Patent Application
20250082656
The present invention provides advantageous therapeutic compositions, combinations and uses thereof for the treatment or prophylaxis of Dengue virus infection.
Dengue fever is caused by four related viruses, known as Dengue virus serotypes 1-4. Dengue viruses (DENV) are members of the Flavivirus genus of the Flaviviridae family. They are enveloped viruses with an 11 kb positive-sense RNA genome which encodes ten proteins. These proteins are the membrane (M) protein, envelope (E) protein, capsid (C) protein, and non-structural (NS) proteins. The NS proteins are NS1, NS2A, NS2B, NS3, NS4A, NS5B and NS5. These proteins are translated as a single polyprotein which is cleaved by proteases into the individual proteins. The NS5 protein is the largest of the non-structural proteins, and acts as the viral RNA dependent RNA polymerase as well as an RNA 2′-O-methyltransferase. The RNA-dependent RNA polymerase (RdRp) activity is responsible for replication of the viral genome.
Dengue is endemic to the tropics, including Eastern Mediterranean, the Americas, South-East Asia, Western Pacific, and Africa. As the range of the Aedes mosquitos which carry Dengue has grown, new cases have been observed in areas where it previously had never been seen. The United States Centers for Disease Control and Prevention has reported recent outbreaks of locally acquired Dengue in Hawaii (2015), Florida (2013, 2020) and Texas (2013) (https://www.cdc.gov/Dengue/areaswithrisk/in-the-us.html). The mosquitos that carry Dengue are now common across much of the southeast United States (Rivera et al. 2020. Travel-associated and Locally Acquired Dengue Cases—United States, 2010-2017. MMWR Morb Mortal Wkly Rep 69:149-154).
Although the distribution of individual Dengue serotypes was previously limited geographically, as the disease has spread around the world the distribution of serotypes has become more pervasive (Nature Scitable “Dengue Viruses”). This is particularly concerning because if a person is infected with one serotype and then later becomes infected with a second, they are at greater risk of severe Dengue. Severe Dengue (or Dengue hemorrhagic fever) is accompanied by severe plasma leakage, severe bleeding, shock and organ impairment. If untreated, severe Dengue has a mortality rate of 13% (Centers for Disease Control and Prevention, Dengue for Healthcare Providers, Clinical Presentation, updated Apr. 13, 2023).
In addition to the geographic spread of Dengue outbreaks, recent outbreaks have been more severe. Worldwide, the incidence has increased 8-fold since the year 2000. Not only has the frequency of outbreaks increased, but also the magnitude. The World Health Organization estimates that half the world's population is at risk of infection, and hundreds of millions of infections are recorded each year (https://www.who.int/news-room/fact-sheets/detail/Dengue-and-severe-Dengue). Nearly half a million cases of severe Dengue are also reported. The majority of those at risk of getting Dengue are children (Aranda C et al., 2018. Arbovirus surveillance: first Dengue virus detection in local Aedes albopictus mosquitoes in Europe, Catalonia, Spain, 2015. Euro Surveill 23, Wilder-Smith et al., 2014. The 2012 Dengue outbreak in Madeira: exploring the origins. Euro Surveill 19:20718).
Vaccines against Dengue have been developed but suffer from several drawbacks. The most significant is a phenomenon known as antibody dependent enhancement (ADE). The only licensed vaccine, Dengvaxia, offers incomplete (https://www.fda.gov/news-events/press-announcements/first-fda-approved-vaccine-prevention-dengue-disease-endemic-regions) and uneven protection against different serotypes, with only 50% protection against DENV1, and less for DENV2 (Sabchareon et al. 2012. Protective efficacy of the recombinant, live-attenuated, CYD tetravalent Dengue vaccine in Thai school children: a randomized, controlled phase 2b trial. Lancet 380:1559-67; Villar et al., 2015. Efficacy of a tetravalent Dengue vaccine in children in Latin America. N Engl J Med. 372:113-23). Vaccination can therefore promote ADE, and consequently severe infection in Dengue naive individuals (Aguiar et al., 2016. The Impact of Newly Licensed Dengue Vaccine in Endemic Countries. PLoS Negl Trop Dis. 10:e0005179). Thus, it is restricted to individuals 9-16 years of age who have had at least one documented previous Dengue virus infection. Despite the current and growing significance of Dengue virus as a worldwide threat to health, the development of a safe and effective pan-Dengue vaccine has yet to be achieved.
Thus, a potential strategy to combat Dengue worldwide is pharmaceuticals, for example, a direct acting antiviral (DAA). Unfortunately, there are no approved DAAs for Dengue fever. In DENV, the viral polymerase is the nonstructural protein NS5 (Karuna et al., 2020. A Cyclic Phosphoramidate Prodrug of 2′-Deoxy-2′-Fluoro-2′C-Methylguanosine for the Treatment of Dengue Virus Infection. Antimicrob Agents Chemother 64). Although the DENV RdRp has been the target of many investigational DAAs, including many nucleoside/nucleotide analogues (Troost et al., 2020. Recent advances in antiviral drug development towards Dengue virus. Curr Opin Virol 43:9-21), to date only one nucleoside, the cytosine analog balapiravir has been tested clinically (Karuna et al., 2020. A Cyclic Phosphoramidate Prodrug of 2′-Deoxy-2′-Fluoro-2′C-Methylguanosine for the Treatment of Dengue Virus Infection. Antimicrob Agents Chemother 64; Nguyen et al., 2013. A randomized, double-blind placebo-controlled trial of balapiravir, a polymerase inhibitor, in adult Dengue patients. J Infect Dis. 207:1442-50). Unfortunately, no differences between compound and placebo treatment were observed regarding antiviral response, cytokine profile and time to fever clearance (Nguyen et al., 2013. A randomized, double-blind placebo-controlled trial of balapiravir, a polymerase inhibitor, in adult Dengue patients. J Infect Dis. 207:1442-50).
