EA049099B1 - PRODRUGS IN THE FORM OF PHOSPHONAMIDE NUCLEOTIDE ANALOGUES AND THEIR PHARMACEUTICAL APPLICATION - Google Patents

Abstract

A compound of formula (I) or a pharmaceutically acceptable salt thereof and the use of said compound or a pharmaceutical composition based on it for the treatment of HIV infection are proposed.

Description

Cross-references to related applications

The present invention claims priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63/062,899, filed August 7, 2020, the contents of which are incorporated herein by reference in their entirety for all purposes.

Field of technology

Described are compounds, compositions and methods useful for treating a viral infection, such as an infection caused by the human immunodeficiency virus (HIV).

Prerequisites for the creation of an invention

Human immunodeficiency virus infection and related diseases are a major public health problem worldwide. Human immunodeficiency virus encodes three enzymes that are required for viral replication: reverse transcriptase, protease, and integrase. Drugs that target reverse transcriptase are widely used and have demonstrated efficacy, particularly when used in combination, such as with protease inhibitors and integrase inhibitors. A cure for HIV has not yet been found, and people infected with HIV may therefore require lifelong treatment. Improved treatments for HIV and other viral infections are desirable.

Statement of the essence of the invention

The present invention provides novel nucleotide analog prodrugs of the reverse transcriptase inhibitor tenofovir and pharmaceutically acceptable salts thereof. In some embodiments, the compounds can be used to treat HIV infections, to inhibit HIV reverse transcriptase activity, and/or to reduce HIV replication. In some embodiments, the compounds described herein can be effective against a number of known drug-resistant mutants of HIV. In some embodiments, the compounds described herein have properties that allow them to be administered at a frequency less than daily, such as at weekly, monthly, or longer intervals. In one embodiment, the invention provides a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein

R ¹ and R ² are independently selected from C _1-12 alkyl, C _6-14 aryl-C _1-4 alkylene, C _3-7 cycloalkyl, C _3-7 cycloalkyl-C _1-4 alkylene, _{C 6-14 aryl} -C _3-7 cycloalkylene, C _7-12 spirocycloalkyl, C _7-12 spirocycloalkyl _- C _1-4 alkylene, bridged C _5-10 bicycloalkyl, bridged _{C 5-10 bicycloalkyl} -C _{1-4 alkylene} , condensed C _{5-10 bicycloalkyl} , _{C 10-16} disspirocycloalkyl, C _10-16 disspirocycloalkyl-C _1-4 alkylene, bridged C _9-12 tricycloalkyl, bridged _C9-12 tricycloalkyl- _C1-4 alkylene, _C3-7 cycloalkyl- _C3-7 cycloalkylene and a 5-7 membered monocyclic heterocyclyl having 1 heteroatom selected from O and S, wherein each C^N alkyl, _C6-14 aryl- _C1-4 alkylene, _C3-7 cycloalkyl, _C3-7 cycloalkyl- _C1-4 alkylene, _C6-14 aryl- _C3-7 cycloalkylene, _C7-12 spirocycloalkyl and _C7-12 spirocycloalkyl- _C1-4 alkylene is optionally substituted with one to three R ^a ;

R ³ , R ⁴ , R ⁵ and R ⁶ are independently selected from C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 aryl-C _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 aryl-C _1-4 alkylene is optionally substituted with one R ^b ; or optionally:

R ³ and R ⁴ , together with the carbon atom to which they are attached, form a 3-6 membered saturated carbocyclic ring optionally substituted with one R ^b ; and R ⁵ and R ⁶ are independently selected from C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 arylC _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 arylC _1-4 alkylene is optionally substituted with one R ^b ; or

R ³ and R ⁴ are independently selected from C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 aryl-C _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 aryl-C _1-4 alkylene is optionally substituted with one R ^b ; and R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a 3- to 6-membered saturated carbocyclic ring optionally substituted with one R ^b ; or

^R3 and ^R4 , together with the carbon atom to which they are attached, form a 3- to 6-membered saturated carbocyclic ring optionally substituted with one ^Rb ; and ^R5 and ^R6 , together with the carbon atom to which they are attached, form a 3- to 6-membered saturated carbocyclic ring optionally substituted with one ^Rb ;

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-R ⁷ HN

or _X R ⁷ N

N x ^R ®

N

B represents

R ⁷ represents hydrogen or R ⁸ ;

R ⁸ is -L1-(L ² )m-(L ³ )nR ^8a ;

L ¹

L ² is selected from the bond, -C(O)- and -C(O)O-; is C _1-6 alkylene;

L ³ is -C(O)O- or

R ^8a is selected from C _1-12 alkyl, C _6-14 aryl, -C(O)-C _1-4 alkyl, ^-C(O)-C _1-4 alkyl, o

' where C _6-14 aryl is optionally substituted with one or two R ^c ;

each R ^a is independently selected from C _1-4 alkyl, halogen, C _1-4 haloalkyl and -OC _1-4 alkyl;

each R ^b is independently C _1-4 alkyl;

each R ^c is independently C _1-4 alkyl or -OC(O)-C _1-4 alkyl;

m and n are independently equal to 0 or 1; and p is equal to 0, 1, or 2.

In another embodiment of the invention there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are independently selected from C1-8 alkyl, C6-14 aryl-C1-4 alkylene, C3-7 cycloalkyl, C3-7 cycloalkyl-C1-4 alkylene, C6-14 aryl-C3-7 cycloalkylene, C7-12 spirocycloalkyl, C7-12 spirocycloalkyl-C1-4 alkylene, bridged C5-10 bicycloalkyl, bridged C5-10 bicycloalkyl-C1-4 alkylene, fused C5-10 bicycloalkyl, C10-16 dispirocycloalkyl, C10-16 dispirocycloalkyl-C1-4 alkylene, bridged C9-12 tricycloalkyl, bridged C9-12 tricycloalkyl-C1-4 alkylene, C3-7cycloalkyl-C3-7cycloalkylene and a 5-7 membered monocyclic heterocyclyl having 1 heteroatom selected from O and S, wherein each C1-8alkyl, C6-14aryl-C1-4alkylene, C3-7cycloalkyl, C3-7cycloalkyl-C1-4alkylene, C6-14aryl-C3-7cycloalkylene, C7-12spirocycloalkyl and C7-12spirocycloalkylC1-4alkylene is optionally substituted with one to three R ^a . In another embodiment of the invention R ¹ and R ² are independently selected from C1-8 alkyl, C _6-14 aryl-C _1-4 alkylene, C _3-7 cycloalkyl, C _3-7 cycloalkyl-C _1-4 alkylene, C _6-14 aryl-C _3-7 cycloalkylene, C _7-12 spirocycloalkyl,

C _7-12 spirocycloalkyl-C _1-4 alkylene, bridged C _5-10 bicycloalkyl-C _1-4 alkylene, fused C ₅₁₀ bicycloalkyl, C _10-16 dispirocycloalkyl-C _1-4 alkylene, bridged C _9-12 tricycloalkyl-C _1-4 alkylene, C _3-7 cycloalkyl-C _3-7 cycloalkylene and a 5-7-membered monocyclic heterocyclyl having 1 heteroatom selected from O and S, each C _1-8 alkyl, C _6-14 aryl-C _1-4 alkylene, C _3-7 cycloalkyl, C _3-7 cycloalkyl-C _1-4 alkylene, C _6-14 aryl-C _3-7 cycloalkylene, C _7-12 spirocycloalkyl and C _7-12 spirocycloalkylC _1-4 alkylene optionally substituted with one to three R ^a .

In another embodiment of the invention there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are independently selected from C 1-8 alkyl, C 3-7 cycloalkyl, C 3-7 cycloalkyl-C 1-4 alkylene, C 7-12 spirocycloalkyl, C 1-12 spirocycloalkyl-C 1-4 alkylene and a bridged C 5-10 bicycloalkyl-C 1-4 alkylene, wherein each C 1-8 alkyl, C 3-7 cycloalkyl, C 3-7 cycloalkyl-C 1-4 alkylene, C 7-12 spirocycloalkyl and C 7-12 spirocycloalkyl-C 1-4 alkylene is optionally substituted with one to three R ^a . In another embodiment of the invention, R ¹ and R ² are independently selected from C5-8 alkyl, C _5-7 cycloalkyl, C _5-7 cycloalkyl-C _1-4 alkylene, C _7-9 spirocycloalkyl, C _7-9 spirocycloalkyl-C _1-4 alkylene, and bridged C _5-7 bicycloalkyl-C _1-4 alkylene, wherein each C _5-8 alkyl, C _5-7 cycloalkyl,

C _5-7 cycloalkyl-C _1-4 alkylene, C _7-9 spirocycloalkyl and C _7-9 spirocycloalkyl-C _1-4 alkylene are optionally substituted with one to three R ^a .

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are different.

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are the same.

In another embodiment of the invention there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are the same and are selected from C _1-8 alkyl, C _6-14 aryl-C _1-4 alkylene, C _3-7 cycloalkyl, C _3-7 cycloalkyl-C _1-4 alkylene, C _6-14 aryl-C _3-7 cycloalkylene, C _7-12 spirocycloalkyl, C _7-12 spirocycloalkyl-C _1-4 alkylene, bridged C _5-10 bicycloalkyl-C _1-4 alkylene, fused C _5-10 bicycloalkyl, C _10-16 dispirocycloalkyl-C _1-4 alkylene, bridged C _9-12 tricycloalkyl-C _1-4 alkylene, C _3-7 cycloalkyl-C _3-7 cycloalkylene and 5-7 membered monocyclic heterocyclyl having 1 heteroatom selected from O and S, each C _1-8 alkyl, C _3-7 cycloalkyl, C _3-7

- 2 049099 cycloalkyl-C _1-4 alkylene, C _7-12 spirocycloalkyl and C _7-12 spirocycloalkyl-C _1-4 alkylene are optionally substituted with one to three R ^a . In another embodiment of the invention, R ¹ and R ² are the same and are selected from C _1-8 alkyl, C _3-7 cycloalkyl, C _3-7 cycloalkyl-C _1-4 alkylene, C _7-12 spirocycloalkyl, C _7-12 spirocycloalkyl-C _1-4 alkylene, and bridged _{C 5-10 bicycloalkyl} -C _1-4 alkylene, wherein each C _1-8 alkyl, _{C 3-7} cycloalkyl, C _3-7 cycloalkyl-C _1-4 alkylene, C _7-12 spirocycloalkyl and C _7-12 spirocycloalkyl-C _1-4 alkylene is optionally substituted with one to three R ^a .

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are independently selected from

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are the same and are selected from

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In another embodiment of the invention there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ³ , R ⁴ , R ⁵ and R ⁶ are independently selected from methyl, ethyl, cyclopropyl and benzyl, wherein each methyl, ethyl, cyclopropyl and benzyl is optionally substituted with one R ^b .

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁵ are the same.

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁵ are methyl.

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ⁴ and R ⁶ are the same.

In another embodiment of the invention there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, characterized in that R ⁴ and R ⁶ are both ethyl or benzyl.

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ are the same.

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ are methyl.

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ⁵ and R ⁶ are the same.

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ⁵ and R ⁶ are methyl.

In another embodiment, the invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ , together with the carbon atom to which they are attached, form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or the cyclobutane ring is optionally substituted with one R ^b ; and R ⁵ and R ⁶ are independently selected from C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 aryl-C _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 aryl-C _1-4 alkylene is optionally substituted with one R ^b . In another embodiment, R ⁵ and R ⁶ are independently selected from methyl, ethyl, cyclopropyl and benzyl, wherein each methyl, ethyl, cyclopropyl and benzyl is optionally substituted with one R ^b .

In another embodiment of the invention there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ are independently selected from C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 aryl-C _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 aryl-C _1-4 alkylene is optionally substituted with one R ^b ; and R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or the cyclobutane ring is optionally substituted with one R ^b . In another embodiment of the invention R ³ and R ⁴ are independently selected from methyl, ethyl, cyclopropyl and benzyl, wherein each methyl, ethyl, cyclopropyl and benzyl is optionally substituted with one R ^b .

In another embodiment of the invention there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ together with the carbon atom to which they are attached form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or cyclobutane ring is optionally substituted with one R ^b ; and R ⁵ and R ⁶ together with the carbon atom to which they are attached form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or cyclobutane ring is optionally substituted with one R ^b .

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein each R ^b is independently methyl or ethyl.

In another embodiment, the invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ³ , R ⁴ , R ⁵ and R ⁶ are the same. In another embodiment, R ³ , R ⁴ , R ⁵ and R ⁶ are each methyl or cyclopropyl. In another embodiment, R ³ , R ⁴ , R ⁵ and R ⁶ are each methyl.

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, having formula (II):

II

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, having formula (III):

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III

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, having formula (IV):

O

IV

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof having formula (V):

V

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein said compound is selected from:

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- 6 049099

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or a pharmaceutically acceptable salt thereof.

In another embodiment of the invention, a compound of the formula is provided:

or a pharmaceutically acceptable salt thereof.

In another embodiment of the invention, there is provided the use of a pharmaceutical composition for the treatment of HIV infection, comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

In another embodiment of the invention there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the treatment of HIV infection, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In another embodiment of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, said compound being

or a pharmaceutically acceptable salt thereof. In another embodiment of the invention, said compound is

or a pharmaceutically acceptable salt thereof. In another embodiment of the invention, said compound

implementation is one variant <

or a pharmaceutically acceptable salt thereof. In another embodiment of the invention, said compound is or a pharmaceutically acceptable salt thereof. In another embodiment of the invention, said compound is

or a pharmaceutically acceptable salt thereof. In another embodiment of the invention, said compound

is or a pharmaceutically acceptable salt thereof. In another embodiment of the invention, said compound is

or a pharmaceutically acceptable salt thereof. In another embodiment of the invention, said compound is a pharmaceutically acceptable salt.

or his

In another embodiment of the invention, there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of HIV infection in humans.

Other embodiments may be set forth in the following detailed description of embodiments, and in part may be obvious from the description of the claimed embodiments, or may be learned by practice. They may be realized and achieved by means of the processes and compositions particularly pointed out in the description and claims thereof. The foregoing Summary of the Invention is to be considered as a brief and general summary of some embodiments described in the present invention, and is not intended to limit in any way the scope or range of equivalents to which the appended disclosure is legally entitled.

- 11 049099 mule invention.

Detailed description

In the following description, certain specific details are set forth to provide a thorough understanding of the various embodiments described herein. However, it will be apparent to one skilled in the art that the embodiments described herein may be practiced without these details. The following description of several embodiments is made with the understanding that the present description is to be considered as an example of the claimed subject matter and is not intended to limit the appended claims to the specific embodiments illustrated. The headings used in the present description are provided for convenience only and are not to be construed as limiting the claims in any way. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

Definitions.

Unless the context otherwise requires, throughout this description and claims, the word "comprise" and its variations such as "comprises" and "containing" are to be construed in an open, inclusive sense, that is, as including but not limiting.

Reference throughout this specification to one embodiment or embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment described herein. Thus, phrases in one embodiment or embodiment in various places throughout this specification do not necessarily all refer to the same embodiment. In addition, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The term "about" or "approximately" when used in connection with an amount includes the stated value and has the meaning determined by the context (e.g., includes the degree of error associated with measuring a particular amount). The term "administering" as used herein refers to administering a composition to a subject to deliver an agent that is or is contained in the composition to a target site or site to be treated. Those skilled in the art will recognize a variety of routes that can be used to administer to a subject, such as a human, under appropriate circumstances. For example, in some embodiments, administration can be parenteral. In some embodiments, administration can be by injection (e.g., intramuscular, intravenous, or subcutaneous injection). In some embodiments, administration can involve only a single dose. In some embodiments, administration can involve the use of a fixed number of doses. In some embodiments, administration can involve intermittent (e.g., multiple doses separated by time) and/or periodic dosing (e.g., individual doses separated by a common period of time). In some embodiments, administration may involve continuous dosing (eg, perfusion) for at least a selected period of time. The term alkyl refers to a straight or branched chain hydrocarbon radical that is saturated, containing from one to twelve carbon atoms (C ₁ . ₁₂ alkyl), in some embodiments from one to eight carbon atoms (O _1-8 alkyl) or from one to six carbon atoms (C _{1 6} alkyl), from one to four carbon atoms (C ^ alkyl), or from five to eight carbon atoms (C 5 ₈ alkyl), and which is attached to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (tert-butyl), n-pentyl, hexyl, 3-methylhexyl, 2-methylhexyl, and the like.

Alkylene refers to a saturated hydrocarbon radical with a straight or branched chain having from one to twelve carbon atoms (N-N-alkylene), in some embodiments from one to eight carbon atoms (N-N-alkylene) or from one to six carbon atoms (N-N-alkylene) or from one to four carbon atoms (O-N-alkylene), and which is divalent. Examples include methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), 1-methylethylene (-CH( _{CH3 ) CH2-} ), _butylene ( _{-CH2CH2CH2CH2-} ), 1-methylpropylene (-CH( _CH3 )CH2CH2-), 1,1-dimethylethylene (-C(CH3 _{)2CH2- )} and 1,2-dimethylethylene (-CH( _CH3 )CH( _CH3 )-). Unless otherwise stated, the definitions of propylene and butylene include all possible isomeric forms of the groups in question with the same number of carbon atoms. Thus, for example, propylene also includes 1-methylethylene, and butylene includes 1-methylpropylene, 1,1-dimethylethylene, and 1,2-dimethylethylene.

The term amino refers to the radical -NH2.

The term antiviral agent as used herein means an agent (compound or biological agent) that is effective in inhibiting the formation and/or replication of a virus in a subject, such as a human, including, but not limited to, agents that affect either the host or the viral mechanisms necessary for the formation and/or replication of a virus in a subject, such as a human.

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Aryl refers to a single aromatic ring or a bicyclic or polycyclic ring. For example, an aryl group can have from 6 to 20 carbon atoms, from 6 to 14 carbon atoms, or from 6 to 12 carbon atoms. Aryl includes a phenyl radical or an ortho-, spirocyclo-, or bridged bicyclic or polycyclic radical containing from about 9 to 14 carbon atoms in which at least one ring is aromatic (e.g., an aryl fused to one or more aryls or carbocycles). Such bicyclic or polycyclic rings can be optionally substituted with one or more (e.g., 1, 2, or 3) oxo groups on any carbocyclic portion of the bicyclic or polycyclic ring. It should be understood that the point of attachment of a bicyclic or polycyclic radical, as defined above, can be at any position on the ring, including the aryl or carbocyclic portion of the ring. Typical examples of aryl groups include, without limitation, phenyl, indenyl, naphthyl, 1, 2, 3, 4-tetrahydronaphthyl, anthracenyl, and the like.

Arylalkylene refers to an alkylene radical, as defined herein, that is bonded to an aryl radical, as defined herein. Accordingly, the point of attachment of the arylalkylene radical is an alkylene group. The alkylene group of an arylalkylene typically contains from 1 to 6 carbon atoms (i.e., aryl-C _1-6 -alkylene). Arylalkylene groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like. An arylalkylene group typically contains from 6 to 20 carbon atoms, for example, the alkylene portion of an arylalkylene group contains from one to six carbon atoms and the aryl portion contains from five to fourteen carbon atoms. Arylcycloalkylene refers to a cycloalkylene radical, as defined herein, that is bonded to an aryl radical, as defined herein. Accordingly, the point of attachment of the arylcycloalkylene radical is the cycloalkylene group. The cycloalkylene group of an arylcycloalkylene typically contains from 3 to 7 carbon atoms (i.e., arylC _3-7 -cycloalkylene).

The term "bicycloalkyl" as used herein refers to a hydrocarbon radical containing a specified number of carbon atoms and two rings, wherein the rings may be bridged, fused, or spirocyclic with respect to one another. In some embodiments, a bicycloalkyl has seven to twelve carbon atoms, six to twelve carbon atoms, six to ten carbon atoms, or five to ten carbon atoms, is saturated, and is attached to the rest of the molecule by a single bond. A bridged bicycloalkyl will have two rings connected through two common atoms that are not adjacent. A fused bicycloalkyl will have two rings connected by a common bond. A spirocyclic bicycloalkyl (referred to as spirocycloalkyl) will have two rings connected through one common atom. For example, bicycloheptyl can be bridged bicycloheptyl, fused bicycloheptyl, or spirocycloheptyl, respectively, as shown below.

Bridged refers to a carbocyclic or heterocyclic ring structure described herein that contains two non-adjacent ring atoms that are linked through a single atom or divalent group. Examples of bridged bicyclic compounds are bicyclo[2,2,1]heptane and 1,4-diazabicyclo[2,2,2]octane, shown below:

The bridged ring structure can be polycyclic (i.e. bicyclic, tricyclic, etc.).

By combination therapy, as used herein, is meant those instances in which a subject is simultaneously exposed to two or more therapeutic or prophylactic regimens (e.g., two or more therapeutic or prophylactic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all doses of the first regimen are administered before any doses of the second regimen are administered); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, administering the combination therapy may involve administering one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents or active fragments thereof may be administered together in a combination composition or even in a combination compound (e.g., as part of a single chemical complex or covalent compound).

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A prefix such as C _uv or (C _u -C _v ) indicates that the following group contains from and to v carbon atoms. For example, the term C _1-6 alkyl indicates that the alkyl group has from one to six carbon atoms.

A carbocyclic ring refers to a non-aromatic hydrocarbon ring having three to fifteen carbon atoms, in some embodiments having three to ten carbon atoms, or three to seven carbon atoms, or three to six carbon atoms, and which is saturated or partially unsaturated and is attached to the rest of the molecule by a single bond. Carbocyclic rings include, for example, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, 1,3-cyclohexadiene, 1,4-cyclohexadiene, cycloheptane, cycloheptene, and cyclooctane.

Cycloalkyl refers to a non-aromatic cyclic hydrocarbon radical that is saturated, has from three to fifteen carbon atoms (C-C cycloalkyl), in certain embodiments, has from three to ten carbon atoms (C-C cycloalkyl), from three to seven carbon atoms (C-C cycloalkyl), from three to six carbon atoms (C _3-6 cycloalkyl), or from five to seven carbon atoms (C-C cycloalkyl), and which is attached to the rest of the molecule by a single bond. Cycloalkyl includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

The term cycloalkylene as used herein refers to a non-aromatic cyclic hydrocarbon radical having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent cycloalkane (i.e., it is divalent). Accordingly, the cyclobutylene radical includes:

a cycloalkylene group which may have from three to fifteen carbon atoms, from three to ten carbon atoms, from three to seven carbon atoms, or from three to six carbon atoms. As used herein, cycloalkylene may be saturated or partially unsaturated. Cycloalkylene groups include, for example, cyclopropylene, cyclobutylene, cyclopentylene, cyclopentenylene, cyclohexylene, cyclohexenylene, 1,3-cyclohexadienylene, 1,4-cyclohexadienylene, cycloheptylene, cycloheptenylene, and cyclooctylene.

The term "dosage form" as used herein refers to a physically discrete unit of an active agent (e.g., a therapeutic, prophylactic, or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of the active agent. In some embodiments, such amount is a unit dose (or integral fraction thereof) suitable for administration according to a dosage regimen that has been determined to correlate with a desired or beneficial result when administered to the appropriate population (i.e., a prophylactic or therapeutic dosage regimen). Those skilled in the art will recognize that the total amount of a composition or agent administered to a particular subject is determined by one or more attending physicians and may involve the administration of multiple dosage forms.

Fused refers to a ring structure described herein that contains carbocyclic, heterocyclic, aromatic, and/or heteroaromatic rings that are linked through two adjacent atoms. For example, the bicyclic compounds depicted below include cyclopropane fused to cyclohexane, pyrrolidine fused to benzene, and thiene fused to furan, respectively:

The fused ring structure can be polycyclic (i.e. bicyclic, tricyclic, etc.).

The term halo or halogen refers to bromine, chlorine, fluorine and iodine.

The term haloalkyl refers to an alkyl as defined above in which one or more hydrogen atoms are each replaced by a halogen. For example, a haloalkyl is an alkyl wherein one or more hydrogen atoms are replaced by a halogen substituent. This ranges from one halogen substituent on the alkyl group to complete halogenation of the alkyl group. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like.

The term heterocyclyl or heterocyclic ring refers to a non-aromatic radical or ring containing from three to fifteen atoms, wherein one to six of the atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, and are attached to the rest of the molecule by a single bond. In some embodiments, a heterocyclyl contains from three to

- 14 049 099 ten atoms, wherein one to four atoms are heteroatoms selected from nitrogen, oxygen and sulfur, or three to seven atoms, wherein one to two atoms are heteroatoms selected from nitrogen, oxygen and sulfur. The nitrogen, carbon or sulfur atoms in a heterocyclyl may be optionally oxidized; the nitrogen atom may be optionally quaternized. The terms heterocyclyl or heterocyclic ring as used herein refer to rings that are saturated unless otherwise indicated, for example, in some embodiments, the term heterocyclyl or heterocyclic ring refers to rings that are saturated or partially saturated, if indicated. Examples of such heterocyclyl include, but are not limited to, dioxolanyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl and 1,1-dioxothiomorpholinyl.

The term hydroxy or hydroxyl refers to the radical -OH.

The term "HIV replication inhibitor" as used herein is intended to mean an agent that is capable of reducing or eliminating the ability of HIV to replicate in a host cell, whether in vitro, ex vivo, or in vivo. The term "HBV replication inhibitor" as used herein is intended to mean an agent that is capable of reducing or eliminating the ability of HBV to replicate in a host cell, whether in vitro, ex vivo, or in vivo.

The term mammal includes humans and animals, both domestic, such as laboratory animals and companion animals (e.g., cats, dogs, pigs, cattle, sheep, goats, horses, rabbits), and non-domestic, such as wild animals, etc.

The term optional or optional means that the event or circumstance described below may or may not occur, and that the description includes instances where the stated event or circumstance occurs and instances where it does not. For example, optionally substituted heterocyclyl means that the heterocyclyl radical may or may not be substituted, and that the description includes both substituted heterocyclyl radicals and heterocyclyl radicals that contain no substitution.

The term oxo refers to the =O substituent.

A pharmaceutical composition refers to a composition of a compound of the embodiments described herein and a medium generally accepted in the art for delivering a biologically active compound to mammals, such as humans. Such a medium includes all pharmaceutically acceptable excipients.

A pharmaceutically acceptable excipient includes, without limitation, any adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye/coloring agent, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, emulsifier or other pharmacologically inactive substance that is formulated in combination with the pharmacologically active ingredient of the pharmaceutical composition and is compatible with the other ingredients of the formulation and is suitable for use in humans or domestic animals without excessive toxicity, irritation, allergic reaction, etc.

As used herein, the term pharmaceutically acceptable salt refers to salts of such compounds that are suitable for pharmaceutical use, i.e., salts that are suitable, within the scope of sound medical judgment, for use in contact with human and/or animal tissues without undue toxicity, irritation, allergic reaction, etc., and the effects of which are characterized by a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. have described several pharmaceutically acceptable salts in detail in the Journal of Pharmaceutical Sciences. S. M. Berge et al., J. Pharma. Sci., 66:119 (1977).

Examples of pharmaceutically acceptable salts of the compounds described herein include salts derived from an appropriate base, such as an alkali metal (e.g., sodium), an alkaline earth metal (e.g., magnesium), ammonium, and NX ₄ ⁺ (where X is C _{1 4} alkyl). Pharmaceutically acceptable salts of a nitrogen atom or an amino group include, for example, salts of organic carboxylic acids such as acetic, trifluoroacetic, adipic, ascorbic, aspartic, butyric, camphoric, cinnamic, citric, digluconic, glutamic, glycolic, glycerophosphoric, formic, hexanoic, benzoic, lactic, fumaric, tartaric, maleic, hydroxymaleic, malonic, malic, mandelic, isethionic, lactobionic, nicotinic, oxalic, pamoic, pectic, phenylacetic, 3-phenylpropionic, pivalic, propionic, pyruvic, salicylic, stearic, sulfanilic, tartaric, undecanoic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, camphorsulfonic, mesitylenesulfonic, benzenesulfonic, p-toluenesulfonic, naphthalenesulfonic and 2-naphthalenesulfonic acids; and inorganic acids such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric and sulfamic acids. Pharmaceutically acceptable salts of a hydroxyl compound

- 15 049099 groups comprise an anion of the specified compound in combination with a suitable cation such as Na ⁺ and NX4' (where X is independently selected from H or a ^^alkyl group).

For therapeutic use, salts of the active ingredients of the compounds described herein will generally be pharmaceutically acceptable, i.e. they will be salts derived from a physiologically acceptable acid or base. However, salts of acids or bases that are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of a compound of formula I or another compound of the embodiments described herein. All salts, whether or not derived from a physiologically acceptable acid or base, are within the scope of the embodiments described herein.

Metal salts are typically prepared by reacting a metal hydroxide with a compound according to the embodiments described herein. Examples of metal salts that have been prepared in this manner are salts containing Li ⁺ , Na ⁺ and K ⁺ . A more soluble metal salt can be precipitated from a solution of a more soluble salt by adding a suitable metal compound.

In addition, salts can be formed by adding an acid, particularly certain organic and inorganic acids, such as HCl, HBr, _H2SO4 , _H3PO4 , or organic sulfonic acids, to basic centers, typically amines. Finally, it should be understood that the compositions of the present document contain the compounds described herein in their non-ionized as well as zwitterionic form.

The term prophylaxis or prevention means any treatment of a disease or condition that prevents the development of clinical symptoms of the disease or condition. In some embodiments, the compositions can be administered to a subject (including a human) who is at risk for the disease or condition. As used herein, the term prophylaxis encompasses the administration of a compound, composition, or pharmaceutically acceptable salt according to the embodiments described herein before or after an individual has been exposed to HIV or HBV, but before symptoms of HIV infection or HBV infection appear and/or before the virus is detected in the blood. The terms also refer to preventing the onset of symptoms of the disease and/or preventing the achievement of detectable levels in the blood. The terms include both pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP), and event-triggered prophylaxis or on-demand prophylaxis. The terms also refer to the prevention of perinatal mother-to-child transmission of HIV by administration to the mother before birth and to the child during the first days of life. The terms also refer to the prevention of HIV transmission by blood transfusion.

Spiro or spirocyclo refers to a ring structure described herein that has two ring structures, each of which may be carbocyclic or heterocyclic, that are linked through a single common atom (this atom is referred to as a spiro atom). Accordingly, spirocyclo refers to a spirocycloalkyl radical or a spiroheterocycloalkyl radical. Dispiro or dispirocyclo refers to a ring structure described herein that has three ring structures, each of which may be carbocyclic or heterocyclic, that are linked through two spiro atoms.

The term stereoisomer refers to a compound consisting of the same atoms that are connected by the same bonds, but have different three-dimensional structures that are not interchangeable. The present invention provides various stereoisomers and mixtures thereof and includes enantiomers, which refer to two stereoisomers whose molecules are non-coincident mirror images of each other. In any of the embodiments described herein, the compounds described herein may be in the form of their stereoisomer.

As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the human subject is an adult, an adolescent, or a child. In some embodiments, the subject suffers from a relevant disease or condition. In some embodiments, the subject is at risk for the disease or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease or condition. In some embodiments, the subject does not exhibit any symptoms or signs of the disease or condition. In some embodiments, the subject is a human with one or more characteristics characteristic of a predisposition to or risk for the disease or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual being diagnosed and/or treated and/or prevented.

As used herein, the term therapeutically effective amount is an amount that produces the desired effect for which the drug is administered. In some embodiments, the term therapeutically effective amount or therapeutically effective dose means an amount that is sufficient when administered per

- 16 049099 a patient suffering from or susceptible to developing a disease or condition according to a therapeutic dosing regimen for treating the disease or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and/or severity, stabilizes one or more characteristics, and/or delays the onset of one or more symptoms of the disease or condition. Those skilled in the art will recognize that the term therapeutically effective amount does not actually require that the treatment be successful in a particular individual. Rather, a therapeutically effective amount may be an amount that provides a specific desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, the term therapeutically effective amount may refer to an amount measured in one or more specific tissues (e.g., tissue affected by the disease or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will recognize that in some embodiments, a therapeutically effective amount may be formulated and/or administered as a single dose. In some embodiments, a therapeutically effective amount may be formulated and/or administered as multiple doses, such as part of a dosing regimen.

The term treatment or treatment is an approach to achieving beneficial or desired results, including clinical results. Beneficial or desired clinical results may include one or more of the following: a) inhibition of a disease or condition (e.g., alleviation of one or more symptoms resulting from a disease or condition and/or reduction in the extent of the disease or condition); b) slowing or halting the progression of one or more clinical symptoms associated with a disease or condition (e.g., stabilization of a disease or condition, prevention or delay in the worsening or progression of a disease or condition, and/or prevention or slowing of the spread (e.g., metastasis) of a disease or condition); and/or c) alleviation of a disease, i.e. initiating a reversal of clinical symptoms (e.g., alleviating the course of a disease, providing partial or complete remission of a disease or condition, enhancing the effect of another drug, slowing the progression of a disease, improving quality of life, and/or increasing survival. The terms "treating" and "treating" as used herein refer to administering a compound, composition, or pharmaceutically acceptable salt according to the embodiments described herein to alleviate or eliminate the symptoms of HIV infection or HBV infection and/or to reduce the viral load in a subject in need thereof. In some embodiments, the subject is a patient.

As used herein, the term tricycloalkyl refers to a hydrocarbon radical containing a specified number of carbon atoms and three rings, wherein the rings may be bridged, fused, spirocyclic, or combinations thereof relative to one another. In some embodiments, the tricycloalkyl has ten to sixteen carbon atoms or nine to twelve carbon atoms, is saturated, and is attached to the rest of the molecule by a single bond. For example, tricyclodecyl can be a bridged tricyclodecyl, a fused tricyclodecyl, a dispirocyclodecyl, or a mixed (e.g., bridged-fused) tricyclodecyl, respectively, as shown below:

Embodiments described herein also encompass all pharmaceutically acceptable compounds of formula I that are isotopically labeled by replacing one or more atoms with an atom having a different atomic mass or mass number. Examples of isotopes that can be included in the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as ^2H , ^3H , 11C ^{, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 36Cl , 123I , and 125I , respectively . In some embodiments ,} these radiolabeled compounds are useful for assisting in determining or measuring the efficacy of the compounds by characterizing, for example, the site or mode of action or the binding affinity to ^a pharmacologically important site of function. Certain isotopically labeled compounds of formula I, such as those incorporating a radioactive isotope, find use in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. ³ H, and carbon-14, i.e. ¹⁴ C, are particularly useful for this purpose because of their ease of incorporation and ease of detection.

In some embodiments, substitution with heavier isotopes such as deuterium, i.e. ² H, may provide certain therapeutic advantages as a result of higher metabolic stability. For example, the in vivo half-life may be increased or the dosage requirements may be reduced. Thus, in some circumstances it may be preferable to

- 17 049099 heavier isotopes are active.

Substitution with positron-active isotopes such as ^nC , ^18F , ^15O and ^13N can be used in positron emission tomography (PET) studies to study receptor occupancy by a substrate. Isotopically labeled compounds of formula I can be prepared by methods known to those skilled in the art or by methods analogous to those described in the examples set forth below, using an appropriate isotopically labeled reagent in place of the previously used unlabeled reagent.

The methods, compositions, kits, and articles of manufacture provided herein utilize or include compounds (e.g., a compound of Formula I) or pharmaceutically acceptable salts thereof in which from 1 to n hydrogen atoms attached to a carbon atom may be replaced by a deuterium atom, or D, where n is the number of hydrogen atoms in the molecule. As is known in the art, a deuterium atom is a non-radioactive isotope of hydrogen. Such compounds may enhance resistance to metabolism and thus may be useful for prolonging the half-life of the compounds described herein or pharmaceutically acceptable salts, isomers, or mixtures thereof when administered to a mammal. See, e.g., Allen B. Foster, Deuterium Isotope Effects in Studies of Drug Metabolism, 5 Trends Pharmacol. Sci. 524, 524-27 (1984). Such compounds can be synthesized by methods well known in the art, for example, using starting materials in which one or more hydrogen atoms have been replaced by deuterium.

Embodiments described herein also contemplate the inclusion of in vivo metabolic products of the disclosed compounds. Such products may be formed, for example, by oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound, especially by enzymatic processes. Accordingly, embodiments described herein include compounds produced by a method comprising administering a compound according to embodiments described herein to a mammal for a period of time sufficient to produce its metabolic product. Such products are typically identified by administering a radiolabeled compound according to embodiments described herein in a detectable dose to an animal, such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism and recovery of its metabolites from urine, blood, or other biological samples.

The compounds of the embodiments described herein, or pharmaceutically acceptable salts thereof, may contain one or more asymmetric centers and thus may give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined in terms of absolute stereochemistry as (R)-, or (S)-, or as (D)-, or (L)for amino acids. The present invention is intended to include all such possible isomers, as well as their racemic, scalemic, and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or separated using methods such as chromatography and fractional crystallization. Methods for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise indicated, the compounds are intended to include both the E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

Connections.

Provided herein are compounds that act as anti-HIV agents or anti-HBV agents, pharmaceutical compositions comprising such compounds, optionally in combination with one or more (e.g., two, three, or four) additional therapeutic agents, and methods of using such compounds and compositions. All embodiments of the compounds described herein include any pharmaceutically acceptable salt, stereoisomer, or mixture of stereoisomers thereof.

In one embodiment, compounds having the following formula (I) or pharmaceutically acceptable salts thereof are provided:

- 18 049099

I where R ¹ and R ² are independently selected from C _1-12 alkyl, aryl-C _1-4 alkylene, C _3-7 cycloalkyl, C _3-7 cycloalkyl-C _1-4 alkylene, aryl-C _3-7 cycloalkylene, C _7-12 spirocycloalkyl, C _7-12 spirocycloalkyl-C _1-4 alkylene, bridged C _5-10 bicycloalkyl, bridged C _5-10 bicycloalkyl-C _1-4 alkylene, condensed C _5-10 bicycloalkyl, C _10-16 dispirocycloalkyl, C _10-16 dispirocycloalkyl-C _1-4 alkylene, bridged C _9-12 tricycloalkyl, bridged C _9-12 tricycloalkyl-C _1-4 alkylene, C _3-7 cycloalkyl-C _3-7 cycloalkylene and a 5-7 membered monocyclic heterocyclyl having from 1 to 3 heteroatoms selected from N, O and S, wherein each C _1-12 alkyl, aryl-C _1-4 alkylene, C _3-7 cycloalkyl, C _3-7 cycloalkyl-C _1-4 alkylene, aryl-C _3-7 cycloalkylene, C _7-12 spirocycloalkyl and C _7-12 spirocycloalkyl-C _1-4 alkylene is optionally substituted with one to three R ^a ;

R ³ , R ⁴ , R ⁵ and R ⁶ are independently selected from C _1-4 alkyl, C _3-6 cycloalkyl and aryl-C _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl and aryl-C _1-4 alkylene is optionally substituted with one to three R ^b ; or optionally:

R ³ and R ⁴ , together with the carbon atom to which they are attached, form a 3- to 6-membered saturated or partially unsaturated carbocyclic ring optionally substituted with one to three R ^b ; and R ⁵ and R ⁶ are independently selected from C _1-4 alkyl, C _3-6 cycloalkyl and aryl-C _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl and aryl-C _1-4 alkylene is optionally substituted with one to three R ^b ; or

R ³ and R ⁴ are independently selected from C _1-4 alkyl, C _3-6 cycloalkyl and aryl-C _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl and aryl-C _1-4 alkylene is optionally substituted with one to three R ^b ; and R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a 3- to 6-membered saturated or partially unsaturated carbocyclic ring optionally substituted with one to three R ^b ; or

R ³ and R ⁴ , together with the carbon atom to which they are attached, form a 3- to 6-membered saturated or partially unsaturated carbocyclic ring optionally substituted with one to three R ^b ; and R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a 3- to 6-membered saturated

R ^b ;

or a partially unsaturated carbocyclic ring optionally substituted with one to three

-R ⁷ HN

N

B represents

N or

-R ⁷ N _X R ⁸ N

R ⁷

R ⁸

L ¹

L ² is hydrogen or R ⁸ ; is -L ¹ -(L ² )m-(L ³ )nR ^8a ; selected from a bond, -C(O)- and -C(O)O-; is C _1-6 alkylene;

is -C(O)O- or

aryl, -C(O)-aryl, -C(O)-C _1-4 alkyl, ^-C(O)-C _1-4 alkyl, o

L ³

R ^8a is selected from C _1-12 alkyl,

_R c _; ' wherein aryl and -C(O)-aryl are optionally substituted with one or two each R ^a each R ^b each R ^c is independently selected from C _1-4 alkyl, halogen, C _1-4 haloalkyl and -O-C _1-4 alkyl;

independently represents C^alkyl;

independently represents C _1-4 alkyl or -OC(O)-C _1-4 alkyl;

m and n are independently equal to 0 or 1; and p is equal to 0, 1, or 2.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, B is

In some embodiments, B is

In some embodiments, the compound of formula I is a compound of formula (II):

NHR ⁷

or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are as defined in formula I.

In some embodiments, the compound of formula I is a compound of formula (III):

^R ⁷

III, or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are as defined in formula I.

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, R ¹ and R ² are independently selected from C 1-8 α-alkyl, aryl-C 1-4 alkylene, C 3-7 cycloalkyl, C 3-7 cycloalkyl-C 1-4 alkylene, aryl-C 3-7 cycloalkylene, C 7-12 spirocycloalkyl, C 7-12 spirocycloalkyl-C 1-4 alkylene, bridged C 5-10 bicycloalkyl, bridged C 5-10 bicycloalkyl-C 1-4 alkylene, fused C 5-10 bicycloalkyl, C 10-16 dispirocycloalkyl, C 10-16 dispirocycloalkyl-C 1-4 alkylene, bridged C 9-12 tricycloalkyl, bridged C 9-12 tricycloalkyl-C 1-4 alkylene, C3-7cycloalkyl-C3-7cycloalkylene and a 5-7-membered monocyclic heterocyclyl having from 1 to 3 heteroatoms selected from N, O and S, wherein each C|-8an<i.t.aryl-C1-4alkylene, C3-7cycloalkyl, C3-7cycloalkyl-C1-4alkylene, aryl-C3-7cycloalkylene, C7-12spirocycloalkyl and C7-12spirocycloalkyl-C1-4alkylene is optionally substituted with one to three R ^a , wherein R ^a is as defined above. In some embodiments _, R ¹ and R ² are independently selected from C _1-8 alkyl, aryl-C _1-4 alkylene, C _3-7 cycloalkyl, _{C 3-7 cycloalkyl} -C _1-4 alkylene, aryl-C _3-7 cycloalkylene, _{C 7-12 spirocycloalkyl} , _{C 7-12} spirocycloalkyl- _{C 1-4} alkylene, bridged C _5-10 bicycloalkyl _- C _1-4 alkylene, fused C _{5-10 bicycloalkyl, C 10-16 dispirocycloalkyl} -C _1-4 alkylene, bridged C _9-12 tricycloalkyl-C _1-4 alkylene, C _3-7 cycloalkyl- _{C 3-7 cycloalkylene ,} and 5-7 membered monocyclic heterocyclyl having from 1 to 3 heteroatoms selected from N, O and S, wherein each C _1-8 alkyl, aryl-C _1-4 alkylene, C _3-7 cycloalkynal, C _3-7 cycloalkyl-C _1-4 alkylene, aryl- _{C 3-7 cycloalkylene} , C _7-12 spirocycloalkyl and _{C 7-12 spirocycloalkyl} -C _1-4 alkylene is optionally substituted with one to three R ^a , wherein R ^a is as defined above. In some embodiments, R ¹ and R ² are independently selected from C1-8 alkyl, C3-7 cycloalkyl, C3-7 cycloalkyl-C1-4 alkylene, C7-12 spirocycloalkyl, C7-12 spirocycloalkyl-C1-4 alkylene, and a bridged C5-10 bicycloalkyl-C1-4 alkylene, wherein each C1-8 alkyl, C3-7 cycloalkyl, C3-7 cycloalkyl-C1-4 alkylene, C7-12 spirocycloalkyl, and C7-12 spirocycloalkyl-C1-4 alkylene is optionally substituted with one to three R ^a , wherein R ^a is as defined above. In some embodiments _, R ¹ and R ² are independently selected from C5-8 alkyl, C _5-7 cycloalkyl, C _5-7 cycloalkyl-C _1-4 alkylene, C _7-9 spirocycloalkyl, C _7-9 spirocycloalkyl-C _1-4 alkylene, and a bridged C _5-7 bicycloalkyl-C _1-4 alkylene, wherein each C _5-8 alkyl, C _5-7 cycloalkyl, C _5-7 cycloalkyl-C _1-4 alkylene, C _7-9 spirocycloalkyl, and _{C 7-9 spirocycloalkyl} -C _1-4 alkylene is optionally

- 20 049099 is placed from one to three Ra, where Ra corresponds to the definition above.

In some embodiments of a compound of formula I, II, or III, or a pharmaceutically acceptable salt thereof, R ¹ and R ² are different. In some embodiments, R ¹ and R ² are the same. In some embodiments, R ¹ and R ² are the same and are independently selected from C 1-8 alkyl, aryl-N-alkylene, C dacloalkyl, C 3-7 cycloalkyl-C 1-4 alkylene, aryl-N-cycloalkylene, C 7-12 spirocycloalkyl, C 7-12 spirocycloalkyl-C 1-4 alkylene, bridged C 5-10 bicycloalkyl-C 1-4 alkylene, fused-N-N-bicycloalkyl, C 10-16 dispirocycloalkyl-C 1-4 alkylene, bridged C 9-12 tricycloalkyl-N-N alkylene, C 3-7 cycloalkyl-C 3-7 cycloalkylene, and a 5-7 membered monocyclic heterocyclyl having from 1 to 3 heteroatoms selected from N, O, and S, wherein each C 1-8 alkyl, C 3-7 cycloalyl, C3-7cycloalkyl-C1-4alkylene, C7-12spirocycloalkyl, and C7-12spirocycloalkyl-C1-4alkylene are optionally substituted with one to three Ra, wherein Ra is as defined above. In some embodiments, R ¹ and R ² are the same and are selected from n-alkyl, n-cycloalkyl, n-cycloalkyl-C1-4alkylene, n-spirocycloalkyl, n-spirocycloalkyl-C1-4alkylene, and a bridged C5-106cycloalkyl-C1-4alkylene, wherein each n-alkyl, n-cycloalkyl, _{C3-7cycloalkyl} - _C1-4alkylene , _{C7-12spirocycloalkyl} , and n-spirocycloalkyl-C1-4alkylene is optionally substituted with one to three Ra, wherein ^Ra is as defined above. In some embodiments, R ¹ and R ² are the same and are selected from C3-7cycloalkyl-C1-4alkylene, C-N-spirocycloalkyl, C7-12spirocycloalkyl-C1-4alkylene, and bridged C-N-bicycloalkylidealkylene, wherein each C3-7cycloalkyl-C1-4alkylene, C7-12spirocycloalkyl, and bridged C-N-spirocycloalkylidealkylene is optionally substituted with one to three Ra, wherein Ra is as defined above. In some embodiments, R ¹ and R ² are the same and are selected from C1-8 alkyl and C-N-cycloalkyl, wherein each _C1-8 alkyl and C-N-cycloalkyl is optionally substituted with one to three ^Ra , wherein ^Ra is as defined above.

In some embodiments of a compound of Formula I, II or III or a pharmaceutically acceptable salt thereof, R ¹ and R ² are independently selected from

In some embodiments of the compound of formula I, II or III or a pharmaceutically acceptable salt thereof, R ¹ is selected from

- 21 049099

In some embodiments of the compound of formula I, II or III or a pharmaceutically acceptable salt thereof, R ² is selected from

- 22 049099

In some embodiments, R ¹ and R ² are the same and are selected from °1 V— Ч νγ _

VT y „ h <^Л t h

Chu 4 ^;A h h h h

In some embodiments, R ¹ and R ² are the same and are selected from

In some embodiments,

R ¹ and R ² are the same and are selected from >> - In some embodiments, R ¹ and R ² are the same and are selected from F

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, ^R3 , ^R4 , ^R5 , and ^R6 are independently selected from _C1-3alkyl , _{C3-5cycloalkyl} , and aryl- _C1-4alkylene , wherein each _C1-3alkyl , C1-4cycloalkyl, and aryl- _C1-4alkylene is optionally substituted with one to three ^Rb , wherein ^Rb is as defined above.

- 23 049099 In some embodiments, R ³ , R ⁴ , R ⁵ and R ⁶ are independently selected from methyl, ethyl, cyclopropyl and benzyl, wherein each methyl, ethyl, cyclopropyl and benzyl is optionally substituted with one to three R ^b , wherein R ^b is as defined above.

In some embodiments of a compound of formula I, II, or III, or a pharmaceutically acceptable salt thereof, R ³ and R ⁵ are the same. In some embodiments, both R ³ and R ⁵ are methyl. In some embodiments, both R ³ and R ⁵ are ethyl. In some embodiments, both R ³ and R ⁵ are cyclopropyl. In some embodiments, both R ³ and R ⁵ are benzyl.

In some embodiments, the compounds of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, R ⁴ and R ⁶ are the same. In some embodiments, both R ⁴ and R ⁶ are ethyl or benzyl. In some embodiments, both ^R4 and ^R6 are methyl. In some embodiments, both R ⁴ and R ⁶ are ethyl. In some embodiments, both R ⁴ and R ⁶ are cyclopropyl. In some embodiments, both R ⁴ and R ⁶ are benzyl.

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, R ³ and R ⁴ are the same. In some embodiments, both R ³ and R ⁴ are methyl. In some embodiments, both R ³ and R ⁴ are ethyl. In some embodiments, both R ³ and R ⁴ are cyclopropyl. In some embodiments, both R ³ and R ⁴ are benzyl.

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, R ⁵ and R ⁶ are the same. In some embodiments, both R ⁵ and R ⁶ are methyl. In some embodiments, both R ⁵ and R ⁶ are ethyl. In some embodiments, both R ⁵ and R ⁶ are cyclopropyl. In some embodiments, both R ⁵ and R ⁶ are benzyl.

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, R ³ and R ⁴ , together with the carbon atom to which they are attached, form a 3-5 membered saturated or partially unsaturated carbocyclic ring optionally substituted with one to three R ^b ; and R ⁵ and R ⁶ are independently selected from C 1-4 alkyl, C 3-6 cycloalkyl, and aryl-C 1-4 alkylene, wherein each C 1-4 alkyl, C 3-6 cycloalkyl, and aryl-C 1-4 alkylene is optionally substituted with one to three R ^b , wherein R ^b is as defined above. In some embodiments, R ³ and R ^4, together with the carbon atom to which they are attached, form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or cyclobutane ring is optionally substituted with one to three R ^b , wherein R ^b is as defined above. In some embodiments, R ³ and R ^4, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted with one to three R ^b . In some embodiments, R ³ and R ⁴ , together with the carbon atom to which they are attached, form a cyclobutane ring optionally substituted with one to three R ^b . In some embodiments, the cyclopropane ring or the cyclobutane ring is unsubstituted. In some embodiments, the cyclopropane ring or the cyclobutane ring is substituted with one R ^b . In some embodiments, the cyclopropane ring or the cyclobutane ring is substituted with two R ^b . In some embodiments, the cyclopropane ring or the cyclobutane ring is substituted with three R ^b . In some embodiments, R ⁵ and R ⁶ are independently selected from C1-3alkyl, _{C3-5cycloalkyl} , and aryl- _C1-4alkylene , wherein each _C1-4alkyl , _{C3-6cycloalkyl} , and aryl- _C1-4alkylene is optionally substituted with one to three R ^b . In some embodiments, ^{R 5} and R ⁶ are independently selected from methyl, ethyl, cyclopropyl, and benzyl, wherein each methyl, ethyl, cyclopropyl, and benzyl is optionally substituted with one to three R ^b . In some embodiments, R ³ and R ^4, together with the carbon atom to which they are attached, form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or cyclobutane ring is optionally substituted with one to three R ^b , and R ⁵ and R ⁶ are independently selected from methyl, ethyl, cyclopropyl, and benzyl, wherein each methyl, ethyl, cyclopropyl, and benzyl is optionally substituted with one to three R ^b .

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, ^R3 and ^R4 are independently selected from _C1-4alkyl , _{C3-6cycloalkyl} , and aryl- _C1-4alkylene , wherein each _C1-4alkyl , _{C3-6cycloalkyl} , and aryl- _C1-4alkylene is optionally substituted with one to three ^Rb ; and ^R5 and ^R6, together with the carbon atom to which they are attached, form a 3-5-membered saturated or partially unsaturated carbocyclic ring optionally substituted with one to three ^Rb , wherein ^Rb is as defined above. In some embodiments, ^R5 and ^R6, together with the carbon atom to which they are attached, form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or the cyclobutane ring is optionally substituted with one to three ^Rb , wherein ^Rb is as defined above. In some embodiments, R ⁵ and R ^6, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted with one to three R ^b . In some embodiments, R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a cyclobutane ring optionally substituted with

- 24 049099 one to three R ^b . In some embodiments, the cyclopropane ring or the cyclobutane ring is unsubstituted. In some embodiments, the cyclopropane ring or the cyclobutane ring is substituted with one R ^b . In some embodiments, the cyclopropane ring or the cyclobutane ring is substituted with two R ^b . In some embodiments, the cyclopropane ring or the cyclobutane ring is substituted with three R ^b . In some embodiments, R ³ and R ⁴ are independently selected from C _1-3 alkyl, C _3-5 cycloalkyl, and aryl-C _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl, and aryl-C _1-4 alkylene is optionally substituted with one to three R ^b ; In some embodiments, ^R3 and ^R4 are independently selected from methyl, ethyl, cyclopropyl and benzyl, wherein each methyl, ethyl, cyclopropyl and benzyl is optionally substituted with one to three ^Rb . In some embodiments, ^R3 and ^R4 are independently selected from methyl, ethyl, cyclopropyl and benzyl, wherein each methyl, ethyl, cyclopropyl and benzyl is optionally substituted with one to three ^Rb ; and ^R5 and ^R6, together with the carbon atom to which they are attached, form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or the cyclobutane ring is optionally substituted with one to three ^Rb .

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, R ³ and R ^4, together with the carbon atom to which they are attached, form a 3- to 5-membered saturated or partially unsaturated carbocyclic ring optionally substituted with one to three R ^b ; and R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a 3- to 5-membered saturated or partially unsaturated carbocyclic ring optionally substituted with one to three R ^b ; In some embodiments, R ³ and R ⁴ , together with the carbon atom to which they are attached, form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or the cyclobutane ring is optionally substituted with one to three R ^b . R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or cyclobutane ring is optionally substituted with one to three R ^b , wherein R ^b is as defined above. In some embodiments, R ³ and R ⁴ , together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted with one to three R ^b . In some embodiments, R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a cyclopropane ring. In some embodiments, R ³ and R ⁴ , together with the carbon atom to which they are attached, form a cyclobutane ring optionally substituted with one to three R ^b . In some embodiments, R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a cyclobutane ring. In some embodiments, R ³ and R ^4, taken together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted with one to three R ^b , and R ⁵ and R ⁶ , taken together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted with one to three R ^b . In some embodiments, R ³ and R ^4, taken together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted with one to three R ^b , and R ⁵ and R ⁶ , taken together with the carbon atom to which they are attached, form a cyclobutane ring optionally substituted with one to three R ^b . In some embodiments, R ³ and R ^4, together with the carbon atom to which they are attached, form a cyclobutane ring optionally substituted with one to three R ^b , and R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted with one to three R ^b . In some embodiments, R ³ and R ^4, together with the carbon atom to which they are attached, form a cyclobutane ring optionally substituted with one to three R ^b , and R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a cyclobutane ring optionally substituted with one to three R ^b .

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, each R ^b is independently methyl, ethyl, propyl, or butyl. In some embodiments, each R ^b is independently methyl, ethyl, or propyl. In some embodiments, each R ^b is independently methyl or ethyl. In some embodiments, each R ^b is methyl. In some embodiments, each R ^b is ethyl.

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, ^R3 , ^R4 , ^R5 , and ^R6 are the same. In some embodiments, ^R3 , ^R4 , ^R5 , and ^R6 are each C6-alkyl, _C3-5 cycloalkyl, or aryl- _C1-4 alkylene, wherein each C6-alkyl, _C3-5 cycloalkyl, or aryl- _C1-4 alkylene is optionally substituted with one to three ^Rb , wherein ^Rb is as defined above. In some embodiments, ^R3 , ^R4 , ^R5 , and ^R6 are each methyl, ethyl, cyclopropyl, and benzyl, wherein each methyl, ethyl, cyclopropyl, and benzyl is optionally substituted with one to three ^Rb , wherein ^Rb is as defined above. In some embodiments, ^R3 , ^R4 , ^R5 , and ^R6 are methyl or cyclopropyl. In some embodiments, each R ³ , R ⁴ , R ⁵ , and R ⁶ is methyl. In some embodiments, each R ³ , R ⁴ , R ⁵ , and R ⁶ is ethyl. In some embodiments, each R ³ , R ⁴ , R ⁵ , and R ⁶ is cyclopropyl. In some embodiments, each R ³ , R ⁴ , R ⁵ , and R ⁶ is

- 25 049099 is benzyl.

In some embodiments of a compound of formula I, II, or III, or a pharmaceutically acceptable salt thereof, R ⁷ is hydrogen. In some embodiments, R ⁷ is R ⁸ .

In some embodiments of the compound of Formula I, II, or III, R ⁸ is -L ¹ L ² -L ³ -R ^8a . In some embodiments, R ⁸ is -L ¹ -(L ³ )mR ^8a . In some embodiments, R ⁸ is -L ¹ -(L ² )mR ^8a . In some embodiments, R ⁸ is -L ¹ -L ³ -R ^8a . In some embodiments, R ⁸ is -L ¹ -L ² -R ^8a . In some embodiments, R ⁸ is -L ¹ -R ^8a . In some embodiments, R ⁸ is R ^8a . In some embodiments, R ⁸ is selected from

In some embodiments, R ⁸ is selected from

- 26 049099

In some embodiments, R ⁸ is selected from

- 27 049099

In some embodiments of a compound of formula I, II, or III, or a pharmaceutically acceptable salt thereof, L ¹ is a bond. In some embodiments, L ¹ is -C(O)-. In some embodiments, L ¹ is -C(O)O-.

