drug.

[0315] "Dose" and "administration" may be used interchangeably herein.

[0316] In general, the daily oral dose of the compound is about 0.01 milligrams / kg per day to 1,000 milligrams / kg per day in human subjects. Oral doses ranging from 0.5 to 50 milligrams / kg per day in one or multiple administrations can produce therapeutic results. The dosage can be appropriately adapted to achieve the desired local or systemic drug levels depending on the method of administration. For example, intravenous administration can vary from one to several orders of magnitude lower per day. If the response in the subject is inadequate at such doses, even higher doses (or higher effective doses by a different, more localized delivery route) can be utilized to the extent that patient tolerance permits. Multiple administrations per day are contemplated to achieve adequate systemic levels of the compound.

[0317] For clinical use, any compound can be administered in an amount equal to or equivalent to 0.2 to 2,000 milligrams (mg) of compound per kilogram (kg) of subject's body weight per day. Compounds can be administered in a dose equal to or equivalent to 2 to 2,000 mg of compound per kg of subject's body weight per day. Compounds can be administered in a dose equal to or equivalent to 20 to 2,000 mg of compound per kg of subject's body weight per day. Compounds can be administered in a dose equal to or equivalent to 50 to 2,000 mg of compound per kg of subject's body weight per day. Compounds can be administered in a dose equal to or equivalent to 100 to 2,000 mg of compound per kg of subject's body weight per day. Compounds can be administered in a dose equal to or equivalent to 200 to 2,000 mg of compound per kg of subject's body weight per day. Where a precursor or prodrug of a compound is to be administered, it is administered in an amount equivalent to, i.e., sufficient to deliver the amounts of the compound described above.

[0318] The compound or a formulation of a pharmacopoietic acceptable salt thereof can be administered to a human subject in an effective amount. A typical dosage range is about 0.01 microgram / kg to about 2 mg / kg of body weight per day. The dosage of the drug to be administered will likely depend on variables such as the type and extent of the disorder, the general health of the particular subject, the particular compound administered, the excipients used to formulate the compound, and its route of administration. Routine experimentation can be used to optimize the dosage and dosing frequency for any particular compound or pharmacopoietic acceptable salt thereof.

[0319] The compound or a medicamentously acceptable salt thereof may be administered at a concentration ranging from about 0.001 micrograms / kg to greater than about 500 mg / kg. For example, the concentrations may be 0.001 micrograms / kg, 0.01 micrograms / kg, 0.05 micrograms / kg, 0.1 micrograms / kg, 0.5 micrograms / kg, 1.0 micrograms / kg, 10.0 micrograms / kg, 50.0 micrograms / kg, 100.0 micrograms / kg, 500 micrograms / 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, 40.0 mg / kg, 50.0 mg / kg, 60.0 mg / kg, 70.0 mg / kg, 80.0 mg / kg, 90.0 mg / kg, 100.0 mg / kg, 15 ... g, 30.0 mg / kg, 35.0 mg / kg, 40.0 mg / kg, 45.0 mg / kg, 50.0 mg / kg, 60.0 mg / kg, 70.0 mg / kg, 80.0 mg / kg, 90.0 mg / kg, 100.0 mg / kg, 150.0 mg / kg, 200.0 mg / kg, 250.0 mg / kg, 300.0 mg / kg, 350.0 mg / kg, 400.0 mg / kg, 450.0 mg / kg to greater than about 500.0 mg / kg, or any increment therein. It will be understood that all values ​​between these values, and all ranges between these ranges, are meant to be encompassed.

[0320] The compound or a medicamentously acceptable salt thereof may be administered at a dosage ranging from about 0.2 milligrams / kg / day to about more than 100 mg / kg / day. For example, dosages may range from 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 10mg / kg / day, 0.25mg / kg / day~7.5mg / kg / day, 0.25mg / kg / day~5mg / kg / day, 0.5mg / kg / day~50mg / kg / day, 0.5mg / kg / day~25mg / kg / day, 0.5mg / kg / day~ 20mg / kg / day, 0.5mg / kg / day ~ 15mg / kg / day, 0.5mg / kg / day ~ 10mg / kg / day, 0.5mg / kg / day ~ 7.5mg / kg / day, 0.5mg / kg / day ~ 5mg / kg / day, 0.75mg / kg / day ~ 50 mg / kg / day, 0.75mg / kg / day ~ 25mg / kg / day, 0.75mg / kg / day ~ 20mg / kg / day, 0.75mg / kg / day ~ 15mg / kg / day, 0.75mg / kg / day ~ 10mg / kg / day, 0.75mg / kg / day ~7.5mg / kg / day, 0.75mg / kg / day ~5mg / kg / day, 1.0mg / kg / day ~50mg / kg / day, 1.0mg / kg / day ~25mg / kg / day, 1.0mg / kg / day ~20mg / kg / day, 1.0mg / kg / day ~1 It may be 5 mg / kg / day, 1.0 mg / kg / day to 10 mg / kg / day, 1.0 mg / kg / day to 7.5 mg / kg / day, 1.0 mg / kg / day to 5 mg / kg / day, 2 mg / kg / day to 50 mg / kg / day, 2 mg / kg / day to 25 mg / kg / day, 2 mg / kg / day to 20 mg / kg / day, 2 mg / kg / day to 15 mg / kg / day, 2 mg / kg / day to 10 mg / kg / day, 2 mg / kg / day to 7.5 mg / kg / day, or 2 mg / kg / day to 5 mg / kg / day.