Other compounds which target NS5 or other viral proteins have been reported in the literature. In 2017, Janssen disclosed a series of compounds which bind to the NS3 protein (WO 2017/167951). In 2020 the Manfroni lab described pyridobenzothiazolones which inhibit NS5 (Cannalire, R. et al. Pyridobenzothiazolones Exert Potent Anti-Dengue Activity by Hampering Multiple Functions of NS5 Polymerase, ACS Med Chem Lett, 2020 11, 773-782). Other publications describing Dengue inhibitors include but are not limited to Moquin et al. NITD-688, a pan-serotype inhibitor of the Dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models. Science Translational Medicine, 2021, 13, 579; Kaptein, S. et al. A pan-serotype Dengue virus inhibitor targeting the NS3-NS4B interaction. Nature, 2021, 598, 504-509; Shimizu, H. et al. Discovery of a small molecule inhibitor targeting Dengue virus NS5 RNA-dependent RNA polymerase. PLoS Negl Trop Dis. 2019, 13, e0007894; Yin, Z. et al. An adenosine nucleoside inhibitor of Dengue virus, PNAS, 2009, 106, 20435-20439; Putra, H. et al. Identification of natural product compounds as NS5 RDRP inhibitor for Dengue virus serotype 1-4 through in silico analysis. AIP Conference Proceedings 2020, 2237; Wang, Q.-Y. et al. A Small-Molecule Dengue Virus Entry Inhibitor. Antimicrobial Agents and Chemotherapy, 2009, 53, 1823-1831; de Oliviera, L. et al. The small molecule AZD6244 inhibits Dengue virus replication in vitro and protects against lethal challenges in a mouse model. 2020, Archives of Virology, doi:10.1007/s00705-020-04524-7; and Celegato, M. et al. Small-Molecule Inhibitor of Flaviviral NS3-NS5 Interaction with Broad-Spectrum Activity and Efficacy In Vivo, Antimicrobial Chemotherapy, 2022 14(1) e03097-22.
Specific antiviral compounds that treat certain RNA viruses include Compound 1 and Compound 2. The free base, Compound 1, is disclosed, for example, in U.S. Pat. No. 9,828,410.
Compound 2, disclosed in U.S. Pat. No. 10,519,186, is the hemi-sulfate salt of isopropyl ((R)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate and is disclosed in particular for the treatment of a viral infection such as hepatitis C virus. Compound 2 has also been reported as active against SARS-CoV2, the causative agent of COVID19 infection (see for example U.S. Pat. No. 10,874,687). The S-phosphorus diastereomer of Compound 2 is currently in human clinical trials for the treatment of HCV and COVID19.
In U.S. Pat. No. 10,946,033 Atea Pharmaceuticals, Inc. disclosed the use of Compound 1 or Compound 2 generally for the treatment of Flaviviruses, with activity data against yellow fever, Dengue (serotype 2), West Nile virus, and Zika virus.
Despite these disclosures, it has proved difficult to effectively treat Dengue due to the challenging issues described above. There remains a strong medical need to determine the proper protocol for the treatment of Dengue that is safe, effective, and well-tolerated.
It is therefore an object of the present invention to provide advantageous regimes to treat infections of Dengue. It is a further object of the present invention to provide new methods of manufacture of Compound 1 and Compound 2, which is the phosphorus R-diastereomer.
It has been unexpectedly discovered that Compound 1 or Compound 2 (and wherein Compound 1 can be a pharmaceutically acceptable salt thereof) in a high dose (i.e., at least about 700 mg or 750 mg, and more generally 700-1000 mg per dose (which can be delivered in one, two or three separate dosage forms for convenience) two, three or more times a day) is advantageous for the treatment of Dengue virus, when administered to a host, such as a human, in need thereof. In typical embodiments, Compound 1 or Compound 2 is provided in one or more pharmaceutical compositions to reach this effective amount
Further, it has been discovered that Compound 1 or Compound 2 are also active against Dengue serotypes 1, 3 and 4. With this new discovery, it is confirmed that Compound 1 and Compound 2 in the high dose regimens described herein possesses potency against all serotypes of Dengue virus (Dengue virus serotype 1, 2, 3, and 4).
The term “Compound 1” as used in the specification can refer to either the free base or a pharmaceutically acceptable salt thereof. Compound 1 is isopropyl ((R)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy)(phenoxy)phosphoryl)-L-alaninate.
