Patent Application
20220110919
The present disclosure relates to methods of modulating leukocytes activation or thrombocyte clearance with inhibitors of specific neuraminidase isoenzymes. More specifically, the present disclosure is concerned with the use of specific inhibitors of neu1, neu3 and neu4 for modulating leukocytes activation or thrombocyte clearance.
N.A.
The response of white blood cells (leukocytes) to infection is critical to human health. Protecting the body from the constant presence of external and internal (gut) microbes requires active surveillance by white blood cells, or leukocytes, which travel from the blood stream to the periphery. The process of leukocyte transmigration to a site of infection is carefully choreographed by chemoattractants (chemokines), cellular receptors, and leukocytes themselves.
The process of leukocyte rolling, extravasation, and homing to sites of inflammation is critical to cellular immunity, and is known as the leukocyte adhesion cascade (Ley et al., 2007). Along each step of this process, different cell adhesion molecules and their ligands mediate recognition between the leukocyte and endothelial cells. The initial attachment of the leukocyte to the endothelial wall, usually referred to as rolling, is mediated by selectins and their carbohydrate ligands (e.g., sialyl Lewis-X; CD15s) (Varki, 1994). Later steps of the process must arrest the cell (firm adhesion) to allow for transmigration. These later steps of the process are largely mediated by integrin receptors and their ligands. The first of these in the cascade is LFA-1 (the αLβ2 integrin; CD11a, CD18), which binds to ICAM-1 (Inter-cellular adhesion molecule-1; CD54) and conveys an outside-in intracellular signal to the leukocyte (Hogg et al., 2004). These and subsequent integrin-mediated processes, including interactions of the very-late antigens (VLA-4, the α4β1 integrin or VLA-5, the α5β1 integrin), allow cells to migrate to the site of inflammation. (Simmons, 2005; Cox et al., 2010; Hogg et al., 2003).
Immune thrombocytopenia purpura (ITP) is an autoimmune condition characterized by a low platelet count in the absence of bone marrow-related abnormalities. It is a frequent cause of thrombocytopenia in children, resulting in significant reduction in quality of life and increased risk of bleeding. ITP is mediated by platelet antibodies that accelerate platelet destruction and inhibit their production. The dominant clinical manifestation is bleeding, which correlates generally with severity of the thrombocytopenia. ITP is a common acquired cause for low platelet counts in childhood affecting between 4-8 per 100,000 children each year with a mean age at presentation of 5.7 yrs (Blanchette, 2010; Yong et al., 2010; Kuhne, 2003). Variability in natural history and response to therapy suggest that ITP comprises heterogeneous disorders arising through diverse mechanisms of the production of platelet autoantibodies. Management of childhood ITP remains controversial. Each patient requires an individualized treatment plan that takes into consideration the platelet count, bleeding symptoms, health-related quality of life, and medication side effects. Traditional first-line agents including corticosteroids, intravenous immunoglobulins (IVIg), and anti-D immunoglobulin (anti-D) are usually effective but may be associated with multiple side effects reviewed in Neunert, 2013. Splenectomy, historically used as second-line therapy for both adults and children with ITP unresponsive to first-line agents, is considered the only “curative” therapy (Neunert, 2013). However, up to 25% of patients may not respond to such therapeutic approaches, and are defined as having refractory ITP (Neunert, 2013). Furthermore, splenectomy leads to a loss of immune protection against encapsulated bacteria and is associated with a lifelong risk of overwhelming sepsis and infection, as well as an increased risk of thromboembolism (Crary et al., 2009).
A host of human diseases involve mis-regulation of the inflammatory response. There is a need for new strategies and mechanisms for anti-adhesive and anti-inflammatory therapeutic.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
The present disclosure provides a method for modulating leukocytes activation in response to infection and inflammatory signals. It also provides compounds for use in the modulation of inflammatory responses in e.g., leukocytes transmigration and adhesion and cytokine response.
The present disclosure also provides a method for modulating thrombocyte clearance, comprising inhibiting of the expression or activity of neuraminidase 1 (neu1), neuraminidase 3 (neu3) or neuraminidase 4 (neu4) in a subject in need thereof.
More specifically, in accordance with the present disclosure, there are provided the following items:
Item 1. A method of modulating leukocyte activation, comprising specifically inhibiting expression or activity of neuraminidase 1 (neu1), neuraminidase 3 (neu3) or neuraminidase 4 (neu4) in a subject in need thereof.
Item 2. The method of item 1, wherein the inhibiting comprises administering a therapeutically effective amount of a specific or a bispecific neu1, neu3, and/or neu4 inhibitor to the subject.
Item 3. The method of item 1 or 2, wherein the leukocyte activation comprises transmigration of monocytes, neutrophils, macrophages, NK cells or T-cells.
Item 4. The method of item 1 or 2, wherein the inhibiting comprises the administration of a therapeutically effective amount of a specific or a bispecific neu1 and/or neu4 inhibitor
Item 5. The method of item 4, wherein the leukocyte activation comprises leukocyte adhesion, leukocyte transmigration and/or cytokine response.
Item 6. The method of item 5, wherein the cytokine is G-CSF/CSF-3, IL-21, IL-6, IFN-γ and/or RANTES.
Item 7. A method of modulating thrombocyte clearance, comprising specifically inhibiting of the expression or activity of neuraminidase 1 (neu1), neuraminidase 3 (neu3) or neuraminidase 4 (neu4) in a subject in need thereof.
Item 8. The method of item 7, wherein the inhibiting comprises the administration of a therapeutically effective amount of a specific or a bispecific neu1, neu3, and/or neu4 inhibitor.
Item 9. The method of items 1 to 8, wherein the inhibitor is (i) compound 5c;
or (ii) a compound of formula I
wherein R1 is H; a C1-C10 alkyl; C1-C10 heteroalkyl; C3-C7 cycloalkyl; C3-C7 heterocycloalkyl; C3-C8 aryl; or C3-C8 heteroaryl; wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted by at least one substituent, each substituent being independently a C1-C10 alkyl, a C3-C8 cycloalkyl, a C3-C7 aryl, a halogen, an amide or a hydroxyl;
R2 is H; —OH, —NHC(═NH)NH2; or azide;
R3 is —NHC(O)(CH2)nR5,
wherein R5 is H; —OH; C1-C10 alkyl; C1-C10 heteroalkyl; C3-C7 cycloalkyl; C3-C7 heterocycloalkyl; C3-C8 aryl or azide; wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, and aryl are optionally substituted by at least one substituent, each substituent being independently a C1-C10 alkyl, a C3-C8 cycloalkyl, a C3-C7 aryl, an halogen, an amide or an hydroxyl; and
n is 0 or 1;
R4 is H; —OH; —O-alkyl; —C(O)-alkyl-NHC(O)-aryl; —NHC(O)R6; or
wherein the alkyl and aryl are optionally substituted by at least one substituent, each substituent being independently a C1-C10 alkyl, a C3-C8 cycloalkyl, a C3-C7 aryl, an halogen, an amine, an amide or an hydroxyl,
with the proviso that when R2 and R4 are OH, R3 is not —NHC(O)CH3,
or is an ester, solvate, hydrate or pharmaceutical salt of the compound of formula I.
Item 10. The method of item 9, wherein R3 is —NHC(O)(CH2)nR5 and n is 1.
Item 11. The method of item 10, wherein R5 is H or azide.
Item 12. The method of any one of items 9 to 11, wherein R2 is —NHC(═NH)NH2.
Item 13. The method of any one of items 9 to 12, wherein R4 is
where R7 and p are as defined in item 9.
Item 14. The method of item 13, wherein p is 2 and R7 is H.
Item 15. The method of item 10, wherein R5 is C1-C5 alkyl.
Item 16. The method of any one of items 9, 10, and 15, wherein R2 is OH.
Item 17. The method of any one of items 9, 10, 15 and 16, wherein R4 is —OH.
Item 18. The method of any one of items 9 to 11 and 16, wherein R4 is —NHC(O)R6.
Item 19. The method of item 18, wherein R6 is C3-C6 alkyl.
Item 20. The method of item 13, wherein p is 0.
Item 21. The method of item 15, wherein R7 is -hydroxy C1-C10 alkyl.
Item 22. The method of any one of items 9, 10 or 11, wherein R4 is
Item 23. The method of any one of items 9 to 22, wherein X is O.
Item 24. The method of any one of items 1 to 6, wherein the inhibiting comprises the administration of a specific or bispecific neu4 inhibitor.
Item 25. The method of item 24, wherein the specific or bispecific inhibitor is compound 28.
Item 26. The method of item 22 or 23, wherein the inhibiting increases monocytes and neutrophils transmigration and decreases T cell transmigration in response to bacterial infection.
Item 27. The method of any one of items 1 to 6, wherein the inhibiting comprises the administration of a specific or bispecific neu3 inhibitor.
Item 28. The method of item 27, wherein the specific or bispecific neu3 inhibitor is compound 8b or 5c.
Item 29. The method of item 27 or 28, wherein the inhibiting increases monocytes and neutrophils transmigration and decreases T cell transmigration in response to bacterial infection.
Item 30. The method of any one of items 1 to 6, wherein the inhibiting comprises the administration of a specific or bispecific neu1 inhibitor.
Item 31. The method of item 30, wherein the specific or bispecific neu1 inhibitor is compound 32 or 50.
Item 32. The method of item 30 or 31, wherein the inhibiting decreases monocytes transmigration and increases neutrophils transmigration in response to bacterial infection.
Item 33. The method of any one of items 1 to 6, wherein the inhibiting comprises the administration of a specific or bispecific neu1 or neu4 inhibitor.
Item 34. The method of item 33, wherein the inhibiting modulates cytokine response.
Item 35. The method of any one of items 1 to 6 and 9 to 34, wherein the inhibiting modulates an inflammatory response to bacterial infection in a subject in need thereof.
Item 36. The method of item 7 or 8, wherein the inhibiting comprises the administration of a therapeutically effective amount of a specific or a bispecific neu1 inhibitor.
Item 37. The method of item 36, wherein the inhibitor is compound 50.
More specifically, in accordance with the present disclosure, there are also provided the following items:
Item 1. A method of preventing or treating an inflammatory response to a bacterial infection, comprising specifically inhibiting the expression or activity of neuraminidase 1 (neu1), neuraminidase 3 (neu3) or neuraminidase 4 (neu4) in a subject in need thereof.
Item 2. The method of item 1, wherein the inhibitor is a compound of formula I
wherein R1 is H; a C1-C10 alkyl; C1-C10 heteroalkyl; C3-C7 cycloalkyl; C3-C7 heterocycloalkyl; C3-C8 aryl; or C3-C8 heteroaryl; wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted by at least one substituent, each substituent being independently a C1-C10 alkyl, a C3-C8 cycloalkyl, a C3-C7 aryl, a halogen, an amide or a hydroxyl;
R2 is H; —OH, —NHC(═NH)NH2; or azide;
R3 is —NHC(O)(CH2)nR5,
wherein R5 is H; —OH; C1-C10 alkyl; C1-C10 heteroalkyl; C3-C7 cycloalkyl; C3-C7 heterocycloalkyl; C3-C8 aryl; or azide; wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, and aryl are optionally substituted by at least one substituent, each substituent being independently a C1-C10 alkyl, a C3-C8 cycloalkyl, a C3-C7 aryl, an halogen, an amide or an hydroxyl; and
n is 0 or 1;
R4 is H; —OH; —O-alkyl; —C(O)-alkyl-NHC(O)-aryl; —NHC(O)R6; or
wherein the alkyl and aryl are optionally substituted by at least one substituent, each substituent being independently a C1-C10 alkyl, a C3-C8 cycloalkyl, a C3-C7 aryl, an halogen, an amine, an amide or an hydroxyl,
with the proviso that when R2 and R4 are OH, R3 is not —NHC(O)CH3,
or is an ester, solvate, hydrate or pharmaceutical salt of the compound of formula I.
Item 3. The method of item 2, wherein R3 is —NHC(O)(CH2)nR5.
Item 4. The method of item 2, wherein n is 0.
Item 5. The method of item 4, wherein R5 is cycloalkyl.
Item 6. The method of item 4, wherein R5 is aryl.
Item 7. The method of item 4, wherein R5 is C1-C10 alkyl.
Item 8. The method of item 4, wherein R5 is C1-C10 alkyl substituted with a C1-C10 alkyl.
Item 9. The method of item 2, wherein n is 1.
Item 10. The method of item 9, wherein R5 is H or azide.
Item 11. The method of item 9, wherein R5 is C1-C5 alkyl.
Item 12. The method of item 11, wherein the C1-C5 alkyl is branched.
Item 13. The method of any one of items 2-12, wherein R2 is OH.
Item 14. The method of any one of items 2-11, wherein R2 is —NHC(═NH)NH2.
Item 15. The method of any one of items 2-11, wherein R2 is azido.
Item 16. The method of any one of items 2-15, wherein R4 is —OH.
Item 17. The method of any one of items 2-15, wherein R4 is —NHC(O)R6.
Item 18. The method of item 17, wherein R6 is C1-C10 alkyl.
Item 19. The method of item 18, wherein the C1-C10 alkyl is branched.
Item 20. The method of item 17, wherein R6 is C3-C7 aryl.
Item 21. The method of item 20, wherein the C3-C7 aryl is substituted with an amine or an amide.
Item 22. The method of any one of items 2-15, wherein R4 is
wherein R7 and p are as defined in item 2.
Item 23. The method of item 22, wherein p is 0.
Item 24. The method of item 23, wherein R7 is —(CH2)qNH(CO)aryl.
Item 25. The method of item 23, wherein R7 is -hydroxy C1-C10 alkyl.
Item 26. The method of item 20, wherein R7 is C1-C10 alkyl.
Item 27. The method of item 22, wherein p is 1.
Item 28. The method of item 27, wherein R7 is halogen.
Item 29. The method of item 27, wherein R7 is O-alkyl.
Item 30. The method of item 27, wherein R7 is —C(O)OH.
Item 31. The method of item 27, wherein R7 is amine.
Item 32. The method of item 27, wherein R7 is acetamide.
Item 33. The method of item 27, wherein R7 is —C1-C10 alkyl.
Item 34. The method of item 27, wherein R7 is —CH2NH(CO)aryl.
Item 35. The method of item 27, wherein R7 is —O—C3-C7 aryl.
Item 36. The method of item 22, wherein p is 2.
Item 37. The method of item 36, wherein R7 is H.
Item 38. The method of any one of items 2-15, wherein R4 is —C(O)-alkyl-NHC(O)-aryl.
Item 39. The method of item 38, wherein the alkyl is C1-C10 alkyl.
Item 40. The method of item 38 or 39, wherein the aryl is C3-C7 aryl.
Item 41. The method of item 38, wherein the C3-C7 aryl is substituted with an amide.
Item 42. The method of item 2, wherein:
wherein p is 0, 1, 2 or 3, and R7 is H, —C(═O)OH, phenyl, or phenyloxy,
wherein p is 1, 2 or 3, and R7 is H, —C(═O)OH, phenyl, or phenyloxy,
or an ester, solvate, hydrate or pharmaceutical salt of the compound of formula I.
Item 47. The method of item 2, wherein the compound is of formula I, wherein X is O, R1 is H, and R3, R2 and R4 are set forth below:
or an ester, solvate, hydrate or pharmaceutical salt of the compound of formula I.
Item 48. The method of any one of items 2 to 47, wherein the compound is of formula I is of formula Ia:
wherein R1, R2, R3, R4 and X are as defined in any one of items 2 to 47.
Item 49. The method of any one of items 2 to 47, wherein the compound of formula I is of formula Ib:
wherein R1, R2, R3, R4 and X are as defined in any one of items 2 to 47.
Item 50. The method of any one of items 1 to 49, wherein the inhibitor is a specific or bispecific inhibitor of neu1.
Item 51. The method of any one of items 1 to 49, wherein the inhibitor is a specific or bispecific inhibitor of neu3.
Item 52. The method of any one of items 1 to 49, wherein the inhibitor is a specific or bispecific inhibitor of neu4.
In another specific embodiment, the specific inhibitor is a compound of formula III
wherein R1 is as defined above or H, a linear alkyl group C1-C12 (i.e. Me, Et, Pr, But, Pent, Hex, etc.), a branched alkyl group C1-C12, or an aryl group; and Ra is the group shown at that position in any one of 7a-7j, and 26 to 28,
or an ester, solvate, hydrate or pharmaceutical salt thereof.
In another specific embodiment, the specific inhibitor is a compound of formula IV
where R1 is as defined above or H, a linear alkyl group C1-C12 (i.e. Me, Et, Pr, But, Pent, Hex, etc.), a branched alkyl group C1-C12, or an aryl group; and Rb is the group shown at that position in any one of compounds 49 to 56,
or an ester, solvate, hydrate or pharmaceutical salt thereof.
In another specific embodiment, the specific inhibitor is a compound of formula V
where R1 is as defined above or H, a linear alkyl group C1-C12 (i.e. Me, Et, Pr, But, Pent, Hex, etc.), a branched alkyl group C1-C12, or an aryl group; and Rc is the group shown at that position in any one of compounds 58-61,
or an ester, solvate, hydrate or pharmaceutical salt thereof.
In another specific embodiment, the specific inhibitor is a compound of formula VI
R1 where R1 is as defined above or H, a linear alkyl group C1-C12 (i.e. Me, Et, Pr, But, Pent, Hex, etc.), a branched alkyl group C1-C12, or an aryl group; and Rd and Re are the groups shown at these positions in any one of compounds 49-52, 54-57, 64-70, 74-74,
or an ester, solvate, hydrate or pharmaceutical salt thereof.
In another specific embodiment, the specific inhibitor is a compound of formula VII
where R1 is as defined above or H, a linear alkyl group C1-C12 (i.e. Me, Et, Pr, But, Pent, Hex, etc.), a branched alkyl group C1-C12, or an aryl group; and Rf is the group shown at that position in any one of compounds 29 to 48,
or an ester, solvate, hydrate or pharmaceutical salt thereof.
In another specific embodiment, the specific inhibitor is a compound of formula VIII
where R1 is as defined above or H, a linear alkyl group C1-C12 (i.e. Me, Et, Pr, But, Pent, Hex, etc.), a branched alkyl group C1-C12, or an aryl group; and Rg is an C3-C7 aryl group substituted or not with a C3-C10 aryl group (substituted or not with an halogen, an amine, a C1-C10 alkyl, a C1-C10 alkyloxy, a trifluoromethyl, a —COOH, a C3-C7 aryl); a C1-C10 alkyl group; or a —COOH group. In a more specific embodiment, it is a group as shown at that position in any one of compounds 8a and 8b,
or an ester, solvate, hydrate or pharmaceutical salt thereof.
In another specific embodiment, there is provided a pharmaceutical composition comprising a neu1/neu3 specific inhibitor that is a compound of any one of formulas I, Ia, Ib, and II-VIII, or any specific inhibitor disclosed in Table I, or an ester, solvate, hydrate or pharmaceutical salt thereof, and a pharmaceutically acceptable carrier. In a specific embodiment, the specific inhibitor has an IC50 against a neu1/neu3 that is lower than 1 μM (e.g., 5c, 7i, 7i, 7j, 8a, 8b, 28 31-32, 50, 67-69, 72, 74 and 75, preferably compound 5c, 8b, 28, 32 or 50).
In another specific embodiment, there is provided a pharmaceutical composition comprising a neu1 specific inhibitor that is a compound of any one of formulas I, Ia, Ib, and II-VIII, or any specific inhibitor disclosed in Table I, or an ester, solvate, hydrate or pharmaceutical salt thereof, and a pharmaceutically acceptable carrier. In a specific embodiment, the specific inhibitor has an IC50 against a neu1 that is lower than 1 μM (e.g., compounds 31-32, 50, 67-69, 72, 74 and 75, preferably compound 32 or 50).
In another specific embodiment, there is provided a pharmaceutical composition comprising a neu3 specific inhibitor that is a compound of any one of formulas I, Ia, Ib, and II-VIII, or any specific inhibitor disclosed in Table I, or an ester, solvate, hydrate or pharmaceutical salt thereof, and a pharmaceutically acceptable carrier. In a specific embodiment, the specific inhibitor has an IC50 against a neu3 that is lower than 1 μM (e.g., compounds 5c, 7i, 8a, 8b, preferably compound 5c or 8b).
In another specific embodiment, there is provided a pharmaceutical composition comprising a neu4 specific inhibitor that is a compound of any one of formulas I, Ia, Ib, and II-VIII, or any specific inhibitor disclosed in Table I, or an ester, solvate, hydrate or pharmaceutical salt thereof, and a pharmaceutically acceptable carrier. In a specific embodiment, the specific inhibitor has an IC50 against a neu4 that is lower than 1 μM (e.g., compounds 7i, 7j, 28, preferably compound 28).
In another specific embodiment, there is provided a method of modulating leukocytes adhesion and/or transmigration (e.g., in response to bacterial infection) comprising administering to a subject in need thereof a therapeutically effective amount of (i) a specific neu1/neu3/neu4 inhibitor of the present disclosure that is a compound of any one of formulas I, Ia, Ib, and II-VIII, or any specific neu1/neu3 inhibitor disclosed in Table I, or an ester, solvate, hydrate or pharmaceutical salt thereof; or (ii) a pharmaceutical composition comprising (i) and a pharmaceutically acceptable carrier. (e.g., 5c, 7i, 7i, 7j, 8a, 8b, 28 31-32, 50, 67-69, 72, 74 and 75, preferably compound 5c, 8b, 28, 32 or 50).
In another specific embodiment, there is provided a method of modulating leukocytes adhesion and/or transmigration (e.g., in response to bacterial infection) comprising administering to a subject in need thereof a therapeutically effective amount of (i) a specific neu1 inhibitor of the present disclosure that is a compound of any one of formulas I, Ia, Ib, and II-VIII, or any specific neu1 inhibitor disclosed in Table I, or an ester, solvate, hydrate or pharmaceutical salt thereof; or (ii) a pharmaceutical composition comprising (i) and a pharmaceutically acceptable carrier. (e.g., compounds 31-32, 50, 67-69, 72, 74 and 75, preferably compound 32 or 50).
In another specific embodiment, there is provided a method of modulating leukocytes adhesion and/or transmigration (e.g., in response to bacterial infection) comprising administering to a subject in need thereof a therapeutically effective amount of (i) a specific neu3 inhibitor of the present disclosure that is a compound of any one of formulas I, Ia, Ib, and II-VIII, or any specific neu3 inhibitor disclosed in Table I, or an ester, solvate, hydrate or pharmaceutical salt thereof; or (ii) a pharmaceutical composition comprising (i) and a pharmaceutically acceptable carrier. (e.g., compounds 5c, 7i, 8a, 8b, preferably compound 5c or 8b).
In another specific embodiment, there is provided a method of modulating leukocytes adhesion and/or transmigration (e.g., in response to bacterial infection) comprising administering to a subject in need thereof a therapeutically effective amount of (i) a specific neu3 inhibitor of the present disclosure that is a compound of any one of formulas I, Ia, Ib, and II-VIII, or any specific neu4 inhibitor disclosed in Table I, or an ester, solvate, hydrate or pharmaceutical salt thereof; or (ii) a pharmaceutical composition comprising (i) and a pharmaceutically acceptable carrier. (e.g., compounds 7i, 7j, 28, preferably compound 28).
Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
The present disclosure relates to the use of specific inhibitors of neuraminidase enzymes for modulating leukocyte activation (e.g., transmigration, adhesion or cytokine response), and in turn, inflammation (e.g., inflammation response to bacterial infection). More specifically, it relates to the use of a therapeutically effective amount of a specific neuraminidase 1 (neu1) inhibitor, a specific neuraminidase 3 (neu3) inhibitor, or a specific neuraminidase 4 (neu4) inhibitor, or a bispecific neu1 inhibitor (e.g., neu1/neu2 or neu1/neu4) or a bispecific neu3 inhibitor (e.g., neu3/neu2 or neu3/neu4); or a bispecific neu4 inhibitor (e.g., neu4/neu1 or neu4/neu3) for modulating leukocyte activation (e.g., transmigration, adhesion or cytokine response), and in turn, inflammation in a subject in need thereof.
The present disclosure also relates to the use of specific inhibitors of neuraminidase enzymes (e.g. neu1, neu3 or neu4 enzyme, more specifically neu1 enzyme) for modulating thrombocyte clearance. Without being so limited, in a more specific embodiment, it relates to the use of specific inhibitors of neuraminidase enzymes (e.g. neu1, neu3 or neu4 enzyme, more specifically neu1 enzyme) for preventing or treating immune thrombocytopenia or a symptom thereof.
As used herein, the term “specific inhibitor” encompasses bispecific inhibitors and refers to any inhibitor that specifically inhibits the activity or expression of one or two of neu1, neu3 and neu4. In another specific embodiment, it relates to any inhibitor that specifically inhibits neu1 or neu4. In another specific embodiment, it relates to any inhibitor that specifically inhibits neu1. In another specific embodiment, it relates to any inhibitor that specifically inhibits neu3. In another specific embodiment, it relates to any inhibitor that specifically inhibits neu1.
As used herein, the term “specific neu1/neu3/neu4 inhibitor” is used herein to refer to “a specific inhibitor of neuraminidase 1 (neu1); neuraminidase 3 (neu3); neuraminidase 4 (neu4); or a bispecific inhibitor of neu1, neu3 or neu4”. It refers to at least one of a “specific neuraminidase 1 inhibitor”, “specific neuraminidase 3 inhibitor”, “specific neuraminidase 4 inhibitor”, “bispecific neuraminidase 1 inhibitor”, “bispecific neuraminidase 3 inhibitor” and “bispecific neuraminidase 4 inhibitor”. For convenience, it refers to an agent able to reduce, the case being, neu1, neu3 and/or neu4 expression and/or activity. Without being so limited, such inhibitors include small molecules including but not limited those of any one of formulas I, Ia, Ib, and II-VIII and those identified as such in in Table I, dsRNA (e.g., RNAi, siRNA, miRNA), peptides, antibodies or antibody fragments (e.g., antibodies that specifically binds to neu1, neu3 or neu4 or are bispecific against neu1, neu3 or neu4 and against another neuraminidase enzyme, and antibody fragments that specifically binds to neu1, neu3 or neu4 or are bispecific against neu1, neu3 or neu4 and another neuraminidase enzyme). In more specific embodiments, such inhibitors are those identified as such in Table I.
Typically, specific inhibitors advantageously avoid certain deleterious side effects that could be present when using inhibitors with less selectivity.
As used herein the terms “specific neuraminidase 1 inhibitor” refer to an inhibitor that is more active against neuraminidase 1 than against neuraminidase 2, 3, or 4. In a specific embodiment, the inhibitor has an IC50 against neu1 that is at least 2× lower than the IC50 against at least one of neu2, neu3, and neu4 (in a specific embodiment, against at least two of neu2, neu3, and neu4 and in another specific embodiment against all three of neu2, neu3, and neu4). In another specific embodiment, its IC50 against neu1 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu2, neu3, and neu4 (in a specific embodiment, against at least two of neu2, neu3, and neu4 and in another specific embodiment against all three of neu2, neu3, and neu4).
As used herein the terms “specific neuraminidase 3 inhibitor” refer to an inhibitor that is more active against neuraminidase 3 than against neuraminidase 1, 2 or 4. In a specific embodiment, it has an IC50 against neu3 that is at least 2× lower than the IC50 against at least one of neu1, neu2 and neu4 (in a specific embodiment, against at least two of neu1, neu2, and neu4 and in another specific embodiment against all three of neu1, neu2, and neu4). In another specific embodiment, its IC50 against neu3 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu1, neu2 and neu4 (in a specific embodiment, against at least two of neu1, neu2, and neu4 and in another specific embodiment against all three of neu1, neu2, and neu4).