[0321] The compound may be administered at a dosage ranging from about 0.25 milligrams / kg / day to about 25 mg / kg / day. For example, dosages may 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, 5.0 mg / kg / day, 6.0 mg / kg / day, 7.0 mg / kg / day, 8.0 mg / kg / day, 9.0 mg / kg / day, 10.0 mg / kg / day, 11.0 mg / kg / day, 12.0 mg / kg / day, 13.0 mg / kg / day, 14.0 mg / kg / day, 15.0 mg / kg / day, 16.0 mg / kg / day, 17.0 mg / kg / day, 18.0 mg / kg / day, 19.0 mg / kg / day, 20.0 mg / kg / day, 21.0 mg / kg / day, 22.0 mg / kg / day, 23.0 mg / kg / day, 24.0 mg / kg / day, 25.0 mg / kg / day, 26.0 mg / kg / day, 27.0 mg / kg / day, 28.0 mg / kg / day, 29.0 mg / kg / day, 30.0 mg / kg / day, 31.0 mg / kg / day, 32.0 mg / kg / day g / 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 / day, 14 mg / 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 / k g / 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, 3 It may be 3 mg / kg / day, 34 mg / kg / day, 35 mg / kg / day, 36 mg / kg / day, 37 mg / kg / day, 38 mg / kg / day, 39 mg / kg / day, 40 mg / kg / day, 41 mg / kg / day, 42 mg / kg / day, 43 mg / kg / day, 44 mg / kg / day, 45 mg / kg / day, 46 mg / kg / day, 47 mg / kg / day, 48 mg / kg / day, 49 mg / kg / day, or 50 mg / kg / day.

[0322] The compound, a pharma- ceutical acceptable salt thereof, or a precursor thereof may be administered at a concentration ranging from 0.01 micromolar to 500 micromolar or more. For example, doses may 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, 50.0 micromolar, 60.0 micromolar, 70.0 micromolar, 80.0 micromolar, 90.0 micromolar, 100.0 micromolar, 110.0 micromolar, 120.0 micromolar, 130.0 micromolar, 140.0 micromolar, 150.0 micromolar, 160.0 micromolar, 170.0 micromolar, 180.0 micromolar, 190.0 micromolar, 210.0 micromolar, 220.0 micromolar, 230.0 micromolar, 240.0 micromolar, 250.0 micromolar, 260.0 micromolar, 270.0 micromolar, 280.0 micromolar, 290.0 micromolar, 300.0 micromolar, 350.0 micromolar, 360.0 micromolar, 370.0 micromolar, 380.0 micromolar, 390.0 micromolar, The range may be from 45.0 micromolar, 50.0 micromolar, 60.0 micromolar, 70.0 micromolar, 80.0 micromolar, 90.0 micromolar, 100.0 micromolar, 150.0 micromolar, 200.0 micromolar, 250.0 micromolar, 300.0 micromolar, 350.0 micromolar, 400.0 micromolar, 450.0 micromolar to greater than about 500.0 micromolar, or any increment therein, and it will be understood that all values ​​between these values, and all ranges between these ranges, are meant to be encompassed.

[0323] The compound, or a pharma- ceutical 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, concentrations may be 0.10 micrograms / mL, 0.50 micrograms / mL, 1 micrograms / 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, 110.0 micrograms / mL, 120.0 micrograms / mL, 130.0 micrograms / mL, 140.0 micrograms / mL, 150.0 micrograms / mL, 160.0 micrograms / mL, 170.0 micrograms / mL, 180.0 micrograms / mL, 190.0 micrograms / mL, 200.0 micrograms / mL, 210.0 micrograms / mL, 220.0 micrograms / mL, 230.0 micrograms / mL, 240.0 micrograms / mL, 250.0 micrograms / mL, 260.0 micrograms / mL, 270.0 micrograms / mL, 280.0 micrograms / mL, 290.0 micrograms / mL, 300.0 micrograms / mL, 350.0 micrograms / mL, 360.0 micrograms / mL, 370.0 micrograms L, 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 increment therein. It will be understood that all values ​​between these values, and all ranges between these ranges, are meant to be encompassed.

[0324] The formulation may be administered in a pharma- ceutically acceptable solution, which may routinely contain pharma- ceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. For use in therapy, an effective amount of the compound may be administered to a subject by any method that delivers the compound to the desired surface. Administering the pharmaceutical composition may be accomplished 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., into a tumor or abscess), mucous membrane (e.g., topical administration to the eye), inhalation, and topical administration.

[0325] For intravenous and other parenteral routes of administration, the compounds can be formulated as lyophilized preparations, as lyophilized preparations of liposome-intercalated or encapsulated active compounds, as lipid complexes in aqueous suspension, or as salt complexes.Lyophilized preparations are generally reconstituted in a suitable aqueous solution, for example, in sterile water or saline, immediately prior to administration.

[0326] For oral administration, the compound can be readily formulated by combining the active compound with pharma- ceutically acceptable carriers known in the art. Such carriers allow the compound to be formulated as 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 by optionally grinding the mixture obtained, processing the mixture of granules after adding suitable auxiliary agents, to obtain tablet cores or dragee cores as solid excipients. Suitable excipients are, in particular, 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, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked PVP, agar, or alginic acid or a salt thereof, such as sodium alginate. Optionally, the oral formulations may also be formulated in saline or buffers, such as ethylenediaminetetraacetic acid (EDTA) for neutralizing internal acid conditions, or may be administered without any carrier.

[0327] Also contemplated are oral dosage forms of the compounds. The compounds can be chemically modified to effect oral delivery of the derivatives. In general, the chemical modifications contemplated are the attachment of at least one moiety to the compound itself, which (a) inhibits acid hydrolysis, and (b) allows uptake from the stomach or intestine into the bloodstream. Also desirable is an increase in the overall stability of the compound and an increase in circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, 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 can be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane.For pharmaceutical uses, as indicated above, polyethylene glycol moieties are preferred.

