Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT ALKOXY-CARBONYLOXY-ALKYL AND ALKYLCARBONYLOXY-ALKYL PRODRUGS OF TAMIFLU CROSS-REFRENCE TO RELATED APPLICATIONS [0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No.63/552,257 filed February 12, 2024, the entirety of which is incorporated by reference for all purposes. FIELD [0002] Described herein are alkoxy-carbonyloxy-alkyl and alkylcarbonyloxy-alkyl prodrugs of oseltamivir and pharmaceutical compositions thereof for the treatment of the influenza virus. BACKGROUND [0003] Seasonal influenza is often characterized by fever, cough, headache, muscle and joint pain, sore throat, and runny nose. While most people recover in less than two weeks, the flu can cause severe illness and even death in high-risk populations. Worldwide, each annual flu epidemic is estimated to result in about 3 to 5 million cases of severe illness, and about 290,000 to 650,000 respiratory deaths. There are four types of seasonal influenza viruses, types A, B, C, and D, with types A and B circulating widely and causing seasonal epidemics. [0004] The most common treatments for the flu include the antiviral medications oseltamivir phosphate (Tamiflu®), zanamivir (Relenza®), peramivir (Rapivap®), and baloxavir marboxil (Xofluza®). While each of these antivirals are effective, peramivir is administered intravenously and zanamivir is administered via an inhaler, but is not recommended for patients with breathing issues. Baloxavir marboxil is not recommended for patients that are pregnant, breastfeeding, hospitalized, or with certain conditions. It is also not currently approved for patients younger than 12 years old. Oseltamivir can be taken orally as a pill or liquid and is most helpful if taken 48 hours after the onset of symptoms. Oseltamivir is approved for the treatment and prevention of influenza type A and type B in patients older than 1 year old. [0005] Oseltamivir (oseltamivir carboxylate ethyl ester (OCEE)) is an ethyl ester prodrug that is mainly hydrolyzed by hepatic carboxylesterases to produce the active agent oseltamivir carboxylate (OC). In humans, OCEE is mainly hydrolyzed by human carboxylesterase-1 (hCES-1), which is highly expressed in the liver, but also in many other tissues albeit at lower levels. OCEE was specifically designed to improve the oral bioavailability of the active metabolite OC. 1 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT
[0006] While OCEE is effective in increasing the oral bioavailability of OC in animals and humans (Li, W. et al. Antimicrobial Agents and Chemotherapy, 1998, 42, 647-653; Wattanagoon, Y., et al. Antimicrobial Agents and Chemotherapy, 2009, 53, 4753-4761), the expression of hCE-1b can vary among individuals due to their age, health, genetic and environmental factors. This variability can affect the hydrolysis of OCEE to OC, resulting in a significant amount of unchanged prodrug remaining in systemic circulation. Also, the molecular properties of OCEE (molecular weight of 312 Daltons, calculated log P of 2.1, and a topological polar surface area (TPSA) of 91) suggest that OCEE may be able to cross the blood-brain barrier, and this has been confirmed in many studies. For example, one study in rats found that after intravenous doses of 30 mg/kg, the brain-to-plasma ratio for OCEE was 30 times higher than for OC (Hoffmann, G., et al. Antimicrobial Agents and Chemotherapy, 2009, 53, 4753-4761). OCEE also showed a longer half-life in the brain than in the plasma, which could lead to accumulation in the brain. [0007] OCEE is an analog of sialic acid and therefore interacts with a number of central nervous system (CNS) targets. The moderate and variable brain penetration is thought to be responsible for a number of adverse CNS adverse effects, including nausea, hypothermia, abnormal behavior (including that with fatal outcome), and death. The side effect profile of OCEE also limits its dosing, which can lead to lower efficacy and potential emergence of viral resistance. There is substantial pharmacological evidence that the sudden-onset type reactions following OCEE dosing in some patients could be linked to OCEE more than to the active parental molecule OC. In particular, it is known that OCEE, but not OC, inhibits nicotinic acetylcholine receptors and monoamine oxidase A. Potential interactions with nicotinic ACh receptors and MAO-A, have been linked to hypothermia, abnormal or excitatory behaviors, nausea, and vomiting. Studies have also shown that OCEE causes central suppression of respiratory function in rats, and thus may be associated with the rare events of sudden death observed in influenza patients after taking OCEE. Interactions with GABA-A, GABA-B, NMDA and their related receptors/channels, including Na+ and Ca2+, channels are thought to be potentially related to respiratory suppression followed by psychotic reactions (both acute 2 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT and chronic) and sudden death. In addition, OCEE may indirectly stimulate dopamine D2 receptors via sialylation of a serum glycolipid, which could be linked to nausea and vomiting, and also to abnormal behavior seen in some adolescents taking OCEE. [0008] In fact, Han et al. (Scientific Reports, 2020, 10: 3116) examined the side effects of neuraminidase inhibitors (NAIs), and analyzing data from FAERS and WebMD, the study identified oseltamivir - the most studied NAI and the only prodrug in its class - as frequently causing psychiatric and gastrointestinal symptoms, like nausea and vomiting, particularly in younger patients. These effects are linked to the drug's interaction with the CNS. Uniquely among NAIs, oseltamivir, as an ethyl prodrug of carboxylic acid, can cross the BBB, leading to neuropsychiatric effects. Other NAIs like zanamivir and peramivir do show varied side effects, but not CNS-related; zanamivir is linked mainly to general disorders and site reactions, while peramivir to severe conditions like cardiac failure in older patients. [0009] On the drug label for oseltamivir, there is a warning of “neurologic and behavioral symptoms that include events such as hallucinations, delirium, and abnormal behavior, in some cases resulting in death.” The drug label notes post-marketing reports (mostly from Japan) of delirium and abnormal behavior, in some cases resulting in fatal outcomes. The drug label also reports that in mice a single oral administration of oseltamivir resulted in toxicity, including death, in juvenile seven-day old rats, but had no effect on adult rats. As stated on the drug label, “clinical relevance of the juvenile rat study finding for young infants in unknown,” and oseltamivir is only approved for patients for one year and older. [0010] One animal study in particular found that the ability of OCEE to enter the brain significantly varies with age (European public assessment report. Scientific Discussion for approval of Tamiflu. European Medicines Agency (2005) p. 6-25). The study compared the distribution of OCEE in 7-day old rats, which are similar in development to neonatal humans, with 42-day old rats (which are similar in development to young adults) (Sengupta, P. (2013) The Laboratory Rat: Relating Its Age with Human’s American Journal of Preventive Medicine, Vol 4, No 6). The study found that the brain-to-plasma ratio of OCEE was considerably higher in the younger rats; 72% in the 7-day old rats compared to 13% in the older rats. This increased brain exposure to OCEE in the younger rats was also associated with higher mortality when given high doses of the drug. For example, after receiving a 500 mg/kg dose, the AUC (area under the curve) for OCEE in the brain was 335 μg.h/mL in the younger rats, which was much higher than the AUC found in adult rats (74 μg.h/ml). It has been suggested that the increased permeability of the brain is a result of the immature blood-brain barrier (BBB). This indicates that adolescent patients may be more susceptible to the central nervous system (CNS) side 3 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT effects of OCEE. The CNS side effects' dependence on the integrity of the BBB may also extend these side effects to additional populations, as the BBB is known to be compromised by many factors, including inflammation, neurodegenerative diseases, and other conditions such as hypertension and diabetes. Therefore, what is needed are oseltamivir prodrugs that quickly metabolize to deliver the active carboxylate form (OC) to systemic circulation and do not penetrate the brain. These prodrugs would avoid the CNS toxicity and gastrointestinal symptoms (nausea and vomiting) seen with oseltamivir, and offer a greater therapeutic window. Further, prodrugs characterized by less CNS toxicity could potentially be offered to a greater range of patient population, including adolescents. SUMMARY [0011] Provided herein are compounds of Formula I:
