Compounds and formulations useful as vaccine adjuvants

Abstract

The present invention relates to novel compounds having a structure set forth in any one of Formulas I, Ia, II, IIa, III, IIIa, IV, or IVa described herein, and formulations comprising such novel compounds. The novel compounds and formulations of the present invention may be useful in vaccine compositions. In some embodiments, the present invention relates to a composition comprising at least one antigen and a compound having a structure set forth in any one of Formulas I, Ia, II, IIa, III, IIIa, IV, or IVa described herein, or one or more pharmaceutically acceptable salts thereof, which is formulated as a stable nanoemulsion (referred to herein as an "SNE adjuvant composition" or "SNE").

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 503,015, filed May 18, 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Many vaccines have low immunogenicity or induce insufficient immune responses that cannot provide sufficient protection against the target infectious agent.Therefore, adjuvants may be needed to help generate immune responses of sufficient magnitude, quality, and durability required for vaccine use.Adjuvants can be used in immunogenic compositions containing a target antigen to enhance the immune response to the antigen, reduce the amount of antigen required to induce a desired immune response, or induce a sustained immune response and reduce the number of injections required in clinical regimens to provide protection from disease.Despite the success of adjuvants for certain compositions, there is a need for additional adjuvants that are safe for use in vaccines and immunogenic compositions and are effective in increasing the immune response to a target antigen. Summary of the Invention

[0003] The present invention relates to compounds of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, and pharmaceutically acceptable salts thereof. Furthermore, the present invention relates to formulations and compositions comprising one or more compounds of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. Furthermore, the present invention relates to formulations and compositions comprising one or more compounds of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier, wherein the formulations and compositions are useful as vaccines.

[0004] The present invention also relates to a formulation comprising i) one or more compounds of the present invention, or pharmaceutically acceptable salt(s) thereof; ii) one or more emulsifying agents; and iii) one or more terpenes.

[0005] The present invention also relates to a formulation comprising i) one or more compounds of the present invention, or pharmaceutically acceptable salt(s) thereof; ii) one or more sorbitan-based surfactants; and iii) one or more terpenes.

[0006] The present invention also relates to formulations comprising one or more compounds of Formula I, Ia, II, IIa, III, IIIa, IV, or IVa, or a pharmaceutically acceptable salt(s) thereof, and further comprising sorbitan trioleate (SPAN-85), polysorbate-20 (PS-20), or polysorbate-80 (PS-80), and squalene. In some embodiments, the formulations are prepared as stable nanoemulsions (referred to herein as "SNE adjuvant formulations" or "SNEs").

[0007] The present invention also relates to compositions comprising the antigens and SNEs disclosed herein. As an example, a specific SNE comprises the compound N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)pyrazin-1-yl)-5-oxopentyl)stearamide, also known as "Compound A-1." This specific SNE is referred to as Compound A-1-SNE and comprises Compound A-1, SPAN-85, PS-20, or PS-80, and squalene. As another example, a specific SNE includes the compound N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, also known as "Compound B-1." This specific SNE is referred to as Compound B-1-SNE and includes Compound B-1, SPAN-85, PS-20, or PS-80, and squalene. As another example, a specific SNE includes the compound N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide, also known as "Compound C-4." This particular SNE is referred to as compound C-4-SNE and includes compounds C-4, SPAN-85, PS-20 or PS-80, and squalene.

[0008] The present invention also relates to immunogenic compositions comprising the antigens and SNEs disclosed herein.

[0009] The present invention also relates to methods of treating or preventing disease in a patient in need thereof by administering to the patient an immunogenic composition of the present invention. [Brief explanation of the drawings]

[0010] [Figure 1]Example 10 shows OD640 readings after incubation of different concentrations of compound B-1-SNE with human TLR7 (top panel) or TLR8 (bottom panel) expressing HEK-Blue™ 293 cells (InvivoGen). The Y-axis represents the total compound B-1-SNE concentration. [Figure 2] Example 11 shows anti-6B IgG titers pre-immunization (day 0, pooled serum), post-dose 1 (day 21), post-dose 2 (day 42), and post-dose 3 (day 70) after immunization of mice with the formulations listed in Table 7. Error bars are geometric means with 95% confidence intervals. Transformed data analyzed by one-way ANOVA with Dunnett's post-hoc test, *p<0.05. [Figure 3] (Example 11) Pre-immune (day 0), post-dose 1 (day 21), post-dose 2 (day 42), and post-dose 3 (day 70) anti-6B OPA titers from pooled sera after immunization of mice with the formulations listed in Table 7. [Figure 4] Example 12 shows the ratios of serotype-specific IgG titers in infant rhesus macaques after immunization with PCV24 containing Compound B-1-SNE (150 μg dose of Compound B-1), PCV24 containing Compound B-1-SNE (50 μg of Compound B-1), and PCV24 containing Compound B-1-SNE (15 μg of Compound B-1) compared to immunization with PCV24 formulated without adjuvant at post-dose 3. Data for serotypes 6C and 15B are included to assess cross-reactivity. [Figure 5] Example 12 shows the ratios of serotype-specific IgG titers in infant rhesus macaques after immunization with PCV24 with Compound B-1-SNE (150 μg dose of Compound B-1), PCV24 with Compound B-1-SNE (50 μg of Compound B-1), and PCV24 with Compound B-1-SNE (15 μg of Compound B-1) compared to immunization with PCV24 formulated with APA after dose 3. Data for serotypes 6C and 15B are included to assess cross-reactivity. [Figure 6](Example 12) Figure showing the ratio of serotype-specific IgG titers in infant rhesus macaques after immunization with PCV24 with Compound B-1-SNE (150 μg dose of Compound B-1), PCV24 with Compound B-1-SNE (50 μg of Compound B-1), and PCV24 with Compound B-1-SNE (15 μg of Compound B-1) compared to immunization with PCV20 at dose 3. [Figure 7] (Example 13) Figure showing the ratio of serotype-specific IgG titers in infant rhesus macaques after immunization with PCV24 with Compound B-1-SNE (100 μg dose of Compound B-1), PCV24 with Compound B-1-SNE (10 μg of Compound B-1), PCV24 with Compound B-1-SNE (1 μg of Compound B-1), and PCV24 with Compound B-1-SNE (0.1 μg of Compound B-1) compared to immunization with PCV20 at dose 3. [Figure 8] Example 14 shows the ratio of serotype-specific IgG titers in mice after immunization with PCV26 containing Compound B-1-SNE (10 μg dose of Compound B-1) compared to immunization with PCV26 formulated with APA at dose 2. Data for serotypes 6C and 15B are included to assess cross-reactivity. [Figure 9] Example 14 shows the IgG2a / IgG1 titer ratios of serotype-specific antibodies in mice after immunization with PCV26 containing Compound B-1-SNE (a 10 μg dose of Compound B-1) or PCV26 formulated with APA at dose 3. Responses from three representative serotypes, 18C, 19F, and 24F, are shown (left to right). [Figure 10A] Example 14: Mice immunized with PCV26 bearing Compound B-1-SNE (a 10 μg dose of Compound B-1) or PCV26 formulated with APA are protected from intratracheal challenge with Streptococcus pneumoniae 24F. Bacteremia was quantified 24 hours after infection with serotype 24F bacteria and expressed as log CFU / mL. [Figure 10B]Example 14: Mice immunized with PCV26 bearing Compound B-1-SNE (a 10 μg dose of Compound B-1) or PCV26 formulated with APA are protected from intratracheal challenge with Streptococcus pneumoniae 24F. Survival was assessed approximately 240 hours after infection with serotype 24F bacteria. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention provides compounds having the structure set forth in Formula I: [ka] (In the formula, R a is selected from H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, and —NR′R″, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted with 1 to 4 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine; R a’ is selected from H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, and —NR′R″, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted with 1 to 4 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine; R a’’is selected from H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, and —NR′R″, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted with 1 to 4 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine; R' and R" are independently selected from H, (C1-C6)alkyl, (C1-C6)alkenyl, and (C1-C6)alkynyl, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, and (C1-C6)alkynyl are optionally substituted with one to four substituents independently selected from the group consisting of -OH, -O(C1-C4)alkyl, -O(C1-C4)alkenyl, -O(C1-C4)alkynyl, chlorine, and fluorine; or R' and R" together with the nitrogen to which they are attached, join together to form a (C3-C6)heterocycloalkyl, wherein said (C3-C6)heterocycloalkyl is optionally substituted with one to four substituents independently selected from the group consisting of -OH, -O(C1-C4)alkyl, -O(C1-C4)alkenyl, -O(C1-C4)alkynyl, chlorine, and fluorine; R bis independently selected from H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, or NR′R″, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted with from 1 to 4 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine; A is (C1-C6) alkyl, (C3-C6) heterocycloalkyl, heterocycloalkyl-C(O)-R z -, (C1-C4) alkyl-N(R z )-R z a carbon or nitrogen bond spacer selected from -, aryl, and heteroaryl, wherein said (C1-C6)alkyl, (C3-C6)heterocycloalkyl, aryl, and heteroaryl are independently selected from the group consisting of -OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, -O(C1-C6)alkyl, -O(C1-C6)alkenyl, -O(C1-C6)alkynyl, chlorine, fluorine, and NR'R''; each of the (C1-C6) alkyl, (C1-C6) alkenyl, (C1-C6) alkynyl, -O(C1-C6) alkyl, -O(C1-C6) alkenyl, and -O(C1-C6) alkynyl is optionally substituted with 1 to 6 substituents independently selected from the group consisting of -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine; R z each occurrence is independently H or (C1-C6) alkyl; B is [ka] is a functional group selected from D is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl, wherein the (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl is optionally substituted with 1 to 6 substituents independently selected from the group consisting of -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine, or D is [ka] and Each occurrence of Z is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 )alkynyl, wherein the (C-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl is optionally substituted with 1 to 6 substituents independently selected from the group consisting of -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine; m is 0, 1, 2, 3, 4, or 5; (n is 0, 1, 2, 3, 4, or 5) or a pharmaceutically acceptable salt thereof.

[0012] The present invention provides compounds having the structure according to formula Ia: [ka] (In the formula, R' and R" are independently selected from H, (C1-C6)alkyl, (C1-C6)alkenyl, and (C1-C6)alkynyl, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, and (C1-C6)alkynyl are optionally substituted with one to four substituents independently selected from the group consisting of -OH, -O(C1-C4)alkyl, -O(C1-C4)alkenyl, -O(C1-C4)alkynyl, chlorine, and fluorine; or R' and R" together with the nitrogen to which they are attached, join together to form a (C3-C6)heterocycloalkyl, wherein said (C3-C6)heterocycloalkyl is optionally substituted with one to four substituents independently selected from the group consisting of -OH, -O(C1-C4)alkyl, -O(C1-C4)alkenyl, -O(C1-C4)alkynyl, chlorine, and fluorine; R b each occurrence of is -O(C1-C4)alkyl, wherein said -O(C1-C4)alkyl is optionally substituted with one or two substituents independently selected from the group consisting of -OH, -O(C1-C4)alkyl, -O(C1-C4)alkenyl, -O(C1-C4)alkynyl, chlorine, and fluorine; A is, [ka] is selected from R z each occurrence is independently H or (C1-C6) alkyl; R d each occurrence of is independently selected from —OH, (C1-C4)alkyl, —O(C1-C4)alkyl, chlorine, and fluorine; B is [ka] and D is [ka] wherein any carbon on the lipid chain may be substituted with -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, or fluorine; [ka] is cis or trans stereochemistry, X 1 -O-, -C(R) 2 - or -NR-, each occurrence of R is independently selected from H, (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, —OH, —O(C-C)alkyl, —O(C-C)alkenyl, —O(C-C)alkynyl, chlorine, and fluorine; m is 0, 1, or 2; n is 0, 1, 2, or 3; p is 0, 1, or 2; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; s is 1, 2, 3, 4, 5, 6, 7, or 8, t is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) or a pharmaceutically acceptable salt thereof.

[0013] The present invention provides compounds having the structure set forth in Formula II: [ka] (In the formula, R 1 is (C1-C6) alkyl, wherein the (C1-C6) alkyl is optionally substituted with 1 to 4 substituents selected from —OH and —O(CH3), R 2 is H, methyl or -O(CH3), R 3each occurrence is independently H, (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, or —O(C-C)alkyl, wherein said (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, or —O(C-C)alkyl is optionally substituted with one or two substituents independently selected from —OH and —O(CH); R 4 each occurrence is independently H, (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, or —O(C-C)alkyl, wherein said (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, or —O(C-C)alkyl is optionally substituted with one or two substituents independently selected from —OH and —O(CH); R 5 teeth, [ka] and R 6 is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 )alkynyl, wherein said (C-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl is optionally substituted with 1 to 6 substituents independently selected from -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine; each occurrence of n is 4) or a pharmaceutically acceptable salt thereof.

[0014] The present invention provides compounds having the structure according to formula IIa: [ka] (In the formula, R 1is butyl, said butyl being optionally substituted with one or two -OH groups; R 3 each occurrence of is independently H or —O(CH), R 5 teeth, [ka] and R 6 is (C 10 -C 20 ) alkyl, (C 10 -C 20 ) alkenyl, and (C 10 -C 20 )alkynyl) or a pharmaceutically acceptable salt thereof.

[0015] The present invention provides compounds having the structure set forth in Formula III: [ka] (In the formula, R 1 is (C1-C6) alkyl, wherein the (C1-C6) alkyl is optionally substituted with 1 to 4 substituents selected from —OH and —O(CH3), R 2 is H, methyl or -O(CH3), R 3 each occurrence is independently H, (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, or —O(C1-C4)alkyl, wherein said (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, or —O(C1-C4)alkyl is optionally substituted with one or two substituents selected from —OH and —O(CH3); R 4 is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 )alkynyl, wherein said (C-C 20) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl is optionally substituted with 1 to 6 substituents selected from -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine; n is 4) or a pharmaceutically acceptable salt thereof.

[0016] The present invention provides compounds having the structure according to formula IIIa: [ka] (In the formula, R 1 is butyl, said butyl being optionally substituted with one or two -OH groups; R 3 each occurrence of is independently H or —O(CH), R 4 is (C 10 -C 20 ) alkyl, (C 10 -C 20 ) alkenyl, and (C 10 -C 20 )alkynyl) or a pharmaceutically acceptable salt thereof.

[0017] The present invention provides compounds having the structure set forth in Formula IV: [ka] (In the formula, R 1 is (C1-C6) alkyl, wherein the (C1-C6) alkyl is optionally substituted with 1 to 4 substituents selected from —OH and —O(CH3), R 2 is H, methyl or -O(CH3), R 3each occurrence is independently H, (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, or —O(C1-C4)alkyl, wherein said (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, and —O(C1-C4)alkyl are optionally substituted with one or two substituents selected from —OH and —O(CH3); R 4 Each occurrence of (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 )alkynyl, wherein the (C-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl is optionally substituted with 1 to 6 substituents selected from -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, or fluorine; n is 4) or a pharmaceutically acceptable salt thereof.

[0018] The present invention provides compounds having the structure according to formula IVa: [ka] (In the formula, R 1 is butyl, said butyl being optionally substituted with one or two -OH groups; R 3 each occurrence of is independently H or —O(CH), R 4 Each occurrence of (C 10 -C 20 ) alkyl, (C 10 -C 20 ) alkenyl, and (C 10 -C 20 )alkynyl) or a pharmaceutically acceptable salt thereof.

[0019] The present invention relates to the following compounds: (N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, also known as compound A-1; (S)—N-(5-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, also known as compound A-2; (S)-1-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)octadecan-1-one, also known as compound A-3 N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, also known as compound B-1; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)tetradecanamide, also known as compound B-2; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)oleamide, also known as compound B-3; (9Z,12Z)-N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)octadeca-9,12-dienamide, also known as compound B-4; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide, also known as compound B-5; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperidin-1-yl)-5-oxopentyl)stearamide, also known as compound B-6; N-(5-(3-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)azetidin-1-yl)-5-oxopentyl)stearamide, also known as compound B-7; 1-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperidine-4-carboxamide, also known as compound B-8; (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-octadecylcyclobutane-1-carboxamide, also known as compound B-9; (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-hexadecylcyclobutane-1-carboxamide, also known as compound B-10; N-(3-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-3-oxopropyl)stearamide, also known as compound B-11; N-(7-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-7-oxoheptyl)stearamide, also known as compound B-12; N-(3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)cyclobutyl)stearamide, also known as compound B-13; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-4,4-dimethyl-5-oxopentyl)stearamide, also known as compound B-14; N-(6-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-methyl-6-oxohexan-2-yl)stearamide, also known as compound B-15; 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-(octadecyloxy)pentan-1-one, also known as compound B-16; 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-(octadecylamino)pentan-1-one, also known as compound B-17; N-(5-(4-(4-((5-amino-7-(butylamino)-3-methyl-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, also known as compound B-18; (9Z,12Z)-N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide, also known as compound C-1; N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)tetradecanamide, also known as compound C-2 N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)oleamide, also known as compound C-3 N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide, also known as compound C-4; N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)-4-oxobutyl)stearamide, also known as compound C-5; (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate, also known as compound D-1; 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperazine-1-carboxamide, also known as compound D-2; 3-stearamidopropyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate, also known as compound D-3 N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-6,6,6-trifluorohexyl)stearamide, also known as compound D-4; N-(4-((4-((7-(butylamino)-5-hydroxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide, also known as compound D-5, and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate, also known as compound D-6; or a pharmaceutically acceptable salt thereof.

[0020] The present invention relates to the following compounds: N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, also known as compound B-1; or a pharmaceutically acceptable salt thereof.

[0021] The present invention also provides stable nanoemulsions comprising: (i) one or more compounds having a structure as set forth in Formula I, Ia, II, IIa, III, IIIa, IV, or IVa, or a pharmaceutically acceptable salt(s) thereof; ii) one or more emulsifying agents; and iii) one or more terpenes.

[0022] The present invention also provides stable nanoemulsions comprising: (i) one or more compounds having a structure set forth in Formula I, Ia, II, IIa, III, IIIa, IV, or IVa, or pharmaceutically acceptable salt(s) thereof; ii) one or more sorbitan-based surfactants; and iii) one or more terpenes.

[0023] The present invention also provides a stable nanoemulsion comprising: (i) N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof; (ii) sorbitan trioleate (SPAN-85); (iii) polysorbate-20 (PS-20) or polysorbate-80 (PS-80); and (iv) squalene.

[0024] The present invention also provides a stable nanoemulsion comprising: (i) at least one antigen; (ii) a compound having a structure set forth in Formula I, Ia, II, IIa, III, IIIa, IV, or IVa, or a pharmaceutically acceptable salt thereof; (ii) SPAN-85; (iii) PS-20 or PS-80; and (iv) squalene.

[0025] The present invention also provides a stable nanoemulsion comprising: (i) at least one antigen; (ii) N-(5,(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof; (ii) SPAN-85; (iii) PS-20 or PS-80; and (iv) squalene.

[0026] The present invention also provides pharmaceutical compositions comprising: (i) at least one antigen; (ii) a compound having a structure set forth in Formula I, Ia, II, IIa, III, IIIa, IV or IVa, or a pharmaceutically acceptable salt thereof; (iii) SPAN-85; (iv) PS-20 or PS-80; (v) squalene; and (vi) a pharmaceutically acceptable carrier.

[0027] The present invention also provides a pharmaceutical composition comprising (i) at least one antigen, (ii) N-(5,(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof, (iii) SPAN-85, (iv) PS-20 or PS-80, (v) squalene, and (vi) a pharmaceutically acceptable carrier.

[0028] The present invention also provides immunogenic compositions comprising: (i) at least one antigen; (ii) a compound having a structure set forth in Formula I, Ia, II, IIa, III, IIIa, IV, or IVa, or a pharmaceutically acceptable salt thereof; (iii) SPAN-85; (iv) PS-20 or PS-80; (v) squalene; and (vi) a pharmaceutically acceptable carrier.

[0029] The present invention also provides an immunogenic composition comprising: (i) at least one antigen; (ii) N-(5,(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof; (iii) SPAN-85; (iv) PS-20 or PS-80; (v) squalene; and (vi) a pharmaceutically acceptable carrier.

[0030] The present invention also provides a single-dose vaccine composition comprising: (i) at least one antigen; (ii) a compound having a structure set forth in Formula I, Ia, II, IIa, III, IIIa, IV or IVa, or a pharmaceutically acceptable salt thereof; (iii) SPAN-85; (iv) PS-20; (v) squalene; and (vi) a pharmaceutically acceptable carrier, wherein a single dose of the vaccine composition is sufficient to elicit a desired immune response against the at least one antigen.

[0031] The present invention also provides a single-dose vaccine composition comprising: (i) at least one antigen; (ii) N-(5,(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof; (iii) SPAN-85; (iv) PS-20; (v) squalene; and (vi) a pharmaceutically acceptable carrier. A single dose of the vaccine composition is sufficient to elicit a desired immune response against the at least one antigen.

[0032] The present invention also provides a method of treating or preventing a disease in a patient, comprising administering to the patient a pharmaceutical composition comprising: (i) at least one antigen; (ii) a compound having a structure set forth in Formula I, Ia, II, IIa, III, IIIa, IV or IVa, or a pharmaceutically acceptable salt thereof; (iii) SPAN-85; (iv) PS-20; (v) squalene; and (vi) a pharmaceutically acceptable carrier.

[0033] The present invention also provides a method of treating or preventing a disease in a patient, comprising administering to the patient a pharmaceutical composition comprising: (i) at least one antigen; (ii) N-(5,(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof; (iii) SPAN-85; (iv) PS-20; (v) squalene; and (vi) a pharmaceutically acceptable carrier.

[0034] In some embodiments of the methods disclosed herein, the patient is a human.

[0035] In some embodiments of the methods disclosed herein, the patient is a non-human animal.

[0036] In some embodiments of the above i) stable nanoemulsion, ii) pharmaceutical composition, iii) immunogenic composition, and iv) single-dose vaccine composition, the concentration of the compound (i.e., (a) a compound having a structure set forth in Formula I, Ia, II, IIa, III, IIIa, IV, or IVa, or a pharmaceutically acceptable salt thereof, or (b) N-(5,(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide or a pharmaceutically acceptable salt thereof) is 0.01 μg / mL to 1000 μg / mL, or 0.1 μg / mL to 100 μg / mL, or 80 μg / mL, or 16 μg / mL, or 4 μg / mL.

[0037] In some embodiments of the above i) stable nanoemulsions, ii) pharmaceutical compositions, iii) immunogenic compositions, and iv) single-dose vaccine compositions, the concentration of SPAN-85 is 0.001 mg / mL to 100 mg / mL, or 0.01 mg / mL to 50 mg / mL, or 0.1 mg / mL to 10 mg / mL, or 0.001 mg / mL to 30 mg / mL, or 0.2 mg / mL to 10 mg / mL.

[0038] In some embodiments of the above i) stable nanoemulsions, ii) pharmaceutical compositions, iii) immunogenic compositions, and iv) single-dose vaccine compositions, the concentration of PS-20 or PS-80 is 0.001 mg / mL to 100 mg / mL, or 0.01 mg / mL to 50 mg / mL, or 0.1 mg / mL to 10 mg / mL, or 0.001 mg / mL to 30 mg / mL, or 0.2 mg / mL to 10 mg / mL.

[0039] In some embodiments of the above i) stable nanoemulsions, ii) pharmaceutical compositions, iii) immunogenic compositions, and iv) single-dose vaccine compositions, the concentration of squalene is 0.01 mg / mL to 100 mg / mL, or 0.02 mg / mL to 20 mg / mL, or 1 mg / mL to 20 mg / mL, or 0.03 mg / mL to 30 mg / mL, or 0.5 mg / mL to 20 mg / mL.

[0040] In some embodiments of the above i) stable nanoemulsion, ii) pharmaceutical composition, iii) immunogenic composition, and iv) single-dose vaccine composition, the concentration of the compound is 80 μg / mL, the concentration of SPAN-85 is 4.8 mg / mL, the concentration of PS-20 or PS-80 is 4.8 mg / mL, and the concentration of squalene is 16 mg / mL.

[0041] In some embodiments of the above i) stable nanoemulsion, ii) pharmaceutical composition, iii) immunogenic composition, and iv) single-dose vaccine composition, the concentration of the compound is 16 μg / mL, the concentration of SPAN-85 is 4.8 mg / mL, the concentration of PS-20 or PS-80 is 4.8 mg / mL, and the concentration of squalene is 16 mg / mL.

[0042] In some embodiments of the above i) stable nanoemulsion, ii) pharmaceutical composition, iii) immunogenic composition, and iv) single-dose vaccine composition, the concentration of the compound is 4 μg / mL, the concentration of SPAN-85 is 4.8 mg / mL, the concentration of PS-20 or PS-80 is 4.8 mg / mL, and the concentration of squalene is 16 mg / mL.

[0043] In some embodiments of the above i) stable nanoemulsions, ii) pharmaceutical compositions, iii) immunogenic compositions, and iv) single-dose vaccine compositions, the nanoemulsions and / or compositions further comprise L-met and EDTA.

[0044] definition The following abbreviations are used herein:

[0045] [Table 1] TIFF2026504779000019.tif236156TIFF2026504779000020.tif234156TIFF2026504779000021.tif179156

[0046] As used throughout this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0047] The term "about," when used herein in reference to a value, refers to a value that is the same as the referenced value or, in context, is similar to the referenced value. Generally, a person skilled in the art, familiar with the context, will understand the absolute amount and / or relative degree of difference encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0048] As defined herein, "adjuvant" refers to a compound or compound formulation or composition that serves to enhance the immunogenicity of the compositions of the invention. An adjuvant may i) enhance the immune response to an antigen (e.g., pneumococcal polysaccharide) that is weakly immunogenic when administered alone, e.g., induces no or a weak antibody titer or cell-mediated immune response, ii) increase the antibody titer against the antigen, and / or iii) reduce the dose of the antigen effective to achieve an immune response in an individual.

[0049] As used herein, the term "administration" refers to the act of providing an active agent, composition, or formulation to a subject. Exemplary routes of administration to the human body can be by eye (ophthalmic), mouth (oral), skin (transdermal), nose (intranasal), lung (inhalant), rectum, vagina, oral mucosa (buccal), ear, injection (e.g., intravenous (IV), subcutaneous, intratumoral, intraperitoneal, intramuscular (IM), intradermal (ID), etc.), etc.

[0050] As used herein, "agent" refers to any chemical class of particle, compound, molecule, or entity.

[0051] As used herein, the term "alkyl" refers to a straight-chain, cyclic, or branched saturated aliphatic hydrocarbon having a specified number of carbon atoms. Numerical ranges may be given, referring to the total chain length. For example, a C1-C6 alkyl has a chain length of 1 to 6 atoms. Alkyl groups may be substituted with one or more "substituents," which may be the same or different and are as defined herein below. Unless otherwise indicated, alkyl groups are unsubstituted.

[0052] As used herein, the term "alkenyl" refers to a straight-chain, cyclic, or branched unsaturated aliphatic hydrocarbon having the specified number of carbon atoms, including, but not limited to, diene, triene, and tetraene unsaturated aliphatic hydrocarbons. Alkenyl groups can be substituted with one or more "substituents," which may be the same or different, and are as defined herein below. Unless otherwise specified, an alkenyl group is unsubstituted.

[0053] As used herein, the term "alkynyl" refers to a straight-chain, cyclic, or branched unsaturated aliphatic hydrocarbon having the specified number of carbon atoms, including, but not limited to, diene, triene, and tetraene unsaturated aliphatic hydrocarbons. Alkynyl groups can be substituted with one or more "substituents," which may be the same or different, and are as defined herein below. Unless otherwise indicated, an alkynyl group is unsubstituted.

[0054] As used herein, the term "antigen" refers to any antigen capable of generating one or more immune responses. An antigen can be a protein, peptide, or polypeptide. In certain embodiments, an antigen is a lipid or carbohydrate. In certain embodiments, an antigen is a polysaccharide. In certain embodiments, an antigen is a pneumococcal polysaccharide. In certain embodiments, a polysaccharide is a Streptococcus pneumoniae polysaccharide. An antigen can be one that generates a humoral and / or CTL immune response.

[0055] As used herein, the term "aryl" refers to a carbocyclic aromatic monocyclic or bicyclic ring system containing from about 6 to about 14 carbon atoms. In one embodiment, an aryl group contains from about 6 to about 10 carbon atoms. Aryl groups can be substituted with one or more "ring system substituents," which may be the same or different, and are as defined herein below. Non-limiting examples of aryl groups include phenyl and naphthyl. In one embodiment, an aryl group is phenyl. Unless otherwise indicated, an aryl group is unsubstituted.

[0056] As used herein, the term "composition" refers to a formulation containing an active pharmaceutical ingredient or biological component (e.g., a pneumococcal polysaccharide carrier protein conjugate and a compound) along with one or more additional ingredients. The term "composition" is used interchangeably with "pharmaceutical composition" and "formulation." The composition may be liquid or solid (e.g., lyophilized). Additional components that may be optionally included include pharmaceutically acceptable excipients, additives, diluents, buffers, sugars, amino acids, chelating agents, surfactants, polyols, bulking agents, stabilizers, cryoprotectants, solubilizers, emulsifiers, salts, adjuvants, tonicity agents, delivery vehicles, and antimicrobial preservatives. The composition is nontoxic to recipients at the dosages and concentrations employed.

[0057] As used herein, the term "comprising" when used in connection with the compositions of the present invention refers to the inclusion of any other ingredients such as adjuvants and excipients, or the addition of one or more polysaccharide carrier protein conjugates not specifically listed.

[0058] As used herein, the term "consisting of" and variations such as "consist of" when used in conjunction with a polyvalent polysaccharide mixture or a polyvalent polysaccharide carrier protein conjugate mixture refers to a mixture having those particular pneumococcal polysaccharides or pneumococcal polysaccharide carrier protein conjugates and not having other pneumococcal polysaccharides or pneumococcal polysaccharide carrier protein conjugates from different serotypes.

[0059] As used herein, the term "consists essentially of" and variations such as "consist essentially of" or "consisting essentially of" refer to the inclusion of any recited element or group of elements, and the optional inclusion of other elements of similar or different nature to the recited elements, which do not materially alter the basic or novel characteristics of the specified dosing regimen, method, or composition.