[0328] The release location of the compound or its pharmacopoietic salt herein can 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 anywhere in the intestine.Release can avoid the adverse effects of the stomach environment, either by protecting the compound or by releasing the compound or its pharmacopoietic salt beyond the stomach environment, for example, in the intestine.

[0329] To ensure complete gastric resistance, a coating impermeable to at least pH 5.0 may be necessary. Examples of the more common inactive ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S and shellac. These coatings can be used as mixed films.

[0330] Coatings, or mixtures of coatings, can also be used on tablets that are not intended for protection against the stomach. This includes sugar coatings, or coatings that make the tablet easier to swallow. Capsules can consist of a hard shell (such as gelatin) for delivery of dry therapeutics (e.g., powder); for liquid forms, a soft gelatin shell can be used. The shell material of cachets can be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet powders, moist sugaring techniques can be used.

[0331] As noted above, the compound or a pharma- ceutical acceptable salt thereof may be included in the formulation as fine multiparticulates, in the form of granules, or pellets about 1 mm in size. The formulation of the material for capsule administration may also be as a powder, lightly compressed plugs, or even tablets.

[0332] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, and soft sealed capsules made of gelatin and plasticizers, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients in admixture with fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, active compounds can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers can be added. Microspheres formulated for oral administration can also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

[0333] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0334] For local administration, the compounds can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, and those designed for transdermal, transmucosal, oral or pulmonary administration.

[0335] For administration by inhalation, the compound can be conveniently delivered in the form of an aerosol spray presentation 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 can be determined by providing a valve to deliver a metered amount. For example, capsules and cartridges of gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0336] Also contemplated is pulmonary delivery of the compound (or salt thereof). The compound is delivered to the lungs of a mammal upon inhalation, across the alveolar epithelial lining, and 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-gamma and tumor necrosis factor alpha), and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor, incorporated herein by reference). Methods and compositions for pulmonary delivery of drugs for systemic effect are described in Wong et al., U.S. Pat. No. 5,451,569, issued Sep. 19, 1995, incorporated herein by reference for its disclosures relating to said methods and compositions.

[0337] Contemplated for use are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

[0338] Nasal delivery of the pharmaceutical composition is also contemplated. Nasal delivery allows the passage of the pharmaceutical composition directly into the bloodstream after administration of the therapeutic product to the nose, without the need for deposition of the product in the lungs. Formulations for nasal delivery include those with dextran or cyclodextran.

[0339] When it is desired to deliver the compounds systemically, they can be formulated for parenteral administration by injection, for example, bolus injection or continuous infusion.The preparations for injection can be provided in unit dosage form, for example, in ampoules or in multi-dose containers with added preservatives.The compositions can take the form of suspension, solution or emulsion in oily or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizing agents and / or dispersing agents.

[0340] Pharmaceutical preparations 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.Suitable lipophilic solvents or vehicles 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 suspensions, such as sodium carboxymethylcellulose, sorbitol or dextran.Optionally, suspensions can also contain suitable stabilizers or agents that increase the solubility of compounds to allow the preparation of highly concentrated solutions.

[0341] Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

[0342] The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0343] In addition to the above-mentioned formulations, compound can also be formulated as depot preparation.Such long-acting preparations can be formulated with suitable polymeric or hydrophobic materials (for example, emulsion in acceptable oil) or ion exchange resins, or can be formulated as sparingly soluble derivatives, for example, as sparingly soluble salts.

[0344] The pharmaceutical compositions may also include suitable solid or gel phase carriers or excipients, examples of which include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0345] Suitable liquid or solid pharmaceutical preparation forms are, for example, microencapsulated, entrapped, coated on microscopic gold particles, contained in liposomes, nebulized, aerosol, pellets for implantation in the skin, or dried on a sharp object for scratching in the skin, aqueous or saline solutions for inhalation.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 auxiliary agents, such as disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, sweeteners or solubilizers, are commonly used as described above.Pharmaceutical compositions are suitable for use in a variety of drug delivery systems.For a brief review of methods for drug delivery, see Langer R, Science 249:1527-1533 (1990).

[0346] Disintegrants may be included in the formulation of the therapeutic agent in solid dosage form. Materials used as disintegrants include, but are not limited to, starch, Explotab, including commercial disintegrants based on starch. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge and bentonite can all be used. Another form of disintegrant is the insoluble cationic exchange resin. Powdered gums may be used as disintegrants and as binders, and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

[0347] Binders can be used to hold the compounds together to form hard tablets and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). Both PVP and hydroxypropylmethylcellulose (HPMC) can be used in alcoholic solutions to granulate the therapeutic agent.

[0348] Antifriction agents may be included in the formulation of therapeutic agent to prevent sticking during formulation process.Lubricants can be used as a layer between therapeutic agent and die wall, including but not limited to stearic acid with its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oil and wax.Soluble lubricants can also be used, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

[0349] Glidants may be added that can improve the flow properties of the drug in the formulation and aid in rearrangement during compression. Glidants can include starch, talc, pyrogenic silica and hydrated aluminum silicate.

[0350] Surfactants may be added as wetting agents to help dissolve the therapeutic agent in the aqueous environment.Surfactants may include anionic detergents, such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.Cationic detergents that may be used include benzalkonium chloride and benzethonium chloride.Potential nonionic detergents that may be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose.These surfactants may be present in the formulation of the compound or its derivatives either alone or as a mixture in different ratios.