As used herein the terms “specific neuraminidase 4 inhibitor” refer to an inhibitor that is more active against neuraminidase 3 than against neuraminidase 1, 2 or 3. In a specific embodiment, it has an IC50 against neu4 that is at least 2× lower than the IC50 against at least one of neu1, neu2 and neu3 (in a specific embodiment, against at least two of neu1, neu2, and neu3 and in another specific embodiment against all three of neu1, neu2, and neu3). In another specific embodiment, its IC50 against neu4 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu1, neu2 and neu3 (in a specific embodiment, against at least two of neu1, neu2, and neu3 and in another specific embodiment against all three of neu1, neu2, and neu3).
As used herein the terms “bispecific neuraminidase 1 inhibitor” refer to “bispecific neuraminidase 1/neuraminidase 2 inhibitor” (or “bispecific neu1/2 inhibitor”), “bispecific neuraminidase 1/neuraminidase 3 inhibitor” (or “bispecific neu1/3 inhibitor”) or “bispecific neuraminidase 1/neuraminidase 4 inhibitor” (or “bispecific neu1/4 inhibitor”).
As used herein the terms “bispecific neuraminidase 1/neuraminidase 2 inhibitor” or “bispecific neu1/2 inhibitor” refer to an inhibitor that has activity against neuraminidase 1 and neuraminidase 2 and is less active against neuraminidase 3 and/or 4. In a specific embodiment, such an inhibitor has an IC50 against neu1 that is of from 3:1 to 1:3 against neu2. In a specific embodiment, such an inhibitor has an IC50 against neu1 and neu2 that is at least 2× lower than the IC50 against at least one of neu3 and neu4 (in a specific embodiment, against both of neu3 and neu4). In another specific embodiment, its IC50 against neu1 and neu2 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu3 and neu4 (in a specific embodiment, against both of neu3 and neu4).
As used herein the terms “bispecific neuraminidase 1/neuraminidase 3 inhibitor” or “bispecific neu1/3 inhibitor” refer to an inhibitor that has activity against neuraminidase 1 and neuraminidase 3 and is less active against neuraminidase 2 and/or 4. In a specific embodiment, such an inhibitor has an IC50 against neu1 that is of from 3:1 to 1:3 against neu3. In a specific embodiment, such an inhibitor has an IC50 against neu1 and neu3 that is at least 2× lower than the IC50 against at least one of neu2 and neu4 (in a specific embodiment, against both of neu2 and neu4). In another specific embodiment, its IC50 against neu1 and neu3 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu2 and neu4 (in a specific embodiment, against both of neu2 and neu4).
As used herein the terms “bispecific neuraminidase 1/neuraminidase 4 inhibitor” or “bispecific neu1/4 inhibitor” refer to an inhibitor that has activity against neuraminidase 1 and neuraminidase 4 and is less active against neuraminidase 2 and/or 3. In a specific embodiment, such an inhibitor has an IC50 against neu1 that is of from 3:1 to 1:3 against neu4. In a specific embodiment, such an inhibitor has an IC50 against neu1 and neu4 that is at least 2× lower than the IC50 against at least one of neu2 and neu3 (in a specific embodiment, against both of neu2 and neu3). In another specific embodiment, its IC50 against neu1 and neu4 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu2 and neu3 (in a specific embodiment, against both of neu2 and neu3).
As used herein the terms “bispecific neuraminidase 3 inhibitor” refer to “bispecific neuraminidase 3/neuraminidase 1 inhibitor” (or “bispecific neu3/1 inhibitor”) or “bispecific neuraminidase 3/neuraminidase 2 inhibitor” (or “bispecific neu3/2 inhibitor”) or “bispecific neuraminidase 3/neuraminidase 4 inhibitor” (or “bispecific neu3/4 inhibitor”).
As used herein the terms “bispecific neuraminidase 3/neuraminidase 1 inhibitor” or “bispecific neu3/1 inhibitor” refer to an inhibitor that has activity against neuraminidase 3 and neuraminidase 1 and is less active against neuraminidase 2 and/or 4. In a specific embodiment, such an inhibitor has an IC50 against neu3 that is of from 3:1 to 1:3 against neu1. In a specific embodiment, such an inhibitor has an IC50 against neu3 and neu1 that is at least 2× lower than the IC50 against at least one of neu2 and neu4 (in a specific embodiment, against both of neu2 and neu4). In another specific embodiment, its IC50 against neu3 and neu1 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu2 and neu4 (in a specific embodiment, against both of neu2 and neu4).
As used herein the terms “bispecific neu3/2 inhibitor” refer to an inhibitor that has activity against neuraminidase 3 and neuraminidase 2 and is less active against neuraminidase 1 and/or 4. In a specific embodiment, such an inhibitor has an IC50 against neu3 that is of from 3:1 to 1:3 against neu2. In a specific embodiment, such an inhibitor has an IC50 against neu3 and neu2 that is at least 2× lower than the IC50 against at least one of neu1 and neu4 (in a specific embodiment, against both of neu1 and neu4). In another specific embodiment, its IC50 against neu3 and neu2 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu1 and neu4 (in a specific embodiment, against both of neu1 and neu4).
As used herein the terms “bispecific neu3/4 inhibitor” refer to an inhibitor that has activity against neuraminidase 3 and neuraminidase 4 and is less active against neuraminidase 1 and/or 2. In a specific embodiment, such an inhibitor has an IC50 against neu3 that is of from 3:1 to 1:3 against neu4. In a specific embodiment, such an inhibitor has an IC50 against neu3 and neu4 that is at least 2× lower than the IC50 against at least one of neu1 and neu2 (in a specific embodiment, against both of neu1 and neu2). In another specific embodiment, its IC50 against neu3 and neu4 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu1 and neu2 (in a specific embodiment, against both of neu1 and neu2).
As used herein the terms “bispecific neuraminidase 4 inhibitor” refer to “bispecific neuraminidase 4/neuraminidase 1 inhibitor” (or “bispecific neu4/1 inhibitor”) or “bispecific neuraminidase 4/neuraminidase 2 inhibitor” (or “bispecific neu4/2 inhibitor”) or “bispecific neuraminidase 4/neuraminidase 3 inhibitor” (or “bispecific neu4/3 inhibitor”).
As used herein the terms “bispecific neuraminidase 4/neuraminidase 1 inhibitor” or “bispecific neu4/1 inhibitor” refer to an inhibitor that has activity against neuraminidase 4 and neuraminidase 1 and is less active against neuraminidase 2 and/or 3. In a specific embodiment, such an inhibitor has an IC50 against neu4 that is of from 3:1 to 1:3 against neu1.
In a specific embodiment, such an inhibitor has an IC50 against neu4 and neu1 that is at least 2× lower than the IC50 against at least one of neu2 and neu3 (in a specific embodiment, against both of neu2 and neu3). In another specific embodiment, its IC50 against neu4 and neu1 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu2 and neu3 (in a specific embodiment, against both of neu2 and neu3).
As used herein the terms “bispecific neu4/2 inhibitor” refer to an inhibitor that has activity against neuraminidase 4 and neuraminidase 2 and is less active against neuraminidase 1 and/or 3. In a specific embodiment, such an inhibitor has an IC50 against neu4 that is of from 3:1 to 1:3 against neu2. In a specific embodiment, such an inhibitor has an IC50 against neu4 and neu2 that is at least 2× lower than the IC50 against at least one of neu1 and neu3 (in a specific embodiment, against both of neu1 and neu3). In another specific embodiment, its IC50 against neu4 and neu2 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu1 and neu3 (in a specific embodiment, against both of neu1 and neu3).
As used herein the terms “bispecific neu4/3 inhibitor” refer to an inhibitor that has activity against neuraminidase 4 and neuraminidase 3 and is less active against neuraminidase 1 and/or 2. In a specific embodiment, such an inhibitor has an IC50 against neu4 that is of from 3:1 to 1:3 against neu3. In a specific embodiment, such an inhibitor has an IC50 against neu4 and neu3 that is at least 2× lower than the IC50 against at least one of neu1 and neu2 (in a specific embodiment, against both of neu1 and neu2). In another specific embodiment, its IC50 against neu4 and neu3 is at least 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, 29×, 30×, 31×, 32×, 33×, 34×, 35×, 36×, 37×, 38×, 39×, 40×, 41×, 42×, 43×, 44×, 45×, or 46× lower than its IC50 against at least one of neu1 and neu2 (in a specific embodiment, against both of neu1 and neu2).
As used herein, the term “leukocyte activation” includes leukocyte transmigration, adhesion and/or cytokine response. As used herein the term “leukocyte transmigration or adhesion” refers to events occurring in inflammation. More specifically, it refers to the specific steps of the inflammatory cascade leading to inflammation. These steps include rolling, firm adhesion, and transmigration. As used herein the term cytokine response, refers to, without being so limited, secretion by e.g., one or more leukocytes of at least one cytokine in the circulation (e.g., blood or plasma). Without being so limited, the response of the following cytokines has been modulated by at least one neuraminidase depletion (e.g., neu1 and neu4) in the present disclosure: CSF/CSF-3, IL-21, IL-6, IFN-γ and RANTES.
The present disclosure also relates to the use of specific neu1, neu3 and neu4 inhibitors, and bispecific neu1 neu3 or neu4 inhibitors (e.g., neu3/neu4 inhibitors) to reduce inflammation in a subject in need thereof.
The present disclosure also relates to the use of specific inhibitors of neuraminidase enzymes (e.g. neu1, neu3 or neu4 enzyme, more specifically neu1 enzyme) for modulating thrombocyte clearance. Without being so limited, in a more specific embodiment, it relates to the use of specific inhibitors of neuraminidase enzymes (e.g. neu1, neu3 or neu4 enzyme, more specifically neu1 enzyme) for preventing or treating immune thrombocytopenia or a symptom thereof.
As used herein, the term “thrombocyte clearance” relates to reduction of thrombocyte in the bloodstream (e.g., through thrombocyte phagocytosis by leukocytes).
As used herein the term “a symptom thereof” in the term “immune thrombocytopenia or a symptom thereof” refers to, without being so limited, a reduced thrombocyte (platelet) level in the blood.
As used herein, the term “prevent/preventing/prevention” or “treat/treating/treatment”, refers to eliciting the desired biological response, i.e., a prophylactic and therapeutic effect, respectively in a subject. In accordance with the present disclosure, the therapeutic effect comprises one or more of a decrease/reduction in the severity, intensity and/or duration of the immune thrombocytopenia or symptom thereof or of the inflammation (following bacterial infection) following administration of the inhibitor of the present disclosure when compared to its severity, intensity and/or duration in the subject prior to treatment or as compared to that/those in a non-treated control subject having the infection or any symptom thereof. In accordance with the disclosure, a prophylactic effect may comprise a delay in the onset of the immune thrombocytopenia or symptom thereof or of the inflammation (following bacterial infection) in an asymptomatic subject at risk of experiencing the immune thrombocytopenia or symptom thereof or of the inflammation (following bacterial infection) at a future time; or a decrease/reduction in the severity, intensity and/or duration of immune thrombocytopenia or symptom thereof or of the inflammation (following bacterial infection) occurring following administration of the inhibitor of the present disclosure, when compared to the timing of their onset or their severity, intensity and/or duration in a non-treated control subject (i.e. asymptomatic subject at risk of experiencing the immune thrombocytopenia or symptom thereof or of the inflammation (following bacterial infection); and/or a decrease/reduction in the progression of any pre-existing immune thrombocytopenia or symptom thereof or of the inflammation (following bacterial infection) in a subject following administration of the agent of the present disclosure when compared to the progression of an immune thrombocytopenia or symptom thereof or of the inflammation (following bacterial infection) in a non-treated control subject having such pre-existing immune thrombocytopenia or symptom thereof or of the inflammation (following bacterial infection). As used herein, in a therapeutic treatment, the inhibitor of the present disclosure is administered after the onset of the immune thrombocytopenia or symptom thereof or of the inflammation (following bacterial infection). As used herein, in a prophylactic treatment, the agent of the present disclosure is administered before the immune thrombocytopenia or symptom thereof or of the inflammation (following bacterial infection) or after the onset thereof but before the progression thereof.
A “therapeutically effective amount” or “effective amount” or “therapeutically effective dosage” of a specific inhibitor of the disclosure or composition thereof can result in a modulation of inflammation (e.g., leukocyte transmigration or adhesion or cytokine response) (e.g., decrease of inflammation) in a subject.
The structure of specific small molecules of the present disclosure are shown in
In specific embodiments of the present disclosure, small molecule inhibitors of the present disclosure (e.g., formulas I, Ia, Ib, and II-VIII) have an IC50 against neu1, neu3 or neu4 that is of 100 μM or lower, 20 μM or lower, 10 μM or lower, 3 μM or lower, 1 μM or lower or lower than 1 μM. In specific embodiments, small molecule inhibitors of the present disclosure are the compounds of Table I that have an IC50 against neu1, neu3 or neu4 that is lower than 1 M (e.g., compounds with neu1 specificity or bispecificity: compounds 31, 32, 67-69, 72, 74 and 75; compounds with neu3 specificity or bispecificity: 5c, 7i, 8a and 8b; and compounds with neu4 specificity or bispecificity: 7i, 7j and 28); 1 M or lower (e.g., the foregoing compounds and compound 7j (neu3 specificity or bispecificity); 3 μM or lower (e.g., the foregoing compounds and compounds 54, 56, 33, 57, 36, 51, 58, 65, 66, 70 and 73 (neu1 specificity or bispecificity), compounds 7h and 27 (neu3 specificity or bispecificity), and compounds 7e, 7f, 7h, 26 and 63); and 10 μM or lower (e.g., the foregoing compounds and compounds 55, 50, 30, 34, 37, 49, 52, 60-61, 64 (neu1 specificity or bispecificity)), compounds 7e, 7c, 7a, 7b, 7d, 7g, zanamivir (6), 40, 58, 60 and 63 (neu3 specificity or bispecificity), and compounds 7d, 7g, 8b, 27, 30 and 40 (neu4 specificity or bispecificity); 20 μM or lower (e.g., the foregoing compounds and compounds 29, 38, 39 and 71 (neu1 specificity or bispecificity)), compounds 7f, 26, and 13 (neu3 specificity or bispecificity), and compounds 7b and 7c (neu4 specificity or bispecificity); or 100 μM or lower (e.g., the foregoing compounds and compounds 35 and 59 (neu1 specificity or bispecificity), compounds 53, 28, 59 and 62 (neu3 specificity or bispecificity), and compounds 7b and 7c (neu4 specificity or bispecificity)).
As used herein, the term “alkyl” refers to a monovalent straight or branched chain, saturated or unsaturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range. Thus, for example, “C1-10 alkyl” (or “C1-C10 alkyl”) refers to any of the hexyl alkyl and pentyl alkyl isomers as well as n-, iso-, sec- and t-butyl, n- and isopropyl, ethyl, and methyl. As another example, “C1-4 alkyl” refers to n-, iso-, sec- and t-butyl, n- and isopropyl, ethyl, and methyl. As another example, “C1-3 alkyl” refers to n-propyl, isopropyl, ethyl, and methyl. Alkyl include unsaturated aliphatic hydrocarbon including alkyne (R—C≡C—R); and/or alkene (R—C═C—R).
The term “halogen” (or “halo”) refers to fluorine, chlorine, bromine and iodine (alternatively referred to as fluoro, chloro, bromo, and iodo). The term “haloalkyl” refers to an alkyl group as defined above in which one or more of the hydrogen atoms have been replaced with a halogen (i.e., F, Cl, Br and/or I). Thus, for example, “C1-10 haloalkyl” (or “C1-C6 haloalkyl”) refers to a C1 to C10 linear or branched alkyl group as defined above with one or more halogen substituents.
The term “fluoroalkyl” has an analogous meaning except that the halogen substituents are restricted to fluoro. Suitable fluoroalkyls include the series (CH2)0-4CF3 (i.e., trifluoromethyl, 2,2,2-trifluoroethyl, 3,3,3-trifluoro-n-propyl, etc.).
The term “heteroalkyl” is given its ordinary meaning in the art and refers to alkyl groups as described herein in which one or more carbon atoms is replaced with a heteroatom (e.g., oxygen, nitrogen, sulfur, or derivatives thereof, and the like). Examples of heteroalkyl groups include, but are not limited to, alkoxy, alkyl-substituted amino, thiol such as methionine side group. Up to two heteroatoms may be consecutive. When a prefix such as C2-6 is used to refer to a heteroalkyl group, the number of carbons (2-6, in this example) is meant to include the heteroatoms as well.
The term “aminoalkyl” refers to an alkyl group as defined above in which one or more of the hydrogen or carbon atoms has been replaced with a nitrogen or an amino derivative. Thus, for example, “C1-6 aminoalkyl” (or “C1-C6 aminoalkyl”) refers to a C1 to C6 linear or branched alkyl group as defined above with one or more amino derivatives (e.g., NH, amide, diazirin, azide, etc.).
The term “thioalkyl” refers to an alkyl group as defined above in which one or more of the hydrogen or carbon atoms has been replaced with a sulfur atom or thiol derivative. Thus, for example, “C1-6 aminoalkyl” (or “C1-C6 aminoalkyl”) refers to a C1 to C6 linear or branched alkyl group as defined above with one or more sulfur atoms or thiol derivatives (e.g., S, SH, etc.).
Aminoalkyl and thioalkyls are specific embodiments of and encompassed by the term “heteroalkyl” or substituted alkyl depending on the heteroatom replaces a carbon atom or an hydrogen atom.
The term “cycloalkyl” refers to saturated alicyclic hydrocarbon consisting of saturated 3-8 membered rings optionally fused with additional (1-3) aliphatic (cycloalkyl) or aromatic ring systems, each additional ring consisting of a 3-8 membered ring. It includes without being so limited cyclopropane, cyclobutane, cyclopentane, and cyclohexane.
The term “heterocyclyl” refers to (i) a 4- to 7-membered saturated heterocyclic ring containing from 1 to 3 heteroatoms independently selected from N, O and S, or (ii) is a heterobicyclic ring (e.g., benzocyclopentyl). Examples of 4- to 7-membered, saturated heterocyclic rings within the scope of this disclosure include, for example, azetidinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, oxazolidinyl, isoxazolidinyl, pyrrolidinyl, imidazolidinyl, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrazolidinyl, hexahydropyrimidinyl, thiazinanyl, thiazepanyl, azepanyl, diazepanyl, tetrahydropyranyl, tetrahydrothiopyranyl, and dioxanyl. Examples of 4- to 7-membered, unsaturated heterocyclic rings within the scope of this disclosure include mono-unsaturated heterocyclic rings corresponding to the saturated heterocyclic rings listed in the preceding sentence in which a single bond is replaced with a double bond (e.g., a carbon-carbon single bond is replaced with a carbon-carbon double bond).
The terms “C(O)” and —CO refer to carbonyl. The terms “S(O)2” and “SO2” each refer to sulfonyl. The term “S(O)” refers to sulfinyl.
The term “aryl” refers to aromatic (unsaturated) compounds consisting of 3-8 membered rings, optionally fused with additional (1-3) aliphatic (cycloalkyl) or aromatic ring systems, each additional ring consisting of 3-8 membered ring. In a specific embodiment, it refers to phenyl, benzocyclopentyl, or naphthyl. The aryl of particular interest is phenyl. The term “heteroaryl” refers to (i) a 3-, 4-, 5- or 6-membered heteroaromatic ring containing from 1 to 4 heteroatoms independently selected from N, O and S, or (ii) is a heterobicyclic ring selected from quinolinyl, isoquinolinyl, and quinoxalinyl. Suitable 3-, 4-, 5- and 6-membered heteroaromatic rings include, for example, diazirin, pyridyl (also referred to as pyridinyl), pyrrolyl, diazine (e.g., pyrazinyl, pyrimidinyl, pyridazinyl), triazinyl, thienyl, furanyl, imidazolyl, pyrazolyl, triazolyl (e.g., 1, 2, 3 triazolyl), tetrazolyl (e.g., 1, 2, 3, 4 tetrazolyl), oxazolyl, iso-oxazolyl, oxadiazolyl, oxatriazolyl, thiazolyl, isothiazolyl, and thiadiazolyl. Heteroaryls of particular interest are pyrrolyl, imidazolyl, pyridyl, pyrazinyl, quinolinyl (or quinolyl), isoquinolinyl (or isoquinolyl), and quinoxalinyl. Suitable heterobicyclic rings include indolyl.
As used herein, and unless otherwise specified, the terms “alkyl”, “haloalkyl”, “aminoalkyl”, “cycloalkyl”, “heterocyclyl”, “aryl”, “heteroalkyl” and “heteroaryl” and the terms designating their specific embodiments (e.g., butyl, fluoropropyl, aminobutyl, cyclopropane, morpholine, phenyl, pyrazole, etc.) encompass the substituted (i.e. in the case of haloalkyl and aminoalkyl, in addition to their halogen and nitrogen substituents, respectively) and unsubstituted embodiments of these groups. Hence for example, the term “phenyl” encompasses unsubstituted phenyl as well as fluorophenyl, hydroxyphenyl, methylsulfonyl phenyl (or biphenyl), trifluoromethyl-diazirin-phenyl, isopropyl-phenyl, trifluorohydroxy-phenyl. Similarly, the term pyrazole, encompass unsubstituted pyrazole as well as methylpyrazole. The one or more substituents may be an amine, halogen, hydroxyl, C1-6 aminoalkyl, C1-6 heteroalkyl, C1-6 alkyl, C3-8 cycloalkyl, C1-6 haloalkyl, aryl, heteroaryl and heterocyclyl groups (etc.).
It is understood that the specific rings listed above are not a limitation on the rings which can be used in the present disclosure. These rings are merely representative.
Unless expressly stated to the contrary in a particular context, any of the various cyclic rings and ring systems described herein may be attached to the rest of the compound at any ring atom (i.e., any carbon atom or any heteroatom) provided that a stable compound results.
As used herein, the term “isomers” refers to optical isomers (enantiomers), diastereoisomers as well as the other known types of isomers.
The compounds of the disclosure have at least five asymmetric carbon atoms and can therefore exist in the form of optically pure enantiomers (optical isomers), as racemates and as mixtures thereof. Some of the compounds have at least two asymmetric carbon atoms and can therefore exist in the form of pure diastereoisomers and as mixtures thereof. It is to be understood, that, unless otherwise specified, the present disclosure embraces the racemates, the enantiomers and/or the diastereoisomers of the small molecule inhibitors of the disclosure as well as mixtures thereof.
In addition, the present disclosure embraces all geometric isomers. For example, when a compound of the disclosure incorporates a double bond or a fused ring, both the cis- and trans-forms, as well as mixtures, are embraced within the scope of the disclosure.
Within the present disclosure, it is to be understood that a compound of the disclosure may exhibit the phenomenon of tautomerism and that the formula drawings within this specification can represent only one of the possible tautomeric forms. It is to be understood that the disclosure encompasses any tautomeric form and is not to be limited merely to any one tautomeric form utilized within the formula drawings.
It is also to be understood that certain small molecule inhibitors of the disclosure may exhibit polymorphism, and that the present disclosure encompasses all such forms.
The present disclosure relates to the small molecule inhibitors of the disclosure as hereinbefore defined as well as to salts thereof. The term “salt(s)”, as employed herein, denotes basic salts formed with inorganic and/or organic bases. Salts for use in pharmaceutical compositions will be pharmaceutically acceptable salts, but other salts may be useful in the production of the compounds of the disclosure. The term “pharmaceutically acceptable salts” refers to salts of compounds of the present disclosure that are pharmacologically acceptable and substantially non-toxic to the subject to which they are administered. More specifically, these salts retain the biological effectiveness and properties of the anti-atherosclerosis compounds of the disclosure and are formed from suitable non-toxic organic or inorganic acids or bases.
For example, where the small molecule inhibitors of the disclosure are sufficiently acidic, the salts of the disclosure include base salts formed with an inorganic or organic base. Such salts include alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts and a cobalt salts; inorganic amine salts such as ammonium or substituted ammonium salts, such as e.g., trimethylammonium salts; and salts with organic bases (for example, organic amines) such as chloroprocaine salts, dibenzylamine salts, dicyclohexylamine salts, dicyclohexylamines, diethanolamine salts, ethylamine salts (including diethylamine salts and triethylamine salts), ethylenediamine salts, glucosamine salts, guanidine salts, methylamine salts (including dimethylamine salts and trimethylamine salts), morpholine salts, morpholine salts, N,N′-dibenzylethylenediamine salts, N-benzyl-phenethylamine salts, N-methylglucamine salts, phenylglycine alkyl ester salts, piperazine salts, piperidine salts, procaine salts, t-butyl amines salts, tetramethylammonium salts, t-octylamine salts, tris-(2-hydroxyethyl)amine salts, and tris(hydroxymethyl)aminomethane salts. Preferred salts include those formed with sodium, lithium, potassium, calcium and magnesium.
Such salts can be formed routinely by those skilled in the art using standard techniques. Indeed, the chemical modification of a pharmaceutical compound (i.e. drug) into a salt is a technique well known to pharmaceutical chemists, (See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457, incorporated herein by reference). Salts of the compounds of the disclosure may be formed, for example, by reacting a compound of the disclosure with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
The present disclosure relates to the small molecule inhibitors of the disclosure as hereinbefore defined as well as to the esters thereof. The term “ester(s)”, as employed herein, refers to compounds of the disclosure or salts thereof in which hydroxy groups have been converted to the corresponding esters using, for example, inorganic or organic anhydrides, acids, or acid chlorides. Esters for use in pharmaceutical compositions will be pharmaceutically acceptable esters, but other esters may be useful in the production of the compounds of the disclosure.
The term “pharmaceutically acceptable esters” refers to esters of the compounds of the present disclosure that are pharmacologically acceptable and substantially non-toxic to the subject to which they are administered. More specifically, these esters retain the biological effectiveness and properties of the anti-atherosclerosis small molecule inhibitors of the disclosure and act as prodrugs which, when absorbed into the bloodstream of a warm-blooded animal, cleave in such a manner as to produce the parent alcohol small molecule inhibitor.
Esters of the small molecule inhibitors of the present disclosure include among others the following groups (1) carboxylic acid esters obtained by esterification of the hydroxy groups, in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, ethyl, n-propyl, t-butyl, n-butyl, methyl, propyl, isopropyl, butyl, isobutyl, or pentyl), alkoxyalkyl (for example, methoxymethyl, acetoxymethyl, and 2,2-dimethylpropionyloxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted with, for example, halogen, C1-4 alkyl, or C1-4 alkoxy, or amino); (2) sulfonate esters, such as alkyl- or aralkylsulfonyl (for example, methanesulfonyl); (3) amino acid esters (for example, L-valyl or L-isoleucyl); (4) phosphonate esters; (5) mono-, di- or triphosphate esters (including phosphoramidic cyclic esters). The phosphate esters may be further esterified by, for example, a C1-20 alcohol or reactive derivative thereof, or by a 2,3-di(C6-24)acyl glycerol. (6) Carbamic acid ester (for example N-methylcarbamic ester); and (7) Carbonic acid ester (for example methylcabonate).
Further information concerning examples of and the use of esters for the delivery of pharmaceutical compounds is available in Design of Prodrugs. Bundgaard H ed. (Elsevier, 1985) incorporated herein by reference. See also, H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 108-109; Krogsgaard-Larsen, et. al., Textbook of Drug Design and Development (2d Ed. 1996) at pp. 152-191; Jarkko Rautio et al., Nat. Rev. Drug Discov., 7, pp. 255-270 (2008); and Pen-Wei Hsieh et al., Curr. Pharm. Des., 15 (19), pp. 2236-2250 (2009), all incorporated herein by reference.