[0060] As used herein, the term "de-O-acetylated-15B" or "de-O-acetyl-15B" or "de-O-Ac-15B" refers to de-O-acetylated serotype 15B having an O-acetyl content of less than 10% per repeat unit. In another embodiment, the O-acetyl content is less than 5% per repeat unit. In another embodiment, the O-acetyl content is less than about 1% per repeat unit. In another embodiment, the O-acetyl content is less than 1% per repeat unit. In another embodiment, the O-acetyl content is less than 0.5% per repeat unit. In another embodiment, the O-acetyl content is less than 0.1% per repeat unit. In another embodiment, the O-acetyl content is 0% per repeat unit. The process of de-O-acetylation is known in the art, for example, as described in Rajam et al., Clinical and Vaccine Immunology, 2007, 14(9):1223-1227.

[0061] As used herein, the term "dose" means the amount of a drug, API (active pharmaceutical ingredient), formulation, composition, pharmaceutical composition, or immunogenic composition that is taken or recommended to be taken at a particular time.

[0062] As used herein, the term "heteroaryl" refers to an aromatic monocyclic or polycyclic ring system containing about 5 to about 14 ring atoms, wherein 1 to 4 of the ring atoms are independently O, N, or S, and the remaining ring atoms are carbon atoms. In one embodiment, a heteroaryl group has 5 to 10 ring atoms. In another embodiment, a heteroaryl group is monocyclic and has 5 or 6 ring atoms. In another embodiment, a heteroaryl group is bicyclic. A heteroaryl group may be substituted with one or more "ring system substituents," which may be the same or different, and are as defined herein below. A heteroaryl group is joined via a ring carbon atom, and any nitrogen atom of a heteroaryl may be oxidized to the corresponding N-oxide. In one embodiment, a heteroaryl group is a 5-membered heteroaryl. In another embodiment, a heteroaryl group is a 6-membered heteroaryl. In another embodiment, a heteroaryl group includes a 5- to 6-membered heteroaryl group fused to a benzene ring. Unless otherwise indicated, a heteroaryl group is unsubstituted.

[0063] As used herein, the term "heterocycloalkyl" refers to a saturated or partially unsaturated non-aromatic monocyclic, bicyclic (including spirocyclic), or bridged carbocyclic ring or ring system containing 3 to about 11 ring atoms, at least one ring heteroatom selected from N, S, and O, with the remaining ring atoms being carbon atoms. A heterocycloalkyl group can be attached through a ring carbon or a ring nitrogen atom, unless otherwise specified. A heterocycloalkyl ring can be substituted on a ring carbon and / or one or more ring nitrogens. In one embodiment, a heterocycloalkyl group is monocyclic and has about 3 to about 7 ring atoms. In another embodiment, a heterocycloalkyl group is monocyclic and has about 4 to about 7 ring atoms. In other embodiments, a heterocycloalkyl group is bicyclic and has 7 to 10 ring atoms, 8 to 10 ring atoms, or 9 or 10 ring atoms. In yet another embodiment, a heterocycloalkyl group is monocyclic and has 5 or 6 ring atoms. In one embodiment, a heterocycloalkyl group is monocyclic. In another embodiment, the heterocycloalkyl group is bicyclic. The heterocycloalkyl group can be substituted. In some embodiments, the heterocycloalkyl group has one to two heteroatoms in the ring selected from nitrogen, sulfur, and oxygen atoms. In some embodiments, the heterocycloalkyl group has one heteroatom in the ring selected from nitrogen, sulfur, and oxygen atoms. In some embodiments, the heteroatom is selected from O, S, S(O), S(O)2, and -NH-, -N(alkyl)-. Non-limiting examples include aliphatic groups containing heteroatoms, such as ethers, thioethers, amines, hydroxymethyl, 3-hydroxypropyl, 1,2-dihydroxyethyl, 2-methoxyethyl, 2-aminoethyl, and 2-dimethylaminoethyl.

[0064] As used herein, the terms "immunogenic" or "immunogenicity" refer to the ability of an antigen (e.g., Streptococcus pneumoniae polysaccharide) to elicit an immune response in a subject. The term "immunogenic composition" refers to the ability of a drug, API, formulation, composition, or pharmaceutical composition to elicit an immune response in a subject.

[0065] As used herein, the phrase "in need of treatment" includes those who have been previously exposed to or infected with a disease-causing agent, those who have previously been vaccinated against a disease-causing agent, as well as anyone who is susceptible to infection or in whom a reduction in the likelihood of infection is desired, such as the immunocompromised, elderly, children, adults, or healthy individuals.

[0066] As used herein, the phrase "indicated for the prevention of disease" means that the vaccine or composition is approved by one or more regulatory agencies, such as the U.S. Food and Drug Administration, for the prevention of one or more diseases caused by infectious agents or antigens, including HIV, HPV, CMV, VZV, EBV, adenovirus, coronavirus, and influenza, among other viruses. Diseases include bacterial diseases such as tuberculosis and pneumococcal pneumoniae.

[0067] As used herein, the term "multiple dose" refers to a vaccine composition, or pharmaceutical composition, or immunogenic composition that requires administration or injection of more than one dose or components therein in a clinical regimen to induce a sustained immune response, provide protection from disease, or reduce the likelihood of infection by an infectious agent. Those skilled in the art will understand how to determine a durable immune response, for example, by measuring antibody titers over a specified period of time.

[0068] As used herein, a "patient" (alternatively referred to herein as a "subject") refers to a mammal that may be infected with a disease-causing agent. In some embodiments, the patient is a human. The patient can be treated prophylactically or therapeutically. Prophylactic treatment provides sufficient protective immunity to reduce the likelihood or severity of infection or its effects. Therapeutic treatment can be performed to reduce the severity of infection or prevent recurrence or its clinical effects. Prophylactic treatment can be performed using the compositions or vaccines, or immunogenic compositions of the invention described herein. The compositions or vaccines, or immunogenic compositions of the invention can be administered to the general population or to those at high risk of infection, such as the elderly, or those living with or caring for the elderly.

[0069] As used herein, the term "PCV1" refers to a monovalent pneumococcal conjugate vaccine or composition comprising one pneumococcal polysaccharide carrier protein conjugate comprising a capsular polysaccharide from a Streptococcus pneumoniae serotype conjugated to a carrier protein. In a specific embodiment, the carrier protein is CRM197.

[0070] As used herein, the term "PCV24" refers to a 24-valent pneumococcal conjugate vaccine or composition comprising 23 pneumococcal polysaccharide carrier protein conjugates, each comprising a capsular polysaccharide from a Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 18C, 19A, 19F, 22F, 23B, 24F, 31, 33F, and 35B, and at least one of the following serotype 15 serotypes: 15B, 15C, or de-O-acetylated-15B. In a specific embodiment, the serotype 15 serotype is serotype 15C or de-O-acetylated-15B. In another embodiment, the serotype 15 serotype is serotype de-O-acetylated-15B. In a specific embodiment, the carrier protein of one or more of the Streptococcus pneumoniae polysaccharide carrier protein conjugates is CRM 197. In a further embodiment, the carrier protein of each of the Streptococcus pneumoniae polysaccharide carrier protein conjugates is CRM 197.

[0071] As used herein, the term "PCV26" refers to a 26-valent pneumococcal conjugate vaccine or composition comprising 25 pneumococcal polysaccharide carrier protein conjugates, each comprising a capsular polysaccharide from a Streptococcus pneumoniae serotype conjugated to a carrier protein, wherein the Streptococcus pneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 16F, 18C, 19A, 19F, 22F, 23A, 23B, 24F, 31, 33F, and 35B, and at least one of the following serotype 15 serotypes: 15B, 15C, or de-O-acetylated-15B. In a specific embodiment, the serotype 15 serotype is serotype 15C or de-O-acetylated 15B. In another embodiment, the serotype 15 serotype is serotype de-O-acetylated 15B. In a specific embodiment, one or more carrier proteins of the Streptococcus pneumoniae polysaccharide carrier protein conjugates is CRM197. In a further embodiment, each carrier protein of the Streptococcus pneumoniae polysaccharide carrier protein conjugate is CRM197.

[0072] As used herein, the term "pharmaceutically acceptable" with respect to a carrier, diluent, or excipient in a pharmaceutical composition indicates that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0073] As used herein, the term "pharmaceutical composition" refers to a composition containing an active pharmaceutical or biological component together with one or more additional ingredients, e.g., a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. As used herein, the terms "pharmaceutical formulation" and "formulation" are used interchangeably with "pharmaceutical composition." In some embodiments, the active agent is present in a unit dosage amount suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. A pharmaceutical composition or formulation may be liquid or solid (e.g., lyophilized). Additional components that may be optionally included include pharmaceutically acceptable excipients, additives, diluents, buffers, sugars, amino acids, chelating agents, surfactants, polyols, bulking agents, stabilizers, cryoprotectants, solubilizers, emulsifiers, salts, adjuvants, tonicity agents, delivery vehicles, and antimicrobial preservatives. The pharmaceutical composition or formulation is nontoxic to recipients at the dosages and concentrations used. In some embodiments, the pharmaceutical composition can be specially formulated for administration in solid or liquid form, including those suitable for: oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., sterile solutions or suspensions, or sustained-release formulations; topical application, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., as a pessary, cream, or foam; sublingually; ophthalmically; transdermally; or intranasally, via the lungs, and other mucosal surfaces. In some embodiments, the term formulation refers to a single dose of vaccine, which can be contained in any volume suitable for injection.

[0074] As used herein, the term "pneumococcal conjugate" or "pneumococcal polysaccharide carrier protein conjugate" refers to a Streptococcus pneumoniae polysaccharide carrier protein conjugate.

[0075] As used herein, the term "pneumococcal conjugate vaccine" (or "PCV") is a pharmaceutical formulation or composition comprising one or more pneumococcal polysaccharide carrier protein conjugates that provide active immunity to a disease or pathological condition caused by one or more serotypes of Streptococcus pneumoniae.

[0076] As used herein, the term "ring system substituent" refers to a substituent attached to an aromatic or non-aromatic ring system that, for example, replaces an available hydrogen on the ring system. Ring system substituents can be the same or different and are each independently selected. Examples of ring system substituents include alkyl, alkenyl, alkynyl, aryl, heteroaryl, -OH, hydroxyalkyl, haloalkyl, -O-alkyl, -O-haloalkyl, -alkylene-O-alkyl, -O-aryl, -O-alkylene-aryl, acyl, -C(O)-aryl, halo, -NO, -CN, -SF, -C(O)OH, -C(O)O-alkyl, -C(O)O-aryl, -C(O)O-alkylene-aryl, -S(O)-alkyl, -S(O)-aryl. Examples of ring system substituents include alkyl, -S(O)-aryl, -S(O)-aryl, -S(O)-heteroaryl, -S(O)-heteroaryl, -S-alkyl, -S-aryl, -S-heteroaryl, -S-alkylene-aryl, -S-alkylene-heteroaryl, -S(O)-alkylene-aryl, -S(O)-alkylene-heteroaryl, cycloalkyl, heterocycloalkyl, -OC(O)-alkyl, -OC(O)-aryl, and -OC(O)-cycloalkyl. Further examples of ring system substituents include (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, -OH, -O(C-C)alkyl, -O(C-C)alkenyl, -O(C-C)alkynyl, chlorine, and fluorine.

[0077] As used herein, the term "single dose" refers to a vaccine composition that requires only one administration or injection in a clinical regimen to induce a durable immune response and provide protection from disease (e.g., pneumococcal disease). One of skill in the art will understand how to determine a durable immune response, for example, by measuring antibody titers over a specified period of time.

[0078] As used herein, the terms "substituent" or "optional substituent" or "optionally substituted" refer to, for example, a substituent attached to an alkyl group, or an alkenyl group, or an alkynyl group that replaces an available hydrogen on the group. The substituents can be the same or different and are each independently selected. Examples of substituents include alkyl, alkenyl, alkynyl, aryl, heteroaryl, -OH, hydroxyalkyl, haloalkyl, -O-alkyl, -O-haloalkyl, -alkylene-O-alkyl, -O-aryl, -O-alkylene-aryl, acyl, -C(O)-aryl, halo, -NO, -CN, -SF, -C(O)OH, -C(O)O-alkyl, -C(O)O-aryl, -C(O)O-alkylene-aryl, -S(O)-alkyl, -S(O)-alkyl.

[0023] Examples of substituents include -C(O)-aryl, -S(O)-aryl, -S(O)-heteroaryl, -S(O)-heteroaryl, -S-alkyl, -S-aryl, -S-heteroaryl, -S-alkylene-aryl, -S-alkylene-heteroaryl, -S(O)-alkylene-aryl, -S(O)-alkylene-heteroaryl, cycloalkyl, heterocycloalkyl, -OC(O)-alkyl, -OC(O)-aryl, and -OC(O)-cycloalkyl. Further examples of substituents include (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, -OH, -O(C-C)alkyl, -O(C-C)alkenyl, -O(C-C)alkynyl, chlorine, and fluorine.

[0079] As used herein, the term "therapeutically effective amount" refers to an amount of an active component (antigen) sufficient to produce a desired therapeutic effect in a human or animal, e.g., the amount necessary to induce an immune response, treat, cure, prevent the onset and progression of, or inhibit a disease or its symptoms, and / or the amount necessary to ameliorate symptoms or cause regression of the disease. The therapeutically effective amount may vary depending on the structure and potency of the active component and the intended mode of administration. One skilled in the art can easily determine the therapeutically effective amount of a given active component in a vaccine.

[0080] As used herein, the term "valency" refers to the presence of a specified number of polysaccharides or polysaccharide carrier protein conjugates in a composition.

[0081] As used herein, the term "vaccine" or "vaccine composition" refers to a biological preparation used to stimulate the production of antibodies and provide immunity against infectious diseases.

[0082] For example, "SNE" refers to a composition comprising a compound of the invention, SPAN-85, PS-20, and squalene, wherein the composition is in the form of a nanoemulsion. In some embodiments, the SNE comprises 0.01 μg / mL to 1000 μg / mL of the compound, 0.001 mg / mL to 60 mg / mL of SPAN-85, 0.001 mg / mL to 60 mg / mL of PS-20, and 0.01 mg / mL to 200 mg / mL of squalene. In further embodiments, the SNE comprises 100 μg / mL or 80 μg / mL of the compound, 0.01 mg / mL to 10 mg / mL of SPAN-85, 0.01 mg / mL to 10 mg / mL of PS-20, and 0.03 mg / mL to 30 mg / mL of squalene. In yet further embodiments, the SNE comprises 80 μg / mL or 16 μg / mL or 4 μg / mL or 0.5 μg / mL of the compound, 0.2 mg / mL to 10 mg / mL of SPAN-85, 0.2 mg / mL to 10 mg / mL of PS-20, and 0.5 mg / mL to 20 mg / mL of squalene.

[0083] An exemplary "Compound B-1-SNE" refers to a composition comprising Compound B-1, SPAN-85, PS-20, and squalene, wherein the composition is in the form of a nanoemulsion. In some embodiments, the Compound B-1 SNE comprises 0.01 μg / mL to 1000 μg / mL of Compound B-1, 0.001 mg / mL to 60 mg / mL of SPAN-85, 0.001 mg / mL to 60 mg / mL of PS-20, and 0.01 mg / mL to 200 mg / mL of squalene. In further embodiments, the SNE of Compound B-1 comprises 100 μg / mL or 80 μg / mL of Compound B-1, 0.01 mg / mL to 10 mg / mL of SPAN-85, 0.01 mg / mL to 10 mg / mL of PS-20, and 0.03 mg / mL to 30 mg / mL of squalene. In still further embodiments, the SNE of Compound B-1 comprises 80 μg / mL, 16 μg / mL, 4 μg / mL, or 0.5 μg / mL of Compound B-1, 0.2 mg / mL to 10 mg / mL of SPAN-85, 0.2 mg / mL to 10 mg / mL of PS-20, and 0.5 mg / mL to 20 mg / mL of squalene.

[0084] Nanoemulsions, also known as nanometer-sized emulsions, are fine oil-in-water (o / w) dispersions of two immiscible fluids. Nanoemulsions are colloidal particle systems in the submicron size range that act as carriers for drug molecules. Their size ranges from 10 to 1,000 nm. These carriers are solid spheres with amorphous, lipophilic surfaces.

[0085] As used herein, the term "carbon or nitrogen linked spacer" refers to any chemistry that links a carbon (-C) or nitrogen (-N) to a benzyl group in the formulae of the present invention (see the bond in variable "A" in Formulae I and Ia). Examples of carbon or nitrogen linked spacers are C1-C6 alkyl, heterocycloalkyl, aryl, and heteroaryl, wherein said alkyl, heterocycloalkyl, aryl, and heteroaryl may be substituted.

[0086] Unless otherwise specified, when describing linkers throughout this disclosure, the first available bond on the linker group connects to the left the portion of the compound adjacent to the linker group, and the terminal available bond of the linker group connects to the right the terminal portion of the compound adjacent to the linker group. For example, if the linker (L) is -CH2-CF2-, the definition of L includes only A-CH2-CF2-B, but not A-CF2-CH2-B.

[0087] As used herein, the term "functional group" refers to any chemical entity that connects a carbon or nitrogen linking spacer to a lipid group. Examples of functional groups include: [ka] where n is 0, 1, 2, 3, 4, or 5.

[0088] Further examples of functional groups include: [ka] is.

[0089] As used herein, the term "lipid" refers to any chemical entity that is insoluble in water but soluble in organic solvents. Examples of lipids are represented by the variable D (see Formulas I and Ia) and include (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl, wherein said alkyl, alkenyl, and alkynyl may be substituted.

[0090] Further examples of lipids include: [ka] and Any carbon on the lipid chain may be substituted, [ka] is cis or trans stereochemistry, X is O, C(R) 2 , or NR, R is independently selected from H, (C1-C4) alkyl, (C1-C4) alkenyl, (C1-C4) alkynyl, OH, O(C1-C4) alkyl, O(C1-C4) alkenyl, O(C1-C4) alkynyl, chlorine, and fluorine.

[0091] Further examples of lipids are represented by the variable D (see Formulas I and Ia), where D is [ka] wherein Z is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 )alkynyl, wherein said (C-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) The alkynyl may be substituted.

[0092] Compounds of the present invention having one or more asymmetric centers may exist as mixtures of stereoisomers or as substantially pure individual diastereomers or enantiomers, unless otherwise specified. The present invention encompasses all stereoisomeric forms of the compounds of Formulas I, Ia, II, IIa, III, IIIa, IV, and IVa. Unless a specific stereochemistry is indicated, the present invention is meant to include all such isomers of these compounds. Any asymmetric centers present in the compounds of Formulas I, Ia, II, IIa, III, IIIa, IV, and IVa can have either the (R) or the (S) configuration, independently of one another. When a bond to a chiral carbon is shown as a straight line in a structural formula of the present invention, it is understood that both the (R) and (S) configurations of the chiral carbon, and thus both enantiomers and mixtures thereof, are encompassed by the formula. Similarly, when a compound name is listed without a chiral designation for the chiral carbon, it is understood that both the (R) and (S) configurations of the chiral carbon, and thus individual enantiomers, diastereomers, and mixtures thereof, are encompassed by the name. The production of specific stereoisomers or mixtures thereof may be identified in the examples where such stereoisomers or mixtures were obtained, but this in no way limits the scope of the invention to the inclusion of all stereoisomers and mixtures thereof.

[0093] When any variable (e.g., n, Rb, etc.) occurs more than one time in any constituent or in formula I, Ia, II, IIa, III, IIIa, IV, and IVa, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0094] A "stable" compound is one that can be prepared and isolated and whose structure and properties remain or remain essentially unchanged for a period of time sufficient to permit use of the compound for the purposes described herein (e.g., use as an adjuvant in an immunogenic composition administered to a subject). The compounds of the present invention are limited to stable compounds encompassed by Formula I, Ia II, IIa, III, IIIa, IV, and IVa.

[0095] Wavy lines as used herein [ka] indicates the point of attachment to the rest of the compound. Lines drawn through ring systems, e.g.: [ka] indicates that the bond may be attached to any of the substitutable ring atoms.

[0096] compound The compounds of the present invention are useful in the adjuvant formulations described herein.These adjuvant formulations are useful for boosting the immunological response of vaccine formulations.For example, the adjuvant formulations described herein are useful for boosting the immunological response of adenovirus vaccine, anthrax vaccine, cholera vaccine, dengue vaccine, diphtheria vaccine, hepatitis vaccine, Haemophilus influenzae type b (Hib) vaccine, human papillomavirus (HPV) vaccine, seasonal influenza (Flu) vaccine, Japanese encephalitis vaccine, measles vaccine, meningitis vaccine, mumps vaccine, pertussis vaccine, pneumococcal vaccine, polio vaccine, rabies vaccine, rotavirus vaccine, rubella vaccine, shingles vaccine, smallpox vaccine, tetanus vaccine, tuberculosis vaccine, typhoid vaccine, chickenpox vaccine and yellow fever vaccine. For example, the adjuvant formulations described herein are useful for boosting the immunological response of a pneumococcal composition or pneumococcal conjugate composition, or a pneumococcal vaccine (PV) or pneumococcal conjugate vaccine (PCV).

[0097] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I: [ka] (In the formula, R a is selected from H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, and —NR′R″, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted with 1 to 4 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine; R a’ is selected from H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, and —NR′R″, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted with 1 to 4 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine; R a’’is selected from H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, and —NR′R″, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted with 1 to 4 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine; R' and R" are independently selected from H, (C1-C6)alkyl, (C1-C6)alkenyl, and (C1-C6)alkynyl, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, and (C1-C6)alkynyl are optionally substituted with one to four substituents independently selected from the group consisting of -OH, -O(C1-C4)alkyl, -O(C1-C4)alkenyl, -O(C1-C4)alkynyl, chlorine, and fluorine; or R' and R" together with the nitrogen to which they are attached, join together to form a (C3-C6)heterocycloalkyl, wherein said (C3-C6)heterocycloalkyl is optionally substituted with one to four substituents independently selected from the group consisting of -OH, -O(C1-C4)alkyl, -O(C1-C4)alkenyl, -O(C1-C4)alkynyl, chlorine, and fluorine; R bis independently selected from H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, or NR′R″, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted with from 1 to 4 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine; A is (C1-C6) alkyl, heterocycloalkyl, heterocycloalkyl-C(O)-R z -, (C1-C4) alkyl-N(R z )-R z a carbon or nitrogen bond spacer selected from -, aryl, and heteroaryl, wherein said (C1-C6)alkyl, heterocycloalkyl, aryl, and heteroaryl are independently selected from the group consisting of -OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, -O(C1-C6)alkyl, -O(C1-C6)alkenyl, -O(C1-C6)alkynyl, chlorine, fluorine, and NR'R''; each of the (C1-C6) alkyl, (C1-C6) alkenyl, (C1-C6) alkynyl, -O(C1-C6) alkyl, -O(C1-C6) alkenyl, and -O(C1-C6) alkynyl is optionally substituted with 1 to 6 substituents independently selected from the group consisting of -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine; R z each occurrence of is independently H or (C1-C6) alkyl; B is [ka] is a functional group selected from D is (C6-C20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl, wherein the (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl is optionally substituted with 1 to 6 substituents independently selected from the group consisting of -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine, or D is [ka] and Each occurrence of Z is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 )alkynyl, wherein the (C-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl is optionally substituted with 1 to 6 substituents independently selected from the group consisting of -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine; m is 0, 1, 2, 3, 4, or 5; where n is 0, 1, 2, 3, 4, or 5).

[0098] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein m is 0, 1, 2, 3, 4, or 5.

[0099] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein m is 0, 1, 2, 3, or 4.

[0100] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein m is 0, 1, 2, or 3.

[0101] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein m is 0, 1, or 2.

[0102] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein m is 2.

[0103] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein m is 1.

[0104] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein m is 0.

[0105] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, where n is 0, 1, 2, 3, 4, or 5.

[0106] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein n is 0, 1, 2, 3, or 4.

[0107] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein n is 0, 1, 2, or 3.

[0108] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein n is 0, 1, or 2.

[0109] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein n is 2.

[0110] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein n is 1.

[0111] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein n is 0.

[0112] In some embodiments, the compounds of the invention are represented by the structure set forth in Formula I, wherein R' and R'' are independently H, (C1-C6) alkyl, (C1-C6) alkenyl, or (C1-C6) alkynyl, wherein said (C1-C6) alkyl, (C1-C6) alkenyl, and (C1-C6) alkynyl are optionally substituted with 1 to 4 substituents independently selected from the group consisting of -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine.

[0113] In some embodiments, the compounds of the invention are represented by the structure set forth in Formula I, wherein R' and R'' are independently H, (C1-C6) alkyl, (C1-C6) alkenyl, or (C1-C6) alkynyl, wherein the (C1-C6) alkyl, (C1-C6) alkenyl, and (C1-C6) alkynyl are optionally substituted with 1 to 4 independently selected -OH or -O(CH3).

[0114] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' and R'' are independently H, (C1-C4) alkyl, (C1-C4) alkenyl, or (C1-C4) alkynyl, wherein the (C1-C4) alkyl, (C1-C4) alkenyl, and (C1-C4) alkynyl are optionally substituted with one or two independently selected -OH or -O(CH3).

[0115] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' and R'' are independently H and (C1-C4) alkyl, wherein the (C1-C4) alkyl is optionally substituted with one or two independently selected -OH or -O(CH3).

[0116] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R′ and R″ are independently H and (C 1 -C 4 ) alkyl.

[0117] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' and R'', together with the nitrogen to which they are attached, are joined together to form a (C3-C6)heterocycloalkyl, wherein said (C3-C6)heterocycloalkyl is optionally substituted with 1 to 4 substituents independently selected from the group consisting of -OH, -O(C1-C4)alkyl, -O(C1-C4)alkenyl, -O(C1-C4)alkynyl, chlorine, and fluorine.

[0118] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a is H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, or NR′R″, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted with 1 to 4 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine.

[0119] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R ais H, —OH, (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, fluorine, or NR′R″, and said (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, and —O(C1-C4)alkynyl are optionally substituted with 1 to 2 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine.

[0120] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a is H, —OH, (C1-C4) alkyl, (C1-C4) alkenyl, (C1-C4) alkynyl, —O(C1-C4) alkyl, —O(C1-C4) alkenyl, —O(C1-C4) alkynyl, chlorine, fluorine, or NR′R″.

[0121] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a is NR'R''.

[0122] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula I, wherein R' is H and R'' is pentyl.

[0123] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' is H and R'' is butyl.

[0124] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula I, wherein R' is H and R'' is propyl.

[0125] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' is H and R'' is ethyl.

[0126] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' is H and R'' is methyl.

[0127] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R′ is H and R″ is H.

[0128] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a is H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, or NR′R″, and the (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted by 1 to 2 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine.

[0129] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a’ is H, —OH, (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, fluorine, or NR′R″, and said (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, and —O(C1-C4)alkynyl are optionally substituted with 1 to 2 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine.

[0130] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a’ is H, —OH, (C1-C4) alkyl, (C1-C4) alkenyl, (C1-C4) alkynyl, —O(C1-C4) alkyl, —O(C1-C4) alkenyl, —O(C1-C4) alkynyl, chlorine, fluorine, or NR′R″.

[0131] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a’ is NR'R''.

[0132] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula I, wherein R' is H and R'' is pentyl.

[0133] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' is H and R'' is butyl.

[0134] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula I, wherein R' is H and R'' is propyl.

[0135] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' is H and R'' is ethyl.

[0136] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' is H and R'' is methyl.

[0137] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R′ is H and R″ is H.

[0138] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a’’is H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, or NR′R″, and the (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted by 1 to 2 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine.

[0139] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a’’ is H, —OH, (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, fluorine, or NR′R″, and said (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, and —O(C1-C4)alkynyl are optionally substituted with 1 to 2 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine.

[0140] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a’’ is H, —OH, (C1-C4) alkyl, (C1-C4) alkenyl, (C1-C4) alkynyl, —O(C1-C4) alkyl, —O(C1-C4) alkenyl, —O(C1-C4) alkynyl, chlorine, fluorine, or NR′R″.

[0141] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a’’ is NR'R''.

[0142] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula I, wherein R' is H and R'' is pentyl.

[0143] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' is H and R'' is butyl.

[0144] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula I, wherein R' is H and R'' is propyl.

[0145] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' is H and R'' is ethyl.

[0146] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R' is H and R'' is methyl.

[0147] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R′ is H and R″ is H.

[0148] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R a’’ is H.

[0149] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R bis H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, or NR′R″, and the (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, and —O(C1-C6)alkynyl are optionally substituted by 1 to 2 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine.

[0150] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R b is H, —OH, (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, fluorine, or NR′R″, and said (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, and —O(C1-C4)alkynyl are optionally substituted with 1 to 2 substituents independently selected from the group consisting of —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine.

[0151] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R b are independently H, —OH, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, —O(C1-C6)alkyl, —O(C1-C6)alkenyl, —O(C1-C6)alkynyl, chlorine, fluorine, or NR′R″.

[0152] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R bEach occurrence of is independently H, —OH, (C1-C4)alkyl, —O(C1-C4)alkyl, chlorine, fluorine, or NH2.

[0153] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein R b Each occurrence of is independently H and O(CH).

[0154] In some embodiments, the compounds of the invention are represented by the structure set forth in Formula I, wherein A is (C-C)alkyl or heterocycloalkyl, and said alkyl or heterocycloalkyl is independently selected from the group consisting of -OH, oxo, (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, -O(C-C)alkyl, -O(C-C)alkenyl, -O(C-C)alkynyl, chlorine, fluorine, and NR'R''. The (C1-C6) alkyl, (C1-C6) alkenyl, (C1-C6) alkynyl, -O(C1-C6) alkyl, -O(C1-C6) alkenyl, and -O(C1-C6) alkynyl may be substituted with 1 to 4 substituents independently selected from the group consisting of -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine.

[0155] In some embodiments, the compounds of the invention are represented by the structure set forth in Formula I, wherein A is (C1-C4) alkyl or heterocycloalkyl, and said alkyl or heterocycloalkyl is independently selected from the group consisting of -OH, oxo, (C1-C4) alkyl, (C1-C4) alkenyl, (C1-C4) alkynyl, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, fluorine, and NR'R''. The (C1-C4) alkyl, (C1-C4) alkenyl, (C1-C4) alkynyl, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, and -O(C1-C4) alkynyl are optionally substituted with 1 to 6 substituents independently selected from the group consisting of -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine.