or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof; wherein R1 is hydrogen or methyl; and R2 is C1-6alkyl optionally substituted with R4; C3-10cycloalkyl optionally substituted with R5; OC1-6alkyl wherein the C1-6alkyl is optionally substituted with R4; OC3-10cycloalkyl wherein the C3-10cycloalkyl is optionally substituted with R5; -CH(NHR7)R6; or -OCH(NHR7)R6; R3 is selected from -NH2, -C(O)NH2, -NHC(O)NH2, -SO2NH2, -OC(O)NH2,
, ; R4 is selected from C3-6cycloalkyl, halogen, hydroxy, amino, cyano, C1-3alkoxy, -COOH, and -C(O)CH(NHR7)R6; R5 is selected from C1-3alkyl, halogen, hydroxy, amino, cyano, C1-3alkoxy, -COOH, and -C(O)CH(NHR7)R6; R6 is the residue of an amino acid sidechain; and R7 is hydrogen; or R6 and R7 are joined together to form proline; 4 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT wherein when R3 is -NH2, R2 is not unsubstituted methyl. [0012] The oseltamivir prodrugs described herein belong to the class of prodrugs classified as self-immolative linker prodrugs (Carl, P. L., et al. Journal of Medicinal Chemistry, 1981, 24, 479-480). A self-immolative linker helps to overcome difficulties associated with electronic or steric features of a parenteral molecule that hinder hydrolysis of the directly linked moiety by a target enzyme. This is accomplished, in part, by spacing the drug away from the enzymatically-activated moiety so that the linker spontaneously separates from the parental drug following enzymatic activation. [0013] It has been surprisingly discovered that these oseltamivir prodrugs have limited systemic exposure and are rapidly metabolized to the active carboxylate (OC) form. For example, as described in Example 2 and shown in FIG. 1, when Compound 2 of Formula I was administered to dogs orally, only trace amounts of the prodrug were detected in plasma immediately following administration. This is in contrast to the pharmacokinetic profile of oseltamivir as described in Li W, et al. (Antimicrobial Agents and Chemotherapy, 1998, 42, 647-653); a dose of oseltamivir resulted in substantial amount of oseltamivir in the plasma. As discussed herein, systemic exposure to oseltamivir has been linked to adverse CNS events. The pharmacokinetic profile of a representative compound of Formula I is surprising and could not have been predicted in advance. [0014] Oral prodrugs of oseltamivir that rapidly metabolize to the active carboxylate (OC) form and are not expected to be brain penetrable could result in reduced or even eliminated CNS-related adverse events. This is advantageous because it allows for more effective dosing regimens, which can increase efficacy and potentially reduce the risk of resistance development. Further, influenza is one of the leading causes of pneumonia and death in adolescents, but OCEE use is currently limited for this population because adolescents are vulnerable to its side effects. Prodrugs with a high safety profile are particularly advantageous for this underserved population. [0015] In one embodiment, the of Formula I is a of Formula I-1:
wherein R1 is hydrogen or methyl; and 5 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT R2 is unsubstituted C2-6alkyl, unsubstituted C3-10cycloalkyl, unsubstituted OC1-6alkyl, or unsubstituted OC3-10cycloalkyl; or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof. [0016] In one embodiment, the compound of Formula I is a compound of Formula I-1A:
or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof. [0017] In one embodiment, the compound of Formula I is a compound of Formula I-1B:
or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof. [0018] Described below is a compound of Formula II:
which can be as a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof; wherein R1 is hydrogen or methyl. [0019] Also provided herein are pharmaceutical compositions comprising a compound of Formula I, Formula IA, Formula II, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, in a pharmaceutically acceptable carrier, excipient, or diluent. In one embodiment, the pharmaceutical composition is suitable for oral administration, for example in the form of a pill, tablet, solution, or suspension. In one embodiment, the pharmaceutical composition is suitable for parenteral administration, including, but not limited to intravenous administration. 6 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT [0020] Also provided herein is a method to treat influenza comprising administering to a patient in need thereof 1) a compound of Formula I, Formula II, Formula IA, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, or 2) a pharmaceutical composition comprising a compound of Formula I, Formula IA, Formula II, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, in a pharmaceutically acceptable carrier, excipient, or diluent. In one embodiment, the influenza is caused by influenza virus type A. In one embodiment, the influenza is caused by influenza virus type B. In one embodiment, the patient is a human, and in a further embodiment, the patient is an adolescent (a human between the ages of about 10 and 19 years). In one embodiment, the patient is a human less than about 13 years of age. In one embodiment, the patient is a human less than about one year of age. In one embodiment, the compound of Formula I, Formula II, Formula IA, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, exhibits less CNS-related adverse events compared to oseltamivir. BRIEF DESCRIPTION OF DRAWINGS [0021] FIG.1 is a pharmacokinetic profile of oseltamivir carboxylate (OC) and Compound 2 in plasma following administration an oral dose of Compound 2 in dogs described in Example 2. DETAILED DESCRIPTION Compounds of Formula I [0022] Provided herein are compounds of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof. In one embodiment of Formula I or Formula I, R1 is hydrogen. In one embodiment of Formula I, R1 is methyl. [0023] In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is unsubstituted C1-6alkyl. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is C1-6alkyl substituted with R4. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is unsubstituted C3-10cycloalkyl. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is C3-10cycloalkyl substituted with R5. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is unsubstituted O-C1-6alkyl. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, R2 is O-C1-6alkyl substituted with R4. In one embodiment of Formula I, 7 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is unsubstituted O-C3-10cycloalkyl. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-C3-10cycloalkyl substituted with R5. In one embodiment of Formula I-1, including any of the foregoing, R2 is unsubstituted C1-6alkyl. [0024] In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is C2-4alkyl, C3-8cycloalkyl, O-C1-3alkyl, or O-C3- 8cycloalkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is C4-6alkyl, C3-6cycloalkyl, O-C3-6alkyl, or O-C3- 6cycloalkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is C6-10cycloalkyl or O-C6-10cycloalkyl. [0025] In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is C1-3alkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is C2-4alkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is C4-6alkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is C3-8cycloalkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is C3-6cycloalkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is C6-10cycloalkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-C1-3alkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-C3-6alkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-C3-8cycloalkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-C3-6cycloalkyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I- 1B, including any of the foregoing, R2 is O-C6-10cycloalkyl. [0026] In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is methyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is ethyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is n-propyl or iso-propyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is n-butyl, sec-butyl, t-butyl, or iso-butyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 8 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT is n-pentyl, iso-pentyl, or neo-pentyl. In one embodiment of Formula I, Formula I-1, Formula I1-A, or Formula I1-B, including any of the foregoing, R2 is n-hexyl, iso-hexyl, or neo-hexyl. [0027] In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-methyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-ethyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-n-propyl or O-iso-propyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-iso-propyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-n-butyl, O-sec-butyl, O-t-butyl, or O-iso-butyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-n-pentyl, O-iso-pentyl, or O-neo-pentyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-n-hexyl, O-iso-hexyl, or O-neo-hexyl. [0028] In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, R2 is cyclopropyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, R2 is cyclobutyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I- 1B, R2 is cyclopentyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, R2 is cyclohexyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, R2 is cycloheptyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, R2 is cyclooctyl. [0029] In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-cyclopropyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-cyclobutyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-cyclopentyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-cyclohexyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-cycloheptyl. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is O-cyclooctyl. [0030] In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is -CH(NHR7)R6. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is -CH(NH2)R6. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the 9 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT foregoing, R2 is selected from ,
, .
embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is selected from
. [0031] In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is -OCH(NHR7)R6. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is -OCH(NH2)R6. In one embodiment of Formula I, Formula I-1, Formula I-1A, or Formula I- 2
of the R is selected from , , ,
,
, , and . In one embodiment of Formula 10 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT I, Formula I-1, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is selected from , [0032] In
I-1B, including any of the foregoing, R3 is -NH2. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R3 is -C(O)NH2. In one embodiment of Formula I, Formula I- 1A, or Formula I-1B, including any of the foregoing, R3 is -NHC(O)NH2. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R3 is -SO2NH2. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R3 is -OC(O)NH2. In one embodiment of Formula I, Formula I-1A, or Formula I- 1B, including any of the foregoing, R3 is
. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R3
. [0033] In one embodiment of Formula I-1A, or Formula I-1B, including any of the foregoing, R4 is C3-6cycloalkyl. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R4 is halogen. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R4 is hydroxy. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R4 is amino. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R4 is cyano. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R4 is, C1-3alkoxy. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R4 is -COOH. [0034] In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R4 is -C(O)CH(NHR7)R6. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R4 is -C(O)CH(NH2)R6. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is –CH2- substituted with -C(O)CH(NH2)R6. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is -CH2- substituted with -C(O)CH(NH2)R6 wherein R6 is the residue or an amino acid sidechain selected from valine, isoleucine, and leucine. 11 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT [0035] In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R5 is C1-3alkyl. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R5 is halogen. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R5 is hydroxy. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R5 is amino. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R5 is cyano. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R5 is C1-3alkoxy. In one embodiment of Formula I, Formula I-1A, or Formula I- 1B, including any of the foregoing, R5 is and -COOH. [0036] In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R5 is -C(O)CH(NHR7)R6. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R5 is -C(O)CH(NH2)R6. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is -OCH2- substituted with -C(O)CH(NH2)R6. In one embodiment of Formula I, Formula I-1A, or Formula I-1B, including any of the foregoing, R2 is -OCH2- substituted with -C(O)CH(NH2)R6 wherein R6 is the residue of an amino acid sidechain selected from valine, isoleucine, and leucine. [0037] In one embodiment, the compound of Formula I, Formula I-1A, or Formula I-1B, or a pharmaceutically acceptable salt thereof is at least about 50% free, at least about 60% free, at least about 70% free, at least about 75% free, at least about 80% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 97% free, at least about 99% free, or even 100% free by weight of other stereoisomers based solely on the weight of the compound of Formula I, Formula I-1A, or Formula I-1B, alone (i.e., excluding the weight of the pharmaceutically acceptable salt, if the compound exists as a pharmaceutically acceptable salt). In one embodiment, the compound of Formula I, Formula I-1A, or Formula I-1B, or a pharmaceutically acceptable salt thereof is about 85% to about 95% free of other stereoisomers based solely on the weight of the compound of Formula I, Formula I-1A, or Formula I-1B. In one embodiment, the compound of Formula I or a pharmaceutically acceptable salt thereof is about 95% to about 100% free of other stereoisomers based solely on the weight of the compound of Formula I, Formula I-1A, or Formula I-1B. 12 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT [0038] In one embodiment, the compound of Formula I is Compound 2: or a pharmaceutically acceptable