The small molecule inhibitors of this disclosure may be esterified by a variety of conventional procedures including reacting the appropriate anhydride, carboxylic acid or acid chloride with an alcohol group of a compound of this disclosure. For example, an appropriate anhydride may be reacted with an alcohol in the presence of a base, such as 1,8-bis[dimethylamino]naphthalene or N,N-dimethylaminopyridine, to facilitate acylation. Also, an appropriate carboxylic acid can be reacted with an alcohol in the presence of a dehydrating agent such as dicyclohexylcarbodiimide, 1-[3-dimethylaminopropyl]-3-ethylcarbodiimide or other water-soluble dehydrating agents which are used to drive the reaction by the removal of water, and, optionally, an acylation catalyst. Esterification can also be effected using the appropriate carboxylic acid. Reaction of an acid chloride with an alcohol can also be carried out. When a compound of the disclosure contains a number of free hydroxy group, those groups not being converted into a prodrug functionality may be protected (for example, using a t-butyl-dimethylsilyl group), and later deprotected. Also, enzymatic methods may be used to selectively phosphorylate or dephosphorylate alcohol functionalities. One skilled in the art would readily know how to successfully carry out these as well as other known methods of esterification of alcohols.
Esters of the small molecule inhibitors of the disclosure may form salts. Where this is the case, this is achieved by conventional techniques as described above.
In a specific embodiment, esters of the present disclosure are compounds of formulas I, Ia, Ib, and II-VIII of the present disclosure with a methyl, ethyl, propyl, or butyl at position R1.
The small molecule inhibitors of the disclosure may exist in unsolvated as well as solvated forms with solvents such as water, ethanol, and the like, and it is intended that the disclosure embrace both solvated and unsolvated forms.
“Solvate” means a physical association of a small molecule inhibitor of this disclosure with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolatable solvates. Solvates for use in pharmaceutical compositions will be pharmaceutically acceptable solvates, but other solvates may be useful in the production of the compounds of the disclosure.
As used herein, the term “pharmaceutically acceptable solvates” means solvates of small molecule inhibitors of the present disclosure that are pharmacologically acceptable and substantially non-toxic to the subject to which they are administered. More specifically, these solvates retain the biological effectiveness and properties of the anti-atherosclerosis small molecule inhibitors of the disclosure and are formed from suitable non-toxic solvents.
Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like, as well as hydrates, which are solvates wherein the solvent molecules are H2O.
Preparation of solvates is generally known. Thus, for example, Ciara, 2004, incorporated herein by reference, describe the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar preparations of solvates, hemisolvate, hydrates and the like are described by van Tonder, 2004; Bingham, 2001, both incorporated herein by reference.
A typical, non-limiting, process for preparing a solvate involves dissolving the inventive compound in desired amounts of the desired solvent (organic or water or mixtures thereof) at a higher than ambient temperature and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example IR spectroscopy, can be used to show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).
The present disclosure also encompasses the use of antibodies that specifically bind to either of neuraminidase 1 (NP_000425.1); to neuraminidase 3 (isoform 1 (Q9UQ49-1); or 2 (Q9UQ49-2) or to neuraminidase 4 (isoform 1 (NP_542779.2); 2 (NP_001161071.1); or 3 (NP_001161072.1). (see
As indicated above, illustrative human neuraminidase amino acid sequences are presented in
Antibodies that specifically bind to neuraminidase 1, 3 or 4 may be devised by targeting epitope regions of these neuraminidases that are specifically found in each of these enzymes. An epitope of a protein/polypeptide is defined as a fragment of said protein/polypeptide of at least about 4 or 5 amino acids in length, capable of eliciting a specific antibody and/or an immune cell (e.g., a T cell or B cell) bearing a receptor capable of specifically binding said epitope. Two different kinds of epitopes exist: linear epitopes and conformational epitopes. A linear epitope comprises a stretch of consecutive amino acids. A conformational epitope is typically formed by several stretches of consecutive amino acids that are folded in position and together form an epitope in a properly folded protein. An immunogenic fragment as used herein refers to either one, or both, of said types of epitopes. Without being so limited, epitopes in a sequence may be predicted with softwares such as BCPred™, AAP™, FBCPred™ and ABCPred™.
Methods for making antibodies are well known in the art. Polyclonal antibodies can be prepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, or other mammal) with the polypeptide/protein of interest or a fragment thereof as an immunogen. A polypeptide/protein “fragment” “portion” or “segment” is a stretch of amino acid residues of at least about 5, 7, 10, 14, 15, 20, 21 or more amino acids of the polypeptide noted above. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized exosomal marker polypeptide or a fragment thereof. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the animal, usually a mouse, and can be used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256: 495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4: 72), the EBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York, N.Y.), pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Coligan et al., eds. (1994) Current Protocols in Immunology, John Wiley & Sons, Inc., New York, N.Y.).
Alternatively, to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a polypeptide or a fragment thereof to thereby isolate immunoglobulin library members that bind the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System™, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).
Without being so limited, anti-neuraminidase 1 (lysosomal sialidase) antibodies include: Anti-NEU1/NEU Antibody (aa172-221) IHC-plus™ from LifeSpan BioScience; Human NEU-1/Sialidase-1 Antibody (MAB6860) from R & D Systems; Human NEU-1/Sialidase-1 Antibody (MAB6860-SP) from R & D systems; NEU-1/Sialidase-1 Antibody (3D4) (NBP2-46152) from Novus Biologicals; NEU-1/Sialidase-1 Antibody (H00004758-B02P-50 ug) from Novus Biologicals; anti-Neuraminidase, NEU (NEU) (Internal Region) antibody (ABIN964880); Monoclonal Antibody to Neuraminidase (NEU) (MAB611Hu21) from Cloud-Clone; Anti-NEU1 (HPA015634) from Atlas antibody.
Without being so limited, anti-neuraminidase 3 (membrane sialidase) antibodies include Anti-NEU3 Antibody (clone 11B) (LS-C179421-100) from Lifespans BioScience; anti-Neu3 antibody (ABIN1449196) from Antibodies online; NEU3 Antibody (NBP2-48694) from Novus Biologicals; Anti-NEU3 Antibody (HPA038730) from Atlas Antibodies; Anti-NEU3 (Human) mAb (D164-3) from MBL International; Sialidase 3 antibody (orb186135) from Biorbyt, etc.
Without being so limited, anti-neuraminidase 4 antibodies include anti-neu4 antibody (ab107258) from Abcam; anti-neu4 antibody (NBP2-32682) from Novus biotechnologicals; anti-neu4 antibody (OAAB00394) from Aviva Systems Biology; anti-neu4 antibody (AP52856PU-N) from Origen; anti-neu4 antibodies ((HPA037394) from Atlas Antibodies, etc.
In a specific embodiment, the specific inhibitor of the present disclosure is a double-stranded RNA (dsRNA) molecule (or a molecule comprising region of double-strandedness). The dsRNA comprises a subsequence of a neu1 and/or neu3 polynucleotide (e.g., a subsequence of the sequence encoding neu1, neu3 or neu4 disclosed in
Generation of Anti-Neu1, Anti-Neu3 or Anti-Neu4 dsRNA Molecules
In some embodiments, an anti-neu1, anti-neu3 or anti-neu4 double stranded RNA molecule with sequences complementary to a target is generated. The synthesis of an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule comprises: (a) synthesis of two complementary strands of the dsRNA molecule; and (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded RNA molecule. In another embodiment, synthesis of the two complementary strands of the RNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the RNA molecule is by solid phase tandem oligonucleotide synthesis. In some embodiments, a nucleic acid molecule described herein is synthesized separately and joined together post-synthetically, for example, by ligation or by hybridization following synthesis and/or deprotection. Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using any suitable method. dsRNA constructs can be purified by gel electrophoresis or can be purified by high pressure liquid chromatography.
In some embodiments, an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is about 20-25 bp. In some embodiments, the 20-25 bp dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) has 2-5 bp overhangs on the 3′ end of each strand, and a 5′ phosphate terminus and a 3′ hydroxyl terminus. In some embodiments, the 20-25 bp dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) has blunt ends.
In some embodiments, an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the anti-sense strand, wherein the anti-sense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the anti-sense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 19 base pairs). In some embodiments, the anti-sense strand of an anti-neu1 or anti-neu3 dsRNA molecule (e.g., siRNA molecules, miRNA molecules, and analogues thereof) comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some embodiments, an anti-neu1 or anti-neu3 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is assembled from a single oligonucleotide, where the self-complementary sense and anti-sense regions of the dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) are linked by means of a nucleic acid-based or non-nucleic acid-based linker(s).
In some embodiments, an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) comprises a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) does not require the presence within the dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide further comprises a terminal phosphate group, such as a 5′-phosphate, or 5′,3′-diphosphate.
In other embodiments, an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) comprises separate sense and anti-sense sequences or regions, wherein the sense and anti-sense regions are covalently linked by nucleotide or non-nucleotide linker molecules, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and/or stacking interactions.
The terminal structure of dsRNA molecules described herein is either blunt or cohesive (overhanging). In some embodiments, the cohesive (overhanging) end structure is a 3′ overhang or a 5′ overhang. In some embodiments, the number of overhanging nucleotides is any length as long as the overhang does not impair gene silencing activity. In some embodiments, an overhang sequence is not complementary (anti-sense) or identical (sense) to the neu1, neu3 or neu4 sequence. In some embodiments, the overhang sequence contains low molecular weight structures (for example a natural RNA molecule such as tRNA, rRNA or tumor or CTC RNA, or an artificial RNA molecule).
The total length of dsRNA molecules having cohesive end structure is expressed as the sum of the length of the paired double-stranded portion and that of a pair comprising overhanging single-strands at both ends. For example, in the exemplary case of a 19 bp double-stranded RNA with 4 nucleotide overhangs at both ends, the total length is expressed as 23 bp.
In some embodiments, the terminal structure of an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) has a stem-loop structure in which ends of one side of the double-stranded nucleic acid are connected by a linker nucleic acid, e.g., a linker RNA. In some embodiments, the length of the double-stranded region (stem-loop portion) is 15 to 49 bp, often 15 to 35 bp, and more commonly about 21 to 30 bp long.
In some embodiments, an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule is a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and anti-sense regions, wherein the anti-sense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
In some embodiments, an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) comprises a circular nucleic acid molecule, wherein the dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.
In some embodiments, a circular dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) contains two loop motifs, wherein one or both loop portions of the dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is biodegradable. In some embodiments, degradation of the loop portions of a circular dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) generates a double-stranded dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) with 3′-terminal overhangs, such as 3-terminal nucleotide overhangs comprising about 2 nucleotides.
The sense strand of a double stranded dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) may have a terminal cap moiety such as an inverted deoxybasic moiety, at the 3-end, 5′-end, or both 3′ and 5′-ends of the sense strand.
In some embodiments, the 3′-terminal nucleotide overhangs of an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In some embodiments, the 3′-terminal nucleotide overhang comprises one or more universal base ribonucleotides. In some embodiments, the 3′-terminal nucleotide overhang comprises one or more acyclic nucleotides.
In some embodiments, an anti- anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) disclosed herein is capable of specifically binding to desired neu1 or neu3 variants while being incapable of specifically binding to non-desired neu1 or neu3 variants.
In some embodiments, an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is selected for use in a method disclosed herein based on predictions of the stability of molecule. In some embodiments, a prediction of stability is achieved by employing a theoretical melting curve wherein a higher theoretical melting curve indicates an increase in the molecule's stability and a concomitant decrease in cytotoxic effects. In some embodiments, stability of an anti-neu1 or anti-neu3 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is determined empirically by measuring the hybridization of a single modified RNA strand containing one or more universal-binding nucleotide(s) to a complementary neu1 or neu3 sequence within, for example, a polynucleotide array. In some embodiments, the melting temperature (i.e., the Tm value) for each modified RNA and complementary RNA immobilized on the array is determined and, from this Tm value, the relative stability of the modified RNA pairing with a complementary RNA molecule determined.
In some embodiments, an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is selected for use in a method disclosed herein based on “off-target” profiling whereby one or more dsRNA molecules is administered to a cell(s), either in vivo or in vitro, and total mRNA is collected, and used to probe a microarray comprising oligonucleotides having one or more nucleotide sequence from a panel of known genes, including non-target genes. The “off-target” profile of the modified dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is quantified by determining the number of non-target genes having reduced expression levels in the presence of the RNAi molecule. The existence of “off target” binding indicates an anti-neu1 or anti-neu3 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) that is capable of specifically binding to one or more non-target gene. Ideally, an anti-neu1 or anti-neu3 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) applicable to therapeutic use will exhibit a high Tm value while exhibiting little or no “off-target” binding.
In some embodiments, an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is selected for use in a method disclosed herein by use of a report gene assay. In some embodiments, a reporter gene construct comprises a constitutive promoter, for example the cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter, operably fused to, and capable of modulating the expression of, one or more reporter gene such as, for example, a luciferase gene, a chloramphenicol (CAT) gene, and/or a β-galactosidase gene, which, in turn, is operably fused in-frame with an oligonucleotide (typically between about 15 base-pairs and about 40 base-pairs, more typically between about 19 base-pairs and about 30 base-pairs, most typically 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 base-pairs) that contains a target sequence for the one or more RNAi molecules. In some embodiments, individual reporter gene expression constructs are co-transfected with one or more RNAi molecules. In some embodiments, the capacity of a given dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) to reduce the expression level of each of the contemplated gene variants is determined by comparing the measured reporter gene activity from cells transfected with and without the modified RNAi molecule.
In some embodiments, an anti-neu1, anti-neu3 or anti-neu4 dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is selected for use in a method disclosed herein by assaying its ability to specifically bind to an mRNA, such as an mRNA expressed by a target cell.
The present disclosure also relates to the use of the above-mentioned inhibitors of the disclosure and in the case of small molecule inhibitors, their pharmaceutically acceptable salts, esters, and solvates thereof in the preparation of a medicament, a combination or a kit.
The present disclosure also relates to pharmaceutical compositions comprising the above-mentioned inhibitors of the disclosure or, in the case of small molecule inhibitors, their pharmaceutically acceptable salts, esters and solvates thereof.
Without being so limited, the medicaments/pharmaceutical compositions of the disclosure may be administered orally, for example in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. Administration can also be carried out rectally, for example using suppositories; locally, topically, or percutaneously, for example using ointments, creams, gels or solutions; or parenterally, e.g., intravenously, intramuscularly, subcutaneously, intrathecally or transdermally, using for example injectable solutions. Furthermore, administration can be carried out sublingually, nasally, or as ophthalmological preparations or an aerosol, for example in the form of a spray, such as a nasal spray.
For the preparation of tablets, coated tablets, dragees or hard gelatin capsules, the compounds of the present disclosure may be admixed with any known pharmaceutically inert, inorganic or organic excipient and/or carrier. Examples of suitable excipients/carriers include lactose, maize starch or derivatives thereof, talc or stearic acid or salts thereof.
Suitable excipients for use with soft gelatin capsules include for example vegetable oils, waxes, fats, semi-solid or liquid polyols etc. According to the nature of the active ingredients it may however be the case that no excipient is needed at all for soft gelatin capsules.
For the preparation of solutions and syrups, excipients which may be used include for example water, polyols, saccharose, invert sugar and glucose.
For injectable solutions, excipients which may be used include for example water, saline, alcohols, polyols, glycerin, vegetable oils and other appropriate excipients.
For suppositories, and local or percutaneous application, excipients which may be used include for example natural or hardened oils, waxes, fats and semi-solid or liquid polyols.
The medicaments/pharmaceutical compositions may also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents or antioxidants. They may also contain other therapeutically active agents.
Intravenous, or oral administrations are preferred forms of use. The dosages in which the inhibitors of the disclosure are administered in effective amounts depend on the nature of the specific active ingredient, the age and the requirements of the patient and the mode of application.
As mentioned above, the pharmaceutical compositions of the disclosure can contain a pharmaceutically acceptable carrier including, without limitation, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters. Aqueous carriers include, without limitation, water, alcohol, saline, and buffered solutions.
Pharmaceutically acceptable carriers also can include physiologically acceptable aqueous vehicles (e.g., physiological saline) or other known carriers appropriate to specific routes of administration.
The inhibitors of the disclosure may be incorporated into dosage forms in conjunction with any of the vehicles which are commonly employed in pharmaceutical preparations, e.g., talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives or glycols. Emulsions such as those described in U.S. Pat. No. 5,434,183, incorporated herein by reference, may also be used in which vegetable oil (e.g., soybean oil or safflower oil), emulsifying agent (e.g., egg yolk phospholipid) and water are combined with glycerol. Methods for preparing appropriate formulations are well known in the art (see e.g., Remington's Pharmaceutical Sciences, 16th Ed., 1980, A. Oslo Ed., Easton, Pa. incorporated herein by reference).
In cases where parenteral administration is elected as the route of administration, preparations containing the inhibitors of the disclosure may be provided to patients in combination with pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose, and the like.
It is a prerequisite that all adjuvants used in the manufacture of the preparations, such as carriers, are non-toxic and more generally pharmaceutically acceptable.
As used herein, “pharmaceutically acceptable” such as pharmaceutically acceptable carrier, excipient, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular inhibitor is administered.
Any amount of a pharmaceutical composition can be administered to a subject. The dosages will depend on many factors including the mode of administration. Typically, the amount of the inhibitor of the disclosure contained within a single dose will be an amount that effectively prevent, delay or treat the disease or condition to be treated, delayed or prevented without inducing significant toxicity.
The effective amount of the inhibitors of the disclosure may also be measured directly. The effective amount may be given daily or weekly or fractions thereof. Typically, a pharmaceutical composition of the disclosure can be administered in an amount from about 0.001 mg up to about 500 mg per kg of body weight per day (e.g., 10 mg, 50 mg, 100 mg, or 250 mg). Dosages may be provided in either a single or multiple dosage regimen. For example, in some embodiments the effective amount may range from about 1 mg to about 25 grams of the composition per day, about 50 mg to about 10 grams of the composition per day, from about 100 mg to about 5 grams of the composition per day, about 1 gram of the composition per day, about 1 mg to about 25 grams of the composition per week, about 50 mg to about 10 grams of the composition per week, about 100 mg to about 5 grams of the composition every other day, and about 1 gram of the composition once a week.
These are simply guidelines since the actual dose must be carefully selected and titrated by the attending physician based upon clinical factors unique to each patient. The optimal daily dose will be determined by methods known in the art and will be influenced by factors such as the age of the patient and other clinically relevant factors. In addition, patients may be taking medications for other diseases or conditions. The other medications may be continued during the time that the pharmaceutical composition of the disclosure is given to the patient, but it is particularly advisable in such cases to begin with low doses to determine if adverse side effects are experienced.
In accordance with another aspect, there is provided a combination of at least one of the inhibitors described herein (e.g., a specific neu1 inhibitor, a specific neu3 inhibitor, a specific neu4 inhibitor, or a bispecific neu1, neu3 or neu4 inhibitor (e.g., neu3/neu4 inhibitor)) with another of the inhibitors described herein and/or with another agent that modulates inflammation (e.g., anti-inflammatory agent) and/or with a non-pharmaceutical treatment/regimen. Without being so limited, such anti-inflammatory agents include NSAIDs, etc.
In accordance with another aspect, when the targeted disease is ITP, there is provided a combination of at least one of the inhibitors described herein (e.g., a specific neu1 inhibitor, a specific neu3 inhibitor, a specific neu4 inhibitor, or a bispecific neu1, neu3 or neu4 inhibitor (e.g., neu3/neu4 inhibitor)) with another of the inhibitors described herein and/or with another agent that treats or prevents ITP or a symptom thereof including a IVIg, a corticosteroid, eltrombopag, romiplostim, and/or fostamatinib.
In accordance with an aspect, there is provided a composition comprising at least one of the inhibitors as defined herein, and (i) another of the inhibitors described herein; (ii) another agent that modulates inflammation; (iii) a pharmaceutically acceptable carrier; or (iv) a combination of at least two of (i) to (iii). In accordance with another aspect, there is provided a method of modulating leukocytes activation (e.g., adhesion and/or transmigration and/or cytokine response (e.g., in response to bacterial infection)) comprising administering an effective amount of at least one of the inhibitors described herein (e.g., a specific neu1 inhibitor, a specific neu3 inhibitor, a specific neu3 inhibitor, or a bispecific neu1, neu3 or neu4 inhibitor (e.g., neu3/neu4 inhibitor)); and (i) another of the inhibitors described herein (e.g., a specific neu1, neu3 or neu4 (or bispecific neu1, neu3 or neu4 inhibitor (e.g., neu3/neu4 inhibitor)); (ii) another that modulates inflammation; and/or (iii) a non-pharmaceutical means.
In a specific embodiment, said composition is a pharmaceutical composition. In another specific embodiment, the composition comprises (i) an inhibitor as defined herein; and (ii) another agent that modulates inflammation.
In accordance with an aspect, there is provided a composition comprising at least one of the inhibitors as defined herein, and (i) another of the inhibitors described herein; (ii) another agent that modulates thrombocyte clearance (or that prevents or treats ITP or a symptom thereof); (iii) a pharmaceutically acceptable carrier; or (iv) a combination of at least two of (i) to (iii). In accordance with another aspect, there is provided a method of modulating inflammation (or preventing or treating ITP or a symptom thereof) comprising administering an effective amount of at least one of the inhibitors described herein (e.g., a specific neu1 inhibitor, a specific neu3 inhibitor, a specific neu3 inhibitor, or a bispecific neu1, neu3 or neu4 inhibitor (e.g., neu3/neu4 inhibitor)); and (i) another of the inhibitors described herein (e.g., a specific neu1, neu3 or neu4 (or bispecific neu1, neu3 or neu4 inhibitor (e.g., neu3/neu4 inhibitor)); (ii) another that modulates thrombocyte clearance (or that prevents or treats ITP or a symptom thereof); and/or (iii) a non-pharmaceutical means.
In a specific embodiment, said composition is a pharmaceutical composition. In another specific embodiment, the composition comprises (i) an inhibitor as defined herein; and (ii) another agent that modulates inflammation. In another specific embodiment, the composition comprises (i) an inhibitor as defined herein; and (ii) another agent that modulates thrombocyte clearance (or that prevents or treats ITP or a symptom thereof).
In accordance with another aspect of the present disclosure, there is provided a kit comprising the inhibitor defined herein or the above-mentioned composition, and instructions to use same in the modulation of leukocytes activation (e.g., leukocytes adhesion and/or transmigration and/or cytokine response (e.g., in response to bacterial infection)) of the instant disclosure.
In a specific embodiment of the kit, the kit comprises: (i) another of the inhibitors described herein; (ii) another agent that modulates inflammation; (iii) instructions to use same in the modulation of leukocytes activation (e.g., leukocytes adhesion and/or transmigration and/or cytokine response (e.g., in response to bacterial infection)); or (iv) a combination of at least two of (i) to (iii).
In accordance with another aspect of the present disclosure, there is provided a kit comprising the inhibitor defined herein or the above-mentioned composition, and instructions to use same in the modulation of thrombocyte clearance (or that prevents or treats ITP or a symptom thereof) of the instant disclosure.
In a specific embodiment of the kit, the kit comprises: (i) another of the inhibitors described herein; (ii) another agent that modulates thrombocyte clearance (or that prevents or treats ITP or a symptom thereof); (iii) instructions to use same in the modulation of thrombocyte clearance (or that prevents or treats ITP or a symptom thereof); or (iv) a combination of at least two of (i) to (iii).
In accordance with another aspect of the present disclosure, there is provided a method of identifying an agent that modulates leukocytes activation (e.g., leukocytes adhesion and/or transmigration and/or cytokine response (e.g., in response to bacterial infection)) (and optionally modulates inflammation), said method comprising contacting a neuraminidase 1, a neuraminidase 3 or a neuraminidase 4 (or a cell expressing same) with a candidate compound (and eventually a neuraminidase 1, neuraminidase 3 or neuraminidase 4 substrate)) and determining the effect of said candidate compound on the neuraminidase 1, 3 or 4 expression and/or activity (e.g., ability of compound to prevent neuraminidase 1, neuraminidase 3 or a neuraminidase 4 modulate leukocyte activation), wherein a decrease in the expression and/or activity of the neuraminidase 1, 3 or 4 in the presence as compared to in the absence of said candidate compound is an indication that said candidate compound may modulate leukocytes activation (e.g., leukocytes adhesion and/or transmigration and/or cytokine response (e.g., in response to bacterial infection)).
As used herein the terms “neuraminidase 1, neuraminidase 3 or neuraminidase 4 activity” refers to ApoB desialylation (e.g., in plasma) by one or more of these enzymes and to events downstream of this desialylation such as LDL uptake by macrophages, formation of foam cells, LDL incorporation in arterial walls, increase of fatty streak regions number on arterial walls, increase of fatty streak regions size on arterial walls, infiltration of T cell in atherosclerotic lesions, infiltration of macrophages, vascular smooth muscle cells or leukocytes in atherosclerotic lesions, production of extracellular matrix molecules, collagen and elastin, formation of a fibrous cap that covers the plaque, cellular necrosis, plaque rupture and thrombosis; and leukocyte transmigration and adhesion. Neuraminidase 1, neuraminidase 4 or neuraminidase 3 activity can further be measured in vitro and in situ using substrates such as sialylated ApoB, 4-Mu-5NeuAc, sialyllactose or other knows substrates of neuraminidases/sialidases. The terms “neuraminidase 1, neuraminidase 3 or neuraminidase 4 activity” may also refer to modification of glycoproteins found on leukocytes and/or thrombocytes leading to hyposialylation of any of these cells, and events downstream of this event including activation of leukocytes and/or or thrombocytes receptors, transmigration (e.g., of leukocytes), adhesion (e.g., of leukocytes), opsonization, cytokines response or clearance of thrombocytes from the bloodstream.
As used herein the terms “subject in need thereof” refer to a subject who would benefit from receiving an effective amount of the inhibitor of the present disclosure (e.g., subject having a bacterial infection or subject having IPT). It refers to an animal and to a human. The inhibitors of the present disclosure may be used for veterinary applications and be used in pets or other animals (e.g., pets such as cats, dogs, horses, etc.; and cattle, fishes, swine, poultry, etc.).
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the following items) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed.
No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.
Herein, the term “about” has its ordinary meaning. In embodiments, it may mean plus or minus 10% of the numerical value qualified.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
The present disclosure is illustrated in further details by the following non-limiting examples.
A series of compounds were designed, synthesized and their inhibitory effects were tested against the four isoenzymes of human neuraminidases. The inventors first varied the aromatic ring of a C9-triazole DANA derivative, including electron-withdrawing and electron-donating groups, negatively and positively charged groups, as well as larger phenyl and phenoxyl groups (7,
The inventors also synthesized compounds with different phenyltriazole groups at C9; nitrogen-containing groups at C4, including guanidine (6), azido, amino groups; and combinations with modifications at both C9 and C4 (8,
Compounds with phenyltriazole groups at C9 were synthesized using C9-azido-DANA methyl ester (9), which could be obtained from Neu5Ac in 6 steps (Zou, 2010). CuCAAC (copper-catalyzed azide-alkyne cycloaddition) was applied with para-substituted phenylalkynes to introduce the various C9 modifications (7a-7j,
For compounds with combined C4 and C9 modifications, two strategies were adopted (synthetic routes shown in
To generate compounds with only C4 amide moieties, compound 19 was treated with different anhydrides or acyl chlorides to form the desired amides (synthetic route shown in
General Synthetic Procedures. All reagents and solvents were purchased from Sigma-Aldrich unless otherwise noted and used without further purification. Reactions were monitored with TLC (Merck TLC Silica gel 60 F254) and spots were visualized under UV light (254 nm) or by charring with 0.5% H2SO4/EtOH. Compounds were purified by flash column chromatography with silica gel (SiliaFlash™ F60, 40-63 μm particle size) or recrystallization with the solvent mixtures specified in the corresponding experiments. Proton (1H) and carbon (13C) NMR spectra were recorded on Varian™ 400 (400 MHz for 1H; 100 MHz for 13C) or Varian™ 500 (500 MHz for 1H; 125 MHz for 13C). High-resolution mass spectrometry (HR-MS) analysis was performed on Agilent Technologies™ 6220 TOF spectrometer. Purity of all final products used for inhibitor assays was determined to be ≥95% by HPLC.
