COMPOUNDS FOR USES IN PHARMACOLOGICAL INDUCTION OF HBF FOR TREATMENT OF SICKLE CELL DISEASE AND ß-THALASSEMIA
20220152036 · 2022-05-19
Inventors
Cpc classification
A61K31/5377
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
International classification
A61K31/5377
HUMAN NECESSITIES
Abstract
Provided herein are compounds of Formula (I). The compounds described herein are useful in treating a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes), and/or anemia (β-thalassemia and/or sickle cell anemia). The compounds described herein are useful in treating, delaying, and/or preventing the adverse effects of β-thalassemia and/or sickle cell disease, inducing γ globin production, and/or inducing the expression of embryonic/fetal globin genes in a subject, cell, tissue, or biological sample. Also provided in the present disclosure are pharmaceutical compositions, kits, and methods of using the compounds for inducing γ globin production described herein and for treating any of the target diseases described herein.
##STR00001##
Claims
1. A compound of Formula (I): ##STR00018## or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein: each instance of R.sup.1 is independently halogen, —C(═O)(alkyl); alkyl optionally substituted with halogen, unsubstituted alkenyl, unsubstituted alkynyl, or a nitrogen protecting group when attached to a nitrogen atom; R.sup.2 is hydrogen, unsubstituted alkyl, or a nitrogen protecting group; each instance of R.sup.3 is independently halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —CN, —OR.sup.D1, —N(R.sup.D1a).sub.2, or —SR.sup.D1; R.sup.4 is hydrogen, unsubstituted alkyl, or a nitrogen protecting group; each instance of R.sup.5 is independently halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —CN, —OR.sup.D1, —N(R.sup.D1a).sub.2, or —SR.sup.D1; R.sup.D1 is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; each occurrence of R.sup.D1a is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally two instances of R.sup.D1a are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring; Y is —O— or —N(R.sup.6)—; R.sup.6 is hydrogen, optionally substituted alkyl, or a nitrogen protecting group; p is 0, 1, 2, 3, 4, or 5; m is 0, 1, or 2; n is 0, 1, 2, 3, or 4; and x is 0, 1, or 2.
2. The compound of claim 1, wherein at least one instance of R.sup.1 is unsubstituted C.sub.1-6 alkyl.
3. The compound of claim 1, wherein at least one instance of R.sup.1 is unsubstituted ethyl.
4. The compound of claim 1, wherein m is 1.
5. The compound of claim 1, wherein R.sup.2 is hydrogen.
6. The compound of claim 1, wherein n is 0.
7. The compound of claim 1, wherein R.sup.4 is hydrogen.
8. The compound of claim 1, wherein at least one instance of R.sup.5 is unsubstituted C.sub.1-6 alkyl.
9. The compound of claim 1, wherein at least one instance of R.sup.5 is unsubstituted methyl.
10. The compound of claim 1, wherein p is 1.
11. The compound of claim 1, wherein x is 0.
12. The compound of claim 1, wherein x is 1.
13. The compound of claim 1, wherein Y is 0.
14. The compound of claim 1, wherein Y is —N(R.sup.6)—, and R.sup.6 is optionally substituted C.sub.1-6 alkyl.
15. The compound of claim 14, wherein Y is —N(R.sup.6)—, and R.sup.6 is unsubstituted C.sub.1-6 alkyl.
16. The compound of claim 15, wherein Y is —N(unsubstituted ethyl)-.
17. The compound of claim 1, wherein Y is —N(R.sup.6)—, and R.sup.6 is C.sub.1-6 alkyl optionally substituted with —OH or —O(unsubstituted C.sub.1-6 alkyl).
18. The compound of claim 17, wherein Y is —N(CH.sub.2CH.sub.2OH)—.
19. The compound of claim 1, wherein the compound is of formula: ##STR00019## or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
20. The compound of claim 1, wherein the compound is of formula: ##STR00020## or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
21. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, and optionally a pharmaceutically acceptable excipient.
22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition comprises a therapeutically effective amount of the compound for treating a disease in a subject in need thereof.
23. The pharmaceutical composition of claim 22, wherein the disease is anemia.
24. A method of treating anemia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
25. The method of claim 24, wherein the subject suffers from or is suspected of having β-thalassemia.
26. The method of claim 24, wherein the subject suffers from or is suspected of having sickle cell anemia.
27. The method of claim 24, wherein the pharmaceutical composition is administered orally.
28. The method of claim 24, wherein the pharmaceutical composition is administered parentally.
29. The method of claim 24, wherein the pharmaceutical composition is administered in combination with an additional therapeutic agent.
30. A method of inducing γ globin production, comprising: contacting a cell with an effective amount of a compound of claim 1, or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
31. The method of claim 30 further comprising contacting the cell, tissue, or biological sample with a therapeutically effective amount of an additional pharmaceutical agent in combination with the compound, or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
32. Use of a compound of claim 1, or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
33. A kit comprising: a compound of claim 1, or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof; and instructions for administering to a subject or contacting a biological sample with the compound, or the pharmaceutically acceptable salt, solvate, hydrate, tautomer, or stereoisomer thereof.
34. A method of treating anemia in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of claim 21.
35. A method of inducing γ globin production, comprising: contacting a cell with an effective amount of a compound of a pharmaceutical composition of 21.
36. The method of claim 35 further comprising contacting the cell, tissue, or biological sample with a therapeutically effective amount of an additional pharmaceutical agent in combination with the compound, or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
37. Use of a pharmaceutical composition of claim 21.
38. A kit comprising: a pharmaceutical composition of any one of claim 21; and instructions for administering the pharmaceutical composition to a subject or contacting a biological sample with the pharmaceutical composition.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0093]
[0094]
[0095]
[0096]
[0097]
[0098]
[0099]
[0100]
[0101]
[0102]
[0103]
DETAILED DESCRIPTION
[0104] The present disclosure provides compounds of Formula (I) for treating a target disease such as a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes). The compounds described herein are useful in treating, delaying, and/or preventing the adverse effects of β-thalassemia and/or sickle cell disease, inducing γ globin production, and/or inducing the expression of embryonic/fetal globin genes in a subject, cell, tissue, or biological sample. In another aspect, the present disclosure provides compounds for treating anemia in a subject in need thereof (e.g., wherein the subject suffers from or is suspected of having β-thalassemia and/or sickle cell anemia). Also provided in the present disclosure are pharmaceutical compositions, kits, and methods of using the compounds for inducing γ globin production described herein and for treating any of the target diseases described herein.