In some embodiments of a compound of formula I, II, or III or a pharmaceutically acceptable salt thereof, L ² is selected from -CH2-, -CH2-CH2-, -CH2-CH2-CH3- -CH2-CH2-CH2-CH2- -CH2-C(CH3)2-CH2-CH2-CH2-CH2-CH2-, -CH2-C(CH3)2-CH3- -CH2-CH2-C(CH3)2-, -CH2-C(CH3)2-CH2-CH2-, and -CH2-CH2CH2-C(CH3)2-. In some embodiments, L ² is selected from -CH2-, -CH ₂ -CH ₂ -, -CH ₂ -CH ₂ -CH ₃ -CH2-C(CH3)2-, -CH2-C(CH3)2-CH2-, -CH2-CH2-C(CH3)2-, and -CH2-C(CH3)2-CH2-CH2-. In some embodiments, L ² is -CH2-. In some embodiments, L ² is -CH2-CH ₂ -. In some embodiments, L ² is -CH2-CH2-CH2-. In some embodiments, L ² is -CH2-C(CH ₃ ) ₂ -. In some embodiments, L ² is -CH2-C(CH3)2-CH2-. In some embodiments, L ² is -CH2-CH ₂ -C(CH ₃ ) ₂ -. In some embodiments, L ² is -CH ₂ -C(CH ₃ ) ₂ CH2-CH2-.

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, L ³ is -C(O)O-. In some embodiments, L ³ is

In some embodiments of a compound of Formula I, II, or III or a pharmaceutically acceptable salt thereof, R ^8a is selected from C1. ₁₂ alkyl, aryl,

-C(O)-C ₁ . ₄ alkyl, -SC(O)-C ₁ . ₄ alkyl, wherein aryl is optionally substituted from one embodiment of R ^8a selected from C1. ₈ alkyl, aryl,

up to three R ^c . In some embodiments, -C(O)-C ₁ . ₄ alkyl, -SC(O)-C ₁ . ₄ alkyl, wherein the aryl is optionally substituted with one or two R ^c .

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, each R ^a is independently selected from C1.4αalkyl, halogen, and C1.4halogenalkyl. In some embodiments, each R ^a is independently _C1.4αalkyl . In some embodiments, each R ^a is independently halogen. In some embodiments, each R ^a is independently _{C1.4halogenalkyl} . In some embodiments, each R ^a is independently -O^^alkyl. In some embodiments, each _R ^a is independently selected from methyl, ethyl, n-propyl, isopropyl, fluoro, methoxy, and trifluoromethyl.

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, each R ^b is independently methyl or ethyl.

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, each R ^b is independently C1.4 alkyl. In some embodiments, each R ^c is independently -OC(O)-C1.4 _alkyl . In some embodiments, each R ^c is independently methyl or -OC(O)-methyl.

In some embodiments of a compound of formula I, II, or III, or a pharmaceutically acceptable salt thereof, the indices m and n are both 0. In some embodiments, the index m is 0 and the index n is 1. In some embodiments, the index m is 1 and the index n is 0. In some embodiments, the index m and the index n are 1.

In some embodiments of a compound of formula I, II, or III, or a pharmaceutically acceptable salt thereof, the index p is 0 or 2. In some embodiments, the index p is 0. In some embodiments, the index p is 1. In some embodiments, the index p is 2.

In some embodiments of a compound of Formula I, II, or III or a pharmaceutically acceptable salt thereof, R ¹ and R ² are the same, and R ³ , R ⁴ , R ⁵ , and R ⁶ are the same. In some embodiments, R ¹ and R ² are the same, and R ³ , R ⁴ , R ⁵ , and R ⁶ are each methyl or cyclopropyl. In some embodiments, R ¹ and R ² are the same, and R ³ , R ⁴ , R ⁵ , and R ⁶ are each cyclopropyl. In some embodiments, R ¹ and R ² are the same, and R ³ , R ⁴ , R ⁵ , and R ⁶ are each methyl.

In some embodiments of a compound of Formula I, II, or III or a pharmaceutically acceptable salt thereof, R ⁷ is hydrogen and R ¹ and R ² are the same.

- 28 049099

In some embodiments, R ⁷ is hydrogen and R ³ , R ⁴ , R ⁵ and R ⁶ are the same. In some embodiments, R ⁷ is hydrogen and R ³ , R ⁴ , R ⁵ and R ⁶ are each methyl or cyclopropyl. In some embodiments, R ⁷ is hydrogen and R ³ , R ⁴ , R ⁵ and R ⁶ are each cyclopropyl. In some embodiments, R ⁷ is hydrogen and R ³ , R ⁴ , R ⁵ and R ⁶ are each methyl.

In some embodiments of a compound of Formula I, II, or III, or a pharmaceutically acceptable salt thereof, R ⁷ is hydrogen, R ¹ and R ² are the same, and R ³ , R ⁴ , R ⁵ , and R ⁶ are the same. In some embodiments, R ⁷ is hydrogen, R ¹ and R ² are the same, and R ³ , R ⁴ , R ⁵ , and R ⁶ are each methyl or cyclopropyl. In some embodiments, R ⁷ is hydrogen, R ¹ and R ² are the same, and R ³ , R ⁴ , R ⁵ , and R ⁶ are each cyclopropyl.

In some embodiments, R ⁷ is hydrogen, R ¹ and R ² are the same, and R ³ , R ⁴ , R ⁵ , and R ⁶ are each methyl. In some embodiments, the compound of formula I or II is a compound of formula (IV):

IV or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are defined in formula I.

In some embodiments of the compound of formula IV, R ¹ and R ² are different.

In some embodiments, R ¹ and R ² are the same. In some embodiments, the compound of formula I or III is a compound of formula (V):

^r ⁷

O

V or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , R ⁷ and R ⁸ are defined above.

In some embodiments, compounds of formula VR ¹ and R ² are different. In some embodiments, R ¹ and R ² are the same. In some embodiments, R ⁷ is H. In some embodiments, R ⁷ is R ⁸ .

In some embodiments, the compounds have the formula:

- 29 049099

- 30 049099

- 31 049099

- 32 049099

- 33 049099

O

or are pharmaceutically acceptable salts thereof.

In some embodiments, the compounds have the formula:

- 34 049099

When any variable represents an unsymmetrical divalent group, both orientations of the group must be included unless otherwise stated. For example, if L ³ is -C(O)O-, both -C(O)O- orientations are included (i.e., if R ⁸ is -L ¹ -L ³ -R ^8a , both -L ¹ -C(O)OR ^8a and -L ¹ -OC(O)-R ^8a are included), or if L ² is -CH2-CH2-C(CH3)2-, both -CH2-CH2C(CH3)2- orientations are included (i.e., if R ⁸ is -L ¹ -L ² -R ^8a , both R ^8s -L ¹ -CH2-CH ₂ C(CH ₃ ) ₂ -R ^8a and -L ¹ -C(CH ₃ ) ₂ -CH ₂ -CH ₂ -R ^8a are included). It should be understood that any embodiment of the compounds of any of Formulas I, II, III, IV, and V, as defined above, and any particular group or substituent described herein (e.g., R ¹ , ^{R 2} , R ³ , R 4 , R ⁵ , R ⁶ , R ⁷ , R ⁸ , and substituents thereof) in the compounds of Formulas I, II, III, IV, and V, as defined above, can be independently combined with other embodiments and/or substituents of the compounds of any of Formulas I, II, III, IV, and V to produce embodiments not specifically described above. Furthermore, if a list of substituents for any particular R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is not listed in a particular embodiment and/or claim, it is to be understood that each individual substituent may be omitted from the particular embodiment and/or claim and that the remaining list of substituents will be considered to be within the scope of the embodiments described herein.

The compounds of formulas I, II, III, IV, and V, or pharmaceutically acceptable salts thereof, are prodrugs, wherein the progroup is rapidly cleaved in the intracellular environment to form tenofovir (TFV). In some embodiments, the compounds of formulas I, II, III, IV, and V, or pharmaceutically acceptable salts thereof, are suitable for use in long-acting formulations. For certain patients, such as those with difficult or limited access to health care, compliance with daily oral treatment or prophylactic regimens for the treatment or prevention of viral infections (e.g., HIV infection) can be challenging. Drugs that have pharmaceutical or physicochemical properties favorable to slow release (e.g., improved potency, prolonged pharmacokinetics, decreased solubility, increased plasma stability, and/or other properties) allow for less frequent dosing and improve patient compliance. Such improvements can, in turn, optimize drug exposure and limit the emergence of drug resistance. Without being bound by theory, it is believed that the low solubility of the compounds of formulas I, II, III, IV, and V, or pharmaceutically acceptable salts thereof, may result in a slow release of the compounds following intramuscular or subcutaneous administration and sustained intracellular levels of the pharmacologically active agent, tenofovir diphosphate (TFV-DP), making them capable of maintaining a therapeutically effective concentration of TFV-DP in the relevant cell types for an extended period of time and being useful as long-acting agents for the treatment or prophylaxis of viral infection. The development of poorly soluble prodrugs for safe and effective long-acting injectable formulations is discussed in Remenar, Mol. Pharmaceutics 2014, 11, 1739-1749.

Pharmaceutical compositions.

In some embodiments, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises one, two, three or

- 35 049099 four additional therapeutic agents as detailed below.

In some embodiments, a composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof in one embodiment does not comprise an agent that affects the rate of metabolism of the active ingredient. Thus, it is understood that compositions comprising a compound of the present invention in one aspect do not comprise an agent that could affect (e.g., slow down, hinder, or retard) the metabolism of the compound of the present invention or any other active ingredient administered separately, sequentially, or simultaneously with the compound of the present invention. It is also understood that any of the methods, kits, articles of manufacture, etc., detailed herein in one aspect do not comprise an agent that could affect (e.g., slow down, hinder, or retard) the metabolism of the compound of the present invention or any other active ingredient administered separately, sequentially, or simultaneously with the compound of the present invention.

In some embodiments, the pharmaceutical compositions described above are intended for use in a human or animal.

The invention further comprises a compound of the present invention for administration as a single active ingredient of a pharmaceutically acceptable composition which can be prepared by conventional methods known in the art, for example by binding the active ingredient to, or mixing with, a pharmaceutically acceptable, therapeutically inert organic and/or inorganic carrier or excipient.

In one aspect, the present document provides the use of a compound of the present invention as a second or other active ingredient having a synergistic effect with other active ingredients in known drugs, or the administration of a compound of the present invention together with such drugs.

Treatment methods.

HIV infection.

The present invention provides methods for treating and/or preventing human immunodeficiency virus (HIV) infection in a subject. In some embodiments, the method for treating and/or preventing HIV infection in a subject comprises administering to the subject a composition provided herein. In some embodiments, the method is for treating and/or preventing HIV-1 infection. In some embodiments, the method is for treating and/or preventing HIV-2 infection.

In some embodiments, a method of treating HIV infection in a subject in need thereof comprises administering to the subject a composition provided herein. In some embodiments, the subject's HIV status is positive. In some such embodiments, the subject's HIV status is unknown. In some such embodiments, the subject's HIV status is not HIV negative.

In some embodiments, a method for preventing HIV infection in a subject at risk of infection comprises administering to the subject a composition provided herein. In some such embodiments, the subject is HIV-negative. In some embodiments, the subject is at risk of acquiring an HIV infection. In some embodiments, the disclosed compositions are combined with one, two, three or four additional therapeutic agents selected from HIV protease inhibitors, non-nucleoside or non-nucleotide HIV reverse transcriptase inhibitors, nucleoside or nucleotide HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV capsid inhibitors, gp41 inhibitors, CXCR4 inhibitors, gp120 inhibitors, CCR5 inhibitors, Nef inhibitors, latency reversing agents, HIV bNAbs, TLR7, TLR8 and TLR9 agonists, HIV vaccines, cytokines, immune checkpoint inhibitors, FLT3 ligands, bispecific antibodies that recruit T cells and NK cells, chimeric T cell receptors targeting HIV antigens, pharmacokinetic enhancers and other HIV treatment drugs and combinations thereof.

In some embodiments, the disclosed compositions are combined with one, two, three or four additional therapeutic agents selected from HIV protease inhibitors, non-nucleoside or non-nucleotide HIV reverse transcriptase inhibitors, nucleoside or nucleotide HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV capsid inhibitors, gp41 inhibitors, CXCR4 inhibitors, gp120 inhibitors, CCR5 inhibitors, Nef inhibitors, latency reversing agents, HIV bNAbs, TLR7, TLR8 and TLR9 agonists, HIV vaccines, cytokines, immune checkpoint inhibitors, FLT3 ligands, bispecific antibodies that recruit T cells and NK cells, chimeric T cell receptors targeting HIV antigens, pharmacokinetic enhancers and other HIV treatment drugs and combinations thereof.

In some embodiments, the disclosed compositions are combined with one, two, three or four additional therapeutic agents selected from dolutegravir, cabotegravir, islatravir, darunavir, bictegravir, elsulfavirine, rilpivirine and lenacapavir and their

- 36 049099 combinations.

HBV infection.

The present invention provides methods for treating and/or preventing hepatitis B virus (HBV) infection in a subject. In some embodiments, a method for treating and/or preventing HBV infection in a subject comprises administering to the subject a composition provided herein.

In some embodiments, a method of treating HBV infection in a subject in need thereof comprises administering to the subject a composition provided herein.

In some embodiments, a method for preventing HBV infection in a subject at risk of infection comprises administering to the subject a composition provided herein. In some such embodiments, the subject is at risk of acquiring HBV infection.

In some embodiments, the disclosed compositions are combined with one, two, three or four additional therapeutic agents selected from combination therapies against HBV, HBV vaccines, HBV polymerase inhibitors, HBV capsid modulators, TLR7, TLR8 and TLR9 agonists, cytokines, immune checkpoint inhibitors, FLT3 ligands, interferon alpha receptor ligands, interferon alpha, interferon lambda, hyaluronidase inhibitors, hepatitis B surface antigen (HBsAg) inhibitors, HBV protein X (HBx) inhibitors, cyclophilin inhibitors, HBV viral entry inhibitors, antisense oligonucleotides, short interfering RNA (siRNA) and DNA-directed RNA interference (ddRNAi), endonuclease modulators, ribonucleotide reductase inhibitors, E-antigen inhibitors HBV antigen (HBeAg), covalently closed circular DNA (cccDNA) inhibitors, farnesoid X receptor agonists, HBV antibodies, bispecific T cell and NK cell recruiting antibodies, chimeric T cell receptors targeting HBV antigens or peptides, CAR T cell therapy, thymosin agonists, retinoic acid-inducible gene 1 stimulators, NOD2 stimulators, phosphatidylinositol 3-kinase (PI3K) inhibitors, indoleamine 2,3-dioxygenase (IDO1) pathway inhibitors, anti-OX40, anti-CD40, anti-CD160, HBV gene editors, PAPD5/PAPD7 inhibitors, ZCCHC14 inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, epigenetic regulators, tertiary lymphoid aggregate inducers, IAP/XIAP antagonists, nucleic acid polymers (eg, NAP and STOPS), modulators of lipid metabolism or transport, arginase inhibitors and other drugs for the treatment of HBV and their combinations.

In some embodiments, the disclosed compositions are combined with one, two, three or four additional therapeutic agents selected from adefovir, entecavir, telbivudine, lamivudine and lenacapavir and combinations thereof.

In some embodiments, the disclosed compositions are combined with one, two, three or four additional therapeutic agents selected from adefovir, entecavir, telbivudine, lamivudine and lenacapavir.

Combination therapy for HIV.

In some embodiments, there is provided a method of treating HIV infection comprising administering to a human a therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt thereof, in combination with a therapeutically effective amount of one, two, three, or four additional therapeutic agents. In one embodiment, there is provided a method of treating HIV infection comprising administering to a human a therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt thereof, in combination with a therapeutically effective amount of one, two, three, or four additional therapeutic agents.

In one embodiment, pharmaceutical compositions are provided comprising a compound described herein, or a pharmaceutically acceptable salt thereof, in combination with one, two, three, or four additional therapeutic agents and a pharmaceutically acceptable carrier, diluent, or excipient.

In some embodiments, the present disclosure provides a method of treating HIV infection comprising administering to a subject in need thereof a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, in combination with a therapeutically effective amount of one, two, three, or four additional therapeutic agents that are suitable for treating HIV infection.

In some embodiments, a compound described herein or a pharmaceutically acceptable salt thereof is in combination with one, two, three or more additional therapeutic agents. In some embodiments, a compound described herein or a pharmaceutically acceptable salt thereof is in combination with one, two, three or four additional therapeutic agents. In some embodiments, a compound described herein or a pharmaceutically acceptable salt thereof is in combination with two additional therapeutic agents. In other embodiments,

- 37 049099 embodiments, the compound described herein or a pharmaceutically acceptable salt thereof are in combination with three additional therapeutic agents. In further embodiments, the compound described herein or a pharmaceutically acceptable salt thereof are in combination with four additional therapeutic agents. The one, two, three, four or more additional therapeutic agents may be different therapeutic agents selected from the same class of therapeutic agents and/or they may be selected from different classes of therapeutic agents.

Introduction of combination therapy directed against HIV.

In some embodiments, a compound described herein is administered with one, two, three, or four additional therapeutic agents. Co-administration of a compound described herein with one, two, three, or four additional therapeutic agents generally refers to the simultaneous or sequential administration of a compound described herein and one, two, three, or four additional therapeutic agents such that therapeutically effective amounts of each agent are present in the patient. When administered sequentially, the combination may be administered in two or more administrations.

Co-administration includes administration of single doses of the compounds described herein before or after administration of single doses of one, two, three, or four additional therapeutic agents. For example, a compound described herein can be administered within seconds, minutes, or hours after administration of one, two, three, or four additional therapeutic agents. In some embodiments, a unit dose of a compound described herein is administered first, followed by a unit dose of one, two, three, or four additional therapeutic agents within seconds or minutes. Alternatively, a unit dose of one, two, three, or four additional therapeutic agents is administered first, followed by a unit dose of a compound described herein within seconds or minutes. In other embodiments, a unit dose of a compound described herein is administered first, followed by a unit dose of one, two, three, or four additional therapeutic agents within hours (e.g., 1-12 hours). In yet other embodiments, a standard dose of one, two, three, or four additional therapeutic agents is administered first, followed by a standard dose of a compound described herein administered several hours later (e.g., 1-12 hours).

In some embodiments, a kit is provided comprising a compound described herein (e.g., a compound of Formula I, II, III, IV, or V), or a pharmaceutically acceptable salt thereof, in combination with one or more (e.g., one, two, three, or four) additional therapeutic agents.

In a specific embodiment, the kit comprises a compound described herein or a pharmaceutically acceptable salt thereof, a nucleoside or nucleotide inhibitor of HIV reverse transcriptase, and an HIV capsid inhibitor or an HIV capsid polymerization inhibitor.

Combination therapy for HIV.

In the above embodiments, the additional therapeutic agent may be an agent or agents directed against HIV. In some cases, the additional therapeutic agent may be HIV protease inhibitors, non-nucleoside or non-nucleotide HIV reverse transcriptase inhibitors, nucleoside or nucleotide HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV integrase non-catalytic site (or allosteric) inhibitors, HIV entry inhibitors, HIV maturation inhibitors, HIV capsid inhibitors, HIV Tat or Rev inhibitors, immunomodulators, immunotherapeutic agents, antibody-drug conjugates, gene modifiers, gene editors (such as CRISPR/Cas9, zinc finger nucleases, homing nucleases, synthetic nucleases, TALENs), cellular therapies (e.g., chimeric antigen receptor T-cell therapy, CART and engineered T-cell receptors, TCR-T, autologous T cells, engineered B cells), latency reversal agents, immune therapy, phosphatidylinositol 3 kinase (PI3K) inhibitors, HIV antibodies, bispecific antibodies and antibody-like therapeutic proteins, HIV p17 matrix protein inhibitors, IL-13 antagonists, peptidyl prolyl cis-trans isomerase A modulators, protein disulfide isomerase inhibitors, complement receptor C5a antagonists, DNA methyltransferase inhibitors, fatty acid synthase inhibitor, HIV vif gene modulators, Vif dimerization antagonists, HIV-1 viral infectivity factor inhibitors, HIV1 Nef modulators, TNF alpha ligand inhibitors, HIV Nef inhibitors, ISC tyrosine kinase modulators, mixed lineage kinase-3 (MLK-3) inhibitors, HIV-1 splicing inhibitors, integrin antagonists, inhibitors nucleoproteins, splicing factor modulators, COMM domain-containing protein 1 modulators, HIV ribonuclease H inhibitors, IFN antagonists, retrocyclin modulators, CD3 antagonists, CDK-4 inhibitors, CDK-6 inhibitors, CDK-9 inhibitors, CXCR4 modulators, ICAM-3 dendritic uptake inhibitors of non-integrin 1, HIV GAG protein inhibitors, HIV POL protein inhibitors, modulators

- 38 049099 complement factor H, ubiquitin ligase inhibitors, deoxycytidine kinase inhibitors, cyclin-dependent kinase inhibitors, proprotein convertase PC9 stimulators, ATP-dependent RNA helicase DDX3X inhibitors, reverse transcriptase priming complex inhibitors, G6PD and NADH oxidase inhibitors, mTOR complex 1 inhibitors, mTOR complex 2 inhibitors, P-glycoprotein modulators, TAT protein inhibitors, prolyl endopeptidase inhibitors, phospholipase A2 inhibitors, pharmacokinetic enhancers, HIV gene therapy, HIV vaccines and their combinations.

In some embodiments, the additional therapeutic agent or agents are selected from combination therapies for the treatment of HIV, other drugs for the treatment of HIV, HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV integrase non-catalytic site (or allosteric) inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors, latency reversing agents, capsid inhibitors, immune therapy, PI3K inhibitors, HIV antibodies and bispecific antibodies and antibody-like therapeutic proteins, and combinations thereof.

In some embodiments, the additional therapeutic agent is selected from the group consisting of combination therapies directed against HIV, other therapies for the treatment of HIV, HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV integrase non-catalytic site (or allosteric) inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors, latency reversing agents, capsid inhibitors, immune therapy, PI3K inhibitors, HIV antibodies and bispecific antibodies and antibody-like therapeutic proteins, and combinations thereof.

In some embodiments, the additional therapeutic agent or agents are selected from HIV protease inhibitors, non-nucleoside or non-nucleotide HIV reverse transcriptase inhibitors, nucleoside or nucleotide HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV capsid inhibitors, gp41 inhibitors, CXCR4 inhibitors, gp120 inhibitors, CCR5 inhibitors, Nef inhibitors, latency reversing agents, HIV bNAbs, TLR7, TLR8, and TLR9 agonists, HIV vaccines, cytokines, immune checkpoint inhibitors, FLT3 ligands, bispecific antibodies that recruit T cells and NK cells, chimeric T cell receptors targeting HIV antigens, pharmacokinetic enhancers, and other HIV treatment agents and combinations thereof.

In some embodiments, the additional therapeutic agent or agents are selected from dolutegravir, cabotegravir, islatravir, darunavir, bictegravir, elsulfavirine, rilpivirine, and lenacapavir, and combinations thereof. In some embodiments, the additional therapeutic agent or agents are selected from dolutegravir, cabotegravir, islatravir, darunavir, bictegravir, elsulfavirine, rilpivirine, and lenacapavir.

Combination drugs directed against HIV.

Examples of combination products include, but are not limited to, ATRIPLA® (efavirenz, tenofovir disoproxil fumarate, and emtricitabine); COMPLERA® (EVIPLERA®; rilpivirine, tenofovir disoproxil fumarate, and emtricitabine); STRIBILD® (elvitegravir, cobicistat, tenofovir disoproxil fumarate, and emtricitabine); TRUVADA® (tenofovir disoproxil fumarate and emtricitabine; TDF+FTC); DESCOVY® (tenofovir alafenamide and emtricitabine); ODEFSEY® (tenofovir alafenamide, emtricitabine, and rilpivirine); GENVOYA® (tenofovir alafenamide, emtricitabine, cobicistat and elvitegravir); darunavir, tenofovir alafenamide hemifumarate, emtricitabine and cobicistat; efavirenz, lamivudine and tenofovir disoproxil fumarate; lamivudine and tenofovir disoproxil fumarate; tenofovir and lamivudine; tenofovir alafenamide and emtricitabine; tenofovir alafenamide hemifumarate and emtricitabine; tenofovir alafenamide hemifumarate, emtricitabine and rilpivirine; tenofovir alafenamide hemifumarate, emtricitabine, cobicistat and elvitegravir; tenofovir analogue; COMBIVIR® (zidovudine and lamivudine; AZT+3TC); EPZICOM® (LIVEXA®; abacavir sulfate and lamivudine; ABC+3TC); KALETRA® (ALUVIA®; lopinavir and ritonavir); TRIUMEQ® (dolutegravir, abacavir and lamivudine); BIKTARVY (bictegravir + emtricitabine + tenofovir alafenamide), DOVATO, TRIZIVIR® (abacavir sulfate, zidovudine and lamivudine; ABC+AZT+3TC); atazanavir and cobicistat; atazanavir sulfate and cobicistat; atazanavir sulfate and ritonavir; darunavir and cobicistat; dolutegravir and rilpivirine; dolutegravir and rilpivirine hydrochloride; dolutegravir, abacavir sulfate and lamivudine; lamivudine, nevirapine and zidovudine; raltegravir and lamivudine; doravirine, lamivudine and tenofovir disoproxil fumarate; doravirine, lamivudine and tenofovir disoproxil; dolutegravir + lamivudine, lamivudine + abacavir + zidovudine, lamivudine + abacavir, lamivudine + tenofovir disoproxil fumarate, lamivudine + zidovudine + nevirapine, lopinavir + ritonavir, lopinavir + ritonavir + abacavir + lamivudine, lopinavir + ritonavir + zidovudine + lamivudine, tenofovir + lamivudine and tenofovir disoproxil fumarate + emtricitabine + rilpivirine hydrochloride, lopinavir, ritonavir, zidovudine, lopinavir + ritonavir + abacavir + lamivudine and lamivudine; cabotegravir + rilpivirine; 3-BNC117 + albuvirtide, elpida (elsulfavirine; VM-1500; VM-1500A.

Other drugs directed against HIV.

- 39 049099

Examples of other HIV treatment drugs include, but are not limited to, aspernigrin C, acemannan, alisporivir, BanLec, deferiprone, gamimune, methenkephalin, naltrexone, prolastin, REP 9, RPI-MN, VSSP, H1viral, Sb-728-T, 1,5-dicaffeoylquinic acid, rHIV7-shl-TAR-CCR5RZ, AAV-eCD4-Ig gene therapy, MazF gene therapy, BlockAide, bevirimate derivatives, ABX-464, AG-1105, APH0812, bryostatin analogs, BIT-225, CYT-107, CS-TATI-1, fluoro-beta-O-arabinose nucleic acid (FANA)-modified antisense oligonucleotides, FX-101, griffithsin, HGTV-43, HPH-116, HS-10234, hydroxychloroquine, IMB-10035, IMO-3100, IND-02, JL-18008, LADAVRU, MK-1376, MK-2048, MK-4250, MK-8507, MK-8558, MK-8591 (islatravir), NOV-205, OB-002H, ODE-Bn-TFV, PA1050040 (PA-040), PC-707, PGN-007, QF-036, S-648414, SCY-635, SB-9200, SCB-719, TR-452, TEV-90110, TEV-90112, TEV-90111, TEV-90113, RN-18, DIACC-1010, Fasnall, Immuglo, peptide 2-CLIPS, HRF-4467, thrombospondin analogues, TBL-1004HI, VG-1177, xl-081, AVI-CO-004, rfhSP-D, [18F]-MC-225, URMC099-C, RES-529, Verdinexor, IMC-M113V, IML-106, fc antiviral conjugate (AVC) and VIR-576.

HIV protease inhibitors.

Examples of HIV protease inhibitors include, but are not limited to, amprenavir, atazanavir, brecanavir, darunavir, fosamprenavir, fosamprenavir calcium, indinavir, indinavir sulfate, lopinavir, nelfinavir, nelfinavir mesylate, ritonavir, saquinavir, saquinavir mesylate, tipranavir, ASC-09 + ritonavir, AEBL-2, DG-17, GS-1156, TMB-657 (PPL-100), T-169, BL-008, MK-8122, TMB-607, GRL02031, and TMC-310911.

HIV ribonuclease H inhibitors.

Examples of HIV ribonuclease H inhibitors include NSC-727447.

HIV Nef inhibitors.

Examples of HIV Nef inhibitors include FP-1.

HIV reverse transcriptase inhibitors.

Examples of non-nucleoside or non-nucleotide HIV reverse transcriptase inhibitors include, but are not limited to, dapivirine, delavirdine, delavirdine mesylate, doravirine, efavirenz, etravirine, lentinan, nevirapine, rilpivirine, ACC-007, ACC-008, AIC-292, F-18, KM-023, PC-1005, M1TFV, M2-TFV, VM-1500A-LAI, PF-3450074, elsulfavirine (oral extended-release formulation, HIV infection), doravirine + islatravir (fixed-dose combination/oral tablet formulation, HIV-1 infection), elsulfavirine (long-acting injectable nanosuspension, HIV infection), and elsulfavirine (VM-1500).

Examples of HIV nucleoside or nucleotide reverse transcriptase inhibitors include, but are not limited to, adefovir, adefovir dipivoxil, azvudine, emtricitabine, tenofovir, tenofovir alafenamide, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir disoproxil, tenofovir disoproxil fumarate, tenofovir octadecyloxyethyl ether (AGX-1009), tenofovir disoproxil hemifumarate, VIDEX® and VIDEX EC® (didanosine, ddl), abacavir, abacavir sulfate, alovudine, apricitabine, censavudine, didanosine, elvutibine, festinavir, fosalvudine tidoxil, CMX-157, dapivirine, doravirine, etravirine, OCR-5753, tenofovir disoproxil orotate, fosivudine tidoxil, lamivudine, phosphazide, stavudine, zalcitabine, zidovudine, rowafovir etalafenamide (GS-9131), GS-9148, MK-8504, islatravir, MK-8583, VM-2500, and KP-1461.

HIV integrase inhibitors.

Examples of HIV integrase inhibitors include, but are not limited to, elvitegravir, elvitegravir (extended-release microcapsules), curcumin, curcumin derivatives, chicoric acid, chicoric acid derivatives, 3,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid derivatives, aurintricarboxylic acid, aurintricarboxylic acid derivatives, caffeic acid phenethyl ester, caffeic acid phenethyl ester derivatives, tyrphostin, tyrphostin derivatives, quercetin, quercetin derivatives, raltegravir, pegylated raltegravir, dolutegravir, JTK-351, bictegravir, AVX-15567, cabotegravir (long-acting injectable), diketoquinoline-4-1 derivatives, interaction inhibitor integrases with LEDGF, ledgins, M-522, M-532, MK-0536, NSC-310217, NSC-371056, NSC-48240, NSC-642710, NSC-699171, NSC-699172, NSC-699173, NSC-699174, stilbenesulfonic acid, T-169, STP-0404, VM-3500 and cabotegravir.

Examples of HIV integrase non-catalytic site inhibitors or allosteric inhibitors (NCINIs) include, but are not limited to, CX-05045, CX-05168, and CX-14442.

Inhibitors of the HIV viral infectivity factor.

Examples of inhibitors of the HIV viral infectivity factor include derivatives of 2-amino-N(2-methoxyphenyl)-6-((4-nitrophenyl)thio)benzamide.

HIV entry inhibitors.

Examples of HIV entry (fusion) inhibitors include, but are not limited to, AAR-501, LBT5001, cenicriviroc, CCR5 inhibitors, gp41 inhibitors, CD4 attachment inhibitors, gp120 inhibitors, gp160 inhibitors, and CXCR4 inhibitors.

Examples of CCR5 inhibitors include, but are not limited to, aplaviroc, vicriviroc, maraviroc, maraviroc (extended-release injectable nanoemulsion), cenicriviroc, leronlimab (PRO-140), adap

- 40 049099 tavir (RAP-101), nifeviroc (TD-0232), bispecific antibodies against GP120/CD4 or CCR5, B-07, MB-66, C25P polypeptide, TD-0680, thioraviroc and vMIP (Haimipu).

Examples of gp41 inhibitors include, but are not limited to, albuvirtide, enfuvirtide, griffithsin (a gp41/gp120/gp160 inhibitor), BMS-986197, enhanced enfuvirtide, biosimilar enfuvirtide, HIV-1 fusion inhibitor (P26-VARS), ITV-1, ITV-2, ITV-3, ITV-4, CPT-31, Cl3hmAb, PIE-12 trimer, and sifuvirtide.

Examples of CD4 receptor inhibitors include, but are not limited to, ibalizumab analogs and CADA.

Examples of gp120 inhibitors include, but are not limited to, the HIV microbicide Radha-108 (Recipol) ZVZ-PE38, BanLec, a bentonite-based nanodrug, fostemsavir tromethamine, IQP-0831, VVX-004, and BMS-663068.

Examples of gp160 inhibitors include fanquinoline.

Examples of CXCR4 inhibitors include, but are not limited to, plerixafor, aLT-1188, N15 peptide, and vMIP (Himipu).

HIV maturation inhibitors.

Examples of HIV maturation inhibitors include, but are not limited to, BMS-955176, GSK-3640254, and GSK-2838232.

Latency reversal agents.

Examples of latency reversal agents include, but are not limited to, toll-like receptor (TLR) agonists (including TLR7 agonists such as GS-9620, TLR8 and TLR9 agonists), histone deacetylase (HDAC) inhibitors, proteasome inhibitors such as Velcade, protein kinase C (PKC) activators, Smyd2 inhibitors, BET bromodomain 4 (BRD4) inhibitors (such as ZL-0580, apabetalone), ionomycin, IAP antagonists (inhibitor of apoptosis proteins such as APG-1387, LBW-242), SMAC mimetics (including TL32711, LCL161, GDC-0917, HGS1029, AT-406, Debio-1143), PMA, SAHA (suberanilohydroxamic acid or suberoyl, anilide and hydroxamic acid), NIZ-985, IL-15 modulating antibodies (including IL-15, IL-15 fusion proteins and IL-15 receptor agonists), JQ1, disulfiram, amphotericin B and ubiquitin inhibitors such as largazole analogs, APH-0812 and GSK-343. Examples of PKC activators include indolactam, prostratin, ingenol B and DAG lactones.

Histone deacetylase (HDAC) inhibitors.

In some embodiments, the agents described herein are combined with an inhibitor of a histone deacetylase, such as histone deacetylase 1, histone deacetylase 9 (HDAC9, HD7, HD7b, HD9, HDAC, HDAC7, HDAC7B, HDAC9B, HDAC9FL, HDRP, MITR; Gene Identification Number: 9734). Examples of HDAC inhibitors include, but are not limited to, abexinostat, ACY-241, AR-42, BEBT-908, belinostat, CKD-581, CS-055 (HBI-8000), CT-101, CUDC-907 (fimepinostat), entinostat, givinostat, mocetinostat, panobinostat, pracinostat, chisinostat (JNJ-26481585), resminostat, ricolinostat, romidepsin, SHP-141, TMB-ADC, valproic acid (VAL-001), vorinostat, tinostamustine, remetinostat, and entinostat.

Capsid inhibitors.

Examples of capsid inhibitors include, but are not limited to, capsid polymerization inhibitors or capsid-disrupting compounds, HIV nucleocapsid p7 (NCp7) inhibitors such as azodicarbonamide, HIV capsid protein p24 inhibitors, lenacapavir (GS-6207), GS-CA1 series, AVI-621, AVI101, AVI-201, AVI-301 and AVI-CAN1-15, PF-3450074, HIV-1 capsid inhibitors (HIV-1 infection, Shandong University) and the compounds described in (GSK WO 2019/087016).

Immune checkpoint modulators.

In various embodiments, the agents described herein are combined with one or more blockers or inhibitors of inhibitory immune checkpoint proteins or receptors and/or one or more stimulators, activators, or agonists of one or more stimulatory immune checkpoint proteins or receptors. Blockade or inhibition of inhibitory immune checkpoints can positively regulate T cell or NK cell activation and prevent immune evasion of infected cells. Activation or stimulation of stimulatory immune checkpoints can enhance the effect of immune checkpoint inhibitors in infectious therapy. In various embodiments, immune checkpoint proteins or receptors regulate T cell responses (e.g., discussed in Xu, et al., J Exp Clin Cancer Res. (2018) 37:110). In various embodiments, immune checkpoint proteins or receptors regulate NK cell responses (e.g., reviewed in Davis, et al., Semin Immunol. (2017) 31:64-75 and Chiossone, et al., Nat Rev Immunol. (2018) 18(R):671-688).

Examples of immune checkpoint proteins or receptors include, but are not limited to, CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3); variable immunoglobulin-like domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set domain-containing immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); cytokine receptor ligand 1

- 41 049099 natural killer cell toxicity 3 (NCR3LG1, B7H6); HERV-H LTR-associated protein 2 (HHLA2, B7H7); inducible T-cell costimulator (ICOS, CD278); inducible T-cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B- and T-lymphocyte-associated protein (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF); TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death protein 1 (PDCD1, PD1, PD-1); cytotoxic T-lymphocyte-associated protein 4 (CTLA4, CD152); CD80 (B7-1), CD28; Nectin cell adhesion molecule-2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); PVR-related immunoglobulin domain-containing protein (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); T-cell immunoglobulin and mucin domain-containing protein 4 (TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); lymphocyte activation gene protein 3 (LAG3, CD223); signaling lymphocyte-activating molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, CD319); UL16-binding protein 1 (ULBP1); UL16-binding protein 2 (ULBP2); UL16-binding protein 3 (ULBP3); retinoic acid early transcript IE (RAET1E; ULBP4); retinoic acid early transcript 1G (RAET1G; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6); lymphocyte activation gene protein 3 (CD223); immunoglobulin-like killer cell receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); lectin-like killer cell receptor C1 (KLRC1, NKG2A, CD159A); lectin-like killer cell receptor K1 (KLRK1, NKG2D, CD314); lectin-like killer cell receptor C2 (KLRC2, CD159c, NKG2C); lectin-like killer cell receptor C3 (KLRC3, NKG2E); lectin-like killer cell receptor C4 (KLRC4, NKG2F); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor D1 (KLRD1); and SLAM family member 7 (SLAMF7).

In various embodiments, the agents described herein are combined with one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. Exemplary T cell inhibitory immune checkpoint proteins or receptors include, but are not limited to, CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 protein (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); variable immunoglobulin-like domain-containing T cell activation inhibitor 1 (VTCN1, B7H4); V-set domain-containing immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B- and T-lymphocyte-associated protein (BTLA)); PVR-related immunoglobulin domain-containing protein (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation gene protein 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3); and immunoglobulin-like killer cell receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1). In various embodiments, the agents as described herein are combined with one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors. Exemplary T cell stimulatory immune checkpoint proteins or receptors include, but are not limited to, CD27, CD70; CD40, CD40LG; inducible T-cell costimulator (ICOS, CD278); inducible T-cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule-2 (NECTIN2, CD112); CD226 (DNAM-1); CD244 (2B4, SLAMF4), poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). See, e.g., Xu, et al., J Exp Clin Cancer Res. (2018) 37:110.

- 42 049099

In various embodiments, the agents described herein are combined with one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. Exemplary NK cell inhibitory immune checkpoint proteins or receptors include, but are not limited to, killer cell receptor immunoglobulin-like, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell receptor immunoglobulin-like, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell receptor immunoglobulin-like, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell receptor immunoglobulin-like, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); and killer cell lectin-like receptor D1 (KLRD1, CD94). In various embodiments, the agents described herein are combined with one or more agonists or activators of one or more NK cell stimulatory immune checkpoint proteins or receptors. Exemplary NK cell stimulatory immune checkpoint proteins or receptors include, but are not limited to, CD16, CD226 (DNAM-1); CD244 (2B4, SLAMF4); killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); SLAM family member 7 (SLAMF7). See, for example, Zhou et al., J. Immunol. (2017) 31:64–75; Fang, et al, Semin Immunol. (2017) 31:37–54; and Chiossone, et al, Nat Rev Immunol. (2018) 18(R):671-688.

In some embodiments, the one or more immune checkpoint inhibitors comprise a protein inhibitor (e.g., an antibody or fragment thereof, or an antibody mimetic) of PD-L1 (CD274), PD-1 (PDCD1), or CTLA4. In some embodiments, the one or more immune checkpoint inhibitors comprise a small organic molecule inhibitor of PD-L1 (CD274), PD1 (PDCD1), or CTLA4. In some embodiments, the small molecule inhibitor of CD274 or PDCD1 is selected from the group consisting of GS-4224, GS-4416, INCB086550, and MAX10181. In some embodiments, the small molecule inhibitor of CTLA4 comprises BPI-002.

Examples of CTLA4 inhibitors that can be co-administered include, but are not limited to, ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884, BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, CG0161, ATOR-1144, PBI-5D3H5, BPI-002, and the polyspecific inhibitors FPT-155 (CTLA4/PDL1/CD28), PF-06936308 (PD-1/CTLA4), MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), XmAb-20717 (PD-1/CTLA4) and AK-104 (CTLA4/PD-1).

Examples of PD-L1 (CD274) or PD-1 (PDCD1) inhibitors that may be co-administered include, but are not limited to, pembrolizumab, nivolumab, cemiplimab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, PF06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX-008), AK-105, CS-1003, HLX10, MGA-012, BI-754091, AGEN-2034, JS-001 (toripalimab), JNJ-63723283, Genolimzumab (CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210 (camrelizumab), Sym-021, ABBV-181 (budigalimab), PD1-PIK, BAT-1306, (MSB0010718C), CX-072, CBT-502, TSR-042 (dostarlimab), MSB-2311, JTX4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308 (sintilimab), HLX-20, KL-A167, STIA1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, GS-4224, GS-4416, INCB086550, MAX10181, as well as polyspecific inhibitors FPT-155 (CTLA4/PD-L1/CD28), PF06936308 (PD-1/CTLA4), MGD-013 (PD-1/LAG-3), FS-118 (LAG-3/PD-L1) MGD-019 (PD-1/CTLA4), KN046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), RO-7121661 (PD-1/TIM-3), XmAb-20717 (PD-1/CTLA4), AK-104 (CTLA4/PD-1), M7824 (PD-L1/TGFP-EC domain), CA-170 (PD-L1/VISTA), CDX-527 (CD27/PDL1), LY-3415244 (TIM3/PDL1), and INBRX-105 (4-1BB/PDL1).

In various embodiments, the agents described herein are combined with TIGIT antibodies such as BMS-986207, RG-6058, AGEN-1307.

Agonists or activators of members of the TNF receptor superfamily (TNFRSF).

In various embodiments, the agents described herein are combined with an agonist of one or more members of the TNF receptor superfamily (TNFRSF), such as an agonist of one or more of TNFRSFIA (NCBI Gene ID: 7132), TNFRSF1B (NCBI Gene ID: 7133), TNFRSF4 (OX40, CD134; NCBI Gene ID: 7293), TNFRSF5 (CD40; NCBI Gene ID: 958), TNFRSF6 (FAS, NCBI Gene ID: 355), TNFRSF7 (CD27, NCBI Gene ID: 939), TNFRSF8 (CD30, NCBI Gene ID: NCBI: 943), TNFRSF9 (4-1BB, CD137, NCBI Gene ID: 3604), TNFRSF10A (CD261, DR4, TRAILR1, NCBI Gene ID: 8797), TNFRSF10B (CD262, DR5, TRAILR2, NCBI Gene ID: 8795), TNFRSF10C (CD263, TRAILR3, NCBI Gene ID: 8794), TNFRSF10D (CD264, TRAILR4, NCBI Gene ID: 8793), TNFRSF11A (CD265, RANK ...1A (CD265, TRAILR1, NCBI Gene ID: 8795), TNFRSF11A (CD265, TRAILR2, NCBI Gene ID: 8795), TNFRSF11A (CD265, TRAILR3, NCBI Gene ID: 8795), TNFRSF11A (CD265, TRAILR4, NCBI Gene ID: 8795), TNFRSF11A (CD265, TRAILR1, NCBI Gene ID: 8795), TNFRSF11A (CD265, TRAILR1, NCBI Gene ID: 8795), TNFRSF11A (CD265, TRAILR1, NCBI Gene ID: 8795), TNFRSF1 NCBI: 8792), TNFRSF11B (NCBI gene accession number: 4982), TNFRSF12A

- 43 049099 (CD266, NCBI Gene ID: 51330), TNFRSF13B (CD267, NCBI Gene ID: 23495), TNFRSF13C (CD268, NCBI Gene ID: 115650), TNFRSF16 (NGFR, CD271, NCBI Gene ID: 4804), TNFRSF17 (BCMA, CD269, NCBI Gene ID: 608), TNFRSF18 (GITR, CD357, NCBI Gene ID: 8784), TNFRSF19 (NCBI Gene ID: 55504), TNFRSF21 (CD358, DR6, NCBI gene accession number: 27242), and TNFRSF25 (DR3, NCBI gene accession number: 8718).

Examples of TNFRSF4 (OX40) antibodies that can be co-administered include, but are not limited to, MEDI6469, MEDI6383, MEDI0562 (tavolyxizumab), MOXR0916, PF-04518600, RG-7888, GSK-3174998, INCAGN1949, BMS-986178, GBR-8383, ABBV-368, and those described in WO2016179517, WO2017096179, WO2017096182, WO2017096281, and WO2018089628.

Examples of anti-TNFRSF5 (CD40) antibodies that may be co-administered include, but are not limited to, RG7876, SEA-CD40, APX-005M, and ABBV-428.

In some embodiments, the anti-TNFRSF7 (CD27) antibody varlilumab (CDX-1127) is co-administered.

Examples of anti-TNFRSF9 antibodies (4-1BB, CD137) that may be co-administered include, but are not limited to, urelumab, utomilumab (PF-05082566), AGEN2373, and ADG-106.

Examples of TNFRSF18 (GITR) antibodies that can be co-administered include, but are not limited to, MEDI1873, FPA-154, INCAGN-1876, TRX-518, BMS-986156, MK-1248, GWN-323, and the antibodies described in WO2017096179, WO2017096276, WO2017096189, and WO2018089628. In some embodiments, an antibody or fragment thereof targeting TNFRSF4 (OX40) and TNFRSF18 (GITR) is co-administered. Such antibodies are described, for example, in WO2017096179 and WO2018089628.

Natural Killer (NK) Cell Attracting Bispecific and Trispecific Activators

In various embodiments, the agents described herein are combined with a bispecific NK cell-engaging activator (BiKE) or a trispecific NK cell-engaging activator (TriKE) (e.g., lacking an Fc) or a bispecific antibody (e.g., having an Fc) against an NK cell activating receptor, e.g., CD16A, C-type lectin receptors (CD94/NKG2C, NKG2D, NKG2E/H, and NKG2F), natural cytotoxicity receptors (NKp30, NKp44 nNKp46), killer cell C-type lectin-like receptors (NKp65, NKp80), the Fc receptor FcyR (which mediates antibody-dependent cellular cytotoxicity), SLAM family receptors (e.g., 2B4, SLAM6, and SLAM7), killer cell immunoglobulin-like receptors (KIR) (KIR-2DS and KIR-3DS), DNAM-1, and CD137 (41BB). Accordingly, bispecific molecules that bind to CD16 may or may not have an Fc. Exemplary bispecific NK cell attracting activators that can be co-administered with a CD16 target and one or more HIV-associated antigens are described herein. BiKE and TriKE are described, for example, in Felices, et al., Methods Mol Biol. (2016) 1441:333–346; Fang, et al., Semin Immunol. (2017) 31:37–54. Examples of NK cell-attracting trispecific activators (TRiKE) include OXS3550, HIV-TriKE, and CD16-IL-15-B7H3 TriKe.

Indoleamine pyrrole-2,3-dioxygenase (IDO1) inhibitors.

In various embodiments, the agents described herein are combined with an inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1; NCBI Gene #3620). Examples of IDO1 inhibitors include, but are not limited to, BLV-0801, epacadostat, F-001287, GBV-1012, GBV-1028, GDC0919, indoximod, NKTR-218, NLG-919 vaccine, PF-06840003, pyranonaphthoquinone derivatives (SN-35837), resminostat, SBLK-200802, BMS-986205, and shIDO-ST, EOS-200271, KHK-2455, LY3381916.

Toll-like receptor (TLR) agonists.

In various embodiments, the agents described herein are combined with a toll-like receptor (TLR) agonist, such as an agonist of TLR1 (NCBI Gene ID: 7096), TLR2 (NCBI Gene ID: 7097), TLR3 (NCBI Gene ID: 7098), TLR4 (NCBI Gene ID: 7099), TLR5 (NCBI Gene ID: 7100), TLR6 (NCBI Gene ID: 10333), TLR7 (NCBI Gene ID: 51284), TLR8 (NCBI Gene ID: 51311), TLR9 (NCBI gene accession number: 54106), and/or TLR10 (NCBI gene accession number: 81793). Examples of TLR7 agonists that can be co-administered include, but are not limited to, AL-034, DSP-0509, GS-9620 (vesatolimod), a vesatolimod analog, LHC-165, TMX101 (imiquimod), GSK-2245035, resiquimod, DSR-6434, DSP-3025, IMO-4200, MCT-465, MEDI-9197, 3M-051, SB-9922, 3M-052, Limtop, TMX-30X, TMX-202, RG-7863, RG-7854, RG-7795, and the compounds described in US 20100143301 (Gilead Sciences), US 20110098248 (Gilead Sciences), and US 20090047249 (Gilead Sciences), US 20140045849 (Janssen), US 20140073642 (Janssen), WO 2014/056953 (Janssen), WO

- 44 049099

2014/076221 (Janssen), WO 2014/128189 (Janssen), US 20140350031 (Janssen), WO 2014/023813 (Janssen), US 20080234251 (Array Biopharma), US 20080306050 (Array Biopharma), US 20100029585 (Ventirx Pharma), US 20110092485 (Ventirx Pharma), US 20110118235 (Ventirx Pharma), US 20120082658 (Ventirx Pharma), US 20120219615 (Ventirx Pharma), US 20140066432 (Ventirx Pharma), US 20140088085 (Ventirx Pharma), US 20140275167 (Novira Therapeutics), and US 20130251673 (Novira Therapeutics). TLR7/TLR8 agonists include NKTR-262, telratolimod, and BDB-001. TLR8 agonists include, but are not limited to, E-6887, IMO-4200, IMO-8400, IMO-9200, MCT-465, MEDI-9197, motolimod, resiquimod, GS-9688, VTX-1463, VTX-763, 3M-051, 3M-052, and the compounds described in US 20140045849 (Janssen), US 20140073642 (Janssen), WO 2014/056953 (Janssen), WO 2014/076221 (Janssen), WO 2014/128189 (Janssen), US 20140350031 (Janssen), WO 2014/023813 (Janssen), US 20080234251 (Array Biopharma), US 20080306050 (Array Biopharma), US 20100029585 (Ventirx Pharma), US 20110092485 (Ventirx Pharma), US 20110118235 (Ventirx Pharma), US 20120082658 (Ventirx Pharma), US 20120219615 (Ventirx Pharma), US 20140066432 (Ventirx Pharma), US 20140088085 (Ventirx Pharma), US 20140275167 (Novira Therapeutics), and US 20130251673 (Novira Therapeutics). TLR9 agonists include, but are not limited to, AST-008, cobitolimod, CMP-001, IMO-2055, IMO-2125, S-540956, litenimob, MGN-1601, BB-001, BB-006, IMO-3100, IMO8400, IR-103, IMO-9200, agatolimod, DIMS-9054, DV-1079, DV-1179, AZD-1419, lefitolimod (MGN1703), CYT-003, CYT-003-QbG10, tilsotolimod, and PUL-042. Examples of TLR3 agonists include rintatolimod, poly-ICLC, RIBOXXON®, Apoxxim, RIBOXXIM®, IPH-33, MCT-465, MCT-475, and ND-1.1. TLR4 agonists include G-100 and GSK-1795091.

CDK inhibitors or antagonists.

In some embodiments, the agents described herein are combined with a CDK inhibitor or antagonist. In some embodiments, the CDK inhibitor or antagonist is selected from the group consisting of VS2-370.

STING agonists, RIG-I and NOD2 modulators.

In some embodiments, the agents described herein are combined with an interferon gene stimulator (STING). In some embodiments, the STING receptor agonist or activator is selected from the group consisting of ADU-S100 (MIW-815), SB-11285, MK-1454, SR8291, AdVCA0848, GSK-532, SYN-STING, MSA-1, SR-8291, a STING agonist (latent HIV), 5,6-dimethylxanthenone-4-acetic acid (DMXAA), cyclic GMP-AMP (cGAMP), and cyclic di-AMP. In some embodiments, the agents described herein are combined with a RIG-I modulator, such as RGT-100, or a NOD2 modulator, such as SB-9200 and IR-103.

LAG-3 and TIM-3 inhibitors.

In certain embodiments, the agents described herein are combined with an anti-TIM-3 antibody such as TSR-022, LY-3321367, MBG-453, INCAGN-2390.

In certain embodiments, the agents described herein are combined with an anti-LAG-3 (lymphocyte activation) antibody such as relatlimab (ONO-4482), LAG-525, MK-4280, REGN-3767, INCAGN2385.

Interleukin agonists.

In certain embodiments, the agents described herein are combined with an interleukin agonist such as IL-2, IL-7, IL-15, IL-10, IL-12 agonists; examples of IL-2 agonists such as proleukin (aldesleukin, IL-2); BC-IL (Cel-Sci), pegylated IL-2 (e.g., NKTR-214); modified IL-2 variants (e.g., THOR-707), bempegaldesleukin, AIC-284, ALKS-4230, CUI-101, Neo-2/15; Examples of IL-15 agonists such as ALT-803, NKTR-255 and hetIL-15, interleukin-15/Fc fusion protein, AM-0015, NIZ-985, SO-C101, syntorin IL-15 (pegylated Il-15), P-22339 and IL-15-PD-1 fusion protein N-809; Examples of IL-7 include CYT-107. Examples of additional immune therapies that can be combined with the agent of the invention include interferon alpha; interferon a-2b; interferon a-n3; pegylated interferon alpha; interferon gamma; FLT3 agonists such as CDX-301 and GS-3583; gepon; normferon, peginterferon alpha-2a, peginterferon alpha-2^ RPI-MN.

Phosphatidylinositol 3-kinase (PI3K) inhibitors.

Examples of PI3K inhibitors include, but are not limited to, idelalisib, alpelisib, buparlisib, CAI orotate, copanlisib, duvelisib, gedatolisib, neratinib, panulisib, perifosin, pictilisib, pilaralisib, puquitinib mesylate, rigosertib, rigosertib sodium, sonolisib, taselisib, AMG-319, AZD-8186, BAY-1082439, CLR-1401, CLR-457, CUDC-907, DS-7423, EN-3342, GSK-2126458, GSK-2269577, GSK2636771, INCB-040093, LY-3023414, MLN-1117, PQR-309, RG-7666, RP-6530, RV-1729, SAR-245409, SAR-260301, SF-1126, TGR-1202, UCB-5857, VS-5584, XL-765 and ZSTK-474.

Alpha-4/beta-7 antagonists.

Examples of alpha-4/beta-7 integrin antagonists include, but are not limited to, PTG-100, TRK-170, abrilumab, etrolizumab, carotegrast methyl, and vedolizumab.

Antibodies targeting HIV.

Examples of HIV antibodies, bispecific antibodies, and antibody-like therapeutic proteins

- 45 049099 include, but are not limited to, DARTs®, DUOBODIES®, BITES®, XmAbs®, TandAbs®, Fab derivatives, bNAbs (broadly neutralizing HIV-1 antibodies), TMB-360 and proteins targeting HIV gp120 or gp41, antibody recruiter molecules targeting HIV, anti-CD63 monoclonal antibodies, anti-GB virus C antibodies, uhtu-GP120/CD4, gp120 bispecific monoclonal antibody, anti-CCR5 bispecific antibody, anti-Nef single domain antibodies, anti-Rev antibody, camelid-derived anti-CD18 antibodies, camelid-derived anti-ICAM-1 antibodies, DCVax-001, gp140 targeted antibodies, gp41-based therapeutic HIV antibodies, human recombinant mAb (PGT-121), PGT121.414.LS, ibalizumab, ibalizumab (second generation), Immuglo, MB-66, human monoclonal antibody clone 3 targeting KLIC (HIV infection), GS-9721, BG-HIV, VRC-BH4MAB091-00-AB.

Various bNAbs can be used. Examples include, but are not limited to, the antibodies described in U.S. Patent Nos. 8,673,307, 9,493,549, 9,783,594, WO 2014/063059, WO 2012/158948, WO 2015/117008 and PCT/US2O15/41272 and WO 2017/096221, including antibodies 12A12, 12A21, NIH45-46, bANC131, 8ANC134, IB2530, INC9, 8ANC195. 8ANC196, 10-259, 10-303, 10-410, 10-847, 10-996, 10-1074, 10-1121, 10-1130, 10-1146, 10-1341, 10-1369, and 10-1074GM. Further examples include the antibodies described in Klein et al., Nature, 492(7427): 118–22 (2012), Horwitz et al., Proc Natl Acad Sci USA, 110(41): 16538–43 (2013), Scheid, et al., Science, 333: 1633–1637 (2011), Scheid, et al., Nature, 458:636–640 (2009), Eroshkin et al, Nucleic Acids Res., 42 (Database issue):Dl 133–9 (2014), Mascola et al., Immunol Rev., 254(1):225–44 (2013), e.g., 2F5, 4E10, M66.6, CAP206-CH12, 10E81 (all of which bind the gp41 MPER); PG9, PG16, CH01-04 (all of which bind the V1V2 glycan), 2G12 (which binds the external domain glycan); b12, HJ16, CH103-106, VRC01-03, VRC-PG04, 04b, VRC-CH30-34, 3BNC62, 3BNC89, 3BNC91, 3BNC95, 3BNC104, 3BNC176, and 8ANC131 (all of which bind the CD4 binding site).

Additional broadly neutralizing antibodies that can be used as a second therapeutic agent in combination therapy are described, for example, in U.S. Patent Nos. 8,673,307; 9,493,549; 9,783,594; and WO 2012/154312; WO 2012/158948; WO 2013/086533; WO 2013/142324; WO 2014/063059; WO 2014/089152, WO 2015/048462; WO 2015/103549; WO 2015/117008; WO 2016/014484; WO 2016/154003; WO 2016/196975; WO 2016/149710; WO 2017/096221; WO 2017/133639; WO 2017/133640, which are incorporated herein by reference in their entireties for all purposes. Additional examples include the antibodies described in Sajadi, et al., Cell. (2018) 173(7): 1783-1795; Sajadi, et al., J Infect Dis. (2016) 213(1): 156-64; Klein et al., Nature, 492(7427): 118-22 (2012), Horwitz et al., Proc Natl Acad Sci USA, 110(41): 16538-43 (2013), Scheid, et al., Science, 333: 1633-1637 (2011), Scheid, et al., Nature, 458:636-640 (2009), Eroshkin et al., Nucleic Acids Res., 42 (Database issue):Dl 133-9 (2014), Mascola et al., Immunol Rev., 254(l):225-44 (2013), e.g. 2F5, 4E10, M66.6, CAP206-CH12, 10E8, 10E8v4, 10E8-5R-100cF, DH511.11P, 7b2, 10-1074, and LN01 (all of which bind the gp41 MPER).