[0351] The compound and, optionally, one or more other therapeutic agents can be administered per se (neat) or in the form of a pharmaceutically acceptable salt.When used in medicine, the salt should be pharmaceutically acceptable, but pharmaceutically unacceptable salts may be conveniently used to prepare pharmaceutically acceptable salts thereof.Such salts include, but are not limited to, those prepared from 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.Such salts can also be prepared as alkali metal or alkaline earth salts, for example, the sodium, potassium, or calcium salts of the carboxylic acid group.

[0352] The volume of the compound may be diluted or increased with inert substances.These diluents may include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextran and starch.Certain inorganic salts may also be used as fillers, including calcium triphosphate, magnesium carbonate and sodium chloride.Some commercially available diluents are Fast-Flo, Emdex, STA-Rx1500, Emcompress and Avicell.

[0353] Suitable buffering agents include acetic acid and salts (1-2% w / v), citric acid and salts (1-3% w / v), boric acid and salts (0.5-2.5% w / v), and phosphoric acid and 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).

[0354] The pharmaceutical composition contains an effective amount of the compound described herein, and one or more other therapeutic agents, optionally contained in a pharma- ceutical acceptable carrier. The term "pharma-ceutical acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans or other vertebrates. The term "carrier" refers to a natural or synthetic, organic or inorganic component with which the active ingredient is combined to facilitate application. The components of the pharmaceutical composition can also be mixed with the compound and each other in a manner that does not cause any interaction that would substantially impair the desired pharmaceutical efficacy.

[0355] Therapeutic agents, including but not limited to compounds, can be provided in particles. As used herein, "particle" refers to nanoparticles or microparticles (or larger particles in some examples) that can be in whole or in part of a compound or other therapeutic agent described herein. The particles can contain the therapeutic agent in a core that is surrounded by a coating, including but not limited to an enteric coating. The therapeutic agent can also be dispersed throughout the particle. The therapeutic agent can also be adsorbed into the particle. The particles can be of any order of release kinetics, including zero order release, first order release, second order release, delayed release, sustained release, immediate release, and any combination thereof. The particles can include any of these materials routinely used in the pharmaceutical and drug arts, including but not limited to erodible, non-erodible, biodegradable, or non-biodegradable substances, or combinations thereof, in addition to the therapeutic agent. The particles can be microcapsules that contain the compound in solution or in a semi-solid state. The particles can be of virtually any shape.

[0356] Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering therapeutic agents. Such polymers can be natural or synthetic. The polymer is selected based on the period over which release is desired. Bioadhesive polymers of particular interest include the biodegradable hydrogels described in Sawhney et al., Macromolecules 26:581-587 (1993), the teachings of which are specifically incorporated herein by reference. These include polyhyaluronic acid, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate) and poly(octadecyl acrylate).

[0357] The therapeutic agent can be contained in a controlled release system. The term "controlled release" is intended to refer to any drug-containing formulation in which the method and profile of drug release from the formulation is controlled. It refers to immediate release and non-immediate release formulations, including but not limited to sustained release and delayed release formulations. The term "sustained release" (also referred to as "extended release") is used in its conventional sense to refer to a drug formulation that can provide a gradual release of drug over an extended period of time to provide a substantially constant blood level of drug over an extended period of time. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of drug from the formulation. "Delayed release" may or may not involve a gradual release of drug over an extended period of time, and therefore may or may not be a "sustained release".

[0358] The use of long-term sustained release implants may be particularly suitable for treating chronic conditions. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and up to 30-60 days. Long-term sustained release implants are well known to those skilled in the art and include some of the release systems described above.

[0359] Depending on the route of administration, approximately 10 6 ~10 11 A wide range of acceptable dosages is contemplated herein, including doses falling within the range of 1000 to 15000 viral particles (VP) / kg. Doses may be single or divided and may be administered according to a wide range of protocols, including qd (once a day), bid (twice a day), tid (three times a day), or even every other day, weekly, monthly, quarterly, etc. In each of these cases, it is understood that the effective amounts described herein correspond to instances of administration, or alternatively correspond to total daily, weekly, monthly, or quarterly doses, as determined by the dosing protocol.

[0360] In addition to the exemplary dosage and administration protocols described herein, the effective amount of any one or mixture of compounds described herein can be determined by the attending diagnostician or physician by using known techniques and / or by observing results obtained under similar circumstances. In determining an effective amount or dose, numerous factors are considered by the attending diagnostician or physician, including, but not limited to, the species of mammal, including human, its size, age and general health, the specific disease or disorder involved, the degree of involvement or severity of the disease or disorder, the response of the individual patient, the specific compound administered, the method of administration, the bioavailability characteristics of the administered preparation, the selected dosage regimen, the use of concomitant drugs, and other relevant circumstances.

[0361] Those skilled in the art will recognize that numerous modifications may be made to the specific implementations described above. Implementations should not be limited to the specific limitations described. Other implementations may be possible.

[0362] While the compounds and pharmaceutical compositions have been illustrated and described in detail in the foregoing description, it is to be understood that the same is considered to be exemplary and not restrictive in nature, that only certain embodiments have been shown and described, and that all changes and modifications that come within the spirit of the invention are desired to be protected.

[0363] It is intended that the spirit of the present compounds, compositions and methods be defined by the scope of the following claims.However, the present disclosure may be carried out other than as specifically described and illustrated without departing from its spirit or scope.Those skilled in the art will understand that various alternatives to the embodiments described herein may be utilized in carrying out the following claims without departing from the spirit and scope defined in the following claims.

[0364] Any use of section headings is intended to aid in the reading of this document and is not to be construed as limiting. Further, information associated with a section heading may be found within or outside of that particular section.