Minichromosome Maintenance Eukaryote Replicative Helicase (MCM) Inhibiting Compounds
[0105] One aspect of the present disclosure relates to the inducers of γ globin production as described herein, as well as their pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, or prodrugs. These compounds are useful in treating diseases associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes), treating, delaying, and/or preventing the adverse effects of β-thalassemia and/or sickle cell disease, inducing γ globin production, and/or inducing the expression of embryonic/fetal globin genes in a subject, cell, tissue, or biological sample, and treating anemia in a subject in need thereof (e.g., wherein the subject suffers from or is suspected of having β-thalassemia and/or sickle cell anemia).
[0106] In certain embodiments, a compound described herein is of Formula (I):
##STR00011##
or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein: R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, Y, m, n, p, and x are as described herein. In certain embodiments, a compound described herein is of Formula (I):
##STR00012##
or a pharmaceutically acceptable salt thereof.
[0107] In some embodiments, Formula (I) includes zero or more instances of substituent R.sup.1. In some embodiments, Formula (I) includes zero instances of substituent R.sup.1. In some embodiments, Formula (I) includes one or more instances of substituent R.sup.1. In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, at least one instance of R.sup.1 is halogen (e.g., F, Cl, Br, or I). In certain embodiments, at least one instance of R.sup.1 is —C(═O)(alkyl). In certain embodiments, at least one instance of R.sup.1 is —C(═O)(optionally substituted alkyl). In certain embodiments, at least one instance of R.sup.1 is —C(═O)(optionally substituted C.sub.1-C.sub.6 alkyl). In certain embodiments, at least one instance of R.sup.1 is alkyl optionally substituted with halogen (e.g., F, Cl, Br, or I). In certain embodiments, at least one instance of R.sup.1 is optionally substituted alkyl. In certain embodiments, at least one instance of R.sup.1 is C.sub.1-C.sub.6 alkyl optionally substituted with halogen (e.g., F, Cl, Br, or I). In certain embodiments, at least one instance of R.sup.1 is unsubstituted C.sub.1-C.sub.6 alkyl (e.g., unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl, unsubstituted butyl). In certain embodiments, at least one instance of R.sup.1 is unsubstituted methyl. In certain embodiments, at least one instance of R.sup.1 is unsubstituted ethyl. In certain embodiments, m is 1; and at least one instance of R.sup.1 is unsubstituted ethyl. In certain embodiments, at least one instance of R.sup.1 is optionally substituted alkenyl (e.g., C.sub.2-C.sub.6 alkenyl). In certain embodiments, at least one instance of R.sup.1 is optionally substituted C.sub.2-C.sub.6 alkenyl. In certain embodiments, at least one instance of R.sup.1 is unsubstituted alkenyl. In certain embodiments, at least one instance of R.sup.1 is unsubstituted C.sub.2-C.sub.6 alkenyl. In certain embodiments, at least one instance of R.sup.1 is optionally substituted alkynyl (e.g., C.sub.2-C.sub.10 alkynyl). In certain embodiments, at least one instance of R.sup.1 is optionally substituted C.sub.2-C.sub.10 alkynyl (e.g., optionally substituted, propynyl or butynyl). In certain embodiments, at least one instance of R.sup.1 is unsubstituted alkynyl (e.g., unsubstituted C.sub.2-C.sub.10 alkynyl). In certain embodiments, at least one instance of R.sup.1 is unsubstituted C.sub.2-C.sub.10 alkynyl (e.g., unsubstituted, propynyl or butynyl). In certain embodiments, at least one instance of R.sup.1 is a nitrogen protecting group when attached to a nitrogen atom (e.g., benzyl (Bn), t-butyl carbonate (BOC or Boc), benzyl carbamate (Cbz), 9-fluorenylmethyl carbonate (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl, or p-toluenesulfonamide (Ts)).
[0108] Formula (I) includes substituent R.sup.2. In some embodiments, R.sup.2 is hydrogen. In some embodiments, R.sup.2 is optionally substituted alkyl. In some embodiments, R.sup.2 is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.2 is C.sub.1-C.sub.6 alkyl optionally substituted with halogen (e.g., F, Cl, Br, or I). In some embodiments, R.sup.2 is unsubstituted alkyl. In some embodiments, R.sup.2 is unsubstituted C.sub.1-C.sub.6 alkyl (e.g., unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl, unsubstituted butyl). In certain embodiments, at least one instance of R.sup.1 is unsubstituted methyl. In certain embodiments, at least one instance of R.sup.1 is unsubstituted ethyl. In some embodiments, R.sup.2 is a nitrogen protecting group (e.g., benzyl (Bn), t-butyl carbonate (BOC or Boc), benzyl carbamate (Cbz), 9-fluorenylmethyl carbonate (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl, or p-toluenesulfonamide (Ts)).
[0109] In some embodiments, Formula (I) includes zero or more instances of substituent R.sup.3. In some embodiments, Formula (I) includes zero instances of substituent R.sup.3. In some embodiments, Formula (I) includes one or more instances of substituent R.sup.3. In certain embodiments, n is 0. In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4. In certain embodiments, at least one instance of R.sup.3 is halogen (e.g., F, Cl, Br, or I). In certain embodiments, at least one instance of R.sup.3 is optionally substituted acyl (e.g., —C(═O)(alkyl)). In certain embodiments, at least one instance of R.sup.3 is optionally substituted alkyl (e.g., optionally substituted C.sub.1-6 alkyl). In certain embodiments, at least one instance of R.sup.3 is optionally substituted C.sub.1-6 alkyl (e.g., optionally substituted, methyl, ethyl, or propyl). In certain embodiments, at least one instance of R.sup.3 is optionally substituted alkenyl (e.g., optionally substituted C.sub.2-C.sub.6 alkenyl). In certain embodiments, at least one instance of R.sup.3 is optionally substituted alkynyl (e.g., optionally substituted C.sub.2-C.sub.10 alkynyl (e.g., optionally substituted, propynyl or butynyl)). In certain embodiments, at least one instance of R.sup.3 can be optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In some embodiments, at least one instance of R.sup.3 can be optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In some embodiments, at least one instance of R.sup.3 can be optionally substituted aryl (e.g., phenyl, or benzyl). In some embodiments, at least one instance of R.sup.3 can be optionally substituted heteroaryl (e.g., 5- to 7-membered monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or optionally substituted 8- to 10-membered bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, at least one instance of R.sup.3 can be —CN. In certain embodiments, at least one instance of R.sup.3 is —OR.sup.D1, —N(R.sup.D1a).sub.2 or —SR.sup.D1, and R.sup.D1 is as defined herein. In some embodiments, at least one instance of R.sup.3 can be —OR.sup.D, —N(R.sup.D1a).sub.2 or —SR.sup.D1; wherein R.sup.D1 is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; each occurrence of R.sup.D1a is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally two instances of R.sup.D1a are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring. In some embodiments, at least one instance of R.sup.3 can be —OR.sup.D1 (e.g., —OH or —OMe). In some embodiments, at least one instance of R.sup.3 can be —N(R.sup.D1a).sub.2 (e.g., —NH.sub.2 or —NMe.sub.2). In some embodiments, at least one instance of R.sup.3 can be —SR.sup.D1 (e.g., —SH or —SMe).