It has also been discovered that Compound 1 (and its hemisulfate salt Compound 2) have an advantageous dual mechanism of action. The active metabolite is an inhibitor of the RNA dependent RNA polymerase (RdRp) domain of the viral protein NS5 as well as an inhibitor of the 2′-O-methyltransferase (MTase) domain of NS5. Both functions of the NS5 protein are required for replication. Inhibition of two key protein functions involved in RNA replication decreases the chance of a resistant mutant forming.
As discussed in Example 2 and shown in Table 1 and
Alternatively, Compound 1 can be used in the free base form or as any desired pharmaceutically acceptable salt in the high dose regimen described herein. Compound 2, the hemisulfate salt of Compound 1, is an advantageous form of Compound 1 with improved in vivo properties. For example, Compound 2 is effective in vivo in a mouse model of Dengue infection (D2Y98P virus strain) as described in Example 18. The use of Compound 2, for example, provides a significant difference in viremia observed on days 6 and 8 post infection (pi) in the blood shown in
In certain aspects Compound 2 or Compound 1 (as a free base or a pharmaceutically acceptable salt thereof) is administered to a patient, typically in the high-dose regimen described herein, wherein the patient has multiple flaviviruses and wherein one of the flaviviruses is Dengue, for example, a patient with Dengue serotype 1, 2, 3, or 4 and another flavivirus. Compound 1 (AT-281) has exhibited potent effects in reducing viral titer against 11 different strains of 7 flaviviruses tested in vitro in 3 different cell types with a range of concentrations to achieve 50% inhibition of the virus-induced cytopathic effect (CPE) of 0.21 to 1.41 μM (Table 1 and
As described in Example 10 and shown in
The chain-terminating efficiency of Compound 5 was measured in competitive nucleotide incorporation assays using all four DENV serotypes. Compound 5 is incorporated into RNA 9 to 12-fold less compared to native substrate guanosine triphosphate (i.e., for every approximately 7-8 GTPs incorporated, one AT-9010 will be incorporated). In comparison, sofosbuvir triphosphate is incorporated into the RNA less than 1 time for every 100 UTP (native uridine triphosphate) incorporations. The discrimination assay data is provided below.
Compound 5 (and thus Compound 1 and Compound 2) is also an inhibitor of the 2′-O-methyltransferase (MTase) domain of Dengue virus NS5. The MTase domain is responsible for capping the 2′-end of the replicated viral RNA. The RNA cap mitigates host immune response to the viral RNA. Inhibition of the MTase domain is a second mechanism of action for Compound 5.
The binding of Compound 5 to the MTase domain was measured in a thermal shift assay. Compound 5 stabilized DENV1, DENV2, DENV3, and DENV4 MTase domain nearly two fold greater than the previously reported MTase inhibitor sinefungin (1.9±0.1° C. for sinefungin compared to 3.8±0.1° C. for Compound 5).
Furthermore, Compound 5 more effectively stabilized the RdRp domain than native substrate guanosine triphosphate (5.6±0.1° C. for Compound 5 compared to 4.5±0.2° C. for GTP), whereas sinefungin negligibly stabilized the RdRp domain (0.1° C.±0.1° C.).
Confirming the activity of Compound 5 against the MTase domain, an IC50 of 29.6+2 μM was measured in a filter-binding assay. As it has been previously shown that Compound 5 reaches hundreds of micromolar concentration in cells (Good, S. et al. Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus, PLoS ONE 15(1): e0227104), an IC50 of 29.6 μM indicates that Compound 5 is an effective inhibitor of the MTase domain.
Compound 5, the active metabolite of Compound 2 and Compound 1, thus has multiple mechanisms of action against Dengue virus. Multiple mechanisms of action may prevent the emergence of resistance, especially against the highly conserved MTase domain.
In certain aspects of the present invention, a compound of Formula I:
or a pharmaceutically acceptable salt thereof optionally in a pharmaceutically acceptable carrier, is administered in a high dose (at least 700 or 750 mg per dose, for example 700-1000 mg per dose (which can be provide in 1, 2, 3 or 4 separate dosage forms for convenience of swallowing), two, three or more times a day) to treat a Dengue virus infection in a host in need thereof, typically a human;
wherein:
R22, R33, R44, and R55 are independently selected at each occurrence from hydrogen, C1-C6alkyl, C3-C7cycloalkyl, alkenyl, alkynyl, F, Cl, Br, I, heterocycle, heterocycloalkyl, haloalkoxy, haloalkyl, C(O)R66, C(O)OR66, C(O)N(R66)2, and cyano;
In certain aspects of the present invention, a compound of Formula II:
or a pharmaceutically acceptable salt thereof optionally in a pharmaceutically acceptable carrier, is administered in a high dose (at least 700-1000, for example, 700-850 mg per dose two, three or more times a day) to treat a Dengue virus infection in a host in need thereof, typically a human;
wherein:
All R groups are intended to be interpreted in a manner that does not include redundancy i.e., as known in the art, alkyl would not be substituted with alkyl; however, for example, alkoxy substituted with alkoxy is not redundant. Aryl substituted with aryl falls within the definition of aryl in a limited scope. R groups are not optionally substituted unless specifically indicated in context.