[0156] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein A is heterocycloalkyl-C(O)-R z - or (C1-C4) alkyl-N(R z ) 2 - and R z Each occurrence of is independently H or (C1-C6) alkyl.

[0157] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein A is [ka] (In the formula, R z is independently selected from H, (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, —O(C-C)alkyl, —O(C-C)alkenyl, and —O(C-C)alkynyl; X is CH or N; R dare independently selected from H, —OH, oxo, (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, —OH, —O(C1-C4)alkyl, —O(C1-C4)alkenyl, —O(C1-C4)alkynyl, chlorine, and fluorine; n is 0, 1, 2, 3, or 4; p is 0, 1, 2, 3, or 4).

[0158] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein A is [ka] (In the formula, R z is independently selected from H, (C-C) alkyl, (C-C) alkenyl, and (C-C) alkynyl; X is CH or N; R d are independently selected from —OH, (C1-C4) alkyl, —O(C1-C4) alkyl, chlorine, and fluorine; n is 0, 1, 2, or 3; p is 0, 1, or 2).

[0159] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein A is [ka] (In the formula, R z are independently H or (C1-C6) alkyl; X is CH or N; R d are independently selected from —OH, (C1-C4) alkyl, —O(C1-C4) alkyl, chlorine, and fluorine; n is 0, 1, 2, or 3; p is 0, 1, or 2).

[0160] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein A is [ka] (In the formula, R z are independently H or (C1-C6) alkyl; R d are independently selected from —OH, (C1-C4) alkyl, —O(C1-C4) alkyl, chlorine, and fluorine; n is 0, 1, 2, or 3; p is 0, 1, or 2).

[0161] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein A is [ka] (In the formula, R z is independently selected from H or (C1-C6) alkyl.

[0162] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein B is: [ka] is selected from.

[0163] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein B is: [ka] is.

[0164] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein D is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, or (C6-C 20 ) alkynyl, wherein the (C6-C 20) alkyl, (C6-C 20 ) alkenyl, or (C6-C 20 ) the alkynyl is optionally substituted with 1 to 6 substituents independently selected from -OH, -O(CH3), chlorine, and fluorine; or D is [ka] (Wherein Z is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 )alkynyl, wherein said (C-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 )Alkynyl is optionally substituted with 1 to 6 substituents independently selected from -OH, -O(CH3), chlorine, and fluorine.

[0165] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein D is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, or (C6-C 20 ) alkynyl, or D is [ka]

[0166] (Wherein Z is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl).

[0167] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein D is [ka] wherein any carbon on the lipid chain may be substituted with -OH, -O(CH), chlorine, or fluorine; [ka] is cis or trans stereochemistry, X is O, C(R) 2 , or NR, R is selected from H, (C1-C4) alkyl, (C1-C4) alkenyl, (C1-C4) alkynyl, —OH, —O(C1-C4) alkyl, —O(C1-C4) alkenyl, —O(C1-C4) alkynyl, chlorine, and fluorine.

[0168] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula I, wherein D is [ka] wherein any carbon on the lipid chain may be substituted with -OH, -O(CH), chlorine or fluorine; [ka] is cis or trans stereochemistry, X 1 -O-, -C(R) 2 -, or -NR-, each occurrence of R is independently selected from H, (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, —OH, —O(C-C)alkyl, —O(C-C)alkenyl, —O(C-C)alkynyl, chlorine, and fluorine; n is 0, 1, 2, 3, 4, or 5; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; s is 0, 1, 2, 3, 4, 5, 6, 7, or 8; t is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18).

[0169] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia: [ka] (In the formula, R' and R" are independently selected from H, (C1-C6)alkyl, (C1-C6)alkenyl, and (C1-C6)alkynyl, wherein said (C1-C6)alkyl, (C1-C6)alkenyl, and (C1-C6)alkynyl are optionally substituted with one to four substituents independently selected from the group consisting of -OH, -O(C1-C4)alkyl, -O(C1-C4)alkenyl, -O(C1-C4)alkynyl, chlorine, and fluorine; or R' and R" together with the nitrogen to which they are attached, join together to form a (C3-C6)heterocycloalkyl, wherein said (C3-C6)heterocycloalkyl is optionally substituted with one to four substituents independently selected from the group consisting of -OH, -O(C1-C4)alkyl, -O(C1-C4)alkenyl, -O(C1-C4)alkynyl, chlorine, and fluorine; R b each occurrence of is -O(C1-C4)alkyl, wherein said -O(C1-C4)alkyl is optionally substituted with one or two substituents independently selected from the group consisting of -OH, -O(C1-C4)alkyl, -O(C1-C4)alkenyl, -O(C1-C4)alkynyl, chlorine, and fluorine; A is, [ka] is selected from R z each occurrence of is independently H or (C1-C6) alkyl; R d each occurrence of is independently selected from —OH, (C1-C4)alkyl, —O(C1-C4)alkyl, chlorine, and fluorine; B is [ka] and D is [ka] wherein any carbon on the lipid chain may be substituted with -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, or fluorine; [ka] is cis or trans stereochemistry, X 1 -O-, -C(R) 2 -, or -NR-, each occurrence of R is independently selected from H, (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, —OH, —O(C-C)alkyl, —O(C-C)alkenyl, —O(C-C)alkynyl, chlorine, and fluorine; m is 0, 1, or 2; n is 0, 1, 2, or 3; p is 0, 1, or 2; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; s is 1, 2, 3, 4, 5, 6, 7, or 8; t is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18).

[0170] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein m is 2.

[0171] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein m is 1.

[0172] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein m is 0.

[0173] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula Ia, wherein R' and R'' are independently selected from H and (C1-C6) alkyl, wherein the alkyl is optionally substituted with 1 to 4 -OH and / or -O(CH3).

[0174] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula Ia, wherein R' and R'' are independently selected from H and (C1-C4) alkyl, wherein the alkyl is optionally substituted with 1 to 4 -OH and -O(CH3).

[0175] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein R' is H and R'' is pentyl.

[0176] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein R' is H and R'' is butyl.

[0177] In some embodiments, the compounds of the present invention are represented by the structure depicted in formula Ia, wherein R' is H and R'' is propyl.

[0178] In some embodiments, the compounds of the present invention are represented by the structure depicted in formula Ia, wherein R' is H and R'' is ethyl.

[0179] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein R' is H and R'' is methyl.

[0180] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein R′ is H and R″ is H.

[0181] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein R a’’is H or (C1-C4) alkyl, said alkyl being optionally substituted with 1 to 2 -OH and / or -O(CH3).

[0182] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein R a’’ is H or (C1-C4) alkyl.

[0183] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein R a’’ is H.

[0184] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein R b is —O(C1-C4)alkyl, and said —O(C1-C4)alkyl may be substituted with 1 to 2 —OH and / or —O(CH3).

[0185] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein R b is -O(CH3).

[0186] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula Ia, wherein A is [ka] (In the formula, R z is independently selected from H or (C1-C6) alkyl.

[0187] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II: [ka] (In the formula, R 1is (C1-C6) alkyl, wherein said (C1-C6) alkyl is optionally substituted with 1 to 4 substituents independently selected from -OH and -O(CH3), R 2 is H, methyl or -O(CH3), R 3 each occurrence is independently H, (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, or —O(C1-C4)alkyl, wherein said (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, or —O(C1-C4)alkyl is optionally substituted with one or two substituents independently selected from —OH and —O(CH); R 4 each occurrence of is H, (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, or —O(C1-C4)alkyl, wherein said (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, or —O(C1-C4)alkyl is optionally substituted with one or two substituents independently selected from —OH and —O(CH3); R 5 teeth, [ka] and R 6 is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 )alkynyl, wherein said (C-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl is optionally substituted with 1 to 6 substituents independently selected from -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine; Each occurrence of n is 4).

[0188] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein n is independently 0, 1, 2, or 3.

[0189] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein n is independently 0, 1, or 2.

[0190] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein n is independently 0 or 1.

[0191] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein n is absent.

[0192] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 1 is ethyl, butyl (branched or straight chain), or pentyl (branched or straight chain), wherein said ethyl, butyl, or pentyl is optionally substituted with 1 to 4 substituents independently selected from -OH, -O(CH), chlorine, and fluorine.

[0193] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 1 is butyl (branched or straight chain), said butyl being optionally substituted with 1 to 4 substituents independently selected from -OH, -O(CH3), chlorine, and fluorine.

[0194] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 1 is ethyl, butyl (branched or straight chain), or pentyl (branched or straight chain), and said ethyl, butyl, or pentyl may be substituted with 1 to 2 substituents independently selected from -OH and -O(CH).

[0195] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 1 is butyl (branched or linear), and said butyl may be substituted with 1 to 2 substituents independently selected from -OH and -O(CH3).

[0196] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 2 is H or methyl.

[0197] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 2 is H.

[0198] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 3 are independently (C1-C4) alkyl, (C1-C4) alkenyl, or —O(C1-C4) alkyl, and the (C1-C4) alkyl, (C1-C4) alkenyl, or —O(C1-C4) alkyl is optionally substituted with 1 to 4 substituents independently selected from —OH or —O(CH3).

[0199] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 3 are independently (C1-C4) alkyl or —O(C1-C4) alkyl, and the (C1-C4) alkyl or —O(C1-C4) alkyl is optionally substituted with 1 to 4 substituents independently selected from —OH or —O(CH3).

[0200] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 3 are independently (C1-C4) alkyl or —O(C1-C4) alkyl, and the (C1-C4) alkyl or —O(C1-C4) alkyl may be substituted with 1 to 2 substituents independently selected from —OH or —O(CH3).

[0201] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 3 are independently methyl or —O(CH3).

[0202] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 3 are independently -O(CH3).

[0203] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 4 are independently (C1-C4) alkyl or —O(C1-C4) alkyl, and said (C1-C4) alkyl or —O(C1-C4) alkyl is optionally substituted with 1 to 4 substituents independently selected from —OH, —O(CH3), chlorine, and fluorine.

[0204] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 4 is independently (C1-C4) alkyl or —O(C1-C4) alkyl.

[0205] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6 is (C6-C 20 ) alkyl and (C6-C 20 ) alkenyl, wherein said (C6-C 20 ) alkyl and (C6-C 20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0206] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6 is (C8-C 20 ) alkyl and (C8-C 20 ) alkenyl, wherein said (C-C 20 ) alkyl and (C8-C 20) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0207] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6 is (C 10 -C 20 ) alkyl and (C 10 -C 20 ) alkenyl, wherein (C 10 -C 20 ) alkyl and (C 10 -C 20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0208] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6 is (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl, wherein (C 12 -C 20 ) alkyl and (C 12 -C 20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0209] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6 is (C 14 -C 20 ) alkyl and (C 14 -C 20 ) alkenyl, (C 14 -C 20 ) alkyl and (C 14 -C 20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0210] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6is (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl, wherein (C 16 -C 20 ) alkyl and (C 16 -C 20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0211] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6 is (C6-C 20 ) alkyl and (C6-C 20 ) alkenyl.

[0212] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6 is (C8-C 20 ) alkyl and (C8-C 20 ) alkenyl.

[0213] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6 is (C 10 -C 20 ) alkyl and (C 10 -C 20 ) alkenyl.

[0214] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6 is (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl.

[0215] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6 is (C 14 -C 20 ) alkyl and (C14 -C 20 ) alkenyl.

[0216] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula II, wherein R 6 is (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl.

[0217] The present invention further provides compounds having the structure according to formula IIa: [ka] (In the formula, R 1 is butyl, said butyl being optionally substituted with one or two -OH groups; R 3 each occurrence of is independently H or —O(CH), R 5 teeth, [ka] and R 6 is (C 10 -C 20 ) alkyl, (C 10 -C 20 ) alkenyl, and (C 10 -C 20 ) alkynyl).

[0218] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIa, wherein R 6 is (C 10 -C 20 ) alkyl and (C 10 -C 20 ) alkenyl, wherein (C 10 -C 20 ) alkyl and (C 10 -C 20) The alkenyl may be substituted with one or two -OH and -O(CH3).

[0219] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIa, wherein R 6 is (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl, wherein (C 12 -C 20 ) alkyl and (C 12 -C 20 ) The alkenyl may be substituted with one or two -OH and -O(CH3).

[0220] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIa, wherein R 6 is (C 14 -C 20 ) alkyl and (C 14 -C 20 ) alkenyl, wherein (C 14 -C 20 ) alkyl and (C 14 -C 20 ) The alkenyl may be substituted with one or two -OH and -O(CH3).

[0221] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIa, wherein R 6 is (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl, wherein (C 16 -C 20 ) alkyl and (C 16 -C 20 ) The alkenyl may be substituted with one or two -OH and -O(CH3).

[0222] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIa, wherein R 6is (C 10 -C 20 ) alkyl and (C 10 -C 20 ) alkenyl.

[0223] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIa, wherein R 6 is (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl.

[0224] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIa, wherein R 6 is (C 14 -C 20 ) alkyl and (C 14 -C 20 ) alkenyl.

[0225] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIa, wherein R 6 is (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl.

[0226] The present invention provides compounds having the structure set forth in Formula III: [ka] (In the formula, R 1 is (C1-C6) alkyl, wherein said (C1-C6) alkyl is optionally substituted with 1 to 4 substituents independently selected from -OH and -O(CH3), R 2 is H, methyl or -O(CH3), R 3each occurrence is independently H, (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, or —O(C1-C4)alkyl, wherein said (C1-C4)alkyl, (C1-C4)alkenyl, (C1-C4)alkynyl, or —O(C1-C4)alkyl is optionally substituted with one or two substituents selected from —OH and —O(CH); R 4 is (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 )alkynyl, wherein said (C-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl is optionally substituted with 1 to 6 substituents selected from -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, and fluorine; n is 4).

[0227] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein n is 0, 1, 2, or 3.

[0228] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein n is 0, 1, or 2.

[0229] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein n is 0 or 1.

[0230] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein n is absent.

[0231] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 1is ethyl, butyl (branched or straight chain), or pentyl (branched or straight chain), wherein said ethyl, butyl, or pentyl is optionally substituted with 1 to 4 substituents independently selected from -OH, -O(CH), chlorine, and fluorine.

[0232] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 1 is butyl (branched or straight chain), said butyl being optionally substituted with 1 to 4 substituents independently selected from -OH, -O(CH3), chlorine, and fluorine.

[0233] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 1 is ethyl, butyl (branched or straight chain), or pentyl (branched or straight chain), and said ethyl, butyl, or pentyl may be substituted with 1 to 2 substituents independently selected from -OH and -O(CH).

[0234] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 1 is butyl (branched or linear), and said butyl may be substituted with 1 to 2 substituents independently selected from -OH and -O(CH3).

[0235] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 2 is H or methyl.

[0236] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 2 is H.

[0237] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 3each occurrence is independently a (C1-C4) alkyl, a (C1-C4) alkenyl, or —O(C1-C4) alkyl, wherein said (C1-C4) alkyl, (C1-C4) alkenyl, or —O(C1-C4) alkyl is optionally substituted with 1 to 4 substituents independently selected from —OH and —O(CH3).

[0238] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 3 each occurrence is independently (C1-C4) alkyl or —O(C1-C4) alkyl, wherein said (C1-C4) alkyl or —O(C1-C4) alkyl is optionally substituted with 1 to 4 substituents independently selected from —OH and —O(CH3).

[0239] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 3 each occurrence is independently (C1-C4) alkyl or —O(C1-C4) alkyl, wherein said (C1-C4) alkyl or —O(C1-C4) alkyl is optionally substituted with 1 to 2 substituents independently selected from —OH and —O(CH3).

[0240] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 3 Each occurrence of is independently methyl or —O(CH 3 ).

[0241] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 3 Each occurrence of is -O(CH3).

[0242] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 4 is (C6-C 20 ) alkyl and (C6-C 20 ) alkenyl, wherein said (C6-C 20 ) alkyl and (C6-C20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0243] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 4 is (C8-C 20 ) alkyl and (C8-C 20 ) alkenyl, wherein said (C-C 20 ) alkyl and (C8-C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0244] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 4 is (C 10 -C 20 ) alkyl and (C 10 -C 20 ) alkenyl, wherein (C 10 -C 20 ) alkyl and (C 10 -C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0245] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 4 is (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl, wherein (C 12 -C 20 ) alkyl and (C 12 -C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0246] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R4 is (C 14 -C 20 ) alkyl and (C 14 -C 20 ) alkenyl, wherein (C 14 -C 20 ) alkyl and (C 14 -C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0247] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 4 is (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl, wherein (C 16 -C 20 ) alkyl and (C 16 -C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0248] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 4 is (C6-C 20 ) alkyl and (C6-C 20 ) alkenyl.

[0249] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 4 is (C8-C 20 ) alkyl and (C8-C 20 ) alkenyl.

[0250] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 4 is (C 10 -C 20 ) alkyl and (C 10 -C 20) alkenyl.

[0251] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 4 is (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl.

[0252] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 4 is (C 14 -C 20 ) alkyl and (C 14 -C 20 ) alkenyl.

[0253] In some embodiments, the compounds of the present invention are represented by the structure set forth in Formula III, wherein R 4 is (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl.

[0254] The present invention also provides compounds having the structure according to formula IIIa: [ka] (In the formula, R 1 is butyl, said butyl being optionally substituted with one or two -OH groups; R 3 each occurrence of is independently H or —O(CH), R 4 is (C 10 -C 20 ) alkyl, (C 10 -C 20 ) alkenyl, and (C 10 -C 20 ) alkynyl).

[0255] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C6-C 20 ) alkyl and (C6-C 20 ) alkenyl, wherein said (C6-C 20 ) alkyl and (C6-C 20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0256] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C8-C 20 ) alkyl and (C8-C 20 ) alkenyl, wherein said (C-C 20 ) alkyl and (C8-C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0257] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C 10 -C 20 ) alkyl and (C 10 -C 20 ) alkenyl, wherein (C 10 -C 20 ) alkyl and (C 10 -C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0258] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl, wherein (C 12 -C 20 ) alkyl and (C 12 -C 20) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0259] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C 14 -C 20 ) alkyl and (C 14 -C 20 ) alkenyl, wherein (C 14 -C 20 ) alkyl and (C 14 -C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0260] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl, wherein (C 16 -C 20 ) alkyl and (C 16 -C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0261] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C6-C 20 ) alkyl and (C6-C 20 ) alkenyl.

[0262] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C8-C 20 ) alkyl and (C8-C 20 ) alkenyl.

[0263] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C 10 -C 20 ) alkyl and (C 10 -C 20 ) alkenyl.

[0264] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl.

[0265] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C 14 -C 20 ) alkyl and (C 14 -C 20 ) alkenyl.

[0266] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IIIa, wherein R 4 is (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl.

[0267] The present invention provides compounds having the structure set forth in Formula IV: [ka] (In the formula, R 1 is (C1-C6) alkyl, wherein said (C1-C6) alkyl is optionally substituted with 1 to 4 substituents independently selected from -OH and -O(CH3), R 2 is H, methyl or -O(CH3), R3 each occurrence is independently H, (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, or —O(C-C)alkyl, wherein said (C-C)alkyl, (C-C)alkenyl, (C-C)alkynyl, and —O(C-C)alkyl are optionally substituted with one or two substituents independently selected from —OH and —O(CH); R 4 Each occurrence of (C6-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 )alkynyl, wherein the (C-C 20 ) alkyl, (C6-C 20 ) alkenyl, and (C6-C 20 ) alkynyl is optionally substituted with 1 to 6 substituents independently selected from -OH, -O(C1-C4) alkyl, -O(C1-C4) alkenyl, -O(C1-C4) alkynyl, chlorine, or fluorine; n is 4).

[0268] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, where n is 0, 1, 2, or 3.

[0269] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein n is 0, 1, or 2.

[0270] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein n is 0 or 1.

[0271] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein n is absent.

[0272] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 1is ethyl, butyl (branched or straight chain), or pentyl (branched or straight chain), wherein said ethyl, butyl, or pentyl is optionally substituted with 1 to 4 substituents independently selected from -OH, -O(CH), chlorine, and fluorine.

[0273] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 1 is butyl (branched or straight chain), said butyl being optionally substituted with 1 to 4 substituents independently selected from -OH, -O(CH3), chlorine, and fluorine.

[0274] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 1 is ethyl, butyl (branched or straight chain), or pentyl (branched or straight chain), and said ethyl, butyl, or pentyl may be substituted with 1 to 2 substituents independently selected from -OH and -O(CH).

[0275] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 1 is butyl (branched or linear), and said butyl may be substituted with 1 to 2 substituents independently selected from -OH and -O(CH3).

[0276] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 2 is H or methyl.

[0277] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 2 is H.

[0278] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 3each occurrence is independently a (C1-C4) alkyl, a (C1-C4) alkenyl, or —O(C1-C4) alkyl, wherein said (C1-C4) alkyl, (C1-C4) alkenyl, or —O(C1-C4) alkyl is optionally substituted with 1 to 4 substituents independently selected from —OH or —O(CH3).

[0279] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 3 each occurrence is independently (C1-C4) alkyl or —O(C1-C4) alkyl, wherein said (C1-C4) alkyl or —O(C1-C4) alkyl is optionally substituted with 1 to 4 substituents independently selected from —OH or —O(CH3).

[0280] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 3 each occurrence is independently (C1-C4) alkyl or -O(C1-C4) alkyl, wherein said (C1-C4) alkyl or -O(C1-C4) alkyl is optionally substituted with 1 to 2 substituents independently selected from -OH or -O(CH3).

[0281] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 3 Each occurrence of is independently methyl or —O(CH 3 ).

[0282] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 3 Each occurrence of is independently —O(CH3).

[0283] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C6-C 20 ) alkyl and (C6-C 20 ) alkenyl, wherein the (C-C 20 ) alkyl and (C6-C20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0284] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C8-C 20 ) alkyl and (C8-C 20 ) alkenyl, wherein the (C-C 20 ) alkyl and (C8-C 20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0285] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C 10 -C 20 ) alkyl and (C 10 -C 20 ) alkenyl, wherein (C 10 -C 20 ) alkyl and (C 10 -C 20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0286] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl, wherein (C 12 -C 20 ) alkyl and (C 12 -C 20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0287] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C 14 -C20 ) alkyl and (C 14 -C 20 ) alkenyl, wherein (C 14 -C 20 ) alkyl and (C 14 -C 20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0288] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl, wherein (C 16 -C 20 ) alkyl and (C 16 -C 20 ) The alkenyl may be substituted with 1 to 6 -OH and -O(CH3).

[0289] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C6-C 20 ) alkyl and (C6-C 20 ) alkenyl.

[0290] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C8-C 20 ) alkyl and (C8-C 20 ) alkenyl.

[0291] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C 10 -C 20 ) alkyl and (C 10 -C 20 ) alkenyl.

[0292] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl.

[0293] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C 14 -C 20 ) alkyl and (C 14 -C 20 ) alkenyl.

[0294] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IV, wherein R 4 Each occurrence of (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl.

[0295] The present invention provides compounds having the structure according to formula IVa: [ka] (In the formula, R 1 is butyl, said butyl being optionally substituted with one or two -OH groups; R 3 each occurrence of is independently H or —O(CH), R 4 Each occurrence of (C 10 -C 20 ) alkyl, (C 10 -C 20 ) alkenyl, and (C 10 -C 20 )alkynyl).

[0296] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 is (C6-C 20 ) alkyl and (C6-C 20 ) alkenyl, wherein the (C-C 20 ) alkyl and (C6-C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0297] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 is (C8-C 20 ) alkyl and (C8-C 20 ) alkenyl, wherein the (C-C 20 ) alkyl and (C8-C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0298] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 is (C 10 -C 20 ) alkyl and (C 10 -C 20 ) alkenyl, wherein (C 10 -C 20 ) alkyl and (C 10 -C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0299] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 is (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl, wherein (C 12 -C 20 ) alkyl and (C12 -C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0300] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 is (C 14 -C 20 ) alkyl and (C 14 -C 20 ) alkenyl, wherein (C 14 -C 20 ) alkyl and (C 14 -C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0301] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 is (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl, wherein (C 16 -C 20 ) alkyl and (C 16 -C 20 ) The alkenyl may be optionally substituted with 1 to 6 substituents independently selected from -OH and -O(CH3).

[0302] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 Each occurrence of (C6-C 20 ) alkyl and (C6-C 20 ) alkenyl.

[0303] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 R 4 Each occurrence of (C8-C 20 ) alkyl and (C8-C20 ) alkenyl.

[0304] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 R 4 Each occurrence of (C 10 -C 20 ) alkyl and (C 10 -C 20 ) alkenyl.

[0305] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 R 4 Each occurrence of (C 12 -C 20 ) alkyl and (C 12 -C 20 ) alkenyl.

[0306] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 R 4 Each occurrence of (C 14 -C 20 ) alkyl and (C 14 -C 20 ) alkenyl.

[0307] In some embodiments, the compounds of the present invention are represented by the structure set forth in formula IVa, wherein R 4 R 4 Each occurrence of (C 16 -C 20 ) alkyl and (C 16 -C 20 ) alkenyl.

[0308] In some embodiments, the present invention provides (N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, (S)—N-(5-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, (S)-1-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)octadecan-1-one, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)tetradecanamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)oleamide, (9Z,12Z)-N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)octadeca-9,12-dienamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperidin-1-yl)-5-oxopentyl)stearamide, N-(5-(3-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)azetidin-1-yl)-5-oxopentyl)stearamide, 1-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperidine-4-carboxamide, (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-octadecylcyclobutane-1-carboxamide, (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-hexadecylcyclobutane-1-carboxamide, N-(3-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-3-oxopropyl)stearamide, N-(7-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-7-oxoheptyl)stearamide, N-(3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)cyclobutyl)stearamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-4,4-dimethyl-5-oxopentyl)stearamide, N-(6-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-methyl-6-oxohexan-2-yl)stearamide, 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-(octadecyloxy)pentan-1-one, 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-(octadecylamino)pentan-1-one, N-(5-(4-(4-((5-amino-7-(butylamino)-3-methyl-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, (9Z,12Z)-N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide, N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)tetradecanamide, N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)oleamide, N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide, N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)-4-oxobutyl)stearamide, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate, 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperazine-1-carboxamide, 3-stearamidopropyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-6,6,6-trifluorohexyl)stearamide, N-(4-((4-((7-(butylamino)-5-hydroxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide, and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate, or a pharmaceutically acceptable salt thereof.

[0309] In some embodiments, the compound of the present invention is (N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0310] In some embodiments, the compound of the present invention is (N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0311] In some embodiments, the compound of the present invention is (S)—N-(5-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0312] In some embodiments, the compound of the present invention is (S)-1-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)octadecan-1-one, or a pharmaceutically acceptable salt thereof.

[0313] In some embodiments, the compound of the present invention is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0314] In some embodiments, the compound of the present invention is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)tetradecanamide, or a pharmaceutically acceptable salt thereof.

[0315] In some embodiments, the compound of the present invention is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)oleamide, or a pharmaceutically acceptable salt thereof.

[0316] In some embodiments, the compound of the present invention is (9Z,12Z)-N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)octadeca-9,12-dienamide, or a pharmaceutically acceptable salt thereof.

[0317] In some embodiments, the compound of the present invention is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0318] In some embodiments, the compound of the present invention is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperidin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0319] In some embodiments, the compound of the present invention is N-(5-(3-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)azetidin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0320] In some embodiments, the compound of the present invention is 1-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof.

[0321] In some embodiments, the compound of the present invention is (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-octadecylcyclobutane-1-carboxamide, or a pharmaceutically acceptable salt thereof.

[0322] In some embodiments, the compound of the present invention is (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-hexadecylcyclobutane-1-carboxamide, or a pharmaceutically acceptable salt thereof.

[0323] In some embodiments, the compound of the present invention is N-(3-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-3-oxopropyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0324] In some embodiments, the compound of the present invention is N-(7-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-7-oxoheptyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0325] In some embodiments, the compound of the present invention is N-(3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)cyclobutyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0326] In some embodiments, the compound of the present invention is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-4,4-dimethyl-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0327] In some embodiments, the compound of the present invention is N-(6-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-methyl-6-oxohexan-2-yl)stearamide, or a pharmaceutically acceptable salt thereof.

[0328] In some embodiments, the compound of the present invention is 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-(octadecyloxy)pentan-1-one, or a pharmaceutically acceptable salt thereof.

[0329] In some embodiments, the compound of the present invention is 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-(octadecylamino)pentan-1-one, or a pharmaceutically acceptable salt thereof.

[0330] In some embodiments, the compound of the present invention is N-(5-(4-(4-((5-amino-7-(butylamino)-3-methyl-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0331] In some embodiments, the compound of the present invention is (9Z,12Z)-N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide, or a pharmaceutically acceptable salt thereof.

[0332] In some embodiments, the compound of the invention is N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)tetradecanamide, or a pharmaceutically acceptable salt thereof.

[0333] In some embodiments, the compound of the invention is N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)oleamide, or a pharmaceutically acceptable salt thereof.

[0334] In some embodiments, the compound of the present invention is N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0335] In some embodiments, the compound of the present invention is N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)-4-oxobutyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0336] In some embodiments, the compound of the present invention is (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl (4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate, or a pharmaceutically acceptable salt thereof.

[0337] In some embodiments, the compound of the present invention is 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperazine-1-carboxamide, or a pharmaceutically acceptable salt thereof.

[0338] In some embodiments, the compound of the present invention is 3-stearamidopropyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate, or a pharmaceutically acceptable salt thereof.

[0339] In some embodiments, the compound of the present invention is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-6,6,6-trifluorohexyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0340] In some embodiments, the compound of the present invention is N-(4-((4-((7-(butylamino)-5-hydroxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide, or a pharmaceutically acceptable salt thereof.

[0341] In some embodiments, the compound of the present invention is (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate, or a pharmaceutically acceptable salt thereof.

[0342] Adjuvants Adjuvant-based approaches are being investigated to enhance vaccine immunogenicity and help address remaining unmet medical needs, particularly for populations that are more susceptible to infectious diseases (i.e., immunocompromised, elderly, or pediatric populations).

[0343] For example, although the incidence of invasive pneumococcal disease (IPD) has dramatically decreased in both children and adults following the introduction of pneumococcal conjugate vaccines, residual disease caused by persistent vaccine serotypes remains. Furthermore, serotype-specific immune responses vary, and the introduction of expanded valency PCVs has resulted in an overall decrease in serotype-specific immune responses. Adjuvant-based strategies to enhance the immunogenicity of PCVs may increase vaccine efficacy against challenging serotypes, provide more sustained immune responses, or allow for a shorter dosing schedule.