Examples of additional antibodies include, but are not limited to, bavituximab, UB-421, BF520.1, BiIA-SG, CH01, CH59, C2F5, C4E10, C2F5+C2G12+C4E10, CAP256V2LS, 3BNC117, 3BNC117-LS, 3BNC60, DH270.1, DH270.6, D1D2, 10-1074-LS, C13hmAb, GS-9722 (elipovimab), DH411-2, BG18, GS9721, GS-9723, PGT145, PGT121, PGT-121.60, PGT-121.66, PGT122, PGT-123, PGT-124, PGT-125, PGT126, PGT-151, PGT-130, PGT-133, PGT-134, PGT-135, PGT-128, PGT-136, PGT-137, PGT-138, PGT-139, MDX010 (ipilimumab), DH511, DH511-2, N6, N6LS, N49P6, N49P7, N49P7.1, N49P9, N49P11, N60P1.1, N60P25.1, N60P2.1, N60P31.1, N60P22, NIH 45-46, PGC14, PGG14, PGT-142, PGT-143, PGT-144, PGDM1400, PGDM12, PGDM21, PCDN-33A, 2Dm2m, 4Dm2m, 6Dm2m, PGDM1400, MDX010 (ipilimumab), VRC01, VRC-01-LS, A32, 7B2, 10E8, VRC-07-523, VRC07-523LS, VRC24, VRC41.01, 10E8VLS, 3810109, 10E8v4, IMC-HIV, iMabm36, eCD4-Ig, IOMA, CAP256-VRC26.25, DRVIA7,VRC-HIVMAB08000-AB, VRC-VICHMAV060-00-AV, P2G12, VRC07, 354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411, 354BG426, VRC29.03, CAP256, CAP256-VRC26.08, CAP256-VRC26.09, CAP256VRC26.25, PCT64-24E and VRC38.01, PGT-151, CAP248-2B, 35022, ACS202, VRC34 and VRC34.01, 10E8, 10E8v4, 10E8-5R-100cF, 4E10,DH511.11P, 2F5, 7b2, and LN01.

Examples of bispecific and trispecific antibodies to HIV include MGD014, B12BiTe, BiIA-SG, TMB bispecific antibody, SAR-441236, VRC-01/PGDM-1400/10E8v4, 10E8.4/iMab, 10E8v4/PGT121-VRC01.

Examples of in vivo delivered bnABs include AAV8-VRC07; mRNA encoding the HIV antibody VRC01; and engineered B cells encoding 3BNC117 (Hartweger et al, J. Exp. Med. 2019, 1301).

Pharmacokinetic enhancers.

Examples of pharmacokinetic enhancers include, but are not limited to, cobicistat and ritonavir. Adjunctive Therapies.

Examples of additional therapeutic agents include, but are not limited to, those described in WO 2004/096286 (Gilead Sciences), WO 2006/015261 (Gilead Sciences), WO 2006/110157 (Gilead Sciences), WO 2012/003497 (Gilead Sciences), WO 2012/003498 (Gilead Sciences), WO 2012/145728 (Gilead Sciences), WO 2013/006738 (Gilead Sciences), WO 2013/159064 (Gilead Sciences), WO 2014/100323 (Gilead Sciences), US

- 46 049099

2013/0165489 (University of Pennsylvania), US 2014/0221378 (Japan Tobacco), US 2014/0221380 (Japan Tobacco), WO 2009/062285 (Boehringer Ingelheim), WO 2010/130034 (Boehringer Ingelheim), WO 2013/006792 (Pharma Resources), US 20140221356 (Gilead Sciences), US 20100143301 (Gilead Sciences) and WO 2013/091096 (Boehringer Ingelheim).

HIV vaccines.

Examples of HIV vaccines include, but are not limited to, peptide vaccines, recombinant protein subunit vaccines, live vector vaccines, DNA vaccines, HIV-MAG DNA vaccine, CD4-based peptide vaccines, combination vaccines, adenovirus vector vaccines (adenovirus vector such as Ad5, Ad26 or Ad35), simian adenovirus (chimpanzee, gorilla, rhesus macaque, i.e. rhAd), adeno-associated virus vaccines, chimpanzee adenovirus vaccines (e.g. ChAdOX1, ChAd68, ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan5, Pan6, Pan7, Pan9), coxsackievirus vaccines, enteric virus vaccines, gorilla adenovirus vaccines, lentiviral vector, arenavirus vaccines (e.g. LCMV, Pichinde), two-segment or three-segment arenavirus vaccine, trimer-based HIV-1 vaccine, measles virus vaccines, flavivirus vector vaccines, tobacco mosaic virus vaccine, varicella zoster virus vaccine, human parainfluenza virus 3 (PIV3) vaccines, poxvirus vaccines (modified vaccinia Ankara (MVA), NYVAC strains derived from orthopoxvirus, and ALVAC (canarypox virus) strains derived from avipoxvirus); fowlpox virus vaccine, rhabdovirus vaccines such as VSV and marabavirus; recombinant human CMV (rhCMV) vaccine, alphavirus-based vaccines such as Semliki Forest virus, Venezuelan equine encephalitis virus, and Sindbis virus; (see Lauer, Clinical and Vaccine Immunology, 2017, DOI: 10.1128/CVI.00298-16); mRNA-based therapeutic vaccines formulated as LNPs; self-replicating RNA/self-amplifying RNA vaccines formulated as LNPs.

Examples of vaccines include the following: AAVLP-HIV vaccine, CD40.Env-gp140 vaccine, Ad4-EnvC150, bG505 SOSIP.664 gp140 adjuvant vaccine, BG505 SOSIP.GT1.1 gp140 adjuvant vaccine, ChAdOx1.tBH4consv1 vaccine, CMV-MVA triplex vaccine, ChAdOx1.HTI, Chimigen HIV vaccine, ConM SOSIP.v7 gp140, rgp120 (AIDSVAX), ALVAC HIV (vCP1521)/AIDSVAX B/E (gp120) (RV144), HIV-1 subtype C monomeric gp120 vaccine, MPER-656 liposomal subunit vaccine, Remune, ITV-1, Contre Vir, Ad5-ENVA-48, DCVax-001 (CDX-2401), Vacc-4x, Vacc-C5, VAC-3S, recombinant adenovirus-5 with multiclade DNA (rAd5), rAd5 gag-pol env A/B/C vaccine, Pennvax-G, Pennvax-GP, Pennvax-G/MVA-CMDR, HIV-TriMixmRNA vaccine, HIV-LAMP-vax, Ad35, Ad35-GRIN, NAcGM3/VSSP ISA-51, poly-ICLC adjuvant vaccine, TatImmune, GTU-multiHIV (FIT-06), ChAdV63.HIVconsv, gp140[delta]V2.TV1+MF-59, rVSVIN HIV-1 vaccine gag, SeV-EnvF, SeV-Gag-based vaccine, AT-20, DNK-4, ad35-Grin/ENV, TBC-M4, HIVАХ, HIVАХ-2, N123-VRC-34.01 HIV epitope-based inducing vaccine, NYVAC-HIVPT1, NYVAC-HIV-PT4, DNA-HIV-PT123, rAAVl-PG9DP, GOVX-B11, GOVX-B21, GOVX-C55, TVI-HIV1, Ad-4 (Ad4-env Clade C+Ad4-mGag), Paxvax, EN41-UGR7C, EN41-FPA2, ENOB-HV-11, PreVaxTat, AEH, MYM-V101, CombiHIVvac, ADVAX, MYM-V201, MVA-CMDR, MagaVax, DNA-Ad5 gag/pol/nef/nev (HVTN505), MVATG-17401, ETV-01, CDX-1401, DNA and Sev vector vaccine expressing SCaVII, rcAD26.MOS1.HIV-Env, Ad26.Mod.HIV vaccine, Ad26.Mod.HIV + MVA and mosaic virus vaccine + gp140, AGS-004, AVX-101, AVX-201, PEP-6409, SAV-001, ThV-01, TL01, TUTI-16, VGX-3300, VIR-1111, IHV-001, and virus-like particle vaccines such as pseudovirion vaccine, CombiVICHvac, hybrid vaccine LFn-p24 B/C, GTU DNA vaccine, gag/pol/nef/env DNA HIV vaccine, anti-TAT HIV vaccine, conjugate polypeptide vaccine, dendritic cell vaccines (e.g. DermaVir), gag DNA vaccine, GI-2010, HIV-1 gp41 vaccine, HIV vaccine (RJA adjuvant), i-key/MHC class II epitope fusion peptide vaccine, ITV-2, ITV-3, ITV-4, LIPO-5, multiclad Env vaccine, MVA vaccine, Pennvax-GP, pp71-deficient HCMV vector with HIV gag, HIV rgp160 vaccine, RNActive HIV vaccine, SCB-703, Tat Oyi vaccine, TBC-M4, UBI HIV gp120, Vacc-4x + romidepsin, gp120 variant polypeptide vaccine, rAd5 gag-pol env A/B/C vaccine, DNA.HTI and MVA.HTI, VRC-HIVDNA016-00-VP + VRC-HIVADV014-00-VP, INO-6145, JNJ-9220, gp145 C.6980; eOD-GT8 60mer vaccine, PD-201401, env (A, B, C, A/E)/gag (C) DNA vaccine, gp120 (A, B, C, A/E) protein vaccine, PDPHV-201401, Ad4-EnvCN54, EnvSeq-1 HIV-1 Env protein vaccine (adjuvanted with GLA-SE), p24gag plasmid DNA prime-boost vaccine, VRC-01 HIV-1 iglb12 neutralizing antibody-based CD4 vaccine, arenavirus vector-based vaccines (Vaxwave, TheraT), MVA-BN HIV-1 vaccination schedule, UBI HIV gp120, mRNA-based prophylactic vaccines, VPI-211, and TBL-1203HI.

Contraceptive (contraceptive) combination therapy.

In certain embodiments, the agents described herein are combined with

- 47 049099 contraceptive or birth control regimen. Therapeutic agents used against conception (for contraception) that can be combined with the agent of this invention include cyproterone acetate, desogestrel, dienogest, drospirenone, estradiol valerate, ethinyl estradiol, ethynodiol, etonogestrel, levomefolate, levonorgestrel, lynestrenol, medroxyprogesterone acetate, mestranol, mifepristone, misoprostol, nomegestrol acetate, norelgestromin, norethindrone, norethinodrel, norgestimate, ormeloxifene, segesterone acetate, ulipristal acetate and any combinations thereof.

In a specific embodiment, a compound described herein or a pharmaceutically acceptable salt thereof is combined with one, two, three or four additional therapeutic agents selected from ATRIPLA® (efavirenz, tenofovir disoproxil fumarate and emtricitabine); COMPLERA® (EVIPLERA®; rilpivirine, tenofovir disoproxil fumarate and emtricitabine); STRIBILD® (elvitegravir, cobicistat, tenofovir disoproxil fumarate and emtricitabine); TRUVADA® (tenofovir disoproxil fumarate and emtricitabine; TDF+FTC); DESCOVY® (tenofovir alafenamide and emtricitabine); ODEFSEY® (tenofovir alafenamide, emtricitabine and rilpivirine); GENVOYA® (tenofovir alafenamide, emtricitabine, cobicistat and elvitegravir); BIKTARVY (bictegravir + emtricitabine + tenofovir alafenamide), adefovir; adefovir dipivoxil; cobicistat; emtricitabine; tenofovir; tenofovir alafenamide and elvitegravir; tenofovir alafenamide + elvitegravir (for rectal use, HIV infection); tenofovir disoproxil; tenofovir disoproxil fumarate; tenofovir alafenamide; tenofovir alafenamide hemifumarate; TRIUMEQ® (dolutegravir, abacavir and lamivudine); dolutegravir, abacavir sulfate, and lamivudine; raltegravir; pegylated raltegravir; raltegravir and lamivudine; maraviroc; tenofovir + emtricitabine + maraviroc, enfuvirtide; ALUVIA® (KALETRA®; lopinavir and ritonavir); COMBIVIR® (zidovudine and lamivudine; AZT+3TC); EPZICOM® (LIVEXA®; abacavir sulfate and lamivudine; ABC+3TC); TRIZIVIR® (abacavir sulfate, zidovudine, and lamivudine; ABC+AZT+3TC); rilpivirine; rilpivirine hydrochloride; atazanavir sulfate and cobicistat; atazanavir and cobicistat; darunavir and cobicistat; atazanavir; atazanavir sulfate; dolutegravir; elvitegravir; ritonavir; atazanavir sulfate and ritonavir; darunavir; lamivudine; prolastin; fosamprenavir; fosamprenavir calcium, efavirenz; etravirine; nelfinavir; nelfinavir mesylate; interferon; didanosine; stavudine; indinavir; indinavir sulfate; tenofovir and lamivudine; zidovudine; nevirapine; saquinavir; saquinavir mesylate; aldesleukin; zalcitabine; tipranavir; amprenavir; delavirdine; delavirdine mesylate; Radha-108 (Receptol); lamivudine and tenofovir disoproxil fumarate; efavirenz, lamivudine and tenofovir disoproxil fumarate; phosphazide; lamivudine, nevirapine and zidovudine; abacavir; and abacavir sulfate.

In some embodiments, the agent described herein or a pharmaceutical composition thereof is combined with a nucleoside or nucleotide inhibitor of HIV reverse transcriptase, a non-nucleoside inhibitor of HIV reverse transcriptase. In another specific embodiment, the agent described herein or a pharmaceutical composition thereof is combined with a nucleoside or nucleotide inhibitor of HIV reverse transcriptase and an HIV protease inhibitor compound. In a further embodiment, the agent described herein or a pharmaceutical composition thereof is combined with a nucleoside or nucleotide inhibitor of HIV reverse transcriptase, a non-nucleoside inhibitor of HIV reverse transcriptase and a pharmacokinetic enhancer. In some embodiments, the agent described herein or a pharmaceutical composition thereof is combined with at least one nucleoside inhibitor of HIV reverse transcriptase, an integrase inhibitor and a pharmacokinetic enhancer. In another embodiment, the agent described herein or a pharmaceutical composition thereof is combined with two nucleoside or nucleotide inhibitors of HIV reverse transcriptase.

In another embodiment, the agent described herein, or a pharmaceutical composition thereof, is combined with a first additional therapeutic agent selected from dolutegravir, cabotegravir, islatravir, darunavir, bictegravir, elsulfavirine, rilpivirine and lenacapavir, and a second additional therapeutic agent selected from emtricitabine and lamivudine.

In some embodiments, the agent described herein or a pharmaceutically acceptable salt thereof is combined with a first additional therapeutic agent (contraceptive) selected from the group consisting of cyproterone acetate, desogestrel, dienogest, drospirenone, estradiol valerate, ethinyl estradiol, ethynodiol, etonogestrel, levomefolate, levonorgestrel, lynestrenol, medroxyprogesterone acetate, mestranol, mifepristone, misoprostol, nomegestrol acetate, norelgestromin, norethindrone, norethinodrel, norgestimate, ormeloxifene, segesterone acetate, ulipristal acetate, and any combinations thereof.

Gene therapy and cell therapy.

In certain embodiments, the agents described herein are combined with a gene or cell therapy regimen. Gene therapy and cell therapy include, but are not limited to,

- 48 049099 genetic modification to suppress a gene; genetic approaches to directly kill infected cells; infusion of immune cells designed to replace a large portion of the patient's own immune system to enhance the immune response to infected cells, or activate the patient's own immune system to kill infected cells, or seek out and kill infected cells; genetic approaches to modify cellular activity to further alter the endogenous immune response against infection. Examples of cell therapies include LB-1903, eNOB-HV-01, gOVX-B01, hSPCs overexpressing ALDH1 (LV-800, HIV infection), AGT103-T and SupT1 cell-based therapies. Examples of dendritic cell-based therapies include AGS-004. CCR5 gene editing agents include SB728T. CCR5 gene inhibitors include Cal-1 and CCR5 shRNA/TRIM5alpha/TAR lentiviral vector, bait-transfected autologous CD34-positive hematopoietic progenitor cells (HIV infection/HIV-associated lymphoma). In some embodiments, C34-CCR5/C34-CXCR4-expressing CD4-positive T cells are co-administered with one or more multispecific antigen-binding molecules. In some embodiments, the agents described herein are used in conjunction with AGT-103-transduced autologous T cell therapy or AAV-eCD4-Ig gene therapy.

Gene editors.

In certain embodiments, the agents described herein are combined with a gene editor, such as an HIV-targeted gene editor. In various embodiments, the genome editing system can be selected from the group consisting of: a CRISPR/Cas9 complex, a zinc finger nuclease complex, a TALEN complex, a homing endonuclease complex, and a meganuclease complex. An exemplary HIV-targeted CRISPR/Cas9 system includes, but is not limited to, EBT-101.

CAR-T cell therapy.

In some embodiments, the agents described herein can be co-administered with a population of effector immune cells engineered to express a chimeric antigen receptor (CAR), wherein the CAR comprises an HIV antigen binding domain. The HIV antigen comprises an HIV envelope protein or a portion thereof, gp120 or a portion thereof, a CD4 binding site on gp120, an inducible CD4 binding site on gp120, an N-glycan on gp120, V2 of gp120, a membrane-proximal region on gp41. The immune effector cell is a T cell or an NK cell. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof. The cells can be autologous or allogeneic. Examples of HIV CAR-T include convertible CAR-T, VC-CAR-T, CMV-N6-CART, anti-CD4 CART cell therapy, CD4 CAR+C34-CXCR4+CCR5 ZFN T cells, anti-CD4 MicAbody antibody + anti-MicAbody CAR T cell therapy (iNKG2D CAR, HIV infection), GP-120 CAR-T therapy, autologous hematopoietic stem cells genetically engineered to express CD4 CAR and C46 peptide.

TCR-T cell therapy.

In certain embodiments, the agents described herein are combined with a population of TCR T cells. The TCR T cells are engineered to target HIV peptides present on the surface of virus-infected cells, such as ImmTAV.

B-cell therapy.

In certain embodiments, the antibodies or antigen-binding fragments described herein are combined with a population of B cells genetically modified to express broadly neutralizing antibodies, such as 3BNC117 (Hartweger et al, J. Exp. Med. 2019, 1301, Moffett et al., Sci. Immunol. 4, eaax0644 (2019) 17 May 2019.

A compound described herein (e.g., any compound of Formula I, II, III, IV, or V) can be administered in combination with one, two, three, or four additional therapeutic agents in any dosage amount of the compound of Formula I, II, III, IV, or V (e.g., from 1 mg to 500 mg of the compound).

In one embodiment, a kit is provided comprising a compound described herein, or a pharmaceutically acceptable salt thereof, in combination with one or more (e.g., one, two, three, one or two, or one to three) additional therapeutic agents.

In one embodiment, the additional therapeutic agent or agents of the kit are an anti-HIV agent selected from HIV protease inhibitors, non-nucleoside or non-nucleotide HIV reverse transcriptase inhibitors, nucleoside or nucleotide HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry inhibitors, HIV maturation inhibitors, immunomodulators, immunotherapeutic agents, antibody-drug conjugates, gene modifiers, gene editors (such as CRISPR/Cas9, zinc finger nucleases, homing nucleases, synthetic nucleases, TALEN), cellular therapies (such as chimeric antigen receptor T-cells, CAR-T and engineered T-cell receptors, TCR-T, autologous

- 49 049099 (genetic T-cell therapy), compounds that target the HIV capsid, latency reversing agents, HIV bNAbs, immune therapies, phosphatidylinositol 3-kinase (PI3K) inhibitors, HIV antibodies, broadly neutralizing HIV antibodies, bispecific antibodies and antibody-like therapeutic proteins, HIV p17 matrix protein inhibitors, IL-13 antagonists, peptidylprolyl-cis-trans-isomerase A modulators, protein disulfide isomerase inhibitors, complement receptor C5a antagonists, DNA methyltransferase inhibitor, HIV vif gene modulators, Vif dimerization antagonists, HIV viral infectivity factor inhibitors, TAT protein inhibitors, HIV Nef modulators, Hck tyrosine kinase modulators, kinase-3 inhibitors of mixed origin (MLK-3), HIV splicing inhibitors, Rev protein inhibitors, integrin antagonists, nucleoprotein inhibitors, splicing factor modulators, COMM domain-containing protein 1 modulators, HIV ribonuclease H inhibitors, retrocyclin modulators, CDK-9 inhibitors, non-integrin 1 inhibitors, dendritic ICAM-3 trapping inhibitors, HIV GAG protein inhibitors, HIV POL protein inhibitors, complement factor H modulators, ubiquitin ligase inhibitors, deoxycytidine kinase inhibitors, cyclin-dependent kinase inhibitors, proprotein convertase PC9 stimulators, ATP-dependent RNA helicase DDX3X inhibitors, reverse transcriptase priming complex inhibitors, G6PD and NADH oxidase inhibitors, pharmacokinetic enhancers, gene therapy HIV, HIV vaccines and their combinations.

In some embodiments, the additional therapeutic agent or agents of the kit are selected from combination therapies for the treatment of HIV, other drugs for the treatment of HIV, HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV integrase non-catalytic site (or allosteric) inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors, latency reversing agents, capsid inhibitors, immune therapy, PI3K inhibitors, HIV antibodies and bispecific antibodies and antibody-like therapeutic proteins, and combinations thereof.

In a specific embodiment, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof and a nucleoside or nucleotide inhibitor of HIV reverse transcriptase. In specific embodiments, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof and a nucleoside or nucleotide inhibitor of HIV reverse transcriptase and a non-nucleoside inhibitor of HIV reverse transcriptase. In another specific embodiment, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof and a nucleoside or nucleotide inhibitor of HIV reverse transcriptase and an HIV protease inhibitor compound. In a further embodiment, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof and a nucleoside or nucleotide inhibitor of HIV reverse transcriptase, a non-nucleoside inhibitor of HIV reverse transcriptase and a pharmacokinetic enhancer. In some embodiments, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof and at least one nucleoside inhibitor of HIV reverse transcriptase, an integrase inhibitor, and a pharmacokinetic enhancer. In another embodiment, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof and two nucleoside or nucleotide inhibitors of HIV reverse transcriptase. In a specific embodiment, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof, a nucleoside or nucleotide inhibitor of HIV reverse transcriptase, and an HIV capsid inhibitor. In a specific embodiment, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof, a nucleoside inhibitor of HIV reverse transcriptase, and an HIV capsid inhibitor. In a specific embodiment, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof and an HIV capsid inhibitor. In a specific embodiment, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof and one, two, three, or four HIV bNAbs. In a specific embodiment, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof and one, two, three, or four HIV bNAbs and an HIV capsid inhibitor. In a specific embodiment, the kit comprises a compound as described herein or a pharmaceutically acceptable salt thereof and one, two, three, or four HIV bNAbs, an HIV capsid inhibitor, and a HIV nucleoside reverse transcriptase inhibitor.

Long-acting therapy against HIV.

Examples of drugs being developed for long-acting regimens include, but are not limited to, cabotegravir, rilpivirine, any integrase LA, VM-1500 LAI, maraviroc (LAI), tenofovir implant, islatravir implant, doravirine, raltegravir, and long-acting dolutegravir.

Combination therapy for HBV.

In some embodiments, a method of treating or preventing HBV infection is provided, comprising administering to a human a therapeutically effective amount of a composition described herein in combination with a therapeutically effective amount of one or more

- 50 049 099 (e.g., one, two, three, four, one or two, one to three, or one to four) additional therapeutic agents. In one embodiment, a method of treating HBV infection is provided, comprising administering to a human a therapeutically effective amount of a composition described herein in combination with a therapeutically effective amount of one or more (e.g., one, two, three, four, one or two, one to three, or one to four) additional therapeutic agents.

In specific embodiments, the present disclosure provides a method of treating an HBV infection comprising administering to a subject in need thereof a therapeutically effective amount of a composition described herein in combination with a therapeutically effective amount of one or more (e.g., one, two, three, four, one or two, one to three, or one to four) additional therapeutic agents that are suitable for treating an HBV infection.

The compounds described herein can be used or combined in one or more of a chemotherapeutic agent, an immunomodulator, an immunotherapeutic agent, a therapeutic antibody, a therapeutic vaccine, a bispecific antibody and antibody-like therapeutic protein (such as DARTs®, Duobodies®, Bites®, XmAbs®, TandAbs®, Fab derivatives), an antibody-drug conjugate (ADC), gene modifiers or gene editors (such as CRISPR Cas9, zinc finger nucleases, homing endonucleases, synthetic nucleases, TALENs), a cell therapy such as a CAR-T (chimeric antigen receptor T cell) and TCR-T (engineered T cell receptor) agent, or any combination thereof.

In some embodiments, the additional therapeutic agent or agents are selected from HBV combination therapies, HBV vaccines, HBV polymerase inhibitors, HBV capsid modulators, TLR7, TLR8, and TLR9 agonists, cytokines, immune checkpoint inhibitors, FLT3 ligands, interferon alpha receptor ligands, interferon alpha, interferon lambda, hyaluronidase inhibitors, hepatitis B surface antigen (HBsAg) inhibitors, HBV protein X inhibitors (HBx), cyclophilin inhibitors, HBV viral entry inhibitors, antisense oligonucleotides, short interfering RNA (siRNA), and DNA-directed RNA interference (ddRNAi), endonuclease modulators, ribonucleotide reductase inhibitors, HBV E antigen (HBeAg) inhibitors, covalently closed circular DNA (cccDNA), farnesoid X receptor agonists, HBV antibodies, bispecific antibodies that recruit T cells and NK cells, chimeric T cell receptors targeting HBV antigens or peptides, CAR T cell therapy, thymosin agonists, retinoic acid-inducible gene 1 stimulators, NOD2 stimulators, phosphatidylinositol 3-kinase (PI3K) inhibitors, indoleamine-2, 3-dioxygenase (IDO1) pathway inhibitors, anti-OX40, anti-CD40, anti-CD160, HBV gene editors, PAPD5/PAPD7 inhibitors, ZCCHC14 inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, epigenetic regulators, tertiary lymphoid aggregate inducers, antagonists IAP/XIAP, nucleic acid polymers (eg, NAP and STOPS), modulators of lipid metabolism or transport, arginase inhibitors and other drugs for the treatment of HBV and their combinations.

In some embodiments, the additional therapeutic agent or agents are selected from adefovir, entecavir, telbivudine, lamivudine, and lenacapavir, and combinations thereof.

In some embodiments, the additional therapeutic agent or agents are selected from adefovir, entecavir, telbivudine, lamivudine, and lenacapavir.

Combination drugs against HBV.

Examples of combination therapies for the treatment of HBV include, but are not limited to, TRUVADA® (tenofovir disoproxil fumarate and emtricitabine); ABX-203, lamivudine and PEG-IFN-alpha; ABX-203, adefovir and PEG-IFN-alpha; and INO-1800 (INO-9112 and RG7944).

Other anti-HBV drugs.

Examples of other drugs for the treatment of HBV include, but are not limited to, alphahydroxytropolone, amdoxovir, beta-hydroxycytosine nucleosides, AL-034, CCC-0975, elvucitabine, ezetimibe, cyclosporine A, gentiopicrin (gentiopicroside), JNJ-56136379, nitazoxanide, birinapant, NJK14047, NOV-205 (molixan, BAM-205), oligotide, mivotylate, feron, GST-HG-131, levamisole, Ka Shu Ning, alloferon, WS-007, Y-101 (Ti Fen Tai), rSIFN-co, PEG-IIFNm, KW-3, BP-Inter-014, oleanolic acid, HepB-nRNA, cTP-5 (rTP-5), HSK-II-2, HEISCO-106-1, HEISCO-106, Hepbarna, IBPB-006IA, Hepuyinfen, DasKloster 0014-01, ISA-204, Jiangantai (Ganxikang), MIV-210, OB-AI-004, PF-06, picroside, DasKloster-0039, hepulantai, IMB-2613, TCM-800B, reduced glutathione, RO-6864018, RG-7834, UB-551, and ZH-2N, and compounds described in US20150210682, (Roche), US 2016/0122344 (Roche), WO2015173164, WO2016023877, US2015252057A (Roche), WO16128335A1 (Roche), WO16120186A1 (Roche), US2016237090A (Roche), WO16107833A1 (Roche), WO16107832A1 (Roche), US2016176899A (Roche), WO16102438A1 (Roche), WO16012470A1 (Roche), US2016220586A (Roche) and US2015031687A (Roche).

HBV vaccines.

- 51 049099

HBV vaccines include both therapeutic and prophylactic vaccines. Examples of prophylactic HBV vaccines include, but are not limited to, Vaxelis, Hexaxim, Heplisav, Mosquirix, DTwP-HBV vaccine, Bio-Hep-B, D/T/P/HBV/M (LBVP-0101; LBVW-0101), DTwP-HepBHib-IPV vaccine, HeberpentaL, DTwP-HepB-Hib, V-419, CVI-HBV-001, Tetrabhay, hepatitis B prophylactic vaccine (Advax Super D), Hepatrol-07, GSK-223192A, ENGERIX B®, recombinant hepatitis B vaccine (intramuscular, Kangtai Biological Products), recombinant hepatitis B vaccine (Hansenual polymorpha yeast, intramuscular, Hualan Biological Engineering), recombinant HBV surface antigen vaccine, Bimmugen, Euforavac, Eutravac, anrix-DTaP-IPV-Hep B, HBAI-20, Infanrix-DTaP-IPV-Hep B-Hib, Pentabio Vaksin DTP-HB-Hib, Comvac 4, Twinrix, Euvax-B, Tritanrix HB, Infanrix Hep B, Comvax, DTP-Hib-HBV vaccine, DTP-HBV vaccine, Yi Tai, Heberbiovac HB, Trivac HB, GerVax, DTwP-Hep B-Hib vaccine, Bilive, Hepavax-Gene, SUPERVAX, Comvac5, Shanvac-B, Hebsulin, Recombivax HB, Revac B mcf, Revac B+, Fendrix, DTwP-HepB-Hib, DNA-001, Shan5, Shan6, rhHBsAG vaccine, HBI pentavaccine, LBVD, Infanrix HeXa and DTaP-rHB-Hib vaccine.

Examples of therapeutic HBV vaccines include, but are not limited to, HBsAG-HBIG complex, ARB-1598, Bio-Hep-B, NASVAC, abi-HB (intravenous), ABX-203, Tetrabhay, GX-110E, GS-4774, peptide vaccine (epsilon PA-44), Hepatrol-07, NASVAC (NASTERAP), IMP-321, BEVAC, Revac B mcf, Revac B+,MGN-1333, KW-2, CVI-HBV-002, AltraHepB, VGX-6200, FP-02, FP-02.2, TG-1050, NU-500, HBVax, im/TriGrid/antigen vaccine, Mega-CD40L adjuvanted vaccine, HepB-v, RG7944 (INO-1800), recombinant therapeutic vaccine based on VLP (HBV infection, VLP Biotech), AdTG-17909, AdTG-17910 AdTG-18202, ChronVac-B, TG-1050 and Lm HBV.

HBV DNA polymerase inhibitors.

Examples of HBV DNA polymerase inhibitors include, but are not limited to, adefovir (HEPSERA®), emtricitabine (EMTRIVA®), tenofovir disoproxil fumarate (VIREAD®), tenofovir alafenamide, tenofovir, tenofovir disoproxil, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir dipivoxil, tenofovir dipivoxil fumarate, tenofovir octadecyloxyethyl ether, CMX-157, besifovir, entecavir (BARACLUDE®), entecavir maleate, telbivudine (TYZEKA®), filocilivir, pradefovir, clevudine, ribavirin, lamivudine (EPIVIR-HBV®), phosphazide, famciclovir, fuzolin, metacavir, SnC-019754, FMCA, aGx-1009, AR-II-04-26, HIP-1302, tenofovir disoproxil aspartate, tenofovir disoproxil orotate, and HS-10234.

Immunomodulators.

Examples of immunomodulators include, but are not limited to, rintatolimod, imidol hydrochloride, ingaron, dermavir, plaquenil (hydroxychloroquine), proleukin, hydroxyurea, mycophenolate mofetil (MPA) and its ester derivative mycophenolate mofetil (MMF), JNJ-440, WF-10, AB-452, ribavirin, IL-12, INO-9112, polyethyleneimine polymer (PEI), Gepon, VGV-1, MOR-22, CRV-431, JNJ0535, TG-1050, ABI-H2158, BMS-936559, GS-9688, RO-7011785, RG-7854, AB-506, RO-6871765, AIC-649 and IR-103.

Toll-like receptor (TLR) modulators.

In various embodiments, the agents described herein are combined with a toll-like receptor (TLR) agonist, such as an agonist of TLR1 (NCBI Gene ID: 7096), TLR2 (NCBI Gene ID: 7097), TLR3 (NCBI Gene ID: 7098), TLR4 (NCBI Gene ID: 7099), TLR5 (NCBI Gene ID: 7100), TLR6 (NCBI Gene ID: 10333), TLR7 (NCBI Gene ID: 51284), TLR8 (NCBI Gene ID: 51311), TLR9 (NCBI gene accession number: 54106) and/or TLR10 (NCBI gene accession number: 81793). Examples of TLR7 agonists that can be co-administered include, but are not limited to, AL-034, DSP-0509, GS-9620 (vesatolimod), a vesatolimod analog, LHC-165, TMX101 (imiquimod), GSK-2245035, resiquimod, DSR-6434, DSP-3025, IMO-4200, MCT-465, MEDI-9197, 3M-051, SB-9922, 3M-052, Limtop, TMX-30X, TMX-202, RG-7863, RG-7854, RG-7795, and the compounds described in US 20100143301 (Gilead Sciences), US 20110098248 (Gilead Sciences), and US 20090047249 (Gilead Sciences), US 20140045849 (Janssen), US 20140073642 (Janssen), WO 2014/056953 (Janssen), WO 2014/076221 (Janssen), WO 2014/128189 (Janssen), US20140350031 (Janssen), WO 2014/023813 (Janssen), US 20080234251 (Array Biopharma), US 20080306050 (Array Biopharma), US 20100029585 (Ventirx Pharma), US 20110092485 (Ventirx Pharma), US 20110118235 (Ventirx Pharma), US 20120082658 (Ventirx Pharma), US 20120219615 (Ventirx Pharma), US 20140066432 (Ventirx Pharma), US 20140088085 (Ventirx Pharma), US 20140275167 (Novira Therapeutics), and US 20130251673 (Novira Therapeutics). Dual TLR7/TLR8 agonists that can be co-administered include NKTR-262, telratolimod, and BDB-001. Examples of TLR8 agonists that can be co-administered include, but are not limited to, E6887, IMO-4200, IMO-8400, IMO-9200, MCT-465, MEDI-9197, motolimod, resiquimod, GS-9688, VTX1463, VTX-763, 3M-051, 3M-052 and the compounds described in US20140045849 (Janssen), US 20140073642 (Janssen), WO 2014/056953 (Janssen), WO 2014/076221 (Janssen), WO 2014/128189 (Janssen), US

- 52 049099

20140350031 (Janssen), WO 2014/023813 (Janssen), US 20080234251 (Array Biopharma), US 20080306050 (Array Biopharma), US 20100029585 (Ventirx Pharma), US 20110092485 (Ventirx Pharma), US 20110118235 (Ventirx Pharma), US 20120082658 (Ventirx Pharma), US 20120219615 (Ventirx Pharma), US 20140066432 (Ventirx Pharma), US 20140088085 (Ventirx Pharma), US 20140275167 (Novira Therapeutics), and US 20130251673 (Novira Therapeutics). Examples of TLR9 agonists that can be co-administered include, but are not limited to, AST-008, cobitolimod, CMP-001, IMO-2055, IMO-2125, S-540956, litenimob, MGN-1601, BB-001, BB-006, IMO-3100, IMO-8400, IR-103, IMO-9200, agatolimod, DIMS-9054, DV-1079, DV-1179, AZD-1419, lefitolimod (MGN-1703), CYT-003, CYT-003-QbG10, tilsotolimod, and PUL-042. Examples of TLR3 agonist include rintatolimod, poly-ICLC, RIBOXXON®, Apoxxim, RIBOXXIM®, IPH-33, MCT-465, MCT-475, and ND-1.1 Examples of TLR4 agonist include interferon-alpha receptor ligands G-100 and GSK-179509.1

Examples of interferon alpha receptor ligands include, but are not limited to, interferon alpha2b (INTRON A®), pegylated interferon alpha-2a (PEGASIS®), pegylated interferon alpha-N, interferon alpha-N (GAPGEN®), weldon, infradur, roferon-A, YPEG-rhIFNalpha-2a, P-1101, alferon, alfarona, ingaron (interferon gamma), rSIFN-co (recombinant super interferon), YPEG-rhIFNalpha-2b, MOR-22, peginterferon alpha-N (PEG-INTRON®), bioferon, novaferon, inmutag (inferon), MULTIFERON®, interferon alpha-n1 (HUMOFERON®), interferon beta-N (AVONEX®), schaferon, interferon alpha^ (Axxo), alphaferon, interferon alpha^ (BioGeneric Pharma), interferon-alpha 2 (CJ), laferon, VIPEG, BLAUFERON-A, m-B, Intermax Alpha, realdiron, lanstion, pegaferon, PDferon-B PDferon-B, interferon alpha^ (IFN, Laboratories Bioprofarma), alphainterferon^, calferon, pegnano, feronsur, PegiHep, interferon alpha^d (Zydus-Cadila), optipeg A, Realfa 2B, reliferon, interferon alpha^ (Amega), interferon alpha-2b (Virchow), ropeginterferon alpha-2b, rHSA-IFN alpha-2a (fusion protein of recombinant human serum albumin and interferon alpha-2a), rHSA-IFN alpha-2b, recombinant human interferon alpha-(1b, 2a, 2b), peginterferon alpha-2b (Amega), peginterferon alpha-2a, reaferon-EC, proquiferon, uniferon, uriferon, interferon alpha-^ (Changchun Institute of Biological Products), anterferon, shanferon, lifeferon, Shang Sheng Lei Tai, INTEFEN, sinogen, Fukangtai, pegstat, rHSA-IFN alpha-2^ SFR-9216, and Interapo (Interapa).

Hyaluronidase inhibitors.

Examples of hyaluronidase inhibitors include, but are not limited to, astodrimer.

Hepatitis B surface antigen (HBsAg) inhibitors.

Examples of HBsAg inhibitors include, but are not limited to, HBF-0259, PBHBV-001, PBHBV-2-15, PBHBV-2-1, REP-9AC, REP-9C, REP-9, REP-2139, REP-2139-Ca, REP-2165, REP-2055, REP-2163, REP2165, REP-2053, REP-2031 and REP-006 and REP-9AC'. Examples of HBsAg secretion inhibitors include, but are not limited to, bM601.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors.

Examples of cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitors include, but are not limited to, AGEN-2041, AGEN-1884, ipilimumab, belatacept, PSI-001, PRS-010, Probody mAb, tremelimumab, and JHL-1155.

Cyclophilin inhibitors.

Examples of cyclophilin inhibitors include, but are not limited to, CPI-431-32, EDP-494, OCB-030, SCY-635, NVP-015, NVP-018, NVP-019, STG-175, and the compounds described in US8513184 (Gilead Sciences), US20140030221 (Gilead Sciences), US20130344030 (Gilead Sciences), and US20130344029 (Gilead Sciences).

Inhibitors of HBV virus entry.

Examples of HBV entry inhibitors include, but are not limited to, Myrcludex B.

Antisense oligonucleotides.

Examples of antisense oligonucleotides targeting viral mRNA include, but are not limited to, ISIS-ВГBRx, IONIS-ВГBRx, IONIS-GSK6-LRx, GSK-3389404, and RG-6004. Antisense oligonucleotides targeting host factors such as PD-L1 are also known.

Short interfering RNAs (siRNAs) and lncRNAi.

Examples of siRNAs include, but are not limited to, TKM-HBV (TKM-NerB), ALN-HBV, SR-008, NeerBnRNA, and ARC-520, ARC-521, ARB-1740, ARB-1467. Examples of DNA-directed RNA interference (dsRNAi) molecules include, but are not limited to, BB-HB-331.

Endonuclease modulators.

Examples of endonuclease modulators include, but are not limited to, PGN-514.

Ribonucleotide reductase inhibitors.

Examples of ribonucleotide reductase inhibitors include, but are not limited to, trimidox.

Inhibitors of antibodies to HBV E-antigen.

Examples of inhibitors of antibodies to HBV E antigen (HBeAg) include, but are not limited to, wogonin.

Inhibitors of covalently closed circular DNA (cccDNA).

- 53 049099

Examples of cccDNA inhibitors include, but are not limited to, BSBI-25 and CHR-101.

Farnesoid X receptor agonist.

Examples of farnesoid X receptor agonists include, but are not limited to, EYP-001, cilofexor, EDP-305, MET-409, tropifexor, AKN-083, RDX-023, BWD-100, LMB-763, INV-3, NTX-023-1, EP024297, and GS-8670.

Antibodies to HBV.

Examples of HBV antibodies targeting hepatitis B surface antigens include, but are not limited to, GC-1102, XTL-17, XTL-19, KN-003, IV Hepabulin SN, fully human monoclonal antibody therapy (hepatitis B virus infection, Humabs BioMed), and anti-HBsAg antibodies (small, medium, large).

Examples of HBV antibodies, including monoclonal antibodies and polyclonal antibodies, include, but are not limited to, Zutectra, Shang Sheng Gan Di, Uman Big (hyperimmune hepatitis B), Omri-Hep-B, Nabi-HB, Hepatect CP, HepaGam B, Igantibe, Niuliva, CT-P24, Hepatitis B Immunoglobulin (IV, pH4, HBV Infection, Shanghai RAAS Blood Products), and Fovepta (BT-088).

Fully human monoclonal antibodies include, but are not limited to, HBC-34.

CCR2 chemokine antagonists.

Examples of CCR2 chemokine antagonists include, but are not limited to, propagermanium.

Thymosin agonists.

Examples of thymosin agonists include, but are not limited to, thymalfasin, recombinant thymosin alpha 1 (GeneScience).

Cytokines.

Examples of cytokines include, but are not limited to, interferon alpha, interferon lambda, recombinant IL-7, CYT-107, interleukin-2 (IL-2, Immunex), recombinant human interleukin-2 (Shenzhen Neptunus), IL-15, IL-21, IL-24, celloleukin, and cytokines targeting CD4, CD8, or B cells, including, but not limited to, IL-2, IL-7, IL-12, IL-15, and IL-21.

Nucleoprotein modulators.

Nucleoprotein modifiers may be inhibitors of the HBV capsid or capsid protein. Examples of nucleoprotein modulators include, but are not limited to, GS-4882, AB-423, AT-130, GLS4, NVR-1221, NVR-3778, AL-3778, BAY 41-4109, morphothiadine mesylate, ARB-168786, ARB-880, JNJ-379, RG-7907, HEC-72702, AB-506, ABI-H0731, JNJ-440, ABI-H2158, and DVR-23.

Examples of capsid inhibitors include, but are not limited to, the compounds described in US 20140275167 (Novira Therapeutics), US 20130251673 (Novira Therapeutics), US 20140343032 (Roche), WO 2014037480 (Roche), US 20130267517 (Roche), WO 2014131847 (Janssen), WO 2014033176 (Janssen), WO 2014033170 (Janssen), WO 2014033167 (Janssen), WO 2015/059212 (Janssen), WO 2015118057 (Janssen), WO 2015011281 (Janssen), WO 2014184365 (Janssen), WO 2014184350 (Janssen), WO 2014161888 (Janssen), WO 2013096744 (Novira), US 20150225355 (Novira), US 20140178337 (Novira), US 20150315159 (Novira), US 20150197533 (Novira), US 20150274652 (Novira), US 20150259324, (Novira), US 20150132258 (Novira), US 9181288 (Novira), WO 2014184350 (Janssen), WO 2013144129 (Roche), WO 2017198744 (Roche), US 20170334882 (Novira), US 20170334898 (Roche), WO 2017202798 (Roche), WO 2017214395 (Enanta), WO 2018001944 (Roche), WO 2018001952 (Roche), WO 2018005881 (Novira), WO 2018005883 (Novira), WO 2018011100 (Roche), WO 2018011160 (Roche), WO 2018011162 (Roche), WO 2018011163 (Roche), WO 2018036941 (Roche), WO 2018043747 (Kyoto Univ), US 20180065929 (Janssen), WO 2016168619 (Indiana University), WO 2016195982 (The Penn State Foundation), WO 2017001655 (Janssen), WO 2017048950 (Assembly Biosciences), WO 2017048954 (Assembly Biosciences), WO 2017048962 (Assembly Biosciences), US 20170121328 (Novira), US 20170121329 (Novira).

Examples of transcript inhibitors include, but are not limited to, the compounds described in WO 2017013046 (Roche), WO 2017016960 (Roche), WO 2017017042 (Roche), WO 2017017043 (Roche), WO 2017061466 (Toyoma chemicals), WO 2016177655 (Roche), WO 2016161268 (Enanta). WO2017001853 (Redex Pharma), WO 2017211791 (Roche), WO 2017216685 (Novartis), WO 2017216686 (Novartis), WO 2018019297 (Ginkgo Pharma), WO 2018022282 (Newave Pharma), US20180030053 (Novartis), WO 2018045911 (Zhejiang Pharma).

Retinoic acid-inducible gene 1 stimulators.

Examples of retinoic acid inducible gene 1 stimulators include, but are not limited to, SB-9200, SB-40, SB-44, ORI-7246, ORI-9350, ORI-7537, ORI-9020, ORI-9198, ORI-7170, and RGT-100.

NOD2 Stimulants.

Examples of NOD2 stimulators include, but are not limited to, SB-9200.

Phosphatidylinositol 3-kinase (PI3K) inhibitors.

Examples of PI3K inhibitors include, but are not limited to, idelalisib, ACP-319, AZD-8186, AZD8835, buparlisib, CDZ-173, CLR-457, pictilisib, neratinib, rigosertib, rigosertib sodium, EN-3342, TGR-1202, alpelisib, duvelisib, IPI-549, UCB-5857, tazelisib, XL-765, gedatolisib, ME-401, VS-5584, copanlisib, CAI orotate, perifosine, RG-7666, GSK-2636771, DS-7423, panulisib, GSK-2269557, GSK2126458, CUDC-907, PQR-309, INCB-40093, pilaralisib, BAY-1082439, piquitinib mesylate, SAR

- 54 049099

245409, AMG-319, RP-6530, ZSTK-474, MLN-1117, SF-1126, RV-1729, sonolisib, LY-3023414, SAR260301, TAK-117, HMPL-689, tenalisib, voxtalisib, and CLR-1401.

Indoleamine 2,3-dioxygenase (IDO1) pathway inhibitors.

In various embodiments, the agents described herein are combined with an inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1; NCBI Gene #3620). Examples of IDO1 inhibitors include, but are not limited to, BLV-0801, epacadostat, F-001287, GBV-1012, GBV-1028, GDC-0919, indoximod, NKTR-218, NLG-919 vaccine, PF-06840003, pyranonaphthoquinone derivatives (SN35837), resminostat, SBLK-200802, BMS-986205 and shIDO-ST, EOS-200271, KHK-2455, LY-3381916.

Recombinant thymosin alpha-1.

Examples of recombinant thymosin alpha-1 include, but are not limited to, NL-004 and PEGylated thymosin alpha-1.

Bruton tyrosine kinase (BTK) inhibitors.

Examples of BTK inhibitors include, but are not limited to, ABBV-105, acalabrutinib (ACP-196), ARQ-531, BMS-986142, dasatinib, ibrutinib, GDC-0853, PRN-1008, SNS-062, ONO-4059, BGB-3111, ML-319, MSC-2364447, RDX-022, X-022, AC-058, RG-7845, spebrutinib, TAS-5315, TP-0158, TP-4207, HM-71224, KBP-7536, M-2951, TAK-020, AC-0025, and the compounds described in US20140330015 (Ono Pharmaceutical), US20130079327 (Ono Pharmaceutical) and US20130217880 (Ono Pharmaceutical).

KDM inhibitors.

Examples of KDM5 inhibitors include, but are not limited to, the compounds described in WO2016057924 (Genentech/Constellation Pharmaceuticals), US20140275092 (Genentech/Constellation Pharmaceuticals), US20140371195 (Epitherapeutics), US20140371214 (Epitherapeutics), US20160102096 (Epitherapeutics), US20140194469 (Quanticel), US20140171432, US20140213591 (Quanticel), US20160039808 (Quanticel), US20140275084 (Quanticel), and WO2014164708 (Quanticel).

Examples of KDM1 inhibitors include, but are not limited to, the compounds described in US 9186337B2 (Oryzon Genomics), GSK-2879552, and RG-6016.

STING agonists.

Examples of STING agonists include, but are not limited to, SB-11285, AdVCA0848, STINGVAX, and the compounds described in WO 2018065360 (Biolog Life Science Institute Forschungslabor und BiochemicaVertrieb GmbH, Germany), WO 2018009466 (Aduro Biotech), WO 2017186711 (InvivoGen), WO 2017161349 (Immune Sensor), WO 2017106740 (Aduro Biotech), US 20170158724 (Glaxo Smithkiline), WO 2017075477 (Aduro Biotech), US 20170044206 (Merck), WO 2014179760 (University of California), WO 2018098203 (Janssn), WO 2018118665 (Merck), WO 2018118664 (Merck), WO 2018100558 (Takeda), WO 2018067423 (Merck) and WO 2018060323 (Boehringer).

Non-nucleoside reverse transcriptase inhibitors (NNRTIs).

Examples of NNRTIs include, but are not limited to, the compounds described in WO2018118826 (Merck), WO2018080903 (Merck), WO2018119013 (Merck), WO2017100108 (Idenix), WO2017027434 (Merck), WO2017007701 (Merck), and WO2008005555 (Gilead).

Inhibitors of HBV replication.

Examples of hepatitis B virus replication inhibitors include, but are not limited to, isothiafludine, IQP-HBV, RM-5038, and Xingantie.

Arginase inhibitors

Examples of arginase inhibitors include, but are not limited to, CB-1158, C-201, and resminostat.

Gene therapy and cell therapy.

Gene therapy and cell therapy include genetic modification to suppress gene expression; genetic approaches to directly kill infected cells; infusion of immune cells designed to replace much of the patient's own immune system to enhance the immune response to infected cells, or activate the patient's own immune system to kill infected cells, or seek out and kill infected cells; and genetic approaches to modify cellular activity to further alter the endogenous immune response against infection.

Gene editors.

Examples of genome editing systems include, but are not limited to, the CRISPR/Cas9 system, the zinc finger nuclease system, the TALEN system, the homing endonuclease system, and the meganuclease system; for example, the removal of cccDNA by targeted cleavage and the alteration of one or more viral genes of the hepatitis B virus (HBV). An alteration (e.g., knockout and/or knockdown) of a PreC, C, X, PreSI, PreS2, S, P, or SP gene refers to (1) a decrease in or abolition of the expression of a PreC, C, X, PreSI, PreS2, S, P, or SP gene, (2) a disruption of the function of a precore protein, core protein, X protein, long surface protein, midsurface protein, S protein (also known as HBs antigen and HBsAg), hepatitis B polymerase protein, and/or spliced protein (HBe, HBc, HBx, PreS1, PreS2, S, Pol, and/or HBSP), or (3) a decrease in or abolition of intracellular, serum, and/or intraparenchymal levels of HBe, HBc, HBx, LHB, MHB, SHB, Pol, and/or HBSP proteins. Knockdown of one or more of the PreC, C, X, PreSI, PreS2, S, P and/or SP genes is accomplished by targeting gene(s) within the HBV cccDNA

- 55 049099 and/or integrated HBV DNA.

CAR-T cell therapy.

CAR-T cell therapy includes a population of immune effector cells engineered to express a chimeric antigen receptor (CAR), wherein the CAR comprises an HBV antigen binding domain. The immune effector cell is a T cell or an NK cell. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof. The cells may be autologous or allogeneic.

TCR-T cell therapy.

TCR-T cell therapy includes T cells expressing HBV-specific T cell receptors. TCR-T cells are engineered to target HBV-derived peptides displayed on the surface of virus-infected cells (e.g., peptides displayed in HLA/pMHC). In some embodiments, the T cells express a TCR specific for HBV surface antigen (HBsAg). Examples of TCR-T therapy directed at treating HBV include, but are not limited to, LTCR-H2-1.

In another specific embodiment, a compound described herein, or a pharmaceutically acceptable salt thereof, is combined with an HBV DNA polymerase inhibitor, one or two additional therapeutic agents selected from: immunomodulators, TLR modulators, HBsAg inhibitors, HBsAg secretion or assembly inhibitors, therapeutic HBV vaccines, HBV antibodies, including HBV antibodies targeting hepatitis B virus surface antigens and bispecific antibodies and antibody-like therapeutic proteins (e.g., DARTs®, DUOBODIES®, BITES®, XmAbs®, TandAbs®, Fab derivatives or TCR-like antibodies), cyclophilin inhibitors, retinoic acid inducible gene 1 stimulators, RIG-I-like receptor stimulators, PD-1 inhibitors, PD-L1 inhibitors, arginase, PI3K inhibitors, IDO inhibitors and NOD2 stimulators, and one or two additional therapeutic agents selected from the group consisting of: HBV entry inhibitors, NTCP inhibitors, HBx inhibitors, cccDNA inhibitors, HBV antibodies targeting hepatitis B surface antigens, siRNA, miRNA-based gene therapy agents, shRNA, KDM5 inhibitors or nucleoprotein modulators (HBV core or capsid protein modulators).

In another specific embodiment, a compound as described herein, or a pharmaceutically acceptable salt thereof, is combined with an HBV DNA polymerase inhibitor and at least a second additional therapeutic agent selected from: immunomodulators, TLR modulators, HBsAg inhibitors, HBV therapeutic vaccines, HBV antibodies including HBV antibodies targeting hepatitis B virus surface antigens including anti-HBsAg antibodies (small, medium, large), bispecific antibodies and antibody-like therapeutic proteins (such as DART®, DUOBODIES®, BITES®, XmAbs®, TandAbs®, Fab derivatives or TCR-like antibodies), cyclophilin inhibitors, retinoic acid inducible gene 1 stimulators, RIG-I like receptor stimulators, PD-1 inhibitors, PD-L1 inhibitors, arginase, PI3K inhibitors, IDO1 inhibitors and NOD2 stimulators.

In another specific embodiment, a compound described herein or a pharmaceutically acceptable salt thereof is combined with an HBV DNA polymerase inhibitor and at least a second additional therapeutic agent selected from: HBV viral entry inhibitors, NTCP inhibitors, HBx inhibitors, cccDNA inhibitors, HBV antibodies targeting hepatitis B surface antigen including anti-HBsAg (small, medium, large), siRNA, miRNA, shRNA, KDM5 inhibitors, and nucleoprotein modulators (HBV core or capsid protein modulators).

In a specific embodiment, a compound described herein, or a pharmaceutically acceptable salt thereof, is combined with compounds such as those described in U.S. Pub. No. 2010/0143301 (Gilead Sciences), U.S. Pub. No. 2011/0098248 (Gilead Sciences), U.S. Pub. No. 2009/0047249 (Gilead Sciences), U.S. Patent No. 8,722,054 (Gilead Sciences), U.S. Pub. No. 2014/0045849 (Janssen), U.S. Pub. No. 2014/0073642 (Janssen), WO 2014/056953 (Janssen), WO2014/076221 (Janssen), WO 2014/128189 (Janssen), U.S. Pub. No. 2014/0350031 (Janssen), WO2014/023813 (Janssen), US Publication No. 2008/0234251 (Array Biopharma), US Publication No. 2008/0306050 (Array Biopharma), US Publication No. 2010/0029585 (Ventirx Pharma), US Publication No. 2011/0092485 (Ventirx Pharma), US 2011/0118235 (Ventirx Pharma), US Publication No. 2012/0082658 (Ventirx Pharma), US Publication No. 2012/0219615 (Ventirx Pharma), US Publication No. 2014/0066432 (Ventirx Pharma), US Publication No. 2014/0088085 (Ventirx Pharma), US Publication No. 2014/0275167 (Novira Therapeutics), US Publication No. 2013/0251673 (Novira Therapeutics), US Patent No. 8,513,184 (Gilead Sciences), US Publication No. 2014/0030221 (Gilead Sciences), US Publication No. 2013/0344030 (Gilead Sciences), US Publication No. 2013/0344029 (Gilead Sciences), US 20140275167 (Novira Therapeutics), US 20130251673 (Novira Therapeutics), US Publication No. 2014/0343032 (Roche), WO 2014037480 (Roche), US Publication No. 2013/0267517 (Roche), WO 2014131847 (Janssen), WO

- 56 049099

2014033176 (Janssen), WO 2014033170 (Janssen), WO 2014033167 (Janssen), WO 2015/059212 (Janssen), WO 2015118057 (Janssen), WO 2015011281 (Janssen), WO 2014184365 (Janssen), WO 2014184350 (Janssen), WO 2014161888 (Janssen), WO 2013096744 (Novira), US 20150225355 (Novira), US 20140178337 (Novira), US 20150315159 (Novira), US 20150197533 (Novira), US 20150274652 (Novira), US 20150259324, (Novira), US 20150132258 (Novira), US 9181288 (Novira), WO 2014184350 (Janssen), WO 2013144129 (Roche), US 20100015178 (Incyte), US 2016137652 (Flexus Biosciences, Inc.), WO2014073738 (Flexus Biosciences, Inc.), WO2015188085 (Flexus Biosciences, Inc.), US publication no. 2014/0330015 (Ono Pharmaceutical), US Publication No. 2013/0079327 (Ono Pharmaceutical), US Publication No. 2013/0217880 (Ono pharmaceutical), WO 2016057924 (Genentech/Constellation Pharmaceuticals), US 20140275092 (Genentech/Constellation Pharmaceuticals), US 20140371195 (Epitherapeutics) and US 20140371214 (Epitherapeutics), US 20160102096 (Epitherapeutics), US 20140194469 (Quanticel), US 20140171432, US 20140213591 (Quanticel), US 20160039808 (Quanticel), US 20140275084 (Quanticel), WO 2014164708 (Quanticel), US 9186337B2 (Oryzon Genomics), and other drugs for the treatment of HBV, as well as combinations thereof.

Introduction.

Routes of administration.

A compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof (also referred to herein as the active ingredient) can be administered by any route appropriate to the condition being treated. Suitable routes of administration include, but are not limited to, oral, rectal, nasal, topical (including buccal and sublingual), transdermal, vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that the appropriate route may vary, for example, depending on the condition of the recipient. In certain embodiments, the compounds of the invention can be administered parenterally. In certain embodiments, the compounds of the invention can be administered intravenously, subcutaneously or intramuscularly. In certain embodiments, the compounds of the invention are orally bioavailable and can be administered orally.

In some embodiments, the compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, is administered using a syringe suitable for administering the compound. In some embodiments, a disposable syringe is used. In some embodiments, a reusable syringe is used. In some embodiments, the syringe is pre-filled with the compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula I, II, III, IV or V or a pharmaceutically acceptable salt thereof is administered using an automatic medical injector. In some embodiments, a disposable syringe is used. In some embodiments, a reusable syringe is used. In some embodiments, the syringe is pre-filled with the compound of formula I, II, III, IV or V or a pharmaceutically acceptable salt thereof.

Dosage regimen.

In some embodiments, a compound, such as a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, is administered to a subject according to an effective dosage regimen for a desired period of time or duration, such as at least about once a day, at least about once a week, at least about once a month, at least about once every 2 months, at least about once every 3 months, at least about once every 4 months, at least about once every 6 months, or at least about once every 12 months or longer. In some embodiments, the compound is administered daily or on an intermittent schedule. In some embodiments, the compound is administered weekly. In some embodiments, the compound is administered monthly. In some embodiments, the compound is administered every two months. In some embodiments, the compound is administered every three months. In some embodiments, the compound is administered every four months. In some embodiments, the compound is administered every five months. In some embodiments, the compound is administered every 6 months.

In some embodiments, a compound, such as a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously or intramuscularly at least about once a month. In some embodiments, a compound (e.g., a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof) is administered to the subject subcutaneously or intramuscularly at least about once every 2 months, or at least about once every 3 months, or at least about once every 4 months, or at least about once every 6 months. In some embodiments, a compound (e.g., a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof) is administered to the subject subcutaneously at least about once a month. In some embodiments, a compound (e.g., a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof) is administered to the subject subcutaneously at least about once every 2 months. In some embodiments, the compound (e.g., a compound of Formula I, II, III, IV, or V

- 57 049099 or a pharmaceutically acceptable salt thereof) is administered to a subject subcutaneously at least approximately once every 3 months.

In some embodiments, the dosage or frequency of dosing of a compound of Formula I, II, III, IV, or V or a pharmaceutically acceptable salt thereof is adjusted during the course of treatment based on the judgment of the treating physician.

In some embodiments, a compound described herein (e.g., a compound of formula I, II, III, IV, or V), or a pharmaceutically acceptable salt thereof, can be administered in an effective dosage volume. For example, the dosage size of the compound can be from 1 mg to 1000 mg.

In some embodiments, the methods described herein comprise administering to a subject a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, on an event-based basis.

As used herein, the terms event-based and event-based administration refer to the administration of a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof (1) prior to an event (e.g., 2 hours, 1 day, 2 days, 5 days, or 7 or more days prior to an event) that exposes an individual to HIV (or otherwise increases the risk of HIV infection); and/or (2) during an event (or more than one recurring event) that may expose the subject to HIV (or otherwise increase the risk of the individual becoming infected with HIV); and/or (3) following an event (or after the last event in a series of recurring events) that may expose the subject to HIV (or otherwise increase the risk of the individual becoming infected with HIV). In some embodiments, event-based administration is performed prior to the subject becoming infected with HIV. In some embodiments, event-based administration is performed after the subject becoming infected with HIV. In some embodiments, event-based administration is performed prior to the subject's exposure to HIV and following the subject's exposure to HIV.

In some embodiments, a compound of Formula I, II, III, IV, or V or a pharmaceutically acceptable salt thereof is administered prior to exposing the subject to HIV.

An example of an event-based dosing regimen comprises administering a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, within 24 hours to 2 hours prior to exposure to HIV (e.g., first sexual contact with a sexual partner known to be HIV-infected, including sexual intercourse), followed by administering a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, every 24 hours during the period of exposure (e.g., sexual contact with a sexual partner known to be HIV-infected), followed by administering a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, after the last exposure (e.g., sexual contact with a sexual partner known to be HIV-infected), and one final administration of a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, 24 hours later.

Another example of an event-based dosing regimen includes administration of a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, within 24 hours prior to exposure to HIV (e.g., sexual contact with a sexual partner known to be HIV-infected), then daily administration during the period of exposure (e.g., sexual activity with a sexual partner known to be HIV-infected, including the last sexual act), followed by a final administration approximately 24 hours after the last exposure (this may be an increased dose, e.g., a double dose).

In some embodiments, such as when administered in the form of PrEP, the compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, is administered daily. In some embodiments, such as when administered in the form of PrEP based on the occurrence of an event, the compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, is administered from 1 hour to 10 days, from 1 hour to 7 days, from 1 hour to 5 days, from 1 to 72 hours, from 1 to 48 hours, from 1 to 24 hours, or 112 hours before an event that may increase the risk of acquiring HIV (e.g., before sexual intercourse or other exposure to the HIV virus). In some embodiments, a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, is administered within 10 days, 7 days, 5 days, 72 hours, 60 hours, 48 hours, 24 hours, 12 hours, 9 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour before an event that may increase an individual's risk of acquiring HIV (e.g., before sexual intercourse or other contact with the HIV virus). In some embodiments, when a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, is administered before an event (e.g., administered prior to an event) that may increase an individual's risk of acquiring HIV, it is administered daily prior to the event (e.g., sexual contact). In some embodiments, when a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, is administered prior to an event that may increase an individual's risk of acquiring HIV, it is administered one to three times prior to the event.

In some embodiments, such as when administered as part of an event-based PrEP regimen, a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable derivative thereof,

- 58 049099 the salt is administered during exposure to HIV. In some embodiments, the compound of formula I, II, III, IV or V is administered before exposure, the compound of formula I, II, III, IV or V or a pharmaceutically acceptable salt thereof is administered daily (e.g., as a single dose) during exposure to HIV (e.g., during sexual intercourse with a partner known to be HIV-infected). In some embodiments, the compound of formula I, II, III, IV or V or a pharmaceutically acceptable salt thereof is administered daily (e.g., for 1-7 days) after the last exposure to HIV (e.g., after a period of sexual contact with a sexual partner known to be HIV-infected). In some embodiments, administration is continued for 1 or 2 days after the last exposure to HIV.

Additional examples of PrEP and/or PEP dosing regimens can be found, for example, in the abstract of a clinical trial entitled On Demand Antiretroviral Pre-exposure Prophylaxis for HIV Infection in Men Who Have Sex With Men (Clinical Trial # NCT01473472); the abstract of a clinical trial entitled Prevention of HIV in Ile-de-France (Clinical Trials # NCT03113123), and in Molina, et al. N. Engl. J. Med. 2015, 353:2237-2246, the disclosures of each of which are incorporated herein by reference in their entireties.

In some embodiments, methods for reducing the risk of acquiring HIV (e.g., HIV-1 and/or HIV-2) comprise administering a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, in combination with protected sexual intercourse. In some embodiments, methods for reducing the risk of acquiring HIV (e.g., HIV-1 and/or HIV-2) comprise administering to an individual at risk of acquiring HIV. Examples of individuals at high risk of acquiring HIV include, but are not limited to, individuals at risk of sexually transmitting HIV.

In some embodiments, the reduction in the risk of acquiring HIV is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In some embodiments, the reduction in the risk of acquiring HIV is at least about 75%. In some embodiments, the reduction in the risk of acquiring HIV is about 80%, about 85%, or about 90%.

Compound.

Formulations suitable for parenteral administration include, but are not limited to, aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the solution isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. In some embodiments, the suspension is a microsuspension. In some embodiments, the suspension is a nanosuspension.

In some embodiments, formulations suitable for parenteral administration (e.g., intramuscular (IM) and subcutaneous (SC) administration) will include one or more excipients. The excipients should be compatible with the other ingredients of the formulation and physiologically safe for the recipient. Examples of suitable excipients are well known to those skilled in the art of parenteral formulations and are listed, for example, in Handbook of Pharmaceutical Excipients (eds. Rowe, Sheskey & Quinn), 6th edition, 2009.

In some embodiments, the active ingredient (e.g., a compound of Formula I, II, III, IV, or V) is present as a free acid.

In some embodiments, the pharmaceutical composition described herein is a parenteral formulation. In some embodiments, the formulation is administered to a subject subcutaneously. In some embodiments, the formulation is administered to a subject intramuscularly.

The amount of active ingredient that can be combined with inactive ingredients to produce a dosage form can vary depending on the intended subject of treatment and the particular route of administration. For example, in some embodiments, a formulation for oral administration to a human can contain from about 1 mg to 1000 mg of the active substance, prepared with a suitable amount of a carrier substance (e.g., an inactive ingredient or excipient). In some embodiments, the carrier substance ranges from about 5% to 95% of the total weight of the composition (weight:weight or w:w).

It should be understood that in addition to the ingredients specifically mentioned above, the compositions of the present embodiments may include other agents common in the art related to the type of composition in question, for example, those suitable for oral administration may contain flavoring agents.

Kits and industrial products.

Kits that contain a compound of the present invention, or an enantiomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing any of the above are also included in the present invention. In one embodiment, a kit up to

- 59 049099 further includes instructions for use. In one aspect, the kit includes a compound of the invention or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug or deuterated analog and a label and/or instructions for using the compounds in the treatment of an indication, such as a disease or condition, described herein. In one embodiment of kits containing a compound of the present invention or a pharmaceutically acceptable salt thereof in combination with one or more (e.g., one, two, three, four, one or two, or one to three, or one to four) additional therapeutic agents are provided.

The present document also provides articles of manufacture that include a compound of the present invention or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug or deuterated analog thereof in a suitable container. The container may be a vial, a vessel, an ampoule, a pre-filled syringe, an implant or an intravenous infusion bag.

In some embodiments, the present invention provides a kit comprising a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt thereof. In one embodiment, the kit may include one, two, three or four additional therapeutic agents as described above. The kit may further comprise instructions for use, such as for use in inhibiting HIV integrase, such as for use in treating HIV infection or AIDS, or as a research tool. The instructions for use are typically printed instructions, although electronic storage media (e.g., magnetic or optical disks) containing the instructions are also acceptable.

In some embodiments, the present invention also provides a pharmaceutical kit comprising one or more containers containing a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof. If desired, associated with such container(s) may be a notification in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which reflects approval by the agency of manufacture, use, or sale for administration to humans. Each component (if more than one component) may be packaged in separate containers, or some components may be combined into a single container if cross-reactivity and shelf life permit. Kits may be unit dosage forms, bulk packaging (e.g., multi-dose packaging), or subunit doses. Kits may also include multiple unit doses of the compounds and instructions for use and be packaged in quantities sufficient for storage and use in pharmacies (e.g., hospital pharmacies and compounding pharmacies).

In some embodiments described herein, articles of manufacture are provided containing a unit dose of a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, in suitable packaging for use in the methods described herein. Suitable packaging is known in the art and includes, for example, vials, vessels, ampoules, bottles, jars, flexible packaging, and the like. The article of manufacture may be further sterilized and/or sealed.

Abbreviations.

ACN acetonitrile Aldrithiol-2 or Aldrithiol™-2 2,2'-dipyridyl disulfide drive. water BLQ below the limit of quantification Vp benzyl Sunday t/?et-butoxycarbonyl CC50 cytotoxic concentration 50% d doublet DCM dichloromethane dd double doublet DMAP dimethylaminopyridine DMF dimethylformamide DMSO dimethyl sulfoxide DP diphosphate

- 60 049099

dt doublet of triplets EC50 half-maximal effective concentration EDCI 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide or N-(3-dimethylaminopropyl)-A-ethylcarbodiimide hydrochloride ELSD evaporative light scattering detector EtOAc Ethyl acetate FBS fetal bovine serum GTD genotype D 4 watch) VGV Hepatitis B virus Hex hexane(s) HIV human immunodeficiency virus HPLC High Performance Liquid Chromatography Hz hertz J jhms liquid chromatography-mass spectrometry interaction constant M molar M Multiplet MeOH methanol mg milligram(s) MHz megahertz min minute(s) ml milliliter(s) mm millimeter(s) mmol millimol(s) MT-4 or MT4 Human T-cell line metallothionein 4 N normal NAP nucleic acid polymer NMP M-methyl-2-pyrrolidone NMR nuclear magnetic resonance And pentet

- 61 049099 mkpc

PBS peripheral blood mononuclear cell phosphate buffered saline

PEG polyethylene glycol

PHH

RMPA primary human hepatocytes (R)-((( 1 -(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphonic acid or (R)-9(2-phosphonomethoxypropyl)adenine or tenofovir prep.

preparative

RUG pyridine

RPMI quartet

Roswell Park Memorial Institute (nutrient medium) room temperature singlet

STOPS™ S-antigen transport inhibitory oligonucleotide polymers td triplet triplet doublets

TEA

TFV triethylamine tenofovir

TFV-DP tenofovir diphosphate

R

TP micron triphosphate µM

ZCCHC micromolar zinc finger protein type CCHC

The following examples are provided for illustrative purposes and not to be limiting.

EXAMPLES

General scheme A

^R3 - ^R35

ABOUT

Conditions:

a. thionyl chloride or

b. 4N HCI/dioxane

CIH Η ₂ Ν' ^Χ γ°'Κ ^{1 2}

Oh a

NH,

About nV

HG-X.C' J he I

Condition: triphenylphosphine, 2,2'-dipyridine^disulfide, triethylamine. pyridine

R ¹ '

JH ₂

RMRA

Π Ν-Ρ^.0.

D ^{N 0} UN

A ^H0 ' _R Conditions;

1.EDCI, DMAP, ACN

2.4N HCI/dioxane, DCM

Compounds of formula A can be prepared in a two- or three-step sequence from geminal disubstituted amino acids according to the general scheme A. The amino acids can be converted to intermediate amino esters of formula a by reaction with alcohols in the presence of thionyl chloride or 4N HCl in dioxane. Alternatively, the protected amino acids can be esterified with alcohols in the presence of EDCI, DMAP and ACN. The resulting N-protected amino esters can be removed by treatment with 4N HCl in dioxane and DCM to give intermediate amino esters of formula a. Intermediates of formula A can be coupled with (((1-(6-amino-9H-purin-9yl)propan-2-yl)oxy)methyl)phosphonic acid (PMPA) using triphenylphosphine, 2,2'dipyridyl disulfide, triethylamine and pyridine to produce compounds of formula A.

- 62 049099

General scheme B

Compounds of formula A in which R ¹ and R ² are different can be prepared in a single step from PMPA, intermediates of formula b, and alcohols other than those used to prepare formula b (R ¹ ≦ R ² ), according to General Scheme B. This can be done by combining PMPA, intermediates of formula b, and an alcohol with triphenylphosphine, 2,2'-dipyridyl disulfide, triethylamine, and pyridine to prepare compounds of formula A in which R ¹ and R ² are different.

General scheme C

Compounds of formula C can be prepared in a single step from compounds of formula A according to General Scheme C. Compounds of formula A can be mixed with anhydrides and pyridine to produce compounds of formula C. Alternatively, compounds of formula A can be mixed with acid chlorides and pyridine to produce compounds of formula C. Alternatively, compounds of formula A can be mixed with carboxylic acids, EDCI, DMAP and ACN to produce compounds of formula C. R ^a is determined by R ⁸ when L ¹ is -C(O)-.

General scheme D

Compounds of formula D can be prepared in three steps from compounds of formula A according to General Scheme D (in addition to the one-step preparation according to General Scheme C with the appropriate RA). Compounds of formula A can be mixed with monobenzyl ester of the dicarboxylic acid, EDCI, DMAP and ACN. The benzyl protecting group of the resulting product can be removed by mixing with 10% Pd/C and EtOAc under H ₂ atmosphere. The carboxylic acid of the resulting product can be alkylated by mixing with an alkyl halide as defined by R ^8a , sodium iodide, potassium carbonate and DMF to give compounds of formula D.

- 63 049099

General scheme E

Conditions: a. O

A E

Compounds of formula E can be prepared in a single step from compounds of formula A according to General Scheme E. Compounds of formula A can be mixed with chloroformates and pyridine to produce compounds of formula E. Alternatively, compounds of formula A can be mixed with alcohols, triphosgene, DMAP and DCM to produce compounds of formula E. R ^B R ⁸ , wherein L ¹ is -C(O)O-.

General scheme F

F-3

Compounds of formulae F-1, F-2 and F-3 can be prepared in a single step from compounds of formula A according to general scheme F. Compounds of formula A can be mixed with alkyl halides as defined by R ^8a , cesium carbonate and DMF to produce compounds of formulae F-1, F-2 and F-3. Alternatively, compounds of formula A can be mixed with alkyl halides as defined by R ^8a , potassium bicarbonate and NMP to produce compounds of formulae F-1, F-2 and F-3.

Example 1. Dihexyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (1)

o o

1a

Synthesis of hexyl 2-amino-2-methylpropanoate hydrochloride (1a).

To a suspension of 2-amino-2-methylpropanoic acid (5 g, 48.5 mmol) in 1-hexanol (49.5 g, 485 mmol) was added thionyl chloride (7.07 mL, 97 mmol) over 10 min at 5 °C under argon in a sealed tube. After complete addition, the reaction mixture was allowed to warm to room temperature and stirred for 30 min, the reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and quenched with water (100 mL). The aqueous layer was washed with 1:1 EtOAc:Hex (50 mL×2) and concentrated. The residue was dissolved in water (20 mL) and concentrated (×2). The residue was taken up in toluene and concentrated to give intermediate 1a. 1H NMR (400 MHz, methanol-d^δ 4.27 (t, J=6.6 Hz, 2H), 1.77-1.67 (m, 2H), 1.59 (s, 6H), 1.461.33 (m, 6H), 0.99-0.89 (m, 3H).

- 64 049099

PMPA at 1

^)-(((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphonic acid (PMPA) (100 mg, 0.35 mmol), intermediate 1a (234 mg, 1.04 mmol), triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol), and triethylamine (0.38 mL, 2.79 mmol) were combined in pyridine (2 mL) under argon. The reaction mixture was heated to 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL), and dried over sodium sulfate. After removal of the drying agent, the resulting solution was concentrated and loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The solution containing the product was concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (1). 1H NMR (400 MHz, DMSO- _d6 ) δ 8.17 (s, 1H), 8.13 (s, 1H), 7.19 (s, 2H), 4.33-4.14 (m, 4H), 4.11-3.91 (m, 5H), 3.63-3.48 (m, 2H), 1.61-1.51 (m, 4H), 1.40 1.461.35 (m, 12H), 1.33-1.19 (m, 12H), 1.06 (d, J=6.2 Hz, 3H), 0.84 (td, J=6.9, 2.1 Hz, 6H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.41 (t, J = 10.2 Hz). LCMS: MS m/z = 626.36 [M+1], tR = 1.78 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex 2.6 μm XB-C18 100A, 50x4.6 mm; solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min, 100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 μL/min HPLC: t _R = 3.35 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0-5.0 min 298% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 2. Bis(2-ethylbutyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (2)

o o

2a

Synthesis of 2-ethylbutyl 2-amino-2-methylpropanoate hydrochloride (2a).

To a suspension of 2-amino-2-methylpropanoic acid (20 g, 194 mmol) in 2-ethylbutan-1-ol (85.3 g, 835 mmol) was added thionyl chloride (28.3 mL, 388 mmol) over 10 min at 5 °C under argon in a sealed tube. After complete addition, the reaction mixture was allowed to warm to room temperature and stirred for 30 min, the reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and quenched with water (100 mL). The aqueous layer was washed with 1:1 HCl/HCl (50 mL x 2) and concentrated. The residue was dissolved in water (20 mL) and concentrated (x 2). The residue was taken up in toluene and concentrated to give intermediate 2a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.77 (s, 3H), 4.08 (d, J=5.5 Hz, 2H), 1.49 (s, 7H), 1.33 (p, J=7.3 Hz, 4H), 0.86 (t, J=7.5 Hz, 6H).

2a 2

PMPA (100 mg, 0.35 mmol), intermediate 2a (195 mg, 0.87 mmol) and triethylamine (0.38 mL, 2.79 mmol) were combined in pyridine (1 mL) and heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL) and dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient of 25-100% acetonitrile in water over 30 min) to afford the title compound (2). 1H NMR (400 MHz, DMSO-de) δ 8.16 (s, 1H), 8.12 (s, 1H), 7.17 (s, 2H), 4.31-4.12 (m, 4H), 4.05-3.89 (m, 5H), 3.66-3.47 (m,

- 65 049099

2H)), 1.54-1.22 (m, 22H), 1.06 (d, J=6.2 Hz, 3H), 0.83 (td, J=7.4, 3.3 Hz, 12H). ³¹ P NMR (162 MHz, DMSO-d6) δ 18.43 (t, J=10.2 Hz). LCMS: MS m/z = 626.38 [M+1], t _R = 1.74 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex 2.6 μm XB-C18 100A, 50x4.6 mm; solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min, 100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 μL/min HPLC: t _R = 3.31 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0-5.0 min 298% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 3. Dibenzyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (3)

For

Synthesis of benzyl 2-amino-2-methylpropanoate hydrochloride (3a).

To a suspension of 2-amino-2-methylpropanoic acid (2 g, 19.4 mmol) in benzyl alcohol (10.4 g, 97.0 mmol) was added thionyl chloride (2.83 mL, 38.8 mmol) over 10 min at 5 °C under argon in a sealed tube. After complete addition, the reaction mixture was allowed to warm to room temperature and stirred for 30 min, the reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and quenched with water (100 mL). The aqueous layer was washed with EtOAc:Iex, 1:1 (50 mL×2), and concentrated. The residue was dissolved in water (20 mL) and concentrated (×2). The residue was taken up in toluene and concentrated to give intermediate 3a. 1H NMR (400 MHz, DMSO- _d6 ) δ 8.46 (s, 3H), 7.46-7.33 (m, 5H), 5.26 (s, 2H), 1.49 (s, 6H).

PMPA (100 mg, 0.35 mmol), intermediate 3a (239 mg, 1.04 mmol) and triethylamine (0.38 mL, 2.79 mmol) were combined in pyridine (1 mL) and heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL) and dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100x30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (3). 1H NMR (400 MHz, DMSO-d6) δ 8.13 (s, 1H), 8.12 (s, 1H), 7.39-7.28 (m, 10H), 7.22 (s, 2H), 5.17-5.03 (m, 4H), 4.28-4.06 (m, 4H), 3.91-3.83 (m, 1H), 3.63-3.45 (m, 2H), 1.39 1.44-1.34 (m, 12H), 1.00 (d, J=6.2 Hz, 3H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.71 (t, J=9.2 Hz). LCMS: MS m/z = 638.13 [M+1], t _R = 1.46 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6 µm XB-C18 100A, 50x4.6 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min, 100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 µL/min HPLC: t _R = 2.91 min; HPLC system: Agilent 1100 series; column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 298% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 4. Dibenzyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (4)

4a

Synthesis of pentyl 2-amino-2-methylpropanoate hydrochloride (4a).

To a suspension of 2-amino-2-methylpropanoic acid (2 g, 19.4 mmol) in 1-pentanol (8.55 g, 97.0 mmol) was added thionyl chloride (2.83 mL, 38.8 mmol) over 10 min at 5 °C under argon in a sealed tube. After complete addition, the reaction mixture was allowed to warm to room temperature.

- 66 049099 temperature and stirred for 30 min. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and quenched with water (100 mL). The aqueous layer was washed with 1:1 BUtDcHex (50 mL x 2) and concentrated. The residue was dissolved in water (20 mL) and concentrated (x 2). The residue was taken up in toluene and concentrated to give intermediate 4a. 1H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 3H), 4.17 (t, J=6.5 Hz, 2H), 1.68-1.57 (m, 2H), 1.48 (s, 6H), 1.36-1.28 (m, 4H), 0.92-0.86 (m, 3H).

4a 4

PMPA (100 mg, 0.35 mmol), intermediate 4a (219 mg, 1.04 mmol) and triethylamine (0.38 mL, 2.79 mmol) were combined in pyridine (1 mL) and heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL) and dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (4). 1H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.12 (s, 1H), 7.19 (s, 2H), 4.32-4.11 (m, 4H), 4.09-3.90 (m, 5H), 3.63-3.47 (m, 2H), 1.56 (k, J=6.7 Hz, 4H), 1.45-1.35 (m, 12H), 1.27 (p, J=3.6 Hz, 8H), 1.06 (d, J=6.2 Hz, 3H), 0.90-0.79 (m, 6H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.43 (t, J=10.1 Hz). LCMS: MS m/z = 598.25 [M+1], t _R = 1.61 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6 µm XBC18 100 A, 50x4.6 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min, 100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 µL/min. HPLC: t _R = 3.11 min; HPLC system: Agilent 1100 series; column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 2-98% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 5. Dibenzyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (5)

5a

Synthesis of neopentyl-2-amino-2-methylpropanoate hydrochloride (5a).

To a suspension of 2-amino-2-methylpropanoic acid (2 g, 19.4 mmol) in 2,2-dimethylpropan-1-ol (8.55 g, 97.0 mmol) was added thionyl chloride (2.83 mL, 38.8 mmol) over 10 min at 5 °C under argon in a sealed tube. After complete addition, the reaction mixture was allowed to warm to room temperature and stirred for 30 min. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and quenched with water (100 mL). The aqueous layer was washed with 1:1 EtOH/HCl (50 mL x 2) and concentrated. The residue was dissolved in water (20 mL) and concentrated (x 2). The residue was taken up in toluene and concentrated to give intermediate 5a. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 3H), 3.88 (s, 2H), 1.51 (s, 6H), 0.94 (s, 9H).

PMPA (100 mg, 0.35 mmol), intermediate 5a (219 mg, 1.04 mmol), and triethylamine (0.38 mL, 2.79 mmol) were combined in pyridine (1 mL) and heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL × 2), water (5 mL), and dried over sulfate for 1 h.

- 67 049099 trioxide. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100x30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (5), 1H NMR (400 MHz, DMSOd6) δ 8.15 (s, 1H), 8.12 (s, 1H), 7.17 (s, 2H), 4.30-3.67 (m, 9H), 3.63-3.50 (m, 2H), 1.48-1.39 (m, 12H), 1.05 (d, J=6.2 Hz, 3H), 0.89 (d, J=5.1 Hz, 18H). ³¹ P NMR (162 MHz, DMSO-dB) δ 18.52-18.37 (m). LCMS: MS m/z = 589.12 [M+1], t _R = 1.57 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex 2.6 μm XB-C18 100A, 50x4.6 mm; solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min, 100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 μL/min. HPLC: t _R = 3.06 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0-5.0 min 2-98% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 6. Disopentyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(K)-bis(2-methylpropanoate) (6)

6a

Synthesis of isopentyl 2-amino-2-methylpropanoate hydrochloride (6a).

To a suspension of 2-amino-2-methylpropanoic acid (2 g, 19.4 mmol) in 3-methylbutan-1-ol (8.55 g, 97.0 mmol) was added thionyl chloride (2.83 mL, 38.8 mmol) over 10 min at 5 °C under argon in a sealed tube. After complete addition, the reaction mixture was allowed to warm to room temperature and stirred for 30 min. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and quenched with water (100 mL). The aqueous layer was washed with 1:1 EtOH/HCl (50 mL × 2) and concentrated. The residue was dissolved in water (20 mL) and concentrated (× 2). The residue was taken up in toluene and concentrated to give intermediate 6a. 1H NMR (400 MHz, DMSO-d6) δ 8.71-8.54 (m, 3H), 4.20 (t, J=6.7 Hz, 2H), 1.75-1.62 (m, 1H), 1.52 (k, J=6.7 Hz, 2H), 1.48 (s, 6H), 0.90 (d, J=6.6 Hz, 6H).

6a 6

PMPA (100 mg, 0.35 mmol) and intermediate 6a (219 mg, 1.04 mmol) were suspended in 2 mL of toluene and concentrated twice under reduced pressure. Pyridine (1 mL) and triethylamine (0.38 mL, 2.79 mmol) were added and the reaction mixture was heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL of EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL) and dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (6). 1H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.13 (s, 1H), 7.17 (s, 2H), 4.32-3.91 (m, 9H), 3.63-3.50 (m, 2H), 1.70-1.58 (m, 2H), 1.51-1.34 (m, 16H), 1.07 (d, J=6.3 Hz, 3H), 0.89-0.84 (m, 12H). ³¹ P NMR (162 MHz, DMSO-d6) δ 18.60-18.39 (m). LCMS: MS m/z = 598.10 [M+1], t _R = 1.60 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6 μm XB-C18 100A, 50x4.6 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min, 100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 μL/min. HPLC: t _R = 3.14 min; HPLC system: Agilent 1100 Series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 2-98% ACN, 5.0-6.0 min 98% ACN at 2 ml/min.

Example 7. Bis(3-ethylpentyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azandiyl))^)-bis(2-methylpropanoate) (7)

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7a 7b

Synthesis of 3-ethylpentyl 2-amino-2-methylpropanoate hydrochloride (7b).

To a solution of 2-amino-2-methylpropanoic acid (2 g, 9.84 mmol), 3-ethylpentan-1-ol (1.37 g, 11.8 mmol), and HATU (2.34 g, 9.84 mmol) in DCM (50 mL) was added TEA (5.37 mL, 39.4 mmol) followed by DMAP (60 mg, 0.49 mmol). After 3 h, the reaction mixture was diluted with DCM (50 mL) and washed with saturated sodium bicarbonate solution (50 mL), water (50 mL), and brine (50 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated. Intermediate 7a was purified by silica gel chromatography (0-50% EtOAc in Hex). Intermediate 7a was dissolved in 4N HCl in dioxane (48.5 mL, 194 mmol). After 2 h, the reaction mixture was concentrated. The residue was taken up in water (20 mL) and concentrated (this process was repeated twice). The residue was taken up in toluene and concentrated to give intermediate 7b. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.57 (s, 3H), 4.20 (t, J=6.8 Hz, 2H), 1.61-1.54 (m, 2H), 1.48 (s, 5H), 1.35-1.26 (m, 6H), 0.87-0.81 (m, 6H).

PMPA (100 mg, 0.35 mmol) and intermediate 7b (335 mg, 1.41 mmol) were suspended in 2 mL of toluene and concentrated twice under reduced pressure. Pyridine (1 mL) and triethylamine (0.38 mL, 2.79 mmol) were added and the reaction mixture was heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL of EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL), and dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (7). 1H NMR (400 MHz, DMSO- _d6 ) δ 8.16 (s, 1H), 8.12 (s, 1H), 7.17 (s, 2H), 4.32-3.90 (m, 9H), 3.63-3.48 (m, 2H), 1.55-1.48 (m, 4H), 1.46-1.35 (m, 12H), 1.29-1.22 (m, 10H), 1.06 (d, J=6.2 Hz, 3H), 0.83-0.77 (m, 12H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.44 (t, J=9.6 Hz). LCMS: MS m/z = 654.17 [M+1], t _R = 1.92 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6 µm XB-C18 100A, 50x4.6 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min, 100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 µL/min. HPLC: t _R = 3.50 min; HPLC system: Agilent 1100 series; column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 298% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 8. Bis(3,3-dimethylbutyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (8)

8a

Synthesis of 3,3-dimethylbutyl 2-amino-2-methylpropanoate hydrochloride (8a).

2-Amino-2-methylpropanoic acid (2 g, 19.4 mmol) and 3,3-dimethylbutan-1-ol (9.91 g, 97.0 mmol) were dissolved in 4N HCl in dioxane (19.4 mL, 77.6 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and concentrated. The residue was dissolved in water (20 mL) and concentrated (×2). The residue was taken up in toluene and concentrated to give intermediate 8a. ¹ H NMR (400 MHz, DMSO-d6) δ 8.47 (s, 3H), 4.23 (t, J=7.1 Hz, 2H), 1.56 (t, J=7.1 Hz, 2H), 1.46 (s, 6H), 0.93 (s, 9H).

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8a 8

PMPA (100 mg, 0.35 mmol) and intermediate 8a (315 mg, 1.41 mmol) were suspended in 2 mL of toluene and concentrated twice under reduced pressure. Pyridine (1 mL) and triethylamine (0.38 mL, 2.79 mmol) were added and the reaction mixture was heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL of EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL), and dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (8). 1H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.12 (s, 1H), 7.17 (s, 2H), 4.32-3.90 (m, 9H), 3.65-3.48 (m, 2H), 1.49 (k, J=7.2 Hz, 4H), 1.45-1.34 (m, 12H), 1.06 (d, J=6.2 Hz, 3H), 0.89 (d, J=3.7 Hz, 18H). ^{9 * * * * * * * * * * * * * * * * * * * * * 31} P NMR (162 MHz, DMSO-d6) δ 18.47 (t, J=9.6 Hz). LCMS: MS m/z = 626.21 [M+1], t _R = 1.73 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6 µm XB-C18 100A, 50x4.6 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min, 100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 µL/min. HPLC: t _R = 3.25 min; HPLC system: Agilent 1100 series; column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 298% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 9. Di-tert-butyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (9)

PMPA (100 mg, 0.35 mmol) and tert-butyl 2-amino-2-methylpropanoate hydrochloride (204 mg, 1.04 mmol) were suspended in 2 mL of toluene and concentrated twice under reduced pressure. Pyridine (1 mL) and triethylamine (0.38 mL, 2.79 mmol) were added and the reaction mixture was heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at

70°C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 ml

EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL), dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18110A 100 x 30 mm column, gradient of 25-100% acetonitrile in water over 30 min) to give the title compound (9). 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.16 (s, 1H), 8.13 (s, 1H), 7.17 (s, 2H), 4.29 (dd,

J=14.3, 3.8 Hz, 1H), 4.18 (dd, J=14.4, 6.0 Hz, 1H), 4.12 (d, J=10.6 Hz, 1H), 4.02 (d, J=10.6 Hz, 1H), 3.98-3.92 (m, 1H), 1.45-1.30 (m, 30H), 1.07 (d, J=6.3 Hz, 3H). ³¹ P NMR (162 MHz, DMSO-d6) δ 18.60-18.39 (m). LCMS:

MS m/z = 570.27 [M+1], t _R = 1.46 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo

LCQ Fleet; column: Kinetex 2.6 µm XB-C18 100A, 50x4.6 mm; solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min,

100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 μL/min. HPLC: t _R = 2.86 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0-5.0 min 2-98% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 10. Dihexyl 2,2'-((((((^)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^,2')-bis(2-methylbutanoate) (10)

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About Dioxane About

10a

Synthesis of hexyl (2S)-2-a\iuiio-2-\ieTu _B i6yTaiioaTa hydrochloride (10a).

(2S)-2-amino-2-methylbutanoic acid (1 g, 8.6 mmol) and 1-hexanol (4.36 g, 42.7 mmol) were dissolved in 4N HCl in dioxane (5.58 mL, 22.3 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and concentrated. The residue was dissolved in water (20 mL) and concentrated (×2). The residue was taken up in toluene and concentrated to give intermediate 10a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 4.02 (t, J=6.5 Hz, 2H), 1.64-1.43 (m, 4H), 1.35-1.24 (m, 6H), 1.17 (s, 3H), 0.90-0.84 (m, 3H), 0.78 (t, J=7.5 Hz, 3H).

10a 10

PMPA (100 mg, 0.35 mmol) and intermediate 10a (248 mg, 1.04 mmol) were suspended in 2 mL of toluene and concentrated twice under reduced pressure. Pyridine (1 mL) and triethylamine (0.38 mL, 2.79 mmol) were added and the reaction mixture was heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL of EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL), and dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (10). 1H NMR (400 MHz, acetonitrile^) δ 8.24 (s, 1H), 8.04 (s, 1H), 6.14 (s, 2H), 4.33 (dd, J=14.5, 3.3 Hz, 1H), 4.23-4.06 (m, 5H), 3.98-3.92 (m, 1H), 3.73-3.43 (m, 4H), 1.90-1.60 (m, 8H), 1.57 (s, 3H), 1.42 (s, 3H), 1.39-1.28 (m, 12H), 1.19 (d, J=6.3 Hz, 3H), 0.95-0.87 (m, 6H), 0.83 (t, J=7.4 Hz, 30H), 0.78 (t, J=7.4 Hz, 3H). ³¹ P NMR (162 MHz, DMSO-d ₃ ) δ 17.48 (t, J=9.8 Hz). LCMS: MS m/z = 654.4 [M+1], t _R = 1.17 min; LC system: Agilent 1260 Infinity II HPLC; MS system: G6124B single quadrupole mass detector; column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-1.00 min 10%-100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 μL/min. HPLC: t _R = 3.55 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0-5.0 min 2-98% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 11. Dihexyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methyl-3-propanoate) (R)

Pa

Synthesis of hexyl ^)-2-amino-2-methyl-3-phenylpropanoate hydrochloride (11a).

(^)-2-Amino-2-methyl-3-phenylpropanoic acid (1 g, 5.6 mmol) and 1-hexanol (2.85 g, 27.9 mmol) were dissolved in 4N HCl in dioxane (5.58 mL, 22.3 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and concentrated. The residue was dissolved in water (20 mL) and concentrated (×2). The residue was taken up in toluene and concentrated to give intermediate 11a. ¹ H NMR (400 MHz, DMSO-d6) δ 7.29-7.19 (m, 3H), 7.16-7.10 (m, 2H), 4.04-3.93 (m, 2H), 2.89 (d, J=12.9 Hz, 1H), 2.76 (d, J=12.9 Hz, 1H), 1.59-1.50 (m, 2H), 1.31-1.24 (m, 6H), 1.22 (s, 3H), 0.90-0.83 (m, 3H).

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NH,

Pa 11

PMPA (100 mg, 0.35 mmol) and intermediate 11a (275 mg, 1.04 mmol) were suspended in 2 mL of toluene and concentrated twice under reduced pressure. Pyridine (1 mL) and triethylamine (0.38 mL, 2.79 mmol) were added and the reaction mixture was heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL of EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate solution (5 mL x 2), water (5 mL), dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient of 25-100% acetonitrile in water over 30 min) to afford the title compound (R). 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.12 (s, 1H), 8.04 (s, 1H), 7.30-7.08 (m, 12H), 4.27-3.82 (m, 9H), 3.61 (d, J=8.7 Hz, 2H), 3.11-2.87 (m, 4H), 1.55-1.39 (m, 10H), 1.27-1.14 (m, 12H), 0.97 (d, J=6.3 Hz, 3H), 0.86-0.78 (m, 6H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.45-18.23 (m). LCMS: MS m/z = 778.4 [M+1], t _R = 1.27 min; LC system: Agilent 1260 Infinity II HPLC; MS system: G6124B single quadrupole mass detector; Column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 0-1.00 min 10%-100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 μL/min. HPLC: tR = 3.86 min; HPLC system: Agilent 1100 series; column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 2-98% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 12. Bis(trans-4-(tert-butyl)cyclohexyl) 2,2'-(((((^)-1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (12)

12a 12b

Synthesis of trans-4-(tert-butyl)cyclohexyl 2-amino-2-methylpropanoate hydrochloride (12b).

To a solution of 2-amino-2-methylpropanoic acid (2 g, 9.84 mmol), trans-4-(tert-butyl)cyclohexan-1-ol (1.69 g, 10.8 mmol), and HATU (2.34 g, 9.84 mmol) in DCM (50 mL) was added TEA (5.37 mL, 39.4 mmol) followed by DMAP (60 mg, 0.49 mmol). After 3 h, the reaction mixture was diluted with DCM (50 mL) and washed with saturated sodium bicarbonate solution (50 mL), water (50 mL), and brine (50 mL). The organic layers were dried over sodium sulfate, filtered, and concentrated. Intermediate 12a was purified by silica gel chromatography (0–50% EtOAc in Hex). Intermediate 12a was dissolved in 4N HCl in dioxane (48.5 mL, 194 mmol). After 2 h, the reaction mixture was concentrated. The residue was dissolved in water (20 mL) and concentrated (×2). The residue was taken up in toluene and concentrated to give intermediate 12a. 1H NMR (400 MHz, DMSO-d6) δ 8.50 (s, 3H), 4.71-4.59 (m, 1H), 2.02-1.93 (m, 2H), 1.81-1.75 (m, 2H), 1.41-1.27 (m, 2H), 1.19-0.95 (m, 3H), 0.85 (s, 9H).

12b 12

PMPA (100 mg, 0.35 mmol) and intermediate 12b (290 mg, 1.04 mmol) were suspended in 2 mL of toluene and concentrated twice under reduced pressure. Pyridine (1 mL) and triethylamine (0.38 mL, 2.79 mmol) were added and the reaction mixture was heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction

- 72 049099 The reaction mixture was cooled to room temperature and diluted with 5 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL), and dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient of 25-100% acetonitrile in water over 30 min) to afford the title compound (12). 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.16 (s, 1H), 8.12 (s, 1H), 7.16 (s, 2H), 4.57-4.44 (m, 2H), 4.28 (dd, J=14.3, 3.8 Hz, 1H), 4.21-4.06 (m, 3H), 3.98-3.91 (m, 1H), 3.63-3.48 (m, 2H), 1.96-1.86 (m, 4H), 1.79-1.68 (m, 4H), 1.44-1.32 (m, 12H), 1.29-1.17 (m, 4H), 1.13-0.90 (m, 9H), 0.82 (s, 18H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.27 (t, J = 9.5 Hz). LCMS: MS m/z = 734.14 [M+1], t _R = 1.99 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex 2.6 μm XB-C18 100A, 50x4.6 mm; solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min, 100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 μL/min. HPLC: t _R = 4.13 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0-5.0 min 2-98% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 13. Bis(4,4-dimethylcyclohexyl)methyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (13) ||_| hydrogen chloride α-Η,Ν^γΟνχΟ^

O L Dioxane θ

13a

Synthesis of (4,4-dimethylcyclohexyl)methyl 2-amino-2-methylpropanoate hydrochloride (13a).

2-Amino-2-methylpropanoic acid (1 g, 9.7 mmol) and (4,4-dimethylcyclohexyl)methanol (2.76 g, 19.4 mmol) were dissolved in 4N HCl in dioxane (9.7 mL, 38.8 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and concentrated. The residue was dissolved in water (20 mL) and concentrated (×2). The residue was taken up in toluene and concentrated to give intermediate 13a. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 3H), 4.04 (d, J=6.0 Hz, 2H), 1.61-1.41 (m, 10H), 1.40-1.31 (m, 2H), 1.25-1.08 (m, 3H), 0.88 (d, J=11.2 Hz, 6H).

PMPA (100 mg, 0.35 mmol) and intermediate 13a (276 mg, 1.04 mmol) were suspended in 2 mL of toluene and concentrated twice under reduced pressure. Pyridine (1 mL) and triethylamine (0.38 mL, 2.79 mmol) were added and the reaction mixture was heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL of EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL), and dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (13). 1H NMR (400 MHz, DMSO- _d6 ) δ 8.16 (s, 1H), 8.13 (s, 1H), 7.17 (s, 2H), 4.32-4.14 (m, 4H), 3.98-3.82 (m, 5H), 3.65-3.49 (m, 2H), 1.55-1.27 (m, 22H), 1.12 (t, J=7.1 Hz, 8H), 1.06 (d, J=6.2 Hz, 3H), 0.87 (d, J=2.2 Hz, 6H), 0.84 (d, J=2.4 Hz, 6H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.35 (t, J=9.7 Hz). LCMS: MS m/z = 706.40 [M+1], t _R = 1.30 min; Agilent. HPLC: t _R = 3.78 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50 x 4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 2-98% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 14. Bis(2-cyclohexylethyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (14)

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14a

Synthesis of 2-cyclohexylethyl 2-amino-2-methylpropanoate hydrochloride (14a).

2-Amino-2-methylpropanoic acid (1 g, 19.4 mmol) and 2-cyclohexylethanol (3.73 g, 29.1 mmol) were dissolved in 4N HCl in dioxane (9.7 mL, 38.8 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and concentrated. The residue was dissolved in water (20 mL) and concentrated (×2). The residue was taken up in toluene and concentrated to give intermediate 14a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.64 (s, 3H), 4.20 (t, J=6.7 Hz, 2H), 1.72-1.58 (m, 5H), 1.56-1.42 (m, 7H), 1.40-1.27 (m, 2H), 1.271.09 (m, 3H), 0.99-0.81 (m, 2H).

14a 14

PMPA (100 mg, 0.35 mmol) and intermediate 14a (223 mg, 1.04 mmol) were suspended in 2 mL of toluene and concentrated twice under reduced pressure. Pyridine (1 mL) and triethylamine (0.38 mL, 2.79 mmol) were added and the reaction mixture was heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL of EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate solution (5 mL x 2), water (5 mL), dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (14). 1H NMR (400 MHz, DMSO- _d6 ) δ 8.17 (s, 1H), 8.13 (s, 1H), 7.17 (s, 2H), 4.32-3.91 (m, 9H), 3.64-3.48 (m, 2H), 1.69-1.56 (m, 10H), 1.51-1.27 (m, 18H), 1.24-1.11 (m, 6H), 1.07 (d, J=6.3 Hz, 3H), 0.96-0.82 (m, 4H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.43 (t, J=9.7 Hz). LCMS: MS m/z = 678.40 [M+1], t _R = 1.32 min; LC System: Agilent 1260 Infinity II HPLC; MS System: G6124B Single Quadrupole Mass Detector; Column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-1.00 min 10%-100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 μL/min. HPLC: t _R = 3.34 min; HPLC system: Agilent 1100 series; column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 2-98% ACN, 5.0-6.0 min 98% ACN at 2 mL/min.

Example 15. Bis(cyclohexylmethyl) 2,2'-(((((^)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^,2^)-bis(2-methyl-3-propanoate) (15)

15a

Synthesis of cyclohexylmethyl^)-2-amino-2-methyl-3-phenylpropanoate hydrochloride (15a).

(2S)-2-amino-2-methyl-3-phenylpropanoic acid (1 g, 5.6 mmol) and cyclohexylmethanol (1.91 g, 16.7 mmol) were dissolved in 4N HCl in dioxane (5.58 mL, 22.3 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and concentrated. The residue was dissolved in water (20 mL) and concentrated (×2). The residue was taken up in toluene and concentrated to give intermediate 15a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.65 (s, 3H), 7.39-7.28 (m, 3H), 7.21-7.16 (m, 2H), 3.98 (dd, J=10.6, 6.2 Hz, 1H), 3.87 (dd, J=10.6, 6.1 Hz, 1H), 3.15 (s, 2H), 1.72-1.46 (m, 9H), 1.27-1.09 (m, 3H), 1.03-0.85 (m, 2H).

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15a 15

PMPA (100 mg, 0.35 mmol) and intermediate 15a (326 mg, 1.04 mmol) were suspended in 2 mL of toluene and concentrated twice under reduced pressure. Pyridine (1 mL) and triethylamine (0.38 mL, 2.79 mmol) were added and the reaction mixture was heated to 70 °C for 5 min under argon. A solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL) was added. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with 5 mL of EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (5 mL x 2), water (5 mL), and dried over sodium sulfate. The product was loaded onto silica gel (12 g). EtOAc (100 mL) was eluted and discarded. The product was extracted with 15% MeOH in DCM (50 mL). The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (15). 1H NMR (400 MHz, acetonitrile^) δ 8.23 (s, 1H), 7.85 (s, 1H), 7.36-7.12 (m, 10H), 5.90 (s, 2H), 4.22 (dd, J=14.5, 3.4 Hz, 1H), 4.06-3.75 (m, 6H), 3.70-3.50 (m, 4H), 3.11-2.93 (m, 4H), 1.79-1.61 (m, 13H), 1.49 (s, 3H), 1.36-1.14 (m, 8H), 1.08 (d, J=6.2 Hz, 3H), 1.04-0.87 (m, 4H). ³¹ P NMR (162 MHz, acetonitrile^) δ 17.81-17.56 (m). LCMS: MS m/z = 802.30 [M+1], t _R = 1.35 min; LC System: LC System: Agilent 1260 Infinity II HPLC; MS System: G6124B Single Quadrupole Mass Detector; Column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-1.00 min 10-100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 μL/min. HPLC: tR = 3.34 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0-5.0 min 298% ACN, 5.0-6.0 min 98% aCN at 2 mL/min.

Example 16. Hexyl (1K,2K)-1-(((((((K)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((1(hexyloxy)-2-methyl-1-oxopropan-2-yl)amino)phosphoryl)amino)-2-ethylcyclopropane-1-carboxylate (16).

Example 17. Dihexyl-1,1 '-((((((R)-1 -(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azandiyl))( 1R,1^,2^2^)-bis(2-ethylcyclopropan-1-carboxylate) (17)

EDCI

DMAP

16a

Synthesis of hexyl (1R,2R)-1-((tert-butoxycarbonyl)amino)-2-ethylcyclopropane-1-carboxylate (16a).

To a mixture of (1R,2R)-1-((tert-butoxycarbonyl)amino)-2-ethylcyclopropane-1-carboxylic acid (1.0 g, 4.36 mmol), n-hexanol (0.82 mL, 6.542 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (813 mg, 5.234 mmol) in acetonitrile (30 mL) was added DMAP (1.07 g, 8.723 mmol). The mixture was stirred at room temperature for 15 h, quenched with water, and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (EtOAc, 0-70% in hexanes) to afford intermediate 16a. 1H NMR (400 MHz, acetonitrile-ds) δ 5.93 (br.s, 1H), 4.08 (t, J=6.5 Hz, 2H), 1.68-1.51 (m, 3H), 1.50-1.28 (m, 18H), 1.13 (dd, J=8.9, 4.8 Hz, 1H), 0.94-0.82 (m, 6H).

16a 16b

Synthesis of hexyl (1R,2R)-1-amino-2-ethylcyclopropane-1-carboxylate hydrochloride (16b).

To a solution of intermediate 16a (600 mg, 1.914 mmol) in DCM (12 mL) was slowly added 4 M HCl in dioxane (1.9 mL) at room temperature. The resulting mixture was stirred at room temperature for 2 h, concentrated in vacuo, co-evaporated with DCM several times, and dried under high vacuum for 15 h to yield intermediate 16b. 1H NMR (400 MHz, acetonitrile^) δ 8.59 (br.s, 3H), 4.21 (m, 2H), 1.97 (m, 1H), 1.87 (dd, J=10.1, 5.8 Hz, 1H), 1.75-1.61 (m, 4H), 1.54 (m, 1H), 1.47-1.25 (m, 6H), 0.98 (t, J=7.4 Hz, 3H), 0.95-0.88 (m, 3H).

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16b la

17

A mixture of PMPA (100 mg, 0.348 mmol), intermediate 16b (174 mg, 0.696 mmol) and triethylamine (0.73 ml, 2.785 mmol) in pyridine (1 ml) was heated to 50 °C for 5 min and a freshly prepared bright yellow solution of 2,2'-dipyridyl disulfide (460 mg, 2.089 mmol) and triphenylphosphine (548 mg, 2.089 mmol) in pyridine (1 ml) was added to the reaction mixture. The reaction mixture was stirred at 90 °C for 5 h. Intermediate 1a (234 mg, 1.045 mmol) was added. The reaction mixture was stirred at 90 °C for 18 h, concentrated in vacuo and purified by silica gel column chromatography (0 to 15% MeOH in DCM) to give a product mixture that was separated by preparative HPLC (0.1% TFA-containing ACN, 35% to 90% in 0.1% TFA-containing water). Each compound was dissolved in ACN (3 mL) and triethylamine (three drops) was added to obtain the free form and re-purified by neutral HPLC (10 to 100% ACN in water for 12 min, 100% ACN for 5 min) to give 16 and 17.

16: 1H NMR (400 MHz, acetonitrile^) δ 8.24-8.21 (m, 1H), 8.05-8.02 (m, 1H), 6.13 (m, 2H), 4.32 (m, 1H), 4.23-4.04 (m, 5H), 4.02-3.84 (m, 2H), 3.78-3.59 (m, 2H), 3.47 (m, 1H), 1.76 (m, 1H), 1.68-1.52 (m, 5H), 1.51-1.22 (m, 21H), 1.17 (d, J=6.22 Hz, 3H), 0.98-0.83 (m, 9H). ³¹ P NMR (162 MHz, acetonitrile^) δ 19.44, 18.94. LCMS: MS m/z = 652.27 [M+1]; tR = 1.66 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 μL/min. HPLC: t _R = 6.64 min (45%) and 6.68 min (55%); HPLC system: 1290 Infinity II; column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min - 8.5 min 298% ACN at 1.5 mL/min.

17: 1H NMR (400 MHz, acetonitrile^) δ 8.23 (s, 1H), 8.03 (s, 1H), 6.07 (s, 2H), 4.35 (dd, J=14.5, 3.2 Hz, 1H), 4.21-4.07 (m, 4H), 4.08-3.84 (m, 4H), 3.73 (dd, J=12.7, 8.4 Hz, 1H), 3.51 (dd, J=12.6, 10.1 Hz, 1H), 1.72-1.09 (m, 26H), 0.98-0.84 (m, 12H). ³¹ P NMR (162 MHz, acetonitrile^) δ 20.76. LCMS: MS m/z = 678.30 [M+1]; t _R = 1.69 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6 µm XB-C18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: tR = 6.81 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100x4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 18. Hexyl-1-(((((((R)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((1-(hexyloxy)2-methyl-1-oxopropan-2-yl)amino)phosphoryl)amino)cyclobutane-1-carboxylate (18).

Example 19. Dihexyl 1,1'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(cyclobutane-1-carboxylate) (19)

18a

Synthesis of hexyl 1-aminocyclobutane-1-carboxylate hydrochloride (18a).

To a mixture of 1-aminocyclobutane-1-carboxylic acid (1.5 g, 13.03 mmol) and n-hexanol (30 mL) was slowly added 4 M HCl in dioxane (6.5 mL, 26.06 mmol) at room temperature. The resulting mixture was heated at 80 °C for 15 h and concentrated at 65 °C under high vacuum. The solid residue was triturated with ether, the filter cake was washed with ether and dried under high vacuum for 15 h to give intermediate 18a. 1H NMR (400 MHz, acetonitrile^) δ 8.74 (br.s, 3H), 4.25 (t, J=6.5 Hz, 2H), 2.84-2.70 (m, 2H), 2.58 (m, 2H), 2.32-2.05 (m, 2H), 1.80-1.62 (m, 2H), 1.53-1.23 (m, 6H), 0.93 (m, 3H).

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Compounds 18 and 19 were prepared from PMPA using a procedure similar to that used for compounds 16 and 17.

18: ^1H NMR (400 MHz, acetonitrile^) δ 8.22 (s, 1H), 8.06 (s, 1H), 6.31 (m, 2H), 4.34 (m, 1H), 4.234.02 (m, 5H), 4.01-3.85 (m, 2H), 3.82-3.60 (m, 2H), 3.47 (m, 1H), 2.68-2.23 (m, 4H), 2.03-1.82 (m, 2H), 1.73-1.56 (m, 4H), 1.53-1.25 (m, 18H), 1.17 (d, J=6.2 Hz, 3H), 0.96-0.84 (m, 6H). ³¹ P NMR (162 MHz, acetonitrile^) δ 18.31, 18.29. LCMS: MS m/z = 638.38 [M+1]; tR = 1.77 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 μL/min. HPLC: t _R = 6.50 min; HPLC system: 1290 Infinity II; column: Phenomenex 2.6 µm C18 100A, 100x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

19: 1H NMR (400 MHz, acetonitrile^) δ 8.22 (s, 1H), 8.04 (s, 1H), 6.23 (s, 2H), 4.34 (dd, J=14.5, 3.2 Hz, 1H), 4.22-4.01 (m, 5H), 4.02-3.86 (m, 2H), 3.82-3.66 (m, 2H), 3.49 (dd, J=12.7, 10.2 Hz, 1H), 2.65-2.30 (m, 8H), 2.01-1.80 (m, 4H), 1.65 (m, 4H), 1.49-1.24 (m, 12N), 1.17 (d, J=6.2 Hz, 3H), 0.98-0.85 (m, 6H). ³¹ P NMR (162 MHz, acetonitrile- ₃ ) δ 18.78. LCMS: MS m/z = 650.37 [M+1]; t _R = 1.79 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: t _R = 6.56 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min 8.5 min 2-98% ACN at 1.5 mL/min.

Example 20. Hexyl-1-(((((((R)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((1-(hexyloxy)2-methyl-1-oxopropan-2-yl)amino)phosphoryl)amino)cyclopropane-1-carboxylate (20)

20a

Synthesis of hexyl 1-aminocyclopropane-1-carboxylate hydrochloride 20a).

Intermediate 20a was prepared from 1-aminocyclopropane-1-carboxylic acid in a manner analogous to that shown for intermediate 18a. 1H NMR (400 MHz, acetonitrile-1- _s13 ) δ 8.52 (br s, 3H), 4.17 (t, J=6.6 Hz, 2H), 1.75-1.58 (m, 4H), 1.52-1.42 (m, 2H), 1.43-1.28 (m, 6H), 1.03-0.82 (m, 3H).

20a 1a

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A mixture of PMPA (170 mg, 0.59 mmol), intermediate 1a (199 mg, 0.89 mmol), 2,2'dipyridyl disulfide (782 mg, 3.55 mmol), triphenylphosphine (931 mg, 3.55 mmol) and triethylamine (0.66 mL, 4.74 mmol) in pyridine (3 mL) was heated to 70 °C for 1 h. Intermediate 20a (197 mg, 0.89 mmol) in pyridine (1 mL) was added and the reaction mixture was stirred at 70 °C for 18 h. After concentration in vacuo, the residue was purified by column chromatography on silica gel (MeOH 0 to 15% in DCM) and prep. HPLC (10 to 100% ACN in water over 12 min, 100% ACN for 5 min) to afford the title compound (20). 1H NMR (400 MHz, acetonitrile _d3 ) δ 8.25-8.20 (m, 1H), 8.10-8.06 (m, 1H), 6.48 (s, 2H), 4.42-4.26 (m, 2H), 4.24-3.82 (m, 7H), 3.71 (m, 1H), 3.57-3.42 (m, 1H), 1.73-1.21 (m, 26H), 1.16 (d, J=6.2 Hz, 3H), 0.95-0.81 (m, 6H). ³¹ P NMR (162 MHz, acetonitrile- ₃ ) δ 19.92, 19.85. LCMS: MS m/z = 624.30 [M+1]; t _R = 1.76 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: t _R = 6.21 min (43%), 6.28 min (54%); HPLC system: 1290 Infinity II; column: phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 21. Bis(trans-4-(trifluoromethyl)cyclohexyl) 2,2'-((((((^)-1-(6-amino-9H-purin-9yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (21).