Non-limiting examples of C1-C6alkyl include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl, isobutyl, —CH2C(CH3)3, —CH(CH2CH3)2, and —CH2CH(CH2CH3)2. Non-limiting examples of C3-C6cycloalkyl include cyclopropyl, CH2-cyclopropyl, cyclobutyl, and CH2-cyclobutyl.
A non-limiting example of aryl(C1-C4alkyl)- is benzyl. Non-limiting examples of aryl are phenyl and naphthyl.
In certain aspects, the compound is of the Formula IIA:
or a pharmaceutically acceptable salt thereof, wherein all variables are as defined herein.
In certain aspects, the compound is of the Formula IIB:
or a pharmaceutically acceptable salt thereof, wherein all variables are as defined herein.
In certain aspects, a selected compound described herein is used to treat Dengue virus infection, which can be serotype 1, 3 and/or 4. In some embodiments, a selected compound described herein is used to treat Dengue virus infection, which may be serotype 1, 2, 3 and/or 4, by administering a dosage of about 700-1000 mg, for example, 700-850 mg per dose two, three or more times a day, (and in some embodiments, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 milligrams two or three times a day) to a patient in need thereof. In certain embodiments, Compound 1 or Compound 2 is administered in a dosage of about 700-1000 mg, for example at least about 700, 750 or 800 mg per dosing (which may be delivered in multiple solid dosage forms to reach the full dose).
In a specific embodiment, Compound 2 is delivered at a dosage of three 250 mg solid dosage forms of free base weight to reach a 750 mg dosage total dosage form twice a day. In other embodiments, Compound 2 is delivered in two 375 mg solid dosage forms to reach a 750 mg dosage total dosage form twice a day.
In certain embodiments Compound 1 or Compound 2 is administered twice, three times or four times a day for four, five, six, seven, eight, nine, or ten days. In certain embodiments this treatment regimen can treat Dengue virus serotypes 1, 3 and/or 4, or even 1, 2 3, and/or 4. For clarity, unless otherwise stated, the milligram dose of active compound administered is based on the weight of the salt of the nucleotide phosphoramidate, if a pharmaceutically acceptable salt is used. For conversion purposes, a dose of about 750 mg of Compound 2 corresponds to a dose of about 692 mg of Compound 1 (which falls within “about 700 mg”), in the free base form.
The present invention includes both treatment and prophylactic or preventative therapies. In some embodiments, the active NS5 RdRp inhibitory compound as described herein is administered as a prophylactic to a host such as a human according to the high dose methods provided herein who has been exposed to and is thus at risk of contracting a Dengue virus infection such as Dengue 1 virus, Dengue 2 virus, Dengue 3 virus, and/or Dengue 4 virus. In another alternative embodiment, a method to prevent transmission is provided that includes administering a high-dose effective amount as described herein of one of the compounds described herein to a human for a sufficient length of time prior to exposure to crowds that can be infected, including during travel or public events or meetings, including for example, up to 3, 5, 7, 10, 12, 14 or more days prior to a communicable situation.
The present invention also provides a stereoselective process for a scalable manufacture of Compound 1 wherein the RP diastereomer is produced in substantially pure form. A substantially pure form of the diastereomer typically refers to at least about 90% or greater of the RP diastereomer over the SP diastereomer. Reaction of a compound of the formula Intermediate A, Intermediate B, and a uronium-based activator, for example 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), preferentially forms the SP phosphoramidate Intermediate C. A similar process was described in WO 2022/040473A1, assigned to Atea Pharmaceuticals, where the desired product was the SP phosphoramidate.
The aryloxy group on the phosphoramidate of Intermediate C is substituted with one, two, or three R6 groups which activate it for displacement, for example, by electron withdrawing function. In a second step, the activated phenoxy leaving group is displaced by phenol, inverting the phosphorus stereochemistry to form Compound 1.
In certain embodiments, the substantially pure R-phosphorus diastereomer is about 93% pure or greater, about 95% pure or greater, about 98% pure or greater or even 99% pure or greater.
In certain embodiments the 700 mg to 1,000 mg dose is administered in three solid dosage forms to a patient in need thereof for the treatment of Dengue, wherein the solid dosage forms totaling about 700 mg to about 1,000 mg of Compound 1 or 2 is administered two times a day (a total of six pills daily). In certain embodiments the three solid dosage forms totaling about 700 mg to about 1,000 mg of Compound 1 or 2 are administered four, five, six, or seven days in a row. In certain embodiments three solid dosage forms totaling about 700 mg to about 1,000 mg of Compound 1 or 2 are administered eight, nine, ten, eleven, twelve, thirteen, or fourteen days in a row.
In certain embodiments Composition B is administered four, five, six, or seven days in a row. In certain embodiments Composition A is administered eight, nine, ten, eleven, twelve, thirteen, or fourteen days in a row.
In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is used in Composition A or Composition B.
In certain embodiments Compound 1 or Compound 2 is administered two times a day in a dose of least about 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 mg wherein the compound is administered, for illustrative example, every 8+/−2 hours.
In certain aspects a dosage regimen described herein is administered within four, three, two, or one day of symptom onset. In certain embodiments a dosage regimen described herein is administered within 48 hours of symptom onset.