Was synthesized as previously reported. (Zou, 2010) 1H NMR (500 MHz, cd3od) δ 5.67 (d, J=2.3 Hz, 1H, H-3), 4.36 (dd, J=8.6, 2.3 Hz, 1H, H-4), 4.10 (dd, J=10.9, 1.1 Hz, 1H, H-6), 3.99 (dd, J=10.9, 8.6 Hz, 1H, H-5), 3.87 (ddd, J=9.1, 5.4, 3.1 Hz, 1H, H-8), 3.80 (dd, J=11.4, 3.1 Hz, 1H, H-9), 3.65 (dd, J=11.4, 5.4 Hz, 1H, H-9′), 3.52 (dd, J=9.1, 1.1 Hz, 1H, H-7), 2.02 (s, 3H, COCH3). 13C NMR (125 MHz, cd3od) δ 174.68, 170.02 (C═O), 149.95 (C-2), 108.34 (C-3), 77.24 (C-6), 71.29 (C-8), 70.22 (C-7), 68.70 (C-4), 64.94 (C-9), 51.96 (C-5), 22.82 (COCH3). HR-MS (ESI) calcd. for C11H16NO8 [M−H]−, 290.0876; found 290.0879.
Was synthesized as previously reported (von Itzstein, 1994; von Itzstein, 1993). 1H NMR (500 MHz, d2o) δ 5.70 (d, J=1.9 Hz, 1H, H-3), 4.54 (dd, J=9.3, 1.9 Hz, 1H, H-4), 4.46 (m, 1H, H-6), 4.29 (dd, J=10.5, 9.3 Hz, 1H, H-5), 4.02 (ddd, J=9.1, 6.2, 2.5 Hz, 1H, H-8), 3.96 (dd, J=11.9, 2.5 Hz, 1H, H-9), 3.77-3.69 (m, 2H, H-7, H-9′), 2.11 (s, 3H, COCH3). 13C NMR (125 MHz, d2o) δ 175.38, 170.10 (C═O), 157.99 (C═N), 150.19 (C-2), 104.79 (C-3), 76.33 (C-6), 70.74 (C-8), 69.11 (C-7), 64.03 (C-9), 52.11 (C-4), 48.71 (C-5), 22.93 (COCH3). HR-MS (ESI) calcd. for C12H21N4O7 [M+H]+, 333.1405; found 333.1400.
To a solution of Methyl 5-acetamido-9-azido-2,6-anhydro-3,5-dideoxy-
Compound 7a was prepared as above in 75% yield (86 mg). 1H NMR (500 MHz, CD3OD) δ 8.11 (s, 1H, Triazole-H5), 7.64 (d, J=8.9 Hz, 2H, Ar—H), 6.81 (d, J=8.9 Hz, 2H, Ar—H), 5.90 (d, J=2.3 Hz, 1H, H-3), 4.49 (dd, J=14.0, 7.5 Hz, 1H, H-9′), 4.40 (dd, J=8.7, 2.3 Hz, 1H, H-4), 4.31-4.25 (m, 1H, H-8), 4.14 (dd, J=10.9, 1.0 Hz, 1H, H-6), 3.99 (dd, J=10.9, 8.7 Hz, 1H, H-5), 3.41 (dd, J=9.2, 1.0 Hz, 1H, H-7), 2.96 (s, 6H, N(CH3)2), 1.99 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 175.16 (C═O), 152.12, 127.59, 120.10, 113.93 (Ar—C), 149.18 (Triazole-C4), 121.83 (Triazole-C5), 77.67 (C-6), 71.23 (C-7), 69.79 (C-4), 68.01 (C-8), 55.13 (C-9), 51.96 (C-5), 40.78 (N—CH3), 22.65 (COCH3). HR-MS (ESI) calcd. for C21H26N5O7 [M−H]−, 460.1832; found 460.1834.
Compound 7b was prepared as above in 79% yield (90 mg). 1H NMR (500 MHz, CD3OD) δ 8.28 (s, 1H, Triazole-H5), 7.76 (d, J=8.2 Hz, 2H, Ar—H), 7.62 (d, J=8.2 Hz, 2H, Ar—H), 5.70 (s, 1H, H-3), 4.50 (dd, J=13.5, 7.7 Hz, 1H, H-9′), 4.35 (d, J=8.6 Hz, 1H, H-4), 4.30-4.27 (m, 1H, H-8), 4.10 (d, J=10.7 Hz, 1H, H-6), 4.04-3.96 (m, 1H, H-5), 3.39 (d, J=7.7 Hz, 1H, H-7), 2.13, 1.98 (2×s, 3H, 2×COCH3). 13C NMR (125 MHz, d6-DMSO) δ 172.10, 168.34, 168.25 (C═O), 165.18 (C═O), 147.63 (C-2), 145.74 (Triazole-C4), 121.67 (Triazole-C5), 138.80, 138.69, 125.72, 125.44, 119.20, 119.11 (Ar—C), 108.60 (C-3), 75.66 (C-6), 69.95 (C-7), 68.10 (C-4), 65.90 (C-8), 53.70 (C-9), 50.81 (C-5), 22.97, 22.49 (COCH3). HR-MS (ESI) calcd. for C21H24N5O8 [M−H]−, 474.1625; found 474.1636.
Compound 7c was prepared as above in 77% yield (80 mg). 1H NMR (500 MHz, CD3OD) δ 8.09 (s, 1H, Triazole-H5), 7.55 (d, J=8.6 Hz, 2H), 6.79 (d, J=8.6 Hz, 2H), 5.88 (d, J=2.4 Hz, 1H, H-3), 4.82 (dd, J=14.1, 2.6 Hz, 1H, H-9), 4.48 (dd, J=14.1, 7.6 Hz, 1H, H-9′), 4.41 (dd, J=8.7, 2.4 Hz, 1H, H-4), 4.28 (ddd, J=9.5, 7.6, 2.6 Hz, 1H, H-8), 4.14 (dd, J=10.9, 1.0 Hz, 1H, H-6), 4.00 (dd, J=10.9, 8.7 Hz, 1H, H-5), 3.41 (dd, J=9.5, 1.0 Hz, 1H, H-7), 1.98 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 175.13, 166.94 (C═O), 149.13, 148.42 (Ar—C, Triazole-C4), 146.69 (C-2), 127.77, 116.96 (Ar—C), 122.01, 121.94 (Ar—C, Triazole-C5), 112.19 (C-3), 77.57 (C-6), 71.24 (C-7), 69.81 (C-4), 68.08 (C-8), 55.14 (C-9), 51.93 (C-5), 22.72 (COCH3). HR-MS (ESI) calcd. for C19H22N5O7 [M−H]−, 432.1529; found 432.1513.
Compound 7d was prepared as above in 86% yield (100 mg). 1H NMR (500 MHz, CD3OD) δ 8.24 (s, 1H, Trizole-H), 7.68 (d, J=8.0 Hz, 2H, Ar—H), 7.22 (d, J=8.0 Hz, 2H, Ar—H), 5.79 (s, 1H, H-3), 4.50 (dd, J=13.9, 7.4 Hz, 1H, H-9′), 4.39 (d, J=8.2 Hz, 1H, H-4), 4.29 (brs, 1H, H-8), 4.13 (d, J=10.9 Hz, 1H, H-6), 4.06-3.97 (m, 1H, H-5), 3.42 (d, J=9.0 Hz, 1H, H-7), 2.34 (s, 3H, PhCH3), 1.98 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 175.00 (C═O), 148.64 (Triazole-C4), 123.08 (Triazole-C5), 139.31, 130.58, 129.01, 126.62 (Ar—C), 110.40 (C-3), 77.27 (C-6), 71.24 (C-7), 69.84 (C-4), 68.35 (C-8), 55.16 (C-9), 51.95 (C-5), 22.76 (COCH3), 21.31 (PhCH3). HR-MS (ESI) calcd. for C20H23N4O7 [M−H]−, 431.1567; found 431.1568.
Compound 7e was prepared as above in 74% yield (80 mg). 1H NMR (500 MHz, CD3OD) δ 8.20 (s, 1H, Triazole-H5), 7.72 (d, J=8.9 Hz, 2H), 6.97 (d, J=8.9 Hz, 2H), 5.77 (d, J=2.0 Hz, 1H, H-3), 4.49 (dd, J=14.0, 7.6 Hz, 1H, H-9′), 4.39 (dd, J=8.7, 2.0 Hz, 1H, H-4), 4.32-4.25 (m, 1H, H-8), 4.12 (d, J=10.8 Hz, 1H, H-6), 4.00 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.81 (s, 3H, COCH3), 3.41 (d, J=9.2 Hz, 1H, H-7), 1.98 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 174.96 (COCH3), 161.27, 128.00, 124.40, 115.37 (Ar—C), 148.49 (Triazole-C4), 122.59 (Triazole-C5), 109.95 (C-3), 77.20 (C-6), 71.26 (C-7), 69.86 (C-4), 68.38 (C-8), 55.80 (PhOCH3), 55.15 (C-9), 51.97 (C-5), 22.74 (COCH3). HR-MS (ESI) calcd. for C20H23N4O8 [M−H]−, 447.1516; found 447.1527.
Compound 7f was prepared as above in 83% yield (80 mg). 1H NMR (500 MHz, CD3OD) δ 8.28 (s, 1H, Triazole-H5), 7.87-7.80 (m, 2H, Ar—H), 7.20-7.13 (m, 2H, Ar—H), 5.92 (d, J=2.4 Hz, 1H, H-3), 4.86 (dd, J=14.0, 2.6 Hz, 1H, H-9), 4.51 (dd, J=14.0, 7.7 Hz, 1H, H-9′), 4.41 (dd, J=8.7, 2.4 Hz, 1H, H-4), 4.29 (ddd, J=9.6, 7.7, 2.6 Hz, 1H, H-8), 4.14 (dd, J=10.8, 1.1 Hz, 1H, H-6), 3.99 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.43 (dd, J=9.6, 1.1 Hz, 1H, H-7), 1.99 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 175.15 (C═O), 164.09 (d, J=245.9 Hz, Ar—C), 147.63 (Triazole-C4), 123.31 (Triazole-C5), 128.61 (d, J=8.2 Hz, Ar—C), 128.34 (d, J=3.2 Hz, Ar—C), 116.77 (d, J=22.0 Hz, Ar—C), 112.86 (C-3), 77.70 (C-6), 71.30 (C-7), 69.80 (C-4), 67.95 (C-8), 55.25 (C-9), 51.96 (C-5), 22.64 (COCH3). HR-MS (ESI) calcd. for C19H20FN4O7 [M−H]−, 435.1316; found 435.1324.
Compound 7g was prepared as above in 45% yield (40 mg). 1H NMR (500 MHz, CD3OD) δ 8.44 (s, 1H, Triazole-C), 8.02 (d, J=8.2 Hz, 2H, Ar—H), 7.72 (d, J=8.2 Hz, 2H, Ar—H), 5.95 (d, J=2.4 Hz, 1H, H-3), 4.90 (dd, J=14.0, 2.6 Hz, 1H, H-9), 4.53 (dd, J=14.0, 7.7 Hz, 1H, H-9′), 4.42 (dd, J=8.7, 2.4 Hz, 1H, H-4), 4.37-4.21 (m, 1H, H-8), 4.16 (dd, J=10.8, 1.0 Hz, 1H, H-6), 4.00 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.45 (dd, J=9.1, 1.0 Hz, 1H, H-7), 1.99 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 175.20 (C═O), 147.04 (Triazole-C4), 124.50 (Triazole-C5), 135.81 (Ar—C), 130.90 (q, J=32.3 Hz, Ar—C), 127.01 (Ar—C), 126.89 (q, J=3.8 Hz), 113.43 (C-3), 77.81 (C-6), 71.35 (C-7), 69.80 (C-4), 67.88 (C-8), 55.34 (C-9), 51.95 (C-9), 22.65 (COCH3). HR-MS (ESI) calcd. for C20H20F3N4O7[M−H]−, 485.1284; found 485.1282.
Compound 7h was prepared as above in 83% yield (100 mg). 1H NMR (500 MHz, CD3OD) δ 8.42 (s, 1H, C═CH—N), 8.06 (d, J=8.5 Hz, 2H, Ar—H), 7.90 (d, J=8.5 Hz, 2H, Ar—H), 5.84 (d, J=1.9 Hz, 1H, H-3), 4.88 (dd, J=14.0, 2.1 Hz, 1H, H-9), 4.54 (dd, J=14.0, 7.6 Hz, 1H, H-9′), 4.43 (dd, J=8.7, 1.9 Hz, 1H, H-4), 4.31 (t, J=7.6 Hz, 1H, H-8), 4.15 (d, J=10.9 Hz, 1H, H-6), 4.03 (dd, J=10.9, 8.7 Hz, 1H, H-5), 3.44 (d, J=7.6 Hz, 1H, H-7), 1.99 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 175.08, 169.69, 167.90 (C═O), 147.50, 124.53 (Triazole-C), 136.19, 131.49, 126.44 (Ar—C), 111.19 (C-3), 77.37 (C-6), 71.31 (C-7), 69.84 (C-4), 68.24 (C-8), 55.29 (C-9), 51.90 (C-5), 22.82 (COCH3). HR-MS (ESI) calcd. for C20H21N4O9 [M−H]−, 461.1309; found 461.1316.
CuAAC reaction gave 77 mg crude protected product, which was deprotected to give the desired final product 52 mg (75%). 1H NMR (500 MHz, CD3OD) δ 8.34 (s, 1H, Triazole-H), 7.88 (d, J=8.1 Hz, 2H, Ar—H), 7.65 (d, J=8.1 Hz, 3H, Ar—H), 7.42 (t, J=7.5 Hz, 2H, Ar—H), 7.32 (t, J=7.5 Hz, 1H, Ar—H), 5.74 (d, J=1.9 Hz, 1H, H-3), 4.88 (dd, J=13.9, 2.2 Hz, 1H, H-9), 4.52 (dd, J=13.9, 7.7 Hz, H-9′), 4.39 (dd, J=8.7, 1.9 Hz, H-4), 4.37-4.21 (m, 1H, H-8), 4.13 (d, J=10.8 Hz, 1H, H-6), 4.02 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.42 (d, J=9.3 Hz, 1H, H-7), 2.00 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 174.93, 169.51 (C═O), 149.28, 123.47 (Triazole-C), 148.20 (C-2), 142.24, 141.76, 130.84, 129.94, 128.54, 128.48, 127.86, 127.10 (Ar—C), 77.14 (C-6), 71.34 (C-7), 69.83 (C-4), 68.49 (C-8), 55.25 (C-9), 52.00 (C-5), 22.77 (COCH3). HR-MS (ESI) calcd. for C25H25N4O7 [M−H]−, 493.1723; found 493.1729.
Click reaction gave 77 mg crude protected product, which was deprotected to give the desired final product 40 mg (43%). 1H NMR (500 MHz, CD3OD) δ 8.24 (s, 1H, Triazole-H5), 8.24 (s, 1H), 7.78 (d, J=8.7 Hz, 2H, Ar—H), 7.35 (dd, J=8.7, 7.4 Hz, 2H, Ar—H), 7.12 (t, J=7.4 Hz, 1H, Ar—H), 7.05-6.98 (m, 4H, Ar—H), 5.93 (d, J=2.4 Hz, 1H, H-3), 4.85 (dd, J=14.0, 2.5 Hz, 1H, H-9), 4.50 (dd, J=14.0, 7.7 Hz, 1H, H-9′), 4.42 (dd, J=8.7, 2.4 Hz, 1H, H-4), 4.30 (ddd, J=10.0, 7.7, 2.5 Hz, 1H, H-8), 4.16 (m, 1H, H-6), 4.01 (dd, J=10.8, 8.8 Hz, 1H, H-5), 3.47-3.41 (m, 1H, H-7), 2.00 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 175.18, 166.12 (C═O), 158.95, 158.31, 131.00, 128.28, 126.98, 124.77, 120.19, 119.98 (Ar—C), 148.06 (Triazole-C4), 123.10 (Triazole-C5), 145.90 (C-2), 113.04 (C-3), 77.73 (C-6), 71.32 (C-7), 69.83 (C-4), 67.95 (C-8), 55.25 (C-9), 51.95 (C-5), 22.70 (COCH3). HR-MS (ESI) calcd. for C25H25N4O8 [M−H]−, 509.1672; found 509.1674.
1H NMR (500 MHz, CD3OD) δ 7.93 (s, 1H, Triazole-H), 7.86-7.80 (m, 2H, Ar—H), 7.53 (t, J=7.4 Hz, 1H, Ar—H), 7.45 (t, J=7.6 Hz, 2H, Ar—H), 5.94 (d, J=2.5 Hz, 1H, H-3), 4.86-4.81 (m, 1H, H-9), 4.64 (s, 2H, N—CH2-Triazole), 4.46-4.38 (m, 2H, H-9′, H-4), 4.22 (td, J=8.4, 2.4 Hz, 1H, H-8), 4.14 (d, J=11.1 Hz, 1H, H-6), 3.97 (dd, J=11.1, 8.8 Hz, 1H, H-5, H-5), 3.43 (d, J=8.4 Hz, 1H, H-7), 2.01 (s, 3H, COCH3). 13C NMR (126 MHz, CD3OD) δ 175.30 (N—C═O), 132.88, 129.62, 128.40 (Ar—C), 113.51 (C-3), 77.73 (C-6), 71.36 (C-7), 69.87 (C-4), 67.85 (C-8), 55.16 (C-9), 51.90 (C-5), 36.21 (N—CH2-Triazole), 22.69 (COCH3). HR-MS (ESI) calcd. for C21H24N5O8 [M−H]−, 474.1630; found 474.1624.
1H NMR (500 MHz, CD3OD) δ 8.31 (d, J=7.4 Hz, 1H, Triazole-H), 7.93 (d, J=7.3 Hz, 2H, Ar—H), 7.86-7.80 (m, 2H, Ar—H), 7.79 (d, J=8.7 Hz, 2H, Ar—H), 7.61-7.57 (m, 1H, Ar—H), 7.53 (d, J=6.5 Hz, 2H, Ar—H), 5.97 (s, 1H, H-3), 4.87 (d, J=11.9 Hz, 1H, H-9), 4.54 (dd, J=14.1, 7.4 Hz, 1H, H-9′), 4.44 (d, J=9.2 Hz, 1H, H-4), 4.35-4.27 (m, 1H, H-8), 4.17 (d, J=10.2 Hz, 1H, H-6), 4.03-3.95 (m, 1H, H-5), 3.44 (d, J=8.7 Hz, 1H, H-7), 2.01 (d, J=3.4 Hz, 3H, COCH3). 13C NMR (126 MHz, CD3OD) δ 133.10, 129.76, 128.66, 127.17, 122.63, 116.42 (Ar—C), 113.69 (C-3), 77.70 (C-6), 71.10 (C-7), 69.76 (C-4), 67.85 (C-8), 55.15 (C-9), 51.85 (C-5), 22.75 (COCH3). HR-MS (ESI) calcd. for C26H26N5O8 [M−H]−, 536.1787; found 536.1782.
A solution of compound 10h (250 mg, 1 eq) in anhydrous pyridine was cooled down to 0° C., followed by dropwise addition of acetic anhydride (230 μl, 4.5 eq). The reaction mixture was allowed to warm to room temperature and kept stirring overnight. After completion, the reaction was quenched with methanol and the solvents were removed under reduced pressure. The residue was dissolved in ethyl acetate and carefully washed with 0.05 M HCl, water, and brine sequentially and dried over Na2SO4. The solution was then concentrated and purified by flash chromatography, providing a crude fully protected product, which was used in the next step without further purification. The obtained crude protected product (800 mg, 1 eq, several batches' product of last step) was dissolved in 10 ml ethyl acetate. The solution was warmed to 40° C. and TMSOTf (408 μl, 3 eq) was added drop wisely. The resulting solution was kept stirring at 50° C. for 4 hours. After completion, the solution was added to a vigorously stirred cold saturated sodium bicarbonate solution. The aqueous phase was separated and extracted with ethyl acetate. The organic phase was combined, dried over Na2SO4, concentrated and purified by flash chromatography to give the desired product (430 mg, 60%). 1H NMR (500 MHz, CD3OD) δ 8.49 (s, 1H, Triazole-H5), 8.03-7.97 (d, J=8.5 Hz, 2H, Ar—H), 7.88 (d, J=8.5 Hz, 2H, Ar—H), 6.39 (d, J=4.0 Hz, 1H, H-3), 5.65-5.57 (m, 2H, H-7, H-8), 5.22 (dd, J=14.8, 2.6 Hz, 1H, H-9), 4.94 (dd, J=9.5, 4.0 Hz, 1H, H-4), 4.79 (m, 1H, H-9′), 4.02 (t, J=9.5 Hz, 1H, H-5), 3.87, 3.79 (2×s, 2×3H, 2×COOCH3), 3.60 (dd, J=9.5, 2.3 Hz, 1H, H-6), 2.17 (s, 3H, oxazole-CH3), 1.97, 1.95 (2×s, 2×3H, 2×COOCH3). 13C NMR (125 MHz, CD3OD) δ 171.55, 171.32, 168.03, 163.34 (C═O), 170.12 (oxazole-O—C═N), 148.09 (Triazole-C4), 124.39 (Triazole-C5), 147.66 (C-2), 136.30, 131.27, 130.78, 126.55 (Ar—C), 109.05 (C-3), 78.35 (C-6), 74.22 (C-4), 73.52 (C-8), 70.86 (C-7), 62.68 (C-5), 53.27, 52.81 (COOCH3), 51.09 (C-9), 20.73, 20.67 (COCH3), 14.06 (oxazole-CH3). HR-MS (ESI) calcd. for C26H28N4NaO10 [M+Na]+, 579.1703; found 579.1697.
To a solution of compound 11 (430 mg, 1 eq) in dry tBuOH, TMSN3 (507 μl, 5 eq) was added and the resulting solution was stirred at 80° C. under a nitrogen atmosphere for 12 hours. After completion, the solution was cooled down to room temperature, concentrated and purified by flash chromatography to give the desired product. 470 mg (quant.). 1H NMR (500 MHz, CD3OD) δ 8.51 (s, 1H, Triazole-H5), 8.07-8.02 (m, 2H, Ar—H), 7.92-7.87 (m, 2H, Ar—H), 5.97 (d, J=2.2 Hz, 1H, H-3), 5.57-5.56 (dd, J=3.3, 2.2 Hz, 1H, H-4), 5.52 (dt, J=9.1, 2.9 Hz, 1H, H-8), 5.29 (dd, J=14.8, 2.7 Hz, 1H, H-9), 4.71 (dd, J=14.8, 9.1 Hz, 1H, H-9′), 4.49 (dd, J=10.3, 1.9 Hz, 1H, H-6), 4.27-4.18 (m, 2H, H-5, H-7), 3.89, 3.80 (2×s, 2×3H, 2×COOCH3), 2.13, 1.93, 1.92 (3×s, 3×3H, 3×COCH3). 13C NMR (125 MHz, CD3OD) δ 173.49, 171.83, 171.42, 168.09, 163.07 (C═O), 147.64 (Triazole-C4), 124.51 (Triazole-C5), 146.32 (C-2), 136.25, 131.26, 130.85, 126.54 (Ar—C), 109.74 (C-3), 78.44 (C-6), 73.96 (C-8), 69.60 (C-7), 60.46 (C-4), 53.21, 52.74 (COOCH3), 51.17 (C-9), 48.39 (C-5), 22.89, 20.88, 20.63 (COCH3). HR-MS (ESI) calcd. for C26H29N7NaO10 [M+Na]+, 622.1874; found 622.1866.
60 mg compound 12 was dissolved in 2 ml 0.5 N NaOH, the solution was stirred under room temperature for 1 hour. After completion, Amberlite™ IR 120 (H+) was added to neutralize the solution. The suspension was then filtered, and the filtrate was concentrated and purified by flash chromatography to give the desired product. 32 mg (66%). 1H NMR (500 MHz, CD3OD) δ 8.43 (s, 1H, Triazole-H5), 8.06 (d, J=8.2 Hz, 2H, Ar—H), 7.91 (d, J=8.2 Hz, 2H, Ar—H), 5.73 (s, 1H), 4.53 (dd, J=14.0, 7.6 Hz, 1H, H-9′), 4.32-4.25 (m, 2H, H-4, H-8), 4.23 (d, J=10.8 Hz, 1H, H-6), 4.18-4.10 (m, 1H, H-5), 3.45 (d, J=9.3 Hz, 1H, H-7), 1.98 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 174.32 (C═O), 147.53 (Triazole-C4), 124.47 (Triazole-C5), 136.17, 131.48, 126.42 (Ar—C), 104.50 (C-3), 77.02 (C-6), 71.03 (C-8), 69.86 (C-7), 60.23 (C-4), 55.25 (C-9), 48.53 (C-5), 22.78 (COCH3). HR-MS (ESI) calcd. for C20H20N7O8 [M−H]−, 486.1373; found 486.1378.
To a solution of compound 12 (50 mg, 1 eq) in THE (2 ml), 0.5 N HCl (200 μl, 2 eq) was added, followed by triphenylphosphine (29 mg, 1.1 eq). The resulting mixture was stirred at room temperature overnight. After completion, solvents were removed under reduced pressure and the residue was purified by flash chromatography, providing the desired product 39 mg (84%). 1H NMR (500 MHz, CD3OD) δ 8.57 (s, 1H, Triazole-H5), 8.05 (d, J=8.4 Hz, 2H, Ar—H), 7.92 (d, J=8.4 Hz, 2H, Ar—H), 6.06 (d, J=2.3 Hz, 1H, H-3), 5.63-5.55 (m, 2H, H-7, H-8), 5.29-5.21 (m, 1H, H-9), 4.74 (dd, J=14.7, 8.4 Hz, 1H, H-9′), 4.64 (dd, J=10.0, 1.1 Hz, 1H, H-6), 4.35 (t, J=10.0 Hz, 1H, H-5), 4.15 (dd, J=10.0, 2.3 Hz, 1H, H-4), 3.90, 3.81 (2×s, 2×3H, 2×COOCH3), 2.12, 1.97, 1.95 (3×s, 3×3H, 3×COCH3). 13C NMR (125 MHz, CD3OD) δ 174.28, 171.71, 171.31, 168.15, 162.80 (C═O), 147.66 (Triazole-C4), 124.59 (Triazole-C5), 147.29, 136.28, 130.84, 126.56 (Ar—C), 107.15 (C-3), 77.96 (C-6), 73.34 (C-8), 69.49 (C-7), 53.34, 52.78 (COOCH3), 51.67 (C-4), 51.31 (C-9), 46.73 (C-5), 23.14, 20.89, 20.66 (COCH3). HR-MS (ESI) calcd. for C26H31N5NaO10 [M+Na]+, 596.1969; found 596.1967.