or mixture of stereoisomers thereof. Compounds of Formula II [0039] Also provided herein are pharmaceutical compositions comprising a compound of Formula II, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, and a pharmaceutically carrier, excipient, or diluent. In one embodiment, the compound of Formula II is
or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof. In one embodiment, the compound of Formula II is
or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof. In certain embodiments, a compound of Formula II, or a pharmaceutically acceptable salt thereof, in the pharmaceutical composition is at least about 50% free, at least about 60% free, at least about 70% free, at least about 75% free, at least about 80% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 97% free, at least about 99% free, or even 100% free by weight of other stereoisomers based solely on the weight of the compound of Formula II alone (i.e., excluding the weight of the pharmaceutically acceptable salt, if the compound exists as a pharmaceutically acceptable salt, and excluding the pharmaceutically carrier, excipient, or diluent). In one embodiment, the compound of Formula II, or a pharmaceutically acceptable salt thereof, in the pharmaceutical composition is about 85% to about 95% free of other stereoisomers based solely on the weight of the compound of Formula II. In one embodiment, 13 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT the compound of Formula II, or a pharmaceutically acceptable salt thereof, in the pharmaceutical composition is about 95% to about 100% free of other stereoisomers based solely on the weight of the compound of Formula II. [0040] Also provided herein are compounds of Formula II, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, optionally in a pharmaceutically carrier, excipient, or diluent, for the treatment of influenza in a patient in need thereof. In one embodiment, the compound of Formula II is
or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof. In one embodiment, the compound of Formula II is
or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof. In certain embodiments, a compound of Formula II, or a pharmaceutically acceptable salt thereof, for the treatment of influenza is at least about 50% free, at least about 60% free, at least about 70% free, at least about 75% free, at least about 80% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 97% free, at least about 99% free, or even 100% free by weight of other stereoisomers based solely on the weight of the compound of Formula II alone (i.e., excluding the weight of the pharmaceutically acceptable salt, if the compound exists as a pharmaceutically acceptable salt and excluding the pharmaceutically carrier, excipient, or diluent, if present). In one embodiment, the compound of Formula II, or a pharmaceutically acceptable salt thereof, is about 85% to about 95% free of other stereoisomers based solely on the weight of the compound of Formula II. In one embodiment, the compound of Formula II, or a pharmaceutically acceptable salt thereof, is about 95% to about 100% free of other stereoisomers based solely on the weight of the compound of Formula II. Definitions [0041] When referring to the compounds and compositions provided herein, the following terms have the following meanings unless indicated otherwise. Unless defined otherwise, all 14 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise. [0042] As used herein, unless specifically defined otherwise, any words of approximation such as without limitation “about,” when used in connection with various terms such as time, temperatures, doses, amounts, or weight percent of ingredients of a composition or a dosage form, mean e.g. a temperature, dose, amount, or weight percent that is recognized by those of ordinary skill in the art to provide an effect equivalent to that obtained from the specified time, temperature dose, amount, or weight percent. Specifically, the terms “about” and “approximately,” when used in this context, contemplate a time, temperature, dose, amount, or weight percent, etc. within 15%, within 10%, within 5%, within 4%, within 3%, within 2%, within 1%, or within 0.5% of the specified time, temperature, dose, amount, or weight percent, etc. In addition, any of the embodiments herein, where ranges or numbers are expressed with “about,” i.e. “about 48 wt/wt% to about 70 wt/wt%,” can be replaced with a range or number that does not recite “about,” i.e. “48 wt/wt% to 70 wt/wt%.” [0043] The term “alkyl,” as used herein, unless otherwise specified, refers to a straight or branched hydrocarbon. An alkyl group can have 1 to 8 carbon atoms (i.e., C1-C8 alkyl) or 1 to 6 carbon atoms (i.e., C1-C6 alkyl). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CHs)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CHs)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (- CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CHs)2CH2CH2CH3), 3-methyl-2- pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CHs)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3, and octyl (-(CH2)7CH3). [0044] The term “alkoxy”, as used herein, unless otherwise specified, refers to the group OR wherein R is alkyl as defined herein. [0045] The term “amino”, as used herein, unless otherwise specified, refers to -NH2. 15 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT [0046] The term “cyano”, as used herein, unless otherwise specified, refers to -CN. [0047] The term “cycloalkyl”, as used herein, unless otherwise specified, refers to a monocyclic or polycyclic saturated carbocyclic ring. Monocyclic carbocycles have 3 to 8 ring atoms or in some embodiments, 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g., arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as a bicyclo [5,6] or [6,6] system, or spiro-fused rings. Non-limiting examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cycIohex-2-enyl, 1-cyclohex-3-enyl, and phenyl. Non-limiting examples of bicyclo carbocycles includes naphthyl, tetrahydronaphthyl, and decalinyl. [0048] The term “halogen”, as used herein, unless otherwise specified, refers to chloro, bromo, fluoro, and iodo. [0049] The term “hydroxy”, as used herein, unless otherwise specified, refers to -OH. [0050] The term “residue of an amino acid sidechain” refers to a D- or L- natural or non- naturally occurring amino acid sidechain. Non-limiting representative amino acid sidechains include, but are not limited to those in the following amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, proline, serine, threonine, valine, tryptophan, and tyrosine. In a preferred embodiment, the amino acid sidechain is the L-amino acid sidechain. [0051] “Patient” or “subject” includes humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In some embodiments the patient is a mammal, and in other embodiments, the patient is human. As used herein, the terms “subject” and “patient” are used interchangeably. The terms “subject” and “subjects” refer to an animal, such as a mammal including a non- primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey such as a cynomolgous monkey, a chimpanzee, and a human), and in certain embodiments, a human. In certain embodiments, the subject is a farm animal (e.g., a horse, a cow, a pig, etc.) or a pet (e.g., a dog or a cat). In preferred embodiments, the subject is a human. [0052] A “pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. It is understood that the pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, which is incorporated herein by reference, or S. M. Berge et al., “Pharmaceutical Salts,” J. Pharm. Sci. 1977; 66, 1-19 which is also incorporated herein by reference. It is also 16 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT understood that the compound can have one or more pharmaceutically acceptable salts associated with it. [0053] Examples of pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2 hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2 naphthalenesulfonic acid, 4- toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4’-methylenebis-(3- hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, p-toluenesulfonic acid, salicylic acid, and the like. [0054] Examples of pharmaceutically acceptable base addition salts include those formed when an acidic proton present in the parent compound is replaced by a metal ion, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum, as salts, and the like. Preferable salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non- toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tromethamine, N-methylglucamine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. [0055] The term “substantially free of” or “substantially in the absence of” stereoisomers with respect to a composition refers to a composition that includes at least 85 or 90% by weight, or in certain embodiments 95%, 98%, 99%, or 100% by weight, of a designated