[0344] Preclinical data indicate that toll-like receptor 7 / 8 (TLR7 / 8) agonists can increase the immunogenicity of PCV. [Dowling, DJ et al.] reported both accelerated and enhanced serotype-specific antibody responses, including both binding antibody titers and functional opsonophagocytic killing, in neonatal and infant rhesus macaques immunized with PCV13 plus the TLR7 / 8 agonist 3M-052 compared with PCV13 alone (PCV13 containing an alum adjuvant). Addition of a TLR7 / 8 agonist to PCV13 also induced Th1-polarized CRM197-specific CD4 T cells and juvenile Streptococcus pneumoniae antigen-specific B cells in infant rhesus macaques and enhanced type II IFN and Th1-polarizing cytokine production after in vitro stimulation of human neonatal umbilical cord blood (Dowling, DJ et al., JCI Insight 2017;2(2)e91020).

[0345] Adjuvant formulations The present disclosure provides an adjuvant formulation comprising 1) one or more compounds of the present invention, or pharmaceutically acceptable salt(s) thereof, 2) one or more sorbitan-based surfactants, and 3) one or more terpenes. The adjuvant formulation of the present invention is also referred to as a stable nanoemulsion or SNE.

[0346] The present disclosure provides an adjuvant formulation comprising: 1) one or more compounds of the invention, or pharmaceutically acceptable salt(s) thereof; 2) sorbitan trioleate (SPAN-85); 3) polysorbate-20 (PS-20) or polysorbate-80 (PS-80); and 4) squalene.

[0347] The present disclosure provides an adjuvant formulation comprising: 1) one or more compounds of the invention, or pharmaceutically acceptable salt(s) thereof; 2) SPAN-85; 3) PS-20; and 4) squalene.

[0348] The present disclosure provides an adjuvant formulation comprising: 1) a compound of the invention, or a pharmaceutically acceptable salt thereof; 2) SPAN-85; 3) PS-20 or PS-80; and 4) squalene.

[0349] The present disclosure provides an adjuvant formulation comprising: 1) a compound of the invention, or a pharmaceutically acceptable salt thereof; 2) SPAN-85; 3) PS-20; and 4) squalene.

[0350] General method for preparing SNE formulations Generally, SNEs can be formed, for example, by first combining and mixing the components. Once mixed and blended, an aqueous buffer is added and mixed with the initial compound components to form a blended emulsion mixture. In some embodiments, the blended emulsion components are first subjected to coarse homogenization, followed by fine homogenization. The resulting formulation is then subjected to a final filtration step and stored at 4°C. The solution can contain one or more compounds, one or more sorbitan surfactants (e.g., PS-20; PS-80; SPAN-85), and one or more terpenes (e.g., squalene) in a specific molar ratio.

[0351] Alternatively, the process for preparing the SNEs of the present invention consists of four major steps: 1) solution preparation of a component mixture containing functional and non-functional components with an aqueous buffer, 2) SNE formation by split-flow mixing, 3) ultrafiltration, and 4) filtration.

[0352] Typically, the components are dissolved in ethanol before being sterile filtered to form a mixture. Some aqueous buffer is also prepared. The mixture and buffer stream are then combined using a T-tube or Y-mixer, then diluted immediately after the outlet and mixed with aqueous buffer to form the SNE intermediate. The SNE intermediate is then subjected to ultrafiltration or dialysis to concentrate the material and exchange it with a suitable buffer to remove residual ethanol. After diafiltration, a final concentration step is performed to achieve the final target concentration. The SNE bulk is then sterile filtered.

[0353] SNE preparation In some embodiments, an SNE formulation is provided that includes: 1) about 10-14 mol% of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 1-4 mol% of SPAN-85; 3) about 1-4 mol% of PS-20 or PS-80; and 4) about 50-80 mol% of squalene.

[0354] In some embodiments, an SNE formulation is provided that includes: 1) about 30-65 mol % of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 5-30 mol % of SPAN-85; 3) about 0.5-4 mol % of PS-20 or PS-80; and 4) about 10-40 mol % of squalene.

[0355] In some embodiments, an SNE formulation is provided that includes: 1) about 55-65 mol % of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 5-15 mol % of SPAN-85; 3) about 1-2.5 mol % of PS-20 or PS-80; and 4) about 25-35 mol % of squalene.

[0356] In some embodiments, an SNE formulation is provided that includes: 1) about 13-45 mol % of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 2-4 mol % of SPAN-85; 3) about 1.5-3 mol % of PS-20 or PS-80; and 4) about 50-82 mol % of squalene.

[0357] In some embodiments, an SNE formulation is provided that includes: 1) about 13-14 mol% of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 1-2 mol% of SPAN-85; 3) about 1-2 mol% of PS-20 or PS-80; and 4) about 79-81 mol% of squalene.

[0358] In some embodiments, an SNE formulation is provided comprising: 1) about 1-60 mol % of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 1-4 mol % of SPAN-85; 3) about 1-4 mol % of PS-20 or PS-80; and 4) about 32-97 mol % of squalene.

[0359] In some embodiments, an SNE formulation is provided that includes: 1) about 0-45 mol % of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, or IVa, or a pharmaceutically acceptable salt thereof; 2) about 1-10 mol % of a non-ionic surfactant; and 3) about 50-85 mol % of squalene. In one aspect of this embodiment, the non-ionic surfactant comprises a mixture of SPAN-85 and PS-20 or PS-80.

[0360] In some embodiments, an SNE formulation is provided that includes: 1) about 10-14 mol% of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 1-4 mol% of SPAN-85; 3) about 1-4 mol% of PS-20; and 4) about 50-80 mol% of squalene.

[0361] In some embodiments, an SNE formulation is provided that includes: 1) about 30-65 mol % of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 5-30 mol % of SPAN-85; 3) about 0.5-4 mol % of PS-20; and 4) about 10-40 mol % of squalene.

[0362] In some embodiments, an SNE formulation is provided that includes: 1) about 55-65 mol% of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 5-15 mol% of SPAN-85; 3) about 1-2.5 mol% of PS-20; and 4) about 25-35 mol% of squalene.

[0363] In some embodiments, an SNE formulation is provided that includes: 1) about 13-45 mol% of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 2-4 mol% of SPAN-85; 3) about 1.5-3 mol% of PS-20; and 4) about 50-82 mol% of squalene.

[0364] In some embodiments, an SNE formulation is provided that includes: 1) about 13-14 mol% of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 1-2 mol% of SPAN-85; 3) about 1-2 mol% of PS-20; and 4) about 79-81 mol% of squalene.

[0365] In some embodiments, an SNE formulation is provided comprising: 1) about 1-60 mol% of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) about 1-4 mol% of SPAN-85; 3) about 1-4 mol% of PS-20; and 4) about 32-97 mol% of squalene.

[0366] In some embodiments, an SNE formulation is provided that includes: 1) about 0-45 mol % of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, or IVa, or a pharmaceutically acceptable salt thereof; 2) about 1-10 mol % of a non-ionic surfactant; and 3) about 50-85 mol % of squalene. In one aspect of this embodiment, the non-ionic surfactant comprises a mixture of SPAN-85 and PS-20.

[0367] In some embodiments, an SNE formulation is provided that includes: 1) 0.01-0.1 mol% of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) 1.5-15.0 mol% of SPAN-85; 3) 1-10.0 mol% of PS-20; and 4) 80.0-98.0 mol% of squalene.

[0368] In some embodiments, an SNE formulation is provided that includes: 1) 0.1-1.0 mol% of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) 8.0-11.9 mol% of SPAN-85; 3) 6-10 mol% of PS-20; and 4) 78-85 mol% of squalene.

[0369] In some embodiments, an SNE formulation is provided that includes: 1) 0.01-0.1 mol% of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) 8.0-11.9 mol% of SPAN-85; 3) 6-10 mol% of PS-20; and 4) 78-85 mol% of squalene.

[0370] In some embodiments, an SNE formulation is provided that includes 200-400 μg of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) 8-16 mg of SPAN-85; 3) 8-16 mg of PS-20; and 4) 40-80 mg of squalene.

[0371] In some embodiments, an SNE formulation is provided that includes 10-20 μg of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) 8-16 mg of SPAN-85; 3) 8-16 mg of PS-20; and 4) 40-80 mg of squalene.

[0372] In some embodiments, an SNE formulation is provided comprising: 1) 0.5-3.0 μg of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) 0.6-3.6 mg of SPAN-85; 3) 0.6-3.6 mg of PS-20; and 4) 2.0-12.0 mg of squalene.

[0373] In some embodiments, an SNE formulation is provided comprising: 1) 0.5-3.0 μg of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) 0.15-0.18 mg of SPAN-85; 3) 0.15-0.18 mg of PS-20; and 4) 0.5-0.6 mg of squalene.

[0374] In some embodiments, an SNE formulation is provided comprising: 1) 0.5-3.0 μg of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) 0.15-0.95 mg of SPAN-85; 3) 0.15-0.95 mg of PS-20; and 4) 0.5-3.0 mg of squalene.

[0375] In some embodiments, an SNE formulation is provided comprising: 4.0-20 μg of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) 0.24-1.3 mg of SPAN-85; 3) 0.24-1.3 mg of PS-20; and 4) 0.8-4.0 mg of squalene.

[0376] In some embodiments, an SNE formulation is provided comprising: 4.0 to 20 μg of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) 1.2 to 6.5 mg of SPAN-85; 3) 1.2 to 6.5 mg of PS-20; and 4) 4.0 to 20.0 mg of squalene.

[0377] In some embodiments, an SNE formulation is provided that includes 20-100 μg of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 2) 1.2-6.5 mg of SPAN-85; 3) 1.2-6.5 mg of PS-20; and 4) 4.0-20.0 mg of squalene.

[0378] In some embodiments, an SNE formulation is provided comprising: 20-40 μg of a compound of Formula I, Ia, II, IIa, III, IIIa, IV, and IVa, or a pharmaceutically acceptable salt thereof; 1) 5-8.0 mg of SPAN-85; 3) 5-8.0 mg of PS-20; and 4) 20.0-40.0 mg of squalene.

[0379] In some embodiments, any one of the formulations provided above comprises: (N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, also known as compound A-1; (S)—N-(5-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, also known as compound A-2; (S)-1-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)octadecan-1-one, also known as compound A-3 N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, also known as compound B-1; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)tetradecanamide, also known as compound B-2; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)oleamide, also known as compound B-3; (9Z,12Z)-N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)octadeca-9,12-dienamide, also known as compound B-4; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide, also known as compound B-5; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperidin-1-yl)-5-oxopentyl)stearamide, also known as compound B-6; N-(5-(3-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)azetidin-1-yl)-5-oxopentyl)stearamide, also known as compound B-7; 1-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperidine-4-carboxamide, also known as compound B-8; (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-octadecylcyclobutane-1-carboxamide, also known as compound B-9; (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-hexadecylcyclobutane-1-carboxamide, also known as compound B-10; N-(3-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-3-oxopropyl)stearamide, also known as compound B-11; N-(7-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-7-oxoheptyl)stearamide, also known as compound B-12; N-(3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)cyclobutyl)stearamide, also known as compound B-13; N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-4,4-dimethyl-5-oxopentyl)stearamide, also known as compound B-14; N-(6-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-methyl-6-oxohexan-2-yl)stearamide, also known as compound B-15; 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-(octadecyloxy)pentan-1-one, also known as compound B-16; 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-(octadecylamino)pentan-1-one, also known as compound B-17; N-(5-(4-(4-((5-amino-7-(butylamino)-3-methyl-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, also known as compound B-18; (9Z,12Z)-N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide, also known as compound C-1; N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)tetradecanamide, also known as compound C-2 N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)oleamide, also known as compound C-3 N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide, also known as compound C-4; N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)-4-oxobutyl)stearamide, also known as compound C-5; (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate, also known as compound D-1; 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperazine-1-carboxamide, also known as compound D-2; 3-stearamidopropyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate, also known as compound D-3 N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-6,6,6-trifluorohexyl)stearamide, also known as compound D-4; N-(4-((4-((7-(butylamino)-5-hydroxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide, also known as compound D-5, and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate, also known as compound D-6; or a pharmaceutically acceptable salt thereof.

[0380] In some embodiments of the present invention, a formulation is provided in which the SNE further comprises one or more additional components selected from a surfactant, a mixture of surfactants, a phospholipid, a terpene, a terpenoid, a triterpene, or a combination thereof.

[0381] In some embodiments of the invention, surfactants include polyoxyethylene sorbitan ester surfactants (commonly referred to as Tweens), particularly PS-20 and PS-80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), such as linear EO / PO block copolymers, sold under the trade name DOWFAX™; octoxynols, which may vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanols (IGEPALCA-630 / NP-40); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl, and oleyl alcohols (known as Brij surfactants), such as triethylene glycol mononitrile. lauryl ether (Brij30); sorbitan trioleate (Span-85, Tween-85, or [2-[(2R,3S,4R)-4-hydroxy-3-[(Z)-octadec-9-enoyl]oxyoxolan-2-yl]-2-[(Z)-octadec-9-enoyl]oxyethyl](Z)-octadec-9-enoate) and sorbitan esters (commonly known as SPAN), such as sorbitan monolaurate.

[0382] In some embodiments of the present invention, a mixture of surfactants is used, such as a PS-20 / Span 85 mixture. A combination of a polyoxyethylene sorbitan ester, such as polyoxyethylene sorbitan monooleate (PS-80), and an octoxynol, such as t-octylphenoxypolyethoxyethanol (Triton X-100), is also suitable. Another useful combination includes laureth 9 and a polyoxyethylene sorbitan ester and / or an octoxynol.

[0383] In some embodiments of the invention, the amount of surfactant or emulsifier is: polyoxyethylene sorbitan ester (such as PS-20) 0.01 to 10 mol %, in particular about 1 to 4 mol %; octyl- or nonylphenoxypolyoxyethanol (such as Triton X-100 or other detergents in the Triton series) 0.001 to 10 mol %, in particular about 1 to 4 mol % w / v, in particular 0.01 to 0.1% w / v; polyoxyethylene ether (such as Laureth 9) 0.1 to 20 mol %, preferably 0.5 to 10 mol %, in particular 1 to 4 mol % or about 10% by weight.

[0384] In some embodiments of the invention, the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), and naturally occurring phospholipids including phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (phosphatidate) (PA), dipalmitoylphosphatidylcholine, monoacyl-phosphatidylcholine (lyso-PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), N-acyl-PE, phosphoinositides, and phosphosphingolipids. Phospholipid derivatives include phosphatidic acid (DMPA, DPPA, DSPA), phosphatidylcholine (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine (DMPE, DPPE, DSPE DOPE), phosphatidylserine (DOPS). Fatty acids include C14:0, palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), and arachidonic acid (C20:4), C20:0, C22:0, and ethynyl. In certain embodiments of the invention, the phospholipid is phosphatidylserine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dilauroylphosphatidylcholine (DLPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0385] In some embodiments of the present invention, the terpene is selected from monoterpenes such as geraniol, terpenol, limonene, myrcene, linalool, or pinene. In some embodiments, the formulation comprises humulene, farnesene, or farnesol; diterpenes such as cafestol, kahweol, cembrene, or taxadiene; triterpenes such as squalene or squalane; tetraterpenes such as acyclic lycopene, monocyclic gamma-carotene, or bicyclic alpha- and beta-carotene; polyterpenes or sesquiterpenes consisting of norisoprenoids.

[0386] Vaccine Composition Vaccines (vaccines or immunogenic compositions) are well known in the art and include adenovirus vaccines, anthrax vaccines, cholera vaccines, dengue vaccines, diphtheria vaccines, hepatitis vaccines, Haemophilus influenzae type b (Hib) vaccines, human papillomavirus (HPV) vaccines, seasonal influenza (Flu) vaccines, Japanese encephalitis vaccines, measles vaccines, meningitis vaccines, mumps vaccines, pertussis vaccines, pneumococcal vaccines, polio vaccines, rabies vaccines, rotavirus vaccines, rubella vaccines, shingles vaccines, smallpox vaccines, tetanus vaccines, tuberculosis vaccines, typhoid vaccines, chickenpox vaccines, and yellow fever vaccines.

[0387] Pneumococcal vaccines or compositions are well known (e.g., PNEUMOVAX®, Merck & Co., Inc., Rahway, New Jersey, USA). Pneumococcal conjugate vaccines or compositions have been previously disclosed. See WO 2011 / 100151, WO 2019 / 139692, and WO 2020 / 131763.

[0388] Bacterial capsular polysaccharides, particularly those that have been used as antigens, are suitable for use in the present invention and can be readily identified by methods for identifying immunogenic and / or antigenic polysaccharides. Exemplary bacterial capsular polysaccharides from Streptococcus pneumoniae are serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20 (20A and 20B), 22F, 23A, 23B, 23F, 24F, 33F, 35B, 35F, or 38, among others.

[0389] Administration / Dosage The compositions and formulations of the invention can be used to protect or treat patients susceptible to infectious diseases by administering the composition, formulation, or vaccine to the patient via systemic or mucosal routes. In one embodiment, the invention provides a method for inducing an immune response to an antigen, comprising administering to a patient an immunologically effective amount of an immunogenic composition of the invention. In another embodiment, the invention provides a method for vaccinating against an infection, comprising administering to a patient an immunologically effective amount of an immunogenic composition, formulation, or vaccine of the invention.

[0390] In some embodiments of the above methods, the patient is a human.

[0391] In other embodiments of the above methods, the patient is a non-human animal.

[0392] The optimal amount of components for a particular composition, formulation, or vaccine can be determined by standard studies involving observation of appropriate immune responses in subjects. For example, in one embodiment, dosages for human vaccination are determined by extrapolating from animal studies to human data. In another embodiment, dosages are determined empirically.

[0393] The methods of the present invention can be used for the prevention and / or reduction of primary clinical symptoms caused by an infectious agent in a subject, ie, a human or non-human animal.

[0394] Administration of the compositions, formulations, or vaccines of the present invention may include one or more of injection via intramuscular, intraperitoneal, intradermal, or subcutaneous routes, or via mucosal administration to the oral / alimentary, respiratory, or genitourinary tracts. In one embodiment, intranasal administration is used for the treatment of disease.

[0395] According to any of the methods of the present invention, and in some embodiments, the subject is a human. In certain embodiments, the human patient is an infant (under 1 year of age), a toddler (approximately 12-24 months of age), or a child (approximately 2-5 years of age). In other embodiments, the human patient is an elderly patient (>65 years of age). The compositions of the present invention are also suitable for use in older children, adolescents, and adults (e.g., 18-45 years of age or 18-65 years of age).

[0396] In one embodiment of the method of the present invention, the composition, formulation or vaccine of the present invention is administered to the subject as a single inoculation.In another embodiment, the composition, formulation or vaccine is administered two, three, or four or more times with sufficient intervals.For example, the composition, formulation or vaccine can be administered at intervals of 1, 2, 3, 4, 5 or 6 months, or any combination thereof.

[0397] formulation In the formulation embodiments below, composition refers to a pharmaceutical composition and / or an immunogenic composition and / or a single-dose vaccine composition. In some embodiments, a composition is provided that includes one or more compounds described herein and one or more antigens.

[0398] In some embodiments, a composition is provided comprising about 1 μg to about 200 mg of a compound of the invention, or a pharmaceutically acceptable salt thereof, and at least one antigen, wherein the antigen is present at a concentration of about 0.01 μg to about 100 μg per 0.5 mL of vaccine formulation.

[0399] In some embodiments, a composition is provided comprising about 0.02 μg to about 40 mg of a compound of the invention, or a pharmaceutically acceptable salt thereof, and at least one antigen, wherein the antigen is present at a concentration of about 0.002 μg to about 20 μg per 0.1 mL of vaccine formulation.

[0400] In some embodiments, a composition is provided comprising about 50 μg to about 2.1 mg of a compound of the invention, or a pharmaceutically acceptable salt thereof, and at least one antigen, wherein the antigen is present at a concentration of about 0.002 μg to about 20 μg per 0.5 mL of vaccine formulation.

[0401] In some embodiments, a composition is provided comprising about 50 μg to about 10 mg of a compound of the invention, or a pharmaceutically acceptable salt thereof, and at least one antigen, wherein the antigen is present at a concentration of about 0.002 μg to about 20 μg per 0.5 mL of vaccine formulation.

[0402] In some embodiments, a composition is provided comprising about 50 μg to about 10 mg of a compound of the invention, or a pharmaceutically acceptable salt thereof, and at least one antigen, wherein the antigen is present at a concentration of about 0.002 μg to about 20 μg per 0.5 mL of vaccine formulation.

[0403] In some embodiments, a composition is provided comprising about 1 μg to about 200 mg of a compound of the invention, or a pharmaceutically acceptable salt thereof, and at least one antigen, wherein the antigen is present at a concentration of about 0.01 μg to about 100 μg per 0.5 mL of vaccine formulation prepared as a lyophilized formulation.

[0404] In some embodiments, a composition is provided comprising about 1 μg to about 200 mg of a compound of the invention, or a pharmaceutically acceptable salt thereof, 1 μg to about 1 mg of aluminum in the form of APA, and at least one antigen, wherein the antigen is present at a concentration of about 0.01 μg to about 100 μg per 0.5 mL of vaccine formulation prepared as a lyophilized formulation.

[0405] In some embodiments, compositions are provided comprising about 0.05 μg to about 200 mg of a compound of the invention, or a pharmaceutically acceptable salt thereof, and further comprising SPAN-85, PS-20, or PS-80, as highlighted in the various embodiments above. In some embodiments, compositions are provided comprising about 0.05 μg to about 200 mg of a compound of the invention, or a pharmaceutically acceptable salt thereof, and further comprising SPAN-85, PS-20, and squalene, as highlighted in the various embodiments above. In another embodiment, the compound is Compound A-1, A-2, or A-3. In another embodiment, the compound is Compound B-1, B-2, B-3, B-4, B-5, B-6, B-7, B-8, B-9, B-10, B-11, B-12, B-13, B-14, B-15, B-16, B-17, or B-18. In another embodiment, the compound is compound C-1, or C-2, or C-3, or C-4, or C-5. In another embodiment, the compound is compound D-1, or D-2, or D-3, or D-4, or D-5, or D-6.

[0406] In some embodiments, the compositions of the present invention are administered subcutaneously, topically, orally, mucosally, intravenously, or intramuscularly. The compositions are administered in an amount sufficient to induce a protective response. The compositions can also be administered by various other routes, such as orally, parenterally, subcutaneously, mucosally, or intramuscularly. The administered dose can vary depending on the patient's general condition, sex, weight, and age, as well as the administration route.

[0407] The compositions of the present invention as highlighted in the various embodiments above may be referred to as immunogenic compositions.

[0408] The compositions of the invention as highlighted in the various embodiments above may be referred to as vaccines or vaccine compositions.

[0409] In each of the above embodiments, the composition further comprises one or more antigens.

[0410] The present invention provides methods for treating or preventing disease by administering the above compositions.

[0411] The present invention provides the use of the above composition for treating or preventing a disease.

[0412] All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing methodology and materials that might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0413] Although particular embodiments of the present invention have been described with reference to the accompanying drawings, it should be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be employed by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0414] The following examples illustrate the present invention but do not limit it.

[0415] [Example 1] General Methods for Making Compounds of Formulas I, Ia, II, IIa, III, IIIa, IV, and IVa The compounds can be prepared from known or readily prepared starting materials according to methods known to those skilled in the art of organic synthesis. Representative methods useful for making the compounds are described in the examples below. Alternative synthetic routes and analogous structures will be apparent to those skilled in the art of organic synthesis.

[0416] Those skilled in the art of organic synthesis will recognize that the synthesis of the polycyclic and / or heterocyclic cores contained in the compounds of the present invention may require protection of certain functional groups (i.e., derivatization for chemical compatibility with certain reaction conditions). Suitable protecting groups for the various functional groups of these compounds and methods for their installation and removal are well known in the field of organic chemistry. A summary of many of these methods can be found in Greene et al., Protective Groups in Organic Synthesis, Wiley-Interscience, New York, (1999).

[0417] Those skilled in the art of organic synthesis will also recognize that one route for synthesis of the polycyclic heterocyclic core of the compounds of the invention may be more desirable depending on the selection of pendant substituents.

[0418] Additionally, one skilled in the art will recognize that in some cases the order of reactions may differ from that presented herein to avoid functional group incompatibilities and adjust the synthetic route accordingly.

[0419] The preparation of polycyclic intermediates useful for making the polycyclic and / or heterocyclic cores of the compounds of the present invention is described in the literature and in compendia such as "Comprehensive Heterocyclic Chemistry," editions I, II, and III, published by Elsevier and edited by A.R. Katrittzky & R.J.K. Taylor. The manipulation of the required substitution patterns is also described in the available chemical literature summarized in compendia such as "Comprehensive Organic Chemistry," published by Elsevier and edited by D.H. R. Barton and W.D. Ollis; "Comprehensive Organic Functional Group Transformations," published by A.R. Katrittzky & R.J.K. Taylor; and "Comprehensive Organic Transformations," published by Wiley-CVH and edited by R.C. Larock.

[0420] The starting materials used and the intermediates prepared using the methods described in the examples below can be isolated and purified if desired using conventional techniques, including, but not limited to, filtration, distillation, crystallization, chromatography, etc. Such materials can be characterized using conventional means, including physical constants and spectral data.

[0421] Those skilled in the art will be aware of standard formulation techniques as described in the published literature and in textbooks such as Zheng, "Formulation and Analytical Development for Low-Dose Oral Drug Products," Wiley, 2009, ISBN.

[0422] Preparation of Compounds and Intermediates The present invention is illustrated by the following examples. All examples can utilize standard work-up and purification methods known to those skilled in the art. Unless otherwise specified, all temperatures are in °C (Celsius). Unless otherwise noted, all reactions are performed at room temperature. The synthetic methodologies presented herein are intended to illustrate applicable chemistry through the use of specific examples and are not indicative of the scope of the present disclosure.

[0423] General method Solvents, reagents and intermediates that are commercially available were used as received. Intermediates that are not commercially available were prepared as described below. 1 H NMR spectra are reported as ppm downfield from Me4Si, with the number of protons, multiplicity, and coupling constants in Hertz in parentheses. Where LC / MS data are presented, the observed parent ion is shown. Flash column chromatography was performed using prepacked normal-phase silica or bulk silica.

[0424] [Example 2] Methods for preparing intermediates of the compounds of the present invention Intermediate 1-1 Preparation of Compound Int.1-1 [ka] Methyl (7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate [ka]

[0425] Step 1: Methyl (7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate To a stirred mixture of methyl 4-amino-1H-pyrazole-5-carboxylate (2 g, 14.17 mmol) and 1,3-bis(methoxycarbonyl)-2-methyl-2-thiopseudoeuracil (2.92 g, 14.17 mmol) in MeOH (20 mL) was added AcOH (4.87 mL, 85 mmol) at ambient temperature. The resulting mixture was stirred for 16 h, and then NaOMe (25.5 g, 142 mmol) in MeOH was added dropwise. After the addition was complete, the reaction mixture was acidified to pH 1-2 with AcOH. The resulting solid was collected by filtration, washed with CHCN (200 mL), and then dried in vacuo to give the title compound. MS m / z (M+H) + : Calculated value 210.1, measured value 210.1. 1 H-NMR (400MHz, DMSO-d6) δ7.83(s,1H), 3.67(s,3H).

[0426] Step 2: Methyl (7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate To a stirred mixture of methyl (7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (5 g, 23.90 mmol), butan-1-amine (4.73 mL, 47.8 mmol), and DBU (3.92 mL, 26.3 mmol) in DMSO (100 mL) was added BOP (12.69 g, 28.7 mmol) at ambient temperature. The resulting mixture was warmed to 60 °C for 3 h. The resulting mixture was cooled to ambient temperature, then filtered and directly purified by reverse-phase chromatography (0-50% CH3CN / water with 0.1% formic acid modifier) ​​to provide the title compound. MS m / z (M+H) + : Calculated value 265.1, measured value 265.1. 1 H-NMR(400 MHz,DMSO-d6)δ 12.94(br.,1H),9.89(s,1H),8.14-7.99(m,2H),3.67(s,3H),3.56-3.51(m,2H),1.66-1.58(m,2H),1.42-1.33(m,2H),0.92(t,J=7.2 Hz,3H).

[0427] Intermediate 2-1 Preparation of Compound Int.2-1 [ka] 5-Azido-N-butyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine [ka]

[0428] Step 1: N-butyl-5-chloro-1H-pyrazolo[4,3-d]pyrimidin-7-amine To a mixture of 5,7-dichloro-1H-pyrazolo[4,3-d]pyrimidine (3 g, 15.87 mmol) in THF (30 mL) was added DIEA (3.08 g, 23.81 mmol) and butan-1-amine (1.741 g, 23.81 mmol) at 0 °C. After the addition was complete, the mixture was warmed to ambient temperature and stirred for 2 h. The resulting mixture was diluted with water (200 mL) and then extracted with EtOAc (3 × 300 mL). The combined organic extracts were washed with brine (2 × 300 mL), then dried (Na SO ), and then filtered. The filtrate was concentrated to give the title compound, which was used directly in the next step without purification. MS m / z (M+H) + : Calculated value 226.1, measured value 226.2.

[0429] Step 2: 5-Azido-N-butyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine To a mixture of N-butyl-5-chloro-1H-pyrazolo[4,3-d]pyrimidin-7-amine (3.5 g, 15.51 mmol) in AcOH (6 mL) and EtOH (24 mL) at ambient temperature was added sodium azide (1.512 g, 23.26 mmol), and the mixture was then heated to 100 °C for 3 h. The resulting mixture was cooled to ambient temperature, diluted with water (200 mL), and extracted with EtOAc (3 × 300 mL). The combined organic extracts were washed with brine (2 × 300 mL), dried (Na SO ), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-20% EtOAc / petroleum ether) to give the title compound. MS m / z (M+H) + : Calculated value 233.1, measured value 233.1.

[0430] Utilizing the procedure described for intermediate 2-1 and substituting the appropriate reagent for butan-1-amine, the following compounds were prepared.