[0365] All publications, patents, patent application publications, journal articles, textbooks, and other publications referred to in this document are indicative of the level of skill of those skilled in the art to which this disclosure pertains.All such publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.If there is a discrepancy in usage between this document and the document so incorporated by reference, the usage in the incorporated reference should be considered as complementary to the usage of this document, and in the case of irreconcilable discrepancies, the usage in this document shall prevail.

[0366] A variety of techniques and mechanisms will sometimes describe the bond or connection between two components. Words such as attached, linked, coupled, bonded, and similar terms with their inflected morphemes are used interchangeably unless a difference is specified or otherwise clear from the context. These words and expressions do not necessarily mean a direct bond, but include a bond through an intermediate component. It should be noted that a bond between two components does not necessarily mean a direct, uninterrupted bond, because a variety of other components may exist between the two components specified. As a result, a bond does not necessarily mean a direct, uninterrupted bond, unless otherwise specified.

[0367] Certain definitions As used herein, the following terms and phrases shall have the meanings set forth below: 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.

[0368] The term "about," when referring to a number or numerical value or range (including, e.g., whole numbers, fractions, and percentages), means that the number or numerical range referred to is approximate within experimental variation (or within statistical experimental error), such that the numerical value or range can vary between 1%-15% of the stated number or numerical range (e.g., + / - 5%-15% of the cited value), but would be considered equivalent (e.g., have the same function or result) by one of ordinary skill in the art. The term "substantially" can allow for variation in value or range, for example, within 90%, within 95%, or within 99% of the stated value of the stated limits of the range.

[0369] The terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods and / or types of steps that are described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0370] The term "or," unless otherwise specified, is used to refer to a non-exclusive "or." In addition, phraseologies or terminologies utilized herein, and not otherwise defined, will be understood to be for purposes of description only and not of limitation.

[0371] "Alkyl" generally refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, such as having 1 to 15 carbon atoms (e.g., C1-C 15 The disclosure of "alkyl" provided herein is intended to include independent references to saturated "alkyl" unless otherwise stated. Alkyl can contain 1 to 13 carbon atoms (e.g., C1 to C6). 13 alkyl). An alkyl can contain 1 to 8 carbon atoms (e.g., C1-C8 alkyl). An alkyl can contain 1 to 5 carbon atoms (e.g., C1-C5 alkyl). An alkyl can contain 1 to 4 carbon atoms (e.g., C1-C4 alkyl). An alkyl can contain 1 to 3 carbon atoms (e.g., C1-C3 alkyl). An alkyl can contain 1 to 2 carbon atoms (e.g., C1-C2 alkyl). An alkyl can contain 1 carbon atom (e.g., C1 alkyl). An alkyl can contain 5 to 15 carbon atoms (e.g., C5-C6 alkyl). 15Alkyl can include 5 to 8 carbon atoms (e.g., C5 to C8 alkyl). Alkyl can include 2 to 5 carbon atoms (e.g., C2 to C5 alkyl). Alkyl can include 3 to 5 carbon atoms (e.g., C3 to C5 alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl is attached to the remainder of the molecule by a single bond.

[0372] "Alkoxy" refers to a group attached through an oxygen atom of the formula -O-alkyl, where alkyl is an alkyl chain as defined above.

[0373] "Alkylene" or "alkylene chain" generally refers to a straight or branched divalent alkyl group that connects the remainder of the molecule to a radical group having from 1 to 12 carbon atoms, such as methylene, ethylene, propylene, i-propylene, n-butylene, and the like.

[0374] "Aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. An aromatic monocyclic or polycyclic hydrocarbon ring system contains only one hydrogen and 5 to 18 carbon atoms, and at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) pi-electron system according to the Hückel rule. Ring systems from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin, and naphthalene.

[0375] "Arylalkyl" refers to a group of the formula -R c -aryl (wherein R crefers to an alkylene chain as defined above, e.g., methylene, ethylene, etc. The alkylene chain part of the aralkyl radical is optionally substituted as described above for an alkylene chain.

[0376] The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude any compound, composition, method, process, etc., embodiments that may "consist of" or "consist essentially of" the recited features. The inventions illustratively described herein may suitably be practiced in the absence of any element or limitation not specifically disclosed herein.

[0377] The term "heteroalkyl" refers to an alkyl group as defined above, where one or more skeletal carbon atoms of the alkyl are replaced with a heteroatom (with the appropriate number of substituents or valences, e.g., -CH2- can be replaced with -NH- or -O-). For example, each substituted carbon atom is independently replaced with a heteroatom, e.g., carbon is replaced with nitrogen, oxygen, selenium, or other suitable heteroatom. In some examples, each substituted carbon atom is independently replaced with oxygen, nitrogen (e.g., -NH-, -N(alkyl)-, or -N(aryl)-, or another substituent contemplated herein), or sulfur (e.g., -S-, -S(=O)-, or -S(=O)2-). The heteroalkyl is attached to the remainder of the molecule at a carbon atom of the heteroalkyl. The heteroalkyl is attached to the remainder of the molecule at a heteroatom of the heteroalkyl. Heteroalkyl is a C1-C 18 Heteroalkyl is a C1-C 12Heteroalkyl is a C1-C6 heteroalkyl. Heteroalkyl is a C1-C4 heteroalkyl. Heteroalkyl can include alkoxy, alkoxyalkyl, alkylamino, alkylaminoalkyl, aminoalkyl, heterocycloalkyl, and heterocycloalkylalkyl, as defined herein.

[0378] 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, farm animals such as dogs, cats or rabbits, agricultural animals such as cows, 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.

[0379] The terms "protein," "polypeptide," and "peptide" refer to compounds containing amino acids joined through peptide bonds, and are used interchangeably. EXAMPLES

[0380] The following examples serve to illustrate the present disclosure and are not intended to limit the scope of the claims in any way.