[0110] In certain embodiments, R.sup.D1 is hydrogen. In certain embodiments, R.sup.D1 is optionally substituted acyl (e.g., —C(═O)Me). In certain embodiments, R.sup.D1 is optionally substituted alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl). In certain embodiments, R.sup.D1 is substituted or unsubstituted methyl. In certain embodiments, R.sup.D1 is substituted or unsubstituted ethyl. In certain embodiments, R.sup.D1 is substituted or unsubstituted propyl. In certain embodiments, R.sup.D1 is optionally substituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain embodiments, R.sup.D1 is optionally substituted alkynyl (e.g., substituted or unsubstituted C.sub.2-6 alkynyl). In certain embodiments, R.sup.D1 is optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In certain embodiments, R.sup.D1 is optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, R.sup.D1 is optionally substituted aryl (e.g., substituted or unsubstituted, 6- to 10-membered aryl). In certain embodiments, R.sup.D1 is benzyl. In certain embodiments, R.sup.D1 is optionally substituted phenyl. In certain embodiments, R.sup.D1 is optionally substituted heteroaryl (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, R.sup.D1 is an oxygen protecting group when attached to an oxygen atom. In certain embodiments, R.sup.D1 is a sulfur protecting group when attached to a sulfur atom.
[0111] In certain embodiments, at least one instance of R.sup.D1a is hydrogen. In certain embodiments, at least one instance of R.sup.D1a is optionally substituted acyl (e.g., —C(═O)Me). In certain embodiments, at least one R.sup.D1a is optionally substituted alkyl (e.g., substituted or unsubstituted C.sub.1-6 alkyl). In certain embodiments, at least one instance of R.sup.D1a is substituted or unsubstituted methyl. In certain embodiments, at least one instance of R.sup.D1a is substituted or unsubstituted ethyl. In certain embodiments, at least one instance of R.sup.D1a is substituted or unsubstituted propyl. In certain embodiments, at least one instance of R.sup.D1a is optionally substituted alkenyl (e.g., substituted or unsubstituted C.sub.2-6 alkenyl). In certain embodiments, at least one instance of R.sup.D1a is optionally substituted alkynyl (e.g., substituted or unsubstituted C.sub.2-6 alkynyl). In certain embodiments, at least one instance of R.sup.D1a is optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In certain embodiments, at least one instance of R.sup.D1a is optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.D1a is optionally substituted aryl (e.g., substituted or unsubstituted, 6- to 10-membered aryl). In certain embodiments, at least one instance of R.sup.D1a is benzyl. In certain embodiments, at least one instance of R.sup.D1a is optionally substituted phenyl. In certain embodiments, at least one instance of R.sup.D1a is optionally substituted heteroaryl (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In certain embodiments, at least one instance of R.sup.D1a is a nitrogen protecting group (e.g., benzyl (Bn), t-butyl carbonate (BOC or Boc), benzyl carbamate (Cbz), 9-fluorenylmethyl carbonate (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl, or p-toluenesulfonamide (Ts)). In certain embodiments, two instances of R.sup.D1a are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic ring (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur) or substituted or unsubstituted heteroaryl ring (e.g., substituted or unsubstituted, 5- to 6-membered, monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or substituted or unsubstituted, 9- to 10-membered, bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur).
[0112] Formula (I) includes substituent R.sup.4. In some embodiments, R.sup.4 is hydrogen. In some embodiments, R.sup.4 is optionally substituted alkyl. In some embodiments, R.sup.4 is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.4 is C.sub.1-C.sub.6 alkyl optionally substituted with halogen (e.g., F, Cl, Br, or I). In some embodiments, R.sup.4 is unsubstituted alkyl. In some embodiments, R.sup.4 is unsubstituted C.sub.1-C.sub.6 alkyl (e.g., unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl, unsubstituted butyl). In certain embodiments, at least one instance of R.sup.4 is unsubstituted methyl. In certain embodiments, at least one instance of R.sup.4 is unsubstituted ethyl. In some embodiments, R.sup.4 is a nitrogen protecting group (e.g., benzyl (Bn), t-butyl carbonate (BOC or Boc), benzyl carbamate (Cbz), 9-fluorenylmethyl carbonate (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl, or p-toluenesulfonamide (Ts)). In some embodiments, R.sup.2 is hydrogen and R.sup.4 is hydrogen.
[0113] In some embodiments, Formula (I) includes zero or more instances of substituent R.sup.5. In some embodiments, Formula (I) includes zero instances of substituent R.sup.5. In some embodiments, Formula (I) includes one or more instances of substituent R.sup.5. In certain embodiments, p is 0. In certain embodiments p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, p is 4. In certain embodiments, p is 5. In certain embodiments, at least one instance of R.sup.5 is halogen (e.g., F, Cl, Br, or I). In certain embodiments, at least one instance of R.sup.5 is optionally substituted acyl (e.g., —C(═O)(alkyl)). In certain embodiments, at least one instance of R.sup.5 is optionally substituted alkyl (e.g., optionally substituted C.sub.1-6 alkyl). In certain embodiments, at least one instance of R.sup.5 is optionally substituted C.sub.1-6 alkyl (e.g., optionally substituted, methyl, ethyl, or propyl). In certain embodiments, at least one instance of R.sup.5 is unsubstituted alkyl. In certain embodiments, at least one instance of R.sup.5 is unsubstituted C.sub.1-6 alkyl (e.g., unsubstituted, methyl, ethyl, or propyl). In certain embodiments, at least one instance of R.sup.5 is unsubstituted methyl. In certain embodiments, at least one instance of R.sup.5 is unsubstituted ethyl. In certain embodiments, at least one instance of R.sup.5 is unsubstituted propyl. In certain embodiments, at least one instance of R.sup.5 is unsubstituted n-propyl. In certain embodiments, at least one instance of R.sup.5 is unsubstituted isopropyl. In certain embodiments, at least one instance of R.sup.5 is optionally substituted alkenyl (e.g., optionally substituted C.sub.2-C.sub.6 alkenyl). In certain embodiments, at least one instance of R.sup.5 is optionally substituted alkynyl (e.g., optionally substituted C.sub.2-C.sub.10 alkynyl (e.g., optionally substituted, propynyl or butynyl)). In certain embodiments, at least one instance of R.sup.5 can be optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In some embodiments, at least one instance of R.sup.5 can be optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In some embodiments, at least one instance of R.sup.5 can be optionally substituted aryl (e.g., phenyl, or benzyl). In some embodiments, at least one instance of R.sup.5 can be optionally substituted heteroaryl (e.g., 5- to 7-membered monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur; or optionally substituted 8- to 10-membered bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, at least one instance of R.sup.5 can be —CN. In certain embodiments, at least one instance of R.sup.5 is —OR.sup.D1, —N(R.sup.D1a).sub.2 or —SR.sup.D1; and R.sup.D1 is as defined herein. In some embodiments, at least one instance of R.sup.5 can be —OR.sup.D1, —N(R.sup.D1a).sub.2 or —SR.sup.D1; wherein R.sup.D1 is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; each occurrence of R.sup.D1a is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally two instances of R.sup.D1a are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring. In some embodiments, at least one instance of R.sup.5 can be —OR.sup.D1 (e.g., —OH or —OMe). In some embodiments, at least one instance of R.sup.5 can be —N(R.sup.D1a).sub.2(e.g., —NH.sub.2 or —NMe.sub.2). In some embodiments, at least one instance of R.sup.5 can be —SR.sup.D1 (e.g., —SH or —SMe).