The present invention thus includes the following features:
It has been unexpectedly discovered that Compound 1 or Compound 2 (and wherein Compound 1 can be a pharmaceutically acceptable salt thereof) in a high dose (i.e., at least 700-1,000 mg per dose two, three or more times a day) is advantageous for the treatment of Dengue virus, when administered to a host, such as a human, in need thereof. In certain embodiments, Compound 1 or Compound 2 can be provided in a pharmaceutical composition.
Further, it has been discovered that Compound 1 or Compound 2 are also active against Dengue serotypes 1, 3 and 4. With this new discovery, it is confirmed that Compound 1 and Compound 2 in the high dose regimens described herein possess potency against all serotypes of Dengue virus (Dengue virus serotype 1, 2, 3, and 4).
The term “Compound 1” as used in the specification can refer to either the free base or a pharmaceutically acceptable salt thereof. Compound 1 is isopropyl ((R)-(((2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy)(phenoxy)phosphoryl)-L-alaninate.
It has also been discovered that Compound 1 (and its hemisulfate salt Compound 2) have an advantageous dual mechanism of action. The active metabolite is an inhibitor of the RNA dependent RNA polymerase (RdRp) domain of the viral protein NS5 as well as an inhibitor of the 2′-O-methyltransferase (MTase) domain of NS5. Both functions of the NS5 protein are required for replication. Inhibition of two key protein functions involved in RNA replication decreases the chance of a resistant mutant forming.
Compound 1 and 2 have an advantageous dual mechanism of action. It is an inhibitor of the RNA dependent RNA polymerase (RdRp) domain of the viral protein NS5 as well as an inhibitor of the 2′-O-methyltransferase (MTase) domain of NS5. Both functions of the NS5 protein are required for replication. Inhibition of two key protein functions involved in RNA replication decreases the chance of a resistant mutant from forming.
Compound 2 (AT-752) is the hemi-sulfate salt of Compound 1 (AT-281), a phosphoramidate protide which forms the L-alanyl metabolite Compound 3 (AT-551) as an intermediate prodrug before being converted to the 5′-monophosphate (MP) metabolite Compound 4 (AT-8001) of the nucleoside 2′-fluoro-2′-C-methylguanosine (Compound 6 (AT-273)). Compound 4 (AT-8001) is then phosphorylated to the active 5′-triphosphate (TP) metabolite Compound 5 (AT-9010). Compound 5 (AT-9010) has been shown to selectively inhibit the viral RNA-dependent RNA polymerase (RdRp) of HCV (Good et al., Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus. PLos One 15: e0227104), and here it is shown that it inhibits serotype 1, 2, 3, and 4 of Dengue. One mechanism is the inhibition of NS5, the RdRp of DENV-2.
In certain aspects, dosage regimens, methods, compositions, and processes of manufacture are provided for the treatment of a host infected with Dengue virus. For example, Compound 1 or 2 or Formula I or II can be administered in a dose of about 700-1000 mg, for example a dose of 750 mg two or three times a day, alone or in combination with another anti-RNA viral agent to treat the infected host in need thereof. In certain embodiments, it is useful to administer a combination of drugs that modulate the same or a different pathway or inhibit a different target in the virus. As Compound 1/2 is a polymerase inhibitor, it can be advantageous to administer Compound 1/2 to a host in combination with a protease inhibitor or an NS3-NS4B interaction inhibitor. Alternatively, Compound 1/2 can be administered in combination with a structurally different polymerase inhibitor.
Compound 1/2 can be administered orally, for example in pill or tablet form, and may be administered via another route which the attending physician considers appropriate, including via intravenous, transdermal, subcutaneous, topical, parenteral, or other suitable route.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting.
Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting.
A “patient” or “host” or “subject” is a human or non-human animal in need of treatment or prevention of a Dengue virus infection. Typically, the host is a human. A “patient” or “host” or “subject” also refers to, for example, a mammal, primate (e.g., human), cow, sheep, goat, horse, dog, cat, rabbit, rat, mice, bird and the like.
The term “prophylactic” or “preventative” when used refers to the administration of an active NS5 RNA dependent RNA polymerase (RdRp) inhibitory compound to prevent, reduce the likelihood of an occurrence or a reoccurrence of a Dengue virus infection such as Dengue 2 virus or Dengue 3 virus, or to minimize a new infection relative to infection that would occur without such treatment or to minimize transmission to another person. The present invention includes both treatment and prophylactic or preventative therapies. In some embodiments, the active NS5 RdRp inhibitory compound is administered to a host who has been exposed to and is thus at risk of contracting a Dengue virus infection such as Dengue 1 virus, Dengue 2 virus, Dengue 3 virus, or Dengue 4 virus. In another alternative embodiment, a method to prevent transmission is provided that includes administering an effective amount of one of the compounds described herein to humans for a sufficient length of time prior to exposure to crowds that can be infected, including during travel or public events or meetings, including for example, up to 3, 5, 7, 10, 12, 14 or more days prior to a communicable situation.