Example 22. Hexyl 2-((((((^)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((2-methyl-1 oxo-1-((trans-4-(trifluoromethyl)cyclohexyl)oxy)propan-2-yl)amino)phosphoryl)amino)-2-methylpropanoate(22)

EDCI

21a

Synthesis of trans-4-(trifluoromethyl)cyclohexyl-2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (21a).

To a mixture of 2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid (650 mg, 3.20 mmol), trans-4-(trifluoromethyl)cyclohexan-1-ol (1076 mg, 6.40 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (993 mg, 6.40 mmol) in acetonitrile (30 mL) was added DMAP (781 mg, 6.40 mmol). The mixture was stirred at room temperature for 15 h, quenched with water, and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (EtOAc, 0-70% in hexanes) to afford intermediate 21a. 1H NMR (400 MHz, acetonitrile^) δ 5.60 (br.s, 1H), 4.66 (m, 1H), 2.27-2.09 (m, 1H), 2.07-1.91 (m, 4H), 1.59-1.30 (m, 19H).

21a 21b

Synthesis of trans-4-(trifluoromethyl)cyclohexyl 2-amino-2-methylpropanoate hydrochloride (21b).

To a solution of intermediate 21a (451 mg, 1.28 mmol) in DCM (10 mL) was slowly added 4 M HCl in dioxane (1.6 mL) at room temperature. The resulting mixture was stirred at room temperature for 15 h, concentrated in vacuo, co-evaporated several times with DCM and dried under high vacuum for 15 h to afford intermediate 21b. 1H NMR (400 MHz, acetonitrile^) δ 8.62 (br s, 3H), 4.79 (m, 1H), 2.20 (s, 1H), 2.10 (d, J=4.9 Hz, 2H), 2.07-1.99 (m, 2H), 1.65 (s, 6H), 1.54-1.44 (m, 4H).

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A mixture of PMPA (100 mg, 0.348 mmol), intermediate 21b (303 mg, 1.04 mmol), 2,2'dipyridyl disulfide (460 mg, 2.089 mmol), triphenylphosphine (548 mg, 2.089 mmol) and triethylamine (0.4 ml, 2.785 mmol) in pyridine (2 ml) was heated at 80 °C for 3 h and intermediate 1a (117 mg, 0.522 mmol) in pyridine (0.5 ml) was added. The mixture was heated at 80°C for 15 h, concentrated in vacuo and purified by column chromatography on silica gel (MeOH 0 to 15% in DCM) to give a mixture of two products, which were separated by preparative HPLC (ACN 10 to 100% in water over 12 min, 100% for 5 min) to give compounds 21 and 22.

21: 1H NMR (400 MHz, acetonitrile^) δ 8.24 (s, 1H), 8.07 (s, 1H), 6.35 (s, 2H), 4.66 (m, 2H), 4.34 (dd, J=14.5, 3.2 Hz, 1H), 4.17 (dd, J=14.5, 7.1 Hz, 1H), 3.94 (m, 1H), 3.77-3.62 (m, 2H), 3.55 (d, J=10.7 Hz, 1H), 3.47 (dd, J=12.6, 9.8 Hz, 1H), 2.18 (m, 2H), 2.10-1.89 (m, 8H), 1.57-1.27 (m, 20H), 1.19 (d, J=6.2 Hz, 3H). ^31P NMR (162 MHz, acetonitrile^) δ 17.89. LCMS: MS m/z = 758.27 [M+1]; t _R = 1.70 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XBC18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: t _R = 6.12 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min 8.5 min 2-98% ACN at 1.5 mL/min.

22: 1H NMR (400 MHz, acetonitrile^) δ 8.23 (s, 1H), 8.06 (s, 1H), 6.17 (s, 2H), 4.75-4.55 (m, 1H), 4.37-4.30 (m, 1H), 4.24-4.01 (m, 3H), 3.93 (m, 1H), 3.75 (m, 1H), 3.67 (m, 1H), 3.56 (m, 1H), 3.45 (m, 1H), 2.262.11 (m, 1H), 2.10-1.90 (m, 4H), 1.63 (m, 2H), 1.56-1.23 (m, 22H), 1.18 (m, 3H), 0.96-0.80 (m, 3H). ³¹ P NMR (162 MHz, acetonitrile^) δ 17.96, 17.90. LCMS: MS m/z = 692.25 [M+1]; tR = 1.74 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XBC18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: tR = 6.25 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min 8.5 min 2-98% ACN at 1.5 mL/min.

Example 23. Hexyl ((((^)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((2-methyl-1-oxo-1((tetrahydro-2H-pyran-4-yl)oxy)propan-2-yl)amino)phosphoryl)^-phenylalaninate (23).

Example 24. Bis(tetrahydro-2H-pyran-4-yl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (24)

Synthesis of tetrahydro-2H-pyran-4-yl-2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (23a).

To a mixture of 2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid (1.0 g, 4.92 mmol), tetrahydro-2H-pyran-4-ol (0.70 mL, 7.381 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.15 g, 7.381 mmol) in acetonitrile (20 mL) was added DMAP (1.20 g, 9.84 mmol). The mixture was stirred at room temperature for 15 h, quenched with water, and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (EtOAc, 0-50% in hexanes) to afford intermediate 23a. 1H NMR (400 MHz, acetonitrile^) δ 5.62 (s, 1H), 4.92 (m, 1H), 3.83 (m, 2H), 3.53 (m, 2H), 1.88 (m, 2H), 1.59 (m, 2H), 1.77-1.59 (m, 15H).

- 79 049099

Synthesis of tetrahydro-2H-pyran-4-yl-2-amino-2-methylpropanoate hydrochloride (23b).

To a solution of intermediate 23a (490 mg, 1.71 mmol) in DCM (10 mL) was slowly added 4 M HCl in dioxane (2 mL) at room temperature. The resulting mixture was stirred at room temperature for 15 h, concentrated in vacuo, co-evaporated with DCM several times and dried under high vacuum for 15 h to afford intermediate 23b. 1H NMR (400 MHz, acetonitrile^) δ 8.48 (br s, 3H), 5.06 (m, 1H), 3.89 (m, 2H), 3.55 (m, 2H), 1.92 (m, 2H), 1.76-1.59 (m, 8H).

Synthesis of hexyl^-phenylalaninate hydrochloride (23 s).

To a mixture of L-phenylalanine (2.5 g, 15.1 mmol) and n-hexanol (30 mL) was slowly added SOCl ₂ (4.4 mL, 60.5 mmol) at room temperature. The resulting mixture was heated at 80 °C for 15 h and concentrated at 65 °C under high vacuum. After cooling, the product precipitated and was filtered. The filter cake was suspended in ether, stirred overnight, filtered and dried under high vacuum for 24 h to give intermediate 23c. 1H NMR (400 MHz, methanol-d^ δ 7.42-7.29 (m, 3H), 7.28-7.22 (m, 2H), 4.29 (t, J=7.0 Hz, 1H), 4.17 (m, 2H), 3.21 (m, 2H), 1.58 (m, 2H), 1.35-1.25 (m, 6H), 0.91 (t, J=6.85 Hz, 3H).

but-f

gryphenylphosphine

2,2'-dipyridyl disulfide Triethylamine pyridine

A mixture of PMPA (100 mg, 0.348 mmol) and intermediate 23b (156 mg, 0.696 mmol) was purged with nitrogen several times and dissolved in pyridine (2 mL). Triphenylphosphine (548 mg, 2.09 mmol), 2,2'-dipyridyl disulfide (460 mg, 2.09 mmol), and triethylamine (0.39 mL, 2.79 mmol) were added. The resulting mixture was heated to 80 °C for 40 min. Intermediate 23c (299 mg, 1.04 mmol) in pyridine (0.5 mL) was added. The mixture was heated at 80°C for 3 h, concentrated in vacuo and purified by silica gel chromatography (MeOH 0 to 25% in DCM) to two products, which were further purified by HPLC (ACN 10 to 100% in water over 12 min, 100% ACN for 5 min for 23 and ACN 10 to 100% in water over 17 min for 24) to give 23 and 24.

23: 1H NMR (400 MHz, acetonitrile^) δ 8.27 (s, 0.5H), 8.25 (s, 0.5H), 8.08 (s, 0.5H), 8.03 (s, 0.5H), 7.33-7.16 (m, 5H), 6.87-6.55 (m, 2H), 4.89 (m, 1H), 4.39-3.95 (m, 4H), 3.94-3.68 (m, 4H), 3.61 (dd, J=12.8, 8.8 Hz, 0.5H), 3.54-3.41 (m, 3.5H), 3.24 (dd, J=12.7, 10.4 Hz, 0.5H), 3.15 (d, J=11.3 Hz, 0.5H), 3.06-2.90 (m, 1.5H), 2.82 (dd, J=13.5, 8.1 Hz, 0.5H), 1.92-1.77 (m, 2H), 1.65-1.46 (m, 4H), 1.43-1.19 (m, 12H), 1.11 (t, J=6.0 Hz, 3H), 0.92-0.83 (m, 3H). ^31P NMR (162 MHz, acetonitrile^) δ 19.52, 19.39. LCMS: MS m/z = 688.31 [M+1]; tR = 1.57 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 1002% acetonitrile, 1.85-2.00 min 2% ACN at 1800 μL/min. HPLC: t _R = 5.57 min; HPLC system: 1290 Infinity II; column: Phenomenex 2.6 μm C18 100A, 100x4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min - 8.5 min 2-98% ACN at 1.5 ml/min.

24: 1H NMR (400 MHz, acetonitrile^ ₃ ) δ 8.23 (s, 1H), 8.05 (s, 1H), 5.99 (s, 2H), 4.92 (m, 2H), 4.34 (dd, J=14.5, 3.3 Hz, 1H), 4.17 (dd, J=14.5, 7.1 Hz, 1H), 3.94 (m, 1H), 3.82 (dt, J=10.6, 5.7 Hz, 4H), 3.75 (d, J=10.7 Hz, 1H), 3.67 (dd, J=12.7, 8.8 Hz, 1H), 3.59-3.42 (m, 6H), 1.89 (m, 4H), 1.62 (m, 4H), 1.54 (s, 3H), 1.47 (s, 6H),

- 80 049099

1.40 (s, 3H), 1.19 (d, J=6.2 Hz, 3H). ³¹ P NMR (162 MHz, acetonitrile-^) δ 17.90. LCMS: MS m/z = 626.18 [M+1]; t _R = 1.21 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 1002% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: t _R = 3.81 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 25. Bis(5,5,5-trifluoropentyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(H)-bis(2-methylpropanoate) (25)

Η^Αγ-θΗ HO^^. _CF o ³ o

25a

Synthesis of 5,5,5-trifluoropentyl-2-amino-2-methylpropanoate hydrochloride (25a).

To a mixture of 2-amino-2-methylpropanoic acid (600 mg, 5.82 mmol) and 5,5,5-trifluoropentan-1-ol (5.0 g, 35.2 mmol) was slowly added SOC12 (0.72 mL, 9.89 mmol) at room temperature. The resulting mixture was heated at 80 °C for 15 h and concentrated at 70 °C under high vacuum. Hexane was added to the resulting residue, which was stirred for 30 min, and the precipitated solid was filtered off. The filter cake was washed several times with ether and dried under high vacuum for 15 h to yield intermediate 25a. 1H NMR (400 MHz, acetonitrile-^3) δ 8.69 (br s, 3H), 4.23 (t, J=6.2 Hz, 2H), 2.25 (m, 2H), 1.86-1.73 (m, 2H), 1.72-1.59 (m, 8H). ^19F NMR (376 MHz, acetonitrile-^3) δ -67.50.

O

25a 25

A mixture of PMPA (500 mg, 1.74 mmol), intermediate 25a (1.51 g, 5.74 mmol), 2,2'dipyridyl disulfide (2.30 g, 10.4 mmol), triphenylphosphine (2.74 g, 10.4 mmol) was purged with nitrogen gas several times, dissolved in pyridine (10 mL), and triethylamine (0.4 mL, 2.785 mmol) was added. The resulting mixture was heated at 90°C for 20 h, concentrated in vacuo and purified by silica gel column chromatography (MeOH 0 to 20% in DCM) and preparative HPLC (ACN 10 to 100% in water for 12 min, ACN 100% for 5 min) to give the title compound (25). 1H NMR (400 MHz, acetonitrile-^) δ 8.24 (s, 1H), 8.06 (s, 1H), 6.15 (s, 2H), 4.34 (dd, J=14.4, 3.2 Hz, 1H), 4.22-4.03 (m, 5H), 3.94 (m, 1H), 3.77-3.61 (m, 2H), 3.56 (d, J=10.6 Hz, 1H), 3.44 (dd, J=12.7, 9.8 Hz, 1H), 2.22 (m, 4H), 1.82-1.56 (m, 8H), 1.53 (s, 3H), 1.46 (s, 3H), 1.45 (s, 3H), 1.39 (s, 3H), 1.19 (d, J=6.3 Hz, 3H). ¹⁹ F NMR (376 MHz, acetonitrile-3) δ -67.52 (d, J=5.3 Hz). ³¹ P NMR (162 MHz, acetonitrile-3) δ 17.83. LCMS: MS m/z = 706.31 [M+1]; t _R = 1.57 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: t _R = 5.53 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 26. Bis(trans-3-(trifluoromethyl)cyclobutyl) 2,2'-(((((((K)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (26)

EDCI

26a

Synthesis of trans-3-(trifluoromethyl)cyclobutyl-2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (26a).

To a mixture of trans-3-(trifluoromethyl)cyclobutan-1-ol (1.0 g, 7.14 mmol), 2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid (2.9 g, 14.3 mmol) and DMAP (1.9 g, 15.7 mmol) in acetonitrile (30 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.44 g, 15.7 mmol) at room temperature. The mixture was stirred at room temperature for 2 h, quenched with water and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (EtOAc, 0-70% in hexanes) to afford intermediate 26a. 1H NMR (400 MHz, acetonite

- 81 049099 ril-dz) δ 5.68 (s, 1H), 5.05 (m, 1H), 3.06 (m, 1H), 2.56 (m, 2H), 2.40 (m, 2H), 1.59-0.92 (m, 15H). ¹⁹ F NMR (376 MHz, acetonitrile^) δ -74.34. LCMS: MS m/z = 325.59 [M+1]; t _R = 1.84 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µl/min.

26a 26b

Synthesis of trans-3-(trifluoromethyl)cyclobutyl 2-amino-2-methylpropanoate hydrochloride (26b).

To a solution of intermediate 26a (1.96 g, 6.02 mmol) in DCM (10 mL) was slowly added 4 M HCl in dioxane (7.53 mL) at room temperature. The resulting mixture was stirred at room temperature for 3 h, concentrated in vacuo, coevaporated with DCM several times, suspended in ether, stirred for 5 min, and filtered. The filter cake was washed several times with ether and dried under high vacuum for 15 h to yield intermediate 26b. 1H NMR (400 MHz, acetonitrile^) δ 8.70 (br s, 3H), 5.18 (t, J = 6.6 Hz, 1H), 3.20 (m, 1H), 2.66-2.49 (m, 2H), 2.40-2.25 (m, 2H), 1.74-1.57 (m, 6H). LCMS: MS m/z = 226.02 [M + 1-HCl]; tR = 0.96 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µl/min.

26b 26

A mixture of PMPA (300 mg, 1.04 mmol), intermediate 26b (600 mg, 2.29 mmol), 2,2'dipyridyl disulfide (768 mg, 3.48 mmol), and triphenylphosphine (914 mg, 3.48 mmol) was purged with nitrogen several times and dissolved in pyridine (5 mL). Triethylamine (0.8 mL, 5.77 mmol) was added. The resulting mixture was stirred at 90 °C for 20 h, concentrated in vacuo, coevaporated several times with toluene, and purified by chromatography on silica gel (MeOH 0 to 20% in DCM) and with prep. HPLC (10 to 100% ACN in water over 12 min, 100% ACN over 5 min) to give the title compound (26). 1H NMR (400 MHz, acetonitrile^) δ 8.24 (s, 1H), 8.03 (s, 1H), 5.91 (s, 2H), 5.06 (m, 2H), 4.34 (dd, J=14.5, 3.2 Hz, 1H), 4.17 (dd, J=14.5, 7.3 Hz, 1H), 3.94 (m, 1H), 3.73-3.63 (m, 2H), 3.55-3.35 (m, 2H), 3.11 (m, 2H), 2.65-2.51 (m, 2H), 2.51-2.36 (m, 2H), 1.53 (s, 3H), 1.47 (s, 3H), 1.43 (s, 3H), 1.40 (s, 3H), 1.20 (d, J=6.2 Hz, 3H). ¹⁹ F NMR (376 MHz, acetonitrile^) δ -74.27. ³¹ P NMR (162 MHz, acetonitrile^) δ 18.10. LCMS: MS m/z = 702.22 [M+1]; t _R = 1.37 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XBC18 100 A 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: t _R = 5.50 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min 8.5 min 2-98% ACN at 1.5 mL/min.

Example 27. Bis(trans-4-propylcyclohexyl) 2,2'-((((((^)-1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (27)

EDCI

27a

Synthesis of trans-4-propylcyclohexyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (27a).

To a mixture of trans-4-propylcyclohexan-1-ol (1.0 g, 7.03 mmol), 2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid (2.14 g, 10.5 mmol), and DMAP (1.72 g, 14.1 mmol) in acetonitrile (30 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.18 g, 14.1 mmol) at room temperature. The mixture was stirred at room temperature for 15 h, quenched with water, and concentrated

- 82 049099 was concentrated in vacuo. The resulting residue was dissolved in EtOAc, washed with brine, dried over sodium sulfate, concentrated in vacuo and purified by silica gel chromatography (EtOAc, 0 to 70% hexanes) to give intermediate 27a. 1H NMR (400 MHz, acetonitrile^) δ 5.58 (br s, 1H), 4.61 (m, 1H), 1.96-1.83 (m, 2H), 1.85-1.73 (m, 2H), 1.45-1.14 (m, 22H), 1.05 (m, 2H), 0.90 (t, >1.3 Hz, 3H). LCMS: MS m/z = 327.77 [M+1]; tR = 1.98 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 μL/min.

27a 27b

Synthesis of trans-4-propylcyclohexyl 2-amino-2-methylpropanoate hydrochloride (27b).

To a solution of intermediate 27a (1.46 g, 4.46 mmol) in DCM (10 mL) was slowly added 4 M HCl in dioxane (6 mL) at room temperature. The resulting mixture was stirred at room temperature for 4 h, concentrated in vacuo, coevaporated with DCM several times, suspended in ether, stirred for 5 min, and filtered. The filter cake was washed several times with ether and dried under high vacuum for 15 h to yield intermediate 27b. 1H NMR (400 MHz, Chloroform-d) δ 8.98 (br s, 3H), 4.76 (m, 1H), 2.02 (m, 2H), 1.80 (m, 2H), 1.72 (s, 6H), 1.48 (m, 2H), 1.38-1.12 (m, 5H), 0.99 (m, 2H), 0.89 (t, J=7.2 Hz, 3H). LCMS: MS m/z = 227.89 [M+1-HCl]; tR = 1.19 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µl/min.

27b 27

A mixture of PMPA (300 mg, 1.04 mmol), intermediate 27b (600 mg, 2.27 mmol), 2,2'dipyridyl disulfide (768 mg, 3.49 mmol) and triphenylphosphine (914 mg, 3.49 mmol) was purged with nitrogen several times and dissolved in pyridine (5 mL). Triethylamine (0.8 mL, 5.77 mmol) was added. The resulting mixture was stirred at 90 °C for 20 h, concentrated in vacuo, co-evaporated several times with toluene and purified by chromatography on silica gel (MeOH 0 to 15% in DCM) to give intermediate (27). 1H NMR (400 MHz, acetonitrile^) δ 8.24 (s, 1H), 8.06 (s, 1H), 6.01 (s, 2H), 4.62 (m, 2H), 4.34 (dd, J=14.5, 3.2 Hz, 1H), 4.17 (dd, J=14.5, 7.0 Hz, 1H), 3.94 (m, 1H), 3.74 (d, J=10.6 Hz, 1H), 3.66 (dd, J=12.7, 8.9 Hz, 1H), 3.55 (d, J=10.7 Hz, 1H), 3.46 (dd, J=12.6, 9.8 Hz, 1H), 1.961.87 (m, 4H), 1.85-1.72 (m, 4H), 1.57-0.95 (m, 33H), 0.91 (t, J=7.3 Hz, 6H). ³¹ P NMR (162 MHz, acetonitrile _{d 3} ) δ 17.59. LCMS: MS m/z = 706.37 [M+1]; tR = 1.89 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-O8 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0-1.8 min 2100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: tR = 7.84 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 28. Dihexyl 2,2'-(((((1-(6-acetamido-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (28)

28

- 83 049099

To a solution of 1 (100 mg, 0.210 mmol) in pyridine (1 mL) was added acetic anhydride (0.03 mL, 0.320 mmol). The resulting mixture was stirred at room temperature for 2 h and purified by prep. HPLC (10-100% ACN in water for 8 min, 100% ACN for 18 min) to afford the title compound (28). 1H NMR (400 MHz, acetonitrile^) δ 9.13 (s, 1H), 8.61 (s, 1H), 8.34 (s, 1H), 4.43 (dd, J=14.5, 3.2 Hz, 1H), 4.26 (dd, J=14.5, 7.1 Hz, 1H), 4.17-4.01 (m, 4H), 3.97 (m, 1H), 3.75 (d, J=10.8 Hz, 1H), 3.69 (dd, J=12.8, 8.6 Hz, 1H), 3.59 (d, J=10.8 Hz, 1H), 3.45 (dd, J=12.7, 9.8 Hz, 1H), 2.48 (s, 3H), 1.62 (m, 4H), 1.52 (s, 3H), 1.45 (s, 6H), 1.42-1.23 (m, 15H), 1.20 (d, J=6.3 Hz, 3H), 0.95-0.88 (m, 6H). ^31P NMR (162 MHz, acetonitrile^ ₃ ) δ 17.75. LCMS: MS m/z = 668.42 [M+1]; tR = 1.90 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XBC18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: tR = 6.66 min; HPLC system: 1290 Infinity II; column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min 8.5 min 2-98% ACN at 1.5 mL/min.

Example 29. Dihexyl 2,2'-(((((1-(6-butyramido-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(K)-bis(2-methylpropanoate) (29)

29

To a solution of 1 (100 mg, 0.210 mmol) in pyridine (1 mL) was added butyryl chloride (0.033 mL, 0.320 mmol). The resulting mixture was stirred at room temperature for 2 h and purified by prep. HPLC (10-100% ACN in water for 8 min, 100% ACN for 15 min) to afford the title compound (29). 1H NMR (400 MHz, acetonitrile^ ₃ ) δ 8.98 (s, 1H), 8.61 (s, 1H), 8.32 (s, 1H), 4.43 (dd, J=14.5, 3.1 Hz, 1H), 4.25 (dd, J=14.5, 7.2 Hz, 1H), 4.17-4.01 (m, 4H), 3.96 (m, 1H), 3.78-3.64 (m, 2H), 3.57 (d, J=10.8 Hz, 1H), 3.44 (dd, J=12.7, 9.9 Hz, 1H), 2.77 (t, J=7.4 Hz, 2H), 1.74 (m, 2H), 1.63 (m, 4H), 1.52 (s, 3H), 1.48-1.42 (m, 6H), 1.43-1.23 (m, 15H), 1.20 (d, J=6.2 Hz, 3H), 1.02 (t, J=7.4 Hz, 3H), 0.96-0.84 (m, 6H). ^31P NMR (162 MHz, acetonitrile^) δ 17.756. LCMS: MS m/z = 696.31 [M+1]; tR = 2.02 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: tR = 7.02 min; HPLC system: 1290 Infinity II; column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 30. Dihexyl-2,2'-(((((1-(6-dodecanamido-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azandiyl))(K)-bis(2-methylpropanoate) (30)

30

To a solution of 1 (100 mg, 0.210 mmol) in pyridine (1 mL) was added lauryl chloride (0.07 mL, 0.320 mmol). The resulting mixture was stirred at room temperature for 2 h, quenched by addition of methanol, concentrated in vacuo and purified by silica gel chromatography (MeOH 0.15% in DCM) to give the title compound (30). 1H NMR (400 MHz, acetonitrile^ ₃ ) δ 8.96 (s, 1H), 8.61 (s, 1H), 8.31 (s, 1H), 4.43 (dd, J=14.5, 3.1 Hz, 1H), 4.30-4.23 (m, 1H), 4.17-4.01 (m, 4H), 3.96 (m, 1H), 3.77-3.64 (m, 2H), 3.54 (d, J=10.8 Hz, 1H), 3.43 (dd, J=12.7, 9.9 Hz, 1H), 2.78 (t, J=7.5 Hz, 2H), 1.781.53 (m, 6H), 1.52 (s, 3H), 1.46-1.40 (m, 6H), 1.40-1.25 (m, 31H), 1.20 (d, J=6.2 Hz, 3H), 0.93-0.83 (m, 9H). ^31P NMR (162 MHz, acetonitrile^) δ 17.71. LCMS: MS m/z = 808.83 [M+1]; HPLC: tR = 2.58 min, LC system: Dionex Ultimate 3000 UHPLC; column: Phenomenex Kinetex 2.6μ C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0 min-0.2 min 40% acetonitrile, 0.2-1.55 min 40-100% acetonitrile, 1.55-2.80 min-100% acetonitrile, 2.80-2.81 min

- 84 049099

100-40% acetonitrile at 1100 µl/min. HPLC: tR = 8.92 min; HPLC system: 1290 Infinity II; column: Phenomenex 2.6 µm C18 100A, 100x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min - 8.5 min 2-98% ACN at 1.5 ml/min.

Example 31. Dihexyl 2,2'-(((((1-(6-((propoxycarbonyl)amino)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate)(31)

31

To a solution of 1 (200 mg, 0.32 mmol) in pyridine (1 mL) was added propyl chloroformate (118 mL, 0.96 mmol). The resulting mixture was stirred at room temperature for 2 h and the reaction was quenched by adding water. The mixture was concentrated and purified by preparative HPLC (ACN 10 to 100% in water for 5 min and ACN 100% for 18 min) to afford the title compound (31). 1H NMR (400 MHz, acetonitrile^) δ 8.88 (s, 1H), 8.61 (s, 1H), 8.31 (s, 1H), 4.43 (dd, J=14.5, 3.2 Hz, 1H), 4.25 (dd, J=14.5, 7.2 Hz, 1H), 4.20-4.01 (m, 6H), 4.01-3.91 (m, 1H), 3.75 (d, J=10.9 Hz, 1H), 3.69 (dd, J=12.8, 8.6 Hz, 1H), 3.58 (d, J=10.9 Hz, 1H), 3.44 (dd, J=12.8, 9.9 Hz, 1H), 1.73 (m, 2H), 1.69-1.58 (m, 4H), 1.52 (s, 3H), 1.45 (s, 6H), 1.41-1.26 (m, 15H), 1.20 (d, J=6.2 Hz, 3H), 1.00 (t, J=7.4 Hz, 3H), 0.93-0.83 (m, 6H). ^31P NMR (162 MHz, acetonitrile^) δ 17.86. LCMS: MS m/z = 712.31 [M+1]; tR = 1.84 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XBC18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: t _R = 7.12 min; HPLC system: 1290 Infinity II; column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min 8.5 min 2-98% ACN at 1.5 mL/min.

Example 32. Dihexyl 2,2'-(((((1-(6-(((pentyloxy)carbonyl)amino)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (32)

32

To a solution of 1 (200 mg, 0.32 mmol) in pyridine (1 mL) was added pentyl chloroformate (144 mL, 0.96 mmol). The resulting mixture was stirred at room temperature for 2 h, quenched by addition of water, and purified by preparative HPLC (10 to 100% acetonitrile in water for 5 min and 100% acetonitrile for 18 min) to give the title compound (32). 1H NMR (400 MHz, acetonitrile^) δ 8.74 (s, 1H), 8.61 (s, 1H), 8.30 (s, 1H), 4.43 (dd, J=14.5, 3.1 Hz, 1H), 4.30-4.17 (m, 3H), 4.17-4.01 (m, 4H), 3.96 (m, 1H), 3.74 (d, J=10.8 Hz, 1H), 3.68 (dd, J=12.8, 8.6 Hz, 1H), 3.56 (d, J=10.8 Hz, 1H), 3.43 (dd, J=12.7, 9.9 Hz, 1H), 1.77-1.56 (m, 6H), 1.52 (s, 3H), 1.47-1.26 (m, 25H), 1.20 (d, J=6.2 Hz, 3H), 0.98-0.85 (m, 9H). ^31P NMR (162 MHz, acetonitrile^) δ 17.80. LCMS: MS m/z = 740.39 [M+1]; t _R = 1.96 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 1002% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: tR = 7.61 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 33. Dihexyl 2,2'-(((((1-(6-(((((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)carbonyl)amino)9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (33)

- 85 049099

O

33

To a mixture of 1 (200 mg, 0.32 mmol) and triphosgene (38 mg, 0.128 mmol) in DCM (4 mL) was slowly added DMAP (234 mg, 1.92 mmol) in portions at room temperature. The resulting mixture was stirred at room temperature for 15 min and 4(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (60 mg, 0.46 mmol) was added at room temperature. The resulting mixture was stirred for 2 h, concentrated in vacuo and purified by silica gel column chromatography (MeOH 0 to 15% in DCM) and preparative HPLC (ACN 10 to 100% in water for 8 min, ACN 100% for 15 min) to give the title compound (33). 1H NMR (400 MHz, acetonitriled3) δ 8.69 (s, 1H), 8.63 (s, 1H), 8.31 (s, 1H), 5.02 (s, 2H), 4.44 (dd, J=14.5, 3.2 Hz, 1H), 4.26 (dd, J=14.5, 7.2 Hz, 1H), 4.16-4.02 (m, 4H), 3.96 (m, 1H), 3.79-3.61 (m, 2H), 3.55 (d, J=10.7 Hz, 1H), 3.44 (dd, J=12.7, 9.8 Hz, 1H), 2.21 (s, 3H), 1.70-1.55 (m, 4H), 1.52 (s, 3H), 1.45 (s, 6H), 1.42-1.26 (m, 15H), 1.20 (d, J=6.2 Hz, 3H), 0.95-0.83 (m, 6H). ³¹ P NMR (162 MHz, acetonitrile^) δ 17.63. LCMS: MS m/z = 782.17 [M+1]; tR = 2.00 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: t _R = 7.05 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 34. Dicyclopentyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azandiyl))(R)-bis(2-methylpropanoate) (34)

34a

Synthesis of cyclopentyl 2-amino-2-methylpropanoate hydrochloride (34a).

2-Amino-2-methylpropanoic acid (1.0 g, 9.70 mmol) was suspended in cyclopentanol (17.6 ml, 194 mmol). Thionyl chloride (1.4 ml, 19.4 mmol) was slowly added over 3 min. The mixture was heated at 70 °C for 3 days. The mixture was diluted with ethyl acetate/hexane (1:1, 20 ml) and water (20 ml). The organic phase was extracted with water (20 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 20 ml), diethyl ether (3 x 20 ml) and dichloromethane (3 x 20 ml). Any residual solvent in the aqueous phase was removed under reduced pressure. The volume was reduced to 20 ml under reduced pressure. The aqueous phase was diluted with acetonitrile (10 mL) and lyophilized. The solid was stirred and washed with hexane for 15 min and isolated by filtration to give intermediate 34a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.54 (s, 3H), 5.18 (m, 1H), 1.94-1.78 (m, 2H), 1.76-1.62 (m, 4H), 1.62-1.52 (m, 2H), 1.45 (s, 6H).

34a 34

PMPA (100 mg, 0.384 mmol), triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) and intermediate 34a (289 mg, 1.39 mmol) were suspended in pyridine (4 ml) under argon. Triethylamine (0.38 ml, 2.79 mmol) was added. The reaction mixture was heated at 75 °C for 3 days. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (2 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 µM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing product were pooled

- 86 049099 li and lyophilized to give the title compound (34). 1H NMR (400 MHz, methanol^) δ 8.30 (s, 1H), 8.23 (s, 1H), 5.20 (m, 1H), 5.15 (m, 1H), 4.43 (dd, J=14.5, 3.1 Hz, 1H), 4.34 (d, J=11.9 Hz, 0.39H), 4.27 (dd, J=14.5, 7.4 Hz, 1H), 4.12 (s, 0.24H), 4.08-3.96 (m, 1H), 3.80 (dd, J=12.8, 8.5 Hz, 1H), 3.55 (dd, J=12.8, 10.2 Hz, 1H), 1.89 (m, 5H), 1.82-1.69 (m, 6H), 1.66 (m, 5H), 1.55 (s, 3H), 1.47 (s, 3H), 1.45 (s, 3H), 1.40 (s, 3H), 1.26 (d, J=6.2 Hz, 3H). ^31P NMR (162 MHz, methanol-bd) δ 20.98-20.75 (m). LCMS: MS m/z = 594.58 [M+1]; tR = 1.145 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: tR = 2.706 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 3 5. Diisobutyl 2,2'-((((( 1 -(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^)-bis(2-methylpropanoate) (35)

Synthesis of isobutyl 2-amino-2-methylpropanoate hydrochloride (35a) 2-amino-2-methylpropanoic acid (2.5 g, 24.2 mmol) was suspended in 2-methylpropan-1-ol (22 ml, 282 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 60 ml, 242 mmol). The mixture was heated at 65 °C for 5 days. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 50 ml) and water (50 ml). The organic phase was extracted with water (50 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 50 ml), diethyl ether (3 x 50 ml) and dichloromethane (3 x 50 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 50 mL under reduced pressure. The aqueous phase was diluted with acetonitrile (20 mL) and water (10 mL) and lyophilized. The solid was stirred with hexane (20 mL) and isolated by filtration with hexane rinses to afford intermediate 35a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.56 (s, 3H), 3.98 (d, J=6.5 Hz, 2H), 1.93 (m, 1H), 1.49 (s, 6H), 0.92 (d, J=6.7 Hz, 6H).

PMPA (100 mg, 0.384 mmol), triphenylphosphine (341 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) and intermediate 35a (289 mg, 1.39 mmol) were suspended in pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The reaction mixture was heated at 90 °C for 18 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (35). 1H NMR (400 MHz, methanol-b4) δ 8.34 (s, 1H), 8.28 (s, 1H), 4.45 (dd, J=14.5, 3.0 Hz, 1H), 4.29 (dd, J=14.5, 7.4 Hz, 1H), 4.05-3.88 (m, 5H), 3.88-3.75 (m, 1H), 3.55 (dd, J=12.8, 10.2 Hz, 1H), 2.03-1.90 (m, 2H), 1.59 (s, 3H), 1.51 (s, 3H), 1.50 (s, 3H), 1.26 (d, J=6.2 Hz, 3H), 1.02-0.92 (m, 12H). ³¹ P NMR (162 MHz, methanol^) δ 20.78 (t, J=9.3 Hz). LCMS: MS m/z = 570.14 [M+1]; t _R = 1.350 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: t _R = 2.673 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

- 87 049099

Example 36. Dipropyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azandiyl))^)-bis(2-methylpropanoate) (36)

about hydrogen chloride

Dioxane

Cl-

36a

Synthesis of 2-amino-2-methylpropanoate hydrochloride (36a).

2-Amino-2-methylpropanoic acid (1 g, 9.7 mmol) was suspended in propan-1-ol (17.6 ml, 278 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 12.1 ml, 48.5 mmol). The mixture was heated at 40 °C for 5 days. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 50 ml) and water (50 ml). The organic phase was extracted with water (50 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 50 ml), diethyl ether (3 x 50 ml) and dichloromethane (3 x 50 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 50 ml under reduced pressure. The aqueous phase was diluted with acetonitrile (20 mL) and water (10 mL) and lyophilized. The solid was stirred with hexane (20 mL) and isolated by filtration with hexane rinses to afford intermediate 36a. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.76-8.33 (m, 3H), 4.14 (t, J=6.5 Hz, 2H), 1.64 (m, 2H), 1.49 (s, 6H), 0.92 (t, J=7.4 Hz, 3H).

36a 36

PMPA (100 mg, 0.384 mmol), triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) and intermediate 36a (341 mg, 1.88 mmol) were suspended in pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The reaction mixture was heated at 90 °C for 16 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (36). 1H NMR (400 MHz, methanol-b ₄ ) δ 8.35 (s, 1H), 8.29 (s, 1H), 4.45 (dd, J=14.5, 3.1 Hz, 1H), 4.29 (dd, J=14.5, 7.5 Hz, 1H), 4.21-4.03 (m, 4H), 4.033.96 (m, 1H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.55 (dd, J=12.8, 10.2 Hz, 1H), 1.77-1.63 (m, 4H), 1.57 (s, 3H), 1.50 (s, 3H), 1.49 (s, 3H), 1.44 (s, 3H), 1.26 (d, J=6.2 Hz, 3H), 1.03-0.92 (m, 6H). ³¹ P NMR (162 MHz, methanol-bd) δ 20.76. LCMS: MS m/z = 542.11 [M+1]; t _R = 1.240 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: t _R = 2.402 min; HPLC system: Agilent 1100 Series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 37. Bis(cyclobutylmethyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (37)

ABOUT

37a

Synthesis of cyclobutylmethyl 2-amino-2-methylpropanoate hydrochloride (37a).

2-Amino-2-methylpropanoic acid (1.0 g, 9.7 mmol) was suspended in cyclobutylmethanol (8.8 mL, 97 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 24.2 mL, 97 mmol). The mixture was heated at 65 °C for 3 days. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 100 mL) and water (100 mL). The organic phase was extracted with water (100 mL). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 100 mL), diethyl ether (3 x 100

- 88 049099 mL) and dichloromethane (3x100 mL). Any residual solvent in the aqueous phase was removed and the volume was reduced to 50 mL under reduced pressure. The aqueous phase was diluted with acetonitrile (20 mL) and water (10 mL) and lyophilized to afford intermediate 37a. 111 NMR (400 MHz, DMSO-d ₆ ) δ 8.52 (s, 3H), 4.16 (d, J=6.4 Hz, 2H), 2.71-2.56 (m, 1H), 2.07-1.95 (m, 2H), 1.95-1.70 (m, 4H), 1.48 (s, 6H).

O

37a 37

PMPA (100 mg, 0.384 mmol), triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) and intermediate 37a (341 mg, 1.64 mmol) were suspended in pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The reaction mixture was heated at 105 °C for 19 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (37). 1H NMR (400 MHz, methanol-dA δ 8.29 (s, 1H), 8.23 (s, 1H), 4.46-4.35 (m, 2H), 4.33-4.24 (m, 1H), 4.11 (m, 5H), 4.02-3.94 (m, 1H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.54 (dd, J=12.8, 10.3 Hz, 1H), 2.77-2.56 (m, 2H), 2.16-2.02 (m, 4H), 2.02-1.88 (m, 4H), 1.88-1.74 (m, 4H), 1.58 (s, 3H), 1.50 (s, 3H), 1.47 (s, 3H), 1.43 (s, 3H), 1.26 (d, J=6.2 Hz, 3H). ^31P NMR (162 MHz, methanol-f) δ 20.87 (t, J=9.4 Hz). LCMS: MS m/z = 594.54 [M+1]; tR = 1.203 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% Acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: tR = 2.811 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 38. Dibutyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (3 8) hydrogen chloride Dioxane

38a

Synthesis of butyl 2-amino-2-methylpropanoate hydrochloride (38a).

2-Amino-2-methylpropanoic acid (2.5 g, 24.2 mmol) was suspended in butan-1-ol (22 ml, 240 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 30.3 ml, 121 mmol). The mixture was heated at 65 °C for 2 days. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 100 ml) and water (100 ml). The organic phase was extracted with water (2 x 100 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 100 ml), diethyl ether (3 x 100 ml) and dichloromethane (3 x 100 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 50 mL under reduced pressure. The aqueous phase was diluted with acetonitrile (20 mL) and water (10 mL) and lyophilized to afford intermediate 38a. ¹ H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 3H), 4.18 (t, J=6.4 Hz, 2H), 1.68-1.54 (m, 2H), 1.48 (s, 6H), 1.42-1.28 (m, 2H), 0.91 (t, J=7.4 Hz, 3H).

- 89 049099

38a 38

PMPA (100 mg, 0.384 mmol), triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) and intermediate 38a (341 mg, 1.74 mmol) were suspended in pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The reaction mixture was heated at 95 °C for 16 h and at 105 °C for 18 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (38). 1H NMR (400 MHz, methanol-^) δ 8.45 (s, 1H), 8.37 (s, 1H), 4.50 (dd, J=14.4, 3.1 Hz, 1H), 4.34 (dd, J=14.5, 7.4 Hz, 1H), 4.26-4.07 (m, 4H), 4.06-3.98 (m, 1H), 3.82 (dd, J=12.9, 8.3 Hz, 1H), 3.56 (dd, J=12.9, 10.2 Hz, 1H), 1.73-1.60 (m, 4H), 1.57 (s, 3H), 1.50 (s, 3H), 1.49 (s, 3H), 1.47-1.36 (m, 7H), 1.26 (d, J=6.2 Hz, 3H), 1.02-0.92 (m, 6H). ^31P NMR (162 MHz, methanol-L ₄ ) δ 20.67 (t, J=9.1 Hz). LCMS: MS m/z = 570.49 [M+1]; tR = 1.124 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: tR = 2.7-10 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 39. Dioctyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(K)-bis(2-methylpropanoate) (39)

39a

Synthesis of octyl 2-amino-2-methylpropanoate hydrochloride (39a).

2-Amino-2-methylpropanoic acid (2.5 g, 24.2 mmol) was suspended in p-octanol (22.0 ml, 137 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 30.3 ml, 121 mmol). The mixture was heated at 65 °C for 40 h. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 200 ml) and water (200 ml). The organic phase was extracted with water (2 x 200 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 200 ml), diethyl ether (3 x 200 ml) and dichloromethane (3 x 200 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 100 mL under reduced pressure. The aqueous phase was diluted with acetonitrile (50 mL) and lyophilized to afford intermediate 39a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.60 (s, 3H), 4.16 (t, J=6.5 Hz, 2H), 1.68-1.57 (m, 2H), 1.48 (s, 6H), 1.40-1.18 (m, 10H), 0.93-0.79 (m, 3H).

39a 39

PMPA (100 mg, 0.384 mmol), triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) and intermediate 39a (341 mg, 1.35 mmol) were suspended in pyridine (4 mL) under argon. Triethylamine (0.475 mL, 3.48 mmol) was added. The reaction mixture was heated at 90 °C for 16 h and 105 °C for 6 h. The pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 mL). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with a gradient of 40% then 100% ethyl

- 90 049099 acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250x30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (39). 1H NMR (400 MHz, methanol^) δ 8.30 (s, 1H), 8.23 (s, 1H), 4.48-4.34 (m, 1.5H), 4.27 (dd, J=14.5, 7.4 Hz, 1H), 4.23-4.04 (m, 5H), 4.04-3.93 (m, 1H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.54 (dd, J=12.8, 10.2 Hz, 1H), 1.76-1.60 (m, 4H), 1.58 (s, 3H), 1.49 (m, 6H), 1.43 (s, 3H), 1.42-1.27 (m, 20H), 1.25 (d, J=6.2 Hz, 3H), 0.96-0.86 (m, 6H). ^31P NMR (162 MHz, methanol^) δ 20.84 (t, J=9.3 Hz). LCMS: MS m/z = 682.71 [M+1]; t _R = 1.751 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: t _R = 3.945 min; HPLC system: Agilent 1100 Series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 40. Dicyclohexyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (40)

40a

Synthesis of cyclohexyl 2-amino-2-methylpropanoate hydrochloride (40a).

2-Amino-2-methylpropanoic acid (2.5 g, 24.2 mmol) was suspended in cyclohexanol (11.2 ml, 97 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 30.3 ml, 121 mmol). The mixture was heated at 65 °C for 3 days. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 200 ml) and water (200 ml). The organic phase was extracted with water (2 x 200 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 200 ml), diethyl ether (3 x 200 ml) and dichloromethane (3 x 200 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 50 mL under reduced pressure. The aqueous phase was diluted with acetonitrile (50 mL) and lyophilized to afford intermediate 40a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.60 (s, 3H), 4.83-4.78 (m, 1H), 1.83-1.73 (m, 2H), 1.73-1.61 (m, 2H), 1.58-1.43 (m, 9H), 1.43-1.18 (m, 3H).

40a 40

PMPA (100 mg, 0.384 mmol), triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol), and intermediate 40a (309 mg, 1.39 mmol) were suspended in pyridine (4 mL) under argon. Triethylamine (0.475 mL, 3.48 mmol) was added. The reaction mixture was heated at 90 °C for 16 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 mL). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with a gradient of 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (40). 1H NMR (400 MHz, methanol^) δ 8.31 (s, 1H), 8.23 (s, 1H), 4.86-4.78 (m, 1H), 4.78-4.69 (m, 1H), 4.43 (dd, J=14.5, 3.0 Hz, 1H), 4.26 (dd, J=14.5, 7.4 Hz, 1H), 4.05-3.95 (m, 1H), 3.81 (dd, J=12.8, 8.5 Hz, 1H), 3.56 (dd, J=12.7, 10.3 Hz, 1H), 1.97-1.79 (m, 4H), 1.79-1.67 (m, 4H), 1.67-1.52 (m, 6H), 1.52-1.46 (m, 8H), 1.46-1.28 (m, 10H), 1.26 (d, J=6.2 Hz, 3H). ³¹ P NMR (162 MHz, methanol-f) δ 20.88 (t, J=9.4 Hz). LCMS: MS m/z = 622.60 [M+1]; t _R = 1.278 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile

- 91 049099 acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: tR = 3.006 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 41. Di(spiro[3,3]heptan-2-yl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (41)

41a

Synthesis of spiro[3,3]heptan-2-yl 2-amino-2-methylpropanoate hydrochloride (41a).

2-Amino-2-methylpropanoic acid (250 mg, 2.42 mmol) was suspended in spiro[3,3]heptan-2-ol (0.671 ml, 4.85 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 3.03 ml, 12.1 mmol). The mixture was heated at 65 °C for 5 days. The reaction mixture was cooled and any solid was removed by filtration. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 25 ml) and water (25 ml). The organic phase was extracted with water (2 x 25 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 25 ml), diethyl ether (3 x 25 ml) and dichloromethane (3 x 25 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 20 ml under reduced pressure. The aqueous phase was diluted with acetonitrile (20 ml) and lyophilized to afford intermediate 41a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.51 (m, 3H), 4.89 (p, J = 7.1 Hz, 1H), 2.46 (m, 2H), 2.08-1.94 (m, 6H), 1.85-1.75 (m, 2H), 1.46 (s, 6H).

41a 41

PMPA (100 mg, 0.384 mmol), triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol), and intermediate 41a (326 mg, 1.39 mmol) were suspended in pyridine (4 mL) under argon. Triethylamine (0.475 mL, 3.48 mmol) was added. The reaction mixture was heated at 90 °C for 16 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 mL). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with a gradient of 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (41). 1H NMR (400 MHz, methanol^) δ 8.42 (s, 1H), 8.35 (s, 1H), 4.88-4.77 (m, 2H), 4.49 (dd, J=14.5, 3.0 Hz, 1H), 4.32 (dd, J=14.5, 7.3 Hz, 1H), 4.07-3.94 (m, 1H), 3.80 (dd, J=12.9, 8.3 Hz, 1H), 3.53 (dd, J=12.9, 10.1 Hz, 1H), 2.48 (m, 4H), 2.121.95 (m, 12H), 1.95-1.80 (m, 4H), 1.54 (s, 3H), 1.47 (m, 6H), 1.42 (s, 3H), 1.25 (d, J=6.2 Hz, 3H). ³¹ P NMR (162 MHz, methanol-dA δ 20.65 (t, J=9.3 Hz). LCMS: MS m/z = 646.55 [M+1]; tR = 1.413 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XBC18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: tR = 3.205 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 mL/min.

Example 42. Bis(3,3-dimethylcyclobutyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (42)

42a

- 92 049099

Synthesis of 3,3-dimethylcyclobutyl 2-amino-2-methylpropanoate hydrochloride (42a).

2-Amino-2-methylpropanoic acid (250 mg, 2.42 mmol) was suspended in 3,3-dimethylcyclobutanol (0.671 ml, 5.43 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 3.03 ml, 12.1 mmol). The mixture was heated at 65 °C for 5 days. The reaction mixture was cooled and any solid was removed by filtration. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 25 ml) and water (25 ml). The organic phase was extracted with water (2 x 25 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3x25 ml), diethyl ether (3x25 ml) and dichloromethane (3x25 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 20 ml under reduced pressure. The aqueous phase was diluted with acetonitrile (20 ml) and lyophilized to afford intermediate 42a. Ή NMR (400 MHz, DMSO-d ₆ ) δ 8.60 (s, 3H), 5.04 (p, J=7.1 Hz, 1H), 2.22 (m, 2H), 1.94-1.79 (m, 2H), 1.47 (m, 6H), 1.15 (s, 3H), 1.13 (s, 3H).

42a 42

PMPA (100 mg, 0.384 mmol), triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol), and intermediate 42a (309 mg, 1.39 mmol) were suspended in pyridine (4 mL) under argon. Triethylamine (0.475 mL, 3.48 mmol) was added. The reaction mixture was heated at 90 °C for 16 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 mL). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with a gradient of 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (42). 1H NMR (400 MHz, methanol^) δ 8.42 (s, 1H), 8.35 (s, 1H), 5.10-5.00 (m, 1H), 5.00-4.93 (m, 1H), 4.49 (dd, J=14.5, 3.0 Hz, 1H), 4.32 (dd, J=14.5, 7.3 Hz, 1H), 4.07-3.93 (m, 1H), 3.80 (dd, J=12.8, 8.3 Hz, 1H), 3.54 (dd, J=12.8, 10.1 Hz, 1H), 2.33-2.19 (m, 4H), 1.97-1.79 (m, 4H), 1.56 (s, 3H), 1.52-1.47 (m, 6H), 1.44 (s, 3H), 1.26 (d, J=6.2 Hz, 3H), 1.18 (m, 12H). ³¹ P NMR (162 MHz, methanol-OOO δ 20.66 (t, J=9.2 Hz). LCMS: MS m/z = 622.56 [M+1]; tR = 1.337 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: tR = 3.057 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 mL/min.

Example 43. Diisopropyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(K)-bis(2-methylpropanoate) (43)

PMPA (100 mg, 0.384 mmol), triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) and isopropyl 2-amino-2-methylpropanoate hydrochloride (341 mg, 1.88 mmol) were suspended in pyridine (4 mL) under argon. Triethylamine (0.475 mL, 3.48 mmol) was added. The reaction mixture was heated at 90 °C for 16 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 mL). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with a gradient of 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM,

- 93 049099

NX-C18, 110A 250x30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to give the title compound (43). 1H NMR (400 MHz, methanol-dA δ 8.31 (s, 1H), 8.23 (s, 1H), 5.11-5.01 (m, 1H), 5.01-4.93 (m, 1H), 4.42 (dd, J=14.5, 3.1 Hz, 1H), 4.35 (d, J=12.2 Hz, 0.18H), 4.27 (dd, J=14.5, 7.4 Hz, 1H), 4.12 (d, J=11.6 Hz, 0.28H), 4.07-3.95 (m, 1H), 3.81 (dd, J=12.8, 8.5 Hz, 1H), 3.55 (dd, J=12.8, 10.2 Hz, 1H), 1.56 (s, 3H), 1.48 (s, 3H), 1.46 (s, 3H), 1.41 (s, 3H), 1.29 (d, J=2.5 Hz, 3H), 1.28-1.23 (m, 12H). ^31P NMR (162 MHz, methanol^) δ 20.81 (t, J=9.3 Hz). LCMS: MS m/z = 542.33 [M+1]; tR = 1.000 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: t _R = 2.411 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 44. Bis(4,4-dimethylcyclohexyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(H)-bis(2-methylpropanoate) (44)

44a

Synthesis of 4,4-dimethylcyclohexyl 2-amino-2-methylpropanoate hydrochloride (44a).

2-Amino-2-methylpropanoic acid (500 mg, 2.42 mmol) was suspended in 4,4-dimethylcyclohexanol (2.15 ml, 13.6 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 6.06 ml, 24.2 mmol). The mixture was heated at 65 °C for 3 days. The reaction mixture was cooled and any solid was removed by filtration. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 50 ml) and water (50 ml). The organic phase was extracted with water (2 x 50 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3x50 ml), diethyl ether (3x50 ml) and dichloromethane (3x50 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 50 ml under reduced pressure. The aqueous phase was diluted with acetonitrile (20 ml) and lyophilized to yield intermediate 44a. 1H NMR (400 MHz, DMSO- _d6 ) δ 8.57 (s, 3H), 4.86-4.72 (m, 1H), 1.79-1.66 (m, 2H), 1.66-1.53 (m, 2H), 1.48 (s, 6H), 1.44-1.38 (m, 2H), 1.24 (m, 2H), 0.93 (s, 3H), 0.91 (s, 3H).

44a 44

PMPA (100 mg, 0.384 mmol), triphenylphosphine (341 mg, 1.36 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol), and intermediate 44a (341 mg, 1.36 mmol) were suspended in pyridine (4 mL) under argon. Triethylamine (0.475 mL, 3.48 mmol) was added. The reaction mixture was heated at 90 °C for 18 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 mL). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with a gradient of 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (44). 1H NMR (400 MHz, metyaol-dd) δ 8.31 (s, 1H), 8.23 (s, 1H), 4.86-4.77 (m, 1H), 4.77-4.68 (m, 1H), 4.43 (dd, J=14.5, 3.0 Hz, 1H), 4.26 (dd, J=14.5, 7.4 Hz, 1H), 4.05-3.94 (m, 1H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.56 (dd, J=12.8, 10.3 Hz, 1H), 1.87-1.70 (m, 4H), 1.70-1.60 (m, 4H), 1.58 (s, 3H), 1.55-1.45 (m, 10H), 1.43 (s, 3H), 1.38-1.28 (m, 4H), 1.26 (d, J=6.3 Hz, 3H), 1.01-0.92 (m, 12H). ³¹ P NMR (162 MHz, methanol-d^ δ 20.85 (t, J=9.4 Hz). LCMS: MS m/z = 678.61 [M+1]; t _R = 1.512 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonite

- 94 049099 ril, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: tR = 3.507 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; Gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 mL/min.

Example 45. Bis(cyclopentylmethyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (45)

45a

Synthesis of cyclopentylmethyl 2-amino-2-methylpropanoate hydrochloride (45a).

2-Amino-2-methylpropanoic acid (1.0 g, 9.99 mmol) was suspended in cyclopentylmethanol (4.9 ml, 40 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 12.5 ml, 49.9 mmol). The mixture was heated at 65 °C for 3 days. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 100 ml) and water (100 ml). The organic phase was extracted with water (100 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 100 ml), diethyl ether (3 x 100 ml) and dichloromethane (3 x 100 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 50 mL under reduced pressure. The aqueous phase was diluted with acetonitrile (20 mL) and water (10 mL) and lyophilized to afford intermediate 45 a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.62 (s, 4H), 4.07 (d, J=7.0 Hz, 2H), 2.20 (m, 1H), 1.79-1.66 (m, 2H), 1.66-1.51 (m, 4H), 1.49 (s, 6H), 1.25 (m, 2H).

45a 45

PMPA (100 mg, 0.384 mmol), triphenylphosphine (344 mg, 1.39 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol) and intermediate 45a (341 mg, 1.54 mmol) were suspended in pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The reaction mixture was heated at 90 °C for 18 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (45). 1H NMR (400 MHz, methanol^ ₄ ) δ 8.30 (s, 1H), 8.23 (s, 1H), 4.42 (dd, J=14.5, 3.0 Hz, 1H), 4.26 (dd, J=14.5, 7.4 Hz, 1H), 4.14-3.94 (m, 5H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.55 (dd, J=12.8, 10.3 Hz, 1H), 2.34-2.15 (m, 2H), 1.86-1.72 (m, 4H), 1.71-1.59 (m, 6H), 1.58 (m, 4H), 1.50 (s, 3H), 1.48 (s, 3H), 1.43 (s, 3H), 1.36-1.27 (m, 3H), 1.27 (s, 3H). ³¹ P NMR (162 MHz, methanol-d) δ 20.86 (t, J=9.3 Hz). LCMS: MS m/z = 622.48 [M+1]; tR = 1.327 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: tR = 3.065 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 46. Bis(cyclohexylmethyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (46)

46a

- 95 049099

Synthesis of cyclohexylmethyl 2-amino-2-methylpropanoate hydrochloride (46a).

2-Amino-2-methylpropanoic acid (1.0 g, 9.70 mmol) was suspended in cyclobutylmethanol (4.79 ml, 34 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 12.1 ml, 48.5 mmol). The mixture was heated at 65 °C for 5 days. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 100 ml) and water (100 ml). The organic phase was extracted with water (100 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 100 ml), diethyl ether (3 x 100 ml) and dichloromethane (3 x 100 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 50 mL under reduced pressure. The aqueous phase was diluted with acetonitrile (20 mL) and water (10 mL) and lyophilized to afford intermediate 46a. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.60 (s, 3H), 3.99 (d, J=6.2 Hz, 2H), 1.68 (m, 6H), 1.49 (s, 6H), 1.31-1.07 (m, 3H), 1.07-0.88 (m, 2H).

46a 46

PMPA (100 mg, 0.384 mmol) and intermediate 46a (328 mg, 1.54 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (344 mg, 1.39 mmol) and 2,2'dipyridyl disulfide (307 mg, 1.39 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 17 h and the pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (46). 1H NMR (400 MHz, methanol^) δ 8.37 (s, 1H), 8.29 (s, 1H), 4.46 (dd, J=14.5, 3.0 Hz, 1H), 4.30 (dd, J=14.5, 7.3 Hz, 1H), 4.06-3.86 (m, 5H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.55 (dd, J=12.9, 10.2 Hz, 1H), 1.88-1.61 (m, 12H), 1.58 (s, 3H), 1.53-1.46 (m, 6H), 1.44 (s, 3H), 1.37-1.16 (m, 9H), 1.12-0.94 (m, 4H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.38 (p, J = 9.4 Hz). LCMS: MS m/z = 650.59 [M+1]; tR = 1.430 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XBC18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.85 min 10-100% acetonitrile, 1.85-2.14 min 100% acetonitrile, 2.14-2.15 min 100-10% acetonitrile, 2.15-2.25 min 10% acetonitrile at 1.8 mL/min. HPLC: tR = 3.351 min; HPLC system: Agilent 1100 Series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 47. Dicyclobutyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^)-bis(2-methylpropanoate) (47) hydrogen chloride H ₂ N^° ^{H +} HO^ ^Dioxane

ABOUT

Synthesis of cyclobutyl 2-amino-2-methylpropanoate hydrochloride (47a).