35 mg compound 14 was dissolved in 400 μL 1N NaOH and the solution was kept stirring at r.t. for 1 h. After completion, the reaction mixture was neutralized with Amberlite™ IR 120 (H+). The suspension was then filtered, and the filtrate was concentrated and purified by flash chromatography to give the desired product. 20 mg (71%). 1H NMR (500 MHz, CD3OD) δ 8.47 (s, 1H, Triazole-H5), 8.08 (d, J=8.2 Hz, 2H, Ar—H), 7.94 (d, J=8.2 Hz, 2H, Ar—H), 5.84 (s, 1H, H-3), 4.89 (d, J=14.4 Hz, 1H, H-9), 4.56 (dd, J=14.0, 7.5 Hz, 1H, H-9′), 4.41-4.26 (m, 3H, H-8, H-6, H-5), 4.18 (d, J=7.1 Hz, 1H, H-4), 3.56 (d, J=9.1 Hz, 1H, H-7), 2.03 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 174.86, 169.48 (C═O), 147.50 (Triazole-C4), 124.66 (Triazole-C5), 136.29, 131.48, 131.25, 126.45 (Ar—C), 103.03 (C-3), 77.01 (C-6), 70.71 (C-8), 70.03 (C-7), 55.19 (C-9), 51.29 (C-4), 47.54 (C-5), 23.03 (COCH3). HR-MS (ESI) calcd. for C20H22N5O8 [M−H]−, 460.1468; found 460.1482.
To a solution of compound 14 (40 mg, 1 eq) in 2 ml anhydrous DCM, TEA (40 μl, 4 eq) was added. The solution was cooled down to 0° C. and N, N′-Di-Boc-1H-pyrazole-1-carboxamidine (42 mg, 2 eq) added. The reaction mixture was allowed to warm up to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, concentrated and purified by flash chromatography to give the desired product. Crude product, 40 mg (72%). 1H NMR (500 MHz, CD3OD) δ 8.55 (s, 1H, Triazole-H5), 8.06 (d, J=8.3 Hz, 2H, Ar—H), 7.92 (d, J=8.3 Hz, 2H), 6.00 (d, J=2.3 Hz, 1H, H-3), 5.58 (d, J=1.5 Hz, 1H, H-7), 5.56-5.52 (m, 1H, H-8), 5.31 (dd, J=14.8, 2.4 Hz, 1H, H-9), 5.02 (dd, J=10.2, 2.3 Hz, 1H, H-4), 4.74 (dd, J=14.8, 9.0 Hz, 1H, H-9′), 4.53 (dd, J=10.2, 1.5 Hz, 1H, H-6), 4.27 (t, J=10.2 Hz, 1H, H-5), 3.91, 3.80 (2×s, 3H, COOCH3), 2.12, 1.94, 1.85 (3×s, 3×3H, 3×COCH3), 1.51, 1.46 (2×s, 2×9H, 2×Boc). 13C NMR (125 MHz, CD3OD) δ 173.57, 171.77, 171.41, 168.07, 164.32, 163.39, 158.01 (C═O), 153.82 (C═N), 147.64 (Triazole-C4), 124.50 (Triazole-C5), 145.61 (C-2), 136.34, 131.27, 131.27, 126.55 (Ar—C), 111.83 (C-3), 84.84, 80.57 (tBoc-C(CH3)3), 78.89 (C-6), 74.00 (C-8), 69.87 (C-7), 53.07, 52.74 (COOCH3), 51.27 (C-9), 50.84 (C-4), 47.90 (C-5), 28.59, 28.26 (tBoc-C(CH3)3), 22.77, 20.86, 20.65 (COCH3). HR-MS (ESI) calcd. for C37H49N7NaO14 [M+Na]+, 838.3235; found 838.3226.
To a solution of compound 16 (40 mg) in 1 ml DCM, 100 μl TFA was added. The solution was then stirred at room temperature for 2 hours. After completion, DCM and TFA were removed under reduced pressure. The residue was dissolved in 2 ml 0.1 N NaOH and stirred at room temperature for 1 hour. After completion, the reaction mixture was added with Amberlite™ IR 120 (H+) to adjust the pH of the solution as 7. The suspension was then filtered, and the filtrate was concentrated and purified by flash chromatography to give the desired product. 10 mg (41%). 1H NMR (500 MHz, D2O) δ 8.49 (s, 1H, Triazole-H), 8.02 (d, J=8.2 Hz, 2H, Ar—H), 7.93 (d, J=8.2 Hz, 2H, Ar—H), 5.68 (d, J=2.1 Hz, 1H, H-3), 4.91 (dd, J=14.4, 2.8 Hz, 1H, H-9), 4.72 (dd, J=14.4, 6.7 Hz, 1H, H-9′), 4.48 (dd, J=9.5, 2.1 Hz, 1H, H-4), 4.46-4.41 (m, 1H, H-8), 4.40 (d, J=11.0 Hz, 1H, H-7), 4.26 (t, J=9.5 Hz, 1H, H-5), 3.53 (d, J=9.5 Hz, 1H, H-6), 1.99 (s, 3H, COCH3). 13C NMR (125 MHz, D2O) δ 175.37, 169.94, 163.66 (C═O), 157.96 (C═N), 150.14 (C-2), 147.81, 124.65 (Triazole-C), 133.00, 130.62, 126.33 (Ar—C), 104.76 (C-3), 76.02 (C-6), 69.79 (C-8), 69.00 (C-7), 54.41 (C-9), 51.83 (C-4), 48.65 (C-5), 22.75 (COCH3). HR-MS (ESI) calcd. for C21H24N7O8 [M−H]−, 502.1686; found 502.1683.
A solution of compound 14 (100 mg, 1 eq) and TEA (56 mg, 2 eq) in anhydrous DCM was cooled down to 0° C. and added with 1,1′-Carbonyldiimidazole (39 mg, 1.2 eq). The reaction mixture was then warmed to room temperature and kept stirring for 2 hours until TLC results showed no amine remained. The solution was then cooled down to 0° C., and -alanine methyl ester (56 mg, 2 eq) was added. The solution was warmed to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, extracted by ethyl acetate. The organic layer was washed with water, brine, dried over Na2SO4. After concentrated, the residue was purified by flash chromatography to give the desired product. 140 mg (quant.). 1H NMR (500 MHz, CD3OD) δ 8.54 (s, 1H, Triazole-H5), 8.07 (d, J=8.7 Hz, 2H, Ar—H), 7.92 (d, J=8.7 Hz, 2H, Ar—H), 5.92 (d, J=2.5 Hz, 1H, H-3), 5.55 (m, 2H, H-8, H-4), 5.31 (dd, J=14.8, 2.6 Hz, 1H, H-9), 4.73 (dd, J=14.8, 8.9 Hz, 1H, H-9′), 4.55 (dd, J=9.9, 2.5 Hz, 1H, H-7), 4.46 (dd, J=10.2, 2.0 Hz, 1H, H-6), 4.10 (t, J=10.2 Hz, 1H, H-5), 3.91, 3.78, 3.66 (3×s, 3×3H, 3×COOCH3), 3.36 (td, J=6.6, 1.9 Hz, 2H, CH2), 2.48 (t, J=6.5 Hz, 2H, CH2), 2.10, 1.93, 1.87 (3×s, 3×3H, 3×COCH3). 13C NMR (125 MHz, CD3OD) δ 174.12, 173.59, 171.79, 171.41, 168.13, 163.56, 160.42 (C═O), 147.66 (C-2), 145.10 (Triazole-C4), 124.46 (Triazole-C5), 136.30, 131.26, 130.88, 126.54 (Ar—C), 114.06 (C-1), 79.10 (C-6), 73.99 (C-8), 69.97 (C-7), 52.96, 52.70, 52.15 (COOCH3), 51.26 (C-9), 50.38 (C-5), 36.92, 35.64 (CH2CH2), 22.85, 20.84, 20.59 (COCH3). HR-MS (ESI) calcd. for C31H39N7N6O13 [M+Na]+, 703.2575; found 703.2571.
140 mg compound 17 was dissolved in 5 ml 0.1 N NaOH and stirred at room temperature for 1 hour. After completion, the reaction mixture was added with Amberlite™ IR 120 (H+) to neutralize the solution. The suspension was then filtered, and the filtrate was concentrated and purified by flash chromatography to give the desired product. 88 mg (77%). 1H NMR (500 MHz, CD3OD) δ 8.44 (s, 1H, Triazole-H5), 8.07 (d, J=8.4 Hz, 2H, Ar—H), 7.93 (d, J=8.4 Hz, 2H, Ar—H), 5.66 (d, J=1.9 Hz, 1H, H-3), 4.57 (dd, J=9.8, 1.9 Hz, 1H, H-4), 4.52 (dd, J=14.0, 7.7 Hz, 1H, H-9′), 4.30 (dd, J=12.1, 4.8 Hz, 1H, H-8), 4.19 (d, J=10.8 Hz, 1H, H-6), 4.02 (t, J=10.3 Hz, 1H, H-5), 3.44 (d, J=9.3 Hz, 1H, H-7), 3.37 (t, J=6.4 Hz, 2H, CH2), 2.46 (t, J=6.4 Hz, 2H, CH2), 1.94 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 175.74, 174.73, 160.76, 169.64 (C═O), 147.51 (Triazole-C4), 124.40 (Triazole-C5), 136.24, 131.68, 131.45, 126.42 (Ar—C), 109.35 (C-3), 77.81 (C-6), 71.36 (C-8), 69.80 (C-7), 55.31 (C-9), 37.00, 35.78 (CH2CH2), 22.74 (COCH3). HR-MS (ESI) calcd. for C24H27N6O11 [M−H]−, 575.1738; found 575.1738.
To a solution of compound 19 (600 mg, 1 eq) and TEA (389 μl, 2 eq) in 20 ml anhydrous DCM at 0° C., di-tert-butyl dicarbonate (456 mg, 1.5 eq) was added in dropwise. The mixture was them warmed up to room temperature and kept stirring overnight. After completion, solvent was removed, and the residue was purified by flash chromatography to give the desired compound 350 mg (crude product, 47%). The crude product (350 mg, 1 eq) was dissolved in 10 ml methanol, and cooled down to 0° C., followed by addition of NaOMe (92 mg, 3 eq). The solution was kept stirring at 0° C. for about 1 hour until no starting material remained. Amberlite™ IR 120 (H+) was added to neutralize the solution. The suspension was then filtered, and the filtrate was concentrated and purified by flash chromatography to give the desired product. 190 mg (71%). 1H NMR (500 MHz, CD3OD) δ 5.82 (d, J=2.2 Hz, 1H, H-3), 4.46 (d, J=10.1 Hz, 1H, H-4), 4.23 (d, J=10.1 Hz, 1H, H-6), 4.05 (t, J=10.1 Hz, 1H, H-5), 3.88 (ddd, J=9.2, 5.4, 2.9 Hz, 1H, H-8), 3.81 (dd, J=11.4, 2.9 Hz, 1H, H-9), 3.65 (dd, J=11.4, 5.4 Hz, 1H, H-9′), 3.58 (dd, J=9.3, 1.1 Hz, 1H, H-7), 1.98 (s, 3H, COOCH3), 1.44 (s, 9H, tBoc-C(CH3)3). 13C NMR (125 MHz, CD3OD) δ 174.65, 164.26, 158.35 (C═O), 145.71 (C-2), 112.19 (C-3), 80.56 (tBoc-C(CH3)3), 78.62 (C-6), 71.13 (C-8), 70.00 (C-7), 64.90 (C-9), 52.79 (C-4), 50.26 (C-5), 28.70 (tBoc- C(CH3)3), 22.70 (COCH3). HR-MS (ESI) calcd. for C17H29N2O9 [M+H]+, 405.1873; found 405.1875.
A solution of compound 20 (190 mg, 1 eq) in anhydrous pyridine was cooled down to 0° C., TsCl (98 mg, 1.1 eq) was then added slowly under stirring. The solution was warmed room temperature and kept stirring overnight. After completion, the reaction was quenched by methanol. The solution was concentrated and purified by flash chromatography to give the desired product. 200 mg (76%). 1H NMR (500 MHz, CDCl3) δ 7.78 (d, J=8.1 Hz, 2H, Ar—H), 7.33 (d, J=8.1 Hz, 2H, Ar—H), 6.93, 5.23, 5.11, 3.59 (4×d, 4H, 2×NH, 2×OH), 5.82 (d, J=2.3 Hz, 1H, H-3), 4.55 (td, J=9.6, 2.2 Hz, 1H, H-8), 4.40-4.31 (m, 1H, H-6), 4.21-4.17 (m, 1H, H-5), 4.15-4.11 (m, 2H, H-9′, H-4), 4.01-3.95 (m, 1H, H-9′), 3.71 (s, 3H, COOCH3), 3.56-3.48 (m, 1H, H-7), 2.43 (s, 3H, PhCH3), 2.00 (s, 3H, COCH3), 1.42 (s, 9H, tBoc-C(CH3)3). 13C NMR (125 MHz, CDCl3) δ 174.01, 162.21, 156.80 (C═O), 145.07 (C-2), 144.86, 132.59, 129.91, 128.02 (Ar—C), 109.92 (C-3), 80.60 (tBoc-C(CH3)3), 72.56 (C-9), 68.53 (C-7), 67.90 (C-8), 52.38 (C-6), 50.19 (C-4), 48.68 (C-5), 28.26 (tBoc-C(CH3)3), 22.86, 21.63 (COCH3, PhCH3). HR-MS (ESI) calcd. for C24H35N2O11S [M+H]+, 559.1962; found 559.1966.
Compound 21 (200 mg, 1 eq) was dissolved in 3 ml acetone-water (2:1) and NaN3 (117 mg, 5 eq) was added. The solution was heated at 67° C. under N2 for two days. After completion, the solution was concentrated and purified by flash chromatography to give 100 mg compound 22 (crude product, 75%) which was used in the next step without further purification. The crude product was dissolved in 2 ml anhydrous DCM, and 200 μl TFA was added. The solution was kept stirring at room temperature until no starting material remained. Solvents was then removed under vacuum and the residue was dissolved in 2 ml anhydrous DCM, and TEA (140 μl, 4 eq) was added. After the solution was cooled down to 0° C., N,N′-Di-Boc-1H-pyrazole-1-carboxamidine (150 mg, 2 eq) was added. The reaction mixture was allowed to warm up to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, concentrated and purified by flash chromatography to give the desired product. 108 mg (82%). 1H NMR (500 MHz, CDCl3) δ 8.63, 8.15, 7.70, 6.43 (2×d, 2×brs, 4H, 2×NH, 2×OH), 5.82 (d, J=2.4 Hz, 1H, H-3), 5.20 (ddd, J=10.2, 8.1, 2.4 Hz, 1H, H-4), 4.22-4.15 (m, 2H, H-6, H-5), 4.02 (td, J=10.2, 6.1 Hz, 1H, H-8), 3.72 (dd, J=12.6, 2.8 Hz, 1H, H-9), 3.60-3.53 (m, 2H, H-9′, H-7), 2.04 (s, 3H, COCH3), 1.52 (2×s, 2×9H, 2×tBoc-C(CH3)3). 13C NMR (125 MHz, CDCl3) δ 174.04, 162.29, 162.08, 157.56 (C═O), 152.70 (C═N), 146.31 (C-2), 107.51 (C-3), 84.39, 80.11 (tBoc-C(CH3)3), 69.14 (C-6), 54.89 (C-9), 52.53 (C-7), 51.53 (C-8), 48.33 (C-5), 28.23, 28.04 (tBoc-C(CH3)3), 22.96 (COCH3). HR-MS (ESI) calcd. for C23H38N7O10 [M+H]+, 572.2680; found 572.2681.
Compound 23 (200 mg, 1 eq) and 4-ethynylbiphenyl (32 mg, 1.5 eq) were taken in to click-reaction as mentioned earlier to give the desired product. 180 mg (69%). 1H NMR (700 MHz, CDCl3) δ 8.55, 8.06 (2×d, 2H, 2×NH), 7.94 (s, 1H, Triazole-H5), 7.81 (d, J=8.3 Hz, 2H, Ar—H), 7.57 (dd, J=13.1, 7.8 Hz, 4H, Ar—H), 7.39 (t, J=7.7 Hz, 2H, Ar—H), 7.31 (t, J=7.4 Hz, 1H, Ar—H), 5.75 (d, J=2.3 Hz, 1H, H-3), 5.51 (brs, 1H, OH), 5.16-5.10 (m, 1H, H-4), 4.90 (dd, J=14.0, 1.7 Hz, 1H, H-9), 4.54 (dd, J=14.0, 6.7 Hz, 1H, H-9′), 4.48-4.43 (m, 1H, H-8), 4.20 (d, J=10.4 Hz, 1H, H-6), 3.96 (td, J=10.4, 6.2 Hz, 1H, H-5), 3.69 (s, 3H, COOCH3), 3.34 (d, J=9.0 Hz, 1H, H-7), 1.90 (s, 3H), 1.47, 1.43 (2×s, 2×9H, 2×tBoc-C(CH3)3). 13C NMR (176 MHz, CDCl3) δ 174.10 (COCH3), 162.26, 162.03 (tBoc-OCO), 157.37 (C-1), 152.66 (C═N), 146.98 (C-2), 146.26 (Triazole-C4), 121.69 (Triazole-C5), 140.63, 140.43, 129.41, 128.77, 127.37, 126.86, 125.99 (Ar—C), 107.54 (C-3), 84.22, 79.96 (tBoc-C(CH3)3), 69.33 (C-6), 68.54 (C-4), 53.90 (C-9), 52.42 (C-8), 51.58 (C-7), 48.37 (C-5), 28.17, 27.99 (tBoc-C(CH3)3), 22.86 (COCH3). HR-MS (ESI) calcd. for C37H48N7O10 [M+H]+, 750.3463; found 750.3454.
300 μl TFA was added to a solution of compound 24 (180 mg) in 3 ml DCM and the solution was then stirred at room temperature for about 2 hours. After completion, DCM and TFA were removed under reduced pressure. The residue was dissolved in 3 ml 0.1 N NaOH and stirred at room temperature for 1 hour. After completion, Amberlite™ IR 120 (H+) was added to neutralize the solution. The suspension was then filtered, and the filtrate was concentrated and purified by flash chromatography to give the desired product. 10 mg (31%). 1H NMR (500 MHz, CD3OD) δ 8.34 (s, 1H, Trizaole-H), 7.87 (d, J=8.1 Hz, 2H, Ar—H), 7.66 (d, J=8.1 Hz, 2H, Ar—H), 7.61 (d, J=7.5 Hz, 2H, Ar—H), 7.41 (t, J=7.6 Hz, 2H, Ar—H), 7.32 (t, J=7.3 Hz, 1H, Ar—H), 5.89 (s, 1H, H-3), 4.88 (d, J=14.3 Hz, 1H, H-9), 4.60-4.47 (m, 2H, H-9′, H-6), 4.42 (d, J=10.0 Hz, 1H, H-4), 4.28 (m, 2H, H-5, H-8), 3.57 (d, J=9.0 Hz, 1H, H-7), 1.98 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 174.56, 165.08 (C═O), 158.96 (C═N), 148.25 (C-2), 146.68, 123.70 (Triazole-C), 142.30, 141.70, 130.71, 129.95, 128.58, 128.50, 127.85, 127.12 (Ar—C), 109.14 (C-3), 77.79 (C-6), 71.21 (C-8), 70.06 (C-7), 55.19 (C-9), 51.53 (C-4), 22.72 (COCH3). HR-MS (ESI) calcd. for C26H28N7O6 [M−H]−, 534.2101; found 534.2105.
A solution of compound 19 (1 eq) and TEA (3 eq) in anhydrous DCM was cooled down to 0° C. and corresponding anhydrides or acyl chlorides (3 eq) was added in dropwise. The resulting mixture was warmed to room temperature and kept stirring overnight. After completion, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was collected and washed with saturated NaHCO3, brine sequentially and dried with NaSO4. Solvents were removed under reduced pressure and the residue was separated by flash chromatography to give desired crude products. For hydrolysis of the C1-methyl ester, the product obtained above was dissolved in MeOH, and 0.5 M NaOH was added. The mixture was kept stirring at room temperature. After completion, the mixture was neutralized with Amberlite™ IR-120 (H+), filtrated and purified by flash chromatography to provide the desired products with yields of 42%-68% (two steps) (
(28 mg, 68%)1H NMR (500 MHz, CD3OD) δ 5.49 (d, J=2.0 Hz, 1H, H-3), 4.75 (dd, J=9.7, 2.0 Hz, 1H, H-4), 4.20 (d, J=10.8 Hz, 1H, H-6), 4.10-4.06 (m, 1H, H-5), 3.86-3.85 (m, 1H, H-8), 3.79 (dd, J=11.4, 3.0 Hz, 1H, H-9), 3.63 (dd, J=11.4, 5.4 Hz, 1H, H-9′), 3.55 (d, J=9.0 Hz, 1H, H-7), 2.17 (q, J=7.6 Hz, 2H, α-CH2), 1.93 (s, 3H, COCH3), 1.09 (t, J=7.6 Hz, 3H, β-CH3). 13C NMR (125 MHz, CD3OD) δ 177.40, 174.17 (C═O), 106.15 (C-3), 77.49 (C-6), 71.46 (C-8), 70.05 (C-7), 64.88 (C-9), 49.64 (C-5), 30.40 (α-CH2), 22.78 (COCH3), 10.59 (β-CH3). HR-MS (ESI) calcd. for C14H21N2O8 [M−H]−, 345.1298; found 345.1302.
(25 mg, 56%). 1H NMR (500 MHz, CD3OD) δ 5.47 (d, J=2.2 Hz, 1H, H-3), 4.76 (dd, J=9.8, 2.2 Hz, 1H, H-4), 4.19 (d, J=10.8 Hz, 1H, H-6), 4.09-4.05 (m, 1H, H-5), 3.87-3.85 (m, 1H, H-8), 3.78 (dd, J=11.5, 3.1 Hz, 1H, H-9), 3.66 (dd, J=11.5, 5.1 Hz, 1H, H-9′), 3.57 (t, J=7.8 Hz, 1H, H-7), 2.17 (t, J=7.5 Hz, 2H, α-CH2), 1.94 (s, 3H, COCH3), 1.60-1.51 (m, 2H, β-CH2), 1.34-1.29 (m, 2H, γ-CH2), 0.90 (t, J=7.4 Hz, 3H, 6-CH3). 13C NMR (125 MHz, CD3OD) δ 176.55, 174.18, 170.01 (3×C═O), 151.02 (C-2), 105.96 (C-3), 77.51 (C-6), 71.55 (C-8), 69.91 (C-7), 64.71 (C-9), 49.60 (C-4), 49.00 (C-5), 37.02 (α-CH2), 29.24 (β-CH2), 23.31 (γ-CH2), 23.31 (COCH3), 14.19 (δ-CH3). HR-MS (ESI) calcd. for C16H25N2O8 [M−H]−, 373.1611; found 373.1612.
(22 mg, 44%). 1H NMR (500 MHz, CD3OD) δ 5.54 (d, J=2.1 Hz, 1H, H-3), 4.77 (dd, J=9.8, 2.1 Hz, 1H, H-4), 4.20 (d, J=10.7 Hz, 1H, H-6), 4.11-4.07 (m, 1H, H-5), 3.90-3.82 (m, 1H, H-8), 3.79 (dd, J=11.4, 3.0 Hz, 1H, H-9), 3.66 (dd, J=11.4, 5.2 Hz, 1H, H-9′), 3.57 (d, J=9.0 Hz, 1H, H-7), 1.94 (s, 3H, COCH3), 1.58-1.53 (m, 1H, α-CH), 0.88-0.78 (m, 2H, β-CH2), 0.74-0.72 (m, 2H, β-CH2). 13C NMR (125 MHz, CD3OD) δ 176.90, 174.36, 169.54 (3×C═O), 150.41 (C-2), 106.92 (C-3), 77.64 (C-6), 71.52 (C-8), 69.95 (C-7), 64.77 (C-9), 49.92 (C-4), 49.28 (C-5), 22.87 (COCH3), 15.05 (α-CH), 7.57, 7.49 (2×β-CH2). HR-MS (ESI) calcd. for C15H21N2O8 [M−H]−, 357.1298; found 357.1305.
(19 mg, 42%). 1H NMR (500 MHz, CD3OD) δ 5.49 (d, J=2.2 Hz, 1H, H-3), 4.75 (dd, J=9.8, 2.2 Hz, 1H, H-4), 4.21 (d, J=10.8 Hz, 1H, H-6), 4.10-4.06 (m, 1H, H-5), 3.88-3.84 (m, m, 1H, H-8), 3.78 (dd, J=11.5, 3.1 Hz, 1H, H-9), 3.66 (dd, J=11.5, 5.2 Hz, 1H, H-9′), 3.56 (d, J=9.3 Hz, 1H, H-7), 3.09-3.02 (m, 1H, α-CH), 2.27-1.77 (m, 6H, 3×CH2), 1.93 (s, 3H, COCH3). 13C NMR (125 MHz, CD3OD) δ 178.06, 174.20, 169.63 (3×C═O), 150.52 (C-2), 106.54 (C-3), 77.53 (C-6), 71.55 (C-8), 69.94 (C-7), 64.77 (C-9), 49.57 (C-4), 49.14 (C-5), 40.90 (α-CH), 26.44, 26.02, 19.09 (3×CH2), 22.88 (COCH3). HR-MS (ESI) calcd. for C16H23N20 [M−H]−, 371.1454; found 371.1458.
C9-azido DANA methyl ester was dissolved in THF-H2O and cooled down to 0° C. with ice water bath. Triphenyl phosphate (TPP) was then added followed with activated carboxylic acids. The solution was allowed to warm to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, concentrated and purified by flash chromatography to give the desired product. The product was then dissolved in MeOH, and 0.5 M NaOH was added (
1H NMR (500 MHz, CD3OD) 5.93 (d, J=2.5 Hz, 1H, H-3), 4.41 (dd, J=8.7, 2.5 Hz, 1H, H-4), 4.20-4.15 (m, 1H, H-6), 3.96 (dd, J=10.7, 8.7 Hz, 1H, H-5), 3.94-3.88 (m, 1H, H-8), 3.57 (dd, J=14.0, 3.3 Hz, 1H, H-9), 3.41 (dd, J=9.1, 1.1 Hz, 1H, H-7), 2.22-2.15 (m, 2H, α-CH2), 2.00 (d, J=9.2 Hz, 3H, COCH3), 1.62 (dd, J=14.8, 7.4 Hz, 2H, β-CH2), 0.93 (t, J=7.4 Hz, 3H, γ-CH3). 13C NMR (126 MHz, CD3OD) δ 177.13, 174.87 ((N—C═O)), 165.51 (C-1), 113.36 (C-3), 77.83 (C-6), 71.49 (C-7), 70.17 (C-4), 67.96 (C-8), 52.01 (C-5), 44.40 (C-9), 38.97 (C-α), 22.73 (COCH3), 20.45 (C-β), 14.05 (C-γ).