[0431] [Table 2]

[0432] Intermediate 2-3 Preparation of Compound Int.2-3 [ka] 5-Azido-N-butyl-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine [ka]

[0433] Step 1: 5-Azido-N-butyl-3-iodo-1H-pyrazolo[4,3-d]pyrimidin-7-amine To a solution of 5-azido-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine (1 g, 4.31 mmol) in DMF (30 mL) was added N-iodosuccinimide (1.937 g, 8.61 mmol), and the mixture was then heated to 40° C. After heating overnight, the mixture was cooled to room temperature and then diluted with 10% NaSO (40 mL). The resulting solid was collected by filtration, washed with water (3×10 mL), and then dried under vacuum to give the title compound. MS m / z (M+H) + :Calculated value 359.0, actual value 359.0.

[0434] Step 2: 5-Azido-N-butyl-3-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine To a solution of 5-azido-N-butyl-3-iodo-2H-pyrazolo[4,3-d]pyrimidin-7-amine (1 g, 2.79 mmol) in 1,4-dioxane (10 mL) and HO (1 mL) was added Pd(dppf)Cl (0.204 g, 0.279 mmol), KPO (1.185 g, 5.58 mmol), and 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (3.51 g, 13.96 mmol) under N, and the mixture was heated to 100 °C overnight. The resulting mixture was cooled to room temperature, diluted with EtOAc (30 mL), dried (NaSO), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (30-70% EtOAc / petroleum ether) to give the title compound. MS m / z (M+H) + : Calculated value 247.1, actual value 247.4.

[0435] Intermediate 3-1 Preparation of Compound Int.3-1 [ka] tert-Butyl 4-(4-(hydroxymethyl)-3,5-dimethoxyphenyl)piperazine-1-carboxylate [ka]

[0436] Step 1: tert-butyl 4-(4-formyl-3,5-dimethoxyphenyl)piperazine-1-carboxylate To a stirred mixture of 4-bromo-2,6-dimethoxybenzaldehyde (5 g, 20.40 mmol) in toluene (20 mL) under an argon atmosphere, tert-butyl piperazine-1-carboxylate (5.70 g, 30.6 mmol), RuPhos (1.904 g, 4.08 mmol), Pd(dba) (1.868 g, 2.040 mmol), and NaOtBu (5.88 g, 61.2 mmol) were added, and the mixture was then heated to 100 °C. After 6 h, the mixture was cooled to ambient temperature, diluted with water (50 mL), and extracted with EtOAc (3 × 80 mL). The combined organic extracts were washed with brine (3 × 50 mL), dried (NaSO), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-100% EtOAc / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 351.2, measured value 351.3.

[0437] Step 2: tert-butyl 4-(4-(hydroxymethyl)-3,5-dimethoxyphenyl)piperazine-1-carboxylate To a stirred mixture of tert-butyl 4-(4-formyl-3,5-dimethoxyphenyl)piperazine-1-carboxylate (3 g, 8.56 mmol) in EtOH (10 mL) was added NaBH (0.389 g, 10.27 mmol) at ambient temperature. After 15 min, the mixture was diluted with water (15 mL) and then extracted with EtO (3 x 50 mL). The combined organic extracts were washed with brine (3 x 50 mL), then dried (NaSO), and then filtered. The filtrate was then concentrated to give the title compound, which was used directly in the next step without purification. MS m / z (M+H) + : Calculated value 353.2, measured value 353.3.

[0438] Intermediate 4-1 Preparation of Compound Int.4-1 [ka] tert-Butyl (4-(chloromethyl)-3,5-dimethoxybenzyl)(methyl)carbamate [ka]

[0439] Step 1: tert-butyl (4-formyl-3,5-dimethoxybenzyl)(methyl)carbamate 4-Bromo-2,6-dimethoxybenzaldehyde (6 g, 24.48 mmol), N-(tert-butoxycarbonyl)-N-methylglycine (9.26 g, 49.0 mmol), Ir[dF(CF3)ppy]2(dtbbpy)PF6 (0.275 g, 0.245 mmol), and [Ni(dtbbpy)(HO)4]Cl2 (0.575 g, 1.224 mmol) were combined in DMSO (120 mL). To this was added BTMG (10 mL, 49.0 mmol). The mixture was sonicated until all solids dissolved. The resulting mixture was evenly divided into four screw-cap vials equipped with stir bars. N2 was bubbled through each mixture for 5 min. The vial was capped, and the mixture was then irradiated overnight in a PennOC Photoreactor® (wavelength: 420 nm; LED intensity: 100%; fan speed: 5000 rpm; stirring: 1200 rpm). The separated mixtures were combined, diluted with HO, and extracted with EtOAc (3x). The combined organic extracts were washed with HO and brine, then dried (MgSO), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-50% 3:1 EtOAc:EtOH / heptane) to provide the title compound. 1 H-NMR(400 MHz,CDCl3)δ 10.47(s,1H),6.44(bs,2H),4.41(s,2H),3.88(s,6H),2.87(bs,3H),1.49(bs,9H).

[0440] Step 2: tert-butyl (4-(hydroxymethyl)-3,5-dimethoxybenzyl)(methyl)carbamate A solution of tert-butyl (4-formyl-3,5-dimethoxybenzyl)(methyl)carbamate (6.98 g, 22.56 mmol) in MeOH (100 mL) was cooled to 0 °C. To this was added NaBH (1.03 g, 27.2 mmol) in portions. After 1 h, gas evolution ceased. The cooling bath was removed, and the mixture was allowed to warm to ambient temperature. After 90 min, the mixture was concentrated. The residue was taken up in DCM and filtered through a pad of Celite®, washing with DCM, and the filtrate was then concentrated. The crude product was subjected to silica gel chromatography (0-15% 3:1 EtOAc:EtOH / heptane) to give the title compound. 1 H-NMR(400 MHz,CDCl3)δ 6.45(bs,2H),4.76(d,J=6.6 Hz,2H),4.39(s,2H),3.83(s,6H),2.86(bs,3H),1.49(s,9H).

[0441] Step 3: tert-butyl (4-(chloromethyl)-3,5-dimethoxybenzyl)(methyl)carbamate A solution of N-chlorosuccinimide (0.472 g, 3.53 mmol) in DCM (1 mL) was cooled to 0 °C. To this, dimethyl sulfide (0.239 g, 3.85 mmol) was added slowly, resulting in a precipitate. The reaction mixture was cooled to -20 °C, and then a solution of tert-butyl (4-(hydroxymethyl)-3,5-dimethoxybenzyl)(methyl)carbamate (1 g, 3.21 mmol) in DCM was added dropwise. The mixture was stirred for 2 h, allowing the temperature to reach 0 °C, during which time all the solid precipitate dissolved to give a clear solution. The solution was poured into cold brine and then extracted with EtO (2x). The combined organic extracts were washed with cold brine, then dried (NaSO), then filtered, and the filtrate was concentrated to give the title compound, which was used directly in the next step without purification. MS m / z (M+H) + : Calculated value 330.1, measured value 329.2.

[0442] Intermediate 5-1 Preparation of Compound Int.5-1 [ka] tert-Butyl (4-oxobutyl)carbamate [ka]

[0443] To a mixture of tert-butyl (4-hydroxybutyl)carbamate (1 g, 5.28 mmol) in CHCN (30 mL) at ambient temperature was added IBX (1.231 g, 6.34 mmol), and the mixture was then heated to 80 °C. After 1 h, the mixture was cooled to ambient temperature, diluted with water (100 mL), and extracted with EtOAc (3 × 200 mL). The combined organic extracts were washed with brine (2 × 200 mL), dried (NaSO), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0–70% EtOAc / petroleum ether) to provide the title compound. 1 H-NMR (400 MHz, CDCl3): δ 3.76-3.73(m,2H),2.53-2.49(t,2H),2.03-1.97(m,2H),1.53-1.45(s,9H).

[0444] Intermediate 6-1 Preparation of Compound Int.6-1 [ka] (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl(4-nitrophenyl)carbonate [ka]

[0445] The mixture of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (300 mg, 0.567 mmol) and hypochlorous acid 4-nitrobenzoic anhydride (229 mg, 1.134 mmol) in DCM (4.5 mL) and pyridine (1.5 mL) was stirred at ambient temperature for 5 hours.The resulting mixture was concentrated and then purified by preparative TLC (petroleum ether: DCM = 1: 1) to obtain the title compound.

[0446] Utilizing the procedure described for intermediate 6-1 and substituting the appropriate reagents for (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol, the following compounds were prepared:

[0447] [ka]

[0448] [Table 3]

[0449] Intermediate 7-1 Preparation of Compound Int.7-1 [ka] 4-Stearamidobutanoic acid [ka]

[0450] A mixture of 4-aminobutanoic acid (1 g, 9.70 mmol) and Na2CO3 (1.542 g, 14.55 mmol) in THF (30 mL) and water (30 mL) was cooled to 0 °C. To this was added stearoyl chloride (2.94 g, 9.70 mmol). After the addition was complete, the mixture was stirred at 25 °C for 16 h. The resulting mixture was quenched with saturated KHSO4 and then extracted with CHCl3 (500 mL). The combined organic layers were washed with brine (3 x 100 mL), then dried (Na2SO4), then filtered, and the filtrate was concentrated to give the title compound. MS m / z (M−H) - : Calculated value 368.6, actual value 368.3.

[0451] Utilizing the procedure described for intermediate 7-1 and substituting the appropriate reagent for 4-aminobutanoic acid, the following compounds were prepared.

[0452] [ka]

[0453] [Table 4]

[0454] Intermediate 8-1 Preparation of Compound Int.8-1 [ka] N-(3-aminopropyl)stearamide [ka]

[0455] A mixture of propane-1,3-diamine (1 g, 13.49 mmol) and pyridine (1.091 mL, 13.49 mmol) in DMF (15 mL) was cooled to 0° C. To this was added stearoyl chloride (4.09 g, 13.49 mmol). After the addition was complete, the mixture was stirred at 25° C. for 16 hours. The resulting mixture was diluted with ice water to give a solid. The solid was collected by filtration, then washed with water (3×100 mL), and then dried to give the title compound. MS m / z (M+H) + : Calculated value 341.6, measured value 341.3.

[0456] Utilizing the procedure described in Intermediate 8-1 and substituting the appropriate reagents for propane-1,3-diamine, the following compounds were prepared:

[0457] [ka]

[0458] [Table 5]

[0459] Intermediate 9-1 Preparation of Compound Int.9-1 [ka] 2-((1s,3s)-3-(octadecylcarbamoyl)cyclobutyl)acetic acid [ka]

[0460] Step 1: tert-Butyl 2-((1s,3s)-3-(octadecylcarbamoyl)cyclobutyl)acetate To a stirred mixture of (1s,3s)-3-(2-(tert-butoxy)-2-oxoethyl)cyclobutane-1-carboxylic acid (0.5 g, 2.334 mmol), HATU (1.78 g, 4.67 mmol), and DIEA (1.22 mL, 7.00 mmol) in DMF (10 mL) was added a solution of octadecan-1-amine (0.818 g, 3.03 mmol) in DMF (10 mL) at 25° C. After 12 h, the mixture was diluted with water (10 mL) and then extracted with EtOAc (3×10 mL). The combined organic layers were washed with brine (3 x 10 mL), then dried (Na2SO4), then filtered, and the filtrate was concentrated to give the title compound as an 80:20 mixture with tert-butyl 2-((1s,3s)-3-(hexadecylcarbamoyl)cyclobutyl)acetate. MS m / z (M+H) + : Calculated value 466.4, actual value 466.2.

[0461] Step 2: 2-((1s,3s)-3-(octadecylcarbamoyl)cyclobutyl)acetic acid To a stirred mixture of tert-butyl 2-(3-(octadecylcarbamoyl)cyclobutyl)acetate (0.932 g, 2 mmol) in 1,4-dioxane (16 mL) at 25° C. was added 4 M HCl in 1,4-dioxane (4 mL). After 6 h, the mixture was diluted with EtOAc and then washed with brine (3×30 mL). The organic layer was dried (NaSO) and then filtered, and the filtrate was concentrated to give the title compound as an 80:20 mixture with 2-((1s,3s)-3-(hexadecylcarbamoyl)cyclobutyl)acetic acid. MS m / z (M+H) + : Calculated value 410.4, measured value 410.4.

[0462] Intermediate 10-1 Preparation of Compound Int.10-1 [ka] 2,2-Dimethyl-5-stearamidopentanoic acid [ka]

[0463] To a mixture of N-(5-hydroxy-4,4-dimethylpentyl)stearamide (Int. 8-3, 500 mg, 1.257 mmol) in acetone (10 mL) was added a solution of chromium trioxide in sulfuric acid (0.691 mL, 1.383 mmol) at 0° C. After 6 h, the mixture was diluted with water. The resulting solid was collected by filtration, washed with water, and then air-dried. MS m / z (M+H) + : Calculated value 412.4, measured value 412.1.

[0464] Intermediate 11-1 Preparation of Compound Int.11-1 [ka] 5-Methyl-5-stearamidohexanoic acid [ka]

[0465] Step 1: tert-Butyl methyl glutarate A solution of 5-(tert-butoxy)-5-oxopentanoic acid (5 g, 26.6 mmol) in DCM (40 mL) and MeOH (40 mL) was cooled to 0 °C. To this was added (diazomethyl)trimethylsilane (39.8 mL, 80 mmol, 2 M in hexanes). After the addition was complete, the mixture was allowed to warm to room temperature. After 3 h, the mixture was concentrated. The crude product was subjected to silica gel chromatography (0-5% EtOAc / petroleum ether) to give the title compound. 1 H NMR(400 MHz,CDCl3)δ 3.68(s,3H),2.37(t,J=7.2 Hz,2H),2.28(t,J=7.6 Hz,2H),1.91(quin,J=7.2 Hz,2H),1.44(s,9H).

[0466] Step 2: tert-Butyl 5-hydroxy-5-methylhexanoate To a solution of tert-butyl methyl glutarate (1 g, 4.94 mmol) in THF (16 mL) was added MeMgBr (4.12 mL, 12.36 mmol, 3 M in THF) at 0 °C. After 2 h, the mixture was quenched with saturated NH4Cl (25 mL), then warmed to room temperature, and then extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (30 mL), then dried (Na2SO4), then filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-20% EtOAc / petroleum ether) to give the title compound. 1 H NMR(400 MHz,CDCl3)δ 2.25(t,J=7.2 Hz,2H),1.69-1.64(m,2H),1.50-1.48(m,2H),1.45(s,9H),1.23(s,6H).

[0467] Step 3: 5-methyl-5-stearamidohexanoic acid To a solution of tert-butyl 5-hydroxy-5-methylhexanoate (300 mg, 1.483 mmol) in AcOH (2 mL) was added stearonitrile (787 mg, 2.97 mmol) at room temperature, and the mixture was then cooled to 0° C. H2SO4 (0.5 mL) was added. After the addition was complete, the cooling bath was removed and the mixture was allowed to warm to room temperature. After stirring overnight, the mixture was quenched with saturated NaHCO3 to pH=5. The resulting mixture was extracted with EtOAc (2 mL x 3). The combined organic layers were dried (Na2SO4) and then filtered, and the filtrate was concentrated. The residue was taken up in MeCN (5 mL). The resulting precipitated solid was collected and then recrystallized from EtOAc to give the title compound. MS m / z (M+H) + : Calculated value 412.7, measured value 412.3.

[0468] Intermediate 12-1 Preparation of Compound Int.12-1 [ka] 5-((tert-butoxycarbonyl)(octadecyl)amino)pentanoic acid [ka]

[0469] Step 1: Methyl 5-((tert-butoxycarbonyl)amino)pentanoate To a solution of 5-((tert-butoxycarbonyl)amino)pentanoic acid (2 g, 9.21 mmol) in MeOH (15 mL) and DCM (15 mL) was added 2 M (trimethylsilyl)diazomethane in hexanes (23.01 mL, 46.0 mmol) at room temperature. After 1 h, the mixture was concentrated to give the title compound, which was used without purification. 1 H NMR(400 MHz,CDCl3)δ 4.56(s,1H),3.68(s,3H),3.13(q,J=6.4 Hz,2H),2.34(t,J=7.2 Hz,2H),1.70-1.62(m,2H),1.49-1.56(m,2H),1.45(s,9H).

[0470] Step 2: Methyl 5-((tert-butoxycarbonyl)(octadecyl)amino)pentanoate To a solution of methyl 5-((tert-butoxycarbonyl)amino)pentanoate (1 g, 4.32 mmol) in DMF (20 mL) was added NaH (0.259 g, 6.49 mmol, 60% in mineral oil) at 0 °C. After the addition was complete, the mixture was warmed to room temperature. After 30 min, 1-bromooctadecane (4.32 g, 12.97 mmol) was added, and the mixture was then heated to 50 °C. After 2 h, the mixture was cooled to room temperature, diluted with water (100 mL), and extracted with EtOAc (3 ×). The combined organic layers were washed with brine (50 mL), dried (Na SO ), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-20% EtOAc / petroleum ether) to provide the title compound. 1 H NMR(400 MHz,CD3OD)δ 3.67-3.64(m,3H),3.23-3.15(m,4H),2.39-2.33(m,2H),1.57(d,J=4.4 Hz,6H),1.46(s,9H),1.29(s,30H),0.92-0.88(m,3H).

[0471] Step 3: 5-((tert-butoxycarbonyl)(octadecyl)amino)pentanoic acid To a stirred solution of methyl 5-((tert-butoxycarbonyl)(octadecyl)amino)pentanoate (240 mg, 0.496 mmol) in THF (6 mL) and HO (2 mL) was added LiOH monohydrate (62.5 mg, 1.488 mmol) at room temperature, and the mixture was then warmed to 40° C. After stirring overnight, the mixture was cooled to room temperature and the pH was adjusted to 7 with 1 N HCl. The resulting mixture was extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (3×5 mL), dried (NaSO), filtered, and the filtrate was concentrated to give the title compound, which was used in the next step without purification. 1 H NMR(400 MHz,CD3OD)δ 3.23-3.14(m,4H),2.35-2.28(m,2H),1.65-1.52(m,6H),1.46(s,9H),1.29(s,30H),0.92-0.88 ppm(m,3H).

[0472] Intermediate 13-1 Preparation of Compound Int.13-1 [ka] 5-(octadecyloxy)pentanoic acid [ka]

[0473] Step 1: 1-(hex-5-en-1-yloxy)octadecane To a solution of octadecan-1-ol (5 g, 18.48 mmol) in DMF (100 mL) was added NaH (3.70 g, 92 mmol) (60% in mineral oil) in portions at room temperature. After stirring for 30 minutes, 6-bromo-1-hexene (24.70 mL, 185 mmol) was added, and the mixture was then heated to 80 °C. After heating overnight, the mixture was cooled to room temperature, diluted with water (200 mL), and extracted with EtOAc (100 mL × 3). The combined organic layers were dried (Na2SO4), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-100% EtOAc / petroleum ether) to give the title compound. 1 H NMR(400 MHz,CDCl3)δ 5.82(ddt,J=17.2,10.4,6.8,6.8 Hz,1H),5.01(dq,J=17.6,1.2 Hz,1H),4.95(d,J=10.0 Hz,1H),3.45-3.36(m,4H),2.08(d,J=7.2 Hz,2H),1.65-1.55(m,5H),1.49-1.44(m,2H),1.39-1.1.17(m,29H),0.85-0.92(m,3H).

[0474] Step 2: 5-(Octadecyloxy)pentanoic acid To a solution of 1-(hex-5-en-1-yloxy)octadecane (1 g, 2.84 mmol) in MeCN (10 mL) was added a solution of RuCl (0.059 g, 0.284 mmol) and NaIO (2.426 g, 11.34 mmol) in HO (10 mL) at room temperature. After 2 hours, the mixture was diluted with NaSO (25 mL) and extracted with EtOAc (25 mL × 3). The combined organic layers were washed with 1N HCl (20 mL), saturated NaHCO (20 mL), and brine (20 mL), then dried (NaSO), filtered, and the filtrate was concentrated to give the title compound, which was used in the next step without purification. 1H NMR(400 MHz,CDCl3)δ ppm 3.39-3.44(m,4 H),2.40(t,J=7.27 Hz,2 H),1.70-1.75(m,2 H),1.65(br d,J=7.51 Hz,2 H),1.55-1.58(m,2 H),1.26(s,30 H),0.87-0.89(m,3 H).

[0475] Intermediate 14-1 Preparation of Compound Int.14-1 [ka] 2-(4-(1,4-diazepan-1-yl)-2-methoxybenzyl)-5-azido-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine [ka]

[0476] Step 1: tert-butyl 1-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperidine-4-carboxylate To a mixture of 5-azido-2-(4-bromo-2-methoxybenzyl)-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine (from compound B-1, Step 1, 500 mg, 1.159 mmol) and tert-butyl 1,4-diazepane-1-carboxylate (302 mg, 1.507 mmol) in toluene (10 mL) was added RuPhos (216 mg, 0.464 mmol), Pd(dba) (212 mg, 0.232 mmol), and CsCO (1133 mg, 3.48 mmol) under N, and the mixture was then heated to 110 °C. After heating overnight, the mixture was cooled to room temperature and then diluted with water (100 mL), and the resulting mixture was then extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (3 x 300 mL), then dried (NaSO), then filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-52% 3:1 EtOAc / petroleum ether) to give the title compound. MS m / z (M+H) + : Calculated value 551.7, measured value 551.1.

[0477] Step 2: 2-(4-(1,4-diazepan-1-yl)-2-methoxybenzyl)-5-azido-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine A solution of tert-butyl 4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-1,4-diazepane-1-carboxylate (350 mg, 0.636 mmol) in DCE (2.00 mL) was cooled to 0° C. To this was added 4 M HCl in 1,4-dioxane (2 mL) at 0° C. After 1 h, the mixture was warmed to room temperature and then concentrated to give the title compound as the HCl salt, which was used without purification. MS m / z (M+H) + : Calculated value 451.3, measured value 451.3.

[0478] Utilizing the procedure described for intermediate 14-1 and substituting the appropriate reagent for tert-butyl 1,4-diazepane-1-carboxylate, the following compounds were prepared.

[0479] [ka]

[0480] [Table 6]

[0481] Intermediate 15-1 Preparation of Compound Int.15-1 [ka] 5-Azido-N-butyl-2-(2-methoxy-4-(piperidin-4-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine [ka]

[0482] Step 1: tert-butyl 4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-3,6-dihydropyridine-1(2H)-carboxylate To a mixture of 5-azido-2-(4-bromo-2-methoxybenzyl)-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine (derived from compound B-1, Step 1, 400 mg, 0.927 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (430 mg, 1.391 mmol) in 1,4-dioxane (4 mL) / HO (0.4 mL) was added CsCO (604 mg, 1.855 mmol) and X-PhosPdG (39.3 mg, 0.046 mmol) under N, and the mixture was then heated to 80 °C. After 2 h, the mixture was cooled to room temperature and then diluted with EtOAc (200 mL). The resulting mixture was washed with brine (3 x 100 mL), then dried (Na2SO4), then filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-50% EtOAc / petroleum ether) to give the title compound. MS m / z (M+H) + : Calculated value 534.3, measured value 534.4.

[0483] Step 2: tert-butyl 4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperidine-1-carboxylate To a solution of tert-butyl 4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-3,6-dihydropyridine-1(2H)-carboxylate (290 mg, 0.543 mmol) in MeOH (10 mL) was added Pd / C (150 mg, 1.410 mmol) under N. The resulting mixture was placed under and atmosphere of H (3 x vacuum / H) and stirred at room temperature. After 16 h, the mixture was degassed (3 x vacuum / N), then filtered, washing with MeOH, and the filtrate was concentrated to give the title compound. MS m / z (M+H) + : Calculated value 536.3, measured value 536.3.

[0484] Step 3: 5-Azido-N-butyl-2-(2-methoxy-4-(piperidin-4-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine To a solution of tert-butyl 4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperidine-1-carboxylate (250 mg, 0.467 mmol) in DCM (2 mL) was added 4 M HCl in 1,4-dioxane (2 mL) at room temperature. After 1 h, the mixture was concentrated to give the title compound as the HCl salt, which was used without purification. MS m / z (M+H) + : Calculated value 436.3, measured value 436.6.

[0485] Intermediate 16-1 Preparation of Compound Int.16-1 [ka] 2-(4-(azetidin-3-yl)-2-methoxybenzyl)-5-azido-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine [ka]

[0486] Step 1: tert-butyl 3-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)azetidine-1-carboxylate To a solution of NiI (36.2 mg, 0.116 mmol) in DMA (1 mL) was added picolinimidamide hydrochloride (36.5 mg, 0.232 mmol), and the mixture was then heated to 50 °C. After 30 min, zinc powder (152 mg, 2.319 mmol) was added, followed by a solution of 5-azido-2-(4-bromo-2-methoxybenzyl)-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine (derived from compound B-1, step 1, 500 mg, 1.159 mmol) and tert-butyl 3-bromoazetidine-1-carboxylate (411 mg, 1.739 mmol) in DMA (10 mL). After stirring overnight at room temperature, the mixture was directly purified by reverse-phase chromatography (CHCN / water with 0.1% TFA modifier) ​​to give the title compound. MS m / z (M+H) + : Calculated value 508.3, measured value 508.4.

[0487] Step 2: 2-(4-(azetidin-3-yl)-2-methoxybenzyl)-5-azido-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine To a solution of tert-butyl 3-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)azetidine-1-carboxylate (60 mg, 0.118 mmol) in DCM (0.5 mL) was added 4 M HCl in 1,4-dioxane (0.5 mL) at room temperature. After 1 h, the mixture was concentrated to give the title compound as the HCl salt, which was used without purification. MS m / z (M+H) + : Calculated value 408.2, measured value 408.3.

[0488] Intermediate 17-1 Preparation of Compound Int.17-1 [ka] (S)-5-Azido-N-(1-((tert-butyldiphenylsilyl)oxy)pentan-2-yl)-2-(2,6-dimethoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine [ka]

[0489] Step 1: (4-bromo-2,6-dimethoxyphenyl)methanol To a solution of 4-bromo-2,6-dimethoxybenzaldehyde (5 g, 20.40 mmol) in EtOH (60 mL) was added NaBH (0.926 g, 24.48 mmol) at room temperature. After 1 h, the mixture was diluted with water (50 mL) and then extracted with EtOAc (30 mL × 2). The combined organic layers were washed with brine (50 mL), then dried (NaSO), then filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (15-50% EtOAc / petroleum ether) to give the title compound. 1H NMR(400 MHz,CDCl3)δ 6.72(s,2H),4.72(d,J=6.8 Hz,2H),3.84(s,6H),2.32(t,J=6.8 Hz,1H).

[0490] Step 2: 5-Bromo-2-(chloromethyl)-1,3-dimethoxybenzene To a solution of (4-bromo-2,6-dimethoxyphenyl)methanol (1 g, 4.05 mmol) in DCM (10 mL) was added SOCl (0.591 mL, 8.09 mmol) at 0 °C. After the addition was complete, the mixture was warmed to room temperature and stirred overnight. The resulting mixture was concentrated to give the title compound, which was used in the next step without purification. 1 H NMR(400 MHz,CDCl3)δ 6.72(s,2H),4.70(s,2H),3.87(s,6H).

[0491] Step 3: (S)-2-((5-azido-2-(4-bromo-2,6-dimethoxybenzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)pentan-1-ol To a solution of (S)-2-((5-azido-2H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)pentan-1-ol (Int. 2-2, 800 mg, 3.05 mmol) in DMF (15 mL) was added 5-bromo-2-(chloromethyl)-1,3-dimethoxybenzene (800 mg, 3.01 mmol) and K2CO3 (1.25 g, 9.04 mmol) at room temperature. After stirring overnight, the mixture was diluted with water (10 mL). The resulting solid was collected by filtration and then dried under vacuum to give the title compound. MS m / z (M+H) + : Calculated value 491.1, measured value 491.2. 1 H NMR(400 MHz,CD3OD)δ 8.53(s,1H),6.93(s,2H),5.64(d,J=1.6 Hz,2H),4.50-4.63(m,1H),3.88(s,6H),3.68-3.76(m,2H),1.61-1.86(m,2H),1.38-1.54(m,2H),0.97(t,J=7.2 Hz,3H).

[0492] Step 4: (S)-5-Azido-2-(4-bromo-2,6-dimethoxybenzyl)-N-(1-((tert-butyldiphenylsilyl)oxy)pentan-2-yl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine To a solution of (S)-2-((5-azido-2-(4-bromo-2,6-dimethoxybenzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-yl)amino)pentan-1-ol (900 mg, 1.832 mmol) in DMF (15 mL) was added imidazole (374 mg, 5.50 mmol) and TBDPSCl (0.565 mL, 2.198 mmol) at room temperature. After stirring overnight, the mixture was diluted with water (15 mL) and then extracted with EtOAc (15 mL x 2). The combined organic layers were washed with brine (20 mL), then dried (Na2SO4), then filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-10% MeOH / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 731.2, measured value 731.2.

[0493] Step 5: Benzyl (S)-4-(4-((5-azido-7-((1-((tert-butyldiphenylsilyl)oxy)pentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazine-1-carboxylate To a solution of (S)-5-azido-2-(4-bromo-2,6-dimethoxybenzyl)-N-(1-((tert-butyldiphenylsilyl)oxy)pentan-2-yl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine (500 mg, 0.685 mmol) in toluene (10 mL) was added benzylpiperazine-1-carboxylate (226 mg, 1.028 mmol), RuPhos Pd G4 (58.3 mg, 0.069 mmol), and Cs2CO3 (670 mg, 2.055 mmol). The mixture was purged with N2 and then heated to 90 °C. After heating overnight, the mixture was cooled to room temperature, diluted with water (10 mL), and extracted with DCM (15 mL × 2). The combined organic layers were dried (Na2SO4) and then filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-5% MeOH / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 869.4, measured value 869.4.

[0494] Step 6: (S)-5-Azido-N-(1-((tert-butyldiphenylsilyl)oxy)pentan-2-yl)-2-(2,6-dimethoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine To a solution of benzyl (S)-4-(4-((5-amino-7-((1-((tert-butyldiphenylsilyl)oxy)pentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazine-1-carboxylate (370 mg, 0.439 mmol) in TFE (8 mL) was added Pd / C (47 mg). The mixture was degassed (3×vacuum / N2) and then placed under an atmosphere of H2 (balloon). After 2 h, the mixture was filtered and the filtrate was concentrated to give the title compound, which was used in the next step without purification. MS m / z (M+H) + : Calculated value 735.4, measured value 735.4.