The terms “coadminister,” “coadministration,” or “in combination” are used to describe the administration of a NS5 RdRp interfering compound in combination with at least one other antiviral active agent. The timing of the coadministration is best determined by the medical specialist treating the patient. It is sometimes desired that the agents are administered at the same time. Alternatively, the drugs selected for combination therapy are administered at different times to the patient. Of course, when more than one viral or other infection or other condition is present, the present compounds may be combined with other agents to treat that other infection or condition as required.
Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the salts and the quaternary ammonium salts of the parent compound formed, for example, from inorganic or organic acids that are not unduly toxic. For example, acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC—(CH2)n-COOH where n is 0-4, and the like, or using a different acid that produces the same counterion. Lists of additional suitable salts may be found, e.g., in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).
The compound can be delivered in any molar ratio of salt that delivers the desired result. For example, the compound can be provided with less than a molar equivalent of a counter ion, such as in the form of a hemi-sulfate salt. Alternatively, the compound can be provided with more than a molar equivalent of counter ion, such as in the form of a di-sulfate salt. Non-limiting examples of molar ratios of the compound to the counter ion include 1:0.25, 1:0.5, 1:1, and 1:2.
In certain embodiments, the term “about” means plus or minus 5% of the listed value. In certain embodiments, the term “about” means plus or minus 10% of the listed value.
Compositions, methods, and dosage forms are provided for the treatment of a host infected with Dengue via administration of an effective amount of Compound 1 or Compound 2.
In an aspect of the invention, pharmaceutical compositions according to the present invention comprise an anti-Dengue virus high-dose of Compound 1 or 2 as described herein, optionally in combination with a pharmaceutically acceptable carrier, additive, or excipient, further optionally in combination or alternation with at least one other active compound. In one embodiment, the invention includes a solid dosage form of Compound 2 in a pharmaceutically acceptable carrier.
In certain embodiments Compound 1 or 2 is administered to a patient in need thereof in a pharmaceutical composition, for example Composition A or Composition B.
In an aspect of the invention, pharmaceutical compositions according to the present invention comprise an anti-Dengue high-dose effective amount of Compound 1 or 2 described herein, optionally in combination with a pharmaceutically acceptable carrier, additive, or excipient, further optionally in combination with at least one other antiviral agent, such as another anti-Dengue agent.
The invention includes pharmaceutical compositions that include an effective amount to treat a Dengue virus infection of Compound 1 or 2 of the present invention or prodrug, in a pharmaceutically acceptable carrier or excipient. In an alternative embodiment, the invention includes pharmaceutical compositions that include an effective amount to prevent a Dengue virus infection of Compound 1 or 2 of the present invention or prodrug, in a pharmaceutically acceptable carrier or excipient.
One of ordinary skill in the art will recognize that a therapeutically effective high dose amount will vary with the infection or condition to be treated, its severity, the treatment regimen to be employed, the pharmacokinetic of the agent used, as well as the patient or subject (animal or human) to be treated, and such therapeutic amount can be determined by the attending physician or specialist.
Compound 1 or 2 according to the present invention can be formulated in a mixture with a pharmaceutically acceptable carrier. In general, it is typical to administer the pharmaceutical composition in orally-administrable form, and in particular, a solid dosage form such as a pill or tablet. Certain formulations may be administered via a parenteral, intravenous, intramuscular, topical, transdermal, buccal, subcutaneous, suppository, or other route, including intranasal spray. Intravenous and intramuscular formulations are often administered in sterile saline. One of ordinary skill in the art may modify the formulations to render them more soluble in water or another vehicle, for example, this can be easily accomplished by minor modifications (salt formulation, esterification, etc.) that are well within the ordinary skill in the art. It is also well within the routineers' skill to modify the route of administration and dosage regimen of Compound 2 in order to manage the pharmacokinetic of the present compounds for maximum beneficial effect in patients, as described in more detail herein.
Compound 1 or 2 may be provided, for example, for oral or parenteral delivery. In certain embodiments, Compound 1 or 2 is provided in a solid, gel, or liquid dosage form. In certain embodiments, Compound 1 or 2 is provided in a liquid dosage form for parenteral administration. In certain embodiments, Compound 1 or 2 is provided in a liquid dosage form for intravenous administration. In certain embodiments, Compound 1 or 2 is provided in a solid dosage form for oral administration. In certain embodiments, Compound 1 or 2 may be provided as a softshell capsule or as a tablet for oral administration.
The high dose amount of Compound 1 or 2 included within the therapeutically active formulation according to the present invention is described in detail herein and is an effective amount to achieve the desired outcome according to the present invention, for example, for treating the DENV infection, reducing the likelihood of a DENV infection or the inhibition, reduction, and/or abolition of DENV or its secondary effects, including disease states, conditions, and/or complications which occur secondary to DENV.
Doses described herein refer to the amount of active pharmaceutical ingredient in the dose with the weight added by pharmaceutically acceptable salts, unless otherwise indicated. For example, a 750 mg dose of Compound 2 comprises about 692 mg of Compound 1 (which falls within “about 700 mg”) when the molecular weight of the hemisulfate salt is factored into the measurement.