1H NMR (500 MHz, CD3OD) δ 5.93 (d, J=1.7 Hz, 1H, H-3), 4.43 (dd, J=8.6, 1.7 Hz, 1H, H-4), 4.18 (d, J=10.8 Hz, 1H, H-6), 4.02-3.95 (m, 1H, H-5), 3.95-3.87 (m, 1H, H-8), 3.59 (dd, J=13.9, 2.9 Hz, 1H, H-9), 3.43 (d, J=9.0 Hz, 1H, H-7), 3.32-3.27 (m, 1H, H-9′), 2.22 (t, J=7.6 Hz, 2H, α-CH2), 2.03 (s, 3H, COCH3), 1.64-1.54 (m, 2H, β-CH2), 1.35 (dd, J=15.0, 7.5 Hz, 2H, γ-CH2), 0.92 (t, J=7.4 Hz, 3H, δ-CH2). 13C NMR (126 MHz, CD3OD) δ 177.26, 174.88 (N—C═O), 165.81 (C-1), 145.81 (C-2), 113.17 (C-3), 77.79 (C-6), 71.50 (C-7), 70.24 (C-4), 68.03 (C-8), 51.97 (C-5), 44.40 (C-9), 36.87 (C-α), 29.28 (C-β), 23.44 (γ), 22.86 (COCH3), 14.22 (C-δ). HRMS (ESI) calcd. for C16H25N2O8 [M−H]−, 373.1616; found 373.1614.
1H NMR (500 MHz, CD3OD) δ 5.92 (d, J=2.5 Hz, 1H, H-3), 4.42 (dd, J=8.7, 2.5 Hz, 1H, H-4), 4.17 (dd, J=10.7, 1.0 Hz, 1H, H-6), 3.97 (dd, J=10.7, 8.7 Hz, 1H, H-5), 3.94-3.88 (m, 1H, H-8), 3.59 (dd, J=13.9, 3.3 Hz, 1H, H-9), 3.42 (dd, J=9.0, 1.0 Hz, 1H, H-7), 3.31-3.27 (m, 1H, H-9′), 2.23-2.16 (m, 2H, α-CH2), 2.01 (s, 3H, COCH3), 1.59 (dt,J=15.0, 7.6 Hz, 2H, β-CH2), 1.39-1.25 (m, 4H, γ-CH2, δ-CH2), 0.89 (t, J=7.1 Hz, 3H, ε-CH3). 13C NMR (126 MHz, CD3OD) δ 177.25, 174.87 (N—C═O), 165.63 (C-1), 113.30 (C-3), 77.82 (C-6), 71.55 (C-7), 70.24 (C-4), 67.98 (C-8), 51.97 (C-5), 44.41 (C-9), 37.09 (C-α), 32.58 (C-β), 26.81 (C-γ), 23.45 (C-δ), 22.81 (COCH3), 14.32 (C-ε). HRMS (ESI) calcd. for C17H27N2O8 [M−H]−, 387.1773; found 387.1766.
1H NMR (500 MHz, CD3OD) δ 5.77 (d, J=2.3 Hz, 1H, H-3), 4.37 (dd, J=8.6, 2.3 Hz, 1H, H-4), 4.16-4.08 (m, 1H, H-6), 3.97 (dd, J=10.7, 8.7 Hz, 1H, H-5), 3.89 (ddd, J=8.7, 7.1, 3.4 Hz, 1H, H-8), 3.58 (dd, J=13.8, 3.4 Hz, 1H, H-9), 3.40 (d, J=8.7 Hz, 1H, H-7), 3.28-3.23 (m, 1H, H-9′), 2.26-2.13 (m, 2H, α-CH2), 2.01 (s, 3H, COCH3), 1.64-1.52 (m, 2H, β-CH2), 1.37-1.23 (m, 6H, γ-CH2, δ-CH2, ε-CH3), 0.88 (dd, J=8.8, 5.1 Hz, 3H, ζ-CH3). 13C NMR (126 MHz, CD3OD) δ 177.04, 174.70 (N—C═O), 110.32 (C-3), 77.35 (C-6), 71.50 (C-7), 70.44 (C-4), 68.36 (C-8), 51.99 (C-5), 44.23 (C-9), 37.15 (C-α), 32.73 (C-β), 30.07 (C-γ), 27.10 (C-δ), 23.58 (C-ε), 22.80 (C-ζ), 14.39 (COCH3). HRMS (ESI) calcd. for C18H29N2O8 [M−H]−, 401.1929; found 401.1931.
1H NMR (700 MHz, CD3OD) δ 5.74 (d, J=2.3 Hz, 1H, H-3), 4.37 (dd, J=8.7, 2.3 Hz, 1H, H-4), 4.14 (dd, J=10.8, 1.0 Hz, 1H, H-6), 3.99 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.91 (ddd, J=9.1, 6.9, 3.4 Hz, 1H, H-8), 3.58 (dd, J=13.8, 3.4 Hz, 1H, H-9), 3.40 (dd, J=9.1, 1.0 Hz, 1H, H-7), 3.33 (dd, J=13.8, 6.9 Hz, 1H, H-9′), 2.49 (dt, J=13.8, 6.9 Hz, 1H, α-CH2), 2.02 (s, 3H, COCH3), 1.12 (dd, J=6.9, 0.5 Hz, 6H, 2×β-CH3). 13C NMR (176 MHz, CD3OD) δ 180.76, 174.56 (N—C═O), 109.51 (C-3), 77.30 (C-6), 71.67 (C-7), 70.20 (C-4), 68.50 (C-8), 52.07 (C-5), 44.23 (C-9), 36.28 (C-α), 22.73, 19.96, 19.90 (2×C-β, COCH3). HRMS (ESI) calcd. for C15H23N2O8 [M−H]−, 359.1460; found 359.1458.
1H NMR (700 MHz, CD3OD) δ 5.74 (d, J=2.3 Hz, 1H, H-3), 4.37 (dd, J=8.7, 2.3 Hz, 1H, H-4), 4.13 (dd, J=10.8, 1.0 Hz, 1H, H-6), 3.99 (dd, J=10.8, 8.7 Hz, 1H, -5), 3.91 (ddd, J=9.0, 6.9, 3.4 Hz, 1H, H-8), 3.60 (dd, J=13.8, 3.4 Hz, 1H, H-9), 3.43-3.39 (dd, J=13.8, 6.9 Hz, 1H, H-9′), 3.33 (m, 1H, H-7), 2.10-2.09 (m, 2H, α-CH2), 2.08-2.03 (m, 1H, β-CH), 2.02 (s, 3H, COCH3), 0.96 (dd, J=6.4, 1.6 Hz, 6H, 2×γ-CH3). 13C NMR (176 MHz, CD3OD) δ 176.27, 174.60 (N—C═O), 109.43 (C-3), 77.30 (C-6), 71.71 (C-7), 70.26 (C-4), 68.51 (C-8), 52.08 (C-5), 46.32 (C-9), 44.18 (C-α), 27.42 (C-β), 22.77, 22.75, 22.74 (2×C-γ, COCH3). HRMS (ESI) calcd. for C16H25N2O8 [M−H]−, 373.1616; found 373.1617.
1H NMR (500 MHz, CD3OD) δ 5.69 (d, J=2.3 Hz, 1H, H-3), 4.36 (dd, J=8.7, 2.3 Hz, 1H, H-4), 4.10 (dd, J=10.8, 0.8 Hz, 1H, H-6), 3.97 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.92-3.85 (m, 1H, H-8), 3.58 (dd, J=13.8, 3.3 Hz, 1H, H-9), 3.38 (dd, J=8.9, 0.8 Hz, 1H, H-7), 3.28-3.21 (m, 1H, H-9′), 2.25-2.18 (m, 2H, α-CH2), 2.01 (s, 3H, COCH3), 1.60-1.44 (m, 3H, β-CH2, γ-CH), 0.90 (d, J=6.5 Hz, 6H, 2×δ-CH3). 13C NMR (126 MHz, CD3OD) δ 177.19, 174.68 (N—C═O), 108.93 (C-3), 77.12 (C-6), 71.55 (C-7), 70.35 (C-4), 68.59 (C-8), 52.00 (C-5), 44.22 (C-9), 36.12 (C-α), 35.23 (C-β), 29.04 (C-γ), 22.86, 22.74 (2×C-β, COCH3). HRMS (ESI) calcd. for C17H27N2O8 [M−H]−, 387.1773; found 387.1765.
1H NMR (500 MHz, CD3OD) δ 7.85-7.79 (m, 2H, Ar—H), 7.54-7.48 (m, 1H, Ar—H), 7.43 (dd, J=10.3, 4.7 Hz, 2H, Ar—H), 5.84 (d, J=2.3 Hz, 1H, H-3), 4.40 (dd, J=8.7, 2.3 Hz, 1H, H-4), 4.18 (d, J=10.8 Hz, 1H, H-6), 4.07-3.96 (m, 2H, H-8, H-5), 3.77 (dd, J=13.8, 3.4 Hz, 1H, H-9), 3.55 (dd, J=13.8, 6.8 Hz, 1H, H-9′), 3.48 (d, J=8.9 Hz, 1H, H-7), 1.96 (s, 3H, COCH3). 13C NMR (126 MHz, CD3OD) δ 174.79, 170.91 (N—C═O), 135.63, 132.71, 129.57, 128.36 (Ar—C), 111.64 (C-3), 77.53 (C-6), 71.60 (C-7), 70.35 (C-4), 68.20 (C-8), 51.92 (C-5), 45.02 (C-9), 22.74 (COCH3). HRMS (ESI) calcd. for C18H21N2O8 [M−H]−, 393.1303; found 393.13.
Compound I-25 was dissolved in anhydrous DCM and TEA was added. The mixture was then cooled down to 0° C. and activated carboxylic acids was added in dropwise. The solution was allowed to warm to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, concentrated and purified by flash chromatography to give the desired product. The product was then dissolved in MeOH, and 0.5 M NaOH was added (
To synthesize compounds 40-48, fully protected 40 (II-82fulpro) was taken to CuAAC as described before with alkynes to give desired product, which was then hydrolyzed using NaOH to give desired final products for enzymatic assay (
1H NMR (500 MHz, CD3OD) δ 5.90 (d, J=2.4 Hz, 1H, H-3), 4.43 (dd, J=8.7, 2.4 Hz, 1H, H-4), 4.15 (dd, J=10.8, 0.9 Hz, 1H, H-6), 4.01-3.95 (m, 1H, H-5), 3.91-3.86 (m, 1H, H-8), 3.81 (dd, J=11.4, 3.0 Hz, 1H, H-9), 3.65 (dd, J=11.4, 5.4 Hz, 1H, H-9′), 3.55 (dd, J=9.2, 0.9 Hz, 1H, H-7), 2.31 (q, J=7.6 Hz, 2H, CH2), 1.15 (t, J=7.6 Hz, 3H, CH3). 13C NMR (126 MHz, CD3OD) δ 178.80 (N—C═O), 166.46 (C-1), 112.52 (C-3), 77.98 (C-6), 71.17 (C-8), 70.17 (C-7), 68.10 (C-4), 64.93 (C-9), 51.80 (C-5), 30.20 (C-α), 10.33 (C-β). HRMS (ESI) calcd. for C12H18NO8 [M−H]−, 304.1032; found 304.1039.
1H NMR (500 MHz, CD3OD) δ 5.89 (d, J=2.4 Hz, 1H, H-3), 4.42 (dd, J=8.7, 2.4 Hz, 1H, H-4), 4.15 (d, J=10.8 Hz, 1H, H-6), 3.99 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.89 (ddd, J=9.0, 5.3, 3.0 Hz, 1H, H-8), 3.81 (dd, J=11.4, 3.0 Hz, 1H, H-9), 3.63 (dd, J=11.4, 5.3 Hz, 1H, H-9′), 3.56 (d, J=9.0 Hz, 1H, H-7), 2.29-2.23 (m, 2H, CH2), 1.66 (dt, J=13.4, 7.0 Hz, 2H, CH2), 0.97 (t, J=7.0 Hz, 3H, CH3). 13C NMR (126 MHz, CD3OD) δ 177.98 (N—C═O), 166.50 (C-1), 112.47 (C-3), 78.00 (C-6), 71.17 (C-7), 70.26 (C-4), 68.09 (C-8), 64.99 (C-9), 51.85 (C-5), 39.04 (C-α), 20.31 (C-β), 14.11 (C-γ). HRMS (ESI) calcd. for C13H20NO8 [M−H]−, 318.1189; found 318.1196.
1H NMR (500 MHz, CD3OD) δ 5.88 (d, J=2.1 Hz, 1H, H-3), 4.44 (dd, J=8.8, 2.1 Hz, 1H, H-4), 4.15 (d, J=10.8 Hz, 1H, H-6), 3.99 (dd, J=10.8, 8.8 Hz, 1H, H-5), 3.89 (m, 1H, H-8), 3.81 (dd, J=11.4, 2.9 Hz, 1H, H-9), 3.64 (dd, J=11.4, 5.3 Hz, 1H, H-9′), 3.57 (d, J=9.0 Hz, 1H, H-7), 2.29 (t, J=7.6 Hz, 2H, α-CH2), 1.62 (m, 2H, β-CH2), 1.37 (dq, J=14.8, 7.4 Hz, 2H, γ-CH2), 0.93 (t, J=7.4 Hz, 3H, CH3). 13C NMR (126 MHz, CD3OD) δ 178.13 (N—C═O), 166.89 (C-1), 146.72 (C-2), 112.33 (C-3), 77.91 (C-6), 71.30 (C-7), 70.18 (C-4), 68.09 (C-8), 64.90 (C-9), 51.76 (C-5), 36.92 (C-α), 29.08 (C-β), 23.46 (C-γ), 14.21 (C-δ). HRMS (ESI) calcd. for C14H22NO8 [M−H]−, 332.1345; found 332.1348.
1H NMR (700 MHz, CD3OD) δ 5.95 (d, J=2.3 Hz, 1H, H-3), 4.43 (dd, J=8.7, 2.3 Hz, 1H, H-4), 4.16 (dd, J=10.8, 0.8 Hz, 1H, H-6), 3.99 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.91 (brs, 1H, H-8), 3.83 (dd, J=11.4, 2.9 Hz, 1H, H-9), 3.63 (dd, J=11.4, 5.5 Hz, 1H, H-9′), 3.56 (dd, J=9.3, 0.7 Hz, 1H, H-7), 2.29 (t, J=7.5 Hz, 2H, α-CH2), 1.69-1.62 (m, 2H, β-CH2), 1.35 (m, 2×2H, γ-CH2, δ-CH2), 0.93 (t, J=7.0 Hz, 3H, CH3). 13C NMR (176 MHz, CD3OD) δ 178.20 (N—C═O), 113.34 (C-3), 78.14 (C-6), 71.06 (C-7), 70.27 (C-4), 67.95 (C-8), 65.03 (C-9), 51.81 (C-5), 37.06 (C-α), 32.56 (C-β), 26.59 (C-γ), 23.44 (C-δ), 14.27 (C-ε). HRMS (ESI) calcd. for C15H25NO8 [M−H]−, 346.1507; found 346.1506.
1H NMR (500 MHz, CD3OD) δ 5.93 (d, J=2.1 Hz, 1H, H-3), 4.40 (dd, J=8.7, 2.1 Hz, 1H, H-4), 4.13 (d, J=10.8 Hz, 1H, H-6), 3.98 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.89 (brs, 1H, H-8), 3.81 (dd, J=11.4, 2.7 Hz, 1H, H-9), 3.61 (dd, J=11.4, 5.5 Hz, 1H, H-9′), 3.53 (d, J=8.8 Hz, 1H, H-7), 2.27 (t, J=7.5 Hz, 2H, α-CH2), 1.69-1.55 (m, 2H, β-CH2), 1.42-1.16 (m, 3×2H, γ-CH2, δ-CH2, ε-CH2), 0.89 (t, J=7.0 Hz, 3H). 13C NMR (126 MHz, CD3OD) δ 178.23 (N—C═O), 113.38 (C-3), 78.18 (C-6), 71.07 (C-7), 70.32 (C-4), 67.98 (C-8), 65.06 (C-9), 51.84 (C-5), 37.12 (C-α), 32.72 (C-β), 30.06 (C-γ), 26.89 (C-δ), 23.58 (C-ε), 14.40 (C-ζ). HRMS (ESI) calcd. for C6H26NO8 [M−H]−, 360.1664; found 360.1665.
1H NMR (500 MHz, CD3OD) δ 5.93 (d, J=2.5 Hz, 1H, H-3), 4.43 (dd, J=8.7, 2.5 Hz, 1H, H-4), 4.15 (dd, J=10.8, 1.1 Hz, 1H, H-6), 3.95 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.88 (s, 1H, H-8), 3.80 (dd, J=11.4, 2.9 Hz, 1H, H-9), 3.62 (dd, J=11.4, 5.4 Hz, 1H, H-9′), 3.52 (dd, J=9.2, 1.1 Hz, 1H, H-7), 2.57-2.46 (m, 1H, α-CH), 1.14 (dd, J=6.9, 2.4 Hz, 6H, 2×β-CH3). 13C NMR (126 MHz, CD3OD) δ 182.10 (N—C═O), 165.49 (C-1), 113.48 (C-3), 78.20 (C-6), 71.05 (C-7), 70.21 (C-4), 67.89 (C-8), 64.98 (C-9), 51.68 (C-5), 36.41 (C-α), 20.10, 19.70 (2×C-β). HRMS (ESI) calcd. for C13H21NO8 [M−H]−, 318.1194; found 318.1193.
1H NMR (500 MHz, CD3OD) δ 5.93 (d, J=2.5 Hz, 1H, H-3), 4.40 (dd, J=8.7, 2.5 Hz, 1H, H-4), 4.14 (dd, J=10.8, 1.1 Hz, 1H, H-6), 3.98 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.89 (s, 1H, H-8), 3.80 (dd, J=11.4, 2.9 Hz, 1H, H-9), 3.61 (dd, J=11.4, 5.5 Hz, 1H, H-9′), 3.57 (dd, J=9.3, 1.1 Hz, 1H, H-7), 2.17-2.04 (m, 3H, α-CH2, β-CH2), 0.96 (dd, J=6.5, 3.8 Hz, 6H, 2×γ-CH3). 13C NMR (126 MHz, CD3OD) δ 177.53 (N—C═O), 113.50 (C-3), 78.18 (C-6), 71.05 (C-7), 70.34 (C-4), 67.95 (C-8), 65.04 (C-9), 51.87 (C-5), 46.33 (C-α), 27.43 (C-β), 22.89, 22.83 (2×C-γ). HRMS (ESI) calcd. for C14H22NO8 [M−H]−, 332.1351; found 333.1348.
1H NMR (500 MHz, CD3OD) δ 5.93 (d, J=2.3 Hz, 1H, H-3), 4.42 (dd, J=8.7, 2.3 Hz, 1H, H-4), 4.14 (d, J=10.8 Hz, 1H, H-6), 3.96 (dd, J=10.8, 8.8 Hz, 1H, H-5), 3.89 (s, 1H, H-8), 3.81 (dd, J=11.4, 2.8 Hz, 1H, H-9), 3.62 (dd, J=11.4, 5.4 Hz, 1H, H-9′), 3.54 (d, J=8.7 Hz, 1H, H-7), 2.32-2.25 (m, 2H, α-CH2), 1.62-1.47 (m, 3H, β-CH2, γ-CH), 0.91 (dd, J=6.4, 1.1 Hz, 6H, 2×δ-CH3). 13C NMR (126 MHz, CD3OD) δ 178.46 (N—C═O), 113.46 (C-3), 78.14 (C-6), 71.07 (C-7), 70.22 (C-4), 67.97 (C-8), 64.99 (C-9), 51.81 (C-5), 35.86 (C-α), 35.19 (C-β), 29.00 (C-γ), 22.75, 22.67 (2×C-β).
HRMS (ESI) calcd. for C15H24NO8 [M−H]−, 346.1507; found 346.1496.
1H NMR (500 MHz, CD3OD) δ 8.26 (d, J=8.4 Hz, 1H, NH), 5.88 (d, J=1.5 Hz, 1H, H-3), 4.47 (d, J=8.7 Hz, 1H, H-4), 4.16 (d, J=10.8 Hz, 1H, H-6), 4.01 (m, 1H, H-5), 3.93-3.85 (m, 1H, H-8), 3.80 (dd, J=11.4, 2.6 Hz, 1H, H-9), 3.69-3.62 (m, 1H, H-9), 3.56 (d, J=9.0 Hz, 1H, H-7), 1.69-1.66 (m, 1H, CH), 0.92-0.88 (m, 2H, CH2), 0.81-0.78 (m, 2H, CH2). 13C NMR (126 MHz, CD3OD) δ 178.35 (N—C═O), 166.87 (C-1), 112.31 (C-3), 78.06 (C-6), 71.28 (C-7), 70.10 (C-4), 68.19 (C-8), 64.84 (C-9), 51.96 (C-5), 15.10 (C-α), 8.10 (C-β), 7.75 (C-β′). HRMS (ESI) calcd. for C13H18NO8 [M−H]−, 316.1032; found 316.1030.
1H NMR (500 MHz, CD3OD) δ 5.89 (d, J=1.7 Hz, 1H, H-3), 4.44 (dd, J=8.9, 1.7 Hz, 1H, H-4), 4.15 (d, J=10.6 Hz, 1H, H-6), 3.99 (dd, J=10.6, 8.9 Hz, 1H, H-5), 3.88 (m, 1H, H-8), 3.80 (dd, J=11.4, 2.7 Hz, 1H, H-9), 3.65 (dd, J=11.4, 5.2 Hz, 1H, H-9′), 3.53 (d, J=9.0 Hz, 1H, H-7), 3.19 (p, J=8.5 Hz, 1H, CH), 2.30-2.22 (m, 2H, CH2), 2.20-2.10 (m, 2H, CH2), 1.89-1.82 (m, 2H, CH2). 13C NMR (126 MHz, CD3OD) δ 179.57 (N—C═O), 166.68 (C-1), 112.52 (C-3), 77.96 (C-6), 71.26 (C-7), 70.10 (C-4), 68.04 (C-8), 64.87 (C-9), 51.69 (C-5), 40.83 (C-α), 26.42 (C-β), 26.09 (C-β′), 19.08 (C-γ). HRMS (ESI) calcd. for C14H20NO8 [M−H]−, 330.1189; found 330.1195.
1H NMR (500 MHz, CD3OD) δ 7.88 (dd, J=8.3, 1.2 Hz, 2H, Ar—H), 7.57-7.50 (m, 1H, Ar—H), 7.49-7.41 (m, 2H, Ar—H), 5.98 (d, J=2.3 Hz, 1H, H-3), 4.64 (dd, J=8.7, 2.3 Hz, 1H, H-4), 4.32 (d, J=11.0 Hz, 1H, H-6), 4.24 (dd, J=11.0, 8.7 Hz, 1H, H-5), 3.92 (s, 1H, H-8), 3.80 (dd, J=11.5, 2.8 Hz, 1H, H-9), 3.68-3.59 (m, 2H, H-9′, H-7). 13C NMR (126 MHz, CD3OD) δ 171.74 (N—C═O), 135.08, 133.05, 129.55, 128.67 (Ar—C), 113.62 (C-3), 78.17 (C-6), 71.16 (C-7), 70.17 (C-4), 68.06 (C-8), 64.86 (C-9), 52.47 (C-5). HRMS (ESI) calcd. for C16H18NO [M−H]−, 352.1038; found 352.1035.
1H NMR (500 MHz, CD3OD) δ 5.86 (d, J=1.5 Hz, 1H, H-3), 4.46 (dd, J=8.7, 1.5 Hz, 1H, H-4), 4.24 (d, J=10.8 Hz, 1H, H-6), 4.07 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.97 (q, J=16.1 Hz, 2H, N—CH2—CO), 3.88 (br, 1H, H-8), 3.81 (dd, J=11.4, 2.6 Hz, 1H, H-9), 3.66 (dd, J=11.4, 5.2 Hz, 1H, H-9′), 3.57 (d, J=9.0 Hz, 1H, H-7). 13C NMR (126 MHz, CD3OD) δ 171.55 (C═O), 111.68 (C-3), 77.43 (C-6), 71.41 (C-8), 70.04 (C-7), 68.23 (C-4), 64.86 (C-9), 53.01 (CH2N), 51.93 (C-5). HRMS (ESI) calcd. for C11H15N4O8 [M−H]−, 331.0890; found 331.0894.
1H NMR (500 MHz, CD3OD) δ 8.47 (s, 1H, N—CH═C), 7.97 (d, J=8.1 Hz, 2H, Ar—H), 7.68 (d, J=8.3 Hz, 2H, Ar—H), 5.76 (s, 1H, H-3), 5.32 (s, 2H, N—CH2—CO), 4.47 (dd, J=8.6, 1.5 Hz, 1H, H-4), 4.27 (d, J=10.8 Hz, 1H, H-6), 4.12 (dd, J=10.8, 8.6 Hz, 1H, H-5), 3.89 (br, 1H, H-8), 3.84-3.76 (m, 1H, H-9), 3.69 (dd, J=11.4, 5.1 Hz, 1H, H-9′), 3.64 (d, J=8.9 Hz, 1H, H-7). 13C NMR (126 MHz, CD3OD) δ 168.79 (C═O), 147.36, 125.07 (Triazole-C), 135.56 (Ar—C), 130.94 (q, J=32.3 Hz, Ar—C), 127.05 (Ar—C), 127.87 (q, J=3.7 Hz, Ar—C), 109.19 (C-3), 76.99 (C-6), 71.60 (C-8), 70.06 (C-7), 68.68 (C-4), 64.80 (9), 53.36 (CH2N3), 52.24 (C-5). HRMS (ESI) calcd. for C20H20F3N4O8 [M−H]−, 501.1233; found 501.1243.
1H NMR (500 MHz, CD3OD) δ 8.31 (s, 1H, NC═CH), 7.70 (d, J=8.1 Hz, 2H, Ar—H), 7.24 (d, J=8.0 Hz, 2H), 5.87 (d, J=2.2 Hz, 1H, H-3), 5.29 (s, 2H, N—CH2—CO), 4.49 (dd, J=8.7, 2.2 Hz, 1H, H-4), 4.29 (d, J=10.8 Hz, 1H, H-6), 4.10 (dd, J=10.7, 8.7 Hz, 1H, H-5), 3.91-3.84 (m, 1H, H-8), 3.81 (dd, J=11.4, 2.9 Hz, 1H, H-9), 3.67 (dd, J=11.4, 5.3 Hz, 1H, H-9′), 3.64 (d, J=9.1 Hz, 1H, H-7), 2.35 (s, 3H, PhCH3). 13C NMR (126 MHz, CD3OD) δ 168.97 (C═O), 167.13 (C-1), 148.95, 123.68 (Triazole-C), 146.97 (C-2), 139.45, 130.64, 128.89, 126.71 (Ar—C), 111.62 (C-3), 77.42 (C-6), 71.51 (C-8), 70.06 (C-7), 68.30 (C-4), 64.86 (C-9), 53.29 (CH2N), 52.18 (C-5), 21.37 (PhCH3). HRMS (ESI) calcd. for C20H23N4O8 [M−H]−, 447.1516; found 447.1520.
1H NMR (500 MHz, CD3OD) δ 8.48 (s, 1H, C═CH—N), 8.10-8.04 (m, 2H, Ar—H), 7.95-7.90 (m, 2H, Ar—H), 5.88 (d, J=2.4 Hz, 1H, H-3), 5.32 (s, 2H, N—CH2—CO), 4.49 (dd, J=8.7, 2.4 Hz, 1H, H-4), 4.30 (dd, J=10.8, 0.6 Hz, 1H, H-6), 4.11 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.91-3.86 (m, 1H, H-8), 3.82 (dd, J=11.4, 3.0 Hz, 1H, H-9), 3.68 (dd, J=11.5, 5.4 Hz, 1H, H-9′), 3.64 (dd, J=9.2, 0.6 Hz, 1H, H-7). 13C NMR (126 MHz, DMSO-d6) δ 169.44, 168.85 (C═O), 167.00 (C-1), 147.83, 125.04 (Triazole-C), 146.85 (C-2), 136.10, 131.60, 131.48, 126.53 (Ar—C), 111.73 (C-3), 77.44 (C-6), 71.45 (C-8), 70.09 (C-7), 68.32 (C-4), 64.88 (C-9), 53.32 (CH2N3), 52.21 (C-5). HRMS (ESI) calcd. for C20H21N4O10[M−H]−, 477.1258; found 477.1268.