[0381] [Example 1] Synthesis of compound S970

[0382] [ka] tert-Butyl (6-bromoimidazo[1,2-a]pyrazin-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 h. After the reaction was completed (progress of the reaction was monitored by thin layer chromatography (TLC)), it was allowed to cool to room temperature and then added to 250 ml of ice-cold water. The resulting precipitate was stirred at room temperature for 30-45 min and filtered through a Buchner funnel. The resulting wet material was washed with water (100 mL) followed by diethyl ether (100 mL) and dried under vacuum at 50° C. for 5 h to give 6-bromo-N-(3,4-dimethoxyphenyl)imidazo[1,2-a]pyrazin-8-amine as a solid.

[0383] The resulting crude intermediate was placed in a 100 ml single-neck RBF and 25 ml of dichloromethane (DCM) was added, followed by (Boc)2O anhydride (0.875 g, 4.0 mmol) and a catalytic amount of 4-dimethylaminopyridine (DMAP) (25 mg, 0.025 equiv.). The resulting solution was heated to 50° C. and stirred for 3 h. After the reaction was completed (the reaction progress was monitored by TLC), the resulting solution was concentrated to dryness under vacuum.

[0384] The crude material obtained was purified by normal phase silica gel chromatography (hexanes: EtOAc) to give an off-white solid (1.00 g, 65% overall yield).

[0385] 1 H NMR (500 MHz, dimethylsulfoxide (DMSO)-d6): δ 8.89 (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; 13C NMR (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 C 19 H 22 Calculated for BrN4O4: 449.0819 Found: 449.0817 (M+1) + .

[0386] tert-Butyl (6-(1H-indol-6-yl)imidazo[1,2-a]pyrazin-8-yl)(3,4-dimethoxyphenyl)carbamate (compound S970-B). 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) followed by addition of H2O (4 mL) in a screw-capped vial with a septum. The reaction mixture was purged with a gentle stream of N2 under stirring. After 5 min of purging, the outlet needle was removed and Pd(PPh3)4 (40 mg, 0.025 mmol) was added followed by the quick addition of Na2CO3 (356 mg, 3.35 mmol). The reaction mixture was heated to 120° C. and stirred at the same temperature for 6 h. The reaction mixture was then concentrated to dryness and the desired product was isolated by normal phase silica gel chromatography (hexane:EtOAc) to give an off-white solid (302 mg, 56%).

[0387] 1 H NMR (500 MHz, DMSO-d6): δ 11.25 (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 C 27 H 28 Calculated value for N5O4: 486.2136 Found value: 486.2133 (M+1) + .

[0388] 1-((6-(8-((3,4-dimethoxyphenyl)amino)imidazo[1,2-a]pyrazin-6-yl)-1H-indol-3-yl)methyl)azetidin-3-ol S970. Compound B (250 mg, 0.56 mmol) was dissolved in DCM (2.5 mL) and trifluoroacetic acid (TFA) (2.5 mL) was added over 10 min at 0° C. The resulting solution was stirred at room temperature under N2 atmosphere for 10 h.

[0389] After the reaction was completed, the reaction mixture was concentrated to dryness under reduced pressure and the crude material was used directly for the next step, N-(3,4-dimethoxyphenyl)-6-(1H-indol-6-yl)imidazo[1,2-a]pyrazin-8-amine, without purification.

[0390] A solution of azetidin-3-ol (45 mg, 0.65 mmol) and 1,4 dioxane (1 mL) was added dropwise to a stirred solution of formaldehyde (30% in H2O, 83 μL) and acetic acid (1 mL) in 1,4 dioxane (1 mL) at 0° C. over 10 min. The resulting solution was stirred at the same temperature for 30 min. A solution of crude N-(3,4-dimethoxyphenyl)-6-(1H-indol-6-yl)imidazo[1,2-a]pyrazin-8-amine in 1,4 dioxane (1 mL) was added dropwise to the reaction mixture at 0° C. over 10 min. The reaction mixture was allowed to reach room temperature and stirred at the same temperature for 24 h. The resulting suspension was concentrated under reduced pressure and the title product was isolated by normal phase silica gel chromatography (dichloromethane:methanol) to give an off-white solid (20 mg, 10%).

[0391] 1H NMR (500 MHz, DMSO) δ 11.11 (s, 1H), 9.47 (s, 1H), 8.56 (s, 1H), 8.12 (d, J = 2.4 Hz, 1H), 8.04 (s, 1H), 8.00 (dd, J = 10.1, 1.0 Hz, 1H), 7.67 (m, 1H), 7.63 (dd, J = 5.6, 0.9 Hz, 3H), 7.27 (d, J = 1.8 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 4.18 (p, J = 6.2 Hz, 1H), 3.83 (s, 3H), 3.77 (s, 3H), 3.74 - 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 C26 H 27 Calculated for N6O3: 471.2139 Found: 471.2139 (M+1) +.

[0392] [Example 2] In vitro analysis of Syk inhibitors Syk kinase inhibition assays were run in 96-well plates using the ADP-Glo ​​assay kit following the manufacturer's specifications. All kinase reactions were run using 1× kinase reaction buffer consisting of 40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg / mL bovine serum albumin, and 0.1 mM dithiothreitol (DTT) as required by the kinase of interest (each of which is listed in Table 2 above) to produce a kinase reaction buffer. Kinase inhibitor stock solutions in DMSO were diluted into 1× 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 25 μL with the following proportions: 8.5 μL 1× kinase reaction buffer, 5 μL diluted kinase inhibitor, 5 μL 1 mg / mL poly-(4:1-Glu,Tyr) kinase substrate peptide, 4 μL kinase in 1× kinase reaction buffer, and 2.5 μL of 100 μM ATP diluted in kinase reaction buffer. The kinase inhibitor was incubated with the kinase and substrate, after which ATP was added for 30 minutes at ambient temperature.