[0114] In Formula (I), Y is part of a six-membered heterocyclic ring. In some embodiments, Y is —O— or —N(R.sup.6)—; wherein R.sup.6 is hydrogen, optionally substituted alkyl, or a nitrogen protecting group. In some embodiments, Y is —O— or —N(R.sup.6)—; wherein R.sup.6 is optionally substituted alkyl. In some embodiments, Y is —O—. In some embodiments, Y is —N(R.sup.6)—, and R.sup.6 is optionally substituted alkyl. In some embodiments, Y is —N(R.sup.6)—, and R.sup.6 is optionally substituted C.sub.1-6 alkyl. In certain embodiments, Y is —N(R.sup.6)—, and R.sup.6 is unsubstituted C.sub.1-6 alkyl (e.g., unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl, unsubstituted butyl). In certain embodiments, Y is —N(unsubstituted ethyl)-. In certain embodiments, Y is —N(R.sup.6)—, wherein R.sup.6 is of formula —(CH.sub.2).sub.n1(R.sup.6a), R.sup.6a is —OH or —O(optionally substituted alkyl), and n1 is 1, 2, 3, 4, 5, 6, or 7. In certain embodiments, n1 is 1. In certain embodiments, n1 is 2. In certain embodiments, n1 is 3. In certain embodiments, n1 is 4. In certain embodiments, n1 is 5. In certain embodiments, n1 is 6. In certain embodiments, n1 is 7. In certain embodiments, Y is —N(R.sup.6)—, wherein R.sup.6 is of formula —(CH.sub.2).sub.n1(R.sup.6a), R.sup.6a is —OH or —O(unsubstituted alkyl), and n1 is 1, 2, 3, 4, or 5. In certain embodiments, Y is —N(R.sup.6)—, and R.sup.6 is C.sub.1-6 alkyl optionally substituted with —OH or —O(unsubstituted C.sub.1-6 alkyl). In some embodiments, Y is —N(CH.sub.2CH.sub.2OH)—.
[0115] In Formula (I), in some embodiments, x is 0, 1, or 2. In some embodiments, x is 0. In certain embodiments, x is 1. In certain embodiments, x is 2.
[0116] In some embodiments, a compound described herein is of Formula (I):
##STR00013##
or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof, wherein:
[0117] each instance of R.sup.1 is independently halogen, —C(═O)(alkyl); alkyl optionally substituted with halogen, unsubstituted alkenyl, or unsubstituted alkynyl;
[0118] R.sup.2 is hydrogen or unsubstituted alkyl;
[0119] each instance of R.sup.3 is independently halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, —CN, —OR.sup.D1, —N(R.sup.D1a).sub.2 or —SR.sup.D1;
[0120] R.sup.4 is hydrogen or unsubstituted alkyl; each instance of R.sup.5 is independently halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, —CN, —OR.sup.D1, —N(R.sup.D1a).sub.2 or —SR.sup.D1;
[0121] R.sup.D1 is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted carbocyclyl; each occurrence of R.sup.D1a is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; or optionally two instances of R.sup.D1a are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring;
[0122] Y is —O— or —N(R.sup.6)—;
[0123] R.sup.6 is hydrogen, optionally substituted alkyl, or a nitrogen protecting group;
[0124] p is 0, 1, 2, or 3;
[0125] m is 0, 1, or 2;
[0126] n is 0 or 1; and
[0127] x is 0, 1, or 2.
[0128] In some embodiments, the compound of Formula (I) can be of one of the following formulae:
##STR00014##
or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof.
[0129] In some embodiments, the compound of Formula (I) can be of one of the following formulae:
##STR00015##
or a pharmaceutically acceptable salt thereof.
[0130] Exemplary compounds of Formula (I) are provided herein. In some embodiments, the compound of Formula (I) can be of one of the following formulae:
##STR00016##
or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative, or prodrug thereof. In some embodiments, the compound of Formula (I) can be of one of the following formulae:
##STR00017##
or a pharmaceutically acceptable salt thereof.
[0131] The compounds described herein can be prepared from readily available starting materials using methods known in the art. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, and pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures. The chemicals used in the above-described synthetic routes may include, for example, solvents, reagents, catalysts, and protecting group and deprotecting group reagents. The methods described above may also additionally include steps, either before or after the steps described specifically herein, to add or remove suitable protecting groups in order to ultimately allow synthesis of the compounds. In addition, various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing applicable compounds are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3.sup.rd Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.
[0132] The compounds of Formula (I) provided herein can be prepared from readily available starting materials using the following general methods and procedures. An exemplary schematic illustration for synthesizing the compounds described herein is provided in the Examples section below. Where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures.
Pharmaceutical Compositions and Kits
[0133] The present disclosure provides pharmaceutical compositions comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient. In certain embodiments, a pharmaceutical composition described herein comprises a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. The pharmaceutical compositions described herein are useful in treating a target disease such as a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes). The pharmaceutical compositions described herein are useful in treating, delaying, and/or preventing the adverse effects of β-thalassemia and/or sickle cell disease, inducing γ globin production, and/or inducing the expression of embryonic/fetal globin genes in a subject, cell, tissue, or biological sample. The pharmaceutical compositions described herein are useful in treating anemia in a subject in need thereof (e.g., wherein the subject suffers from or is suspected of having β-thalassemia and/or sickle cell anemia).
[0134] In certain embodiments, a subject being treated herein is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject is a human. In certain embodiments, the subject is a mammal (e.g., non-human mammal). In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, a subject being treated herein is a companion animal such as a dog or cat. In certain embodiments, a subject being treated herein is a livestock animal such as a cow, pig, horse, sheep, or goat. In certain embodiments, a subject being treated herein is a zoo animal. In another embodiment, a subject being treated herein is a research animal such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic or genetically engineered animal (e.g., transgenic mice and transgenic pigs). In certain embodiments, the subject suffers from or is suspected of having β-thalassemia and/or sickle cell anemia.