1H NMR (500 MHz, CD3OD) δ 8.27 (s, 1H, C═CH—N), 7.74 (d, J=8.7 Hz, 2H, Ar—H), 7.01 (d, J=8.7 Hz, 2H, Ar—H), 5.89 (d, J=1.8 Hz, 1H, H-3), 5.31 (s, 2H, N—CH2—CO), 4.56-4.45 (m, 1H, H-4), 4.33 (d, J=10.8 Hz, 1H, H-6), 4.11 (dd, J=10.5, 8.9 Hz, 1H, H-5), 3.92-3.86 (m, 1H, H-8), 3.85-3.83 (m, 4H, H-9, OCH3), 3.70-3.63 (m, 2H, H-7, H-9′). 13C NMR (126 MHz, CD3OD) δ 169.15 (C═O), 167.27 (C-1), 148.92, 123.53 (Triazole-C), 146.62 (C-2), 161.19, 128.28, 124.06 115.61 (Ar—C), 112.08 (C-3), 77.26 (C-6), 71.38 (C-8), 69.81 (C-7), 68.41 (C-4), 64.77 (C-9), 56.20 (PhOCH3), 53.38 (CH2N3), 52.02 (C-5). HRMS (ESI) calcd. for C20H23N4O9 [M−H]−, 463.1465; found 463.1471.
1H NMR (500 MHz, CD3OD) δ 8.33 (s, 1H, C═CH—N), 7.81 (dd, J=8.6, 5.4 Hz, 2H, Ar—H), 7.14 (t, J=8.7 Hz, 2H, Ar—H), 5.80 (s, 1H, H-3), 5.29 (s, 2H, N—CH2—CO), 4.48 (d, J=8.2 Hz, 1H, H-4), 4.27 (d, J=10.7 Hz, 1H, H-6), 4.14-4.07 (m, 1H, H-5), 3.88 (m, 1H, H-8), 3.81 (d, J=11.0 Hz, 1H, H-9), 3.73-3.60 (m, 2H, H-9′, H-7). 13C NMR (126 MHz, CD3OD) δ 168.94 (C-1), 168.64 (C═O), 165.11, 163.15 (d, J=246.3 Hz), 148.28 (C-2), 147.97, 123.91 (Triazole-C), 128.70 (d, J=8.2 Hz, Ar—C), 128.11 (d, J=3.2 Hz, Ar—C), 116.78 (d, J=22.0 Hz, Ar—C), 110.10 (C-3), 77.12 (C-6), 71.64 (C-8), 69.99 (C-7), 68.50 (C-4), 64.75 (C-9), 53.32 (CH2N), 52.18 (C-5). HRMS (ESI) calcd. for C19H20N4O8 [M−H]−, 451.1265; found 451.1271.
1H NMR (500 MHz, CD3OD) δ 8.18 (s, 1H, C═CH—N), 7.57 (d, J=8.6 Hz, 2H, Ar—H), 6.81 (d, J=8.5 Hz, 2H, Ar—H), 5.70 (d, J=2.2 Hz, 1H, H-3), 5.28 (s, 2H, N—CH2—CO), 4.47 (dd, J=8.7, 2.2 Hz, 1H, H-4), 4.25 (d, J=10.8 Hz, 1H, H-6), 4.09 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.93-3.86 (m, 1H, H-8), 3.83 (dd, J=11.6, 2.8 Hz, 1H, H-9), 3.66 (dd, J=11.6, 5.7 Hz, 1H, H-9′), 3.60 (d, J=9.3 Hz, 1H, H-7). 13C NMR (126 MHz, CD3OD) δ 169.93 (C-1), 169.04 (C═O), 149.58, 122.78 (Triazole-C), 148.93 (C-2), 127.94, 121.52, 117.07 (Ar—C), 108.57 (C-3), 76.73 (C-6), 71.42 (C-8), 70.02 (C-7), 68.81 (C-4), 64.87 (C-9), 53.35 (CH2N), 52.19 (C-5). HRMS (ESI) calcd. for C19H22N5O8 [M−H]−, 448.1468; found 448.1481.
1H NMR (500 MHz, CD3OD) δ 8.18 (s, 1H, C═CH—N), 7.57 (d, J=8.6 Hz, 2H, Ar—H), 6.81 (d, J=8.5 Hz, 2H, Ar—H), 5.70 (d, J=2.2 Hz, 1H, H-3), 5.28 (s, 2H, N—CH2—CO), 4.47 (dd, J=8.7, 2.2 Hz, 1H, H-4), 4.25 (d, J=10.8 Hz, 1H, H-6), 4.09 (dd, J=10.8, 8.7 Hz, 1H, H-5), 3.93-3.86 (m, 1H, H-8), 3.83 (dd, J=11.6, 2.8 Hz, 1H, H-9), 3.66 (dd, J=11.6, 5.7 Hz, 1H, H-9′), 3.60 (d, J=9.3 Hz, 1H, H-7). 13C NMR (126 MHz, CD3OD) δ 169.93 (C-1), 169.04 (C═O), 149.58, 122.78 (Triazole-C), 148.93 (C-2), 127.94, 121.52, 117.07 (Ar—C), 108.57 (C-3), 76.73 (C-6), 71.42 (C-8), 70.02 (C-7), 68.81 (C-4), 64.87 (C-9), 53.35 (CH2N), 52.19 (C-5). HRMS (ESI) calcd. for C19H22N5O8 [M−H]−, 448.1468; found 448.1481.
1H NMR (500 MHz, CD3OD) δ 8.30 (s, 1H, C═CH—N), 7.75 (d, J=8.6 Hz, 2H, Ar—H), 7.62 (d, J=8.6 Hz, 2H, Ar—H), 5.69 (d, J=2.2 Hz, 1H, H-3), 5.26 (s, 2H, N—CH2—CO), 4.43 (dd, J=8.6, 2.2 Hz, 1H, H-4), 4.23 (d, J=10.8 Hz, 1H, H-6), 4.10 (dd, J=10.8, 8.6 Hz, 1H, H-5), 3.90-3.84 (m, 1H, H-8), 3.81 (dd, J=11.4, 2.9 Hz, 1H, H-9), 3.67 (dd, J=11.4, 5.3 Hz, 1H, H-9′), 3.57 (d, J=9.2 Hz, 1H, H-7), 2.13 (s, 3H, NAc). 13C NMR (126 MHz, CD3OD) δ 171.70 (C═O), 169.98 (C-1), 168.75 (C═O), 150.14 (C-1), 148.58, 123.59 (Triazole-C), 140.08, 127.37, 127.17, 121.36 (Ar—C), 108.11 (C-3), 76.80 (C-6), 71.44 (C-8), 70.17 (C-7), 68.78 (C-4), 64.92 (C-9), 53.30 (CH2N3), 52.23 (C-5), 23.90 (CH3). HRMS (ESI) calcd. for C21H25N5O9 [M−H]−, 490.1574; found 490.1584.
Fully protected C9-azido DANA (II-34) was dissolved in THF-H2O and cooled down to 0° C. with ice water bath. Triphenyl phosphate was then added followed with activated carboxylic acids. The solution was allowed to warm to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, concentrated and purified by flash chromatography to give the desired C9-modified product. The product was then dissolved in anhydrous DCM and TEA and then cooled down to 0° C. Corresponding activated carboxylic acids for C5 modifications was added in dropwise. The solution was allowed to warm to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, concentrated and purified by flash chromatography to give the desired C5-modified product. The product was then dissolved in MeOH, and 0.5 M NaOH was added (
1H NMR (500 MHz, CD3OD) δ 5.74 (d, J=1.9 Hz, 1H, H-3), 4.38 (dd, J=8.7, 1.9 Hz, 1H, H-4), 4.11 (d, J=10.8 Hz, 1H, H-6), 3.97 (dd, J=10.7, 8.8 Hz, 1H, H-5), 3.93-3.85 (m, 1H, H-8), 3.57 (dd, J=13.4, 3.1 Hz, 1H, H-9), 3.38 (d, J=8.7 Hz, 1H, H-7), 3.26 (dd, J=13.4, 6.0 Hz, 1H, H-9′), 2.30-2.23 (m, 2H, α-CH2), 2.23-2.15 (m, 2H, α′-CH2), 1.59 (tdd, J=15.3, 11.2, 7.5 Hz, 4H, β-CH2, β′-CH2), 1.34 (dq, J=22.0, 7.4 Hz, 4H, γ-CH2, γ′-CH2), 0.92 (q, J=7.4 Hz, 6H, δ-CH2, δ′-CH2). 13C NMR (126 MHz, CD3OD) δ 177.78, 176.97 (N—C═O), 109.98 (C-3), 77.36 (C-6), 71.51 (C-7), 70.36 (C-4), 68.40 (C-8), 51.87 (C-5), 44.27 (C-9), 36.96, 36.89 (C-α, C-α′), 29.24, 29.11 (C-β, C-β′), 23.49, 23.45 (C-γ, C-γ′), 14.19 (COCH3). HRMS (ESI) calcd. for C19H31N2O8 [M−H]−, 415.2086; found 415.2081.
C9-azido DANA methyl ester was dissolved in THF-H2O and cooled down to 0° C. with ice water bath. Triphenyl phosphate was then added followed with anhydrides or acyl chlorides. The solution was allowed to warm to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, concentrated and purified by flash chromatography to give the desired product. The product was then dissolved in MeOH, and 0.5 M NaOH was added. The mixture was kept stirring at room temperature. After completion, the mixture was neutralized with Amberlite™ IR-120 (H+ form), filtered and purified by flash chromatography to provide the desired products.
Compound 58 was synthesized from C9-azido DANA methyl ester using 4-acetamidobenzyl chloride. 45 mg (28%(36%×77%, over two steps). 1H NMR (500 MHz, D2O) δ 7.80 (d, J=8.2 Hz, 2H, Ar—H), 7.58 (d, J=8.2 Hz, 2H, Ar—H), 5.95 (s, 1H, H-3), 4.54 (d, J=7.8 Hz, 1H, H-4), 4.33 (d, J=10.9 Hz, 1H, H-6), 4.14 (t, J=9.5 Hz, 2H, H-5, H-8), 3.86-3.80 (m, 1H, H-9), 3.68-3.58 (m, 2H, H-7, H-9′), 2.22 (s, 3H, COCH3), 2.07 (s, 3H, COCH3). 13C NMR (126 MHz, D2O) δ 175.64, 173.90, 171.46 (3×N—C═O), 168.07 (C-1), 141.49, 130.56, 129.13, 121.72 (Ar—C), 111.40 (C-3), 76.57 (C-6), 70.37 (C-7), 69.56 (C-4), 68.16 (C-8), 50.72 (C-5), 44.12 (C-9), 24.04, 23.02 (2×COCH3). HRMS (ESI) calcd. for C18H21N2O8 [M−H]−, 450.1518; found 450.1525.
Compound 59 was synthesized from C9-azido DANA methyl ester using N-hydroxysuccinimidyl-4-((tert-butoxycarbonyl) amino) benzoate. 30 mg (20%(45%×45% (yields for two steps of deprotection), over three steps). 1H NMR (500 MHz, CD3OD) δ 7.91 (d, J=7.3 Hz, 2H), 7.33 (d, J=7.3 Hz, 2H), 5.94 (s, 1H, H-3), 4.46 (d, J=8.0 Hz, 1H, H-4), 4.21 (d, J=10.6 Hz, 1H, H-6), 4.08-3.96 (m, 2H, H-5, H-8), 3.77 (d, J=12.9 Hz, 1H, H-9), 3.51 (m, 2H, H-9′, H-7), 1.97 (s, 3H, COCH3). 13C NMR (126 MHz, CD3OD) δ 165.66 (C-1), 145.21, 130.38, 122.41 (Ar—C), 113.68 (C-3), 77.75 (C-6), 71.40 (C-7), 70.28 (C-4), 67.93 (C-8), 51.73 (C-5), 45.05 (C-9), 22.92 (COCH3). HRMS (ESI) calcd. for C18H21N2O8 [M−H]−, 408.1412; found 408.1415.
Compound 60 was synthesized from C9-azido DANA methyl ester using 3-acetamidobenzyl chloride. 40 mg (30%(36%×82%, over two steps). 1H NMR (500 MHz, CD3OD) δ 7.97 (t, J=1.5 Hz, 1H, Ar—H), 7.69 (dd, J=7.9, 1.5 Hz, 1H, Ar—H), 7.52 (d, J=7.9 Hz, 1H, Ar—H), 7.37 (t, J=7.9 Hz, 1H, Ar—H), 5.78 (d, J=2.0 Hz, 1H, H-3), 4.38 (dd, J=8.6, 2.0 Hz, 1H, H-4), 4.16 (d, J=10.8 Hz, 1H, H-6), 4.00 (m, 2H, H-5, H-8), 3.76 (dd, J=13.8, 3.3 Hz, 1H, H-9), 3.53 (dd, J=13.8, 6.8 Hz, 1H, H-9′), 3.47 (d, J=8.8 Hz, 1H, H-7), 2.12 (s, 3H, COCH3), 1.97 (s, 3H, COCH3). 13C NMR (126 MHz, CD3OD) δ 174.74, 171.81, 170.56 (3×N—C═O), 140.21, 136.51, 130.03, 124.15, 123.74, 120.14 (Ar—C), 110.38 (C-3), 77.38 (C-6), 71.78 (C-7), 70.21 (C-4), 68.40 (C-8), 51.96 (C-5), 45.07 (C-9), 23.86, 22.77 (2×COCH3). HRMS (ESI) calcd. for C18H21N2O8 [M−H]−, 450.1518; found 450.1515.
Compound 61 was synthesized from C9-azido DANA methyl ester using 3-amidobenzyl chloride. 1H NMR (500 MHz, CD3OD) 30 mg (18%(46%×40%, over two steps). δ 7.94 (d, J=7.8 Hz, 1H, Ar—H), 7.89 (s, 1H, Ar—H), 7.63 (t, J=7.8 Hz, 1H, Ar—H), 7.60-7.56 (m, 1H, Ar—H), 5.95 (d, J=2.4 Hz, 1H, H-3), 4.45 (dd, J=8.7, 2.4 Hz, 1H, H-4), 4.21 (d, J=10.9 Hz, 1H, H-6), 4.08-3.97 (m, 2H, H-5, H-8), 3.84-3.79 (m, 1H, H-9), 3.57-3.48 (m, 2H, H-7, H-9′), 1.99 (s, 3H, COCH3). 13C NMR (126 MHz, CD3OD) δ 175.00, 168.86 (2×N—C═O), 165.60 (C-1), 145.27 (C-2), 137.83, 132.62, 131.55, 128.67, 127.16, 123.51 (Ar—C), 113.61 (C-3), 77.79 (C-6), 71.72 (C-7), 70.03 (C-4), 67.90 (C-8), 51.81 (C-5), 45.29 (C-9), 22.80 (COCH3). HRMS (ESI) calcd. for C18H21N2O8 [M−H]−, 408.1412; found 408.1411.
Compound 62 was synthesized from C9-azido DANA methyl ester using N-hydroxysuccinimidyl-5-(4-acetamidobenzamido) pentanoate. 30 mg (22%(42%×53%, over two steps). 1H NMR (500 MHz, CD3OD) δ 7.77 (d, J=8.7 Hz, 2H, Ar—H), 7.64 (d, J=8.7 Hz, 2H, Ar—H), 5.90 (d, J=2.3 Hz, 1H, H-3), 4.42 (dd, J=8.7, 2.3 Hz, 1H, H-4), 4.19-4.13 (d, J=10.7, 1H, H-6), 3.98 (dd, J=10.7, 8.7 Hz, 1H, H-5), 3.93-3.86 (m, 1H, H-8), 3.59 (dd, J=13.8, 3.1 Hz, 1H, H-9), 3.42 (d, J=8.9 Hz, 1H, H-7), 3.37 (t, J=6.0 Hz, 2H, δ-CH2), 2.27 (t, J=7.1 Hz, 2H, α-CH2), 2.13, 2.01 (2×s, 2×3H, 2×COCH3), 1.73-1.56 (m, 4H, β-CH2, γ-CH2). 13C NMR (126 MHz, CD3OD) δ 176.77, 174.82, 171.90, 169.57 (4×N—C═O), 143.07 (C-2), 130.74, 129.15, 120.26 (Ar—C), 112.90 (C-3), 77.69 (C-6), 71.35 (C-8), 70.24 (C-7), 51.81 (C-4), 49.88 (C-5), 44.28 (C-9), 40.53 (δ-CH2), 36.59 (α-CH2), 30.04 (γ-CH2), 24.42 (α-CH2), 24.03, 22.86 (2×COCH3). HRMS (ESI) calcd. for C25H33N4O10 [M−H]−, 549.2202; found 549.2207.
Compound 63: To a solution of methyl 5-acetamido-9-azido-2,6-anhydro-3,5-dideoxy-d-glycero-d-galacto-non-2-enonate (50 mg, 1 eq) and the heptyne (30 mg, 2 eq) in THF-H2O (2:1), sodium L-ascorbate (5 mg, 0.3 eq) and copper (II) sulfate (3 mg, 0.2 eq) were added sequentially. The reaction mixture was kept stirring at room temperature and monitored by TLC until no azide remained. Silica gel was then added to the reaction mixture and the solvent was removed under reduced pressure. The residue was separated by flash chromatography to provide the desired product 59 mg (92%). To hydrolyze the C1-methyl ester, the product was dissolved in MeOH, and 0.5 M NaOH was added. The mixture was kept stirring at room temperature. After completion, the pH of the solution was adjusted to 2 with Amberlite™ IR-120 (H+). The solution was filtered, concentrated and purified by flash chromatography or recrystallization to provide the desired product 40 mg (70%). 1H NMR (500 MHz, CD3OD) δ 7.71 (s, 1H, Triazole-H), 5.91 (d, J=2.3 Hz, 1H, H-3), 4.77 (dd, J=14.0, 2.5 Hz, 1H, H-4), 4.44-4.33 (m, 2H, H-6, H-5), 4.24-4.16 (m, 1H, H-8), 4.13 (d, J=10.9 Hz, 1H, H-9), 3.98 (dd, J=10.7, 8.7 Hz, 1H, H-9′), 3.39 (d, J=9.1 Hz, 1H, H-7), 2.66 (t, J=7.7 Hz, 2H, α-CH2), 2.01 (s, 3H, COCH3), 1.71-1.59 (m, 2H), 1.39-1.27 (m, 4H, β-CH2, γ-CH2), 0.89 (dd, J=9.7, 4.3 Hz, 3H, δ-CH3). 13C NMR (126 MHz, CD3OD) δ 175.10, 166.11, 145.91, 124.31, 112.97, 77.69, 71.34, 69.86, 67.93, 55.00, 51.92, 32.51, 30.36, 26.29, 23.46, 22.72, 14.35). HRMS (ESI) calcd. for C18H27N4O7 [M−H]−, 411.1885; found 411.1889.
Compound methyl 5-(tert-butoxycarbonyl)amino-9-azido-4,7,8-di-O-acetyl-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonate was dissolved in THF-H2O, and cooled down to 0° C. with ice water bath. Triphenyl phosphate was then added followed with activated carboxylic acids. The solution was allowed to warm to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, concentrated and purified by flash chromatography to give the desired C9-modified product. The product was then dissolved in anhydrous TFA-DCM (10%) and the solution was stirred for 2-4 hours at r.t. Solvents were removed under vacuum and the residue was dissolved in anhydrous DCM and TEA was added. The solution was cooled down to 0° C. and corresponding activated carboxylic acids for C5 modifications was added in dropwise. The solution was allowed to warm to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, concentrated and purified by flash chromatography to give the desired C5-modified product. The product was then dissolved in MeOH, and 0.5 N NaOH was added. The mixture was kept stirring at room temperature. After completion, the mixture was neutralized with Amberlite™ IR-120 (H+ form), filtered and purified by flash chromatography to provide the final C5, C9-double modified compounds.
Compound 64 was synthesized from compound methyl 5-(tert-butoxycarbonyl)amino-9-azido-4,7,8-di-O-acetyl-2,6-anhydro-3,5-dideoxy-
Compound 65 was synthesized from compound methyl 5-(tert-butoxycarbonyl)amino-9-azido-4,7,8-di-O-acetyl-2,6-anhydro-3,5-dideoxy-
Compound 66 was synthesized from methyl 5-(tert-butoxycarbonyl)amino-9-azido-4,7,8-di-O-acetyl-2,6-anhydro-3,5-dideoxy-
Compound 67 was synthesized from compound methyl 5-(tert-butoxycarbonyl)amino-9-azido-4,7,8-di-O-acetyl-2,6-anhydro-3,5-dideoxy-
Compound 68 was synthesized from compound methyl 5-(tert-butoxycarbonyl)amino-9-azido-4,7,8-di-O-acetyl-2,6-anhydro-3,5-dideoxy-
Compound 69 was synthesized from compound methyl 5-(tert-butoxycarbonyl)amino-9-azido-4,7,8-di-O-acetyl-2,6-anhydro-3,5-dideoxy-
Compound 70 was synthesized from compound methyl 5-(tert-butoxycarbonyl)amino-9-azido-4,7,8-di-O-acetyl-2,6-anhydro-3,5-dideoxy-
Compound 73 was synthesized from compound methyl 5-(tert-butoxycarbonyl)amino-9-azido-4,7,8-di-O-acetyl-2,6-anhydro-3,5-dideoxy-d-glycero-d-galacto-non-2-enonate using acetic anhydride and 2-ethylhexanoyl chloride. The product was obtained as a mixture of diastereoisomers at a position of the hexanamido group. 6.0 mg (24%(96%×67%×38%), over three steps). 1H NMR (500 MHz, CD3OD) δ 5.67 (2H), 4.36-4.33 (2H), 4.14-4.07 (2H), 4.01-3.97 (2H), 3.90-3.87 (2H), 3.64-3.56 (2H), 3.41-3.39 (2H), 3.26-3.14 (2H), 2.22-2.13 (4H), 1.93 (3H), 1.94 (3H) 1.63-1.26 (8H), 1.50-1.24 (m, 7H), 0.97-0.84 (6H). 13C NMR (126 MHz, CD3OD) δ 180.53, 173.75, 173.65, 108.90, 79.80, 77.38, 72.33, 72.01, 70.03, 69.94, 68.58, 68.54, 51.97, 51.95, 50.21, 50.14, 44.78, 44.54, 33.69, 33.51, 31.07, 30.78, 27.27, 27.12, 23.77, 22.60, 14.31, 12.68, 12.45. HRMS (ESI) calcd. for C19H31N2O8 [M−H]−, 415.2086; found 415.2088.
Compound 74 was synthesized from compound methyl 5-(tert-butoxycarbonyl)amino-9-azido-4,7,8-di-O-acetyl-2,6-anhydro-3,5-dideoxy-d-glycero-d-galacto-non-2-enonate using acetic anhydride and 2-methylhexanoyl chloride. The product was obtained as a mixture of diastereoisomers at a position of the hexanamido group. 10.2 mg (54%(96%×70%×60%), over three steps). 1H NMR (500 MHz, CD3OD) δ 5.76 (d, J=2.2 Hz, 1H), 5.72 (d, J=2.3 Hz, 1H), 4.43 (dd, J=8.8, 2.3 Hz, 1H), 4.40-4.35 (m, 1H), 4.32 (dd, J=11.0, 1.0 Hz, 1H), 4.17-4.14 (m, 1H), 4.11 (dd, J=10.9, 3.0 Hz, 1H), 3.98-3.93 (m, 1H), 3.92-3.85 (m, 2H), 3.64-3.53 (m, 3H), 3.39-3.30 (m, 2H), 3.20 (dd, J=13.7, 7.5 Hz, 1H), 2.41-2.32 (m, 2H), 1.68-1.58 (m, 2H), 1.42-1.24 (m, 10H), 1.14-1.09 (m, 6H), 0.93-0.86 (m, 6H). 13C NMR (126 MHz, CD3OD) δ 181.38, 174.09, 173.80, 168.70, 110.49, 110.37, 109.65, 77.58, 77.54, 76.27, 72.09, 71.74, 71.39, 70.24, 70.00, 69.96, 68.31, 68.28, 68.21, 52.30, 51.88, 51.69, 44.75, 44.52, 44.35, 42.25, 42.21, 35.05, 34.90, 30.93, 30.74, 23.74, 22.60, 22.57, 22.45, 18.57, 18.30, 14.40, 14.33. HRMS (ESI) calcd. for C13H29N2O8 [M−H]−, 401.1929; found 401.1926.
A solution of compound methyl 5-amino-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-d-glycero-d-galacto-non-2-enonate in anhydrous dichloromethane and triethylamine (4 eq) was cooled down to 0° C. and anhydrides or acyl chlorides (1.5 eq) was added in dropwise. The solution was allowed to warm to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, concentrated and purified by flash chromatography to give the protected product. The protected product was then dissolved in methanol and 0.5 N NaOH was added. The solution was stirred under room temperature until completion. After completion, the solution was neutralized by Amberlite™ IR 120 (H). The suspension was then filtered, and the filtrate was concentrated and purified by flash chromatography or precipitated in a mixture of methanol and ethyl acetate to give the desired product.
Compound 75 was synthesized from compound methyl 5-amino-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-d-glycero-d-galacto-non-2-enonate using 2-ethylhexanoyl chloride, and the product was obtained as a mixture of diastereoisomers at a position of the hexanamido group. 18.6 mg (25% (45%×55%), over two steps). 1H NMR (500 MHz, CD3OD) δ 5.64 (2H), 4.42 (dd, J=8.8, 2.2 Hz, 1H), 4.34 (dt, J=8.6, 2.6 Hz, 1H), 4.29 (dd, J=11.0, 0.8 Hz, 1H), 4.15 (dd, J=11.0, 8.8 Hz, 1H), 4.09 (ddd, J=10.9, 2.4, 1.1 Hz, 1H), 3.99 (ddd, J=10.8, 8.6, 7.0 Hz, 1H), 3.90-3.77 (m, 4H), 3.63 (dd, J=11.5, 5.5 Hz, 1H), 3.58-3.50 (m, 2H), 3.44 (dd, J=9.2, 0.9 Hz, 1H), 2.17 (dq, J=9.4, 5.0 Hz, 1H), 1.65-1.22 (m, 17H), 0.95-0.84 (m, 12H). 13C NMR (126 MHz, CD3OD) δ 180.56, 180.54, 170.35, 170.19, 159.62, 159.32, 150.18, 150.08, 118.72, 108.41, 107.90, 77.41, 76.07, 71.44, 71.36, 71.30, 70.80, 70.63, 70.16, 68.63, 68.59, 68.50, 65.45, 65.24, 64.84, 52.30, 51.89, 51.83, 50.23, 50.20, 33.70, 33.56, 31.11, 30.79, 27.33, 27.11, 23.83, 23.80, 14.39, 14.32, 12.61, 12.44. HRMS (ESI) calcd. for C17H28NO8 [M−H]−, 374.1820; found 374.1811.