[0393] After adding ATP to the wells, the plate was incubated at 37°C for 30 minutes. After incubation, 25 μL of ADP-Glo ​​reagent was added immediately and incubated at ambient temperature for 30 minutes. After 30 minutes, 50 μL of Kinase Detection Reagent was added and the plate was incubated at ambient temperature for 45 minutes, protected from light. After 45 minutes, luminescence was recorded using a Neo2 plate reader and 1 second integration of the signal. Background signal was subtracted from all readings and the signal was normalized to the maximum signal obtained in the absence of inhibitor. Data was plotted and inhibition curves were fitted using Graphpad software, and the results are shown in Tables 2 and 3.

[0394] [Example 3] Synthesis of compound S1115A

[0395] [ka] A solution of 3,5-dichloropyrazine-2-carboxamide (1.0 equiv.) was added to a stirred solution of 3,4,-dimethoxyoxyaniline (1.0 equiv.) in THF (20V) and lithium bis(trimethylsilyl)amide (LHMDS) (3.0 equiv.) at -78°C. The resulting reaction mixture was stirred at the same temperature for 2 h, then warmed to room temperature and stirred for 2 h. After the reaction was completed (progress was monitored by TLC), the reaction mixture was poured into ice-cold 2N hydrochloric acid (HCl), extracted with DCM, and dried over sodium sulfate.

[0396] The solvent was then removed under vacuum. The resulting crude intermediate was triturated with hexane to give compound S1115A (5-chloro-3-((3,4-dimethoxyphenyl)amino)pyrazine-2-carboxamide) as a deep yellow solid (70% yield).

[0397] [Example 4] Synthesis of compound S1115B

[0398] [ka] Compound S1115-A (1.0 equiv.) and (1H-indol-5-yl)boronic acid (1.2 equiv.) were dissolved in 1,4 dioxane (20 V) followed by the addition of H2O (20 V) in a screw-capped vial with a septum. The reaction mixture was stirred and purged with a gentle stream of N2. 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.).

[0399] 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 obtain compound S1115B (3-((3,4-methoxyphenyl)amino)-5-(1H-indol-6-yl)pyrazine-2-carboxamide) as a yellow solid (yield 58%).

[0400] [Example 5] Synthesis of compound S1115

[0401] [ka] A solution of azetidin-3-ol (3.0 equiv.) and 1,4 dioxane (20 V) was added dropwise over 10 min at 0 °C to a stirred solution of formaldehyde (30% in H2O, 3.0 equiv.) and acetic acid (10 equiv.) in 1,4 dioxane (10 V).

[0402] The resulting solution was stirred at the same temperature for 30 min, and a solution of compound S1115B in 1,4 dioxane (20V) was added dropwise to the reaction mixture over 10 min at 0° C. The reaction mixture was allowed to reach room temperature and stirred at the same temperature for 24 h. The resulting suspension was concentrated under reduced pressure, and compound S1115 (3-((3,4,-methoxyphenyl)amino)-5-(3-((3-hydroxyazetidin-1-yl)methyl)-1H-indol-6-yl)pyrazine-2-carboxamide) was isolated by reverse phase silica gel chromatography (pH 7.4, ammonium acetate:CAN) to give compound S1115 as a yellow solid (yield 10-15%).

Claims

1. Formula X: A-B-C (X) A compound of or a pharmaceutically acceptable salt thereof, wherein the formula is A is a first ring that is optionally substituted and linked by nitrogen, the first ring includes a fused bicyclic ring or a monocyclic ring, and each ring of the first ring includes a 4-6 member heterocyclic ring or a carbon ring. B is a second ring optionally substituted with at least one substituent, the second ring including a monocyclic ring or a fused bicyclic or tricyclic ring, and each of the rings in the second ring including a five-membered or six-membered heterocyclic or carbon ring. C is a third bicyclic ring comprising a five-membered or six-membered carbon ring or a five-membered or six-membered carbon ring fused with a heterocycle, wherein the third bicyclic ring is 【Chemistry 1】 Alternatively, it is substituted with at least one first substituent, which includes an amine, or a ketone, or a carboxamide, or an alcohol. During the ceremony, X ４ CH ２ , NH, CO, O or S, Y ５ is CH or N, Z is OH, SH, NH ２ CO ２ H, NHCH ３ , and 【Chemistry 2】 Selected from, 【Transformation 3】 The compound is the attachment site of the first substituent to the third bicyclic ring.

2. A, 【Chemistry 4】 And, During the ceremony, Each X' is independently selected from CH and NH. Each Y ２ CH is independent. ２ Selected from NH, O and S, Z ２ is CH or N, Each R ３ These are independently H, F, Cl, OH, and -Y-(CH ２ ) ｎ - Selected from, and in the formula, n = 1 to 14, or B, 【Transformation 5】 And, During the ceremony, Each X2 is independently selected from CH and N. Y3 is selected from O, NH, and S. Z3 is selected from OH, NH2, and NHCH3. The compound according to claim 1.

3. However, B 【Transformation 6】 Isn't that right? Can one or more hydrogen atoms be optionally substituted with deuterium? or B is a pyrazine substituted with at least one substituent, and optionally, the at least one substituent of B is an amide. The compound according to claim 1.

4. The compound according to claim 1, wherein the N connected to the first ring of A forms a nitrogen bridge together with B.