[0135] In certain embodiments, the cell contacted with an effective amount of a compound or pharmaceutical composition described herein is in vitro. In certain embodiments, the contacted cell is ex vivo. In certain embodiments, the cell described herein is in vivo.
[0136] In certain embodiments, the compound described herein is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount (e.g., amount effective for treating a target disease such as a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes), or treating anemia (e.g., wherein the subject suffers from or is suspected of having β-thalassemia and/or sickle cell anemia) in a subject in need thereof). In certain embodiments, the disease is associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes. In certain embodiments, the disease is anemia. In certain embodiments, the disease is β-thalassemia and/or sickle cell disease. In certain embodiments, the disease is β-thalassemia and/or sickle cell anemia. In certain embodiments, the disease is treated through at least the induction of the globin gene expression in erythrocytes. In certain embodiments, the effective amount is a prophylactically effective amount (e.g., amount effective for preventing, delaying a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., anemia (e.g., β-thalassemia and/or sickle cell anemia), or preventing the adverse effects of β-thalassemia and/or sickle cell disease). In certain embodiments, the effective amount is a therapeutically effective amount (e.g., amount effective for treating a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., anemia (e.g., β-thalassemia and/or sickle cell anemia)).
[0137] Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the compound described herein (i.e., the “active ingredient”) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.
[0138] Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.
[0139] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.
[0140] Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.
[0141] Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof. In certain embodiments, the composition of the instant disclosure is encapsulated in a carrier vehicle, which may be rigid vesicles, elastic vesicles, monolayer vesicles, multi-layer vesicles, liposomes, niosomes, proniosomes, Transfersomes®, ethosomes, L-595-PEG-8-L vesicles, nanoemulsions, nanosomes, nanoparticles, or a combination thereof.
[0142] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
[0143] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
[0144] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.
[0145] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
[0146] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.
[0147] Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.
[0148] The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.
[0149] Dosage forms for topical and/or transdermal administration of a compound described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.
[0150] Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Ballistic powder/particle delivery devices which use compressed gas to accelerate the compound in powder form through the outer layers of the skin to the dermis are suitable.
[0151] Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
[0152] A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
[0153] Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
[0154] Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.
[0155] Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
[0156] Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
[0157] A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are also contemplated as being within the scope of this disclosure.
[0158] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.
[0159] The compounds provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
[0160] Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.
[0161] In certain embodiments, a kit described herein includes a first container comprising a compound or pharmaceutical composition described herein. In certain embodiments, a kit described herein is useful in treating a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes), and/or anemia (e.g., β-thalassemia and/or sickle cell anemia). In certain embodiments, the disease is anemia (e.g., β-thalassemia and/or sickle cell anemia) in a subject in need thereof. In some embodiments, the compounds described herein are useful in treating, delaying, and/or preventing the adverse effects of β-thalassemia and/or sickle cell disease, inducing γ globin production, and/or inducing the expression of embryonic/fetal globin genes in a subject, cell, tissue, or biological sample.
[0162] In certain embodiments, a kit described herein further includes instructions for using the compound or pharmaceutical composition included in the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for treating a treating a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes), and/or anemia (e.g., β-thalassemia and/or sickle cell anemia) in a subject in need thereof, and/or treating, delaying, and/or preventing the adverse effects of β-thalassemia and/or sickle cell disease, inducing γ globin production, and/or inducing the expression of embryonic/fetal globin genes in a subject, cell, tissue, or biological sample. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.
Methods of Treatment
[0163] As shown in the Examples below, exemplary compounds described herein successfully induce γ-globin gene expression (e.g., induce γ-globin gene expression in hydroxyurea (HU)-resistant primary erythroid cells), reduce expression levels of the exemplary γ-globin gene repressor BCL11A, may target γ globin production, and contribute to inducing γ globin production in cells such as erythrocytes (e.g., primary erythroid cells).
[0164] Accordingly, the present disclosure provides methods of treating a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes) in a subject in need thereof, the methods comprising administering to the subject an effective amount (e.g., therapeutically effective amount) of a compound, or pharmaceutical composition thereof, described herein. The present disclosure provides methods of treating anemia in a subject in need thereof (e.g., wherein the subject suffers from or is suspected of having β-thalassemia and/or sickle cell anemia).
[0165] Another aspect of the present disclosure relates to methods of preventing proliferative disease and/or infectious disease in a subject in need thereof, the methods comprising administering to the subject an effective amount (e.g., prophylactically effective amount) of a compound, or pharmaceutical composition thereof, described herein.
[0166] The compounds and pharmaceutical compositions described herein are useful in treating, delaying, and/or preventing the adverse effects of β-thalassemia and/or sickle cell disease, inducing γ globin production, and/or inducing the expression of embryonic/fetal globin genes in a subject, cell, tissue, or biological sample. In certain embodiments, the disease is anemia. In certain embodiments, the disease is β-thalassemia and/or sickle cell disease. In certain embodiments, the disease is β-thalassemia and/or sickle cell anemia. In certain embodiments, the disease is treated through at least the induction of the globin gene expression in erythrocytes.
[0167] In certain embodiments, the method described herein further includes administering to the subject an additional pharmaceutical agent. In certain embodiments, the method described herein further includes contacting the biological sample with an additional pharmaceutical agent. In certain embodiments, the method described herein further includes contacting the tissue with an additional pharmaceutical agent. In certain embodiments, the method described herein further includes contacting the cell with an additional pharmaceutical agent. In certain embodiments, the method described herein further includes a therapeutic for treating anemia (e.g., β-thalassemia and/or sickle cell disease).
[0168] The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intracranial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), by any means that facilitate in vivo or ex vivo transport of the compound or composition as described herein in, into, or through tissue/skin of a subject (such as iontophoresis), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. In some embodiments, the pharmaceutical composition is administered orally or parentally. In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered parentally. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), transfusion, perfusion, regional administration via blood and/or lymph supply, and/or direct administration to an affected site, such as intra-tumoral. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.
[0169] The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, any two doses of the multiple doses include different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, biological sample, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, biological sample, tissue, or cell. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a compound described herein.
[0170] Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower, higher, or the same as that administered to an adult.