of a compound in the composition based on the weight of the designated stereoisomer alone (i.e., excluding the weight of a pharmaceutically acceptable salt, if the compound exists as a pharmaceutically acceptable salt). In some embodiments, the total weight % constitutes the 17 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT weight % of the designated stereoisomer plus the weight % of the undesignated stereoisomer in the composition based on weight of the designated stereoisomer (excluding the weight of a pharmaceutically acceptable salt, if the compound exists as a pharmaceutically acceptable salt) plus the weight of the undesignated stereoisomer (excluding the weight of a pharmaceutically acceptable salt, if the compound exists as a pharmaceutically acceptable salt). In certain embodiments, in the methods and compounds provided herein, the compounds are substantially free of stereoisomers. [0056] Similarly, the term “isolated” with respect to a composition refers to a composition that includes at least 85%, 90%, 95%, 98%, or 99% to 100% by weight, of a specified compound alone (i.e., excluding the weight of a pharmaceutically acceptable salt, if the compound exists as a pharmaceutically acceptable salt), the remainder comprising other chemical species or stereoisomers based on the weight of the specified compound alone (i.e., excluding the weight of a pharmaceutically acceptable salt, if the compound exists as a pharmaceutically acceptable salt). [0057] The term “isotopic composition,” as used herein, and unless otherwise specified, refers to the amount of each isotope present for a given atom, and “natural isotopic composition” refers to the naturally occurring isotopic composition or abundance for a given atom. Atoms containing their natural isotopic composition may also be referred to herein as “non-enriched” atoms. Unless otherwise designated, the atoms of the compounds recited herein are meant to represent any stable isotope of that atom. For example, unless otherwise stated, when a position is designated specifically as "H" or "hydrogen,” the position is understood to have hydrogen at its natural isotopic composition. [0058] “Administration” and variants thereof (e.g., in some embodiments, “administering” a compound) in reference to a compound described herein means introducing the compound as described herein into the system of the animal in need of treatment. When a compound as described herein is provided in combination with one or more other active agents (e.g., in some embodiments, surgery, radiation, and chemotherapy, etc.), “administration” and its variants are each understood to include concurrent and sequential introduction of the compound and other agents. [0059] “Therapeutically effective amount” is an amount of a compound or composition, that when administered to a patient, is sufficient to affect such treatment for the viral infection, e.g., to ameliorate a symptom of the influenza. The amount of a prodrug or dual prodrug described herein which constitutes a “therapeutically effective amount” will vary depending on the compound, the influenza infection and its severity, the age of the patient to be treated, and the 18 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT like. The therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their knowledge and to this disclosure. [0060] As used herein, the terms “therapeutic agent” and “therapeutic agents” refer to any agent(s) which can be used in the treatment of an influenza infection or one or more symptoms thereof. In certain embodiments, the term “therapeutic agent” includes a compound provided herein. In certain embodiments, a therapeutic agent is an agent that is known to be useful for, or has been or is currently being used for the treatment of influenza or one or more symptoms thereof. [0061] “Treating” or “treatment” of influenza, as used herein, includes (i) preventing the influenza from occurring in a human, i.e., causing the clinical symptoms of the influenza not to develop in an animal that may be exposed to or predisposed to the influenza, but does not yet experience or display symptoms of the influenza; (ii) inhibiting the influenza infection, i.e., arresting its development and/or its replication; and (iii) relieving influenza, e.g., relieving or reducing a symptom thereof. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the viral infection may be necessary, and will be ascertainable with routine experimentation by one of ordinary skill in the art. “Treating” or “treatment” of influenza refers, in certain embodiments, to ameliorating influenza that exists in a subject. In another embodiment, “treating” or “treatment” includes ameliorating at least one physical parameter, which may be indiscernible in the subject, for example, viral load. In yet another embodiment, “treating” or “treatment” includes modulating influenza, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. In yet another embodiment, “treating” or “treatment” includes delaying the onset of the influenza. [0062] “Preventing” or “prevent” of an influenza infection, as used herein, includes the administration of a compound as described herein to reduce the likelihood of an occurrence or reoccurrence of the influenza infection, or to minimize a new influenza infection relative to an influenza infection that would have occurred without such treatment. In one embodiment, prevention includes administering a compound as described herein to a host who has been exposed to and is thus at risk of contracting an influenza infection. [0063] As used herein, the terms “prophylactic agent” and “prophylactic agents” refer to any agent(s) which can be used in the prevention of an influenza infection, or one or more symptoms thereof. In certain embodiments, the term “prophylactic agent” includes a compound as provided herein. In certain other embodiments, the term “prophylactic agent” does not refer 19 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT a compound provided herein. In certain embodiments, a prophylactic agent can be an agent that is known to be useful for, or has been or is currently being used to prevent or impede the onset, development, progression, and/or severity of influenza/an influenza infection. [0064] As used herein, the phrase “prophylactically effective amount” refers to the amount of a therapy (e.g., prophylactic agent) which is sufficient to result in the prevention or reduction of the development, recurrence or onset of one or more symptoms associated with an influenza infection, or to enhance or improve the prophylactic effect(s) of another therapy (e.g., another prophylactic agent). Methods of Treatment [0065] Provided herein is a method to treat influenza comprising administering a compound of Formula I, Formula II, Formula IA, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, to a patient in need thereof. In