[0495] [Example 3] Preparation of Compound A-1 [ka] TIFF2026504779000105.tif179170N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide (Compound A-1)

[0496] Step 1: tert-butyl 4-(4-((7-(butylamino)-5-((methoxycarbonyl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazine-1-carboxylate To a stirred mixture of tert-butyl 4-(4-(hydroxymethyl)-3,5-dimethoxyphenyl)piperazine-1-carboxylate (Int. 3-1, 13.87 g, 39.4 mmol) in benzene (80 mL) under an argon atmosphere was added methyl (7-(butylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl)carbamate (Int. 1-1, 8 g, 30.3 mmol) and cyanomethylenetributylphosphorane (10.96 g, 45.4 mmol), and the mixture was then heated to 80° C. After 3 hours, the mixture was cooled to ambient temperature, diluted with water (10 mL), and extracted with EtOAc (3×20 mL). The combined organic extracts were washed with brine (3×10 mL), dried (NaSO), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-100% EtOAc / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 599.3, actual value 599.3.

[0497] Step 2: tert-butyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazine-1-carboxylate To a stirred mixture of tert-butyl 4-(4-((7-(butylamino)-5-((methoxycarbonyl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazine-1-carboxylate (2.6 g, 4.34 mmol) in 1,4-dioxane (5 mL) was added NaOH (10 M, 20 mL, 200 mmol) and the mixture was heated to 60° C. After 12 h, the mixture was cooled to ambient temperature, diluted with water (20 mL), and extracted with EtOAc (3×80 mL). The combined organic extracts were washed with brine (50 mL), dried (NaSO), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-10% MeOH / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 541.3, measured value 541.4.

[0498] Process 3:N 7 -butyl-2-(2,6-dimethoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine To a stirred mixture of tert-butyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazine-1-carboxylate (1.8 g, 3.33 mmol) in DCM (25 mL) was added a solution of phenol (9.40 g, 100 mmol) in DCM (5 mL) at ambient temperature. The mixture was cooled to 5° C., and then a solution of chlorotrimethylsilane (0.24 ml, 1.878 mmol) in DCM (3 mL) was added dropwise. After the addition was complete, the mixture was allowed to warm to ambient temperature. After 25 minutes, the mixture was poured into ice-cold 2N NaOH (30 mL) and then extracted with ethyl acetate (5×150 mL). The combined organic extracts were dried (NaSO) and then filtered, and the filtrate was concentrated. The crude product was subjected to reverse phase chromatography (0-60% MeCN / water with 0.5% NH4OH modifier) ​​to give the title compound. MS m / z (M+H) + : Calculated value 441.3, actual value 441.2.

[0499] Step 4: tert-butyl (5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate To a stirred mixture of 5-((tert-butoxycarbonyl)amino)pentanoic acid (225 mg, 1.035 mmol) in DMF (2 mL) was added a solution of HATU (738 mg, 1.941 mmol) in DMF at ambient temperature. After 30 min, N 7 To the resulting mixture was added 2H-butyl-2-(2,6-dimethoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (570 mg, 1.294 mmol) and DIEA (0.452 mL, 2.59 mmol). After 5 h, the mixture was cooled to ambient temperature, diluted with water (30 mL), and extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with brine (3 x 25 mL), dried (NaSO), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (1-10% MeOH / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 640.4, actual value 640.5.

[0500] Step 5: 5-amino-1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)pentan-1-one A solution of tert-butyl (5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate (550 mg, 0.860 mmol) in 10% TFA / DCM (2 mL) was stirred at ambient temperature. After 30 min, the mixture was diluted with toluene (30 mL) and then concentrated. The crude product was subjected to reverse phase chromatography (0-100% MeCN / water with 1% NH4OH modifier) ​​to give the title compound. MS m / z (M+H) + : Calculated value 540.3, actual value 540.5.

[0501] Step 6: N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide To a stirred mixture of stearic acid (274 mg, 0.964 mmol) in DMF (7 mL) was added a solution of HATU (423 mg, 1.112 mmol) in DMF at ambient temperature. After 30 minutes, 5-amino-1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)pentan-1-one (400 mg, 0.741 mmol) and DIEA (0.259 mL, 1.482 mmol) were added. After 2 hours, the mixture was cooled to ambient temperature, diluted with water (20 mL), and extracted with EtOAc (3 × 50 mL). The combined organic extracts were washed with brine (3 × 20 mL), dried (NaSO), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (1-10% MeOH / DCM) to give the title compound. Compound A-1: ​​MS m / z (M+H) + : Calculated value 806.6, measured value 806.6. 1H-NMR(400 MHz, CDCl3)δ 12.41(s,1H),7.41(s,1H),6.71-6.68(m,1H),6.11(s,2H),5.78-5.76(m,1H),5. 38(s,2H),3.85-3.82(m,9H),3.65-3.60(m,5H),3.28-3.25(m,7H),2.41(t,J=7.2 Hz,2H),2.16(t,J=7.2Hz,2H),1.70-1.56(m,18H),1.51-1.49(m,4H),1.25(s,9H),1.00-0.90(m,4H),0.89-0.80(m,6H).

[0502] Preparation of Compound A-2 [ka] (S)—N-(5-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide (Compound A-2)

[0503] Step 1: (S)—N-(5-(4-(4-((5-azido-7-((1-((tert-butyldiphenylsilyl)oxy)pentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide To a solution of 5-stearamidopentanoic acid (Int. 7-4, 129 mg, 0.337 mmol) in DMF (10 mL), DIEA (109 mg, 0.842 mmol) and PyBOP (175 mg, 0.337 mmol) were added at room temperature. After 10 min, (S)-5-azido-N-(1-((tert-butyldiphenylsilyl)oxy)pentan-2-yl)-2-(2,6-dimethoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine (Int. 17-1, 300 mg, 0.281 mmol) was added at room temperature. After 30 min, the mixture was diluted with water (10 mL) and then extracted with EtOAc (15 mL × 2). The combined organic layers were dried (NaSO) and then filtered, and the filtrate was concentrated. The crude product was purified by preparative TLC (DCM / MeOH=10:1) to give the title compound and (S)-1-(4-(4-((5-azido-7-((1-((tert-butyldiphenylsilyl)oxy)pentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)octadecan-1-one (Int.18-1). Title compound: MS m / z (M+H) + Calculated value 1100.7, Measured value 1100.7. Int.18-1: MS m / z (M+H) + : Calculated value 1001.6, measured value 1001.7.

[0504] Step 2: (S)—N-(5-(4-(4-((5-azido-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide To a solution of (S)—N-(5-(4-(4-((5-azido-7-((1-((tert-butyldiphenylsilyl)oxy)pentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide (150 mg, 0.075 mmol) in THF (2 mL) was added TBAF (0.112 mL, 0.112 mmol, 1 M in THF) at room temperature. After 1 h, the mixture was diluted with water (10 mL) and then extracted with EtOAc (15 mL×2). The combined organic layers were dried (NaSO) and then filtered, and the filtrate was concentrated to give the title compound, which was used in the next step without purification. MS m / z (M+H) + : Calculated value 862.6, measured value 862.5.

[0505] Step 3: (S)—N-(5-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide To a solution of (S)—N-(5-(4-(4-((5-azido-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide (55 mg, 0.064 mmol) in THF (1.5 mL) and HO (0.3 mL) was added BuP (0.079 mL, 0.319 mmol), and the mixture was then heated to 50° C. After 1 hour, the mixture was cooled to room temperature and then concentrated. The crude product was purified by reverse-phase chromatography (CHCN / water with 0.2% FA modifier) ​​to give the title compound. Compound A-2: MS m / z (M+H) + : Calculated value 836.6, actual value 836.5. 1H NMR(400 MHz,CD3OD)δ 7.57(s,1H),6.27(s,2H),5.42(s,2H),4.49-4.40(m,1H),3.84(s,6H),3.72(dd,J=5.2,10.0 Hz,4H),3.68-3.61(m,2H),3.27-3.18(m,4H),2.48(t,J=7.2 Hz,2H),2.17(t,J=7.2 Hz,2H),1.73-1.53(m,10H),1.50-1.36(m,4H),1.30-1.24(m,27H),0.97(t,J=7.2 Hz,4H),0.89(t,J=6.8 Hz,3H).

[0506] Utilizing the procedure described for compound A-2, substituting the appropriate reagent for 5-stearamidopentanoic acid in step 1, the following compounds were prepared:

[0507] [ka]

[0508] [Table 7]

[0509] [Example 4] Preparation of Compound B-1 [ka] TIFF2026504779000110.tif134169N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide (Compound B-1)

[0510] Step 1: 5-Azido-2-(4-bromo-2-methoxybenzyl)-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine 5-Azido-N-butyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine (Int. 2-1, 938 mg, 4.04 mmol), 4-bromo-1-(bromomethyl)-2-methoxybenzene (1.36 g, 4.86 mmol), and K2CO3 (1.2 g, 8.68 mmol) were combined in DMF (20 mL) at ambient temperature. After stirring overnight, the mixture was diluted with HO and then extracted with EtOAc (3x). The combined organic extracts were washed with brine, then dried (Na2SO4), then filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-50% 3:1 EtOAc:EtOH / heptane) to give the title compound. MS m / z (M+H) + : Calculated value 431.3, actual value 431.0.

[0511] Step 2: tert-butyl 4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate 5-Azido-2-(4-bromo-2-methoxybenzyl)-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine (1.38 g, 3.20 mmol), tert-butyl piperazine-1-carboxylate (0.775 g, 4.16 mmol), and CsCO (3.13 g, 9.60 mmol) were combined in 1,4-dioxane (20 mL). The mixture was degassed (3× pump / N). RuPhos Pd G (0.25 g, 0.322 mmol) was added, and the mixture was heated to 100° C. After stirring at 100° C. overnight, the mixture was cooled to ambient temperature, diluted with EtOAc, and filtered through a pad of Celite®, washing with EtOAc. The filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-50% 3:1 EtOAc:EtOH / heptane) to give the title compound. MS m / z (M+H) + : Calculated value 537.3, measured value 537.1.

[0512] Step 3: 5-Azido-N-butyl-2-(2-methoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine To a solution of tert-butyl 4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate (799 mg, 1.489 mmol) in DCM (8 mL) was added TFA (1.5 mL, 19.47 mmol) at ambient temperature. After 2 h, the mixture was concentrated. The residue was taken up in 1:1 DCM:heptane and then concentrated (twice) and dried under vacuum to give the TFA salt of the title compound, which was used directly in the next step. MS m / z (M+H) + : Calculated value 437.2, measured value 437.1.

[0513] Step 4: tert-butyl (5-(4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate 5-((tert-Butoxycarbonyl)amino)pentanoic acid (421 mg, 1.936 mmol) and HATU (736 mg, 1.936 mmol) were combined in DCM (5 mL), and then DIEA (1.300 ml, 7.45 mmol) was added at ambient temperature. After 1 h, a solution of crude 5-azido-N-butyl-2-(2-methoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine (TFA salt, 650 mg, 1.489 mmol) in DCM (10 mL) was added. After stirring overnight at ambient temperature, the mixture was concentrated. The crude product was subjected to silica gel chromatography (0-100% 9:1 DCM:MeOH / DCM) to give an impure product which was further purified by silica gel chromatography (0-100% 3:1 EtOAc:EtOH / heptane) to give the title compound. MS m / z (M+H) + : Calculated value 636.4, measured value 636.2.

[0514] Step 5: 5-amino-1-(4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)pentan-1-one tert-Butyl (5-(4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate (848 mg, 1.334 mmol) was taken up in DCM (10 mL) and then TFA (1.1 mL, 14.28 mmol) was added at ambient temperature. After 90 min, the mixture was concentrated to give the TFA salt of the title compound, which was used directly in the next step. MS m / z (M+H) + : Calculated value 536.3, measured value 536.3.

[0515] Step 6: N-(5-(4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide Stearic acid (493 mg, 1.734 mmol) and HATU (659 mg, 1.734 mmol) were combined in DCM (5 mL), and then DIEA (1.2 mL, 6.87 mmol) was added at ambient temperature. After 30 minutes, the mixture was transferred to a flask containing crude 5-amino-1-(4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)pentan-1-one (TFA salt, 715 mg, 1.334 mmol) and DCM (10 mL). After stirring overnight at ambient temperature, the mixture was concentrated. The crude product was subjected to silica gel chromatography (0-100% 9:1 DCM:MeOH / DCM) to give an impure product which was further purified by silica gel chromatography (0-100% 3:1 EtOAc:EtOH / heptane) to give the title compound. MS m / z (M+H) + : Calculated value 802.6, measured value 802.9.

[0516] Step 7: N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide N-(5-(4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide (641 mg, 0.799 mmol) was taken up in THF (8 mL):HO (0.800 mL) to give a thick suspension. The mixture was heated to 50° C., and the suspension went into solution upon heating. To this was added 1 M trimethylphosphine in THF (2.4 mL, 2.400 mmol) and heating was continued at 50° C. After 3 hours, the mixture was cooled to ambient temperature and then quenched with MeOH and concentrated. The crude product was subjected to silica gel chromatography (0-15% MeOH:DCM) to give the title compound. Compound B-1: MS m / z (M+H) + : Calculated value 776.6, measured value 776.5. 1 H-NMR(400 MHz,DMSO-d6)δ 11.5(br.,1H),8.11(s,1H),7.72-7.65(m,2H),7.06-6.80(m,1H),6.60(s,1H),6.49-6.47(m,1H),6.32(s,1H),5.32(s,2H),3.81(s,3H) ),3.57-3.42(m,6H),3.32-3.02(m,4H),2.36-2.32(m,2H),2.07-1.99(m,4H),1.47-1.34(m,8H),1.29-1.18(m,30H),0.91-0.83(m,6H). Utilizing the procedure described for compound B-1, substituting the appropriate reagent for stearic acid, the following compounds were prepared:

[0517] [ka]

[0518] [Table 8]

[0519] Preparation of Compound B-5 [ka] N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide [ka]

[0520] Step 1: N-(5-(4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide To a mixture of 2-(4-(1,4-diazepan-1-yl)-2-methoxybenzyl)-5-azido-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine hydrochloride (Int. 14-1, 300 mg, 0.666 mmol) and 5-stearamidopentanoic acid (Int. 7-4, 383 mg, 0.999 mmol) in DMF (5 mL) was added HATU (506 mg, 1.332 mmol) and DIEA (0.349 mL, 1.998 mmol) at room temperature. After 2 hours, the mixture was diluted with water (100 mL) and extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (3 × 100 mL), dried (NaSO), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-6% MeOH / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 816.6, measured value 816.7.

[0521] Step 2: N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide To a solution of N-(5-(4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide (290 mg, 0.355 mmol) in THF (3 mL):HO (0.60 mL) was added PMe (1.066 mL, 1.066 mmol), and the mixture was heated to 50 °C. After heating overnight, the mixture was cooled to room temperature and quenched with MeOH (5 mL), and the resulting mixture was concentrated. The crude product was subjected to silica gel chromatography (0-6% MeOH / DCM) to give the title compound. Compound B-5: MS m / z (M+H) + : Calculated value 790.6, observed value 790.6. 1 H-NMR(400 MHz,DMSO-d6)δ 8.16(s,1H),7.72(d,J=7.2 Hz,1H),7.60-7.56(m,1H),6.93(d,J=8.0 Hz,1H),6.31-6.27(m,2H),6.19(s,2H),5.26(s,2H),3.79(s,3H),3.66-3.39(m,8H),3.29-3.27(m,2H),2.99-2.95(m,2H),2.26(t,J=7.2 Hz,1H),2.16(t,J=7.2 Hz,1H),2.03-1.98(m,2H),1.87-1.76(m,2H),1.59-1.55(m,2H),1.47-1.22(m,36H),0.90(t,J=7.2 Hz,3H),0.85(t,J=7.2 Hz,3H).

[0522] Utilizing the procedure described for compound B-5, substituting the appropriate reagents for 2-(4-(1,4-diazepan-1-yl)-2-methoxybenzyl)-5-azido-N-butyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine hydrochloride and 5-stearamidopentanoic acid in step 1, the following compounds were prepared:

[0523] [ka]

[0524] [Table 9]

[0525] Preparation of Compounds B-9 and B-10 [ka] (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-octadecylcyclobutane-1-carboxamide (B-9) [ka] and (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-hexadecylcyclobutane-1-carboxamide (B-10) [ka]

[0526] Step 1: (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-octadecylcyclobutane-1-carboxamide and (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-hexadecylcyclobutane-1-carboxamide To a stirred mixture of 2-(3-(octadecylcarbamoyl)cyclobutyl)acetic acid (Int. 9-1 as an 80:20 mixture with 2-((1s,3s)-3-(hexadecylcarbamoyl)cyclobutyl)acetic acid, 0.3 g, 0.732 mmol), DMAP (8.95 mg, 0.073 mmol), DIEA (0.192 mL, 1.098 mmol), EDC (0.211 g, 1.098 mmol), and HOBt (0.148 g, 1.098 mmol) in DMF (10 mL) was added N 7A solution of 2-butyl-2-(2-methoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (0.391 g, 0.952 mmol) in DMF (10 mL) was added at 25 °C. After 6 h, the mixture was diluted with HO and then extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (3 × 10 mL), then dried (NaSO), then filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0–20% MeOH / DCM) to afford a purified mixture of the title compounds. Further purification by chiral chromatography (CHIRALPAK OD, 30% EtOH in hexanes containing 0.1% TFA modifier) ​​afforded P1 and P2.

[0527] P1: (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-hexadecylcyclobutane-1-carboxamide Compound B-10: MS m / z (M+H) + : Calculated value 774.6, measured value 774.6. 1 H-NMR(400 MHz,DMSO-d6)δ 8.37(br.,1H),7.67(s,1H),7.58(t,J=5.6 Hz,1H),6.96(d,J=8.4 Hz,1H),6.60(d,J=2.4 Hz,1H),6.50-6.43(m,1H),5.32(s,2H),3.80(s,3H),3.55-3.43(m,5H),3.16-3.11(m,2H),2.99-2.95(m,2H),2.85-2. 73(m,1H),2.46-2.43(m,4H),2.14-2.13(m,2H),1.76-1.75(m,2H),1.58-1.55(m,2H),1.23(s,32H),0.95-0.85(m,6H). P2: (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-octadecylcyclobutane-1-carboxamide Compound B-9: MS m / z (M+H) + : Calculated value 802.6, observed value 802.6. 1 H-NMR(400 MHz,DMSO-d6)δ 8.11(br.,1H),7.67(s,1H),7.58(t,J=5.6 Hz,1H),6.96(d,J=8.4 Hz,1H),6.60(d,J=2.4 Hz,1H),6.50-6.43(m,1H),6.18(br.,1H),5.32(s,2H),3.80(s,3H),3.55(t,J=5.2 Hz,4H),3.43(d,J=6.8 Hz,1H),3.16-3.11(m,4H),2.99-2.95(m,2H),2.85-2.73(m,1H),2.46-2.43(m,4H),2. 14-2.13(m,2H),1.76-1.75(m,2H),1.58-1.55(m,2H),1.23(s,34H),0.93-0.80(m,6H).

[0528] Utilizing the procedure described for compounds B-9 and B-10, substituting the appropriate reagent for 2-(3-(octadecylcarbamoyl)cyclobutyl)acetic acid, the following compounds were prepared:

[0529] [ka]

[0530] [Table 10] TIFF2026504779000122.tif143154

[0531] Utilizing the procedure described for compound B-1, substituting the appropriate reagent for 5-azido-N-butyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine in step 1, the following compounds were prepared: [ka]

[0532] [Table 11]

[0533] [Example 5] Preparation of Compound C-1 [ka]

[0534] (9Z,12Z)-N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide (compound C-1)

[0535] Step 1: tert-butyl (4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)carbamate To a mixture of tert-butyl (4-(chloromethyl)-3,5-dimethoxybenzyl)(methyl)carbamate (Int. 2-1, 1 g, 3.03 mmol) in DMF (20 mL) was added 5-azido-N-butyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine (Int. 4-1, 0.634 g, 2.73 mmol) and K2CO3 (0.503 g, 3.64 mmol) at ambient temperature. After 2 h, the mixture was diluted with HO (20 mL) and extracted with EtOAc (3 × 30 mL). The combined organic extracts were washed with brine (2 × 30 mL), dried (Na2SO4), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-70% EtOAc / petroleum ether) to give the title compound. MS m / z (M+H) + : Calculated value 526.3, measured value 526.3.

[0536] Step 2: 5-Azido-N-butyl-2-(2,6-dimethoxy-4-((methylamino)methyl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine To a solution of tert-butyl (4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)carbamate (1.2 g, 2.283 mmol) in DCM (10 mL) was added TFA (2 mL) at ambient temperature. After 3 h, the mixture was concentrated to give the TFA salt of the title compound, which was used directly in the next step. MS m / z (M+H) + : Calculated value 426.2, measured value 426.3.

[0537] Step 3: tert-butyl (4-((4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate To a mixture of crude 5-azido-N-butyl-2-(2,6-dimethoxy-4-((methylamino)methyl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine (TFA salt, 1.1 g, 2.163 mmol) and tert-butyl (4-oxobutyl)carbamate (Int. 5-1, 0.608 g, 3.24 mmol) in DCM (8 mL) was added NaBH(OAc)3 (0.917 g, 4.33 mmol) at ambient temperature. After 3 h, the mixture was concentrated. The crude product was subjected to silica gel chromatography (0-20% MeOH / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 597.4, actual value 597.5.

[0538] Process 4:N 1 -(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)-N 1 -Methylbutane-1,4-diamine To a solution of tert-butyl (4-((4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate (790 mg, 1.324 mmol) in DCM (10 mL) was added TFA (2 mL) at room temperature. After 3 h, the mixture was concentrated to give the TFA salt of the title compound, which was used directly in the next step. MS m / z (M+H) + : Calculated value 497.3, measured value 497.4.

[0539] Step 5: (9Z,12Z)-N-(4-((4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide coarse N 1 -(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)-N 1 To a mixture of 1,4-methylbutane-1,4-diamine (TFA salt, 447 mg, 0.9 mmol) in DCM (6 mL) was added DIEA (0.314 mL, 1.800 mmol) and (9Z,12Z)-octadeca-9,12-dienoyl chloride (296 mg, 0.990 mmol) at 0 °C. After the addition was complete, the mixture was warmed to ambient temperature. After 2 h, EtOAc (30 mL) was added, and the mixture was then washed with saturated NaHCO (2 × 30 mL). The organic layer was dried (NaSO) and filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-15% MeOH / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 759.5, actual value 759.6.

[0540] Step 6: (9Z,12Z)-N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide To a mixture of (9Z,12Z)-N-(4-((4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide (430 mg, 0.566 mmol) in THF (5 mL) and water (0.5 mL) was added trimethylphosphane (1 M in THF, 1.699 mL, 1.699 mmol), and the mixture was heated to 50° C. After 16 h, the mixture was cooled to ambient temperature and diluted with EtOAc (30 mL). The resulting mixture was washed with water (30 mL), dried (NaSO), filtered, and the filtrate was concentrated. The crude product was purified by preparative TLC developed with MeOH / DCM / ammonia (1 / 10 / 0.05) to give the title compound. Compound C-1: MS m / z (M+H) + : Calculated value 733.5, measured value 733.5. 1H-NMR(400 MHz,CD3OD):δ 7.49(s,1H),6.70(s,2H),5.46(s,2H),5.33-5.28(m,4H),3.85(s,6H),3.52-3.48(m,4H),3.16(t,J=6.4 Hz,2H),2.74(t,J=6.2 Hz,2H),2.41(t,J=7.1 Hz,2H),2.22(s,3H),2.14(t,J=7.5 Hz,2H),2.04-2.01(m 4H),1.66-1.41(m,10H),1.36-1.29(m,14H),0.97(t,J=7.4 Hz,3H),0.88(t,J=6.8 Hz,3H).

[0541] Utilizing the procedure described for compound C-1, substituting the appropriate reagent for (9Z,12Z)-octadeca-9,12-dienoyl chloride, the following compounds were prepared:

[0542] [ka]

[0543] [Table 12]

[0544] Preparation of compound C-5 [ka] N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)-4-oxobutyl)stearamide [ka]

[0545] Step 1: N-(4-((4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)-4-oxobutyl)stearamide A mixture of 4-stearamidobutanoic acid (Int. 7-1, 132 mg, 0.357 mmol), 5-azido-N-butyl-2-(2,6-dimethoxy-4-((methylamino)methyl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine hydrochloride (Compound C-1 Step 2, 110 mg, 0.238 mmol), HATU (181 mg, 0.476 mmol), and DIEA (0.208 mL, 1.191 mmol) in DMF (3 mL) was stirred at room temperature. After stirring overnight, the mixture was directly purified by reverse-phase chromatography (CHCN / water with 0.05% FA modifier) ​​to give the title compound. MS m / z (M+H) + : Calculated value 777.5, measured value 777.6.

[0546] Step 2: N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)-4-oxobutyl)stearamide To a solution of N-(4-((4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)-4-oxobutyl)stearamide (160 mg, 0.206 mmol) in THF (3 mL):HO (0.60 mL) was added PMe (1 M in THF, 0.618 mL, 0.618 mmol), and the mixture was heated to 50° C. After 5 h, the mixture was cooled to room temperature and quenched with MeOH (5 mL), and the resulting mixture was concentrated. The crude product was purified by reverse-phase chromatography (1:3 CHCN:MeOH / water) to give the title compound. Compound C-5: MS m / z (M+H) + : Calculated value 751.6, measured value 751.6. 1H-NMR(400 MHz,DMSO-d6)δ 7.99-7.54(m,2H),7.37(d,J=3.6 Hz,1H),6.54(d,J=19.6 Hz,2H),5.92(s,2H),5.36(s,2H),4.54(d,J=16.8 Hz,2H),3.79(d,J=8.4 Hz,6H),3.44-3.39(m,2H),3.10-3.00(m,2H),2.93(s,2H),2.84(s,1H),2.38-2.33(m,2H) ,2.03-1.98(m,2H),1.70-1.65(m,2H),1.60-1.55(m,2H),1.34-1.30(m,2H),1.22(d,J=3.2 Hz,30H),0.90-0.82(m,6H).

[0547] [Example 6] Preparation of Compound D-1 [ka] (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate (Compound D-1)

[0548] Step 1: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl (4-((4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)-amino)butyl)carbamate (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(4-nitrophenyl)carbonate (Int. 6-1, 355 mg, 0.511 mmol) and crude N 1 -(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)-N 1To a mixture of 1,4-methylbutane-1,4-diamine (TFA salt, 274 mg, 0.460 mmol) in THF (5 mL) was added K2CO3 (0.058 mL, 1.023 mmol), and the mixture was then heated to 65 °C. After 15 h, the mixture was cooled to ambient temperature and then concentrated. The crude product was subjected to silica gel chromatography (0-10% MeOH / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 1051.8, actual value 1052.0.

[0549] Step 2: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl (4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)-amino)butyl)carbamate To a mixture of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl (4-((4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)-amino)butyl)carbamate (500 mg, 0.475 mmol) in THF (10 mL) and HO (1.0 mL), trimethylphosphane (1 M in THF, 1.426 mL, 1.426 mmol) was added at ambient temperature. After 15 hours, the mixture was quenched with MeOH (1.42 mL) and then concentrated. The crude product was purified by preparative TLC (DCM:MeOH=10:1) to obtain the title compound. Compound D-1:MS m / z(M+H) + : Calculated value 1025.8, measured value 1025.7. 1 H-NMR(300 MHz,CD3OD):δ 7.65(s,1H),6.71(s,2H),5.49(s,2H),5.35-5.32(m,8H),4.86-4.85 (m,1H),3.86(s,6H),3.56-3.55(m,4H),3.12-3.10(m,2H),2.77-2.7 5(m,4H),2.45-2.43(m,2H),2.25(s,3H),2.05-2.03(m,8H),1.68-1. 66(m,6H),1.46-1.44(m,6H),1.32-1.27(m,36H),0.97-0.94(m,9H).

[0550] Preparation of Compound D-2 [ka] 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperazine-1-carboxamide [ka]

[0551] Step 1: 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperazine-1-carboxamide A mixture of N-(3-aminopropyl)stearamide (Int. 8-1, 457 mg, 1.342 mmol), CDI (136 mg, 0.839 mmol), DMAP (13.67 mg, 0.112 mmol), and DIEA (0.586 mL, 3.36 mmol) in DCM (10 mL) was stirred at 0° C. After 30 min, N 7 To the resulting mixture was added 2H-butyl-2-(2-methoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine hydrochloride (500 mg, 1.119 mmol). The mixture was warmed to room temperature and then heated to 50° C. After 3 hours, the mixture was cooled to room temperature and then concentrated. The crude product was purified by reverse phase chromatography (CHCN / water with 0.1% TFA modifier) ​​to give the title compound. Compound D-2: MS m / z (M+H) + : Calculated value 777.6, measured value 777.6. 1 H-NMR(300 MHz,DMSO-d6)δ 8.89(s,1H),7.83-7.55(m,2H),7.16-6.98(m,2H),6.60-6.48(m,2H),5.34(s ,2H),3.80(s,3H),3.47(s,6H),3.12(s,4H),3.04-3.01(m,4H),2.03(t,J=7.2 Hz,2H),1.60-1.48(m,6H),1.22(s,28H),0.96-0.77(m,6H).

[0552] Preparation of Compound D-3 [ka] 3-Stearamidopropyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate [ka]

[0553] Step 1: 3-Stearamidopropyl 4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate A mixture of 4-nitrophenyl(3-stearamidopropyl)carbonate (Int. 6-2, 130 mg, 0.257 mmol), 5-azido-N-butyl-2-(2-methoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine (Compound B-1, Step 3, 56 mg, 0.128 mmol), and DIEA (0.045 mL, 0.257 mmol) in DCM (0.5 mL) was stirred at room temperature. After 1 hour, the mixture was directly purified by preparative TLC (DCM / MeOH=15:1) to give the title compound. MS m / z (M+H) + : Calculated value 804.6, measured value 804.7.