Compound 72 was synthesized from compound methyl 5-amino-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-dideoxy-d-glycero-d-galacto-non-2-enonate using 2-methylhexanoyl chloride, and the product was obtained as a mixture of diastereoisomers at a position of the hexanamido group. 8.7 mg (10% (18%×55%), over two steps). 1H NMR (500 MHz, CD3OD) δ 5.70 (d, J=1.5 Hz, 1H), 5.68 (d, J=2.2 Hz, 1H), 4.43 (dd, J=8.8, 2.2 Hz, 1H), 4.36 (ddd, J=8.6, 4.1, 2.4 Hz, 1H), 4.31 (dd, J=11.0, 0.7 Hz, 1H), 4.18-4.15 (m, 1H), 4.12-4.08 (m, 1H), 3.96 (dd, J=10.8, 8.7 Hz, 1H), 3.90-3.78 (m, 4H), 3.65-3.53 (m, 3H), 3.52-3.47 (m, 1H), 3.45 (dd, J=9.3, 0.8 Hz, 1H), 2.40-2.32 (m, 1H), 1.69-1.57 (m, 1H), 1.40-1.21 (m, 12H), 1.15-1.05 (m, 6H), 0.89 (dt, J=7.0, 4.4 Hz, 6H). 13C NMR (126 MHz, CD3OD) δ 181.39, 181.37, 169.55, 169.33, 116.42, 109.43, 109.33, 108.68, 77.57, 76.22, 71.43, 71.26, 71.20, 70.67, 70.41, 70.17, 68.51, 68.46, 68.41, 65.38, 65.11, 64.87, 52.25, 51.81, 51.69, 42.32, 42.23, 35.13, 34.98, 30.96, 30.74, 23.79, 23.76, 18.47, 18.36, 14.38, 14.33. HRMS (ESI) calcd. for C6H26NO[M−H]−, 360.1664; found 360.1663.
Compound methyl 5-acetamido-4-azido-7,8,9-tri-O-acetyl-2,6-anhydro-3,4,5-trideoxy-
A solution of compound 76 (140 mg, 1 eq) in anhydrous pyridine was cooled down to 0° C. and acetic anhydride (400 μl, 10 eq) was added in dropwise. The mixture was then allowed to warm to temperature and kept stirring overnight. After completion, the reaction was quenched with methanol and solvents were removed under reduced pressure. The residue was dissolved in 200 ml ethyl acetate and carefully washed with 0.1 M HCl, water, brine and dried over Na2SO4.
The solution was concentrated to give 160 mg yellow oil, which was dissolved in 20 ml anhydrous DCM and 2 ml TFA was added slowly. The solution was then stirred at room temperature for 2 hours. After completion, DCM and TFA were removed under reduced pressure. The residue was dissolved in 10 ml anhydrous DCM and TEA (124 μl, 3 eq) was added. The mixture was then cooled down to 0° C. and 4-methylpentanoyl chloride (75 mg, 1.2 eq) was added. The solution was allowed to warm to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, concentrated and purified by flash chromatography to give the desired product. 105 mg (52%, over three steps). 1H NMR (500 MHz, CDCl3) δ 6.05 (d, J=8.3 Hz, 1H, NH), 5.94 (d, J=2.1 Hz, 1H, H-3), 5.41 (d, J=5.2 Hz, 1H, H-7), 5.29 (td, J=6.5, 2.7 Hz, 1H, H-8), 4.59 (dd, J=12.4, 2.6 Hz, 1H, H-9), 4.54-4.50 (m, H5, H-4), 4.16 (dd, J=12.4, 6.6 Hz, 1H, H-9′), 3.78 (s, 3H, COOCH3), 2.17 (t, J=7.8 Hz, 2H, α-CH2), 2.11, 2.03, 2.02 (3×s, 9H, 3×COCH3), 1.62-1.43 (m, 3H, β-CH2, γ-CH), 0.88 (d, J=6.3 Hz, 6H, 2×δ-CH3). 13C NMR (126 MHz, CDCl3) δ 174.00, 170.69, 170.27, 170.08 (4×C═O), 161.55 (C-1), 145.05 (C-2), 107.66 (C-3), 75.58 (C-6), 70.66 (C-8), 67.74 (C-7), 62.00 (C-9), 57.51 (C-4), 52.56 (COOCH3), 48.74 (C-5), 34.71 (C-β), 34.00 (C-γ), 27.72, 22.24, 22.20 (3×COCH3), 20.82, 20.72 (2×C-δ). HRMS (ESI) calcd. for C22H32N4NaO8 [M−H]−, 535.2011; found 535.2003.
To a solution of compound 77 (50 mg, 1 eq) in THE (2 ml), 1 N HCl (200 μl, 2.2 eq) was added, followed by triphenylphosphine (29 mg, 1.2 eq). The resulting mixture was stirred at room temperature overnight. After completion, solvents were removed under reduced pressure and the residue was purified by flash chromatography, providing crude product 50 mg. The residue was dissolved in 5 ml anhydrous DCM, and TEA (50 μl, 4 eq) was added. The solution was cooled down to 0° C. and N, N′-Di-Boc-1H-pyrazole-1-carboxamidine (600 mg, 2 eq) added. The reaction mixture was allowed to warm up to room temperature and kept stirring overnight. After completion, the reaction was quenched with water, extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, concentrated and purified by flash chromatography to give the desired product. 60 mg (87%, over two steps). 1H NMR (500 MHz, CDCl3) δ 8.46 (d, J=8.9 Hz, 1H, NH), 7.76 (brs, 1H, NH), 6.16 (d, J=9.2 Hz, 1H, NH), 5.88 (d, J=2.4 Hz, 1H, H-3), 5.42 (dd, J=4.9, 1.7 Hz, 1H, H-7), 5.28 (ddd, J=7.4, 4.9, 2.7 Hz, 1H, H-8), 5.19 (td, J=9.7, 2.4 Hz, 1H, H-4), 4.67 (dd, J=12.4, 2.7 Hz, 1H, H-9), 4.31 (dd, J=10.5, 9.7 Hz, 1H, H-5), 4.26 (dd, J=10.5, 1.7 Hz, 1H, H-6), 4.15 (dd, J=12.4, 7.4 Hz, 1H, H-9′), 3.79 (s, 3H, COOCH3), 2.17-1.96 (m, 11H, 3×COCH3, α-CH2), 1.56-1.33 (m, 21H, 2×tBoc, β-CH2, γ-CH), 0.85 (dd, J=6.5, 2.7 Hz, 6H, 2×δ-CH3). 13C NMR (126 MHz, CDCl3) δ 174.01, 170.56, 170.24, 170.07 (4×C═O), 161.70 (tBoc-OCO), 157.23 (C-1), 152.62 (C═N), 145.07 (C-2), 109.71 (C-3), 83.87, 79.77 (tBoc-C(CH3)3), 78.11 (C-6), 71.57 (C-8), 67.76 (C-7), 62.29 (C-9), 52.45 (COOCH3), 48.88 (C-4), 47.60 (C-5), (COCH3), 34.67 (C-α), 34.12 (C-β), 28.27, 28.03 (tBoc-C(CH3)3), 27.70 (C-γ), 22.32, 22.13 (C-δ), 20.91, 20.87, 20.79 (COCH3). HRMS (ESI) calcd. for C33H52N4NaO14 [M+Na]+, 751.3372; found 751.3378.
To a solution of compound 78 (60 mg) in 5 ml DCM, 500 μl TFA was added. The solution was then stirred at room temperature for 2 hours. After completion, DCM and TFA were removed under reduced pressure. The residue was dissolved in methanol and 2 ml 1 N NaOH was added. The solution was stirred at room temperature for 1 hour. After completion, the reaction mixture was added with Amberlite™ IR 120 (H) to make the pH of the solution as 7. The suspension was then filtered, and the filtrate was concentrated to give a light-yellow oil. The residue was dissolved in minimum methanol and the product was precipitated by ethyl acetate. The product was obtained by filtering as a white solid. 15 mg (48%, over two steps). 1H NMR (700 MHz, CD3OD) δ 5.50 (s, 1H, H-3), 4.37 (d, J=8.5 Hz, 1H, H-4), 4.33 (d, J=10.0 Hz, 1H, H-6), 4.19 (t, J=9.4 Hz, 1H, H-5), 3.89-3.79 (m, 2H, H-8, H-9), 3.65 (dd, J=11.3, 5.4 Hz, 1H, H-9′), 3.58 (d, J=9.2 Hz, 1H, H-7), 2.26 (t, J=7.5 Hz, 2H, α-CH2), 1.61-1.49 (m, 3H, β-CH2, γ-CH), 0.92 (d, J=6.4 Hz, 6H, 2×δ-CH3). 13C NMR (176 MHz, CD3OD) δ 177.19 (COCH3), 169.63 (C-1), 158.78 (C═N), 151.58 (C-2), 103.33 (C-3), 77.04 (C-6), 71.33 (C-8), 70.31 (C-7), 64.88 (C-9), 52.26 (C-4), 35.85 (C-α), 35.21 (C-β), 28.86 (C-γ), 22.72, 22.63 (2×C-δ). HRMS (ESI) calcd. for C33H52N4NaO14 [M+Na]+, 387.1885; found 387.1879.
Inhibition assays against 4MU-NANA (2′-(4-methylumbellifery)-α-D-N-acetylneuraminic acid) cleavage and GM3 cleavage was performed using protocols reported previously (Zhang, 2013). NEU3 and NEU2 were expressed as N-terminal MBP fusion protein in E. coli and purified as previously reported (Albohy, 2010). NEU4 was expressed as a GST fusion protein in E. coli and purified as previously reported (Albohy, 2011). NEU1 was expressed as His fusion protein in HEK293 cells and cell lysate was used without further purification (Pshezhetsky, 1996). All assays were conducted in 0.1 M sodium acetate buffer at optimum pH for each enzyme (4.5 for NEU1, NEU3 and NEU4; 5.5 for NEU2) (Zhang, 2013). To get comparable IC50 among the four isoenzymes, similar activity units of each enzyme were used in the assay.
For assays using 4MU-NANA as the substrate, inhibitors with of a 3-fold serial dilution of concentrations were incubated with enzyme at 0° C. for 15 min. 4MU-NANA was then added to the mixture, making the final concentration of 4MU-NANA as 50 μM and the total volume of the reaction mixture as 20 μL. After incubation at 37° C. for 30 min, the reaction was quenched with 100 μL of 0.2 M sodium glycine buffer (pH 10.2). The reaction mixture was transferred to 386-well plate and the enzyme activity was determined by measuring fluorescence (λex=365 nm; λem=445 nm) using a plate reader (Molecular Devices, Sunnyvale Calif.). Assays were performed with duplicates for each point and IC50 was obtained by plotting the data with Graphpad™ Prism 5.0. For curves that showed less than a 50% decrease in signal, fits were conducted using maximum inhibition values found for DANA.
For inhibition assays against GM3 cleavage, a method developed by Markely and coworkers was adopted (Markely, 2010). The assay was conducted in 0.1 M sodium acetate buffer (pH 4.5). After serial concentrations of inhibitors were incubated with enzyme at 0° C. for 15 min, GM3 was added, making the final concentration of GM3 as 500 μM and the total volume of the reaction mixture as 20 μL. The reaction mixture was incubated at 37° C. for 30 min and quenched with 100 μL of freshly made 0.2 M sodium borate buffer (pH 10.2). 0.8% malononitrile solution (40 μL) was added to form a fluorescent adduct with the free sialic acids. Fluorescence was obtained (λex=357 nm; λem=434 nm) and the data was processed using Graphpad™ Prism 5.0. For curves that showed less than a 50% decrease in signal, fits were conducted using maximum inhibition values found for DANA.
Enzymes were incubated with serial concentrations of inhibitors at 0° C. for 15 min and serial concentrations of 4MU-NANA were added. The reaction mixture was transferred to 386-well plate immediately and the rate of product formation was obtained by measuring fluorescence (λex=315 nm; λem=450 nm) every 30 s for 30 min. The obtained data was processed with Graphpad™ Prism 5.0 for Ki determination.
To evaluate the inhibitory effects of the compounds against individual isoenzymes, each enzyme was produced recombinantly or purification (Albohy, 2013; Zhang, 2013; Albohy, 2010) and the IC50 tested using an artificial substrate, 2′-(4-methylumbelliferyl)-α-
In Table I, the specificity of a compound it designated as dual (bispecific) e.g., Neu3/Neu4, when the ratio of IC50 of this compound against an enzyme over the IC50 of this compound against another enzyme is of about 3 or less. Compounds 72-75 were tested as a mixture of diasteroisomers. All the others are pure compounds of a single stereochemistry.
Adhesion of Jurkat cells (T cells) to ICAM-1 was determined using flow cytometry and ICAM-1-coated fluorescent beads (1 μm) under the indicated conditions. Control samples were treated with DMSO (0.05%), used as negative control, phorbol myristate acetate (PMA), known to induce leukocyte adherence and used as a positive control, or the nonspecific neu inhibitor DANA for 30 min (see Dana structure in Table I above). Results are presented in
Then, adhesion of Jurkat cells to ICAM-1 was determined using flow cytometry and ICAM-1-coated fluorescent beads; samples were treated with buffer (DMSO) or enzyme (Neu3; and NanI sialidase, another sialidase used as a control) for 3 hrs, followed by incubation with PMA for 30 min. All cytometry data were normalized to the appropriate control after background subtraction (BSA coated beads). Results are shown in
Then, homotypic aggregation of Jurkat cells was determined using microscopy. Cells were incubated under the indicated conditions for 3 hrs. Aggregation was determined by imaging and analysis with CellProfiler™ (version 2.1.1) to determine the total number of cells and the number of cells found within aggregates. Aggregation is expressed in
To confirm the effect of NEU3 on primary cells, PBMC were tested using the static adhesion assay as described in Example 94. Results are shown in
The inventors have then tested the effect of NEU enzymes on LFA-1-ICAM-1 adhesion. Lymphocyte function-associated antigen 1 (LFA-1) is an integrin found on lymphocytes and other leukocytes and is known to bind to ICAM-1. Jurkat T cells (
A transwell assay was implemented with a porous (3 μm pores) membrane coated with fibronectin (FN). The bottom well of the transmigration plate was coated with FBS, TNFα and IL-4 as chemo attractants. Neu3 inhibitors (compound 5c—see structure below; and compound 8b, see structure in Table I above); neu1 inhibitors (compounds 32 and 50, see structures in Table I above) and a neu4 inhibitor (compound 28, see structure in Table I above) were added to the upper well along with T cells (Jurkat) at the indicated concentrations. DMSO buffer was used as the negative control (control on left panel and control (DMSO) on right panel), and Cytochalasin D (Cyto D) was used as a positive control for inhibition of migration. Cells were then incubated for 21 h.
The number of cells migrating into the lower chamber were quantified using FACs and normalized to the control. Results are shown in
Mice were maintained at the Canadian Council on Animal Care (CCAC)-accredited animal facilities of the CHU Sainte-Justine Research Centre, according to the CCAC guidelines.
To induce an air pouch, mice (6-wk-old, male Neu1−/− (KO), Neu3−/− (KO), Neu4−/ (KO)−, Neu3−/− (KO), Neu4−/− (KO) and C57Bl/6 mice) were injected subcutaneously with 3 cc of air (passed through 0.2 μm filter) on the back two times per week using a 26-gauge needle. Before the procedure, mice were anesthetized with isoflurane (2-3% with oxygen 0.4 L/min) and checked for proper anesthesia initiation. After injections mice were returned to their cages and checked for restoration of normal functions after recovery from anesthesia.
After one week, the air pouch was injected with 1 ml of an inflammatory substance LPS (Sigma-Aldrich, USA) at 1 ug/ml concentration in saline to induce inflammation. 1 ml of saline were injected on the air pouches to the control group of mice. Mice were returned to their cages, where they were allowed to run free for 6 h. At the end of 6 h, the animals were sacrificed using CO2 asphyxiation. The contents of the pouch were aspirated. Then the air pouches were washed twice with 2 ml of HBSS containing 10 mM EDTA. The exudates were collected and centrifuged at 100 g for 10 min at room temperature. The supernatant were collected and frozen for later analysis. The cells were resuspended in 1 ml of HBSS-EDTA and taken for cell count. The air pouch were dissected from the subcutaneous tissue and stained (hematoxylin and eosin and Giemsa stains) using standard methods. The spleen, bones and other organs were collected and frozen for future analysis.
The cells collected by aspiration (in 4 ml of HBSS containing 10 mM EDTA) were aliquoted according to the following segmentation for hemocytometer, cytokine, FACS and Glycolipid analysis.
For hemocytometer analysis, cells were centrifuged and spread onto microscopy slides, and stained with Hema stain for quantification of granulocyte and monocytes.
For cytokine and glycolipid analysis fraction was centrifuged 100×g for 10 min at RT. Plasma was separated from the cells, and frozen in liquid N2. The cells were reconstituted in freezing buffer (RPMI+50% FBS+5% DMSO) and frozen in liquid nitrogen.
For further identification of other subpopulations, 300 μl of the cells were centrifuged 100×g for 10 min. The supernatant was washed once with HBSS. Cells were suspended in blocking solution containing 25 μl of mouse serum (prepared by 1000×g centrifugation of mouse blood collected by cardiac puncture and clotted undisturbed for 30 min at RT) and the pellet was mixed gently with it. For staining 25 μl cell suspension from the previous step was taken and added 25 μl master mix of antibodies (see Table II below for the antibodies and concentration used) and stained at RT for 30 min. Then tubes were centrifuged once at 100×g for 10 min and the supernatant was discard. The pellet was reconstituted to 100 μl using FACS buffer (2% FBS in HBSS) with 4% PFA. FACS analysis was used to quantify different cell types.
1:100
Femurs and tibias of analyzed mice were flushed and plated into 30% L929 supernatant, 70% DMEM (10% FBS, 4.5 g/L glucose, L-glutamine, Sodium Pyruvate, 1% Pen/Strep), at a concentration of 100 ml/mouse, 10 ml/10 cm tissue culture-treated dish (day 0). Dishes were incubated at 37 C, 5% CO2 and 1 ml of L929 supernatant is added to each dish on days 2 & 4. Cells were mature by day 7.
In vivo infiltration of leukocytes was performed using a murine air pouch (as described in Example 98, Sin et al, 1986), as an acute model of bacterial infection with LPS stimulation.
First, murine WT C57BL/6 mice or NEU KO or DKO (NEU1, NEU3, NEU4, NEU3/NEU4) animals were analyzed for changes in leukocyte migration. The air pouch was loaded with saline or LPS to induce leukocyte migration. Cells were washed out of the pouch and counted by hemocytometer and FACs. Significant leukocyte count (migration) increases were observed for NEU4 KO mice after LPS treatment. NEU1 KO mice showed a significant decrease in leukocytes count (migration). Results are shown in
Then, using the same air pouch model, murine WT C57BL/6 mice were administered saline or LPS in their pouches to induce leukocyte migration. Cells were washed out of the pouch and counted. The mice were pre-treated with inhibitors of human neuraminidase (i.e. Compounds 32 (neu1-specific), 8b (neu3-specific) and 28 (neu4-specific), see compounds structures in Table I above) 48 h, 24 h prior and in conjunction to injection of LPS or saline. Results are shown in
The cells isolated from the air pouch of Example 99 after stimulation with saline or LPS were sorted through flow cytometry with the use of appropriate lineage-specific markers shown in the Table II above to determine the cell subpopulations. The results show that neu3/4, neu4, neu3 and neu1 KO mice along with inhibitors for those neuraminidases modulate (increases or decreases) migrations of members of the leukocytes in saline or in the context of bacterial infection. Results are shown in
The Passive Antibody Transfer Model of ITP has been extensively used to understand the progression of chronic ITP and to rapidly evaluate the efficacy of various therapeutics (Neschadim et al., 2015). The model is characterized by a rapid onset of the ITP and clear involvement of phagocytic monocytes in platelet destruction. In addition, it provides a possibility to use self-antigens relevant to the human condition. Depending on the type, quantity, and frequency of the administered antibody it is possible to vary the severity and the persistence of the induced ITP by fine-tuning the rate and the level of platelet decline whereas the repeated administration of the monoclonal antibody simulates chronic human IT.
Female 6-week old C57Bl/6NCrI mice and genetically-modified C57Bl/6NCrI mice with neu1 deficiency (Passive Antibody Transfer Model of ITP, Neschadim et al., 2015) received escalating daily doses (68 μg/kg on the 1st and 2d days, 102 μg/kg on the third day) of anti-CD41 anti-platelet monoclonal mouse antibody (clone MWReg30) by intraperitoneal injection. The reticulated platelets were measured daily for three days by flow cytometric analysis. Control mice (C57B16) received injections of saline. In the wt mice antibody injection resulted in rapid (over 2 days) reduction of reticulated platelets from about 1000×109 to less than 50×109 counts/L. No increase in reticulated platelets was observed in this model over time, indicating thrombopoiesis suppression. Lower platelet reduction rates in NEU1-deficient CathAS190A-Neo mice (˜10% of residual neu1 activity) (
Of note, the CathAS190A-neo knock-in mouse is not clinically affected (Seyrantepe et al., 2008), suggesting that a partial pharmacological inhibition of NEU1 may have a preventive or therapeutic effect for ITP without affecting catabolism of sialoglycoconjugates catalyzed by NEU1 in the lysosome.
The recognition of LPS by the immune system involves TLR4/MD2, CD14 and LPS-binding protein (Park et al., 2013). Results shown in Examples 94-96 suggest that NEU regulates integrin function.
The mouse model of pulmonary Pseudomonas aeruginosa infection is used. Five mouse strains (wild-type C57Bl6 (WT), Neu3−/−, Neu4−/−; Neu1ΔEx3-Geo (constitutive NEU1 KO); and Neu1MΘΔEx3 (macrophage-specific NEU1 KO) are infected intratracheally with a 50 μL suspension of 1.5×107 P. aeruginosa strain PAO1 which produces a severe pneumonia (pulmonary bacterial burden of ˜1×105 colony forming units). Mouse survival remains >90% at 48 hours in this model, permitting adequate sampling of all experimental groups and obviating a healthy survivor bias. Forty-eight hours after infection, mice are sacrificed following bronchoalveolar lavage. Analysis of bronchoalveolar lavage fluid inflammatory markers are performed, including flow cytometry enumeration of leukocyte populations and multiplex ELISA for cytokine levels. Bronchoalveolar lavage fluid LDH levels are quantified as a measure of pulmonary injury. Pulmonary bacterial burden is determined by quantitative culture as the primary virulence outcome. A cut-off of 0.5 log difference in bacterial burden is considered biologically significant and the Wilcoxon-rank sum is used to compare bacterial burden between groups, performed in duplicate with 10 mice the minimum group size.
Pharmacological inhibition of NEU1-4 is tested pulmonary Pseudomonas aeruginosa infection. Selective inhibitors of human and mouse NEU isoenzymes with potency in sub-micromolar range are used. The inhibitors are given intraperitoneally once/day before and during the infection. Inhibitors of NEU1, NEU3 and NEU4 in mice have been tested for the period of up to month which demonstrated that compounds are well tolerated by animals and result in almost complete inhibition in of NEU enzymes in the mouse tissues.
Female 6-week old C57Bl/6NCrI mice (Passive Antibody Transfer Model of ITP) received escalating daily doses (68 μg/kg on the 1st and 2d days, 102 μg/kg on the third day) of anti-CD41 anti-platelet monoclonal mouse antibody (clone MWReg30) by intraperitoneal injection. Experimental group received daily intraperitoneal injections of neu1 inhibitor compound 50 in saline at a dose of 30 μg/kg while control mice (Nov C57B16) received daily intraperitoneal injections of saline. The reticulated platelets were measured daily by flow cytometric analysis.
Cell-specific NEU1 KO strains are generated by crossing a conditional Cre NEU1 gene-targeted mouse strain with mice expressing Cre recombinase in the cells of interest.
The inventors have acquired mouse ES line ENSMUSE00000141558 which is targeted with the PG00096_Z_3_G09 vector that allows production of conditional and tissue specific NEU1 KO. They have generated gene-targeted C57Bl6 mice heterozygous for the targeted NEU1 allele. The targeted allele contains LacZ/BactPNeo cassette flanked with FRT sites inserted into the intron 2 of the mouse NEU1 gene. In addition, the exon 3 of the gene is flanked with LoxP sites. The inventors have produced mice homozygous for the ENSMUSE00000141558 allele and showed that they do not express WT NEU1 mRNA. Similarly to previously described NEU1 KO mice (de Geest, 2002) NEU1ENSMUSE00000141558 mice showed almost complete deficiency of NEU1 mRNA and sialidase activity in kidney, where the NEU1 is a predominant neuraminidase. These data demonstrate that the NEU1 gene in NEU1ENSMUSE00000141558 mice is correctly targeted.
NEU1ENSMUSE00000141558 strain was first crossed with “FLP deleter” strain with a global expression of flippase (FLP) recombinase (JaxLab) to remove the FRT-flanked gene trap-cassette from the targeted NEU1 allele allowing normal expression of the gene. Then the mice were crossed with the available mice (JaxLab) expressing Cre recombinase under control of the macrophage specific Cx3cr1 (chemokine C-X3-C motif receptor 1) promoter (Yona et al, 2013) or and will be crossed with the available mice (JaxLab) expressing Cre recombinase under control of the platelet-specific Pf4 (platelet factor 4) promoter (Tiedt et al, 20107). This causes a recombination removing the entire exon 3 from the NEU1 gene only in MO (Neu1−/−MΘ, see
Heterozygous mice are mated to obtain homozygous and WT siblings (embryonic lethality is not expected since it is not observed in the full NEU1 KO). Expression of NEU1 mRNA in purified platelets and peritoneal and spleen MO as well as in control tissues (liver, kidney, brain, heart) of the homozygous animals are studied by qPCR and the level of neuraminidase activity measured by the fluorescent assay to confirm efficiency of the splicing outcome. The expression of NEU1 protein is studied by Western blot in the same tissues using available antibodies against mouse NEU1. Monocytes and monocyte-derived cells (spleen resident MΘ, liver Kupfer cells, brain microglia (Yona et al., 2013) as well as tissues of visceral organs, lungs, and different areas of brain are studied by light and electron microscopy to assess vacuolization of cells and presence of lysosomal storage bodies. Phenotypic characterization of mice is performed to determine if they show neurological symptoms previously described for NEU1 KO mice (de Geest et al., 2002). General observation, weight measurements, and estimation of life span of animals are performed. Neurological assessment and behaviour study by the open field test are performed to detect signs of neurodegeneration (Martins et al., 2015).
ITP in macrophage-specific NEU1 KO is studied using the Passive Antibody Transfer Model as described in Example 101. ITP in platelet-specific KO is induced by anti-GPIb antibodies that specifically trigger NEU1 induction. If an effect is observed in vivo experiments are performed to decipher the cellular/molecular mechanism by which NEU1 triggers ITP. In particular, the surface sialylation of platelets and MO are studied by SNA/PNA lectin binding and FACS, sialylation of FcγR receptor and platelet surface antigens, by immunoprecipitation and SNA/PNA lectin blot, and platelet activation, by selectin binding and FACS. Platelet uptake by spleen MΘ are directly studied by injecting mice with fluorescently labeled antibody against murine platelets followed by IHC analysis to detect opsonized platelets in spleen tissues stained with anti-F4/80 antibodies specific for MΘ. Profiles of circulating inflammatory cytokines (IL-1 α, -2, -6,-17, -23, GM-CSF, MCP-1, MIP-1 α and β, RANTES, TNFα and IFN γ) are also studied because by inducing their production through activation of TLR-3,4 receptors on MΘ NEU1 can also contribute to severity of ITP.
This application claims the benefit of priority from U.S. Provisional patent application No. 62/772,704, filed on Nov. 29, 2018, which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2019/051715 | 11/29/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62772704 | Nov 2018 | US |