[0171] A compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents) useful in treating disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating 3-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes) in a subject in need thereof, and/or in treating, delaying, and/or preventing the adverse effects of a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes) and/or anemia (e.g., β-thalassemia and/or sickle cell disease) in a subject in need thereof). The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes) in a subject in need thereof, and/or in treating, delaying, and/or preventing the adverse effects of a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., treating β-thalassemia and/or sickle cell disease through at least the induction of the globin gene expression in erythrocytes) and/or anemia (e.g., β-thalassemia and/or sickle cell disease) in a subject in need thereof), improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject, biological sample, tissue, or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, for different disorders, and/or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including a compound described herein is administered to a patient in need thereof, to advantageously treat one or more diseases. In certain embodiments, said one or more diseases is a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., β-thalassemia and/or sickle cell disease), anemia (e.g., β-thalassemia and/or sickle cell anemia), or a combination thereof. In a preferred embodiment, said one or more diseases is β-thalassemia, anemia, or a combination thereof.
[0172] The compound or composition may be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies in treating, delaying, and/or preventing the adverse effects of a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes (e.g., β-thalassemia and/or sickle cell disease). In certain embodiments, the compound or composition described herein can be administered to a patient in need thereof, wherein the anemia (e.g., β-thalassemia and/or sickle cell anemia) of the patient is resistant to at least one pharmaceutical agent. In certain embodiments, the disease is anemia (e.g., β-thalassemia and/or sickle cell anemia) and said anemia (e.g., β-thalassemia and/or sickle cell anemia) is resistant to one or more anemia treatment agents including but not limited to hydroxyurea (HU). Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful in treating a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes, and/or anemia (e.g., β-thalassemia and/or sickle cell anemia). In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful in preventing a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful in treating anemia (e.g., β-thalassemia and/or sickle cell anemia). In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent approved by a regulatory agency (e.g., the US FDA) for treating and/or preventing a disease associated with the expression of endogenous embryonic/fetal globin (e.g., γ globin) in erythrocytes and/or anemia (e.g., β-thalassemia and/or sickle cell anemia). In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent approved by a regulatory agency (e.g., the US FDA) for treating and/or preventing anemia (e.g., β-thalassemia and/or sickle cell anemia). Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
[0173] In certain embodiments, the additional pharmaceutical agent is an anti-angiogenesis agent, anti-inflammatory agent, immunosuppressant, anti-bacterial agent, anti-viral agent, cardiovascular agent, cholesterol-lowering agent, anti-diabetic agent, anti-allergic agent, pain-relieving agent, or a combination thereof. In certain embodiments, the compounds described herein or pharmaceutical compositions can be administered in combination with anemia therapy.
Methods of Synthesis
[0174] An exemplary synthetic scheme for making the compounds described herein is as described previously. (See, Nam T-G et al., Identification and Characterization of Small-Molecule Inducers of Fetal Hemoglobin. ChemMedChem. 2011; 6(5):777-780; and Imbach P, et al., PCT document: WO/2000/049018A1, 2000; Published patents: EP1153024B1, 2004; U.S. Pat. No. 6,767,906B2, 2004). In one aspect, the present invention provides methods for preparing compounds of Formula (I).
EXAMPLES
[0175] In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.
Example 1: Preparation of Exemplary Compounds
[0176] In a previous study, six asymmetric heterocyclic compounds that efficiently induce the expression of γ-globin in primary human erythroid cells were identified. These compounds shared an identical pharmacophore, which was previously described as a yellow dye.sup.19,20. Given its higher efficacy and specificity in the induction of γ-globin chains, compound II has the potential to be developed into a new generation of therapeutics for SCD and β-thalassemia. However, the solubility of asymmetric heterocyclic compound II is poor and it can only be dissolved in DMSO. Moreover, pharmacokinetic data revealed that the oral bioavailability of compound II is only 2.34% (
Materials and Methods
Compound Synthesis
[0177] The 2-amino-6-anilino-purine compounds described in
[0178] 3-p-Tolyl-propynoic acid {3-[9-ethyl-2-(4-ethylpiperazin-1-yl)-9H-purin-6-ylamino]phenyl}amide HCl salt (AS-28). .sup.1H NMR (300 MHz, DMSO-d6): δ 10.92 (s, 1H), 10.77 (s, 1H), 10.39 (s, 1H), 8.83 (s, 1H), 8.56 (s, 1H), 7.56 (d, J=8.1 Hz, 2H), 7.44 (d, J=8.1 Hz, 1H), 7.35-7.30 (m, 3H), 7.16 (d, J=8.4 Hz, 1H), 4.82-4.77 (m, 2H), 4.21 (q, J=7.2 Hz, 2H), 3.56-3.38 (m, 4H), 3.19-2.98 (m, 4H), 2.37 (s, 3H), 1.46 (t, J=7.4 Hz, 3H), 1.27 (t, J=7.2 Hz, 3H); MS (ES.sup.+) m/z calculated for C.sub.29H.sub.32N.sub.80: 508.27; found: 509.4 (M+H.sup.+).
[0179] 3-p-Tolyl-propynoic acid (3-{9-ethyl-2-[4-(2-hydroxyethyl)-piperazin-1-yl]-9H-purin-6-ylamino}phenyl)amide HCl salt (AS-061; AS-61). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 11.02 (s, 1H), 10.77 (bs, 2H), 9.28 (s, 1H), 8.59 (s, 1H), 7.56 (d, J=8.0 Hz, 2H), 7.43 (d, J=8.8 Hz, 1H), 7.36-7.29 (m, 3H), 7.20 (d, J=8.0 Hz, 1H), 4.79-4.75 (m, 2H), 4.24 (q, J=7.2 Hz, 2H), 3.82 (t, J=5.0 Hz, 2H), 3.63-3.48 (m, 4H), 3.24-3.15 (m, 4H), 2.36 (s, 3H), 1.47 (t, J=7.2 Hz, 3H); MS (ES.sup.+) m/z calculated for C.sub.29H.sub.32N.sub.8O.sub.2: 524.26; found: 525.2 (M+H.sup.+).
[0180] 3-p-Tolyl-propynoic acid {3-[9-ethyl-2-(2-morpholin-4-yl-ethoxy)-9H-purin-6-ylamino]phenyl}amide (AS-29). .sup.1H NMR (300 MHz, DMSO-d.sub.6): 10.87 (s, 1H), 9.97 (s, 1H), 8.35 (s, 1H), 8.19 (s, 1H), 7.59 (d, J=7.5 Hz, 2H), 7.63 (d, J=7.8 Hz, 2H), 7.32 (d, J=6.6 Hz, 1H), 4.47 (t, J=6.0, 5.9 Hz, 2H), 4.18 (q, J=14.4, 7.2 Hz, 2H), 3.60 (t, J=4.4, 4.2 Hz, 4H), 3.39 (s, 4H), 2.72 (t, J=5.9, 5.7 Hz, 2H), 2.54 (d, J=6.6 Hz, 2H), 2.42 (s, 3H), 1.46 (t, J=7.4, 7.2 Hz, 3H); MS (ES.sup.+) m/z calcd. for C.sub.29H.sub.31N.sub.7O.sub.3: 525.25; found: 526.3 (M+H.sup.+).