one embodiment, the method is for inhibiting an influenza infection. In one embodiment, the method is for relieving influenza. In one embodiment, the method is for the prevention of an influenza infection. In one embodiment, the influenza virus is type A. In one embodiment, the influenza virus is type B. In one embodiment, the patient is a human, and in a further embodiment, the patient is an adolescent (a human between the ages of about 10 and 19 years). In one embodiment, the patient is a human less than about 13 years of age. In one embodiment, the patient is a human less than about one year of age. [0066] In one embodiment, the compound of Formula I, Formula II, Formula IA, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, is for the treatment of oseltamivir-resistant influenza caused by type A or type B influenza virus. [0067] In one embodiment, the compound of Formula I, Formula II, Formula IA, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, exhibits fewer CNS-related adverse events compared to oseltamivir. [0068] In one embodiment, the compound of Formula I, Formula II, Formula IA, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, is for the treatment of influenza, type A or type B, in patients who have been symptomatic for less than five days, less than four days, less than three days, less than two days, or less than one day. [0069] Also provided herein is a compound of Formula I, Formula II, Formula IA, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, for the use in the treatment of influenza in a subject in need thereof. 20 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT [0070] Also provided herein is the use of a compound of Formula I, Formula II, Formula IA, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, in the manufacture of a medicament for the treatment of influenza. Pharmaceutical Compositions [0071] Compounds of Formula I, Formula IA, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, and compounds of Formula II, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, for the treatment of influenza, can be formulated into pharmaceutical compositions using methods available in the art and those disclosed herein. Any of the compounds disclosed herein can be provided in the appropriate pharmaceutical composition and be administered by a suitable route of administration. [0072] The methods provided herein encompass administering pharmaceutical compositions containing at least one compound as described herein, including a compound of Formula I, Formula II, Formula IA, or Formula IB, or a pharmaceutically acceptable salt thereof and/or a stereoisomer or mixture of stereoisomers thereof, either used alone or in the form of a combination with one or more compatible and pharmaceutically acceptable carriers, such as diluents or adjuvants, or with another agent for the treatment of influenza. [0073] In certain embodiments, a composition provided herein is a pharmaceutical composition or a single unit dosage form. Pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., a compound provided herein, or other prophylactic or therapeutic agent), and typically one or more pharmaceutically acceptable carriers. In a specific embodiment and in this context, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” includes a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water can be used as a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22 edition (September 15, 2012). 21 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT [0074] In clinical practice the active agents provided herein may be administered by any conventional route, in particular orally, parenterally, rectally or by inhalation (e.g. in the form of aerosols). In certain embodiments, the compound provided herein is administered orally. In certain embodiments, the compound provided herein is administered parenterally, for example intravenously. [0075] Typical pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well-known to those skilled in the art of pharmacy, and in certain embodiments, suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a subject and the specific active ingredients in the dosage form. The composition or single unit dosage form, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. [0076] Lactose free compositions provided herein can comprise excipients that are well known in the art and are listed, in certain embodiments, in the U.S. Pharmacopeia (USP 36–NF 31 S2). In general, lactose free compositions comprise an active ingredient, a binder/filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. Exemplary lactose free dosage forms comprise an active ingredient, microcrystalline cellulose, pre gelatinized starch, and magnesium stearate. [0077] Further provided are pharmaceutical compositions and dosage forms that comprise one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, which are referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers. [0078] The pharmaceutical compositions and single unit dosage forms can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions and dosage forms will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent, in certain embodiments, in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulation should suit the mode of administration. In a certain embodiment, the pharmaceutical compositions or single unit 22 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT dosage forms are sterile and in suitable form for administration to a subject, in certain embodiments, an animal subject, such as a mammalian subject, in certain embodiments, a human subject. [0079] A pharmaceutical composition is formulated to be compatible with its intended route of administration. In certain embodiments, routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, buccal, sublingual, inhalation, intranasal, transdermal, topical, transmucosal, intra-tumoral, intra-synovial and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to human beings. In an embodiment, a pharmaceutical composition is formulated in accordance with routine procedures for subcutaneous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection. [0080] In certain embodiments, dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a subject, including suspensions (e.g., aqueous or non- aqueous liquid suspensions, oil in water emulsions, or a water in oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a subject; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a subject. [0081] The composition, shape, and type of dosage forms provided herein will typically vary depending on their use. In certain embodiments, a dosage form used in the initial treatment of viral infection may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the maintenance treatment of the same infection. Similarly, a parenteral dosage form may contain smaller amounts of one or more of the active ingredients it comprises than an oral dosage form used to treat influenza. These and other ways in which specific dosage forms encompassed herein will vary from one another will be readily apparent to those skilled in the art. See, e.g., Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22 edition (September 15, 2012). 