2-Amino-2-methylpropanoic acid (3.0 g, 29.1 mmol) was suspended in cyclobutanol (20.7 ml, 233 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 36.3 ml, 145 mmol). The mixture was heated at 65 °C for 24 h. The reaction mixture was cooled and any solid was removed by filtration. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 100 ml) and water (100 ml). The organic phase was extracted with water (2 x 100 ml). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 100 ml), diethyl ether (3 x 100 ml) and dichloromethane (3 x 100 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 50 mL under reduced pressure. The aqueous phase was diluted with acetonitrile (20 mL) and lyophilized to afford intermediate 47a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.65 (s, 3H), 5.06-4.94 (m, 1H), 2.39-2.25 (m, 2H), 2.17-2.01 (m, 2H), 1.85-1.71 (m, 1H), 1.71-1.59 (m, 1H), 1.48 (s, 6H).

- 96 049099

PMPA (200 mg, 0.696 mmol) and intermediate 47a (547 mg, 3.48 mmol) were azeotroped with toluene (2 x 10 mL). Triphenylphosphine (688 mg, 2.79 mmol) and 2,2'dipyridyl disulfide (614 mg, 2.79 mmol) were added, followed by pyridine (4 mL) under argon. Triethylamine (0.950 mL, 7.05 mmol) was added. The mixture was heated at 90 °C for 18 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 mL). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (47). 1H NMR (400 MHz, methanol^) δ 8.27 (s, 1H), 8.23 (s, 1H), 5.07-4.99 (m, 1H), 4.99-4.91 (m, 1H), 4.41 (dd, J=14.5, 3.1 Hz, 1H), 4.32 (d, J=12.0 Hz, 0.87H), 4.26 (dd, J=14.5, 7.5 Hz, 1H), 4.07 (d, J=11.4 Hz, 0.92H), 4.03-3.93 (m, 1H), 3.79 (dd, J=12.8, 8.4 Hz, 1H), 3.52 (dd, J=12.8, 10.1 Hz, 1H), 2.44-2.29 (m, 4H), 2.19-1.99 (m, 4H), 1.91-1.76 (m, 2H), 1.76-1.60 (m, 2H), 1.56 (s, 3H), 1.48 (s, 3H), 1.46 (s, 3H), 1.43 (s, 3H), 1.25 (d, J=6.2 Hz, 3H). ^31P NMR (162 MHz, methanol^) δ 20.82 (t, J=10.4, 9.5 Hz). LCMS: MS m/z = 566.21 [M+1]; tR = 1.253 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 2.468 min; HPLC system: Agilent 1100 Series; Column: Gemini 5 μm C18 110Α, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 48. Bis(bicyclo[2.2.2]octan-1-ylmethyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azandiyl))(^)-bis(2-methylpropanoate) (48) __ 1. EDCI, DMAP, dichloromethane \| [\| xj /~Ζ\ ² · ^4Ν NS/dioxane, dichloromethane q| μ

BoeHN>Y ^OH _HO ^V7 ------------------ J

ABOUT

48a

Synthesis of bicyclo[2,2,2]octan-1-ylmethyl 2-amino-2-methylpropanoate hydrochloride (48a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.0 g, 4.92 mmol), 1-bicyclo[2,2,2]octanylmethanol (875 mg, 6.24 mmol), 4-dimethylaminopyridine (1.8 g, 14.8 mmol) and 3-(ethyliminomethyleneamino)-Na-d-dimethylpropan-1-amine hydrochloride (2.8 g mg, 14.8 mmol) were dissolved in dichloromethane (10 mL). After 16 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3 x 10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 6.2 ml, 24.6 mmol). After 90 min, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to afford intermediate 48a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.56 (s, 3H), 3.79 (s, 2H), 1.61-1.50 (m, 7H), 1.49 (s, 6H), 1.42-1.34 (m, 6H).

PMPA (100 mg, 0.348 mmol) and intermediate 48a (328 mg, 1.25 mmol) were azeotroped with toluene (2x10 mL). Triphenylphosphine (344 mg, 1.39 mmol) and 2,2'dipyridyl disulfide (307 mg, 1.39 mmol) were added, followed by pyridine (4 mL) under argon. Three

- 97 049099 ethylamine (0.475 mL, 3.48 mmol). The mixture was heated at 90 °C for 16 h. The pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 mL). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material retained on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were combined and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250x30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were combined and lyophilized to give the title compound (48). 1H NMR (400 MHz, methanol^) δ 8.29 (s, 1H), 8.23 (s, 1H), 4.42 (dd, J=14.5, 3.0 Hz, 1H), 4.36 (d, J=12.1 Hz, 0.29H), 4.26 (dd, J=14.5, 7.3 Hz, 1H), 4.12 (d, J=11.5 Hz, 0.37H), 4.03-3.92 (m, 1H), 3.863.64 (m, 6H), 3.56 (dd, J=12.8, 10.3 Hz, 1H), 1.69-1.53 (m, 17H), 1.49 (d, J=5.2 Hz, 6H), 1.48-1.37 (m, 15H), 1.25 (d, J=6.1 Hz, 3H). ³¹ P NMR (162 MHz, methanol^) δ 20.84 (t, J=10.5, 9.6 Hz). LCMS: MS m/z = 702.25 [M+1]; tR = 1.680 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 3.582 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 49. Bis(3,3-diethylcyclobutyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azandiyl))(^-bis(2-methylpropanoate) (49)

1. EDCI, DMAP, dichloromethane 2.4N НС/dioxane, dichloromethane

ABOUT

49a

Synthesis of 3,3-diethylcyclobutyl 2-amino-2-methylpropanoate hydrochloride (49a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.0 g, 4.92 mmol), 3,3-diethylcyclobutanol (875 mg, 6.83 mmol), 4-dimethylaminopyridine (1.8 g, 14.8 mmol), and 3-ethyliminomethyleneamino)-N-dimethylpropan-1-amine hydrochloride (2830 mg, 14.8 mmol) were dissolved in dichloromethane (10 mL). After 18 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3-10 mL), saturated ammonium chloride solution (10 mL), and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 mL) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 6.2 mL, 24.6 mmol). After 90 min, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 mL). The residue was subjected to high vacuum for 5 h to afford intermediate 49a. 1H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 3H), 5.07-4.88 (m, 1H), 2.26-2.14 (m, 2H), 1.85-1.71 (m, 2H), 1.47 (s, 6H), 1.46-1.40 (m, 4H), 0.80-0.73 (m, 6H).

49a 49

PMPA (100 mg, 0.348 mmol) and intermediate 49a (366 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 174 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 16 h and the pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (49). 1H NMR (400 MHz, methanol-Od) δ 8.28 (s, 1H), 8.23 (s, 1H), 5.06-4.95 (m, 1H), 4.95-4.90 (m, 1H), 4.42 (dd, J=14.5, 3.1 Hz, 1H), 4.36 (d, J=12.1 Hz, 0.06H) 4.26 (dd, J=14.5, 7.3 Hz, 1H), 4.12 (d, J=11.5 Hz, 0.06H), 4.05-3.92 (m, 1H), 3.80 (dd, J=12.8, 8.4 Hz, 1H), 3.53 (dd, J=12.8, 10.2 Hz, 1H), 2.32-2.16 (m, 4H),

- 98 049099

1.85-1.67 (m, 4H), 1.57 (s, 3H), 1.54-1.43 (m, 14H), 1.42 (s, 3H), 1.24 (d, J=6.2 Hz, 3H), 0.90-0.81 (m, 6H), 0.81-0.72 (m, 6H). ^31P NMR (162 MHz, methanol-^) δ 20.75 (t, J=9.2 Hz, 1P). LCMS: MS m/z = 678.18 [M+1]; t _R = 1.600 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 3.470 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 50. Bis(spiro[3,3]heptan-2-ylmethyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azandiyl))^)-bis(2-methylpropanoate) (50)

1. EDCI, DMAP, dichloromethane।

I y\ /\ 2.4N HC/dioxane, dichloromethane si n A/ '

Ж/ОН + ~ CIH ₃ N^Y

BocHN HOθ

ABOUT

50a

Synthesis of spiro[3,3]heptan-2-ylmethyl 2-amino-2-methylpropanoate hydrochloride (50a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.5 g, 7.38 mmol), spiro[3,3]heptan-2-ylmethanol (1313 mg, 10.4 mmol), 4-dimethylaminopyridine (2.7 g, 22.1 mmol) and 3(ethyliminomethyleneamino)-^^-dimethylpropan-1-amine hydrochloride (4.25 g, 22.1 mmol) were dissolved in dichloromethane (10 mL). After 90 min, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3-10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 9.2 ml, 36.9 mmol). After 90 min, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to yield intermediate 50a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.59 (s, 3H), 4.12 (d, J=6.6 Hz, 2H), 2.44 (m, 1H), 2.10-2.01 (m, 2H), 2.01-1.96 (m, 2H), 1.92-1.84 (m, 2H), 1.81-1.70 (m, 4H), 1.48 (s, 6H).

50a 50

PMPA (100 mg, 0.348 mmol) and intermediate 50a (363 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 16 h and the pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (50). 1H NMR (400 MHz, methanol^) δ 8.28 (s, 1H), 8.23 (s, 1H), 4.42 (dd, J=14.5, 3.1 Hz, 1H), 4.26 (dd, J=14.5, 7.3 Hz, 1H), 4.19-3.92 (m, 5H), 3.80 (dd, J=12.8, 8.4 Hz, 1H), 3.54 (dd, J=12.8, 10.2 Hz, 1H), 2.59-2.36 (m, 2H), 2.18-1.97 (m, 8H), 1.97-1.87 (m, 4H), 1.87-1.70 (m, 8H), 1.57 (s, 3H), 1.49 (s, 3H), 1.47 (s, 3H), 1.42 (s, 3H), 1.24 (d, J=6.2 Hz, 3H). ³¹ P NMR (162 MHz, methanol-^) δ 20.72 (t, J=9.2 Hz). LCMS: MS m/z = 674.19 [M+1]; t _R = 1.600 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 3.41 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1%

- 99 049099

TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 51. Bis((^^)-3-(tert-butyl)cyclobutyl) 2,2'-((((((R)-1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (51)

1. EDCI, DMAP, dichloromethane

2. 4N HC/dioxane, dichloromethane

51a

Synthesis of (^^)-3-(tert-butyl)cyclobutyl 2-amino-2-methylpropanoate hydrochloride (51a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.0 g, 4.92 mmol), (^^)-3-(tert-butyl)cyclobutan-1-ol (875 mg, 6.83 mmol), 4-dimethylaminopyridine (1.8 g, 14.8 mmol) and 3-(ethylimethyleneamino)-N,N-dimethylpropan-1-amine hydrochloride (2.8 g, 14.8 mmol) were dissolved in dichloromethane (10 mL). After 45 min, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3 x 10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 6.1 ml, 24.6 mmol). After 1 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to afford intermediate 51a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.46 (s, 3H), 4.84 (p, J=7.4 Hz, 1H), 2.25 (m, 2H), 1.87-1.65 (m, 3H), 1.46 (s, 6H), 0.82 (s, 9H).

51a 51

PMPA (100 mg, 0.348 mmol) and intermediate 51a (366 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 21 h and the pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (51). 1H NMR (400 MHz, methanol^ ₄ ) δ 8.29 (s, 1H), 8.24 (s, 1H), 7.08 (s, 0.10H), 4.87-4.80 (m, 1H), 4.80-4.71 (m, 1H), 4.42 (dd, J=14.5, 3.1 Hz, 1H), 4.35 (d, J=12.0 Hz, 0.07H), 4.26 (dd, J=14.5, 7.4 Hz, 1H), 4.11 (d, J=11.4 Hz, 0.12H), 4.03-3.93 (m, 1H), 3.80 (dd, J=12.9, 8.4 Hz, 1H), 3.53 (dd, J=12.8, 10.1 Hz, 1H), 2.38-2.18 (m, 4H), 1.89-1.68 (m, 6H), 1.57 (s, 3H), 1.48 (s, 3H), 1.46 (s, 3H), 1.41 (s, 3H), 1.24 (d, J=6.2 Hz, 3H), 0.85 (s, 9H), 0.84 (s, 9H). ^31P NMR (162 MHz, methanol- ₄ ) δ 20.71 (t, J=9.2 Hz). LCMS: MS m/z = 678.18 [M+1]; t _R = 1.647 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 3.504 min; HPLC system: Agilent 1100 Series; Column: Gemini 5 μm C18 110Α, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 52. Bis((1R,3R)-3-(tert-butyl)cyclobutyl) 2,2'-((((((R)-1-(6-amino-9H-purin-9-yl)propan2-yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (52) __ _L OH 1. EDCI, DMAP, dichloromethane I . I /7 2.4N HC/dioxane, dichloromethane %% LH, _

X >OH + ----------------------* CIH ₃ N y 'GP

BocHN 0 ⁰

52a

Synthesis of (1R,3R)-3-(tert-butyl)cyclobutyl 2-amino-2-methylpropanoate hydrochloride (52a).

- 100 049099

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.5 g, 7.38 mmol), (1R,3R)-3-(tert-butyl)cyclobutan-1-ol (1.14 mg, 8.86 mmol), 4-dimethylaminopyridine (2.7 g, 22.1 mmol) and 3-(ethylimethyleneamino)-X,X-dimethylpropan-1-amine hydrochloride (4.2 g, 22.1 mmol) were dissolved in dichloromethane (10 mL). After 3 days, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3-10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 9.2 ml, 36.9 mmol). After 90 min, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to afford intermediate 52a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.50 (s, 3H), 5.02-4.88 (m, 1H), 2.35-2.18 (m, 3H), 2.15-1.97 (m, 2H), 1.48 (s, 6H), 0.83 (s, 9H).

PMPA (100 mg, 0.348 mmol) and intermediate 52a (366 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 21 h and the pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (52). 1H NMR (400 MHz, methanol- _b4 ) δ 8.28 (s, 1H), 8.23 (s, 1H), 4.99-4.94 (m, 1H), 4.94-4.89 (m, 1H), 4.47-4.35 (m, 1.32H), 4.26 (dd, J=14.5, 7.3 Hz, 1H), 4.16-4.12 (m, 0.41H), 4.04-3.95 (m, 1H), 3.86-3.76 (m, 1H), 3.62-3.49 (m, 1H), 2.34-2.19 (m, 6H), 2.18-1.99 (m, 4H), 1.59 (s, 3H), 1.51 (s, 3H), 1.49 (s, 3H), 1.44 (s, 3H), 1.25 (d, J=6.2 Hz, 3H), 0.87 (s, 9H), 0.86 (s, 9H). ³¹ P NMR (162 MHz, methanol-bd) δ 20.75 (t, J=9.4 Hz). LCMS: MS m/z = 678.14 [M+1]; t _R = 1.627 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 3.468 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 53. Bis(spiro[3,5]nonan-7-ylmethyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (53)

it

1. EDCI, OMAR, dichloromethane OH 2. 4N HC/dioxane, dichloromethane

53a

Synthesis of spiro[3,5]nonan-7-ylmethyl 2-amino-2-methylpropanoate hydrochloride (53a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (2.0 g, 9.84 mmol), spiro[3,5]nonan7-ylmethanol (1.75 g, 11.3 mmol), 4-dimethylaminopyridine (3.6 g, 29.5 mmol) and 3(ethyliminomethyleneamino)-X,X-dimethylpropan-1-amine hydrochloride (5.65 g, 29.5 mmol) were dissolved in dichloromethane (10 mL). After several days, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3 × 10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0–40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

A solution of hydrogen chloride in 1,4-dioxane was added to a solution of the residue in dichloromethane (5 ml).

- 101 049099 (4N, 12.3 mL, 49.2 mmol). After 1 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 mL). The residue was subjected to high vacuum for 5 h to afford intermediate 53a. ¹ H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 3H), 3.99 (d, J=6.0 Hz, 2H), 1.88-1.77 (m, 2H), 1.74 (m, 2H), 1.70-1.62 (m, 4H), 1.62-1.50 (m, 3H), 1.48 (s, 6H), 1.22 (m, 2H), 1.00 (m, 2H).

53a 53

PMPA (100 mg, 0.348 mmol) and intermediate 53a (404 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 21 h and the pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to give the title compound (53). 1H NMR (400 MHz, methanol^) δ 8.29 (s, 1H), 8.23 (s, 1H), 4.48-4.35 (m, 1.54H), 4.26 (dd, J=14.5, 7.2 Hz, 1H), 4.16 (dd, J=11.6, 1.5 Hz, 0.46H), 4.06-3.92 (m, 4H), 3.92-3.85 (m, 1H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.55 (dd, J=12.8, 10.2 Hz, 1H), 1.94-1.82 (m, 4H), 1.82-1.65 (m, 11H), 1.65-1.52 (m, 10H), 1.50 (s, 3H), 1.49 (s, 3H), 1.43 (s, 3H), 1.35-1.15 (m, 7H), 1.15-0.97 (m, 4H). ^31P NMR (162 MHz, methanol-^) δ 20.77 (t, J=10.3, 9.7 Hz). LCMS: MS m/z = 730.2 [M+1]; tR = 1.800 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 3.955 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 54. Bis(3,3-dimethylpentyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(K)-bis(2-methylpropanoate) (54). I hydrogen chloride I

H ₂ ->V ^OH - ^he D=----.

o O ^/x

54a

Synthesis of 3,3-dimethylpentyl 2-amino-2-methylpropanoate hydrochloride (54a).

2-Amino-2-methylpropanoic acid (0.50 g, 4.85 mmol) was suspended in 3,3-dimethylpentan-1ol (2.08 mL, 14.5 mmol) and a solution of hydrogen chloride in 1,4-dioxane (4N, 6.06 mL, 24.2 mmol). The mixture was heated at 65 °C for 3 days. The reaction mixture was cooled and any solid was removed by filtration. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 100 mL) and water (100 mL). The organic phase was extracted with water (2 x 100 mL). The combined aqueous phases were washed with ethyl acetate/hexane (1:1, 3 x 100 ml), diethyl ether (3 x 100 ml) and dichloromethane (3 x 100 ml). Any residual solvent in the aqueous phase was removed and the volume was reduced to 10 ml under reduced pressure. The aqueous phase was diluted with acetonitrile (8 ml) and lyophilized to yield intermediate 54a. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.57 (m, 3H), 4.21 (t, J=7.3 Hz, 2H), 1.54 (t, J=7.3 Hz, 2H), 1.47 (s, 6H), 1.24 (k, J=7.5 Hz, 2H), 0.87 (s, 6H), 0.81 (t, J=7.5 Hz, 3H).

- 102 049099

PMPA (100 mg, 0.348 mmol) and intermediate 54a (349 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 20 h and the pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (54). 1H NMR (400 MHz, methanol^) δ 8.29 (s, 1H), 8.23 (s, 1H), 4.42 (dd, J=14.5, 3.1 Hz, 1H), 4.33-4.08 (m, 5H), 4.05-3.94 (m, 1H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.55 (dd, J=12.8, 10.1 Hz, 1H), 1.65-1.53 (m, 7H), 1.49 (d, J=4.8 Hz, 6H), 1.42 (s, 3H), 1.35-1.27 (m, 4H), 1.25 (d, J=6.3 Hz, 3H), 0.92 (s, 6H), 0.91 (s, 6H), 0.90-0.82 (m, 6H). ³¹ P NMR (162 MHz, methanol-d^ δ 20.80 (t, J=9.2 Hz). LCMS: MS m/z = 654.19 [M+1]; tR = 1.617 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 3.416 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 mL/min.

Example 55. Bis((^)-1-cyclohexylethyl) 2,2'-(((((((^)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (55)

1. EDCI, DMAP, dichloromethane ।

I 2.4N HC/dioxane, dichloromethane

BOCHN^Y ⁰⁴ ' A - ^CIH ' ^N J T

55a

Synthesis of (K)-1-cyclohexylethyl 2-amino-2-methylpropanoate hydrochloride (55 a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.0 g, 4.92 mmol), (R)-1-cyclohexylethane-1-ol (757 mg, 5.90 mmol), 4-dimethylaminopyridine (1.8 g, 14.8 mmol), and 3-ethyliminomethyleneamino)-N-dimethylpropan-1-amine hydrochloride (2.8 g, 14.8 mmol) were dissolved in dichloromethane (10 mL). After 19 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3-10 mL), saturated ammonium chloride solution (10 mL), and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 6.1 ml, 24.6 mmol). After 2 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to afford intermediate 55a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.46 (s, 3H), 4.74 (p, J=6.3 Hz, 1H), 1.81-1.69 (m, 4H), 1.69-1.54 (m, 3H), 1.47 (m, 7H), 1.29-1.11 (m, 4H), 1.11-0.83 (m, 2H).

55a 55

PMPA (100 mg, 0.348 mmol) and intermediate 55a (366 mg, 1.39 mmol) were subjected to

- 103 049099 was azeotroped with toluene (2 x 10 mL). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 mL) under argon. Triethylamine (0.475 mL, 3.48 mmol) was added. The mixture was heated at 90 °C for 24 h and the pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 mL). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material retained on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). The product containing fractions were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250x30 mm column, gradient 40-100% acetonitrile/water over 20 min). The product containing fractions were pooled and lyophilized to give the title compound (55). 1H NMR (400 MHz, methanol-d^ δ 8.32 (s, 1H), 8.23 (s, 1H), 4.78 (p, J=6.4 Hz, 1H), 4.70 (p, J=6.3 Hz, 1H), 4.48-4.35 (m, 1.56H), 4.27 (dd, J=14.5, 7.2 Hz, 1H), 4.16 (dd, J=11.4, 1.2 Hz, 0.62H), 4.09-3.94 (m, 1H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.58 (dd, J=12.8, 10.2 Hz, 1H), 1.90-1.63 (m, 11H), 1.60 (s, 3H), 1.58-1.40 (m, 11H), 1.36-1.13 (m, 14H), 1.13-0.93 (m, 4H). ^31P NMR (162 MHz, methanol-d^ δ 20.74 (t, J=10.4, 9.8 Hz). LCMS: MS m/z = 678.21 [M+1]; tR = 1.650 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 3.534 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110Α, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 982% B at 2 ml/min.

Example 56. Bis(^)-1-cyclohexylethyl) 2,2'-(((((((((R.)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (56)

1. EDCI, DMAP, dichloromethane ι p|

2.4N HC/dioxane, dichloromethane 'sL η 1 J

----------------------CIH ₃ N'^'

About ^Ξ

56a

Synthesis of (K)-1-cyclohexylethyl 2-amino-2-methylpropanoate hydrochloride (56a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.0 g, 4.92 mmol), (S)-1-cyclohexyl-ethan-1-ol (757 mg, 5.90 mmol), 4-dimethylaminopyridine (1.8 g, 14.8 mmol) and 3-ethyliminomethyleneamino)-1-N-dimethylpropan-1-amine hydrochloride (2.8 g, 14.8 mmol) were dissolved in dichloromethane (10 mL). After 19 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3 x 10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 6.1 ml, 24.6 mmol). After 2 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to yield intermediate 56a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.44 (s, 3H), 4.74 (p, J=6.3 Hz, 1H), 1.81-1.67 (m, 4H), 1.67-1.55 (m, 3H), 1.55-1.35 (m, 7H), 1.31-1.11 (m, 4H), 1.11-0.83 (m, 2H).

56a 56

PMPA (100 mg, 0.348 mmol) and intermediate 56a (366 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 24 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250x30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fraction

- 104 049099 product-containing residues were combined and lyophilized to give the title compound (56). 1H NMR (400 MHz, methanol^) δ 8.31 (s, 1H), 8.23 (s, 1H), 4.82-4.62 (m, 2H), 4.47-4.36 (m, 1.71H), 4.27 (dd, J=14.5, 7.2 Hz, 1H), 4.18 (d, J=11.8 Hz, 0.83H), 4.07-3.93 (m, 1H), 3.81 (dd, J=12.8, 8.7 Hz, 1H), 3.56 (dd, J=12.8, 9.9 Hz, 1H), 1.89-1.61 (m, 11H), 1.57 (s, 3H), 1.55-1.45 (m, 7H), 1.43 (s, 3H), 1.33-1.13 (m, 14H), 1.13-0.90 (m, 5H). ^31P NMR (162 MHz, methanol^) δ 20.71 (p, J=10.5, 9.7 Hz). LCMS: MS m/z = 678.20 [M+1]; tR = 1.657 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 3.527 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 57. Bis(dispiro[3,1,3 ⁶ ,1 ⁴ ]decan-2-ylmethyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (57)

57a

Synthesis of dispiro[3.1.36.14]decane-2-ylmethyl 2-amino-2-methylpropanoate hydrochloride (57a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.5 g, 7.38 mmol), dispiro[3,1,36,14]decan-2-ylmethanol (1.47 g, 8.86 mmol), 4-dimethylaminopyridine (2.7 g, 22.1 mmol) and 3-(ethyliminomethyleneamino)-N-dimethylpropan-1-amine hydrochloride (4.2 g, 22.1 mmol) were dissolved in dichloromethane (10 mL). After 16 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3 x 10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 9.2 ml, 36.9 mmol). After 1 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to afford intermediate 57a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.52 (s, 3H), 4.11 (d, J=6.6 Hz, 2H), 2.50-2.41 (m, 1H), 2.06-1.95 (m, 4H), 1.95-1.83 (m, 6H), 1.81-1.70 (m, 4H), 1.47 (s, 6H).

57a 57

PMPA (100 mg, 0.348 mmol) and intermediate 57a (422 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 18 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (57). 1H NMR (400 MHz, methanol^) δ 8.29 (s, 1H), 8.23 (s, 1H), 4.42 (dd, J=14.5, 3.1 Hz, 1H), 4.27 (dd, J=14.5, 7.3 Hz, 1H), 4.17-3.92 (m, 5H), 3.80 (dd, J=12.8, 8.4 Hz, 1H), 3.54 (dd, J=12.8, 10.2 Hz, 1H), 2.56-2.42 (m, 2H), 2.12-1.97 (m, 8H), 1.97-1.85 (m, 12H), 1.85-1.70 (m, 8H), 1.57 (s, 3H), 1.49 (s, 3H), 1.47 (s, 3H), 1.42 (s, 3H), 1.24 (d, J=6.2 Hz, 3H). ^31P NMR (162 MHz, methanol^) δ 20.73 (t, J=9.2 Hz). LCMS: MS m/z = 754.23 [M+1]; tR = 1.880 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing;

- 105 049099 MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 4.210 min; HPLC system: Agilent 1100 Series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 58. Bis(6,6-difluorospiro[3,3]heptan-2-yl)methyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(H)-bis(2-methylpropanoate) (58)

Synthesis of 6,6-difluorospiro[3,3]heptan-2-yl)methyl 2-amino-2-methylpropanoate hydrochloride (58a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.5 g, 7.38 mmol), (2,2-difluorospiro[3,3]heptan-6-yl)methanol (1.47 g, 9.08 mmol), 4-dimethylaminopyridine (2.7 g, 22.1 mmol) and 3-(ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine hydrochloride (4.2 g, 22.1 mmol) were dissolved in dichloromethane (10 mL). After 16 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3 x 10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 9.2 ml, 36.9 mmol). After 1 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to afford intermediate 58a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.57 (s, 3H), 4.14 (d, J=6.5 Hz, 2H), 2.75-2.58 (m, 2H), 2.58-2.52 (m, 3H), 2.23-2.10 (m, 2H), 2.03-1.91 (m, 2H), 1.48 (s, 6H). ¹⁹ P NMR (376 MHz, DMSO-d6) δ -89.58 (p, J=12.6 Hz).

F F

58a 58

PMPA (100 mg, 0.348 mmol) and intermediate 58a (416 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 mL). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 mL) under argon. Triethylamine (0.475 mL, 3.48 mmol) was added. The mixture was heated at 90 °C for 18 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 mL). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (58). 1H NMR (400 MHz, methanol^) δ 8.27 (s, 1H), 8.24 (s, 1H), 4.42 (dd, J=14.5, 3.1 Hz, 1H), 4.26 (dd, J=14.5, 7.4 Hz, 1H), 4.22-4.02 (m, 4H), 4.02-3.93 (m, 1H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.54 (dd, J=12.8, 10.2 Hz, 1H), 2.69-2.54 (m, 6H), 2.54-2.41 (m, 4H), 2.27-2.12 (m, 4H), 2.04-1.86 (m, 4H), 1.57 (s, 3H), 1.50 (s, 3H), 1.47 (s, 3H), 1.42 (s, 3H), 1.25 (d, J=6.2 Hz, 3H). ³¹ P NMR (162 MHz, methanol-^) δ 20.77 (t, J=9.4 Hz). ¹⁹ P NMR (376 MHz, methanol-f) δ -92.77 (dd, J=12.4, 4.8 Hz). LCMS: MS m/z = 746.23 [M+1]; tR = 1.467 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 3.072 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A

- 106 049099 acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 59. Bis(((^^)-adamantan-1-yl)methyl) 2,2'-(((((((R)-1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azandiyl))bis(2-methylpropanoate) (59)

O

1. EDCI, DMAP, dichloromethane

2. 4N HC/dioxane, dichloromethane

SP o

59a

Synthesis of 1-adamantylmethyl 2-amino-2-methylpropanoate hydrochloride (59a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (2.0 g, 9.84 mmol), 1-adamantimethanol (1.96 g, 11.8 mmol), 4-dimethylaminopyridine (3.6 g, 29.5 mmol) and 3-ethyliminomethyleneamino)-1-N-dimethylpropan-1-amine hydrochloride (5.6 g, 29.5 mmol) were dissolved in dichloromethane (10 mL). After 1 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3 x 10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 12.3 ml, 49.2 mmol). After 1 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to afford intermediate 59a. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.54 (s, 3H), 3.78 (s, 2H), 2.00-1.91 (m, 3H), 1.77-1.60 (m, 6H), 1.53 (m, 6H), 1.50 (s, 6H).

NH _Z

nh ₂ , W triphenylphosphine

2,2'-dipyridyl disulfide cih ₃ n y o

59a

PMPA (100 mg, 0.348 mmol) and intermediate 59a (416 mg, 1.37 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 18 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (59). 1H NMR (400 MHz, methanol-dA δ 8.30 (s, 1H), 8.23 (s, 1H), 4.48-4.36 (m, 1.34H), 4.26 (dd, J=14.5, 7.2 Hz, 1H), 4.16 (d, J=11.5 Hz, 0.42H), 4.06-3.95 (m, 1H), 3.88-3.63 (m, 5H), 3.57 (dd, J=12.7, 10.3 Hz, 1H), 2.03-1.92 (m, 6H), 1.85-1.74 (m, 6H), 1.74-1.64 (m, 6H), 1.63-1.58 (m, 9H), 1.58-1.54 (m, 6H), 1.54-1.49 (m, 6H), 1.46 (s, 3H), 1.24 (d, J=6.2 Hz, 3H). ³¹ P NMR (162 MHz, methanol^) δ 20.78 (t, J=9.8 Hz). LCMS: MS m/z = 754.33 [M+1]; t _R = 1.825 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 3.948 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 60. Di((1R,3R)5(S-bicyclo[3,1,0]hexan-3-yl)2,2'-((((((R)-1-(6-amino-9H-purin-9yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (60)

It

OH g- 1. EDCI, OMAR, dichloromethane \ 2. 4N HC/dioxane, dichloromethane

CIH ₃ I o

60a

- 107 049099

Synthesis of (1R,3R,5S)-bicyclo[3,1,0]hexan-3-yl 2-amino-2-methylpropanoate hydrochloride (60a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (5.0 g, 24.6 mmol), (1R,5S)bicyclo[3,1,0]hexan-3-ol (2.89 g, 29.5 mmol), 4-dimethylaminopyridine (6.01 g, 29.5 mmol) and 3-(ethyliminomethyleneamino)-N-dimethylpropan-1-amine hydrochloride (9.4 g, 49.2 mmol) were dissolved in dichloromethane (20 mL). After 2 h, the reaction mixture was diluted with DCM (20 mL). The solution was washed with water (3 x 20 mL), saturated ammonium chloride solution (20 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 15.3 ml, 61.0 mmol). After 90 min, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to give intermediate 60a, which was used in the next reaction.

PMPA (100 mg, 0.348 mmol) and intermediate 60a (322 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 24 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (60). 1H NMR (400 MHz, methanol^) δ 8.28 (s, 1H), 8.23 (s, 1H), 5.26 (t, J=6.8 Hz, 1H), 5.19 (t, J=6.8 Hz, 1H), 4.42 (dd, J=14.5, 3.1 Hz, 1H), 4.26 (dd, J=14.5, 7.4 Hz, 1H), 4.04-3.94 (m, 1H), 3.80 (dd, J=12.8, 8.5 Hz, 1H), 3.54 (dd, J=12.8, 10.2 Hz, 1H), 2.32-2.12 (m, 5H), 1.90-1.77 (m, 4H), 1.53 (s, 3H), 1.45 (s, 3H), 1.43 (s, 4H), 1.38 (s, 3H), 1.37-1.29 (m, 3H), 1.25 (d, J=6.2 Hz, 3H), 0.58-0.46 (m, 2H), 0.38-0.34 (m, 1H), 0.330.30 (m, 1H). ³¹ P NMR (162 MHz, methanol-d^ δ 20.90 (t, J=9.4 Hz). LCMS: MS m/z = 618.09 [M+1]; t _R =1.360 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 2.756 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 mL/min.

Example 61. Di(spiro[4,5]decan-8-yl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))^)-bis(2-methylpropanoate) (61)

1. EDCI, DMAP, DCM IYI ^NH 2 /gh/ ⁰

2. HCI/dioxane, DCM HCI

61a

Synthesis of spiro[4,5]decan-8-yl 2-amino-2-methylpropanoate hydrochloride (61a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.0 g, 4.92 mmol), spiro[4,5]decan-8-ol (835 mg, 5.41 mmol), 4-dimethylaminopyridine (1.8 g, 14.8 mmol) and 3-ethyliminomethyleneamino)-N-dimethylpropan-1-amine hydrochloride (2.8 g, 14.8 mmol) were dissolved in dichloromethane (10 mL). After 20 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3 x 10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 6.1 ml, 24.6 mmol). After 1 h, the solvent was removed under reduced pressure. The residue was evaporated

- 108 049099 or together with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to give intermediate 61a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.48 (s, 3H), 4.81 (m, 1H), 1.75 (m, 2H), 1.63-1.48 (m, 7H), 1.47 (s, 6H), 1.43-1.27 (m, 7H).

PMPA (100 mg, 0.348 mmol) and intermediate 61a (404 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 24 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (61). 1H NMR (400 MHz, methanol-d^δ 8.30 (s, 1H), 8.23 (s, 1H), 4.85-4.77 (m, 1H), 4.77-4.67 (m, 1H), 4.42 (dd, J=14.5, 3.0 Hz, 1H), 4.37 (d, J=12.0 Hz, 0.83H), 4.27 (dd, J=14.5, 7.3 Hz, 1H), 4.13 (d, J=11.4 Hz, 0.85H), 4.03-3.96 (m, 1H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.57 (dd, J=12.8, 10.2 Hz, 1H), 1.87-1.69 (m, 4H), 1.69-1.51 (m, 18H), 1.51-1.48 (m, 6H), 1.48-1.28 (m, 16H), 1.25 (d, J=6.2 Hz, 3H). ^31P NMR (162 MHz, methanol-bd) δ 20.80 (q, J=10.4 Hz). LCMS: MS m/z = 730.20 [M+1]; tR = 1.790 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 3.823 min; HPLC system: Agilent 1100 series; column: Gemini 5 μmC18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 62. Bis(2-(bicyclo[1,1,1]pentan-1-yl)ethyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azandiyl))^)-bis(2-methylpropanoate) (62)

1.EDCI,DMAP,DCM

---------------► ΟΙΉβΝ ξ

2. HCI/dioxane, DCM O

ABOUT

62a

Synthesis of 2-(bicyclo[1,1,1]pentan-1-yl)ethyl-2-amino-2-methylpropanoate hydrochloride (62a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.8 g, 8.86 mmol), 2-(bicyclo[1,1,1]pentan-1-yl)ethan-1-ol (993 mg, 8.86 mmol), 4-dimethylaminopyridine (2.1 g, 17.7 mmol) and 3-(ethyliminomethyleneamino)-X,X-dimethylpropan-1-amine hydrochloride (3.4 g, 17.7 mmol) were dissolved in dichloromethane (10 mL). After 90 min, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3-10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 11.1 ml, 44.3 mmol). After 90 min, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to afford intermediate 62a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.56 (s, 3H), 4.16 (t, J=6.3 Hz, 2H), 2.47 (s, 1H), 1.77 (t, J=6.2 Hz, 2H), 1.70 (s, 6H), 1.48 (s, 6H).

- 109 049099

PMPA (100 mg, 0.348 mmol) and intermediate 62a (343 mg, 1.39 mmol) were azeotroped with toluene (2 x 10 ml). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 ml) under argon. Triethylamine (0.475 ml, 3.48 mmol) was added. The mixture was heated at 90 °C for 18 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 ml). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material remaining on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to afford the title compound (62). 1H NMR (400 MHz, methanol-d^δ 8.29 (s, 1H), 8.24 (s, 1H), 4.42 (dd, J=14.5, 3.0 Hz, 1H), 4.26 (dd, J=14.5, 7.4 Hz, 1H), 4.22-4.03 (m, 4H), 4.03-3.92 (m, 1H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.55 (dd, J=12.8, 10.2 Hz, 1H), 2.46 (s, 1H), 2.45 (s, 1H), 1.84-1.76 (m, 2H), 1.75 (m, 7N), 1.73 (m, 7H), 1.57 (s, 3H), 1.49 (s, 3H), 1.48 (s, 3H), 1.42 (s, 3H), 1.25 (d, J=6.2 Hz, 3H). ^31P NMR (162 MHz, methanol^) δ 20.78 (t, J=9.2 Hz). LCMS: MS m/z = 646.11 [M+1]; tR = 1.537 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 3.115 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 982% B at 2 ml/min.

Example 63. Di(spiro[3,5]nonan-7-yl)2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(N-bis(2-methylpropanoate) (63) ₀ H - 2O° ^{H 1EDCI} ' ^DMAP ' ^DCM » CIH ₃ N^Y° BocHN'^Y' 2. HCI/dioxane, DCM O

ABOUT

63a

Synthesis of spiro[3,5]nonan-7-yl 2-amino-2-methylpropanoate hydrochloride (63a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (2.9 g, 14.3 mmol), spiro[3,5]nonan-7-ol (2.0 g, 14.3 mmol), 4-dimethylaminopyridine (2.6 g, 21.4 mmol) and 3-ethyliminomethyleneamino)-N-dimethylpropan-1-amine hydrochloride (4.1 g, 21.4 mmol) were dissolved in dichloromethane (25 mL). After 18 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3 x 10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 17.8 ml, 71.3 mmol). After 3 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to afford intermediate 63a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.50 (s, 3H), 4.77 (d, J=8.4 Hz, 1H), 1.94-1.77 (m, 2H), 1.77-1.60 (m, 8H), 1.57-1.34 (m, 10H).

63a 63

PMPA (100 mg, 0.348 mmol) and intermediate 63a (384 mg, 1.39 mmol) were subjected to

- 110 049099 azeotroped with toluene (2 x 10 mL). Triphenylphosphine (430 mg, 1.74 mmol) and 2,2'dipyridyl disulfide (384 mg, 1.74 mmol) were added, followed by pyridine (4 mL) under argon. Triethylamine (0.475 mL, 3.48 mmol) was added. The mixture was heated at 90 °C for 18 h. Pyridine was removed under reduced pressure. The residue was co-evaporated with toluene (3 x 10 mL). The residue was absorbed through a silica plug using an ISCO solid loading cartridge with 40% then 100% ethyl acetate/hexane. The material retained on the solid loading cartridge was flash chromatographed (0-20% methanol/dichloromethane). The product containing fractions were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250x30 mm column, gradient 40-100% acetonitrile/water over 20 min). The product containing fractions were pooled and lyophilized to give the title compound (63). 1H NMR (400 MHz, methanol^ ₄ ) δ 8.31 (s, 1H), 8.25 (s, 1H), 7.20 (s, 0.09H), 4.81-4.73 (m, 1H), 4.73-4.64 (m, 1H), 4.43 (dd, J=14.5, 3.0 Hz, 1H), 4.36 (d, J=12.0 Hz, 0.05H), 4.27 (dd, J=14.5, 7.3 Hz, 1H), 4.12 (d, J=11.5 Hz, 0.05H), 4.05-3.95 (m, 1H), 3.81 (dd, J=12.8, 8.4 Hz, 1H), 3.57 (dd, J=12.8, 10.2 Hz, 1H), 1.97-1.82 (m, 4H), 1.82-1.62 (m, 16H), 1.57 (s, 3H), 1.54-1.44 (m, 13H), 1.44-1.36 (m, 4H), 1.24 (d, J=6.2 Hz, 3H). ³¹ P NMR (162 MHz, methanol-d^ δ 20.74 (t, J=9.4 Hz). LCMS: MS m/z = 702.22 [M+1]; tR = 1.703 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 3.557 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110Α, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 mL/min.

Example 64. Dihexyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2,2-dicyclopropyl acetate) (64)

64a

Synthesis of hexyl 2-amino-2,2-dicyclopropyl acetate hydrochloride (64a).

2-Amino-2,2-dicyclopropylacetic acid (2 g, 10.4 mmol) and 1-hexanol (5.3 g, 52.2 mmol) were dissolved in 4N HCl in 1,4-dioxane (13 mL, 52 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature, the solids were filtered off, and the solution was concentrated under reduced pressure. The residue was dissolved in water (40 mL) and concentrated under reduced pressure, dissolved in water (40 mL), and concentrated under reduced pressure. This residue was taken up in toluene and concentrated under reduced pressure. The residue was taken up in water and lyophilized over the weekend to afford intermediate 64a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.43 (br.s, 3H), 4.16 (t, J=6.4 Hz, 2H), 1.69-1.51 (m, 2H), 1.43-1.23 (m, 5H), 1.21-1.05 (m, 3H), 0.92-0.81 (m, 3H), 0.77-0.42 (m, 8H). LCMS: MS m/z = 240.2 [M+1], tR = 0.70 min; LC system: Agilent 1260 Infinity II HPLC; MS system: single quadrupole mass detector G6124B; Column: Kinetix 2.6 µm, C18, 100Α, 50 mm x 2.1 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-1.00 min 10% 100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 µL/min.

64a 64

PMPA (100 mg, 0.35 mmol), intermediate 64a (384 mg, 1.39 mmol) and triethylamine (0.30 mL, 2.09 mmol) were combined in pyridine (1 mL) under argon and heated at 50 °C for 5 min. To the above reaction mixture was added a freshly prepared bright yellow solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL). The reaction mixture was stirred at 85 °C overnight. Then the reaction mixture was cooled to room temperature and diluted with 20 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (10 mL x 2), brine (10 mL) and dried over sodium sulfate. Hexane (0.5 volume equivalent) was added, the mixture was loaded onto a 3 cm pad of silica gel and washed with ethyl acetate, and the desired compound was eluted with 10% methanol in DCM. The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm column).

- 111 049099

C18-110A 100x30 mm, gradient 25-100% acetonitrile in water over 30 min) to give the title compound (64). ¹ H NMR (400 MHz, DMSO-d6) δ 8.17-7.97 (m, 2H), 7.15 (br.s, 2H), 4.32-4.12 (m, 2H), 4.10-3.89 (m, 5H), 3.80-3.58 (m, 4H), 1.69-1.47 (m, 4H), 1.41-1.20 (m, 14H), 1.15-0.95 (m, 5H), 0.910.77 (m, 6H), 0.71-0.56 (m, 2H), 0.55-0.20 (m, 14H). ³¹ P NMR (162 MHz, DMSO-d6) δ 19.18 (m). LCMS: MS m/z = 730.4 [M+1], tR = 1.28 min; LC system: Agilent 1260 Infinity II HPLC; MS system: G6124B single quadrupole mass detector; Column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-1.00 min 10% 100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 μL/min. HPLC: t _R = 3.73 min; HPLC system: Agilent 1100 series; column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 2-98% acetonitrile, 5.0-6.0 min 98% acetonitrile at 2 mL/min.

Example 65. Dicyclopentyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azandiyl))(^-bis(2-ethylbutanoate) (65)

65a

Synthesis of hexyl 2-amino-2-ethylbutanoate hydrochloride (65a).

2-Amino-2-ethylbutanoic acid (1.7 g, 10.1 mmol) and 1-hexanol (5.2 g, 50.7 mmol) were dissolved in 4N HCl in 1,4-dioxane (12.7 mL, 50.7 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature, the solids were filtered off, and the solution was concentrated under reduced pressure. The residue was dissolved in water (40 mL) and concentrated under reduced pressure, dissolved in water (40 mL), and concentrated under reduced pressure. This residue was taken up in toluene and concentrated under reduced pressure. The residue was taken up in water and lyophilized over the weekend to provide intermediate 65a. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.59 (br.s, 3H), 4.19 (t, J=6.5 Hz, 2H), 1.97-1.76 (m, 4H), 1.71-1.52 (m, 2H), 1.43-1.16 (m, 6H), 0.98-0.77 (m, 9H). LCMS: MS m/z = 216.2 [M+1], tR = 0.68 min; LC system: Agilent 1260 Infinity II HPLC; MS system: single-quadrupole mass detector G6124B; column: Kinetix 2.6 µm, C18, 100A, 50 mmx2.1 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 0-1.00 min 10%-100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 µl/min.

65a 65

PMPA (100 mg, 0.35 mmol), intermediate 65a (350 mg, 1.39 mmol) and triethylamine (0.30 mL, 2.09 mmol) were combined in pyridine (1 mL) under argon and heated at 50 °C for 5 min. To the above reaction mixture was added a freshly prepared bright yellow solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL). The reaction mixture was stirred at 85 °C overnight. Then the reaction mixture was cooled to room temperature and diluted with 20 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (10 mL x 2), brine (10 mL) and dried over sodium sulfate. Hexane (0.5 volume equivalent) was added, the mixture was loaded onto a 3 cm pad of silica gel and washed with ethyl acetate, and the desired compound was eluted with 10% methanol in DCM. The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (65). ^1H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H), 8.10 (s, 1H), 7.16 (br.s, 2H), 4.30-4.15 (m, 2H), 4.13-4.01 (m, 4H), 4.01-3.92 (m, 2H), 3.84 (d, J=11.0 Hz, 1H), 3.67-3.47 (m, 2H), 2.18-1.94 (m, 2H), 1.93-1.48 (m, 10H), 1.38-1.17 (m, 12H), 1.07 (d, J=6.2 Hz, 3H), 0.90-0.58 (m, 18H). ³¹ P NMR (162 MHz, DMSO-d6) δ 16.75 (m). LCMS: MS m/z = 682.4 [M+1], tR = 1.31 min; LC system: Agilent 1260 Infinity II HPLC; MS system: G6124B single quadrupole mass detector; Column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-1.00 min 10%-100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 μL/min. HPLC: t _R = 3.76 min; HPLC system: Agilent 1100 Series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0-5.0 min 2-98% acetonitrile, 5.0-6.0 min 98% acetonitrile at 2 mL/min.

- 112 049099

Example 66. Diheptyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (66)

H _? N ^> V ^0H ₊ hydrogen chloride

II οι·η ₃ ν ηρ ^u 1,4-dioxane q

66a

Synthesis of heptyl 2-amino-2-methylpropanoate hydrochloride (66a).

2-Amino-2-methylpropanoic acid (1.5 g, 10.7 mmol) and 1-heptanol (6.2 g, 53.7 mmol) were dissolved in 4N HCl in 1,4-dioxane (13.4 mL, 53.7 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature, the solids were filtered off, and the solution was concentrated under reduced pressure. The residue was dissolved in water (40 mL) and concentrated under reduced pressure, dissolved in water (40 mL), and concentrated under reduced pressure. This residue was taken up in toluene and concentrated under reduced pressure. The residue was taken up in water and lyophilized over the weekend to provide intermediate 66a. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.53 (s, 3H), 4.16 (t, J=6.5 Hz, 2H), 1.711.47 (m, 2H), 1.47 (s, 6H), 1.35-1.18 (m, 8H), 0.89-0.81 (m, 3H). LCMS: MS m/z = 202.2 [M+1], tR = 0.66 min; LC system: Agilent 1260 Infinity II HPLC; MS system: single-quadrupole mass detector G6124B; column: Kinetix 2.6 µm, C18, 100A, 50 mmx2.1 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 0-1.00 min 10%-100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 µl/min.

66a 66

PMPA (100 mg, 0.35 mmol), intermediate 66a (331 mg, 1.39 mmol) and triethylamine (0.30 mL, 2.09 mmol) were combined in pyridine (1 mL) under argon and heated at 50 °C for 5 min. To the above reaction mixture was added a freshly prepared bright yellow solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL). The reaction mixture was stirred at 85 °C overnight. Then the reaction mixture was cooled to room temperature and diluted with 20 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (10 mL x 2), brine (10 mL) and dried over sodium sulfate. Hexane (0.5 volume equivalent) was added, the mixture was loaded onto a 3 cm pad of silica gel and washed with ethyl acetate, the desired compound was eluted with 10% methanol in DCM. The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to give the title compound (66). 1H NMR (400 MHz, DMSO- _d6 ) δ 8.16 (s, 1H), 8.12 (s, 1H), 7.16 (s, 2H), 4.364.09 (m, 4H), 4.09-3.90 (m, 5H), 3.67-3.43 (m, 2H), 1.67-1.50 (m, 4H), 1.46-1.33 (m, 12H), 1.32-1.17 (m, 16H), 1.06 (d, J=6.2 Hz, 3H), 0.87-0.74 (m, 6H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.42 (m). LCMS: MS m/z = 654.4 [M+1], tR = 1.25 min; LC system: Agilent 1260 Infinity II HPLC; MS system: G6124B single quadrupole mass detector; Column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-1.00 min 10-100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 μL/min. HPLC: tR = 3.59 min; HPLC system: Agilent 1100 series; column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 2-98% acetonitrile, 5.0-6.0 min 98% acetonitrile at 2 mL/min.

Example 67. Bis(4-methylpentyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (67)

Ηχίγ-ΟΗ, HO^A. chlorovedoro^ _α . _Η3 Αγ°Χ^Α O 1,4-dioxane q

67a

Synthesis of 4-methylpentyl 2-amino-2-methylpropanoate hydrochloride (67a).

2-Amino-2-methylpropanoic acid (1.5 g, 10.7 mmol) and 4-methylpentan-1-ol (5.49 g, 53.7 mmol) were dissolved in 4N HCl in 1,4-dioxane (13.4 mL, 53.7 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature, the solids were filtered off, and the solution was concentrated under reduced pressure.

- 113 049099 nii. The residue was dissolved in water (40 mL) and concentrated under reduced pressure, dissolved in water (40 mL) and concentrated under reduced pressure. This residue was taken up in toluene and concentrated under reduced pressure. The residue was taken up in water and lyophilized over the weekend to afford intermediate 67a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.65 (br s, 3H), 4.15 (t, J=6.5 Hz, 2H), 1.67-1.48 (m, 3H), 1.48 (s, 6H), 1.27-1.14 (m, 2H), 0.87 (d, J=6.6 Hz, 6H). LCMS: MS m/z = 188.2 [M+1], tR = 0.61 min; LC system: Agilent 1260 Infinity II HPLC; MS system: G6124B single quadrupole mass detector; Column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-1.00 min 10%-100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 μL/min.

PMPA (100 mg, 0.35 mmol), intermediate 67a (312 mg, 1.39 mmol) and triethylamine (0.30 mL, 2.09 mmol) were combined in pyridine (1 mL) under argon and heated at 50 °C for 5 min. To the above reaction mixture was added a freshly prepared bright yellow solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL). The reaction mixture was stirred at 85 °C overnight. Then the reaction mixture was cooled to room temperature and diluted with 20 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (10 mL x 2), brine (10 mL) and dried over sodium sulfate. Hexane (0.5 volume equivalent) was added, the mixture was loaded onto a 3 cm pad of silica gel and washed with ethyl acetate, and the desired compound was eluted with 10% methanol in DCM. The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (67). 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.16 (s, 1H), 8.12 (s, 1H), 7.16 (br.s, 2H), 4.35-4.11 (m, 4H), 4.09-3.89 (m, 5H), 3.66-3.47 (m, 2H), 1.61-1.46 (m, 6H), 1.45-1.32 (m, 12H), 1.26-1.12 (m, 4H), 1.06 (d, J=6.2 Hz, 3H), 0.87-0.80 (m, 12H). ³¹ P NMR (162 MHz, DMSO-d6) δ 18.42 (m). LCMS: MS m/z = 626.3 [M+1], tR = 1.11 min; LC System: Agilent 1260 Infinity II HPLC; MS System: G6124B Single Quadrupole Mass Detector; Column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-1.00 min 10% 100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 μL/min. HPLC: tR = 3.29 min; HPLC System: Agilent 1100 Series; Column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 2-98% acetonitrile, 5.0-6.0 min 98% acetonitrile at 2 ml/min.

Example 68. Bis(5-methylhexyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(K)-bis(2-methylpropanoate) (68)

IU kAAdEN but ^x / hydrogen chloride O /x /

Η ₂ Ν γ + CI'H ₃ N'^[| ^X

O I 1,4-dioxane q I

68a

Synthesis of 5-methylhexyl 2-amino-2-methylpropanoate hydrochloride (68a).

2-Amino-2-ethylpropanoic acid (1.5 g, 10.7 mmol) and 5-methylhexan-1-ol (6.24 g, 53.7 mmol) were dissolved in 4N HCl in 1,4-dioxane (13.4 mL, 53.7 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature, the solids were filtered off, and the solution was concentrated under reduced pressure. The residue was dissolved in water (40 mL) and concentrated under reduced pressure, dissolved in water (40 mL), and concentrated under reduced pressure. This residue was taken up in toluene and concentrated under reduced pressure. The residue was taken up in water and lyophilized over the weekend to provide intermediate 68a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.61 (br.s, 3H), 4.16 (t, J=6.5 Hz, 2H), 1.65-1.49 (m, 3H), 1.48 (s, 6H), 1.38-1.26 (m, 2H), 1.22-1.11 (m, 2H), 0.86 (d, J=6.6 Hz, 6H). LCMS: MS m/z = 202.2 [M+1], tR = 0.63 min; LC system: Agilent 1260 Infinity II HPLC; MS system: single-quadrupole mass detector G6124B; column: Kinetix 2.6 µm, C18, 100A, 50 mmx2.1 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 0-1.00 min 10%100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 µl/min.

- 114 049099

PMPA (100 mg, 0.35 mmol), intermediate 68a (331 mg, 1.39 mmol) and triethylamine (0.30 mL, 2.09 mmol) were combined in pyridine (1 mL) under argon and heated at 50 °C for 5 min. To the above reaction mixture was added a freshly prepared bright yellow solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL). The reaction mixture was stirred at 85 °C overnight. Then the reaction mixture was cooled to room temperature and diluted with 20 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (10 mL x 2), brine (10 mL) and dried over sodium sulfate. Hexane (0.5 volume equivalent) was added, the mixture was loaded onto a 3 cm pad of silica gel and washed with ethyl acetate, and the desired compound was eluted with 10% methanol in DCM. The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (68). 1H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.12 (s, 1H), 7.16 (br.s, 2H), 4.33-4.11 (m, 4H), 4.10-3.86 (m, 5H), 3.66-3.46 (m, 2H), 1.60-1.45 (m, 6H), 1.45-1.34 (m, 12H), 1.33-1.21 (m, 4H), 1.18-1.09 (m, 4H), 1.06 (d, J=6.2 Hz, 3H), 0.85-0.79 (m, 12H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.41 (m). LCMS: MS m/z = 654.4 [M+1], tR = 1.21 min; LC system: Agilent 1260 Infinity II HPLC; MS system: G6124B single quadrupole mass detector; Column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 0-1.00 min 10%100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 µl/min. HPLC: tR = 3.52 min; HPLC system: Agilent 1100 series; Column: Gemini 5 µm C18 110A,

50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 2-98% acetonitrile, 5.0-6.0 min 98% acetonitrile at 2 ml/min.

Example 69. Bis(2-propylpentyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^)-bis(2-methylpropanoate) (69)

69a

Synthesis of 2-propylpentyl 2-amino-2-methylpropanoate hydrochloride (69a).

2-Amino-2-ethylpropanoic acid (1.5 g, 10.7 mmol) and 2-propylpentan-1-ol (6.3 g, 48.4 mmol) were dissolved in 4N HCl in 1,4-dioxane (13.4 mL, 53.7 mmol) under argon in a sealed tube. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature, the solids were filtered off, and the solution was concentrated under reduced pressure. The residue was dissolved in water (40 mL) and concentrated under reduced pressure, dissolved in water (40 mL), and concentrated under reduced pressure. This residue was taken up in toluene and concentrated under reduced pressure. The residue was taken up in water and lyophilized over the weekend to provide intermediate 69a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.62 (br s, 3H), 4.08 (d, J = 5.4 Hz, 2H), 1.77-1.60 (m, 1H), 1.48 (s, 6H), 1.35-1.20 (m, 8H), 0.94-0.80 (m, 6H). LCMS: MS m/z = 216.2 [M+1], tR = 0.62 min; LC system: Agilent 1260 Infinity II HPLC; MS system: G6124B single quadrupole mass detector; Column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 0-1.00 min 10%-100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 µl/min.