Example 2: Biological Assays of Exemplary Compounds
[0181] Primary Erythroid Cell Culture
[0182] Peripheral blood samples were purchased from the Taipei Blood Center. The concentrated blood was diluted 1:5 (V/V) in phosphate-buffered saline (PBS) and distributed in a thin layer on Ficoll-paque PLUS (d=1.007 g/ml) (GE Healthcare) in a SepMate column (STEMCELL). After centrifugation at 1200 g for 10 minutes, the cells in the inter-phase region were collected. The collected cells were washed with PBS and centrifuged at low speed three times. The remaining mononuclear cells were expanded in Phase I medium containing 1×SFEM (STEMCELL), 100 ng/ml SCF, 20 ng/ml IL-3, 20 ng/ml IL-6, and 100 ng/ml Flt3-L at 37° C. in an incubator with 5% CO.sub.2 for 7 days. The expanded mononuclear cells were further differentiated in Phase II medium containing 1×SFEM (StemSpan), 20 ng/ml SCF, 5 ng/ml IL-3, 1 U/ml EPO for another 7 days. The differentiated erythroid cells were treated with indicated compounds of different dosages with a seeding density of 5×10.sup.5 cells/ml for another 3 days.
[0183] Quantitative RT-PCR
[0184] After 3 days of compound treatment, total RNA was extracted by a Quick-RNA miniprep kit (Zymo) and reverse-transcription was performed using Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific) according to the manufacturer's instructions. Quantitative PCR was performed on a LightCycler system with SYBR green master mix following the manufacturer's instructions (Roche). The relative quantitative RT-PCR data was normalized to the Cq number of 3-actin and compared to a mock control.
Cell Viability Assay
[0185] Cell viability was assessed using AlamarBlue reagent (Invitrogen). After 3 days of treatment, 100 μl of treated cell culture was transferred into a 96-well plate and 1/10 volume of AlamarBlue reagent was added and the cell culture was incubated overnight at 37° C. Cell viability was evaluated using a multi-label counter (Ex 530-560 nm, Em 590 nm) (PerkinElmer).
Western Blot Analysis
[0186] After 3 days of treatment, total protein was extracted by modified RIPA (50 mM Tris-HCl pH7.8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EGTA, 5 mM EDTA, 10 mM NaF, 1 mM NaV.sub.3O.sub.4 and 1× complete EDTA-free Protease Inhibitor Cocktail). 30 μg of protein extracts were separated by 10% SDS-PAGE and the resulting gel was blotted onto PVDF membrane. After blocking with 5% non-fat milk in TBST, the membrane was incubated overnight with primary antibody against BCL11A or β-actin at 4° C. After incubating with horseradish peroxidase-conjugated secondary antibodies and washing the blot, the signals of indicated proteins were visualized by ECL (PerkinElmer) following the manufacturer's protocol.
Mice
[0187] Sickle cell disease mice (B6; 129-Hba.sup.tm1(HBA)Tow, Hbb.sup.tm2(HBG1,HBB*)Tow/J) were purchased from Jackson Laboratory and were bred at the AS Core, Academia Sinica. Mice of 6-8 weeks old were used for experiments. Treatment groups received vehicle [20% (2-Hydroxypropyl)-β-cyclodextrin] plus AS-28 (50 mg/kg, 75 mg/kg or 100 mg/kg), which was administered by oral gavage twice per day, 5 days per week, for 4 weeks.
Complete Blood Count Analysis
[0188] Blood was collected by submandibular blood collection and analysis was performed using a ProCyte Dx automatic analyzer.
F-Cell Quantitation
[0189] 10 μl of whole blood was washed twice with PBS and then fixed with 0.05% glutaraldehyde for 10 minutes. After fixation, the cells were washed twice with PBS and then permeabilized by adding cold 0.1% Triton X-100 for 5 minutes. Cells were washed twice with 0.1% BSA/PBS and stained with PE-HbF antibody (BD) for 40 minutes at room temperature (with protection from light). Finally, the cells were washed three times with 0.1% BSA/PBS and passed through a 0.3 μM cell strainer (Falcon). F-cell % was analyzed by flow cytometry (LSRII-18P, BD).
Hemoglobin High-Performance Liquid Chromotography (HPLC)
[0190] 50 μl of whole blood was lysed in 200 μl ddH.sub.2O and centrifuged for 5 minutes at 13,000 rpm to extract the hemolysate. HPLC was performed according to a previously described protocol.sup.22.
Blood Smear
[0191] For hypoxia treatment, whole blood was incubated in a hypoxic incubator chamber (3% 02) for 30 minutes. 2 l of whole blood (with or without hypoxia) was used to make blood smears on slides. Slides were air-dried and then stained with Liu-stain before quantifying the percentage of sickle-shaped cells.
Exemplary Effects of Exemplary Compound AS-28 in Erythroid Cells
[0192] AS-28 can Induce γ-Globin Expression in Primary Human Erythroid Cell Culture
[0193] Compound AS-28 was designed based on the pharmacophore of the HbF-inducing compound TN1 (
[0194] AS-28 Activates γ-Globin Expression in Hydroxyurea-Resistant Primary Human Erythroid Cells
[0195] Since there are currently limited therapeutic options for SCD patients that are poorly- or non-responsive to HU, next the HbF-inducing capability of AS-28 in primary human erythroid cells that are non-responsive to HU treatment were examined. As shown in
[0196] BCL11A Protein Levels are Down-Regulated by AS-28 Treatment
[0197] BCL11A has been reported as a key negative regulator of γ-globin gene expression.sup.23. Knockdown of BCL11A dramatically increases expression of the γ-globin gene. To investigate whether HbF-inducing agents activate γ-globin gene expression by modulating BCL11A protein levels, primary human erythroid cells were treated with known HbF-inducing agents (HU, NaB, and AS-28 (0.5 μM and 1 μM)), and also Compound II, SS-2394, Compound 3, Compound 8, and then harvested for Western blot analysis (
[0198] Oral Administration of AS-28 Significantly Relieves Symptoms of Anemia in SCD Mice.
[0199] To further evaluate the therapeutic potential of AS-28 for treating hemoglobinopathies, SCD mice (an SCD disease animal model generated by the laboratory of Prof. Tim Townes, University of Alabama) were used to examine whether AS-28 treatment could relieve anemia in SCD mice. Different dosages of AS-28 were administered to 6-8 week-old SCD mice over four weeks. After four weeks of oral gavage, blood samples from the treated SCD mice were collected for several biochemical analyses (
[0200] Analysis of AS-28 and Cellular Toxicity
[0201] In order to examine whether AS-28 would cause cellular toxicity, the gene expression profiles of human primary erythroid cells in culture were compared by RNA-sequence analysis. Several candidate genes, encoding known 7 globin gene regulators were selected from the gene list of RNA-sequence data (fold change >1.4) for validation by RT-qPCR (
[0202] To reveal the global effect of AS-28 on human primary erythroid cells, the differentially expressed genes (DEGs) from the RNA-sequence data (fold change ≥1.4 and p<0.05) were selected for analysis by Ingenuity Pathways Analysis (IPA). As seen in
[0203] Derivatives of AS-28
[0204] The invention described herein is based on the unexpected discovery that certain 2-amino-6-anilino-purine compounds efficiently induce γ-globin expression.