23 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT [0082] In a preferred embodiment, the composition is suitable for oral administration. In one embodiment, the composition is a solid composition for oral administration, including, but not limited to, a tablet, pill, hard gelatin capsule, powder or granule. In these compositions, in certain embodiments, the active product is mixed with one or more inert diluents or adjuvants, such as sucrose, lactose or starch. These compositions can also comprise substances other than diluents, for example a lubricant, such as magnesium stearate, or a coating intended for controlled release. [0083] In one embodiment, the composition is a liquid composition for oral administration, including, but not limited to, a suspension, emulsion, syrup, or elixir containing inert diluents, such as water or liquid paraffin. These compositions can also comprise substances other than diluents, for example, wetting, sweetening or flavoring products. [0084] Pharmaceutical compositions that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy; Pharmaceutical Press; 22 edition (September 15, 2012). [0085] In certain embodiments, the oral dosage forms are solid and prepared under anhydrous conditions with anhydrous ingredients, as described in detail herein. However, the scope of the compositions provided herein extends beyond anhydrous, solid oral dosage forms. As such, further forms are described herein. [0086] Typical oral dosage forms are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. In certain embodiments, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. In certain embodiments, excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents. [0087] Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or non-aqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and 24 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary. [0088] In certain embodiments, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. [0089] In certain embodiments, excipients that can be used in oral dosage forms include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos.2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof. [0090] In certain embodiments, fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form. [0091] In certain embodiments, suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL PH 101, AVICEL PH 103 AVICEL RC 581, AVICEL PH 105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, PA), and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC 581. Suitable anhydrous or low moisture excipients or additives include AVICEL PH 103™ and Starch 1500 LM. [0092] Disintegrants are used in the compositions to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to 25 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT form solid oral dosage forms. The amount of disintegrant used varies based upon the type of formulation and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, specifically from about 1 to about 5 weight percent of disintegrant. [0093] Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pre gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof. [0094] Lubricants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, in certain embodiments, a syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, MD), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Plano, TX), CAB O SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, MA), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated. [0095] The composition can also be suitable for parenteral administration. The compositions for parenteral administration can be emulsions or sterile solutions. Non-limiting examples of solvents and vehicles include propylene glycol, a polyethylene glycol, vegetable oils, in particular olive oil, or injectable organic esters, for example, ethyl oleate. These compositions can also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterilization can be carried out in several ways, for example, using a bacteriological filter, by radiation or by heating. The compositions can also be prepared in the form of sterile solid compositions which can be dissolved at the time of use in sterile water or any other injectable sterile medium. [0096] Parenteral dosage forms can be administered to subjects by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intra-arterial. Because their administration typically bypasses subjects’ natural defenses against contaminants, parenteral dosage forms are typically, sterile or capable of being sterilized prior to administration to a subject. In certain embodiments, parenteral dosage forms include, but are 26 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. [0097] Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. In certain embodiments, suitable vehicles include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer’s Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer’s Injection; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. [0098] Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into the parenteral dosage forms. [0099] The compositions can also be formulated for inhalation. Formulations suitable for inhalation can be delivered by a wide range of passive breath driven and active power driven single/-multiple dose dry powder inhalers (DPI). The devices most commonly used for respiratory delivery include nebulizers, metered- dose inhalers, and dry powder inhalers. Several types of nebulizers are available, including jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. [0100] Compositions for rectal administration are suppositories or rectal capsules which contain, in addition to the active principle, excipients such as cocoa butter, semi-synthetic glycerides or polyethylene glycols. [0101] Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, in certain embodiments, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. [0102] Typical dosage forms comprise a compound provided herein, or a pharmaceutically acceptable salt, solvate or hydrate thereof lie within the range of from about 0.1 mg to about 1000 mg per day, given as a single once-a-day dose in the morning or as divided doses throughout the day taken with food. Particular dosage forms can have about 0.1, 0.2, 0.3, 0.4, 27 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100, 200, 250, 500 or 1000 mg of the active compound. EXAMPLES [0103] Abbreviations: ACN: acetonitrile Boc: tert-butyloxycarbonyl CH3CN: acetonitrile DCM: dichloromethane DIEA: N,N-diisopropylethylamine DMF: dimethylformamide HCl: hydrochloric acid HPLC: high-pressure liquid chromatography K2CO3: potassium carbonate K2EDTA: dipotassium ethylenediaminetetraacetic acid LC-MS: liquid chromatography–mass spectrometry MeOH: methanol N2: nitrogen NaOH: sodium hydroxide OC: oseltamivir carboxylate OCEE: oseltamivir carboxylate ethyl ester TFA: trifluoroacetic acid THF: tetrahydrofuran TLC: thin-layer chromatography TPGS: tocopherol polyethylene glycol succinate TPSA: total polar surface area [0104] General Analytical Methods: 1H was recorded at 400 MHz on a Varian Mercury 400 spectrometer. 13C NMR was recorded at 100 MHz. Proton chemical shifts were internally referenced to the residual proton resonance in CDCl3 (7.26 ppm). Carbon chemical shifts were internally referenced to the deuterated solvent signals in CDCl3 (77.20 ppm). LC-MS spectra were recorded on a Shimadzu LC-MS2020 using Agilent C18 column (Eclipse XDB-C18, 5um, 2.1 x 50mm) with flow rate of 1 mL/min. Mobile phase A: 0.1% of formic acid in water; mobile phase B: 0.1% of formic acid in acetonitrile. A generic gradient method was used. Analytical HPLC was performed on Agilent 1200 HPLC with a Zorbax Eclipse XDB C18 column (2.1 x 150 mm) with flow rate of 1 mL/min. Mobile phase A: 0.1% of TFA in water; mobile phase B: 0.1% of TFA in acetonitrile. A generic gradient method was used. Preparative HPLC was performed on Varian ProStar using Hamilton C18 PRP-1 column (15 x 250 mm) with flow rate of 20 mL/min. Mobile phase A: 0.1% of TFA in water; mobile phase B: 0.1% of TFA in acetonitrile. A generic gradient method was used. 28 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT [0105] Example 1. Synthesis of Compound 1 and Compound 2