- 115 049099

PMPA (100 mg, 0.35 mmol), intermediate 69a (351 mg, 1.39 mmol) and triethylamine (0.30 mL, 2.09 mmol) were combined in pyridine (1 mL) under argon and heated at 50 °C for 5 min. To the above reaction mixture was added a freshly prepared bright yellow solution of triphenylphosphine (457 mg, 1.74 mmol), 2,2'-dipyridyl disulfide (384 mg, 1.74 mmol) in pyridine (1 mL). The reaction mixture was stirred at 85 °C overnight. Then the reaction mixture was cooled to room temperature and diluted with 20 mL EtOAc. The organic layers were washed with saturated aqueous sodium bicarbonate (10 mL x 2), brine (10 mL) and dried over sodium sulfate. Hexane (0.5 volume equivalent) was added, the mixture was loaded onto a 3 cm pad of silica gel and washed with ethyl acetate, and the desired compound was eluted with 10% methanol in DCM. The organic layers were concentrated under reduced pressure. The residue was purified by HPLC (Gemini 5 μm C18-110A 100 x 30 mm column, gradient 25-100% acetonitrile in water over 30 min) to afford the title compound (69). 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.16 (s, 1H), 8.13 (s, 1H), 7.20 (br.s, 2H), 4.37-4.10 (m, 4H), 4.05-3.82 (m, 5H), 3.66-3.46 (m, 2H), 1.72-1.51 (m, 2H), 1.48-1.35 (m, 12H), 1.30-1.19 (m, 16H), 1.06 (d, J= 6.2 Hz, 3H), 0.87-0.80 (m, 12H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ 18.34 (m). LCMS: MS m/z = 682.3 [M+1], tR = 1.29 min; LC system: Agilent 1260 Infinity II HPLC; MS system: G6124B single quadrupole mass detector; Column: Kinetix 2.6 μm, C18, 100A, 50 mm x 2.1 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-1.00 min 10% 100% acetonitrile, 1.00-1.35 min 100% acetonitrile, 1.35-1.36 min 100-10% acetonitrile at 2 μL/min. HPLC: t _R = 3.78 min; HPLC system: Agilent 1100 series; column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 2-98% acetonitrile, 5.0-6.0 min 98% acetonitrile at 2 mL/min.

Example 70. Bis(2-ethylbutyl) 1,1'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azandiyl))(^)-bis(cyclopropane-1-carboxylate) (70)

Example 71. 2-Ethylbutyl 1-(((((((E)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((1-(2-ethylbutoxy)-2-methyl-1-oxopropan-2-yl)amino)phosphoryl)amino)cyclopropane-1-carboxylate (71)

Example 72. 2-Ethylbutyl 1-(((((((E)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((1-(2-ethylbutoxy)-2-methyl-1-oxopropan-2-yl)amino)phosphoryl)amino)cyclopropane-1-carboxylate (72)

70a

Synthesis of 2-ethylbutyl 2-amino-2-methylpropanoate hydrochloride (70a).

To 2-amino-2-methylpropanoic acid (5 g, 48.5 mmol) in a 250 mL round-bottomed flask equipped with a stir bar was added 2-ethylbutan-1-ol (29.7 mL, 291 mmol). To the well-stirred mixture at room temperature was added dropwise thionyl chloride (7.08 mL, 97.0 mmol) over 5 min under argon (temperature was maintained below 25 °C). The reaction mixture was heated under reflux at 90 °C for 24 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure at 65 °C, coevaporated with THF (50 mL x 2) and once with DCM (50 mL). The crude product was taken up in water (100 mL) and washed with EtOAc:hexanes, 1:1 (50 mL x 2) and once with DCM (40 mL). The aqueous solution was frozen and concentrated using a lyophilizer (30 h) to give intermediate 70a as a white solid. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.80 (s, 3H), 4.09 (d, J = 5.5 Hz, 2H), 1.50 (s, 7H), 1.37-1.30 (m, 4H), 0.86 (t, J = 7.4 Hz, 6H).

70b

Synthesis of 2-ethylbutyl 1-aminocyclopropane-1-carboxylate hydrochloride (70b).

Intermediate 70b was synthesized in the same manner as intermediate 70a using 1-aminocyclopropanecarboxylic acid (2.5 g, 24.7 mmol), 2-ethylbutan-1-ol (18.6 mL, 148 mmol) and thionyl chloride (3.61 mL, 49.5 mmol) to afford intermediate 70b. 1H NMR (400 MHz, DMSO-d ₆ ) δ 9.11 (s, 3H), 4.05 (d, J=5.6 Hz, 2H), 1.53-1.46 (m, 3H), 1.39-1.25 (m, 6H), 0.85 (t, J=7.4 Hz, 7H).

- 116 049099

70a 70b

71 72

PMPA (100 mg, 0.348 mmol), intermediate 70a (156 mg, 0.696), intermediate 70b (154 mg, 0.696) were placed in an 8 mL vial with a stirring bar. To this mixture was added triethylamine (0.5 mL) followed by pyridine (1.2 mL), capped and stirred at 70 °C for 10 min. 2,2'-Dipyridyl disulfide (307 mg, 1.39 mmol) and triphenylphosphine (365 mg, 1.39 mmol) were mixed in another 8 mL vial, pyridine (1.4 mL) was added, sonicated until completely dissolved under argon, and the clear yellow solution was transferred to the stirred suspension. The reaction mixture was stirred at 70 °C overnight. The reaction mixture was cooled to room temperature, concentrated and combined with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g column, dried by flushing nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture of 3 products. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 µM, nX-C18, 110A 250 x 30 mm column, 20-100% acetonitrile/water gradient, 20 min cycle) to isolate 70, 71 and 72.

70: 1H NMR (400 MHz, acetonitrile^) δ 8.23 (s, 1H), 8.04 (s, 1H), 6.14 (s, 2H), 4.43-4.09 (m, 4H), 4.04-3.85 (m, 5H), 3.79 (dd, J=12.8, 8.0 Hz, 1H), 3.52 (dd, J=12.8, 10.4 Hz, 1H), 1.46 (bq, J=10.4, 6.0 Hz, 2H), 1.38-1.24 (m, 14H), 1.23-1.10 (m, 6H), 0.94-0.80 (m, 12H). LCMS: MS m/z = 622.3 [M+1]; tR = 1.5 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 2.98 min; HPLC system: Agilent 1100 Series; Column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

71: 1H NMR (400 MHz, acetonitrile-b ₃ ) δ 8.23 (s, 1H), 8.06 (s, 1H), 6.09 (s, 2H), 4.39-4.23 (m, 2H), 4.17 (dd, J=14.6, 7.2 Hz, 1H), 4.09-3.93 (m, 4H), 3.93-3.83 (m, 2H), 3.73 (dd, J=12.8, 8.2 Hz, 1H), 3.45 (dd, J=12.7, 10.4 Hz, 1H), 1.58-1.43 (m, 3H), 1.41 (d, J=1.7 Hz, 6H), 1.38-1.27 (m, 10H), 1.19 (d, J=6.2 Hz, 3H), 1.15-1.07 (m, 1H), 0.89 (td, J=7.5, 3.8 Hz, 12H). LCMS: MS m/z = 624.3 [M+1]; tR = 1.52 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 2.81 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

72: 1H NMR (400 MHz, acetonitrile-b ₃ ) δ 8.23 (s, 1H), 8.05 (d, J=8.0 Hz, 1H), 6.15 (s, 2H), 4.48-4.12 (m, 3H), 4.12-3.83 (m, 6H), 3.79-3.61 (m, 1H), 3.59-3.34 (m, 1H), 1.63-1.24 (m, 18H), 1.22-1.07 (m, 4H), 0.930.72 (m, 12H). LCMS: MS m/z = 624.3 [M+1]; tR = 1.53 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 2.85 min; HPLC system: Agilent 1100 Series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 73. Dipentyl-1,1'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azandiyl))(K)-bis(cyclopropane-1-carboxylate) (73).

- 117 049099

Example 74. Pentyl 1-((((((^)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((2-methyl-1-oxo1-(pentyloxy)propan-2-yl)amino)phosphoryl)amino)cyclopropane-1-carboxylate (74)

73a

Synthesis of pentyl 1-aminocyclopropanecarboxylate hydrochloride (73 a).

Intermediate 73a was synthesized in the same manner as intermediate 70a using 1-aminocyclopropanecarboxylic acid (2.5 g, 24.7 mmol), pentan-1-ol (16.1 mL, 148 mmol) and _SOCl2 (3.61 mL, 49.5 mmol) to give intermediate 73a. 1H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 3H), 4.11 (t, J=6.5 Hz, 2H), 1.58 (dk, J=10.7, 6.8, 5.2 Hz, 2H), 1.53-1.47 (m, 2H), 1.39-1.33 (m, 2H), 1.29 (h, J=3.7 Hz, 4H), 0.93-0.79 (m, 3H).

I am thionyl chloride I

N ₂ gu° ⁿ ₊ _ ΟΙΉ ₃ Ν· ^> γ ^Ο ^ ^χΧχ/ ^

Oh oh

73b

Synthesis of pentyl 2-amino-2-methylpropanoate hydrochloride (73b).

Intermediate 73b was synthesized in the same manner as intermediate 70a using 2-amino-2-methylpropanoic acid (2.5 g, 24.7 mmol), pentan-1-ol (15.8 mL, 145 mmol) and _SOCl2 (3.54 mL, 48.5 mmol) to afford intermediate 73b. 1H NMR (400 MHz, DMSO-d6) δ 9.06 (s, 3H), 3.92 (d, J=6.5 Hz, 2H), 1.89 (dk, J=13.3, 6.6 Hz, 1H), 1.60-1.28 (m, 4H), 0.90 (d, J=6.7 Hz, 6H).

PMPA (100 mg, 0.348 mmol), intermediate 73a (145 mg, 0.696 mmol), intermediate 73b (146 mg, 0.696 mmol) were placed in an 8 mL vial with a stirring bar. To this mixture was added triethylamine (0.5 mL) followed by pyridine (1.2 mL), capped and stirred at 70 °C for 10 min. 2,2'-Dipyridyl disulfide (307 mg, 1.39 mmol) and triphenylphosphine (365 mg, 1.39 mmol) were mixed in another 8 mL vial, pyridine (1.4 mL) was added, sonicated until completely dissolved under argon, and the clear yellow solution was transferred to the stirred suspension. The reaction mixture was stirred at 70 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and co-evaporated with toluene (20 mL x 2). The residue was dissolved in dichloromethane, loaded onto a 24 g column, dried by flushing nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture of 3 products. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 µM, nX-C18, 110A 250 x 30 mm column, 20-100% acetonitrile/water gradient, 20 min cycle) to isolate 73 and 74.

73: 1H NMR (400 MHz, methanol-BD) δ 8.30 (s, 1H), 8.20 (s, 1H), 4.41 (dd, J=14.5, 3.0 Hz, 1H), 4.21 (dd, J=14.5, 7.7 Hz, 1H), 4.08 (tt, J=7.9, 4.0 Hz, 3H), 4.02-3.78 (m, 5H), 3.59 (dd, J=12.8, 11.7 Hz, 1H), 1.65-1.50 (m, 4H), 1.40-1.26 (m, 10H), 1.24 (d, J=6.2 Hz, 3H), 1.20-1.08 (m, 4H), 0.90 (q, J=6.7 Hz, 6H). ³¹ P NMR (162 MHz, methanol-6 ₄ ) δ 25.55. LCMS: MS m/z = 594.25 [M+1]; t _R = 1.38 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

74: 1H NMR (400 MHz, methanol-b ₄ ) δ 8.27 (d, J=6.0 Hz, 1H), 8.20 (d, J=2.3 Hz, 1H), 4.40 (dt, J=14.4, 3.0

- 118 049099

Hz, 1H), 4.23 (dt, J=14.4, 7.1 Hz, 1H), 4.18-4.10 (m, 2H), 4.07 (qg, J=6.6, 2.2 Hz, 2H), 4.02-3.91 (m, 2H), 3.82 (ddd, J=13.1, 7.9, 5.4 Hz, 1H), 3.56 (dd, J=15.8, 12.8, 10.7 Hz, 1H), 1.69-153 (m, 4H), 1.48 (d, J=10.1 Hz, 4H), 1.40 (d, J=6.3 Hz, 4H), 1.38-1.26 (m, 6H), 1.23 (dd, J=6.2, 1.9 Hz, 4H), 1.20-1.10 (m, 1H), 0.90 ^ d, J=7.1, 4.2 Hz, 7H). ³¹ P NMR (162 MHz, methanol-a ₄ ) δ 23.09 (d, J=12.3 Hz). LCMS: MS m/z = 596.23 [M+1]; tR = 1.43 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 2.88 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 7 5. Diisobutyl 1,1'-(((((1 -(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^)-bis(cyclopropane-1-carboxylate) (75).

Example 76. Isobutyl-1-(((((((R)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((1-(hexyloxy)-2-methyl-1-oxopropan-2-yl)amino)phosphoryl)amino)cyclopropane-1-carboxylate (76)

75a

Synthesis of isobutyl 1-aminocyclopropane-1-carboxylate hydrochloride 75a).

Intermediate 75a was synthesized in the same manner as intermediate 1a using 1-aminocyclopropanecarboxylic acid (2.5 g, 24.7 mmol), 2-methylpropan-1-ol (13.5 mL, 148 mmol) and thionyl chloride (3.61 mL, 49.5 mmol) to afford intermediate 75a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 9.06 (s, 3H), 3.92 (d, J=6.5 Hz, 2H), 1.93-1.84 (m, 1H), 1.60-1.28 (m, 4H), 0.90 (d, J=6.7 Hz, 6H).

OH thionyl chloride Xl η ~ ~ .

η ₂ ν^ ^0Η ₊ o o

75b

Synthesis of hexyl 2-amino-2-methylpropanoate hydrochloride (75b).

To a suspension of 2-amino-2-methylpropanoic acid (5 g, 48.5 mmol) in 1-hexanol (49.5 g, 485 mmol) was added thionyl chloride (7.07 mL, 97 mmol) over 10 min at 5 °C under argon in a sealed tube. After complete addition, the reaction mixture was allowed to warm to room temperature and stirred for 30 min. The reaction mixture was heated to 90 °C for 16 h. The reaction mixture was cooled to room temperature and quenched with water (100 mL). The aqueous layer was washed with 1:1 EtOAc:Hex (50 mL×2) and concentrated. The residue was dissolved in water (20 mL) and concentrated (×2). The residue was taken up in toluene and concentrated to give intermediate 75b. 1H NMR (400 MHz, methanol-d^δ 4.27 (t, J=6.6 Hz, 2H), 1.77-1.67 (m, 2H), 1.59 (s, 6H), 1.46-1.33 (m, 6H), 0.99-0.89 (m, 3H).

PMPA (100 mg, 0.348 mmol), isobutyl 1-aminocyclopropanecarboxylate hydrochloride (75a) (135 mg, 0.696 mmol), hexyl 2-amino-2-methylpropanoate hydrochloride (75b) (146 mg, 0.696 mmol) were placed in an 8 mL microwave vial with a stir bar. To this mixture was added TEA (0.5 mL) followed by pyridine (1.2 mL), capped and stirred at 70 °C for 10 min. 2,2'-Dipyridyl disulfide (307 mg, 1.39 mmol) and triphenylphosphine (365 mg, 1.39 mmol) were mixed in another 8 mL vial, pyridine (1.4 mL) was added, sonicated until completely

- 119 049099 dissolved under argon, the clear yellow solution was transferred to a stirred suspension of the above mixture. The reaction mixture was stirred at 70 °C overnight. The reaction mixture was cooled to room temperature, concentrated and combined with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g column, dried the column by bubbling nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture of 3 products. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 µM, nX-C18, 110A 250x30 mm column, gradient 20-100% acetonitrile/water, 20 min cycle) to isolate 75 and 76.

75: 1H NMR (400 MHz, methanol^) δ 8.31 (s, 1H), 8.20 (s, 1H), 4.41 (dd, J=14.5, 2.9 Hz, 1H), 4.20 (dd, J=14.5, 7.9 Hz, 1H), 4.03-3.93 (m, 1H), 3.93-3.81 (m, 4H), 3.68 (d, J=6.6 Hz, 2H), 3.65-3.54 (m, 1H), 1.93-1.77 (m, 2H), 1.42-1.29 (m, 6H), 1.24 (d, J=6.3 Hz, 3H), 1.21-1.06 (m, 3H), 0.91 (d, J=6.7 Hz, 6H), 0.86 (dd, J=6.7, 1.9 Hz, 6H). ³¹ P NMR (162 MHz, methanol-O ₄ ) δ 25.67. LCMS: MS m/z = 566.22 [M+1]; tR = 1.27 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 2.70 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110Α, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

76: 1H NMR (400 MHz, methanol^) δ 8.27 (d, J=6.1 Hz, 1H), 8.20 (d, J=2.1 Hz, 1H), 4.40 (dt, J=14.5, 3.1 Hz, 1H), 4.27-4.19 (m, 1H), 4.17-4.11 (m, 1H), 4.09-4.03 (m, 1H), 4.02-3.89 (m, 1h), 3.88-3.68 (m, 3H), 3.613.48 (m, 1H), 1.94-1.79 (m, 1H), 1.70-1.54 (m, 2H), 1.48 (d, J=10.0 Hz, 4H), 1.40 (d, J=7.9 Hz, 4H), 1.37-1.26 (m, 5H), 1.25-1.11 (m, 5H), 0.97-0.82 (m, 10H). ³¹ P NMR (162 MHz, methanol-O ₄ ) δ 23.19, 23.09. LCMS: MS m/z = 596.23 [M+1]; tR = 1.43 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 77. Bicyclo[1,1,1]pentan-1-ylmethyl 2-((((((K.)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((1-(hexyloxy)-2-methyl-1-oxopropan-2-yl)amino)phosphoryl)amino)-2-methylpropanoate (77)

PMPA (100 mg, 0.348 mmol), 1-bicyclo[1,1,1]pentanylmethanol (103 mg, 1.04 mmol), intermediate 1a (156 mg, 0.696 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol), triphenylphosphine (365 mg, 1.39 mmol) were placed in an 8 mL vial with a stir bar, triethylamine (0.5 mL) and pyridine (2.4 mL) were added. The mixture was purged with argon, capped and stirred at 70 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and co-evaporated with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g column, the column was dried by flushing nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture of 3 products. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 µM, NX-C18, 110A 250x30 mm column, gradient 20-100% acetonitrile/water over 20 min) to give the title compound (77) 1H NMR (400 MHz, methanol- _O4 ) δ 8.21 (d, J=1.1 Hz, 1H), 8.17 (d, J=4.1 Hz, 1H), 4.45-4.33 (m, 1H), 4.26 (ddd, J=14.5, 7.2, 2.7 Hz, 1H), 4.07 (q, J=6.7 Hz, 3H), 3.92-3.61 (m, 4H), 2.48 (d, J=9.1 Hz, 1H), 1.75 (s, 3H), 1.71 (s, 3H), 1.66-1.53 (m, 2H), 1.51-1.40 (m, 14H), 1.38-1.27 (m, 4H), 1.23 (t, J=5.8 Hz, 3H), 0.96-0.82 (m, 3H). ³¹ P NMR (162 MHz, methanol-OOO δ 26.62, 26.47. LCMS: MS m/z = 622.32 [M+1]; tR = 1.41 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2

- 120 049099 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 78. (1-Fluorocyclopropyl)methyl 2-((((((^)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((1-(hexyloxy)-2-methyl-1-oxopropan-2-yl)amino)phosphoryl)amino)-2-methylpropanoate (78)

PMPA (100 mg, 0.348 mmol), (1-fluorocyclopropyl)methanol (94 mg, 1.04 mmol), intermediate 1a (156 mg, 0.696 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol), triphenylphosphine (365 mg, 1.39 mmol) were placed in an 8 mL microwave vial with a stir bar and triethylamine (0.5 mL) and pyridine (2.4 mL) were added. The mixture was purged with argon, capped and stirred at 70 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and co-evaporated with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g column, dried by flushing nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture of 3 products. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A 250x30 mm column, gradient 20-100% acetonitrile/water, 20 min cycle) to give the title compound (78). 1H NMR (400 MHz, methanol^ ₄ ) δ 8.20 (s, 1H), 8.18 (d, J=3.4 Hz, 1H), 4.57 (s, 1H), 4.39 (dt, J=14.6, 3.0 Hz, 1H), 4.31-4.11 (m, 3H), 4.10-3.96 (m, 2H), 3.90 (td, J=12.7, 12.2, 8.4 Hz, 1H), 3.73 (ddd, J=18.2, 13.5, 9.1 Hz, 1H), 1.59 (d, J=8.7 Hz, 3H), 1.51-1.40 (m, 12H), 1.39-1.26 (m, 6H), 1.23 (dd, J=6.2, 1.6 Hz, 3H), 1.07 (dd, J=18.1, 8.4 Hz, 2H), 0.89 (t, J=6.6 Hz, 3H), 0.84-0.74 (m, 2H). ³¹ P NMR (162 MHz, methanol^) δ 26.94, 26.80. LCMS: MS m/z = 614.29 [M+1]; t _R = 1.32 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 79. Hexyl 2-((((((^)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((1-(2-methoxy-2methylpropoxy)-2-methyl-1-oxopropan-2-yl)amino)phosphoryl)amino)-2-methylpropanoate (79)

MH ₂ triphenylphosphine

Ϊ 2,2'-dipyridyl disulfide \/ Triethylamine o ' - -οΎ ^ΟΗ .

NO'R^O^ O he |

1a

PMPA (100 mg, 0.348 mmol), 2-methoxy-2-methyl-propan-1-ol (109 mg, 1.04 mmol), intermediate 1a (156 mg, 0.696 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol), triphenylphosphine (365 mg, 1.39 mmol) were placed in an 8 mL microwave vial with a stir bar, triethylamine (0.5 mL) and pyridine (2.4 mL) were added. The mixture was flushed with argon, capped and stirred at 70 °C overnight. The reaction mixture was cooled to room temperature.

- 121 049099 , concentrated under reduced pressure and co-evaporated with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g column, dried by bubbling nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture of 3 products. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A 250 x 30 mm column, gradient 20-100% acetonitrile/water, 20 min cycle) to give the title compound (79). 1H NMR (400 MHz, methanol-d ₄ ) δ

8.21 (s, 1H), 8.19 (d, J=2.6 Hz, 1H), 7.96 (d, J=8.6 Hz, 1H), 4.39 (ddd, J=14.5, 6.0, 3.4 Hz, 1H), 4.26 (ddd, J=14.6, 7.1, 3.0 Hz, 1H), 4.12-3.96 (m, 3H), 3.94-3.63 (m, 5H), 3.20 (d, J=10.5 Hz, 3H), 1.68-1.54 (m, 2H), 1.501.44 (m, 8H), 1.42 (d, J=3.2 Hz, 4H), 1.38-1.26 (m, 3H), 1.23 (dd, J=6.3, 2.6 Hz, 3H), 1.13 (dd, J=16.2, 7.4 Hz, 7H), 0.94-0.83 (m, 3H). ³¹ P NMR (162 MHz, methanol-dd) δ 26.76, 26.57. LCMS: MS m/z = 628.32 [M+1]; tR = 1.32 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 80. Bicyclo[2,2,1]hepatn-1-ylmethyl 2-(((((^)-1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)((1-(hexyloxy)-2-methyl-1-oxopropan-2-yl)amino)phosphoryl)amino)-2-methylpropanoate (80). . gA ⁴ "triphenylphosphine \/ q 1/1 2,2'-dipyridyl disulfide

ΟΙΉ ₃ Ν^%Τ'' Triethylamine

O I pyridine____________ he

1a

PMPA (100 mg, 0.348 mmol), norbornane-1-ylmethanol (132 mg, 1.04 mmol), intermediate 1a (156 mg, 0.696 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol), triphenylphosphine (365 mg, 1.39 mmol) were placed in an 8 mL microwave vial with a stir bar and triethylamine (0.5 mL) and pyridine (2.4 mL) were added. The mixture was purged with argon, capped and stirred at 70 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and co-evaporated with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g column, dried by flushing nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture of 3 products. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A 250x30 mm column, gradient 20-100% acetonitrile/water, 20 min cycle) to give the title compound (80). 1H NMR (400 MHz, methanol- _d4 ) δ 8.20 (s, 1H), 8.17 (d, J=3.3 Hz, 1H), 7.97 (d, J=9.7 Hz, 1H), 4.38 (ddd, J=14.5, 7.2, 3.2 Hz, 1H), 4.25 (ddd, J=14.4, 7.2, 3.5 Hz, 1H), 4.11-3.97 (m, 4H), 3.96-3.80 (m, 2H), 3.69 (td, J=13.4, 9.1 Hz, 1H), 2.18 (dt, J=9.7, 4.3 Hz, 1H), 1.711.53 (m, 5N), 1.52-1.41 (m, 13H), 1.38-1.27 (m, 7H), 1.26-1.08 (m, 6H), 0.95-0.78 (m, 4H). ³¹ P NMR (162 MHz, methanol-d ₄ ) δ 26.38, 26.16. LCMS: MS m/z = 650.37 [M+1]; t _R = 1.49 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 81. 1-Cyclopropylethyl 2-((((((^)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)((1(hexyloxy)-2-methyl-1-oxopropan-2-yl)amino)phosphoryl)amino)-2-methylpropanoate (81)

- 122 049099

PMPA (100 mg, 0.348 mmol), 1-cyclopropylethanol (90 mg, 1.04 mmol), intermediate 1a (156 mg, 0.696 mmol), 2,2'-dipyridyl disulfide (307 mg, 1.39 mmol), triphenylphosphine (365 mg, 1.39 mmol) were placed in an 8 mL microwave vial with a stir bar and triethylamine (0.5 mL) and pyridine (2.4 mL) were added. The mixture was purged with argon, capped and stirred at 70 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and co-evaporated with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g column, the column was dried by flushing nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture of 3 compounds. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 µM, nX-C18, 110A 250x30 mm column, 20-100% acetonitrile/water gradient, 20 min cycle) to isolate 81. 1H NMR (400 MHz, acetonitrile^) δ 8.24 (s, 1H), 8.02 (s, 1H), 5.98 (s, 2H), 4.41-4.10 (m, 3H), 4.05-3.96 (m, 3H), 3.92-3.70 (m, 2H), 3.68-3.45 (m, 2H), 1.70-1.51 (m, 3H), 1.47-1.25 (m, 20H), 1.24-1.14 (m, 4H), 0.95-0.82 (m, 4H), 0.56-0.17 (m, 4H). ³¹ P NMR (162 MHz, acetonitrile^) δ 24.75-23.19 (m). LCMS: MS m/z = 610.19 [M+1]; tR = 1.37 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 82. Dihexyl 2,2'-(((((1-(6-(((((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)amino)-9H-purin-9yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (82)

Compound 1 (50 mg, 0.079 mmol), 4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (31 mg, 0.16 mmol), _Cs2CO3 (130 mg, 0.4 mmol), NaI (59.9 mg, 0.4 mmol) were placed in _an 8 mL microwave vial with a stir bar and DMF (1 mL) was added. The mixture was flushed with argon, capped and stirred at room temperature for 4 h and heated at 80 °C for 4 h. The reaction mixture was cooled to room temperature, quenched with water, extracted with DCM, dried over sodium sulfate and concentrated. The residue was dissolved in DCM, loaded onto a 12 g column and eluted with 0-20% MeOH/DCM for 15 min to give a title compound enriched mixture. This mixture was dissolved in MeOH (2 mL), filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A 250x30 mm column, gradient 40-100% acetonitrile/water, 20 min cycle) to give the title compound (82). 1H NMR (400 MHz, acetonitrile^) δ 8.33 (s, 1H), 8.06 (s, 1H), 6.61 (s, 1H), 4.60 (s, 2H), 4.35 (dd, J=14.5, 3.2 Hz, 1H), 4.24-3.99 (m, 5H), 3.97-3.90 (m, 1H), 3.81-3.60 (m, 2H), 3.58-3.31 (m, 2H), 2.20 (s, 3H), 1.71-1.56 (m, 5H), 1.52 (s, 3H), 1.44 (d, J=4.4 Hz, 6H), 1,401.26 (m, 14H), 1.19 (d, J=6.2 Hz, 3H), 0.98-0.80 (m, 6H). ³¹ P NMR (162 MHz, acetonitrile-a ₃ ) δ 17.71. LCMS: MS m/z = 738.30 [M+1]; tR = 2.01 min, LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gr.

- 123 049099 dient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 3.80 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 83. Dihexyl 2,2'-(((((1-(6-imino-1-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)-1,6-dihydro9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^)-bis(2-methylpropanoate) (83).

Example 84. Dihexyl 2,2'-(((((1-(1-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)-6-(((5-methyl-2-oxo1,3-dioxol-4-yl)methyl)imino)-1,6-dihydro-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(N,E)-bis(2-methylpropanoate) (84)

82

84

Compound 1 (200 mg, 0.32 mmol) and _KHCO3 (64 mg, 0.64 mmol) were transferred to a 20 mL microwave vial with a stirring bar and NMP (4 mL) was added. To the well-stirred mixture, 4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (71 μL, 0.64 mmol) was added dropwise and stirred at 70 °C for 15 h. At this point, excess KHCO3 (32 mg, 0.32 mmol) and 4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (36 µL, 0.32 mmol) were added and stirring was continued at 70 °C for an additional 12 h. The reaction mixture was cooled to 0 °C, quenched with water by dropwise addition and extracted with EtOAc (40 mL x 2). The ethyl acetate layer was washed once with saturated aqueous ammonium chloride solution, once with water and once with brine. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was dissolved in DCM, loaded onto a 12 g gold column, dried, eluted with 0-20% MeOH/DCM for 15 min to give a mixture of 3 compounds. This mixture was dissolved in MeOH (4 mL), filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A column 250x30 mm, gradient 40-100% acetonitrile/water, 20 min cycle) to isolate 82, 83 and 84.

83: 1H NMR (400 MHz, acetonitrile^) δ 7.87 (s, 1H), 7.81 (s, 1H), 7.10 (s, 1H), 5.01 (s, 2H), 4.23 (dd, J=14.4, 3.3 Hz, 1H), 4.17-4.05 (m, 5H), 3.92-3.85 (m, 1H), 3.77-3.61 (m, 2H), 3.60-3.35 (m, 2H), 2.23 (s, 3H), 1.73-1.58 (m, 4H), 1.53 (s, 3H), 1.46 (d, J=3.2 Hz, 6H), 1.42-1.26 (m, 12H), 1.17 (d, J=6.2 Hz, 3H), 0.97-0.85 (m, 6H). ^31P NMR (162 MHz, acetonitrile^) δ 17.69. LCMS: MS m/z = 738.15 [M+1]; tR = 1.56 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 3.79 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

84: 1H NMR (400 MHz, acetonitrile^) δ 7.92 (s, 1H), 7.87 (s, 1H), 5.13-4.97 (m, 2H), 4.92 (s, 2H), 4.25 (dd, J=14.4, 3.2 Hz, 1H), 4.18-3.99 (m, 5H), 3.96-3.82 (m, 1H), 3.75-3.62 (m, 2H), 3.58-3.37 (m, 2H), 2.19 (s, 3H), 2.12 (s, 3H), 1.69-1.50 (m, 4H), 1.53 (s, 3H), 1.46 (d, J=1.6 Hz, 6H), 1.40 (s, 4H), 1.34-1.25 (m, 7H), 1.19 (d, J=6.2 Hz, 3H), 0.95-0.83 (m, 6H). ^31P NMR (162 MHz, acetonitrile^) δ 17.65, LCMS: MS m/z = 850.07 [M+1]; tR = 1.34 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% ace

- 124 049099 acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 3.81 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 85. Bis((^^)-3-phenylcyclobutyl) 2,2'-((((((^-1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (85) . 1.EDCI, DMAP, DCM Γγ*°γ^ΝΗ ₃ ΌΙ ^Η °Ίι ^NHBoc + ⁰

Ο |Ι J 2. HCI/dioxane, DCM

85a

Synthesis of (^^)-3-phenylcyclobutyl 2-amino-2-methylpropanoate hydrochloride (85a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (0.50 g, 2.46 mmol), 3-phenylcyclobutanol (438 mg, 2.95 mmol), 4-dimethylaminopyridine (0.90 g, 7.38 mmol) and 3-ethyliminomethyleneamino)-1-d-dimethylpropan-1-amine hydrochloride (1.4 g, 7.38 mmol) were dissolved in dichloromethane (10 mL) and stirred at room temperature. After 17 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3 x 10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 3.0 ml, 12.3 mmol). After 18 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to yield intermediate 85a. 1H NMR (400 MHz, DMSO- _d6 ) δ 8.60 (s, 3H), 7.387.30 (m, 2H), 7.30-7.25 (m, 2H), 7.25-7.18 (m, 1H), 5.11-5.00 (m, 1H), 3.24-3.10 (m, 1H), 2.89-2.76 (m, 2H), 2.23-2.09 (m, 2H), 1.49 (s, 6H).

PMPA (100 mg, 0.348 mmol) and intermediate 85a (276 mg, 1.39 mmol) were collected in an 8 mL vial with a stirring bar. To this mixture was added triethylamine (0.5 mL), then pyridine (1.2 mL), capped and stirred at 70 °C for 10 min (not completely soluble, suspension). 2,2'-Dipyridyl disulfide (384 mg, 1.74 mmol) and triphenylphosphine (457 mg, 1.74 mmol) were mixed in another 8 mL vial, pyridine (1.4 mL) was added, sonicated until completely dissolved under argon, and the clear yellow solution was transferred to the stirred suspension of the above mixture. The reaction mixture was stirred at 80 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and co-evaporated with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g gold column, dried by flushing nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0.15% MeOH/DCM for 15 min to give a mixture. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water, 20 min cycle) to give the title compound (85). 1H NMR (400 MHz, acetonitrile^) δ 8.24 (s, 1H), 8.07 (s, 1H), 7.34-3.19 (m, 10H), 6.12 (s, 2H), 4.97 (k, J=8.8 Hz, 2H), 4.47-4.02 (m, 2H), 3.99-3.33 (m, 5H), 3.17 (s, 2H), 2.94-2.68 (m, 4H), 2.53 (s, 1H), 2.18-2.07 (m, 6H), 1.46 (t, J=28.0 Hz, 10H), 1.17 (d, J=6.6 Hz, 3H). ³¹ P NMR (162 MHz, acetonitrile^) δ 17.95. LCMS: MS m/z = 718.34 [M+1]; tR = 1.51 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid;

- 125 049099 gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 2.28 min; HPLC system: Agilent 1100 series; column: Gemini 5 μm C18 110Α, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 982% B at 2 ml/min.

Example 86. Di(spiro[3,5]nonan-2-yl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(K)-bis(2-methylpropanoate) (86)

BUT

NHBoc

ABOUT

2. HCI/dioxane, DCM he 1-EDCI, DMAP, DCM

_NH3CI

86a

Synthesis of spiro[3,5]nonan-2-yl 2-amino-2-methylpropanoate hydrochloride (86a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.0 g, 4.92 mmol), spiro[3,5]nonan-2-ol (1.03 g, 7.38 mmol), 4-dimethylaminopyridine (1.8 g, 14.8 mmol) and 3(ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine hydrochloride (2.8 g, 14.8 mmol) were dissolved in dichloromethane (10 mL) and stirred at room temperature. After 17 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (3 x 10 mL), saturated ammonium chloride solution (10 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-40% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure.

To a solution of the residue in dichloromethane (5 ml) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 6.1 ml, 24.6 mmol). After 1 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (10 ml). The residue was subjected to high vacuum for 5 h to afford intermediate 86a. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.59 (s, 3H), 5.04-4.97 (m, 1H), 2.32-2.19 (m, 2H), 1.82-1.71 (m, 2H), 1.46 (d, J=6.1 Hz, 10H), 1.43-1.26 (m, 6H).

O

II

ΗΟ'Ρχ/ ⁰ ·

HE

N

N

86a triphenylphosphine

2,2'-dipyridyl disulfide Triethylamine pyridine

PMPA (100 mg, 0.348 mmol) and intermediate 86a (273 mg, 1.04 mmol) were taken into an 8 mL microwave vial with a stir bar. To this mixture was added TEA (0.5 mL), then pyridine (1.2 mL), capped and stirred at 70 °C for 10 min (not completely soluble, suspension). 2,2'-Dipyridyl disulfide (384 mg, 1.74 mmol) and triphenylphosphine (457 mg, 1.74 mmol) were mixed in another 8 mL vial, pyridine (1.4 mL) was added, sonicated until completely dissolved under argon, and the clear yellow solution was transferred to the stirred suspension of the above mixture. The reaction mixture was stirred at 80 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure, co-evaporated with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g gold column, dried by flushing nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0.15% MeOH/DCM for 15 min to give a mixture. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water, 20 min cycle) to give the title compound (86). 1H NMR (400 MHz, acetonitrile^) δ 8.24 (s, 1H), 8.06 (s, 1H), 6.06 (s, 2H), 4.99-4.88 (m, 2H), 4.34 (dd, J=14.5, 3.3 Hz, 1H), 4.17 (dd, J=14.5, 7.0 Hz, 1H), 3.96-3.89 (m, 1H), 3.80-3.60 (m, 2H), 3.59-3.31 (m, 2H), 2.27 (t, J=6.8 Hz, 2H), 1.79-1.70 (m, 5H), 1.56-1.41 (m, 21N), 1.37 (d, J=5.0 Hz, 13H), 1.19 (d, J=6.2 Hz, 3H). ³¹ P NMR (162 MHz, acetonitrile^) δ 17.81. LCMS: MS m/z = 702.23 [M+1]; tR = 1.55 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10%

- 126 049099 acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 87. Bis(bicyclo[2,2,1]heptan-1-ylmethyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azandiyl))(K)-bis(2-methylpropanoate) (87)

O

87a

Synthesis of bicyclo[2,2,1]heptan-1-ylmethyl 2-amino-2-methylpropanoate hydrochloride (87a).

In a sealed 40 mL tube with a stirring bar were placed 2-amino-2-methylpropanoic acid (0.5 g, 4.85 mmol) and bicyclo[2.2.1]heptan-1-ylmethanol (2.44 g, 19.4 mmol) and a solution of hydrogen chloride in 1,4-dioxane (20 mL, 4N solution in dioxane). The mixture was stirred at 90 °C for 3 days. 1,4-dioxane was removed under reduced pressure. The mixture was diluted with ethyl acetate/hexane (1:1, 50 mL) and water (50 mL). The organic phase was extracted with water (50 mL). The combined aqueous phase was washed with ethyl acetate/hexanes (1:1, 2 x 50 mL). Any residual solvent in the aqueous phase was removed under reduced pressure. The aqueous phase was diluted with acetonitrile (10 ml) and water (10 ml) and lyophilized to yield intermediate 87a.

PMPA (100 mg, 0.348 mmol) and intermediate 87a (259 mg, 1.04 mmol) were collected in an 8 mL vial with a stirring bar. To this mixture was added triethylamine (0.5 mL), then pyridine (1.2 mL), capped and stirred at 70 °C for 10 min (not completely soluble, suspension). 2,2'-Dipyridyl disulfide (384 mg, 1.74 mmol) and triphenylphosphine (457 mg, 1.74 mmol) were mixed in another 8 mL vial, pyridine (1.4 mL) was added, sonicated until completely dissolved under argon, and the clear yellow solution was transferred to the stirred suspension of the above mixture. The reaction mixture was stirred at 80 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure, co-evaporated with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g gold column, dried by flushing nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0.15% MeOH/DCM for 15 min to give a mixture. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water, 20 min cycle) to give the title compound (87). 1H NMR (400 MHz, acetonitrile^) δ 8.24 (s, 1H), 8.06 (s, 1H), 6.05 (s, 2H), 4.43-4.10 (m, 8H), 3.97-3.90 (m, 1H), 3.77 (d, J=10.8 Hz, 1H), 3.71-3.54 (m, 2H), 3.48 (dd, J=12.6, 9.8 Hz, 1H), 1.72-1.56 (m, 6H), 1.54 (s, 3H), 1.50-1.45 (m, 10H), 1.42 (s, 3H), 1.38-1.27 (m, 6H), 1.24 (d, J=8.1 Hz, 5H), 1.18 (d, J=6.2 Hz, 3H). ³¹ P NMR (162 MHz, acetonitrile-^) δ 17.71. LCMS: MS m/z = 674.19 [M+1]; tR = 1.59 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 2.89 min; HPLC system: Agilent 1100 series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; Solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 88. Bis(4,4-difluorocyclohexyl)methyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azanediyl))(K)-bis(2-methylpropanoate) (88)

- 127 049099

Synthesis of 4,4-difluorocyclohexyl)methyl-2-amino-2-methylpropanoate hydrochloride (88a).

Intermediate 88a was synthesized in the same manner as intermediate 87a using 2-amino-2-methylpropanoic acid (0.5 g, 4.85 mmol), (4,4-difluorocyclohexyl)methanol (1.22 g, 8.15 mmol) and 4N HCl//dioxane (10 mL) to afford intermediate 88a. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.48 (s, 3H), 4.07 (d, J=6.0 Hz, 2H), 2.09-1.69 (m, 6H), 1.48 (s, 6H), 1.36-1.17 (m, 3H).

PMPA (100 mg, 0.348 mmol) and intermediate 88a (246 mg, 1.05 mmol) were collected in an 8 mL vial with a stirring bar. To this mixture was added triethylamine (0.475 mL) followed by pyridine (1.2 mL), capped and stirred at 70 °C for 10 min. 2,2' Dipyridyl disulfide (384 mg, 1.74 mmol) and triphenylphosphine (430 mg, 1.74 mmol) were mixed in another 8 mL vial, pyridine (1.4 mL) was added, and sonicated under argon until completely dissolved. The clear yellow solution was transferred to a stirred suspension of the above mixture. The reaction mixture was stirred at 80 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and co-evaporated with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g column and dried on the column by bubbling nitrogen for 2 min (to dry the DCM). The column was eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water, 20 min cycle) to give the title compound (88). 1H NMR (400 MHz, acetonitrile^) δ 8.25 (d, J=1.9 Hz, 1H), 8.05-7.91 (m, 1H), 5.92 (s, 2H), 4.44-4.04 (m, 3H), 4.05-3.92 (m, 4H), 3.84-3.44 (m, 4H), 2.09 (d, J=23.7 Hz, 3H), 1.89-1.65 (m, 4H), 1.55-1.09 (m, 22H). ³¹ P NMR (162 MHz, acetonitrile^) δ 17.86. LCMS: MS m/z = 722.34 [M+1]; tR = 1.34 min; LC system:

Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min.

Example 89. Bis(cycloheptylmethyl) 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (89)

89a

Synthesis of cycloheptylmethyl 2-(chloro-15-azanyl)-2-methylpropanoate (89a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (2.5 g, 12.3 mmol), cycloheptylmethanol (2.18 g, 17.1 mmol), 4-dimethylaminopyridine (4.5 g, 36.9 mmol) and 3-ethyliminomethyleneamino)-N-dimethylpropan-1-amine hydrochloride (7.07 g, 36.9 mmol) were dissolved in dichloromethane (30 mL) and stirred at room temperature. After 17 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (2 x 50 mL), saturated ammonium chloride solution (50 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-50% ethyl acetate/hexanes with ELSD). The product-containing fractions were combined and the solvent was removed under reduced pressure. To a solution of the residue in dichloromethane (10 mL) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 10 mL, 24.6 mmol). After 18 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (40 mL). The residue was subjected to high vacuum for 5 h to afford intermediate 89a. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.67 (s, 3H), 3.98 (d, J=6.5 Hz, 2H), 1.88-1.78 (m, 1H), 1.74-1.60 (m, 3H), 1.58-128 049099

1.50 (m, 2H), 1.49 (s, 6H), 1.46-1.12 (m, 6H).

89a 89

PMPA (150 mg, 0.522 mmol) and intermediate 89a (417 mg, 1.67 mmol) were collected in an 8 mL vial with a stirring bar. To this mixture was added triethylamine (0.755 mL) followed by pyridine (3 mL), capped and stirred at 70 °C for 10 min. 2,2' Dipyridyl disulfide (575 mg, 2.61 mmol) and triphenylphosphine (685 mg, 2.61 mmol) were mixed in another 8 mL vial, pyridine (3 mL) was added, and the mixture was sonicated under argon until completely dissolved. The clear yellow solution was transferred to a stirred suspension of the above mixture. The reaction mixture was stirred at 80 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and co-evaporated with toluene (30 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g flash column, dried by flushing nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A 250 x 30 mm column, gradient 40-100% acetonitrile/water, 20 min cycle) to give the title compound (89). 1H NMR (400 MHz, acetonitrile^) δ 8.24 (s, 1H), 8.07 (s, 1H), 6.18 (s, 2H), 4.35 (dd, J=14.5, 3.2 Hz, 1H), 4.17 (dd, J=14.5, 7.0 Hz, 1H), 3.99-3.82 (m, 6H), 3.78 (d, J=10.8 Hz, 1H), 3.73-3.56 (m, 2H), 3.46 (dd, J=12.7, 9.9 Hz, 1H), 2.27 (s, 8H), 1.92-1.56 (m, 10H), 1.53 (s, 4H), 1.46 (d, J=1.7 Hz, 8H), 1.40 (s, 3H), 1.25 (ddt, J=15.0, 9.1, 2.7 Hz, 4H), 1.18 (d, J=6.3 Hz, 3H). ³¹ P NMR (162 MHz, acetonitrile^) δ 17.80. LCMS: MS m/z = 678.16 [M+1]; tR = 1.66 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100Α, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 90. Di([1,1'-bi(cyclobutane)]-3-yl)2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^)-bis(2-methylpropanoate) (90).

Synthesis of [1,1'-bi(cyclobutan)1-3-yl 2-amino-2-methylpropanoate (90a).

2-(tert-Butoxycarbonylamino)-2-methylpropanoic acid (1.2 g, 5.9 mmol), [1,1'bi(cyclobutane)]-3-ol (1.05 g, 8.32 mmol), 4-dimethylaminopyridine (2.16 g, 17.7 mmol) and 3-(ethyliminomethyleneamino)-1-dimethylpropan-1-amine hydrochloride (3.39 g, 17.7 mmol) were dissolved in dichloromethane (30 mL) and stirred at room temperature. After 17 h, the reaction mixture was diluted with DCM (10 mL). The solution was washed with water (2 x 20 mL), saturated ammonium chloride solution (20 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue was flash chromatographed (0-50% ethyl acetate/hexanes with ELSD). Fractions containing product were combined and the solvent was removed under reduced pressure. To a solution of the residue in dichloromethane (6 mL) was added a solution of hydrogen chloride in 1,4-dioxane (4N, 6.2 mL, 24.6 mmol). After 18 h, the solvent was removed under reduced pressure. The residue was co-evaporated with dichloromethane (40 mL). The residue was subjected to high vacuum for 5 h to afford intermediate 90a. ^1H NMR (400 MHz, DMSO- _d6 ) δ 8.64 (s, 3H), 4.91-4.84 (m, 1H), 2.45-2.25 (m, 3H), 2.08-1.86 (m, 3H), 1.83-1.52 (m, 6H), 1.47 (s, 6H).

- 129 049099

PMPA (150 mg, 0.522 mmol) and intermediate 90a (410 mg, 1.66 mmol) were collected in an 8 mL vial with a stirring bar. To this mixture was added triethylamine (0.755 mL) followed by pyridine (3 mL), capped and stirred at 70 °C for 10 min. 2,2' Dipyridyl disulfide (575 mg, 2.61 mmol) and triphenylphosphine (685 mg, 2.61 mmol) were mixed in another 8 mL vial, pyridine (3 mL) was added, and the mixture was sonicated under argon until completely dissolved. The clear yellow solution was transferred to a stirred suspension of the above mixture. The reaction mixture was stirred at 80 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and co-evaporated with toluene (30 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g column, the column was dried by purging with nitrogen for 2 min (to dry the DCM) and eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 uM, NX-C18, 110A 250x30 mm column, 50-100% acetonitrile/water gradient, 20 min cycle) to afford 90. 1H NMR (400 MHz, acetonitrile-6 ₃ ) δ 8.24 (s, 1H), 8.05 (s, 1H), 5.93 (s, 2H), 4.86-4.75 (m, 2H), 4.34 (dd, J=14.5, 3.3 Hz, 1H), 4.17 (dd, J=14.5, 7.1 Hz, 1H), 4.00-3.83 (m, 1H), 3.74-3.57 (m, 2H), 3.55-3.35 (m, 2H), 2.46-2.29 (m, 6H), 2.21-2.01 (m, 5H), 1.90-1.73 (m, 5H), 1.72-1.57 (m, 8H), 1.51 (s, 3H), 1.44 (s, 6H), 1.38 (s, 3H), 1.19 (d, J=6.2 Hz, 3H). ³¹ P NMR (162 MHz, acetonitrile-^) δ 17.69. LCMS: MS m/z = 674.14 [M+1]; tR = 1.63 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 91. Bis((trans-4-methylcyclohexyl)methyl) 2,2'-(((((((K)-1-(6-amino-9H-purin-9-yl)propan2-yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (91)

Synthesis of (trans-4-methylcyclohexyl)methyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (91a).

To a mixture of (trans-4-methylcyclohexyl)methanol (1.0 g, 7.80 mmol), 2-((tert-butoxycarbonyl)amino)2-methylpropanoic acid (2.4 g, 11.7 mmol), and DMAP (1.91 g, 15.6 mmol) in acetonitrile (30 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.42 g, 15.6 mmol) at room temperature. The mixture was stirred at room temperature for 15 h, quenched with water, and concentrated in vacuo. The resulting residue was dissolved in EtOAc, washed with brine, dried, concentrated in vacuo, and the residue was purified by silica gel chromatography (EtOAc, 0% to 50% hexanes) to give intermediate 91a. 1H NMR (400 MHz, acetonitrile-6 ₃ ) δ 5.62 (s, 1H), 3.88 (d, J = 6.4 Hz, 2H), 1.83-1.67 (m, 4H), 1.56 (m, 1H), 1.45-1.26 (m, 16H), 1.10-0.83 (m, 7H). LCMS: MS m/z = 313.83 [M+1]; tR = 2.12 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-O18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µl/min.

91a 91b

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Synthesis of trans-4-methylcyclohexyl)methyl 2-amino-2-methylpropanoate hydrochloride (91b).

To a solution of intermediate 91a (2.1 g, 6.70 mmol) in DCM (10 mL) was slowly added 4 M HCl in dioxane (10 mL) at room temperature. The resulting mixture was stirred at room temperature for 3.5 h, concentrated in vacuo, co-evaporated with DCM several times and dried under high vacuum for 15 h to afford intermediate 91b. 1H NMR (400 MHz, Chloroform-d) δ 9.00 (br s, 3H), 4.04 (d, J=6.5 Hz, 2H), 1.91-1.58 (m, 11H), 1.32 (m, 1H), 1.09-0.85 (m, 7H). LCMS: MS m/z = 214.02 [M+1-HCl]; tR = 1.89 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0.1 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 μL/min.

91b 91

A mixture of PMPA (300 mg, 1.04 mmol), intermediate 91b (800 mg, 3.20 mmol), 2,2'dipyridyl disulfide (1.15 g, 5.22 mmol), and triphenylphosphine (1.37 g, 5.22 mmol) was purged with nitrogen several times and dissolved in pyridine (7 mL). TEA (1.16 mL, 8.36 mmol) was added. The resulting mixture was stirred at 90 °C for 20 h, concentrated in vacuo, coevaporated with toluene several times, dissolved in EtOAc, washed vigorously with sat. sodium bicarbonate several times, dried over sodium sulfate, concentrated in vacuo, and purified by chromatography on silica gel (MeOH 0 to 15% in DCM) and with prep. HPLC (ACN 0 to 100% over 5 min in water and 100% over 18 min) to give the title compound (91). 1H NMR (400 MHz, acetonitrile^) δ 8.24 (s, 1H), 8.05 (s, 1H), 5.94 (s, 2H), 4.34 (dd, J=14.5, 3.3 Hz, 1H), 4.17 (dd, J=14.5, 7.0 Hz, 1H), 4.01-3.82 (m, 5H), 3.74 (d, J=10.7 Hz, 1H), 3.66 (dd, J=12.7, 8.9 Hz, 1H), 3.56 (d, J=10.8 Hz, 1H), 3.45 (dd, J=12.7, 9.9 Hz, 1H), 1.83-1.65 (m, 8H), 1.63-1.55 (m, 2H), 1.53 (s, 3H), 1.49-1.44 (m, 6H), 1.40 (s, 3H), 1.37-1.24 (m, 2H), 1.18 (d, J=6.2 Hz, 3H), 1.11-0.83 (m, 14H). ³¹ P NMR (162 MHz, acetonitrile^) δ 17.60. LCMS: MS m/z = 678.36 [M+1]; tR = 1.94 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-O8 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: tR = 6.95 min; HPLC system: 1290 Infinity II; column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 92. Bis(trans-4-propylcyclohexyl)methyl) 2,2'-(((((((K)-1-(6-amino-9H-purin-9-yl)propan2-yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (92)

EDCI

92a

Synthesis of (trans-4-propylcyclohexyl)methyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (92a).

To a mixture of (trans-4-propylcyclohexyl)methanol (1.0 g, 6.40 mmol), 2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid (1.951 g, 9.60 mmol), and DMAP (1.56 g, 12.8 mmol) in acetonitrile (30 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.99 g, 12.8 mmol) at room temperature. The mixture was stirred at room temperature for 15 h, quenched with water, and concentrated in vacuo. The resulting residue was dissolved in EtOAc, washed with brine, dried over sodium sulfate, concentrated in vacuo and purified by silica gel chromatography (EtOAc, 0 to 50% hexanes) to give intermediate 92a. ¹ H NMR (400 MHz, acetonitrile^) δ 5.55 (br s, 1H), 3.88 (d, J = 6.4 Hz, 2H), 1.83-1.70 (m, 4H), 1.67-1.49 (m, 1H), 1.45-1.29 (m, 17H), 1.28-1.13 (m, 3H), 1.10-0.80 (m, 7H). LCMS: MS m/z = 341.80 [M+1]; ]; tR = 2.07 min; LC system: Dionex Ultimate 3000 UHPLC; column: Phenomenex Kinetex 2.6μ C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0 min-0.2 min 40% acetonitrile, 0.2-1.55 min 40-100% acetonitrile, 1.55-2.80 min 100% acetonitrile, 2.80-2.81 min 100-40% acetonitrile at 1100 μL/min.

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Synthesis of trans-4-propylcyclohexyl)methyl 2-amino-2-methylpropanoate hydrochloride (92b).

To a solution of intermediate 92a (2.0 g, 5.86 mmol) in DCM (5 mL) was slowly added 4 M HCl in dioxane (4.83 mL) at room temperature. The resulting mixture was stirred at room temperature for 3.5 h, concentrated in vacuo, co-evaporated several times with DCM and dried under high vacuum for 15 h to give intermediate 92b. 1H NMR (400 MHz, Chloroform-d) δ 9.00 (br s, 3H), 4.04 (d, J=6.6 Hz, 2H), 1.88-1.59 (m, 11H), 1.33 (m, 2H), 1.26-1.11 (m, 3H), 1.05-0.81 (m, 7H). LCMS: MS m/z = 242.05 [M+1-HCl]; tR = 1.38 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XBC18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 μL/min.

O

92b 92

A mixture of PMPA (400 mg, 1.39 mmol), intermediate 92b (1.16 g, 4.18 mmol), 2,2'dipyridyl disulfide (1.53 g, 6.96 mmol), and triphenylphosphine (1.83 g, 6.96 mmol) was purged with nitrogen several times and dissolved in pyridine (10 mL). Triethylamine (1.54 mL, 11.10 mmol) was added. The resulting mixture was stirred at 90 °C for 20 h, concentrated in vacuo, coevaporated with toluene several times, dissolved in EtOAc, washed vigorously with sat. sodium bicarbonate several times, dried over sodium sulfate, concentrated, and purified by chromatography on silica gel (MeOH 0 to 10% in DCM) and with prep. HPLC (ACN 100% for 43 min) to give the title compound (92). 1H NMR (400 MHz, acetonitrile) δ 8.24 (s, 1H), 8.06 (s, 1H), 5.99 (s, 2H), 4.34 (dd, J=14.5, 3.3 Hz, 1H), 4.17 (dd, J=14.5, 7.0 Hz, 1H), 4.01-3.83 (m, 5H), 3.75 (d, J=10.7 Hz, 1H), 3.66 (dd, J=12.7, 8.9 Hz, 1H), 3.57 (d, J=10.8 Hz, 1H), 3.45 (dd, J=12.7, 9.8 Hz, 1H), 1.82-1.68 (m, 8N), 1.60 (m, 2H), 1.53 (s, 3H), 1.50-1.42 (m, 6H), 1.40 (s, 3H), 1.38-1.29 (m, 4H), 1.24-1.10 (m, 9H), 1.09-0.80 (m, 14H). ³¹ P NMR (162 MHz, acetonitrile^ ₃ ) δ 17.58. LCMS: MS m/z = 734.42 [M+1]; ]; t _R = 2.15 min; LC system: Dionex Ultimate 3000 UHPLC; column: Phenomenex Kinetex 2.6μ C18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0 min-0.2 min 40% acetonitrile, 0.2-1.55 min 40-100% acetonitrile, 1.55-2.80 min 100% acetonitrile, 2.80-2.81 min 100-40% acetonitrile at 1100 µL/min. HPLC: t _R = 8.35 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100x4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min - 8.5 min 2-98% ACN at 1.5 ml/min.

Example 93. Bis(trans-4-phenylcyclohexyl) 2,2'-((((((^)-1-(6-amino-9H-purin-9-yl)propan-2yl)oxy)methyl)phosphoryl)bis(azanediyl))bis(2-methylpropanoate) (93)

93a

Synthesis of trans-4-phenylcyclohexyl-2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (93a).

To a solution of trans-4-phenylcyclohexanol (1.0 g, 5.67 mmol), 2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid (1.73 g, 8.51 mmol), and DMAP (1.39 g, 11.3 mmol) in acetonitrile (30 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.76 g, 11.3 mmol) at room temperature. The mixture was stirred at room temperature for 5 h, quenched with water, and concentrated in vacuo. The resulting residue was dissolved in EtOAc, washed with brine, dried over sodium sulfate, concentrated in vacuo, and purified by silica gel chromatography (EtOAc, 0 to 50% hexanes) to give intermediate 93a. 1H NMR (400 MHz, acetonitrile^) δ 7.36-7.11 (m, 5H), 5.55 (br s, 1H), 4.76 (m, 1H), 2.59 (m, 1H), 2.10-2.01 (m, 2H), 1.96-1.86 (m, 2H), 1.72-1.58 (m, 2H), 1.57-1.45 (m, 2H), 1.42-1.32 (m, 15H). LCMS: MS m/z = 361.84 [M+1]; ]; tR = 1.85 min; LC system: Dionex Ultimate 3000 UHPLC; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 0 min-0.2 min

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40% acetonitrile, 0.2-1.55 min 40-100% acetonitrile, 1.55-2.80 min 100% acetonitrile, 2.80-2.81 min 100-40% acetonitrile at 1100 µl/min

Synthesis of trans-4-phenylcyclohexyl 2-amino-2-methylpropanoate hydrochloride (93b).