[0205] Several pharmacophores were combined to design an exemplary new compound, AS-28. AS-28 exhibited improved water solubility and bioavailability. The in vitro assay demonstrated that AS-28 can efficiently induce γ-globin expression at a non-toxic concentration in primary erythroid cells. It was also found that AS-28 can induce γ-globin expression in HU-resistant cells. These findings support that AS-28 could represent a good candidate for development into new therapeutic compounds. The molecular mechanism by which AS-28 induces γ-globin expression was identified. According to a Western blot analysis, AS-28 reduces expression levels of BCL11A, which is a key repressor of γ-globin. To test the γ-globin-inducing efficacy of AS-28 in vivo, SCD mice were used as an animal model and AS-28 was designed as an orally-administered compound for drug development purposes. AS-28 was administered by oral gavage to SCD mice for 4 weeks. The results show that AS-28 improved several blood parameters in SCD mice. Thus, AS-28 is an exemplary compound as a candidate for treating SCD and β-thalassemia.
REFERENCES
[0206] 1. Weatherall D J. Phenotype[mdash]genotype relationships in monogenic disease: lessons from the thalassaemias. Nat Rev Genet. 2001; 2(4):245-255. [0207] 2. Modell B. Global epidemiology of haemoglobin disorders and derived service indicators. Bulletin of the World Health Organization. 2008; 2008(6):480-487. [0208] 3. Patrinos G P, Grosveld F G. Pharmacogenomics and therapeutics of hemoglobinopathies. Hemoglobin. 2008; 32(1-2):229-236. [0209] 4. Schechter A N. Hemoglobin research and the origins of molecular medicine. Blood. 2008; 112(10):3927-3938. [0210] 5. Platt O S, Brambilla D J, Rosse W F, et al. Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med. 1994; 330(23):1639-1644. [0211] 6. Scott M D, van den Berg J J, Repka T, et al. Effect of excess alpha-hemoglobin chains on cellular and membrane oxidation in model beta-thalassemic erythrocytes. J Clin Invest. 1993; 91(4):1706-1712. [0212] 7. Aljurf M, Ma L, Angelucci E, et al. Abnormal assembly of membrane proteins in erythroid progenitors of patients with beta-thalassemia major. Blood. 1996; 87(5):2049-2056. [0213] 8. Natta C L, Niazi G A, Ford S, Bank A. Balanced globin chain synthesis in hereditary persistence of fetal hemoglobin. J Clin Invest. 1974; 54(2):433-438. [0214] 9. Noguchi C T, Rodgers G P, Serjeant G, Schechter A N. Levels of fetal hemoglobin necessary for treatment of sickle cell disease. N Engl J Med. 1988; 318(2):96-99. [0215] 10. Ley T J, Nienhuis A W. Induction of hemoglobin F synthesis in patients with beta thalassemia. Annu Rev Med. 1985; 36:485-498. [0216] 11. Humphries R K, Dover G, Young N S, et al. 5-Azacytidine acts directly on both erythroid precursors and progenitors to increase production of fetal hemoglobin. J Clin Invest. 1985; 75(2):547-557. [0217] 12. Olivieri N F, Weatherall D J. The therapeutic reactivation of fetal haemoglobin. Hum Mol Genet. 1998; 7(10):1655-1658. [0218] 13. McCaffrey P G, Newsome D A, Fibach E, Yoshida M, Su M S. Induction of gamma-globin by histone deacetylase inhibitors. Blood. 1997; 90(5):2075-2083. [0219] 14. Witt O, Monkemeyer S, Ronndahl G, et al. Induction of fetal hemoglobin expression by the histone deacetylase inhibitor apicidin. Blood. 2003; 101(5):2001-2007. [0220] 15. Constantoulakis P, Knitter G, Stamatoyannopoulos G. On the induction of fetal hemoglobin by butyrates: in vivo and in vitro studies with sodium butyrate and comparison of combination treatments with 5-AzaC and AraC. Blood. 1989; 74(6):1963-1971. [0221] 16. Grigg A. Effect of hydroxyurea on sperm count, motility and morphology in adult men with sickle cell or myeloproliferative disease. Intern Med J. 2007; 37(3):190-192. [0222] 17. Kinney T R, Helms R W, O'Branski E E, et al. Safety of hydroxyurea in children with sickle cell anemia: results of the HUG-KIDS study, a phase I/II trial. Pediatric Hydroxyurea Group. Blood. 1999; 94(5):1550-1554. [0223] 18. Steinberg M H, Lu Z H, Barton F B, Terrin M L, Charache S, Dover G J. Fetal hemoglobin in sickle cell anemia: determinants of response to hydroxyurea. Multicenter Study of Hydroxyurea. Blood. 1997; 89(3):1078-1088. [0224] 19. Rule H G, Thompson S B. Acenaphthenone and acenaphthenequtnone. Journal of the Chemical Society. 1937:1761-1763. [0225] 20. Bistrzycki A, Risi J. On the effect of various diamines on naphthalic acid anhydride. Helvetica Chimica Acta. 1925; 8:810-820. [0226] 21. Nam T-g, Lee J, Walker J R, Brinker A, Cho C Y, Schultz P G. Identification and Characterization of Small-Molecule Inducers of Fetal Hemoglobin. ChemMedChem. 2011; 6(5):777-780. [0227] 22. Fibach E, Prus E. Differentiation of Human Erythroid Cells in Culture. Current Protocols in Immunology. 2005; 69(1):22F.27.21-22F.27.10. [0228] 23. Basak A, Sankaran V G. Regulation of the fetal hemoglobin silencing factor BCL11A. Annals of the New York Academy of Sciences. 2016; 1368(1):25-30. [0229] 24. Akinsheye I, Alsultan A, Solovieff N, et al. Fetal hemoglobin in sickle cell anemia. Blood. 2011; 118:19-27. [0230] 25. Imbach P, Capraro H G, Zimmermann J, Caravatti G, Furet P, Brill W, (Novartis-Erfindungen; Vienna, Austria), PCT document: WO/2000/049018A1, 2000; Published patents: EP1153024B1, 2004; U.S. Pat. No. 6,767,906B2, 2004.
EQUIVALENTS AND SCOPE
[0231] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
[0232] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
[0233] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
[0234] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.