[0554] Step 2: 3-Stearamidopropyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate To a solution of 4-(4-((5-azido-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate (80 mg, 0.099 mmol) in THF (2 mL):HO (0.5 mL) was added PMe (1 M in THF, 0.298 mL, 0.298 mmol), and the mixture was heated to 50° C. After 5 hours, the mixture was cooled to room temperature and quenched with MeOH (5 mL), and the resulting mixture was concentrated. The crude product was purified by preparative TLC (DCM:MeOH=10:1) to give the title compound. Compound D-3: MS m / z (M+H) + : Calculated value 778.6, measured value 778.6. 1H-NMR(400 MHz,DMSO-d6)δ 7.80(t,J=5.6 Hz,1H),7.64(s,1H),7.55(s,1H),6.91(d,J=8.0 Hz,1H),6.60(d,J=2.0 Hz,1H),6.49-6.45(m,1H),5.56(s,2H),5.29(s,2H),4.01(t,J=6.4 Hz,2H),3.80(s,3H),3.49-3.38(m,6H),3.14-3.11(m,6H),2.03(t,J=7.2 Hz,2H),1.71-1.68(m,2H),1.58-1.53(m,2H),1.47-1.26(m,4H),1.28-1.15(m,28H),0.91-0.83(m,6H).

[0555] Preparation of compound D-4 [ka] N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-6,6,6-trifluorohexyl)stearamide [ka]

[0556] Step 1: 6-chloro-1,1,1-trifluorohexan-2-ol To a solution of 5-chloropentanal (4.5 g, 37.3 mmol) and trimethyl(trifluoromethyl)silane (6.37 g, 44.8 mmol) in THF (80 mL) was added tetrabutylammonium fluoride (0.485 mL, 0.485 mmol, 1 M in THF) at 0 °C. After 1 h, the cooling bath was removed and the mixture was allowed to warm to room temperature. After 2 h, the mixture was quenched with 1 N HCl (60 mL). After stirring for 2 h, the mixture was diluted with water (60 mL) and then extracted with EtOAc (35 mL × 2). The combined organic layers were dried (Na SO ) and filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-5% EtOAc / petroleum ether) to give the title compound.1 H NMR(400 MHz,CDCl3)δ 3.99-3.83(m,1H),3.56(t,J=6.4 Hz,2H),2.85-2.66(m,1H),1.90-1.79(m,2H),1.78-1.68(m,2H),1.67-1.54(m,2H).

[0557] Step 2: 6-chloro-1,1,1-trifluorohexan-2-yl trifluoromethanesulfonate To a solution of 6-chloro-1,1,1-trifluorohexan-2-ol (1.6 g, 8.39 mmol) and pyridine (1.013 mL, 12.59 mmol) in DCM (25 mL) was added trifluoromethanesulfonic anhydride (1.702 mL, 10.07 mmol) dropwise at 0 °C. After 1 h, the cooling bath was removed and the mixture was warmed to room temperature. After 2 h, the mixture was diluted with water (10 mL) and then extracted with EtOAc (15 mL × 2). The combined organic layers were washed with 1 N HCl (20 mL), then washed with saturated NaHCO , then dried (Na SO ), then filtered, and the filtrate was concentrated to give the title compound, which was used in the next step without purification. 1 H NMR(400 MHz,CDCl3)δ 5.11-4.96(m,1H),3.58(t,J=6.4 Hz,2H),2.06-1.96(m,2H),1.94-1.82(m,2H),1.79-1.62(m,2H).

[0558] Process 3:N 7 -butyl-2-(4-(4-(6-chloro-1,1,1-trifluorohexan-2-yl)piperazin-1-yl)-2-methoxybenzyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine N 7To a solution of 2-butyl-2-(2-methoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (1.4 g, 3.41 mmol) in 1,4-dioxane (16 mL) was added DIEA (1.787 mL, 10.23 mmol) and 6-chloro-1,1,1-trifluorohexan-2-yl trifluoromethanesulfonate (1.650 g, 5.12 mmol), and the mixture was then heated to 80° C. After heating overnight, the mixture was cooled to room temperature and then concentrated. The crude product was purified by reverse-phase chromatography (CHCN / water with 10 mM NHHCO modifier) ​​to give the title compound. MS m / z (M+H) + : Calculated value 583.3, measured value 583.4. 1 H NMR(400 MHz,CD3OD)δ 7.52(s,1H),7.08(d,J=8.4 Hz,1H),6.57(d,J=2.0 Hz,1H),6.50(dd,J=2.0,8.4 Hz,1H),5.32(s,2H),3.81(s,3H),3.58(t,J=6.4 Hz,2H),3.52-3.45(m,2H),3.23-3.03(m,7H),2.82-2.75(m,2H),1.84-1.70(m,4H),1.66-1.55(m,4H),1.47-1.38(m,2H),0.96(t,J=7.2 Hz,3H).

[0559] Step 4: 2-(4-(4-(6-azido-1,1,1-trifluorohexan-2-yl)piperazin-1-yl)-2-methoxybenzyl)-N 7 -butyl-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine N 7To a solution of -butyl-2-(4-(4-(6-chloro-1,1,1-trifluorohexan-2-yl)piperazin-1-yl)-2-methoxybenzyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (160 mg, 0.274 mmol) in DMF (2 mL) was added sodium azide (80 mg, 1.231 mmol), and the mixture was heated to 100° C. After 2 h, the mixture was cooled to room temperature and then diluted with water (5 mL). The pH was adjusted to >9 with saturated NaHCO (15 mL), and the mixture was extracted with EtOAc (15 mL × 2). The combined organic layers were dried (NaSO), then filtered, and the filtrate was concentrated. The crude product was purified by preparative TLC (DCM / MeOH=10:1) to give the title compound. MS m / z (M+H) + : Calculated value 590.3, measured value 590.3.

[0560] Step 5: 2-(4-(4-(6-amino-1,1,1-trifluorohexan-2-yl)piperazin-1-yl)-2-methoxybenzyl)-N 7 -butyl-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine 2-(4-(4-(6-azido-1,1,1-trifluorohexan-2-yl)piperazin-1-yl)-2-methoxybenzyl)-N 7 To a solution of 2H-butyl-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (100 mg, 0.170 mmol) in MeOH (2 mL) was added Pd / C (18.05 mg). The mixture was purged with H2 (15 psi). After 2 h, the mixture was concentrated. The crude product was purified by preparative TLC (DCM / MeOH = 10:1) to give the title compound. MS m / z (M+H) + : Calculated value 564.3, measured value 564.3.

[0561] Step 6: N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-6,6,6-trifluorohexyl)stearamide 2-(4-(4-(6-amino-1,1,1-trifluorohexan-2-yl)piperazin-1-yl)-2-methoxybenzyl)-N 7To a solution of 2H-butyl-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (60 mg, 0.106 mmol) in DCM (1 mL) was added DIEA (0.056 mL, 0.319 mmol) and stearoyl chloride (32.2 mg, 0.106 mmol) at room temperature. After 2 hours, the mixture was concentrated. The crude product was purified by reverse-phase chromatography (CHCN / water with 0.2% FA modifier) ​​to obtain the title compound. Compound D-4: MS m / z (M+H) + : Calculated value 830.6, actual value 830.5. 1 H NMR(400 MHz,CD3OD)δ 7.63(s,1H),7.14(d,J=8.4 Hz,1H),6.58(d,J=2.0 Hz,1H),6.52(dd,J=2.0,8.4 Hz,1H),5.37(s,2H),3.83(s,3H),3.58(t,J=7.2 Hz,2H),3.25-3.12(m,7H),3.10-3.03(m,2H),2.84-2.75(m,2H),2.19-2.1 1(m,2H),1.78-1.51(m,9H),1.40(s,3H),1.32-1.20(m,28H),0.98(t,J=7.2 Hz,3H), 0.89(t,J=6.8 Hz,3H).

[0562] Preparation of compound D-5 [ka] N-(4-((4-((7-(butylamino)-5-hydroxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide [ka]

[0563] Step 1: tert-butyl (4-((7-(butylamino)-5-chloro-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)carbamate To a solution of N-butyl-5-chloro-2H-pyrazolo[4,3-d]pyrimidin-7-amine (Int. 2-1, Step 1, 800 mg, 3.54 mmol) in DMF (15 mL) was added tert-butyl (4-(chloromethyl)-3,5-dimethoxybenzyl)(methyl)carbamate (Int. 4-1, 1169 mg, 3.54 mmol) and K2CO3 (1470 mg, 10.63 mmol) at room temperature. After stirring overnight, the mixture was diluted with water (80 mL) and extracted with EtOAc (15 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried (Na2SO4), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-40% EtOAc / petroleum ether) to give the title compound. MS m / z (M+H) + : Calculated value 519.2, measured value 519.3. 1 H NMR(400 MHz,CD3OD)δ 7.88(s,1H),6.59(s,2H),5.57(s,2H),4.44(s,2H),3.85(s,6H),3.55(t,J=7.2 Hz,2H),2.86(s,3H),1.66(quin,J=7.2 Hz,2H),1.48(br s,9H),1.45-1.39(m,2H),0.98(t,J=7.6 Hz,3H).

[0564] Step 2: N-butyl-5-chloro-2-(2,6-dimethoxy-4-((methylamino)methyl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine To a solution of tert-butyl (4-((7-(butylamino)-5-chloro-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)carbamate (1 g, 1.927 mmol) in DCM (15 mL) was added TFA (3 mL) at room temperature. After 3 h, the mixture was concentrated to give the TFA salt of the title compound, which was used without purification. MS m / z (M+H) + : Calculated value 419.2, measured value 419.3. 1H NMR(400 MHz,CD3OD)δ 7.99(s,1H),6.85(s,2H),5.65(s,2H),4.19(s,2H),3.91(s,6H),3.63(t,J=7.2 Hz,2H),2.74(s,3H),1.68(quin,J=7.2 Hz,2H),1.44(qd,J=7.6,15.2 Hz,2H),0.98(t,J=7.6 Hz,3H).

[0565] Step 3: tert-butyl (4-((4-((7-(butylamino)-5-chloro-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate To a solution of N-butyl-5-chloro-2-(2,6-dimethoxy-4-((methylamino)methyl)benzyl)-2H-pyrazolo[4,3-d]pyrimidin-7-amine (300 mg, 0.716 mmol) in DMF (10 mL) was added DIEA (0.375 mL, 2.148 mmol) and tert-butyl(4-bromobutyl)carbamate (217 mg, 0.859 mmol), and the mixture was heated to 40° C. After heating overnight, the mixture was cooled to room temperature, diluted with water (50 mL), and extracted with EtOAc (10 mL×3). The combined organic layers were washed with brine (30 mL), dried (NaSO), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-5% MeOH / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 590.3, measured value 590.3.

[0566] Process 4:N 1 -(4-((7-(butylamino)-5-chloro-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)-N1-methylbutane-1,4-diamine To a solution of tert-butyl (4-((4-((7-(butylamino)-5-chloro-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate (250 mg, 0.424 mmol) in DCM (5 mL) was added TFA (1 mL) at room temperature. After stirring overnight, the mixture was concentrated to give the TFA salt of the title compound, which was used without purification. MS m / z (M+H) + : Calculated value 490.3, measured value 490.3.

[0567] Step 5: N-(4-((4-((7-(butylamino)-5-chloro-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide N 1 -(4-((7-(butylamino)-5-chloro-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)-N 1 To a solution of 200 mg (0.408 mmol) of 1,4-methylbutane-1,4-diamine in 5 mL of DCM was added DIEA (0.214 mL, 1.224 mmol) followed by stearoyl chloride (124 mg, 0.408 mmol) at 0° C. After the addition was complete, the mixture was allowed to warm to room temperature. After 2 h, the mixture was diluted with water (15 mL) and extracted with DCM (3 mL×3). The combined organic layers were washed with brine (15 mL), dried (NaSO), filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-5% MeOH / DCM) to give the title compound. MS m / z (M+H) + : Calculated value 756.5, actual value 756.4.

[0568] Step 6: N-(4-((4-((7-(butylamino)-5-hydroxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide To a solution of N-(4-((4-((7-(butylamino)-5-chloro-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide (90 mg, 0.119 mmol) in DMSO (7.5 mL) was added KCO (49.3 mg, 0.357 mmol) and acetohydroxamic acid (17.86 mg, 0.238 mmol), and the mixture was then heated to 140° C. by microwave irradiation for 2 hours. Acetohydroxamic acid (17.86 mg, 0.238 mmol) was added, and the mixture was then heated to 140° C. by microwave irradiation for 2 hours. The mixture was diluted with water (20 mL) and then extracted with DCM (3 mL×3). The combined organic layers were washed with brine (15 mL), then dried (NaSO), then filtered, and the filtrate was concentrated. The crude product was purified by reverse phase chromatography (CHCN / water with 0.2% FA modifier) ​​to give the title compound. Compound D-5: MS m / z (M+H) +: Calculated value 738.6, measured value 738.4. 1 H NMR(400 MHz,CD3OD)δ 7.41(s,1H),6.76(s,2H),5.46(s,2H),3.88(s,6H),3.77(br s,2H),3.53(t,J=7.2 Hz,2H),3.18(t,J=6.8 Hz,2H),2.71-2.61(m,2H),2.41(s,3H),2.16(t,J=7.2 Hz,2H),1.68-1.61(m,4H),1.60-1.50(m,4H),1.43(dd,J=7.2,15.2 Hz,2H),1.27(br s,28H),0.97(t,J=7.2 Hz,3H),0.92-0.86(m,3H).

[0569] Preparation of compound D-6 [ka] (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate [ka]

[0570] Step 1: tert-butyl (5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate To a solution of 5-((tert-butoxycarbonyl)amino)pentanoic acid (0.699 g, 3.22 mmol) in DMF (20 ml) was added TEA (0.678 g, 6.70 mmol) and HATU (1.528 g, 4.02 mmol) at room temperature. After 20 min, N 7-butyl-2-(2-methoxy-4-(piperazin-1-yl)benzyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7-diamine (1.1 g, 2.68 mmol) was added. After stirring overnight, the mixture was filtered and then directly purified by reverse phase chromatography (CHCN / water with 10 mM NHHCO modifier) ​​to give the title compound. MS m / z (M+H) + : Calculated value 610.4, measured value 610.5. 1 H NMR(400 MHz,CD3OD)δ 7.53(s,1H),7.11(d,J=8.4 Hz,1H),6.60(d,J=2.0 Hz,1H),6.53(dd,J=2.0,8.4 Hz,1H),5.34(s,2H),3.83(s,3H),3.75-3.65(m,4H),3.50(t,J=7.2 Hz,2H),3.25-3.21(m,2H),3.20-3.16(m,2H),3.06(t,J=6.8 Hz,2H),2.45(t,J=7.6 Hz,2H),1.68-1.59(m,4H),1.57-1.49(m,2H),1.48-1.43(m,2H),1.41(s,9H),0.97(t,J=7.2 Hz,3H).

[0571] Step 2: 5-amino-1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)pentan-1-one To a solution of tert-butyl (5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate (800 mg, 1.312 mmol) in DCM (10 ml) was added TFA (2 ml) at room temperature. After 1 h, the mixture was concentrated to give the TFA salt of the title compound, which was used without purification. MS m / z (M+H) + : Calculated value 510.3, measured value 510.3.

[0572] Step 3: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate To a solution of 5-amino-1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)pentan-1-one (660 mg, 1.295 mmol) in DMF (8 mL) and THF (8 mL), DIEA (4.52 mL, 25.9 mmol) and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(4-nitrophenyl)carbonate (Int. 6-1, 1168 mg, 1.683 mmol) were added at room temperature. After 30 minutes, the mixture was diluted with water (20 mL) and then extracted with EtOAc (20 mL × 2). The combined organic layers were dried (Na2SO4) and then filtered, and the filtrate was concentrated. The crude product was subjected to silica gel chromatography (0-10% MeOH / DCM) to obtain the impure title compound. The compound was further purified by reverse phase chromatography (CH3CN / water with 0.1% TFA modifier) ​​to obtain the title compound as a TFA salt. The salt was taken up in saturated NaHCO3 (20 mL) and then extracted with EtOAc (20 mL x 2). The combined organic layers were dried (Na2SO4) and then filtered, and the filtrate was concentrated to obtain the title compound. Compound D-6: MS m / z (M+H) + : Calculated value 1064.8, measured value 1064.8. 1H NMR(400 MHz,CD3OD)δ 7.61(s,1H),7.14(d,J=8.4 Hz,1H),6.60(d,J=1.6 Hz,1H),6.53(dd,J=2.0,8.4 Hz,1H),5.39-5.26(m,10H),4.67(td,J=6.0,12.0 Hz,1H),3.84(s,3H),3.74-3.66(m,4H),3.55(t,J=7.2 Hz,2H),3.25-3.21(m,2H),3.20-3.16(m,2H),3.13(br t,J=6.4 Hz,2H),2.76(t,J=6.0 Hz,4H),2.46(br t,J=7.2 Hz,2H),2.09-2.00(m,8H),1.70-1.61(m,4H),1.56-1.47(m,5H),1.45-1.39(m,2H),1.35-1.25(m,36H),0.97(t,J=7.2 Hz,3H),0.90(t,J=6.8 Hz,6H).

[0573] [Example 7] Preparation of pneumococcal polysaccharide-carrier protein conjugates Polysaccharide(s) (highlighted below and in the tables and examples) were dissolved, sized to a target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 (carrier protein) were individually lyophilized and redissolved in DMSO. The redissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before final 0.2 micron filtration. Several process parameters within each step, such as pH, temperature, concentration, and time, were controlled to obtain conjugates with desired attributes.

[0574] Polysaccharide size reduction Purified pneumococcal capsular polysaccharide (Ps) powder was dissolved in water. Except for ST-19A (serotype ST), the dissolved polysaccharide was sized and unreduced. The dissolved polysaccharide was then filtered to 0.45 microns and homogenized or acid hydrolyzed to reduce the molecular weight of Ps. The target Ps size for homogenization was achieved by controlling the pressure and number of passes. The target Ps size for acid hydrolysis was achieved by controlling the temperature and time. The polysaccharide was then filtered to 0.2 microns, concentrated, and diafiltered against water using a 5 or 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0575] De-O-acetylation (ST-15B only) The size-reduced ST-15B Ps solution was heated to 60°C and sodium bicarbonate pH 9.4 buffer was added to a final concentration of 50 mM. The batch was incubated at 60°C to release the O-acetyl groups. Potassium phosphate pH 6 buffer was added to neutralize the pH, and the solution was cooled to ambient temperature. The solution was then concentrated and diafiltered against water using a 5 or 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0576] Deketalization (ST-4 only) The sized and reduced ST-4Ps solution was adjusted to 50°C and pH 4.1 with sodium acetate buffer to partially deketalize the polysaccharide, and the polysaccharide solution was then cooled to 22°C before activation.

[0577] polysaccharide oxidation Polysaccharide solutions were adjusted to 22°C for all serotypes except ST-5, 7F, and 19F, which were adjusted to 4°C. The pH of the solutions was adjusted to 4-5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Polysaccharide activation was initiated by the addition of sodium metaperiodate solution. The amount of sodium metaperiodate added was controlled to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeating unit).

[0578] The activated product of all serotypes except ST-5 was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water using a 5 or 10 kDa NMWCO tangential flow ultrafiltration membrane. For ST-5, the activated product was diafiltered against 10 mM sodium acetate, pH 4.1, followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration for all serotypes was performed at 2–8°C.

[0579] Polysaccharide conjugation to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered to 0.2 microns. The activated polysaccharide was formulated for lyophilization using water and sucrose. CRM197 was formulated for lyophilization at 6 mg Pr / mL (the CRM197 protein is also known as "Pr") with a 1% w / v sucrose concentration. The formulated Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were separately redissolved in equal volumes of DMSO. For some serotypes, additives such as salt were added to the Ps-DMSO. The polysaccharide and CRM197 solutions were blended to achieve the target polysaccharide concentration and polysaccharide-to-CRM197 mass ratio. The mass ratio was chosen to control the polysaccharide to CRM197 ratio in the resulting conjugate. For most serotypes, a reducing agent such as sodium cyanoborohydride was added and conjugation was allowed to proceed at 22°C.

[0580] Final Rebate A reducing agent such as sodium borohydride was added after the conjugation reaction and incubated at 22°C for all serotypes. Batches were diluted to 150 mM sodium chloride containing approximately 0.025% (w / v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. Some lots were concentrated and diafiltered against 150 mM sodium chloride, 25 mM potassium phosphate pH 7 at approximately 4°C using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

[0581] Final Filtration and Product Storage Each batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride (pH 7.0) containing 0.015% (w / v) PS-20 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane. Specifically, for ST-5, midway through the diafiltration step, the ST-5 conjugate was collected and incubated with 50 mM sodium bicarbonate (pH 9.3) for 3 hours. Before completing the diafiltration, the ST-5 solution was neutralized with 1.5 M potassium phosphate (pH 6.0).

[0582] Each retentate batch was 0.2 micron filtered (using a 0.5 micron prefilter) and then diluted with an additional 10 mM histidine in 150 mM sodium chloride (pH 7.0) containing 0.015% (w / v) PS-20, aliquoted, and frozen at ≦−60° C. Details of the serotype-specific conjugates can be found previously (WO 2011 / 100151, WO 2019 / 139692, and WO 2020 / 131763).

[0583] [Example 8] Formulation of pneumococcal conjugate compositions Individual pneumococcal polysaccharide carrier protein conjugates prepared using different chemistries as described in Example 7 were used in the formulation of monovalent or 24-valent pneumococcal conjugate compositions, designated PCV1 or PCV24, respectively.

[0584] The PCV1 formulations added to Compound A-1-SNE, Compound B-1-SNE, or Compound D-1-SNE contained serotype 6B conjugated using reductive amination as described in the examples above and were formulated in 20 mM L-histidine pH 5.8, 150 mM NaCl, and 0.1% (w / v) PS-20 to a final concentration of 0.4 μg / mL (w / v) pneumococcal polysaccharides (PnPs) in the vaccine. A PCV1 vaccine formulation prepared using APA conjugated using reductive amination in aprotic (DMSO) solvent and serotype 6B as described in Example 7 was formulated in 20 mM L-histidine pH 5.8, 150 mM NaCl, and 0.2% (w / v) PS-20 and 250 μg (Al) / mL in the form of APA, resulting in a final concentration of 0.4 μg / mL (w / v) pneumococcal polysaccharides (PnPs) in the vaccine.

[0585] The PCV24 formulations added to compound A-1-SNE for IRM studies contained serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-Ac15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B, all conjugated to CRM197 using reductive amination and formulated in 20 mM L-histidine pH 5.8, 150 mM NaCl, and 0.1% PS-20. Each polysaccharide carrier protein conjugate was formulated at 0.8 μg / mL (w / v) PnPs, resulting in a final vaccine concentration of 19.2 μg / mL pneumococcal polysaccharide (PnPs).

[0586] PCV24 formulations with and without APA for IRM studies contained serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-Ac15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B, all conjugated to CRM197 using reductive amination as described in the previous examples and formulated in the form of APA at 20 mM L-histidine pH 5.8, 150 mM NaCl, 0.2% PS-20, and 250 μg (AI) / mL (where required). Each polysaccharide carrier protein conjugate was formulated at 4.0 μg / mL (w / v) PnPs, resulting in a final vaccine concentration of 96 μg / mL pneumococcal polysaccharide (PnPs).

[0587] PCV20 containing the following serotypes (1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F), all conjugated to CRM197, were purchased from Myonex.

[0588] The PCV26 formulations added to compound B-1-SNE for mouse studies contained serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-Ac15B, 16F, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B, all conjugated to CRM197 using reductive amination and formulated in 20 mM L-histidine pH 5.8, 150 mM NaCl, and 0.1% PS-20. Each polysaccharide carrier protein conjugate was formulated at 0.8 μg / mL (w / v) PnPs, resulting in a final vaccine concentration of 19.2 μg / mL pneumococcal polysaccharide (PnPs).

[0589] To prepare the PCV formulations, the required volume of monovalent bulk conjugate needed to obtain the indicated final concentration (w / v) of pneumococcal polysaccharides (also called PnPs) was calculated based on the batch volume or mass and bulk polysaccharide concentration.

[0590] The formulation process consisted of conjugate bulk blend preparation at twice the final concentration of the PnPs blend in 20 mM histidine, 0.05–0.15% (w / v) PS-20, and 150 mM sodium chloride, pH 5.8.

[0591] Histidine pH 5.8, PS-20, and sodium chloride solutions were prepared and added to the formulation container. Individual frozen pneumococcal polysaccharide carrier protein conjugates were thawed at 2-8°C and then added to the formulation container. During the addition of the polysaccharide carrier protein conjugates to the formulation buffer (conjugate blend), the container was mixed to ensure uniformity using a magnetic server or magnetic impeller. After all additions were made and the solution was stirred, the conjugate blend was passed through a sterile filter and collected in a container with or without APA. In some cases, the sterile filter was chased with 150 mM sodium chloride to adjust the batch to the target concentration.

[0592] The formulations were filled into plastic syringes, glass syringes, or vials.

[0593] [Example 9] Preparation of stable nanoemulsion (SNE) adjuvant systems with and without compounds SNE adjuvant formulations are prepared as described in the examples above using the following compounds:

[0594] (N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide (compound A-1), (S)—N-(5-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide (compound A-2), (S)-1-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)octadecan-1-one (compound A-3), N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide (compound B-1), N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)tetradecanamide (compound B-2), N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)oleamide (compound B-3), (9Z,12Z)-N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)octadeca-9,12-dienamide (compound B-4), N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide (compound B-5), N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperidin-1-yl)-5-oxopentyl)stearamide (compound B-6), N-(5-(3-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)azetidin-1-yl)-5-oxopentyl)stearamide (compound B-7), 1-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperidine-4-carboxamide (compound B-8), (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-octadecylcyclobutane-1-carboxamide (compound B-9), (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-hexadecylcyclobutane-1-carboxamide (compound B-10), N-(3-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-3-oxopropyl)stearamide (compound B-11), N-(7-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-7-oxoheptyl)stearamide (compound B-12), N-(3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)cyclobutyl)stearamide (compound B-13), N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-4,4-dimethyl-5-oxopentyl)stearamide (compound B-14), N-(6-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-methyl-6-oxohexan-2-yl)stearamide (compound B-15), 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-(octadecyloxy)pentan-1-one (compound B-16), 1-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-(octadecylamino)pentan-1-one (compound B-17), N-(5-(4-(4-((5-amino-7-(butylamino)-3-methyl-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide (compound B-18), (9Z,12Z)-N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide (compound C-1), N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)tetradecanamide (compound C-2), N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)oleamide (compound C-3), N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide (compound C-4), N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)-4-oxobutyl)stearamide (compound C-5), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate (compound D-1), 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperazine-1-carboxamide (compound D-2), 3-stearamidopropyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate (compound D-3), N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-6,6,6-trifluorohexyl)stearamide (compound D-4), N-(4-((4-((7-(butylamino)-5-hydroxy-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide (compound D-5), and (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)carbamate (compound D-6).

[0595] SNE is a multi-component emulsion formulation consisting of three stabilizing components, SPAN-85, PS-20, and squalene, with and without a compound, e.g., Compound A-1 (referred to as Compound A-1-SNE, see Table 1) and without the compound (referred to as SNE, see Table 2). The formulation is prepared by dissolving the compound, SPAN-85, PS-20, and squalene components together in 100% EtOH and then mixing them together (Table 1).

[0596] The process for making compound-stable nanoemulsions (compound-SNEs) consists of five steps: 1) solution preparation of a component mixture containing the three stabilizing components SPAN-85, PS-20, and squalene along with the compound; 2) SNE formation by T-blending; 3) ultrafiltration; 4) bioburden reduction filtration; and 5) sterile filtration and vial filling.

[0597] Solution preparation of stabilizer / compound mixture The stabilizer / compound components were weighed, combined, dissolved in ethanol, and then sterile filtered to form a component mixture. A histidine buffer (20 mM histidine pH 5.8) was prepared.

[0598] SNE formation by T mixing The component mixture and histidine buffer were then mixed together at adjacent ends of a T-tube mixer. The flow exiting the T-mix device was immediately diluted 1:1 with 20 mM histidine, 0.05% PS-20, and 75 mM NaCl and then collected as the formed SNE.

[0599] Ultrafiltration The SNE intermediate was then subjected to dialysis or ultrafiltration with a 500 kDA NMWCO to concentrate the material approximately 10-fold and to buffer exchange the material against 20 mM histidine, 0.05% (w / v) PS-20, and 75 mM NaCl, pH 5.8, or 20 mM histidine pH 5.8, or 20 mM histidine, 0.05% PS-20 pH 5.8. After dialysis or diafiltration, there was a final concentration step performed to achieve the final target concentration.

[0600] Bioburden reduction filtration The adjuvant bulk was then pre-filtered through a 0.45 μm cellulose acetate (CA) filter followed by a 0.2 μm CA bioburden reduction filter and the liquid was stored at 4°C.

[0601] Sterile filtration and vial filling The adjuvant bulk was passed through a 0.45 μm polyvinylidene fluoride (PVDF) bioburden reduction filter and a 0.22 μm PVDF sterile glazing filter. The filtered adjuvant bulk was then diluted to the target SNE adjuvant concentration with 20 mM histidine, 0.05% (w / v) PS-20, and 75 mM NaCl pH 5.8. This diluted final bulk adjuvant was then filled into glass vials and stored at 4°C.

[0602] [Table 13]

[0603] [Table 14]

[0604] [Table 15]

[0605] [Table 16]

[0606] Example 9A Alternative preparation of a stable nanoemulsion (compound B-1-SNE) adjuvant system Compound B-1-SNE is a multi-component emulsion formulation consisting of three stabilizing components: SPAN-85, PS-20, and squalene and Compound B-1 (see Table 5). The formulation is prepared by dissolving the Compound B-1, SPAN-85, PS-20, and squalene components together in 100% EtOH and then mixing them together.

[0607] The process for making compound-stable nanoemulsion (Compound B-1-SNE) consists of five steps: 1) solution preparation of a component mixture containing three stabilizing components: SPAN-85, PS-20, and squalene, and Compound B-1; 2) SNE formation by T-mixing or controlled precipitation process utilizing a mixing system to drive self-assembly of the nanoemulsion; 3) ultrafiltration; 4) bioburden reduction filtration; and 5) sterile filtration and vial filling.

[0608] Solution preparation of stabilizer / compound mixture The stabilizer / compound components were weighed, combined, and dissolved in ethanol, then heated at 40° C. for 30 minutes and sterile filtered to form a component mixture.