To a mixture of intermediate 93a (1.1 g, 3.04 mmol) in DCM (5 mL) was slowly added 4 M HCl in dioxane (2.51 mL) at room temperature. The resulting mixture was stirred at room temperature for 4 h, concentrated in vacuo, co-evaporated with DCM several times, and dried under high vacuum for 15 h to yield intermediate 93b. 1H NMR (400 MHz, Chloroform-d) δ 9.06 (br s, 3H), 7.34-7.26 (m, 2H), 7.24-7.13 (m, 3H), 4.92 (m, 1H), 2.57 (m, 1H), 2.18 (dd, J=12.9, 4.2 Hz, 2H), 1.98 (m, 2H), 1.89-1.50 (m, 10H).

93b 93

A mixture of PMPA (320 mg, 1.11 mmol), intermediate 93b (929 mg, 3.12 mmol), 2,2'dipyridyl disulfide (1.23 g, 5.57 mmol) and triphenylphosphine (1.46 g, 5.57 mmol) was purged with nitrogen several times and dissolved in pyridine (10 mL). TEA (1.54 mL, 11.10 mmol) was added. The resulting mixture was stirred at 90 °C for 20 h, concentrated in vacuo, co-evaporated with toluene several times, dissolved in EtOAc, washed vigorously with saturated sodium bicarbonate solution several times, dried with sodium sulfate, concentrated and purified by silica gel chromatography (MeOH 0 to 10% in DCM) to afford compound (93). 1H NMR (400 MHz, acetonitrile^) δ 8.26 (s, 1H), 8.09 (s, 1H), 7.36-7.14 (m, 10H), 6.12 (s, 2H), 4.78 (m, 2H), 4.36 (dd, J=14.5, 3.2 Hz, 1H), 4.19 (dd, J=14.5, 7.0 Hz, 1H), 3.96 (m, 1H), 3.78 (d, J=10.6 Hz, 1H), 3.69 (dd, J=12.6, 8.9 Hz, 1H), 3.60 (d, J=10.7 Hz, 1H), 3.49 (dd, J=12.6, 9.8 Hz, 1H), 2.57 (m, 2H), 2.12-2.01 (m, 4H), 1.96-1.84 (m, 4H), 1.73-1.44 (m, 17H), 1.42 (s, 3H), 1.21 (d, J=6.2 Hz, 3H). ^31P NMR (162 MHz, acetonitrile^) δ 17.68. LCMS: MS m/z = 774.31 [M+1]; tR = 1.90 min; LC system: Thermo Accela 1250 UHPlC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 1002% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µL/min. HPLC: tR = 6.99 min; HPLC system: 1290 Infinity II; column: Phenomenex 2.6 µm C18 100A, 100x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 94. Dihexyl 2,2'-(((((1-(6-(((2-(benzoyloxy)ethoxy)carbonyl)amino)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (94)

O

94

To a mixture of 1 (200 mg, 0.32 mmol) and triphosgene (38 mg, 0.128 mmol) in DCM (4 mL) was slowly added DMAP (234 mg, 1.92 mmol) in portions at room temperature. The resulting mixture was stirred for 15 min at room temperature and 2-benzoyloxyethanol (60 mg, 0.46 mol) was added at room temperature. The resulting mixture was stirred for 2 h, concentrated in vacuo and purified by column chromatography on silica gel to give the title compound (94). 1H NMR (400 MHz, acetonitrile^) δ 9.67 (br.s, 1H), 8.61 (s, 1H), 8.32 (s, 1H), 8.01-7.92 (m, 2H), 7.65-7.57 (m, 1H), 7.47-7.40 (m, 2H), 4.59-4.53 (m, 4H), 4.36 (dd, J=14.5, 3.2 Hz, 1H), 4.19 (dd, J=14.5, 7.1 Hz, 1H), 4.16

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3.99 (m, 4H), 3.93 (m, 1H), 3.73 (d, J=10.7 Hz, 1H), 3.67 (dd, J=12.8, 8.7 Hz, 1H), 3.55 (d, J=10.8 Hz, 1H), 3.43 (dd, J=12.8, 9.7 Hz, 1H), 1.68-1.55 (m, 4H), 1.49 (s, 3H), 1.44-1.37 (m, 6H), 1.38-1.23 (m, 15H), 1.16 (d, J=6.2 Hz, 3H), 0.92-0.82 (m, 6H). ³¹ P NMR (162 MHz, acetonitrile^) δ 17.71. LCMS: MS m/z = 818.30 [M+1]; tR = 1.88 min. LC system: Dionex Ultimate 3000 UHPLC; column: Phenomenex Kinetex 2.6μ C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0 min-0.2 min 40% acetonitrile, 0.2-1.55 min 40-100% acetonitrile, 1.55-2.80 min 100% acetonitrile, 2.80-2.81 min. 100-40% acetonitrile at 1100 µL/min. HPLC: tR = 7.42 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100x4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 95. Dihexyl 2,2'-(((((1-(6-(((2-((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)-2oxoethoxy)carbonyl)amino)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate) (95)

Synthesis of (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl-2-hydroxyacetate (95a).

To a suspension _{of K2CO3 (} 716 mg, 5.18 mmol) in DMF (5 mL) was added glycolic acid (197 mg, 2.59 mmol) at room temperature. The resulting mixture was stirred at room temperature for 15 min and NaI (39 mg, 0.259 mmol) and 4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (500 mg, 2.59 mmol) were added. The mixture was stirred at room temperature for 3 h, filtered, the filter cake was washed with DCM, the combined filtrate was concentrated in vacuo and the resulting residue was purified by silica gel chromatography (EtOAc from 0 to 70% in hexane) to give intermediate 95a. 1H NMR (400 MHz, acetonitrile^) δ 4.95 (s, 2H), 4.13 (d, J=6.5 Hz, 2H), 3.25 (t, J=6.5 Hz, 1H), 2.16 (s, 3H).

NH-

triphosgene DMAP DCM o

To a mixture of 1 (100 mg, 0.160 mmol) and triphosgene (31.3 mg, 0.105 mmol) in DCM (2 mL) was slowly added DMAP (117 mg, 0.959 mmol) in portions at room temperature. The resulting mixture was stirred at room temperature for 15 min and intermediate 95a (60 mg, 0.32 mmol) was added at room temperature. The resulting mixture was stirred for 2 h and additional triphosgene (30 mg) was added. The mixture was stirred for 1 h at room temperature, concentrated in vacuo, dissolved in ACN and purified by preparative HPLC (10 to 100% ACN in water for 5 min and in 100% ACN for 10 min) and silica gel chromatography (MeOH 0 to 10% in DCM) to give the title compound (95). 1H NMR (400 MHz, acetonitrile^) δ 9.10 (br.s, 1H), 8.64 (s, 1H), 8.35 (s, 1H), 5.01 (s, 2H), 4.77 (s, 2H), 4.48-4.42 (m, 1H), 4.27 (dd, J=14.6, 7.1 Hz, 1H), 4.17-4.01 (m, 4H), 3.97 (m, 1H), 3.82-3.66 (m, 2H), 3.57 (d, J=10.8 Hz, 1H), 3.45 (dd, J=12.8, 9.7 Hz, 1H), 2.16 (s, 3H), 1.69-1.55 (m, 4H), 1.52 (s, 3H), 1.45 (s, 6H), 1.41-1.26 (m, 15H), 1.20 (d, J=6.2 Hz, 3H), 0.95-0.84 (m, 6H). ^31P NMR (162 MHz, acetonitrile^) δ 17.67. LCMS: MS m/z = 840.22 [M+1]; tR = 1.99 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 1002% acetonitrile, 1.85-2.00 min 2% ACN at 1800 µl/min. HPLC: tR = 7.10 min; HPLC system: 1290

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Infinity II; column: Phenomenex 2.6 µm C18 100A, 100x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min - 8.5 min 2-98% ACN at 1.5 mL/min.

Example 96. Dihexyl-2,2'-((((((K)-1-(6-((K)-5-oxotetrahydrofuran-2-carboxamido)-9H-purin-9yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azandiyl))bis(2-methylpropanoate) (96)

To a mixture of (N)-5-oxotetrahydrofuran-2-carboxylic acid (41.6 mg, 3.2 mmol) and DMAP (39 mg, 0.32 mmol) in ACN (4 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (61.3 mg, 3.20 mmol) at room temperature. The resulting mixture was stirred at room temperature for 15 min and 1 (100 mg, 0.16 mmol) in DCM (1 mL) was added. The resulting mixture was stirred at room temperature for 4 h, quenched by addition of water (0.5 mL) and purified by preparative HPLC (ACN 10 to 100% in water for 8 min and ACN 100% for 10 min) to give the title compound (96). 1H NMR (400 MHz, acetonitrile-0 ₃ ) δ 9.27 (s, 1H), 8.66 (s, 1H), 8.37 (s, 1H), 5.55 (dd, J=8.6, 5.3 Hz, 1H), 4.45 (dd, J=14.6, 3.2 Hz, 1H), 4.27 (m, 1H), 4.17-4.02 (m, 4H), 3.97 (m, 1H), 3.79-3.62 (m, 2H), 3.58 (d, J=10.8 Hz, 1H), 3.45 (dd, J=12.7, 9.8 Hz, 1H), 2.78-2.66 (m, 1H), 2.62-2.54 (m, 2H), 2.46-2.36 (m, 1H), 1.70-1.56 (m, 4H), 1.52 (s, 3H), 1.45 (s, 6H), 1.41-1.25 (m, 15H), 1.20 (d, J=6.2 Hz, 3H), 0.95-0.85 (m, 6H). ^31P NMR (162 MHz, acetonitrile-a ₃ ) δ 17.73. LCMS: MS m/z = 738.24 [M+1]; tR = 1.92 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6 μm XB-C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0-1.8 min 2-100% acetonitrile, 1.8-1.85 min 100-2% acetonitrile, 1.85-2.00 min 2% ACN at 1800 μL/min. HPLC: tR = 6.32 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 μm C18 100A, 100x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0 min - 8.5 min 2-98% ACN at 1.5 ml/min.

Example 97. Dihexyl 2,2'-(((((1-(6-(4-(acetylthio)butanamido)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(K)-bis(2-methylpropanoate) (97)

To a mixture of 4-(acetylthio)butanoic acid (41.6 mg, 0.26 mmol) and DMAP (39 mg, 0.32 mmol) in ACN (4 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (61.3 mg, 0.32 mmol) at room temperature. The resulting mixture was stirred at room temperature for 15 min and 1 (100 mg, 0.16 mmol) in DCM (1 mL) was added. The resulting mixture was stirred at room temperature for 4 h, quenched by adding water (0.5 mL), and purified by preparative HPLC (10 to 100% acetonitrile in water for 5 min and 100% acetonitrile for 13 min) and gas chromatography

- 135 049099 by silica gel chromatography (MeOH 0 to 10% in DCM) to give the title compound (97). ¹ H NMR (400 MHz, acetonitrile-a ₃ ) δ 9.02 (s, 1H), 8.62 (s, 1H), 8.32 (s, 1H), 4.43 (dd, J=14.5, 3.1 Hz, 1H), 4.25 (dd, J=14.5, 7.2 Hz, 1H), 4.18-4.02 (m, 4H), 3.96 (m, 1H), 3.80-3.64 (m, 2H), 3.58 (d, J=10.8 Hz, 1H), 3.44 (dd, J=12.7, 9.9 Hz, 1H), 2.98 (t, J=7.2 Hz, 2H), 2.88 (t, J=7.3 Hz, 2H), 2.33 (s, 3H), 1.97 (m, 2H), 1.63 (m, 4H), 1.52 (s, 3H), 1.45 (s, 6H), 1.42-1.23 (m, 15H), 1.20 (d, J=6.2 Hz, 3H), 0.96-0.85 (m, 6H). ³¹ P NMR (162

MHz, acetonitrile-0 ₃ ) δ 17.79. LCMS: MS m/z = 770.36 [M+1]; tR = 1.78 min, LC system: Dionex Ultimate 3000 UHPLC; column: Phenomenex Kinetex 2.6μ C18 100A, 50x3.0 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0 min-0.2 min 40% acetonitrile, 0.2-1.55 min 40-100% acetonitrile, 1.55-2.80 min. 100% acetonitrile, 2.80-2.81 min 100-40% acetonitrile at 1100 µL/min. HPLC: tR = 7.09 min; HPLC system: 1290 Infinity II; Column: Phenomenex 2.6 µm C18 100A, 100 x 4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 0 min 8.5 min 2-98% ACN at 1.5 mL/min.

Example 98. Dihexyl 2,2'-((((( 1 -(6-(((2,2-dimethyl-3-((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)-3oxopropoxy)carbonyl)amino)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))adabis(2-methylpropanoate) (98)

98a

Synthesis of (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl-3-hydroxy-2,2-dimethylpropanoate (98a).

4-(Bromomethyl)-5-methyl-1,3-dioxol-2-one (1.20 g, 5.93 mmol) was added to a mixture of 3-hydroxy-2,2-dimethylpropanoic acid (700 mg, 5.93 mmol), potassium carbonate (983 mg, 7.11 mmol) and sodium iodide (888 mg, 5.93 mmol) in N,N-dimethylformamide (10 mL) at 0 °C. After 16 h, the solids were removed by filtration and washed with ethyl acetate (20 mL). The other solution was diluted with ethyl acetate (20 mL). The solution was washed with aqueous lithium chloride solution (5%, 2 x 25 mL) and brine (25 mL). The organic phase was dried over sodium sulfate and the solvent was removed under reduced pressure. The residue was flash chromatographed (0-100% ethyl acetate/hexanes with ELSD). Fractions containing the product were combined and the solvent was removed under reduced pressure to give intermediate 98a. ¹ H NMR (400 MHz, acetonitrile-03) δ 4.88 (s, 2H), 3.56-3.46 (m, 2H), 2.94 (s, 1H), 2.15 (s, 3H), 1.15 (s, 6H).

4-Dimethylaminopyridine (58.6 mg, 0.479 mmol) was added to a solution of triphosgene (14.2 mg, 0.0479 mmol) in dichloromethane (4 mL), forming a solid. After 1 min, 1 (100 mg, 0.160 mmol) was added and the solids dissolved. After 15 min, intermediate 98a (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl 3-hydroxy-2,2-dimethylpropanoate (73.6 mg, 0.160 mmol) was added. After 20 h, the mixture was diluted with DCM (5 mL). The mixture was washed with water (3 x 5 mL) and saturated ammonium chloride (5 mL). The organic phase was dried over sodium sulfate and the solvent was removed under reduced pressure. The residue was subjected to flash chromatography (0-30% methanol/dichloromethane). Fractions containing the product were pooled and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A column 250 x 30 mm, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were pooled and lyophilized to give the title compound (98). ¹ H NMR (400 MHz, acetonitrile-0 ₃ ) δ 8.71 (s, 1H), 8.61 (s, 1H), 8.30 (s, 1H), 4.90 (s, 2H), 4.44 (dd, J=14.5, 3.2 Hz, 1H), 4.32-4.21 (m, 3H), 4.18-4.01 (m,

- 136 049099

4H), 4.01-3.91 (m, 1H), 3.78-3.63 (m, 2H), 3.57 (d, J=10.8 Hz, 1H), 3.45 (dd, J=12.7, 9.8 Hz, 1H), 2.11 (s, 3H), 1.71-1.57 (m, 4H), 1.52 (s, 3H), 1.49-1.42 (m, 6H), 1.42-1.24 (m, 21H), 1.20 (d, J=6.2 Hz, 3H), 0.96-0.85 (m, 6H). ³¹ P NMR (162 MHz, acetonitrile^) δ 17.63 (n, J=10.0 Hz). LCMS: MS m/z = 882.18 [M+1]; tR = 1.743 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: t _R = 3.888 min; HPLC system: Agilent 1100 series; column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 mL/min.

Example 99

99a

Synthesis of benzyl 5-[[9-[(2R)-2-[bis[(2-hexoxy-1,1-dimethyl-2-oxo-ethyl)amino]phosphorylmethoxy]propyl]purin-6-yl]amino]-5-oxo-pentanoate (99a).

A solution of 1 (200 mg, 0.320 mmol), 5-benzyloxy-5-oxopentanoic acid (85.2 mg, 0.384 mmol), 4-dimethylaminopyridine (78.1 mg, 0.639 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (123 mg, 0.639 mmol) in dichloromethane (4 mL) was stirred for 3 days. The reaction mixture was diluted with dichloromethane (20 mL). The mixture was washed with water (2 x 10 mL) and saturated ammonium chloride (10 mL). The organic phase was dried over sodium sulfate and the solvent was removed under reduced pressure. The residue was flash chromatographed (0-100% ethyl acetate/hexanes). The product-containing fractions were pooled and the solvent was removed under reduced pressure to yield intermediate 99a. LCMS: MS m/z = 830.24 [M+1]; t _R = 1.760 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

99a

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Synthesis of (^-5-((9-(2-((bis((1-(hexyloxy)-2-methyl-1-oxopropan-2-yl)amino)phosphoryl)methoxy)propyl)-9H-purina-6-yl)amino)-5-oxopentanoic acid (99b).

Palladium on carbon (10%, 90 mg, 0.084 mmol) was added to a solution of intermediate 99a (235 mg, 0.283 mmol) in ethyl acetate (5 mL). The atmosphere was saturated with hydrogen using a balloon. The mixture was stirred at room temperature under hydrogen atmosphere. After 16 h, the hydrogen was removed. Palladium on carbon was removed by filtration. The solvent was removed under reduced pressure, yielding intermediate 99b. LCMS: MS m/z = 740.22 [M+1]; tR = 1.573 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

o o \ . potassium carbonate x I O N + Dimethylformamide

-O ⁿ NH I 0o o

4-(Bromomethyl)-5-methyl-1,3-dioxol-2-one (21.9 mg, 0.114 mmol) was added to a mixture of intermediate 99b (70.0 mg, 0.0946 mmol) and potassium carbonate (19.6 mg, 0.142 mmol) in dimethylformamide (4 ml). After 90 min, the solids were removed by filtration. The filtrate was diluted with ethyl acetate (20 ml) and washed with 5% lithium chloride (3 x 10 ml) and brine (10 ml). The organic phase was dried over sodium sulfate and the solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Gemini, 10 μM, NX-C18, 110A 250x30 mm column, gradient 40-100% acetonitrile/water over 20 min). Fractions containing the product were combined and lyophilized to give the title compound (99). 1H NMR (400 MHz, acetonitrile^ ₃ ) δ 8.95 (s, 1H), 8.61 (s, 1H), 8.32 (s, 1H), 4.88 (s, 2H), 4.43 (dd, J=14.5, 3.2 Hz, 1H), 4.26 (dd, J=14.6, 7.1 Hz, 1H), 4.18-4.01 (m, 4H), 4.01-3.92 (m, 1H), 3.77-3.64 (m, 2H), 3.57 (d, J=10.8 Hz, 1H), 3.45 (dd, J=12.7, 9.8 Hz, 1H), 2.88 (t, J=7.2 Hz, 2H), 2.48 (t, J=7.4 Hz, 2H), 2.14 (s, 3H), 2.04-1.98 (m, 2H), 1.70-1.56 (m, 4H), 1.52 (s, 3H), 1.49-1.41 (m, 6H), 1.41-1.23 (m, 12H), 1.20 (d, J=6.2 Hz, 3H), 0.97-0.83 (m, 6H). ^31P NMR (162 MHz, acetonitrile^) δ 17.69 (p, J=10.0 Hz). LCMS: MS m/z = 852.27 [M+1]; tR = 1.680 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; Gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 mL/min. HPLC: tR = 3.608 min; HPLC system: Agilent 1100 Series; Column: Gemini 5 μm C18 110A, 50x4.6 mm; solvents: A - acetonitrile with 5% water and 0.1% TFA, B - water with 5% acetonitrile and 0.1% TFA; gradient: 0-4.0 min 2-95% B, 4.0-5.0 min 95% B, 5.0-5.25 min 95-98% B, 5.25-5.50 min 98-2% B at 2 ml/min.

Example 100. Dihexyl 2,2'-(((((1-(6-(3-(2-acetoxy-4,6-dimethylphenyl)-3-methylbutanamido)-9Hpurin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(R.)-bis(2-methylpropanoate) (100)

- 138 049099

100

Compound 1 (240 mg, 0.384 mmol), 3-(2-acetoxy-4,6-dimethylphenyl)-3-methylbutanoic acid (406 mg, 1.53 mmol), 4-dimethylaminopyridine (141 mg, 1.15 mmol), 3(ethyliminomethyleneamino)-N-dimethylpropan-1-amine hydrochloride (221 mg, 1.15 mmol) were placed in a 40 mL vial with a stir bar and DCM (4 mL) was added. The mixture was flushed with argon, capped and stirred at room temperature for 15 h. The reaction mixture was diluted with DCM (15 mL). The solution was washed with water (2 x 15 mL) and once with saturated ammonium chloride solution (15 mL) and dried over sodium sulfate. The solution was concentrated under reduced pressure. This residue was dissolved in acetonitrile (4 mL), filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A 250x30 mm column, gradient 40-100% acetonitrile/water in 20 min) to give the title compound (100). 1H NMR (400 MHz, acetonitrile-b ₃ ) δ 8.62 (s, 1H), 8.58 (s, 1H), 8.28 (s, 1H), 6.84 (d, J=2.0 Hz, 1H), 6.60 (d, J=2.0 Hz, 1H), 4.42 (dd, J=14.5, 3.1 Hz, 1H), 4.24 (dd, J=14.5, 7.1 Hz, 1H), 4.16-4.00 (m, 4H), 3.98-3.91 (m, 1H), 3.76-3.63 (m, 2H), 3.56 (d, J=10.8 Hz, 1H), 3.44 (dd, J=12.7, 9.8 Hz, 1H), 3.31 (s, 2H), 2.58 (s, 3H), 2.25 (d, J=2.8 Hz, 9H), 2.21 (s, 3H), 1.70-1.56 (m, 12H), 1.52 (s, 3H), 1.45 (d, J=2.1 Hz, 6H), 1.38 (s, 6H), 1.35-1.25 (m, 7H), 1.19 (d, J=6.3 Hz, 3H), 0.96-0.83 (m, 6H). LCMS: MS m/z = 872.4 [M+1]; tR = 1.82 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 101. Dihexyl 2,2'-(((((1-(6-((((2-methyl-4-((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)-4oxobutan-2-yl)oxy)carbonyl)amino)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(I)-bis(2-methylpropanoate) (101)

Synthesis of (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl-3-hydroxy-3-methylbutanoate (101a).

To an ice-cooled mixture of 3-hydroxy-3-methylbutanoic acid (1 g, 8.47 mmol), K ₂ CO ₃ (1.4 g, 10.2 mmol), NaI (1.27 g, 8.47 mmol) and DMF (15 mL) was added 4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (1.7 mL, 8.47 mmol) dropwise. After 5 min, the ice was removed and the mixture was stirred at room temperature for 15 h. The reaction mixture was diluted with EtOAc, washed with water (2 x 25 mL) and once with 5% LiCl solution (40 mL). The combined organic mixture was dried over sodium sulfate and concentrated to give intermediate 101a, which was used in subsequent reactions without purification. 1H NMR (400 MHz, acetonitrile^) δ 4.90 (s, 2H), 3.06 (s, 1H), 2.51 (s, 2H), 2.15 (d, J=3.6 Hz, 3H), 1.27 (s, 6H).

- 139 049099

101

Compound 1 (75 mg, 0.12 mmol), triphosgene (35.6 mg, 0.12 mmol), 4-dimethylaminopyridine (43.9 mg, 0.36 mmol) were placed in a 40 mL vial with a stirring bar, DCM (3 mL) was added and stirred at room temperature for 15 min. After 15 min, (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl 3-hydroxy-3-methylbutanoate (101a, 66.2 mg, 0.14 mmol) was added and stirred at room temperature for 2 h. The solvent was concentrated under reduced pressure. This residue was dissolved in acetonitrile (4 ml), filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A column 250x30 mm, gradient 50%-100% acetonitrile/water in 20 min) to give the title compound (101). 1H NMR (400 MHz, acetonitrile^) δ 8.59 (s, 1H), 8.29 (s, 1H), 8.28 (s, 1H), 4.88 (s, 2H), 4.43 (dd, J=14.5, 3.2 Hz, 1H), 4.25 (dd, J=14.5, 7.1 Hz, 1H), 4.15-4.05 (m, 4H), 3.99-3.92 (m, 1H), 3.79-3.62 (m, 2H), 3.62-3.35 (m, 2H), 3.03 (s, 2H), 2.08 (s, 3H), 1.71-1.58 (m, 12H), 1.53 (s, 3H), 1.46 (d, J=1.9 Hz, 7H), 1.39 (s, 4H), 1.32 (td, J=6.0, 3.6 Hz, 8H), 1.20 (d, J=6.2 Hz, 3H), 0.94-0.87 (m, 6H). ³¹ P NMR (162 MHz, acetonitrile^ ₃ ) δ 17.67. LCMS: MS m/z = 882.1 [M+1]; tR = 1.72 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; Solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 102. Dihexyl 2,2'-(((((1-(6-(3,3-dimethyl-5-((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)-5oxopentanamido)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(^-bis(2-methylpropanoate)(102)

102a

Synthesis of 3,3-dimethyl-5-((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)-5-oxopentanoic acid (102a)

To an ice-cooled mixture of 3,3-dimethylpentanedic acid (1 g, 6.24 mmol), K ₂ CO ₃ (1.03 g, 7.49 mmol) in DMF (15 mL) was added dropwise 4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (1.26 g, 6.24 mmol). After 5 min, the ice was removed and the mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with EtOAc, washed with water (2 x 25 mL) and once with 5% LiCl solution (40 mL). The combined organic mixture was dried over sodium sulfate and concentrated. The residue was purified by flash chromatography (0-20% MeOH/DCM with ELSD). The fractions containing the product were combined and the solvent was removed under reduced pressure to yield intermediate 102a. 1H NMR (400 MHz, acetonitrile^) δ 8.90 (s, 1H), 4.87 (s, 2H), 2.47 (s, 2H), 2.45 (s, 3H), 2.38 (d, J=3.9 Hz, 2H), 1.11 (s, 6H).

- 140 049099

102

Compound 1 (200 mg, 0.32 mmol), intermediate 102a (338 mg, 1.24 mmol), 4-dimethylaminopyridine (117 mg, 0.95 mmol), 3-(ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine hydrochloride (184 mg, 0.95 mmol) were placed in a 40 mL vial with a stir bar and DCM (6 mL) was added. The mixture was flushed with argon, capped and stirred at room temperature for 15 h. The reaction mixture was diluted with DCM (15 mL). The solution was washed with water (2 x 15 mL) and once with saturated ammonium chloride solution (15 mL) and dried over sodium sulfate. The solution was concentrated under reduced pressure. This residue was dissolved in acetonitrile (6 mL), filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A 250x30 mm column, gradient 50-100% acetonitrile/water, 20 min cycle) to give the title compound (102). 1H NMR (400 MHz, acetonitrile^) δ 8.94 (s, 1H), 8.62 (s, 1H), 8.31 (s, 1H), 4.91 (s, 2H), 4.44 (dd, J=14.5, 3.1 Hz, 1H), 4.25 (dd, J=14.5, 7.2 Hz, 1H), 4.17-4.02 (m, 4H), 4.00-3.93 (m, 1H), 3.78-3.63 (m, 2H), 3.55 (d, J=10.8 Hz, 1H), 3.44 (dd, J=12.7, 9.8 Hz, 1H), 2.76 (s, 2H), 2.57 (s, 2H), 2.14 (s, 3H), 1.68-1.59 (m, 5H), 1.52 (s, 3H), 1.45 (d, J=3.2 Hz, 6H), 1.38 (s, 5H), 1.36-1.25 (m, 8H), 1.21-1.17 (m, 10H), 0.96-0.84 (m, 6H). ^3| P NMR (162 MHz, acetonitrile^) δ 17.67. LCMS: MS m/z = 880.2 [M+1]; tR = 1.73 min; LC system: Thermo Dionex Ultimate 3000 UHPLC+ focusing; MS system: Thermo LCQ Fleet; Column: Phenomenex Kinetex 2.6μ C18 100A, 50x2.1 mm; solvents: acetonitrile with 0.1% trifluoroacetic acid, water with 0.1% trifluoroacetic acid; gradient: 0-0.2 min 10% acetonitrile, 0.2-1.55 min 10-100% acetonitrile, 1.55-2.20 min 100% acetonitrile at 1.0 ml/min.

Example 103. Diphenethyl 2,2'-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(azanediyl))(K)-bis(2-methylpropanoate) (103)

103a

Synthesis of phenethyl 2-amino-2-methylpropanoate hydrochloride (103a).

Intermediate 103a was synthesized in the same manner as intermediate 87a using 2-amino-2-methylpropanoic acid (1 g, 9.7 mmol), 2-phenylethan-1-ol (3.88 g, 31.8 mmol) and 4N HCl/dioxane (15 mL) to afford intermediate 103a. LCMS: MS m/z = 208.04 [M+1], tR = 1.04 min; LC System: Thermo Accela 1250 UHPLC; MS System: Thermo LCQ Fleet; Column: Kinetex 2.6 μm XB-C18 100A, 50x4.6 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min, 100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 µL/min.

103a 103

PMPA (100 mg, 0.348 mmol) and intermediate 103a (339 mg, 1.39 mmol) were collected in an 8 mL vial with a stirring bar. Triethylamine (0.5 mL) was added to this mixture, followed by

- 141 049099 then pyridine (2.5 mL), capped and stirred at 70 °C for 10 min. 2,2' Dipyridyl disulfide (384 mg, 1.74 mmol) and triphenylphosphine (457 mg, 1.74 mmol) were mixed in another 8 mL vial, pyridine (2.5 mL) was added, sonicated until completely dissolved under argon. The clear yellow solution was transferred to a stirred suspension of the above mixture. The reaction mixture was stirred at 80 °C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure and co-evaporated with toluene (20 mL x 2). The residue was dissolved in DCM, loaded onto a 24 g flash column, dried by flushing nitrogen for 2 min (to dry DCM) and eluted with 100% EtOAc for 6 min, 0-15% MeOH/DCM for 15 min to give a mixture. This mixture was dissolved in MeOH, filtered and purified by preparative HPLC (Gemini, 10 μM, nX-C18, 110A column 250x30 mm, gradient 40-100% acetonitrile/water, 20 min cycle) to give the title compound (103). LCMS: MS m/z = 666.15 [M+1], t _R = 1.41 min; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6 μm HB-C18 100Α, 50x4.6 mm; Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 0-2.0 min, 2-100% acetonitrile, 2.0-3.05 min, 100% acetonitrile, 3.05-3.2 min, 100-2% acetonitrile, 3.2-3.5 min, 2% ACN at 2 μL/min. HPLC: t _R = 5.19 min; HPLC system: 1290 Infinity II series; Column: Phenomenex Kinetex 2.6μ C18 100Α, 100x4.6 mm; Solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 8.5 min, 2-98% ACN, at 1.5 ml/min.

Example 104. Analysis of antiviral (HIV) activity in MT-4 cells.

Compounds were tested in a high-throughput 384-well assay format for their ability to inhibit HIV-1(IIIB) replication in MT-4 cells. Compounds were serially diluted (1:3) in DMSO in 384-well polypropylene plates and further diluted 200-fold in complete RPMI medium (10% FBS, 1% P/S) using a Biotek Micro Flow acoustic dispenser and Labcyte ECHO. Each plate contained up to 8 test compounds with negative (no drug) and positive (5 pM AZT) controls. MT-4 cells were pretreated with 10 pL of either RPMI (mock-infected) or a fresh 1:250 dilution of HIV-1 IIIB virus stock. Infected and uninfected MT-4 cells were further diluted in complete RPMI medium and added to each plate using the Micro Flow dispenser. After 5 days of incubation in a temperature-controlled, humidified incubator (37°C), Cell Titer Glo (Promega) was added to the assay plates and chemiluminescence data were read with an Envision plate reader. EC ₅₀ values were defined as the concentration of compound that caused a 50% decrease in luminescence and were calculated using a sigmoidal dose-response model to fit the curves as shown in Table 1.

Example 105. Cytotoxicity analysis in MT-4 cells.

The assays were performed as described in Example 104, except that uninfected MT-4 cells were added to each well containing the test compound. In addition, 10 μM puromycin was added to the last column of each assay plate to assess the baseline level of cytotoxicity. The EC ₅₀ values are shown in Table 1.

Example 106. Analysis of antiviral (HBV) activity in primary human hepatocytes (PHH).

Cryopreserved RNNs were thawed, recovered by centrifugation at 100 g in cryopreserved hepatocyte recovery medium (Thermo Fisher Scientific; CM7500), and plated on a collagen scaffold (Life Technology; catalog # R-011-K) in a T225 flask (Corning CellBIND, catalog # 3293) at a density of 25E6 live cells in 60 mL of medium. Cells were seeded in William E medium (Thermo Fisher Scientific; A1217601) supplemented with 3.6% Hepatocyte Thawing and Seeding Supplement (Thermo Fisher Scientific, A15563), 5% fetal bovine serum (Thermo Fisher Scientific; 16000-036), 1 μM dexamethasone (Thermo Fisher Scientific, a15563), and 0.2% Torpedo antibiotic mixture (Bioreclamation; Z990008). The seeding medium was removed after 14 h, and the cells were then cultured in maintenance medium: William E medium supplemented with 4% hepatocyte maintenance supplement (Thermo Fisher Scientific; AI15564), 2% fetal bovine serum, 0.1 μM dexamethasone, 1.5% DMSO (Sigma-Aldrich, St. Louis, MO; D8418), 0.2% Torpedo antibiotic cocktail. Approximately 24 h after seeding, RNNs were infected with HepAD38-derived GTD virus at 500 viral genome equivalents per cell in RNN maintenance medium supplemented with 4% PEG 8000 (Promega, Madison, WI; V3011). Infections were allowed to develop for 24 h before residual extracellular virions were removed by washing three times with maintenance medium. After exposure to viral infection for 3 days, infected cells were harvested from flasks by trypsinization (0.25X in Ca/Mg-free PBS, 25 min) followed by 4 min treatment with 0.5X Versene (Life Technologies, catalog number: 15040-066) and two washes with PBS. Trypsinized cells were inactivated in maintenance medium containing 10% FBS, centrifuged for 6 min at 50 g, resuspended in hepatocyte maintenance medium, filtered through a 40 μm filter and adjusted to a density of 0.25E6 cells/ml. Infected cells were seeded in 384-well collagen-coated plates (Greiner, Austria) at a density of 20,000 cells/well containing serially diluted

- 142 049099 compounds of the present invention or DMSO (0.5%) in a final volume of 80 μl. The assay plates were incubated for 5 days and the antiviral activity of the test compounds was analyzed by determining the presence of HBV DNA in the culture supernatant using the QuantiGeneTM 2.0 Nucleic Acid Quantification Kit (Affymetrix, Santa Clara, CA).

Example 107. Cytotoxicity assay with primary human hepatocytes (PHH).

HBV-infected primary human hepatocytes were seeded (20,000 cells/well in 80 μl of medium) in 384-well collagen-coated plates pre-coated with compounds. After 5 days, 40 μl of cell supernatant was removed for quantification of HBV DNA levels as described in Example 106. Then, 40 μl of Cell Titer Glo Reagent (Promega, Cat. # G7572) was added to the remaining cells and luminescence was measured on an EnVision Microplate Reader.

Example 108. Kinetic analysis of solubility.

Buffer preparation: 0.1 N HCl:hydrochloric acid, 0.1 N standard solution (MP Biomedicals catalog #199569). 1x PBS, pH 7.4: 10x phosphate buffered saline (PBS) solution (Fisher Bioreagent, catalog #BP399-500) 50 mL was added to approximately 450 mL of HPLC grade IDO. The solution was then made up to 500 mL for a total dilution factor of 1:10 and a final concentration of 1x PBS. The pH of the resulting solution was measured and found to be 7.4.

Kinetic solubility analysis of compound stock solutions in DMSO: 100-fold dilutions of each compound stock solution in DMSO were prepared in duplicate by combining 3 μL of DMSO stock with 297 μL of the appropriate medium in a Millipore solubility filter plate with a 0.45 μM polycarbonate membrane using a Hamilton STARlet liquid handling system. The final DMSO concentration was 1.0% (v/v) and the maximum theoretical compound concentration was 100 μM (for a compound stock concentration of 10 mM). The filter plate was sealed. After 24 h incubation at ambient temperature (24.2-27.5°C), samples were vacuum filtered and filtrates were collected in a 96-well polypropylene assay plate. The plate was sealed for assay. The filtrates were injected into an Antek 8060 chemiluminescent nitrogen detector for quantitative determination. Results are presented in µM.

Results calculation: The equimolar nitrogen response of the detector was calibrated using standards covering the dynamic range of the instrument from 0.08 to 4500 µg/mL nitrogen. Filtrates were quantified using this calibration curve. Each calculated solubility value was corrected for background nitrogen present in the DMSO and in the medium used for sample preparation. All reported values for compounds containing adjacent nitrogen atoms in a ring structure were increased by approximately 25%.

Example 109. TFV-DP formation in peripheral blood mononuclear cells (PBMCs)

Compounds were tested in a 96-well assay format for TFV-DP levels in PBMCs in the presence of human serum. PBMCs were activated with 100 units/mL recombinant human IL-2 and 2 μg/mL phytohemagglutinin (PHA) for 36 h. Following activation, PBMCs (2 million) were incubated with compounds (1 μM) and cell culture medium with 50% serum in a 96-well plate for 2 h at 37°C. Cells were transferred to a 96-well deep-well plate and centrifuged. The supernatant was removed and the cells were washed twice with 1 mL 0.9% saline, centrifuging between washes. After final washing and removal of liquid, 500 μl MeOH + 200 nM Cl-ATP + 200 nM [adenine- ¹³ C5]-tenofovir diphosphate (tetraammonium salt) were added and mixed thoroughly with the cells. The plate was then centrifuged and 450 μl of sample was transferred to a new 96-well plate. The supernatants were then evaporated, rediluted in 80 μl 1 mM ammonium phosphate and vortexed thoroughly. Blanks were pooled for serial dilution to generate calibration standards for TFV, TFV-DP and TFV-TP in pmol/million cells. Samples were then transferred to a small-well 96-well plate, centrifuged and injected into the LC-MS/MS system for quantification. Values are presented in µM using an intracellular volume of 0.2 pL/cell.

Example 110. Formation of TFV-DP in primary human hepatocytes (PHH).

Compounds were tested in a 24-well assay for TFV-DP production in RNNs. Cells were seeded (4.0 x 10 ⁵ cells/well) in collagen-coated plates and incubated at 37°C. After 4-6 h, the fluid was removed, 500 μl of new medium (2% FBS, 1.5% DMSO) was added, and the cells were incubated at 37°C overnight. The medium was then replaced (2% FBS, 1.0% DMSO), compounds (1 μM) were added, and the cells were incubated at 37°C in the presence of compounds for 1 h. The medium was then removed and the cells were washed twice with 500 μl of 0.9% saline. After the final wash and removal of liquid, 500 μl MeOH + 200 nM Cl-ATP + 200 nM [adenine- ¹³ C5]-tenofovir diphosphate (tetraammonium salt) were added and the cells were incubated at -80 ° for 30 min. The cells were kept on water ice, while the MeOH/standard supernatant was pipetted and released several times,

- 143 049099 and then transferred to a 96-well deep-well plate. The supernatants were then evaporated and the samples were diluted in 80 µl of 1 mM ammonium phosphate and then vortexed thoroughly. Blanks were pooled for serial dilution to generate calibration standards for TFV, TFV-DP and TFV-TP in pmol/million cells. The samples were then transferred to a 96-well shallow-well plate, centrifuged and injected into the LC-MS/MS system for quantification. Values are presented in µM using an intracellular volume of 3 pL/cell.

The compounds of the present invention exhibit potent antiviral activity as shown in Table 1 below.

The compounds of the present invention exhibit kinetic solubility as shown in Table 2 below. Consistent with their antiviral activity, the compounds of the present invention exhibit efficacy in intracellular TFV-DP formation in MCGF and PFIH as shown in Table 2.

Table 1 ____________ Results of analyses in examples 104-107 ____________

Example MT4 HIV ECso (nM) ¹ MT4 SS ₅ o (nM) ¹ RIN vgv ECso (nM) ¹ RIN SS ₅ o (nM) ¹ 1 5 4990 1.96 > 100 2 4 6087 0.69 > 1000 3 10 4460 > 100 4 6 8417 > 100 5 5 6 2 7 9 8 5 9 169 10 37 AND 17 12 172

- 144 049099

13 16 5473 14 7 3191 2.57 > 100 15 17 4359 16 35 6408 17 >500 8853 18 13 6567 19 30 17 278 20 12 21 7 4467 3.55 > 100 22 9 24 >500 25 8 26 6 27 26 > 100 28 65 29 16 30 68 > 100 31 >500 9014 > 100 32 >500 33 7 6619 2.62 > 100 34 3 3666 35 5 4346 36 14 8965 37 4 38 10 39 24 7271 4.50 > 100 40 6 41 3 42 2 43 20 44 7 45 3

- 145 049099

46 5 4158 2.51 > 100 47 7 48 6 3340 4.63 > 100 49 5 5215 5.45 > 100 50 7 8059 3.10 > 100 51 8 7250 6.00 > 100 52 8 7187 14.36 > 100 53 6 7547 8.38 > 100 54 4 6721 4.45 > 100 55 101 9419 6.26 > 100 56 177 9654 3.99 > 100 57 23 6618 4.71 > 100 58 5 3676 1.26 > 100 59 47 6955 6.53 > 100 60 3 3306 1.44 > 100 61 28 6002 > 100 62 7 3192 > 100 63 7 2217 64 >500 7343 65 >500 6715 > 100 66 9 4766 > 100 67 8 68 9 69 24 70 >500 71 233 72 16 77 >500 > 50,000 > 100 78 >500 79 271 80 >500 81 >500

- 146 049099

82 345 83 6 6599 2.14 > 100 84 229 10594 3.37 > 100 85 5 86 5 3729 3.16 > 100 87 6 5052 > 100 88 3 7290 2.50 > 100 89 6 5104 2.42 > 100 90 7 5514 2.95 > 100 91 5 4924 3.76 > 100 92 23 7986 4.56 > 100 93 97 5471 94 130 8222 > 100 95 27 14780 96 AND 10974 97 13 10740 98 213 24523 99 91 17240 100 22 101 12 7681 102 >500 45125 103 >500 > 42 540

¹ Values represent the mean of at least two independent experiments.

Table 2

Results of the analyses in examples 108-110

Example Kinetic solubility at pH 7.4 PBS (µg/mL) Mean TFV-DP MIC (µM) ¹ Average RNN TFV-DP (µM) ¹ 1 33 5.2 2,1 2 65 4,4 2,5

- 147 049099

z 50 z,o 4 53 5.0 5 53 3.2 6 49 4,5 7 7 5.4 8 50 6.4 9 0,2 10 7 0.8 AND < 1 1,4 12 < 1 BLQ 13 < 1 2.6 14 27 15 2 ι,ο 16 12 17 18 22 4.1 19 18 1,8 20 49 4.0 21 39 5.8 22 36 5.4 24 69 BLQ 25 84 4.9 26 78 8,8 27 1 ι,ο 28 55 0,7 2.93 29 26 2,2 30 < 1 0.4 31 12 o,z 32 < 1 o,z 0.26 33 14 4.9 4.85 34 59 0.9

- 148 049099

35 54 0.9 36 60 o,z 37 1,5 38 45 1,1 39 2,1 0.9 40 57 z,o 41 54 8.1 42 54 7.1 43 53 0,6 44 10 2.0 45 64 5.3 46 < 1 6.1 47 72 1,0 48 < 1 5,6 49 4 4.8 50 < 1 4,5 51 6 2.9 52 < 1 2.0 53 < 1 2,2 54 12 5.7 55 < 1 0.4 0.88 56 5 o,z 0.91 57 < 1 1,2 58 51 7,8 5.75 59 < 1 0,5 0.68 60 76 1,2 5.42 61 < 1 1,2 1.08 62 < 1 3.6 5.01 63 2,2 2.40 64 < 1 o,z 65 0,7 0.26

- 149 049099

66 3.9 67 38 5.5 68 3 5.1 69 1 1.9 70 71 72 35 2,1 77 57 78 60 79 68 80 56 81 61 82 10 o,z 83 33 4.0 2.66 84 8 0.4 1.81 85 6 9.4 86 2 5.7 87 < 1 8.4 88 69 8.1 89 < 1 7.6 3.46 90 7 5.7 3.72 91 1 4.6 92 < 1 1,2 0.75 93 1 0.4 0.21 94 2 0,2 0.64 95 60 0,5 0.23 96 92 3.1 2.53 97 13 1,4 1.60 98 7 1,5 0,12 99 25 0.4 1.78 100 1.9 4.73 101 23 3.2 0.66 102 10 BLQ 0.16 103 64 0,4

¹ Values represent the mean of at least two independent experiments.

Claims (46)

1. Compound of formula (I): or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are independently selected from C _1-12 alkyl, ^-aryl^-alkylene, ^-cycloalkyl, C _3-7 cycloalkyl^-alkylene, C _6-14 aryl-C _3-7 cycloalkylene, ^-spirocycloalkyl, ^-spirocycloalkyl^-alkylene, bridged ^-bicycloalkyl, bridged ^-bicycloalkyl^-alkylene, condensed - 150 049099 C _5-10 bicycloalkyl, C _10-16 dispirocycloalkyl, C _10-16 dispirocycloalkyl-C _1-4 alkylene, bridged C _9-12 tricycloalkyl, bridged C _9-12 tricycloalkyl-C _1-4 alkylene, C _3-7 cycloalkyl-C _3-7 cycloalkylene and a 5- to 7-membered monocyclic heterocyclyl having 1 heteroatom selected from O and S, each C _1-12 alkyl, C _6-14 aryl-C _1-4 alkylene, C _3-7 cycloalkyl, C _3-7 cycloalkyl-C _1-4 alkylene, C _6-14 aryl-C _3-7 cycloalkylene, C _7-12 spirocycloalkyl and C _7-12 spirocycloalkyl—C _1-4 alkylene optionally substituted with one to three R ^a ; R ³ , R ⁴ , R ⁵ and R ⁶ are independently selected from C _1-4 alkyl, C _3-6 cycloalkyl and _{C 6-14 aryl} -C _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl and C _{6-14 aryl} -C _1-4 alkylene is optionally substituted with one R ^b ; or optionally: R ³ and R ⁴ together with the carbon atom to which they are attached form a 3- to 6-membered saturated carbocyclic ring optionally substituted with one R ^b ; and R ⁵ and R ⁶ are independently selected from C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 aryl-C _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 arylC _1-4 alkylene is optionally substituted with one R ^b ; or R ³ and R ⁴ are independently selected from C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 aryl-C _1-4 alkylene, wherein each C _1-4 alkyl, C _3-6 cycloalkyl and C _6-14 aryl-C _1-4 alkylene is optionally substituted with one R ^b ; and R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a 3- to 6-membered saturated carbocyclic ring optionally substituted with one R ^b ; or ^R3 and ^R4 , together with the carbon atom to which they are attached, form a 3- to 6-membered saturated carbocyclic ring optionally substituted with one ^Rb ; and ^R5 and ^R6 , together with the carbon atom to which they are attached, form a 3- to 6-membered saturated carbocyclic ring optionally substituted with one ^Rb ; B is R ⁷ represents hydrogen or R ⁸ ; R ⁸ is -L ¹ -(L ² )m-(L ³ ) _n -R ^8a ; L ¹ is selected from the bond, -C(O)- and -C(O)O-; L ² is C _1-6 alkylene; 3 P · L ³ is -C(O)O- or ' R ^8a is selected from C _1-12 alkyl, C _6-14 aryl, -C(O)-C _1-4 alkyl, ^-C(O)-C _1-4 alkyl, where C _6-14 aryl is optionally substituted with one or two R ^c ; each R ^a is independently selected from C _1-4 alkyl, halogen, C _1-4 haloalkyl and -O-C _1-4 alkyl; each R ^b is independently C _1-4 alkyl; each R ^c is independently C _1-4 alkyl or -OC(O)-C _1-4 alkyl; m and n are independently equal to 0 or 1; and p is equal to 0, 1, or 2. 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are independently selected from C _1-8 alkyl, C _6-14 aryl-C _1-4 alkylene, C _3-7 cycloalkyl, C _3-7 cycloalkyl-C _1-4 alkylene, C _6-14 aryl-C _3-7 cycloalkylene, C _7-12 spirocycloalkyl, C _7-12 spirocycloalkyl-C _1-4 alkylene, bridged C _5-10 bicycloalkyl, bridged C _5-10 bicycloalkyl-C _1-4 alkylene, condensed C _5-10 bicycloalkyl, C _10-16 dispirocycloalkyl, C _10-16 dispirocycloalkyl-C _1-4 alkylene, bridged C 9-12 tricycloalkyl, bridged C 9-12 tricycloalkyl-C 1-4 alkylene, C 3-7 cycloalkyl-C 3-7 cycloalkylene and a 5-7 membered monocyclic heterocyclyl having 1 heteroatom selected from O and S, each C 1-8 alkyl, C 6-14 aryl-C 1-4 alkylene, C 3-7 cycloalkyl, C 3-7 cycloalkyl-C 1-4 alkylene, C6-14aryl-C3-7cycloalkylene, C7-12spirocycloalkyl and C7-12spirocycloalkyl-C1-4alkylene are optionally substituted with one to three R ^a ; 3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are independently selected from C _1-8 alkyl, C _6-14 aryl-C _1-4 alkylene, C _3-7 cycloalkyl, C _3-7 cycloalkyl-C _1-4 alkylene, C _6-14 aryl-C _3-7 cycloalkylene, C _7-12 spirocycloalkyl, C _7-12 spirocycloalkyl-C _1-4 alkylene, bridged C _5-10 bicycloalkyl-C _1-4 alkylene, condensed C _5-10 bicycloalkyl, C _10-16 dispirocycloalkyl-C _1-4 alkylene, bridged C _9-12 tricycloalkyl-C _1-4 alkylene, C _3-7 cycloalkyl-C _3-7 cycloalkylene and 5-7 membered monocyclic heterocyclyl having 1 heteroatom selected from O and S, each C _1-8 alkyl, C _6-14 - 151 049099 aryl- _{C1_4} alkylene, _{C3_7} cycloalkyl, _{C3_7 cycloalkyl - C1_4} alkylene, C6.14 _aryl - _C3.7 cycloalkylene, _{C7_12} spirocycloalkyl and _C7.12 spirocycloalkyl- _{C1.4 alkylene} are optionally substituted with one to _three R ^a . 4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are independently selected from C _1-8 alkyl, C 3_ ₇ cycloalkyl, C _{3. 7} cycloalkyl-C _{1. 4} alkylene, C _{7_ 12} spirocycloalkyl, C _{7_ 12} spirocycloalkyl-C 1. ₄ alkylene, and a bridged C _{5. 10} bicycloalkyl-C 1. ₄ alkylene, wherein each C _1-8 alkyl, C _{3. 7} cycloalkyl-C _{1. 4} alkylene, C _{7. 12} spirocycloalkyl, and C _{7. 12} spirocycloalkyl-C _{1. 4} alkylene is optionally substituted with one to three R ^a . 5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are independently selected from C _{5.8 alkyl, C 5.7 cycloalkyl} , C _5.7 cycloalkyl-C _1.4 alkylene, _{C 7.9 spirocycloalkyl} , C _7-9 spirocycloalkyl-C _1-4 alkylene _, and a bridged C _5.7 bicycloalkyl-C _1.4 alkylene, wherein each C _{5.8 alkyl} , C _5.7 cycloalkyl, C _5.7 cycloalkyl-C _1.4 alkylene _, C _7.9 spirocycloalkyl, and _{C 7.9 spirocycloalkyl} -C _{1.4 alkylene is} optionally substituted with one to three R ^a . 6. A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are different. 7. A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are the same. 8. _The compound according to any one of claims 1 to 5 or _a pharmaceutically acceptable salt thereof, wherein R ¹ _{and R 2 are the same and are selected from C 1-8 alkyl, C 6_14 aryl-C 1_4 alkylene, C 3_7 cycloalkyl, C 3_7 cycloalkyl-C 1_4 alkylene, C 6_14 aryl-C 3_7 cycloalkylene, C 7_12 spirocycloalkyl, C 7_12} ^{spirocycloalkyl} _{- C 1_4 alkylene , bridged C 5_10 bicycloalkyl - C 1_4 alkylene , condensed C 5_10 bicycloalkyl , C 10_16 dispirocycloalkyl - C 1_4 alkylene} , bridged C _{9_ 12} tricycloalkyl-C _{1_4} alkylene, C _{3_7} cycloalkyl- _{C 3_7 cycloalkylene} and _{5-7 -} membered monocyclic heterocyclyl having 1 heteroatom selected from O and S, wherein each C _1-8 alkyl, C _{3_7} cycloalkyl, _{C 3_7 cycloalkyl} - _{C 1_4 alkylene} , C _{7_12} spirocycloalkyl and _{C 7_12} spirocycloalkyl- _{C 1_4 alkylene} is optionally substituted _with one to three R ^a . 9. A compound according to any one of claims 1 to ₅ , or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are the same and are selected from C _1-8 alkyl, C _3-7 cycloalkyl, _{C 3_7 cycloalkyl} - _{C 1_4 alkylene} , C _{7_12 spirocycloalkyl} , _{C 7_12 spirocycloalkyl} -C _{1_4} alkylene and _a bridged _{C 5_10} bicycloalkyl- _{C 1_4 alkylene} , wherein each _{C 1-8 alkyl} , C _3-7 cycloalkyl, C _{3_7} cycloalkyl-C _{1_4} alkylene, _{C 7_12} spirocycloalkyl _{and C 7_12 spirocycloalkyl} -C _{1_4} alkylene is optionally substituted with one to three R ^a . 10. A compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are independently selected from - 152 049099 11. A compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are the same and are selected from 12. A compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein R ³ , R ⁴ , R ⁵ and R ⁶ are independently selected from methyl, ethyl, cyclopropyl and benzyl, wherein each methyl, ethyl, cyclopropyl and benzyl is optionally substituted with one R ^b . - 153 049099 13. A compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁵ are the same. 14. A compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁵ are methyl. 15. A compound according to any one of claims 1 to 12, wherein R ⁴ and R ⁶ are the same. 16. A compound according to any one of claims 1 to 12 and 15, characterized in that R ⁴ and R ⁶ both represent ethyl or benzyl. 17. A compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ are the same. 18. A compound according to any one of claims 1 to 12 and 17, or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ are methyl. 19. A compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, wherein R ⁵ and R ⁶ are the same. 20. A compound according to any one of claims 1 to 12 or 19, or a pharmaceutically acceptable salt thereof, wherein R ⁵ and R ⁶ are methyl. 21. A compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ together with the carbon atom to which they are attached form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or the cyclobutane ring is optionally substituted with one R ^b ; and R ⁵ and R ⁶ are independently selected from C ₁ . ₄ alkyl, C ₃ . ₆ cycloalkyl and C ₆ . ₁₄ aryl-C ₁ . ₄ alkylene, wherein each C 1 . ₄ alkyl, C ₃ . ₆ cycloalkyl and C ₆ . ₁₄ aryl-C ₁ . ₄ alkylene is optionally substituted with one R ^b . 22. A compound according to claim 21 or a pharmaceutically acceptable salt thereof, wherein R ⁵ and R ⁶ are independently selected from methyl, ethyl, cyclopropyl and benzyl, wherein each methyl, ethyl, cyclopropyl and benzyl is optionally substituted with one R ^b . 23. A compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ are independently selected from _C1.4 alkyl, C _{3.6 cycloalkyl} and C _{6.14 aryl} - _C1.4 alkylene, wherein each _C1.4 alkyl, C _3.6 cycloalkyl and C _6.14 aryl-C1.4 _alkylene is optionally substituted with one R ^b ; and R ⁵ and R ⁶ , together with the carbon atom to which they are attached, form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or the cyclobutane ring is optionally substituted with one R ^b . 24. A compound according to claim 23 or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ are independently selected from methyl, ethyl, cyclopropyl and benzyl, wherein each methyl, ethyl, cyclopropyl and benzyl is optionally substituted with one R ^b . 25. A compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein R ³ and R ⁴ together with the carbon atom to which they are attached form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or the cyclobutane ring is optionally substituted with one R ^b ; and R ⁵ and R ⁶ together with the carbon atom to which they are attached form a cyclopropane ring or a cyclobutane ring, wherein the cyclopropane ring or the cyclobutane ring is optionally substituted with one R ^b . 26. A compound according to any one of claims 1 to 25, wherein each R ^b is independently methyl or ethyl. 27. A compound according to any one of claims 1 to 11, wherein R ³ , R ⁴ , R ⁵ and R ⁶ are the same. 28. The compound of claim 27 or a pharmaceutically acceptable salt thereof, wherein each of R ³ , R ⁴ , R ⁵ and R ⁶ is methyl or cyclopropyl. 29. The compound of claim 27 or a pharmaceutically acceptable salt thereof, wherein each of R ³ , R ⁴ , R ⁵ and R ⁶ is methyl. 30. A compound according to any one of claims 1 to 29 or a pharmaceutically acceptable salt thereof, having the formula (P): NHR ⁷ II 31. A compound according to any one of claims 1 to 29 or a pharmaceutically acceptable salt thereof, having the formula (III): - 154 049099 32. A compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, having the formula (IV): 33. A compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, having the formula (V): 34. The compound according to claim 1, wherein said compound is selected from: - 155 049099 - 156 049099 - 157 049099 - 158 049099 - 159 049099 or a pharmaceutically acceptable salt thereof. 35. Compound of formula: or a pharmaceutically acceptable salt thereof. 36. Use of a pharmaceutical composition for treating HIV infection, containing a therapeutically effective amount of a compound according to any one of claims 1-35 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. 37. The use of a compound according to any one of claims 1 to 35 or a pharmaceutically acceptable salt thereof for the treatment of HIV infection, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 35 or a pharmaceutically acceptable salt thereof. 38. The compound according to claim 1, wherein said compound is ’ or a pharmaceutically acceptable salt thereof. according to item 1, wherein said compound is 39. Connection or a pharmaceutically acceptable salt thereof. - 160 049099 p.1, wherein said compound is 40. Connection by or a pharmaceutically acceptable salt thereof. p.1, wherein said compound is 41. Connection by ’ or a pharmaceutically acceptable salt thereof. 42. The connection according to item 1, wherein said compound is a pharmaceutically acceptable salt. wherein the said compound is and or its 43. The connection according to item 1, pharmaceutically acceptable salt. and the said connection or its 44. The connection according to item 1, is or a pharmaceutically acceptable salt thereof. 45. The compound according to claim 1, wherein said compound is ’ or a pharmaceutically acceptable salt thereof. 46. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicinal product for the treatment of HIV infection in humans.