In general, a therapeutically effective amount of Compound 2 in a pharmaceutical dosage form may range from about 600 mg to about 1,000 mg, about 700 mg to about 1,000 mg, or about 700 mg to about 800 or 850 mg when measured as the free base. In certain embodiments, Compound 1 is administered in the same range as the free base.
In certain embodiments, Compound 2 is administered in dose amounts of at least about 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1,000 mg, 1,150 mg, 1,200 mg, 1,250 mg, 1,300 mg, 1,350 mg, 1,400 mg, 1,450 mg, or 1,500 mg when measured as the free base two or three times a day. In some embodiments, the dose is provided in several dosage forms to reduce the size of the solid dosage form such as a pill, tablet or capsule.
In certain embodiments, about 700 mg to about 800 or 850 mg of Compound 1 or 2 is administered once, twice or three times per day (QD). In certain embodiments, about 700 mg to about 800 mg of Compound 1 or 2 is administered once, twice or three times per day (QD). In certain embodiments, about 800 mg to about 900 mg of Compound 2 is administered once per day (QD). In certain embodiments, about 900 mg to about 1,000 mg of Compound 2 is administered once per day (QD).
In certain embodiments, about 700 mg to about 800 mg of Compound 2 is administered twice per day (BID). In certain embodiments, about 700 mg to about 900 mg of Compound 2 is administered twice per day (BID). In certain embodiments, about 800 mg to about 900 mg of Compound 2 is administered twice per day (BID). In certain embodiments, about 900 mg to about 1,000 mg of Compound 2 is administered twice per day (BID).
In certain embodiments, about 600 mg to about 700 mg of Compound 2 is administered three times per day (TID). In certain embodiments, about 700 mg to about 800 mg of Compound 2 is administered three times per day (TID). In certain embodiments, about 800 mg to about 900 mg of Compound 2 is administered three times per day (TID). In certain embodiments, about 900 mg to about 1,000 mg of Compound 2 is administered three times per day (TID).
In certain embodiments, about 700 mg to about 900 mg of Compound 2 is administered four times per day (QID). In certain embodiments, about 600 mg to about 700 or 750 mg of Compound 2 is administered four times per day (QID). In certain embodiments, about 700 mg to about 800 mg of Compound 2 is administered four times per day (QID). In certain embodiments, about 800 mg to about 900 mg of Compound 2 is administered three four per day (QID). In certain embodiments, about 900 mg to about 1,000 mg of Compound 2 is administered four times per day (QID).
For purposes of the present invention, a prophylactically or preventive effective amount of the compositions according to the present invention falls within the same high-dose concentration range as set forth above for therapeutically effective amount and is usually the same as a therapeutically effective amount.
In certain embodiments, the Dengue virus is serotype 1, 3, or 4. In certain embodiments, the Dengue virus is serotype 1, 3, or 4 and about 700 mg to about 1000 mg of Compound 1 or 2 is administered once per day (QD). In certain embodiments, the Dengue virus is serotype 1, 3, or 4 and about 550 mg to about 750 mg of Compound 2 is administered once per day (QD). In certain embodiments, the Dengue virus is serotype 1, 3, or 4 and about 600 mg of Compound 1 or 2 is administered once per day (QD).
In certain embodiments, the Dengue virus is serotype 1, 3, or 4 and about 500 mg to about 800 mg of Compound 1 or 2 is administered twice per day (BID). In certain embodiments, the Dengue virus is serotype 1, 3, or 4 and about 550 mg to about 750 mg of Compound 1 or 2 is administered twice per day (BID). In certain embodiments, the Dengue virus is serotype 1, 3, or 4 and about 600 mg of Compound 1 or 2 is administered twice per day (BID).
In certain embodiments, the Dengue virus is serotype 1, 3, or 4 and about 500 mg to about 800 mg of Compound 1 or 2 is administered three times per day (TID). In certain embodiments, the Dengue virus is serotype 1, 3, or 4 and about 550 mg to about 750 mg of Compound 1 or 2 is administered three times per day (TID). In certain embodiments, the Dengue virus is serotype 1, 3, or 4 and about 600 mg of Compound 1 or 2 is administered three times per day (TID).
Administration of Compound 1 or 2 may range from continuous (intravenous drip) to several oral or intranasal administrations per day (e.g., two, three, four, or five times per day) or transdermal administration and may include oral, topical, parenteral, intramuscular, intravenous, sub-cutaneous, transdermal (which may include a penetration enhancement agent), buccal, and suppository administration, among other routes of administration. Enteric coated oral tablets may also be used to enhance bioavailability of the compounds for an oral route of administration. The most effective dosage form will depend upon the bioavailability/pharmacokinetic of the particular agent chosen as well as the severity of disease in the patient. Oral dosage forms are particularly preferred, because of ease of administration and prospective favorable patient compliance.
To prepare the pharmaceutical compositions according to the present invention, a therapeutically effective amount of Compound 1 or 2 according to the present invention can be intimately admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose. A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral. In preparing pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical excipients and components may be used. Thus, for liquid oral preparations such as suspensions, elixirs, and solutions, suitable carriers and additives including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like may be used. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives including starches, sugar carriers, such as dextrose, manifold, lactose, and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used. If desired, the tablets or capsules may be enteric-coated or sustained release by standard techniques. The use of these dosage forms may significantly enhance the bioavailability of the compounds in the patient.