[0609] SNE formation by T-mixing or controlled precipitation processes utilizing a mixing system to drive nanoemulsion self-assembly The component mixture and histidine buffer were then mixed together at adjacent ends of a T-tube mixer or fluidic assembly. The flow exiting the device was immediately diluted 1:1 with 20% histidine, pH 5.8, and then collected as the SNE formed.

[0610] Ultrafiltration The SNE intermediate was then subjected to dialysis or ultrafiltration with a 500 kDA NMWCO to concentrate the material approximately 10-fold and buffer exchange it into 20 mM histidine, 0.05% (w / v) PS-20, and 75 mM NaCl, pH 5.8, or 20 mM histidine pH 5.8, or 20 mM histidine, 0.05% PS-20 pH 5.8. After dialysis or diafiltration, there was a final concentration step performed to achieve the final target concentration.

[0611] Histidine pH 5.8, PS-20, sodium chloride, L-met, and EDTA solutions were prepared and added to the formulation container. The adjuvant bulk SNE was added to the formulation container. During the addition of SNE to the formulation buffer, the container was mixed to ensure homogeneity using a magnetic server or magnetic impeller. After all additions were made and the solution was agitated, the formulation was filled into plastic syringes, glass syringes, or vials.

[0612] [Table 17]

[0613] [Example 10] Cell-based TLR7 / 8 assay to assess compound-SNE activity As shown in Figure 1, a cell-based activation system was used to evaluate the activity of the compound-formulated nanoemulsion system (Compound B-1-SNE) prepared as described in the previous example. HEK-Blue™ TLR7 and TLR8 cells (InvivoGen) were designed to evaluate stimulation of TLR7 and TLR8 by monitoring activation of NF-κB and AP-1. Compound B-1-SNE samples were diluted 1:2 over an 8-point titration in diluent buffer histidine / PS-20 in a sterile V-bottom plate. 20 μL of sample was transferred to corresponding wells of two flat-bottom assay plates. Media was aspirated from HEK-Blue™ human TLR7 and TLR8 cells (T-75 flasks) and gently rinsed with prewarmed (37°C) DPBS for a total volume of 5 mL / flask. DPBS was gently removed from the cells, and the cells were gently manually dislodged and resuspended in 2 mL of DPBS. A cell assay suspension was then prepared at approximately 2.2 x 10 cells per mL of HEK-Blue™ detection medium (InvivoGen). 180 μL of the cell suspension in detection medium was added to each well of two flat-bottom assay plates containing diluted Compound B-1-SNE samples (180 μL cell suspension and 20 μL sample in detection medium) for a final sample dilution equal to 1:10. The cells were then incubated at 37°C and 5% CO for 20 hours. Upon stimulation, the reporter gene NF-Kb induces the production of secreted embryonic alkaline phosphatase (SEAP), which can be monitored using a vendor-supplied SEAP detection kit. The SEAP colorimetric change was read at absorbance at 640 nm using a SpectroMax Stakmax plate reader. As shown in Figure 1, compound B-1-SNE activates both TLR7 and TLR8 receptors with an EC50 of 65.4 μg / mL for TLR7 and 82.3 μg / mL for TLR8. EC50 values ​​were calculated based on total compound B-1-SNE concentrations using GraphPad Prism software and analyzed using a four-parameter least-squares fit of the agonist versus response variable slope.

[0614] Example 10A Additional cell-based TLR7 / 8 assay information for assessing compound-SNE activity In addition to the description in Example 10, a cell-based activation system was used to evaluate the activity of the compound-formulated nanoemulsion system prepared as described in the previous examples. HEK-Blue™ TLR7 and TLR8 cells (InvivoGen) were designed to evaluate stimulation of TLR7 and TLR8 by monitoring activation of NF-κB and AP-1. Compound-SNE samples were diluted 1:2 over an 8-point titration in diluent buffer histidine / PS-20 in a sterile V-bottom plate. 20 μL of sample was transferred to corresponding wells of two flat-bottom assay plates. Media was aspirated from HEK-Blue™ human TLR7 and TLR8 cells (T-75 flasks) and gently rinsed with prewarmed (37°C) DPBS for a total volume of 5 mL per flask. DPBS was gently removed from the cells, and the cells were gently manually dislodged and resuspended in 2 mL of DPBS. A cell assay suspension was then prepared at approximately 2.2 x 10 cells per mL of HEK-Blue™ detection medium (InvivoGen). 180 μL of the cell suspension in detection medium was added to each well of two flat-bottom assay plates containing diluted compound-SNE samples for a final sample dilution equal to 1:10 (180 μL cell suspension and 20 μL sample in detection medium). Cells were then incubated at 37°C and 5% CO for 20 hours. Upon stimulation, the reporter gene NF-Kb induces the production of secreted embryonic alkaline phosphatase (SEAP), which can be monitored using a vendor-supplied SEAP detection kit. The SEAP colorimetric change was read at 640 nm absorbance using a SpectroMax Stakmax plate reader. EC50 values ​​were calculated based on compound concentration alone, rather than total compound-SNE concentration, using GraphPad Prism software (Table 6) and analyzed using a four-parameter least-squares fit of the agonist versus response variable slope. As shown in Table 6, compounds formulated as SNEs activate both the TLR7 and TLR8 receptors.

[0615] [Table 18]

[0616] [Example 11] PCV1 immunogenicity in mice: evaluation of adjuvant systems Young female Balb / C mice (6–8 weeks old, n = 10 / group) were immunized intramuscularly (IM) with 0.2 mL of PCV1 formulated with different adjuvants (Table 7) on days 0, 28, and 56. PCV1 was administered at 0.08 μg of PnPs (6B conjugated to CRM197) per immunization. Mice were observed at least daily by trained animal care staff for any signs of illness or distress. The vaccine formulation in mice was considered safe and well-tolerated, as no vaccine-related adverse events were observed. All animal experiments were performed in strict accordance with the recommendations in the National Institutes of Health's Guide for Care and Use of Laboratory Animals. The mouse experimental protocol was approved by the Animal Care and Use Committee of Merck & Co., Inc. (Rahway, NJ, USA).

[0617] [Table 19]

[0618] Mouse sera were evaluated for serotype 6B (ST-6B)-specific IgG antibody titers using an ELISA immunoassay (Figure 2). PCV1 immunization of Balb / C mice produced ST-6B-specific IgG antibody titers when formulated with APA, compound A-1-SNE (either 0.15, 1.5, or 15 μg of compound A-1 per dose), compound B-1-SNE (either 0.3, 3, or 30 μg of compound B-1 per dose), or compound C-4-SNE (0.3, 3, or 30 μg of compound C-4 per dose). All formulations were found to be immunogenic, resulting in increased antibody titers after boosting and generally demonstrating a dose-dependent response profile.

[0619] Functional antibody titers were determined by opsonophagocytosis assay (OPA, Figure 3) based on previously described protocols at www.vaccine.uab.edu and Opsotiter® 3 software, owned and licensed by the University of Alabama (UAB) Research Foundation (see Caro-Aguilar I. et al., Vaccine (2017) 35(6):865-72 and Burton RL and Nahm MHClin. Vaccine Immunol. (2006) 13(9):1004-9). ST-6B-specific functional antibody titers were generated in Balb / C mice, and all formulations were confirmed to be immunogenic.

[0620] [Example 12] PCV24 immunogenicity study in infant rhesus macaques (IRM) PCV24 (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B, each individually conjugated to CRM197) and adjuvant formulations were prepared or obtained as described in the above examples (PCV20). Serotype "15C" is the de-O-acetylated 15B serotype. PCV24 serotypes can also be defined as (serotypes -1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-acetylated 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B). Infant rhesus macaques (n = 5 per group) were immunized intramuscularly on days 0, 28, and 56 with either 0.1 mL (SNE unadjuvanted or aluminum-adjuvanted) or 0.5 mL (compound B-1-SNE) vaccine, as described in Table 8 below. Serum was collected before the start of the study (pre), and on days 14 (PD1), 42 (PD2), and 70 (PD3). IRMs were observed at least daily by trained animal care staff for any signs of illness or distress. The vaccine formulation in IRM appeared to be safe and well tolerated, with no vaccine-related adverse events observed.

[0621] [Table 20]

[0622] Serotype-specific IgG responses were assessed using a multiplexed electrochemiluminescence (ECL) assay developed for use with rhesus macaque sera and based on the human assay described in Marchese et al., Clin Vaccine Immunol (2009) 16(3):387-96.

[0623] At post-dose 3 (day 70), PCV24 formulated with compound B-1-SNE at all dose levels demonstrated significantly higher antibody titers compared to PCV24 without adjuvant (Figure 4). Statistical significance was defined as a GMT ratio with a lower 95% CI greater than 1.0. As shown in Figure 5, PCV24 formulated with compound B-1-SNE demonstrated significantly higher antibody titers compared to PCV24 formulated with APA at all dose levels for the majority of post-dose 3 serotypes. PCV24 formulated with compound B-1-SNE at all dose levels demonstrated significantly higher antibody titers compared to PCV20 for the majority of shared serotypes at post-dose 3 (Figure 6).

[0624] [Example 13] PCV24 immunogenicity study in infant rhesus macaques (IRM) PCV24 (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B, each individually conjugated to CRM197) and adjuvant formulations were prepared or obtained as described in the above examples (PCV20). Serotype "15C" is the de-O-acetylated 15B serotype. PCV24 serotypes can also be defined as (serotypes -1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-acetylated 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B). On days 0, 28, and 56, IRM (infant rhesus macaques, n = 5 per group) were immunized intramuscularly with 0.1 mL of PCV20 (aluminum adjuvant) or 0.5 mL of PCV24 adjuvanted with compound B-1-SNE, as described in Table 9 below. Serum was collected before the start of the study (Pre), and on days 14 (PD1), 42 (PD2), and 70 (PD3). IRM were observed at least once daily by trained animal care staff for any signs of illness or distress. The vaccine formulation in IRM appeared to be safe and well tolerated, with no vaccine-related adverse events observed.

[0625] [Table 21]

[0626] Serotype-specific IgG responses were assessed using a multiplexed electrochemiluminescence (ECL) assay developed for use with rhesus macaque sera and based on the human assay described in Marchese et al., Clin Vaccine Immunol (2009) 16(3):387-96.

[0627] At post-dose 3 (day 70), PCV24 formulated with compound B-1-SNE at the 100 μg dose level demonstrated significantly higher antibody titers for all shared serotypes compared to PCV20 (Figure 7). Compared to PCV20, statistically higher antibody titers were observed for 17 of 19 shared serotypes at the 10 μg dose level, 7 of 19 shared serotypes at the 1 μg dose level, and 1 of 19 shared serotypes at the 0.1 μg dose level. All other titers were statistically comparable between the two groups. Statistical significance was defined as a GMT ratio with a lower 95% CI greater than 1.0.

[0628] [Example 14] PCV26 immunogenicity and functional antibody responses in mice Female Balb / c mice (6-8 weeks old, n = 15 / group) were immunized intramuscularly with 0.1 mL of 26-valent pneumococcal conjugate vaccine (PCV) on days 0, 28, and 56. PCV26 was administered with 0.4 μg of each pneumococcal polysaccharide (1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-acetylated 15B, 16F, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, 35B), all individually conjugated to CRM197 and adjuvanted with aluminum phosphate adjuvant (APA) or compound B-1-SNE. Adjuvants were utilized at the concentrations listed in Table 10. Mice were observed at least daily by trained animal care staff for any signs of illness or distress.

[0629] [Table 22]

[0630] Mouse sera were collected before the start of the study (pre-immunization, day 0) and on days 14 (PD1), 42 (PD2), and 70 (PD3). Sera were evaluated for IgG immunogenicity using a multiplexed electrochemiluminescence (ECL) assay. This assay was developed for use with mouse sera based on the human assay described by Marchese et al. (Marchese RD, et al., Clin. Vaccine Immunol. (2009) 16(3):387-396) using technology developed by MesoScale Discovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, MD), which utilizes SULFO-TAG™ labels that emit light upon electrochemical stimulation. SULFO-TAG™-labeled anti-mouse IgG was used as the secondary antibody for testing mouse serum samples. Functional antibody titers were determined by multiplexed opsonophagocytosis assay (MOPA) based on previously described protocols at www.vaccine.uab.edu and Opsotiter® 3 software owned and licensed by the University of Alabama (UAB) Research Foundation (Caro-Aguilar, I. et al., Vaccine (2017) 35(6):865-872; and Burton RL, and Nahm MH, Clin. Vaccine Immunol. (2006) 19(9):1004-1009).

[0631] Mouse sera were tested individually in the ECL assay to determine antibody titers and generated antibody titers for all serotypes in the vaccine. Day 0 sera were pooled by group prior to testing in the ECL assay. Antibody titers in mice immunized with PCV26 / Compound B-1-SNE were comparable to or higher than those in mice immunized with PCV26 / APA, with the greatest difference observed by day 42 (Figure 8).

[0632] It should also be noted that PCV26 containing polysaccharide conjugates 15A-CRM197, deOAc15B-CRM197, 6A-CRM197, and 6B-CRM197 also provided cross-reactivity to 15B and 6C, and these titers in mice immunized with PCV26 / compound B-1-SNE were comparable to or higher than those in mice immunized with PCV26 / APA (Figure 8).

[0633] For all time points, mouse sera were pooled by group before testing with MOPA to determine functional antibody titers in mice immunized with killed vaccine-type bacterial serotypes and PCV26-generated functional antibody titers. Similar to the results from the ECL assay, functional antibody titers in mice immunized with PCV26 / compound B-1-SNE tended to be similar to or higher than those in mice immunized with PCV26 / APA, with the greatest difference observed by day 42 (PD2).

[0634] To determine whether the immune response was driven toward Th1 or Th2, antibody subclassification was performed. Day 70 (PD3) mouse sera were individually tested in an IgG subclassification assay to quantify IgG2a and IgG1 antibody titers, and the IgG2a / IgG1 ratio was calculated. A higher ratio indicates immunity more driven toward a Th1 response, while a lower ratio indicates immunity more driven toward a Th2 response. Data from three representative serotypes (18C, 19F, and 24F) show that PCV26 / compound B-1-SNE-immunized mice had a more Th1-like response, while PCV26 / APA-immunized mice had a more Th2-like response (Figure 9). Unpaired t-tests showed significant differences in the IgG2a / IgG1 ratio between the PCV26 / APA-immunized and PCV26 / compound B-1-SNE-immunized groups across all three representative serotypes (P<0.0001). The addition of compound B-1-SNE to pneumococcal antigen compositions is beneficial not only for the response but also for the quality of the response. This data demonstrates differences in the quality of the immune response induced by compound B-1-SNE adjuvant and APA adjuvant.

[0635] On day 77, 10 mice per group were inoculated with 10 0.1 mL of PBS. 5 Mice were challenged intratracheally with 10 ... One-way ANOVA with Dunnett's multiple comparison test showed that the PCV26 / APA and PCV26 / compound B-1-SNE immunized groups were significantly protected from bacteremia compared with the naive control group (P < 0.0001) (Figure 10A). Mantel-Cox log-rank tests of survival curves showed that the PCV26 / APA and PCV26 / compound B-1-SNE immunized groups were significantly protected from challenge compared with the naive control group (P < 0.0001) (Figure 10B).

Claims

1. A compound having the structure according to Formula I: 【Chemistry 1】 (In the formula, R ａ is H, -OH, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, (C １ -C ６ ) alkynyl, —O(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkenyl, —O(C １ -C ６ ) selected from alkynyl, chlorine, fluorine, and —NR′R″, １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, (C １ -C ６ ) alkynyl, —O(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkenyl, and —O(C １ -C ６ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 4 substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine; R ａ’ is H, -OH, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, (C １ -C ６ ) alkynyl, —O(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkenyl, —O(C １ -C ６ ) selected from alkynyl, chlorine, fluorine, and —NR′R″, １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, (C １ -C ６ ) alkynyl, —O(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkenyl, and —O(C １ -C ６ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 4 substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine; R ａ’’ is H, -OH, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, (C １ -C ６ ) alkynyl, —O(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkenyl, —O(C １ -C ６ ) selected from alkynyl, chlorine, fluorine, and —NR′R″, １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, (C １ -C ６ ) alkynyl, —O(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkenyl, and —O(C １ -C ６ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 4 substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine; R' and R'' are H, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, and (C １ -C ６ ) alkynyl, wherein (C １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, and (C １ -C ６ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 4 substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine, or R′ and R″, together with the nitrogen to which they are attached, are bonded together to form (C ３ -C ６ ) heterocycloalkyl, ３ -C ６ ) Heterocycloalkyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 4 substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine; R ｂ Each occurrence of １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, (C １ -C ６ ) alkynyl, —O(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkenyl, —O(C １ -C ６ ) independently selected from alkynyl, chlorine, fluorine, or NR′R″; １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, (C １ -C ６ ) alkynyl, —O(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkenyl, and —O(C １ -C ６ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 4 substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine; A is (C １ -C ６ ) alkyl, heterocycloalkyl, heterocycloalkyl-C(O)-R ｚ -, (C １ -C ４ ) alkyl-N(R ｚ )-R ｚ a carbon or nitrogen bond spacer selected from -, aryl, and heteroaryl, １ -C ６ ) alkyl, heterocycloalkyl, aryl, and heteroaryl are —OH, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, (C １ -C ６ ) alkynyl, —O(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkenyl, —O(C １ -C ６ ) optionally substituted with 1 to 6 substituents independently selected from the group consisting of alkynyl, chlorine, fluorine, and NR'R''; １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, (C １ -C ６ ) alkynyl, —O(C １ -C ６ ) alkyl, —O(C １ -C ６ ) alkenyl, and —O(C １ -C ６ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 6 substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine; R ｚ Each occurrence of is independently H or (C １ -C ６ ) alkyl, B is, 【Chemistry 2】 is a functional group selected from D is (C ６ -C ２０ ) alkyl, (C ６ -C ２０ ) alkenyl, and (C ６ -C ２０ ) alkynyl, wherein (C ６ -C ２０ ) alkyl, (C ６ -C ２０ ) alkenyl, and (C ６ -C ２０ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 6 substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine, or D is 【Transformation 3】 and Each occurrence of Z is ６ -C ２０ ) alkyl, (C ６ -C ２０ ) alkenyl, and (C ６ -C ２０ ) alkynyl, wherein (C ６ -C ２０ ) alkyl, (C ６ -C ２０ ) alkenyl, and (C ６ -C ２０ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 6 substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine; m is 0, 1, 2, 3, 4, or 5; n is 0, 1, 2, 3, 4, or 5. or a pharmaceutically acceptable salt thereof.

2. 10. The compound of claim 1 having the structure according to formula Ia: 【Chemistry 4】 (In the formula, R' and R'' are H, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, and (C １ -C ６ ) alkynyl, wherein (C １ -C ６ ) alkyl, (C １ -C ６ ) alkenyl, and (C １ -C ６ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 4 substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine, or R′ and R″, together with the nitrogen to which they are attached, are bonded together to form (C ３ -C ６ ) heterocycloalkyl, ３ -C ６ ) Heterocycloalkyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 4 substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine; R ｂ Each occurrence of is -O(C １ -C ４ ) alkyl, and said —O(C １ -C ４ ) alkyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with one or two substituents independently selected from the group consisting of alkynyl, chlorine, and fluorine; A is, 【Transformation 5】 is selected from R ｚ Each occurrence of is independently H or (C １ -C ６ ) alkyl, R ｄ Each occurrence of is -OH, (C １ -C ４ ) alkyl, —O(C １ -C ４ ) independently selected from alkyl, chlorine, and fluorine; B is, 【Transformation 6】 and D is 【Transformation 7】 and a lipid chain selected from Any carbon on the lipid chain can be replaced with -OH, -O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with alkynyl, chlorine, or fluorine; 【Transformation 8】 is cis or trans stereochemistry, X １ is -O-, -C(R) ２ - or -NR-; Each occurrence of R is H, (C １ -C ４ ) alkyl, (C １ -C ４ ) alkenyl, (C １ -C ４ ) alkynyl, —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) independently selected from alkynyl, chlorine, and fluorine; m is 0, 1, or 2; n is 0, 1, 2, or 3; p is 0, 1, or 2; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; s is 1, 2, 3, 4, 5, 6, 7, or 8; t is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1 having the structure according to Formula II: 【Chemistry 9】 (In the formula, R １ is (C １ -C ６ ) alkyl, and the (C １ -C ６ ) alkyl is —OH and —O(CH ３ and optionally substituted with 1 to 4 substituents independently selected from R ２ is H, methyl or —O(CH ３ ) and R ３ Each occurrence of １ -C ４ ) alkyl, (C １ -C ４ ) alkenyl, (C １ -C ４ ) alkynyl, or —O(C １ -C ４ ) alkyl, and the (C １ -C ４ ) alkyl, (C １ -C ４ ) alkenyl, (C １ -C ４ ) alkynyl, or —O(C １ -C ４ ) alkyl is —OH and —O(CH ３ and optionally substituted with one or two substituents independently selected from R ４ Each occurrence of １ -C ４ ) alkyl, (C １ -C ４ ) alkenyl, (C １ -C ４ ) alkynyl, or —O(C １ -C ４ ) alkyl, and the (C １ -C ４ ) alkyl, (C １ -C ４ ) alkenyl, (C １ -C ４ ) alkynyl, or —O(C １ -C ４ ) alkyl is —OH and —O(CH ３ and optionally substituted with one or two substituents independently selected from R ５ teeth, 【Chemistry 10】 and R ６ is (C ６ -C ２０ ) alkyl, (C ６ -C ２０ ) alkenyl, and (C ６ -C ２０ ) alkynyl, wherein (C ６ -C ２０ ) alkyl, (C ６ -C ２０ ) alkenyl, and (C ６ -C ２０ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 6 substituents independently selected from alkynyl, chlorine, and fluorine; each occurrence of n is 4) or a pharmaceutically acceptable salt thereof.

4. 4. The compound of claim 3 having the structure according to formula IIa: 【Chemistry 11】 (In the formula, R １ is butyl, said butyl being optionally substituted with one or two —OH groups; R ３ Each occurrence of is independently H or —O(CH ３ ) and R ５ teeth, 【Chemistry 12】 and R ６ is (C １０ -C ２０ ) alkyl, (C １０ -C ２０ ) alkenyl, and (C １０ -C ２０ ) alkynyl) or a pharmaceutically acceptable salt thereof.

5. 10. The compound of claim 1 having the structure according to Formula III: 【Chemistry 13】 (In the formula, R １ is (C １ -C ６ ) alkyl, and the (C １ -C ６ ) alkyl is —OH and —O(CH ３ and optionally substituted with 1 to 4 substituents independently selected from R ２ is H, methyl or —O(CH ３ ) and R ３ Each occurrence of １ -C ４ ) alkyl, (C １ -C ４ ) alkenyl, (C １ -C ４ ) alkynyl, or —O(C １ -C ４ ) alkyl, and the (C １ -C ４ ) alkyl, (C １ -C ４ ) alkenyl, (C １ -C ４ ) alkynyl, or —O(C １ -C ４ ) alkyl is —OH and —O(CH ３ and optionally substituted with one or two substituents independently selected from R ４ is (C ６ -C ２０ ) alkyl, (C ６ -C ２０ ) alkenyl, and (C ６ -C ２０ ) alkynyl, wherein (C ６ -C ２０ ) alkyl, (C ６ -C ２０ ) alkenyl, and (C ６ -C ２０ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 6 substituents selected from alkynyl, chlorine, and fluorine; n is 4) or a pharmaceutically acceptable salt thereof.

6. The compound of claim 5 having the structure according to Formula IIIa: 【Chemistry 14】 (In the formula, R １ is butyl, said butyl being optionally substituted with one or two —OH groups; R ３ Each occurrence of is independently H or —O(CH ３ ) and R ４ is (C １０ -C ２０ ) alkyl, (C １０ -C ２０ ) alkenyl, and (C １０ -C ２０ ) alkynyl) or a pharmaceutically acceptable salt thereof.

7. 10. The compound of claim 1 having the structure according to Formula IV: 【Chemistry 15】 (In the formula, R １ is (C １ -C ６ ) alkyl, and the (C １ -C ６ ) alkyl is —OH and —O(CH ３ and optionally substituted with 1 to 4 substituents selected from R ２ is H, methyl or —O(CH ３ ) and R ３ Each occurrence of １ -C ４ ) alkyl, (C １ -C ４ ) alkenyl, (C １ -C ４ ) alkynyl, or —O(C １ -C ４ ) alkyl, and the (C １ -C ４ ) alkyl, (C １ -C ４ ) alkenyl, (C １ -C ４ ) alkynyl, and —O(C １ -C ４ ) alkyl is —OH and —O(CH ３ and optionally substituted with one or two substituents independently selected from R ４ Each occurrence of (C ６ -C ２０ ) alkyl, (C ６ -C ２０ ) alkenyl, and (C ６ -C ２０ ) alkynyl, wherein (C ６ -C ２０ ) alkyl, (C ６ -C ２０ ) alkenyl, and (C ６ -C ２０ ) alkynyl is —OH, —O(C １ -C ４ ) alkyl, —O(C １ -C ４ ) alkenyl, —O(C １ -C ４ ) optionally substituted with 1 to 6 substituents independently selected from alkynyl, chlorine, or fluorine; n is 4) or a pharmaceutically acceptable salt thereof.

8. 8. The compound of claim 7 having the structure according to formula IVa: 【Chemistry 16】 (In the formula, R １ is butyl, said butyl being optionally substituted with one or two —OH groups; R ３ Each occurrence of is independently H or —O(CH ３ ) and R ４ Each occurrence of (C １０ -C ２０ ) alkyl, (C １０ -C ２０ ) alkenyl, and (C １０ -C ２０ ) alkynyl) or a pharmaceutically acceptable salt thereof.

9. (N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, (S)—N-(5-(4-(4-((5-amino-7-((1-hydroxypentan-2-yl)amino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)tetradecanamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)oleamide, (9Z,12Z)-N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)octadeca-9,12-dienamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-1,4-diazepan-1-yl)-5-oxopentyl)stearamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperidin-1-yl)-5-oxopentyl)stearamide, N-(5-(3-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)azetidin-1-yl)-5-oxopentyl)stearamide, 1-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperidine-4-carboxamide, (1s,3s)-3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)-N-hexadecylcyclobutane-1-carboxamide, N-(3-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-3-oxopropyl)stearamide, N-(7-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-7-oxoheptyl)stearamide, N-(3-(2-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-2-oxoethyl)cyclobutyl)stearamide, N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-4,4-dimethyl-5-oxopentyl)stearamide, N-(5-(4-(4-((5-amino-7-(butylamino)-3-methyl-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, (9Z,12Z)-N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)octadeca-9,12-dienamide, N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)tetradecanamide, N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)oleamide, N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)stearamide, N-(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)-4-oxobutyl)stearamide, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl(4-((4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3,5-dimethoxybenzyl)(methyl)amino)butyl)carbamate, 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)-N-(3-stearamidopropyl)piperazine-1-carboxamide, and 3-stearamidopropyl 4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazine-1-carboxylate, 2. The compound of claim 1 selected from: or a pharmaceutically acceptable salt thereof.

10. N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide or a pharmaceutically acceptable salt thereof.

11. 11. A formulation comprising: (i) a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof; (ii) one or more emulsifying agents; and (iii) a terpene.

12. 11. A formulation comprising: (i) a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof; (ii) sorbitan trioleate (SPAN-85); (iii) polysorbate-20 (PS-20) or polysorbate-80 (PS-80); and (iv) squalene.

13. 13. The formulation of claim 12, wherein the compound is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

14. 11. A pharmaceutical composition comprising: (i) at least one antigen; (ii) a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof; (iii) one or more emulsifiers; (iv) a terpene; and (v) a pharmaceutically acceptable carrier.

15. 11. A pharmaceutical composition comprising: (i) at least one antigen; (ii) the compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof; (iii) sorbitan trioleate (SPAN-85); (iv) polysorbate-20 (PS-20) or polysorbate-80 (PS-80); (v) squalene; and (vi) a pharmaceutically acceptable carrier.

16. 16. The pharmaceutical composition of claim 15, wherein the compound is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

17. 11. An immunogenic composition comprising: (i) at least one antigen; (ii) a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof; (iii) one or more emulsifying agents; (iv) a terpene; and (v) a pharmaceutically acceptable carrier.

18. 11. An immunogenic composition comprising: (i) at least one antigen; (ii) the compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof; (iii) sorbitan trioleate (SPAN-85); (iv) polysorbate-20 (PS-20) or polysorbate-80 (PS-80); (v) squalene; and (vi) a pharmaceutically acceptable carrier.

19. 19. The immunogenic composition of claim 18, wherein the compound is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

20. 11. A single-dose vaccine composition comprising: (i) at least one antigen; (ii) a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof; (iii) one or more emulsifying agents; (iv) a terpene; and (v) a pharmaceutically acceptable carrier, wherein a single dose of the vaccine composition is sufficient to elicit a desired immune response against the at least one antigen.

21. 11. A single-dose vaccine composition comprising: (i) at least one antigen; (ii) a compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof; (iii) sorbitan trioleate (SPAN-85); (iv) polysorbate-20 (PS-20) or polysorbate-80 (PS-80); (v) squalene; and (vi) a pharmaceutically acceptable carrier, wherein a single dose of the vaccine composition is sufficient to elicit a desired immune response against the at least one antigen.

22. 22. The single-dose vaccine composition of claim 21, wherein the compound is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

23. 11. A method of treating or preventing a disease in a patient in need thereof, comprising administering to the patient a pharmaceutical composition comprising: (i) at least one antigen; (ii) a compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof; (iii) one or more emulsifying agents; (iv) a terpene; and (v) a pharmaceutically acceptable carrier.

24. 11. A method of treating or preventing a disease in a patient in need thereof, comprising administering to the patient a pharmaceutical composition comprising: (i) at least one antigen; (ii) a compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof; (iii) sorbitan trioleate (SPAN-85); (iv) polysorbate-20 (PS-20) or polysorbate-80 (PS-80); (v) squalene; and (vi) a pharmaceutically acceptable carrier.

25. 25. The method of claim 24, wherein the compound is N-(5-(4-(4-((5-amino-7-(butylamino)-2H-pyrazolo[4,3-d]pyrimidin-2-yl)methyl)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentyl)stearamide, or a pharmaceutically acceptable salt thereof.

26. The method of any one of claims 23 to 25, wherein the patient is a human.

27. The method of any one of claims 23 to 25, wherein the patient is a non-human animal.

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