5. The compound according to claim 4, wherein A is a first monocyclic ring substituted with a bulky substituent that is para with respect to the nitrogen bridge, and optionally the bulky substituent is morpholine.

6. The compound according to any one of claims 1, 2, 4, and 5, wherein each of the rings of the first ring of A is a six-membered, optionally substituted, open or closed heterocycle or carbon ring.

7. The compound according to claim 1, wherein A is a first monocyclic ring substituted with two methoxy groups.

8. The compounds according to claims 1, 2, 4, and 5, wherein B is a monocyclic ring substituted with an amide.

9. C is, 【Transformation 7】 And, During the ceremony, Each X ３ These are independently selected from CH and N, Y ４ CH ２ , NH, O or SH The compound according to claim 1.

10. The first substituent of C is 【Transformation 8】 The compound according to claim 9.

11. The Z of the first substituent is -OR (wherein R is 【Chemistry 9】 The compound according to claim 1, wherein n = 1 to 14.

12. Is the third biring ring of C indole? or The third bicyclic ring of C is indole, and the first substituent of C includes a 4- to 6-membered, optionally substituted carbon ring or heterocyclic substituent. The compound according to claim 1.

13. The compound according to claim 12, wherein the substituent of the carbon ring or heterocycle includes a lipophilic ester.

14. The Z of the first substituent of C contains the phosphoric acid or phosphonic acid ester, The first substituent of C is 【Chemistry 10】 In the formula, Q is either phosphoric acid or a phosphonic acid ester, or The first substituent of C is 【Chemistry 11】 In the formula, Q is either RC(=O)O- (where R is alkyl), or, B can form a hydrogen bond with the amino acid residue Ala451 in the catalytic domain of Syk tyrosine kinase, and the first substituent of C can form a hydrogen bond with the amino acid residue Asp512 in the catalytic domain of Syk tyrosine kinase. The compounds according to claims 1, 2, 4, and 5.

15. structure: 【Chemistry 12】 It has, During the ceremony, R １ teeth, 【Chemistry 13】 And, Y is independently CH or N. R ２ teeth, 【Chemistry 14】 or an amine or an alcohol, in the formula, X ４ CH ２ , NH, CO, O or S, Y ５ is CH or N, Z is OH, SH, NH ２ CO ２ H, NHCH ３ , and 【Chemistry 15】 Selected from, 【Chemistry 16】 This is the attachment point, R ４ H, CH ３ , amine, or methanol, or, structure: 【Chemistry 17】 It has, During the ceremony, R 1 is, [Chemistry 18] And, R2 is, 【Chemistry 19】 or amine or propanol, X is independently CH₂, NH, CO, O, or S. Y is independently either CH or N. Z is OH, SH, NH₂, CO₂H, NHCH₃, and 【Chemistry 20】 Selected from, 【Chemistry 21】 This is the attachment point. The compound according to claim 1.

16. CLogP values ​​from 2 to 5, Polar surface area of ​​250 square Å or less, H bond donors of 5 or less, H-binding receptors of 15 or less, and 20 or fewer rotatable connections Having one or more of the following characteristics The compound according to any one of claims 1, 2, 4, 5, and 15.

17. structure: 【Chemistry 22】 or structure: 【Chemistry 23】 Do you have, Alternatively, a pharmaceutically acceptable salt of any of the aforementioned structures, The compound according to claim 1.

18. structure: 【Chemistry 24】 The compound according to claim 1, which is a pharmaceutically acceptable salt having or any of the above-described structures.

19. The hydroxyl (HO-) group of the aforementioned compound is replaced with phosphoric acid or a phosphonic acid ester, or The hydroxyl (HO-) group of the above compound is replaced by RC(=O)O- (wherein R is alkyl), and optionally the alkyl is a C1 to C6 alkyl. The compound according to claim 18.

20. A pharmaceutical composition comprising the compound described in claim 1, and a pharmaceutically acceptable carrier or excipient.

21. It further comprises one or more of cremohole, polysorbate, and nanoparticles, or, Further comprising a polymer or hydrogel, The pharmaceutical composition according to claim 20.

22. The pharmaceutical composition according to claim 20 or 21 for use in treating red blood cell (RBC)-mediated diseases in a subject.

23. The RBC-mediated disease is selected from the group consisting of sickle cell disease, thalassemia, glucose-6-phosphate dehydrogenase deficiency, glutathione reductase deficiency, and diseases involved in the release of microvesicles from blood cells, or RBC-mediated diseases, either alone or in combination with sickle cell disease, are alpha-thalassemia or beta-thalassemia. The pharmaceutical composition according to claim 22.

24. A pharmaceutical composition according to claim 20 or 21, used to block or inhibit the phosphorylation of human erythrocyte anion transporter band 3 in a subject.

25. IC with a mass of 200 nM or less ５０ The pharmaceutical composition according to claim 20 or 21, wherein the SYK tyrosine kinase is inhibited.

26. The pharmaceutical composition according to claim 20 or 21, wherein the subject is a human child.

27. ​​The pharmaceutical composition according to claim 20 or 21, wherein the pharmaceutical composition is used to be administered sequentially or simultaneously with at least one second active agent selected from the group consisting of hostamatinib, PRT062607, TAK-659, TAE-684, entospretinib, lanraprenib, celduratinib, piceatannol, S701, SykII, SykIV, TAS05567, GSK143, Syk-IN-3, Syk-IN-4, SRX3207, RO9021, gusacitinib, R112, PRT-060318 and OXSI-2.

28. The pharmaceutical composition according to claim 20 or 21, wherein the pharmaceutical composition is used to be administered sequentially or simultaneously with hydroxyurea.

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