[0106] Step 1: Synthesis of Compound 1-2: A solution of compound 1-1 (2.0 g, 4.87 mmol, 1 eq), Boc anhydride (2.1 g, 9.74 mmol, 2 eq) and triethylamine (2 mL, 19.48 mmol, 4 eq) in methanol (10 mL) under nitrogen was stirred at room temperature for 2 hours. LC-MS showed the reaction was completed. The methanol was evaporated to dryness under reduced pressure, diluted with water (15 mL), and extracted with ethyl acetate (30 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford compound 1-2 (2 g, 4.80 mmol, 99%) as a yellow solid. [0107] Step 2: Synthesis of Compound 1-3: To a solution of compound 1-2 (1.8 g, 4.36 mmol, 1 eq) in THF (25 mL) was treated with aq. NaOH (5N, 13 mL). The resulting reaction mixture was stirred at room temperature overnight. The LC-MS showed the 29 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT reaction was completed. Then the solution was acidified to pH 3~4 with citric acid (6 N). A large amount of solid was precipitated, which was filtered and washed with water (5 mL) and dried in vacuo to afford compound 1-3 (1.6 g, 4.16 mmol, 95%) as a white solid. [0108] Step 3A: Synthesis of Compound 1-5: A mixture of compound 1-3 (3.1 g, 8.1 mmol, 1 eq), compound 1-4 (2.0 g, 12.1 mmol, 1.5 eq) and DIEA (1.6 g, 12.1 mmol，1.5 eq) in CH3CN (62 mL) under N2 was heated to 90 °C overnight. LC-MS showed the reaction was completed. The reaction solution was concentrated under reduced pressure, diluted with water (50 mL) and the mixture was extracted with ethyl acetate (30mL x 3). The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford a crude product. It was purified by Combi-flash to get compound 1-5 (2.7 g, 5.74 mmol, 70%) as purple solid. [0109] Step 4A: Synthesis of Compound 1: To a solution of compound 1-5 (550 mg, 1.2 mmol, 1.0 eq) in CH3CN (11 mL) was added iodotrimethylsilane (260 mg, 1.3 mmol, 1.1 eq). The mixture was stirred at room temperature for 1h. TLC (DCM: MeOH, 10:1) and LC-MS showed the reaction was completed. Then the reaction was concentrated under reduced pressure (keeping the bath temperature below 30 °C) to a volume of approximately 3-4 mL. The salt was filtered and the filtrate was concentrated to get a crude product, which was purified by reverse-phase prep-HPLC to get Compound 1 (50 mg, 11%) as a light-yellow oil. LC-MS: 371.2 [M+1]+; 1H NMR (400 MHz, CD3OD) ^ 6.88-6.83 (m, 1H), 6.79 (s, 1H), 4.08 (s, 1H), 3.72 (t, J=9.8 Hz, 1H), 3.41-3.35 (m, 1H), 3.03-2.96 (m, 1H), 2.80-2.71 (m, 1H), 2.22-2.15 (m, 1H), 2.03-1.99 (m, 5H), 1.52-1.46 (m, 6H), 0.92-0.84 (m, 6H). [0110] Step 3B: Synthesis of Compound 1-7: A mixture of compound 1-3 (3.1 g, 8.1 mmol, 1 eq), compound 1-6 (1.8 g, 12.1 mmol, 1.5 eq) and K2CO3 (2.2 g, 16.2 mmol, 2 eq) in DMF (50 mL) under N2 was heated to 90 °C for 1h. LC-MS showed the reaction was completed. The reaction solution was poured into water (150 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 1-7 (1.8 g, 45%) as light-yellow solid. [0111] Step 4B: Synthesis of Compound 2: To a solution of compound 1-7 (203 mg, 0.41 mmol, 1 eq) in MeOH (3 mL) was added diluted HCl (2N, 3 mL). The reaction was stirred at room temperature for 30 minutes. TLC and LC-MS showed the reaction was completed. It was then concentrated under reduced pressure to obtain a crude product, which was purified by reverse phase prep-HPLC to get Compound 2 (51 mg, 30%) as a white solid. LC-MS: 401.4 [M+1]+; 1H NMR (400 MHz, CDCl3) ^ 8.31 (s, 2H), 8.11 (s, 1H), 6.85 (s, 1H), 5.78 (s, 1H), 30 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT 4.88 (s, 1H), 4.37 (s, 1H), 4.12 (s, 1H), 3.80 (s, 1H), 3.37 (s, 1H), 2.17-2.14 (m, 3H), 2.10 (s, 1H),1.48 (s, 4H), 1.28 (d, 6H), 0.86-0.82 (m, 6H). [0112] Example 2: Pharmacokinetics assay in dogs [0113] Compound 2 was dosed orally by gavage to male Beagle dogs (n = 2 per timepoint) at 0.94 mg/kg in 2% TPGS in 50 mM citric acid. Blood samples were collected into pre-chilled collection tubes containing K2EDTA and processed to plasma at 9 time points over a span of pre-dose to 24 h post-administration. Plasma samples were subject to protein precipitation with a 12.5-fold volume of methanol, vortexed and centrifuged. [0114] An aliquot of 20 μL plasma samples or 20 μL working solutions mixed with 20 μL 100% ACN (100 ng/mL internal standard working solution) and 170 μL precipitant (40% methanol/40% acetonitrile/20% isopropyl alcohol (v/v/v)) were dispensed into a 96-well plate. The mixture was vortexed for 5 min and centrifuged at 4000 rpm for 5 min. An aliquot of 100 µL of each supernatant was transferred to a deep 96-well plate, then 200 µL of deionized water was added, mixed well and 5 μL was injected into LC-MS/MS for analysis. Separation was achieved on a Waters CORTECS® T32.7 μm, 4.6x50 mm column, a mobile phase A of 0.1% TFA in water and a mobile phase B of 0.1% TFA in 100% acetonitrile with a step-wise linear gradient from 20 to 95% mobile phase B over 5 min at flow rate of 0.8 mL/min. An LC-MS/MS method was used to measure the concentrations of Compound 2 or the active metabolite of Compound 2, oseltamivir carboxylate (OC) in plasma. Data for OC and Compound 2 following oral administration of Compound 2 in dogs are shown in Table 1. FIG.1 shows the pharmacokinetic profile of OC and Compound 2 in plasma after administration of Compound 2. 31 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT Table 1. Pharmacokinetic Parameters for OC and Compound 2 Following PO Administration of Compound 2 to Dogs Dose Dose Cmax T1/2 AUC(0-24h)
[0115] Compound 2 was also tested for chemical stability in the aqueous solution. Incubation mixtures were prepared in a total volume of 400 μL with final Compound 2 concentrations of 1 μM in 0.1M PBS (pH 2 and 7). The samples were shaken at 23°C for 360 min. At 360 min, the solutions were mixed with 300 µL of acetonitrile with IS and analyzed on LC/MS system. Separation was achieved on a Gemini 3μm C6-Phenyl 110A 50x3mm, a mobile phase A of 0.1% formic acid in water and a mobile phase B of 100% acetonitrile with a linear gradient from 0 to 100% mobile phase B over 2.5 min at flow rate 0.8 m/min. An LC-MS/MS method was used to measure the concentrations of Compound 2. The results are shown in Table 2. Table 2. Chemical Stability of Compound 2 in Aqueous Solution
[0116] The prodrugs are sufficiently chemically stable at GI tract pH. Table 2 shows that Compound 2 is very stable at pH 2 and moderately stable at pH 7.4, the pH range encountered in the GI tract. Upon an oral dose, the prodrugs are expected to be fully hydrolyzed in the brush border intestinal mucosa with only traces of unchanged prodrug available in the systemic circulation. [0117] FIG.1 shows the pharmacokinetic plasma profile of Compound 2 and OC following an oral dose of Compound 2. As shown in FIG.1 and Table 1, an oral dose of Compound 2 produced only OC with just a trace amount of unchanged prodrug. As described in Li W, et al., Antimicrobial Agents and Chemotherapy, 1998, 42, 647-653, an oral dose of oseltamivir (OCEE) afforded a comparable amount of OC to the amount of OC when Compound 2 was administered, but a dose of OCEE also produced a substantial amount of the unchanged OCEE. As described herein, OCEE is thought to lead to CNS-adverse events. A prodrug, for example Compound 2, that rapidly converts to OC, which is not thought to cause these adverse events, 32 1103806668\2\AMERICAS
Attorney Ref. No.127531.00010 Client Ref. No. DMPK100PCT is advantageous compared to OCEE. Further, as described in Hoffmann, G., et al. Antimicrobial Agents and Chemotherapy, 2009, 53, 4753-4761, following intravenous administration of OCEE, it was shown that OCEE was able to penetrate the brain. The pharmacokinetic profile of Compound 2 suggests that only small amounts Compound 2 can reach the brain, representing another advantage over OCEE. [0118] The embodiments and examples described above are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials and procedures. All such equivalents are considered to be within the scope and are encompassed by the appended claims. 33 1103806668\2\AMERICAS