For parenteral formulations, the carrier will usually comprise sterile water or aqueous sodium chloride solution, though other ingredients, including those which aid dispersion, also may be included. Of course, where sterile water is to be used and maintained as sterile, the compositions and carriers must also be sterilized. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, and the like may be employed.
Liposomal suspensions (including liposomes targeted to viral antigens) may also be prepared by conventional methods to produce pharmaceutically acceptable carriers. This may be appropriate for the delivery of free nucleosides, acyl/alkyl nucleosides or phosphate ester pro-drug forms of the nucleoside compounds according to the present invention.
In typical embodiments according to the present invention, Compound 1 or 2 and the compositions described are used to treat, prevent or delay a Dengue infection or a secondary disease state, condition or complication of Dengue.
The present invention includes compounds and the use of Compound 1 or Compound 2 with desired isotopic substitutions of atoms at amounts above the natural abundance of the isotope, i.e., enriched. Isotopes are atoms having the same atomic number but different mass numbers, i.e., the same number of protons but a different number of neutrons. By way of general example and without limitation, isotopes of hydrogen, for example, deuterium (2H) and tritium (3H) may be used anywhere in described structures. Alternatively, or in addition, isotopes of carbon, e.g., 13C and 14C, may be used. A preferred isotopic substitution is deuterium for hydrogen at one or more locations on the molecule to improve the performance of the drug. The deuterium can be bound in a location of bond breakage during metabolism (an α-deuterium kinetic isotope effect) or next to or near the site of bond breakage (a β-deuterium kinetic isotope effect). Achillion Pharmaceuticals, Inc. (WO/2014/169278 and WO/2014/169280) describes deuteration of nucleotides to improve their pharmacokinetics or pharmacodynamics, including at the 5-position of the molecule.
Substitution with isotopes such as deuterium can afford certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements. Substitution of deuterium for hydrogen at a site of metabolic break-down can reduce the rate of or eliminate the metabolism at that bond. At any position of the compound that a hydrogen atom may be present, the hydrogen atom can be any isotope of hydrogen, including protium (1H), deuterium (H) and tritium (3H). Thus, reference herein to a compound encompasses all potential isotopic forms unless the context clearly dictates otherwise.
The term “isotopically-labeled” analog refers to an analog that is a “deuterated analog”, a “13C-labeled analog,” or a “deuterated/13C-labeled analog.” The term “deuterated analog” means a compound described herein, whereby an H-isotope, i.e., hydrogen/protium (1H), is substituted by a H-isotope, i.e., deuterium (H). Deuterium substitution can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted by at least one deuterium. In certain embodiments, the isotope is 90, 95 or 99% or more enriched in an isotope at any location of interest. In some embodiments it is deuterium that is 90, 95 or 99% enriched at a desired location. Unless indicated to the contrary, the deuteration is at least 80% at the selected location. Deuteration of the nucleoside can occur at any replaceable hydrogen that provides the desired results.
In certain aspects of the present invention, a compound of Formula I:
or a pharmaceutically acceptable salt thereof optionally in a pharmaceutically acceptable carrier, is administered in a high dose effective amount as described herein to treat a Dengue virus infection in a host in need thereof, typically a human;
wherein:
R22, R33, R44, and R55 are independently selected at each occurrence from hydrogen, C1. C6alkyl, C3-C7cycloalkyl, alkenyl, alkynyl, F, Cl, Br, I, heterocycle, heterocycloalkyl, haloalkoxy, haloalkyl, C(O)R66, C(O)OR66, C(O)N(R66)2, and cyano;
All R groups are intended to be interpreted in a manner that does not include redundancy i.e., as known in the art, alkyl would not be substituted with alkyl; however, for example, alkoxy substituted with alkoxy is not redundant. Aryl substituted with aryl falls within the definition of aryl in a limited scope. R groups are not optionally substituted unless specifically indicated in context.
Non-limiting examples of C1-C6alkyl include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl, isobutyl, —CH2C(CH3)3, —CH(CH2CH3)2, and —CH2CH(CH2CH3)2. Non-limiting examples of C3-C6cycloalkyl include cyclopropyl, CH2-cyclopropyl, cyclobutyl, and CH2-cyclobutyl.
A non-limiting example of aryl(C1-C4alkyl)- is benzyl. Non-limiting examples of aryl are phenyl and naphthyl.
In certain embodiments, Formula I is:
In certain aspects of the present invention, a compound of Formula II:
or a pharmaceutically acceptable salt thereof optionally in a pharmaceutically acceptable carrier, is administered in an effective amount to treat a Dengue virus infection in a host in need thereof, typically a human; and all variables are as defined herein.
In certain aspects, the compound is of the Formula IIA:
or a pharmaceutically acceptable salt thereof, wherein all variables are as defined herein.
In certain embodiments, the compound of Formula IIA is selected from:
In certain aspects, the compound is of the Formula IIB:
or a pharmaceutically acceptable salt thereof, wherein all variables are as defined herein.
In certain embodiments, the compound of Formula IIB is selected from:
In certain embodiments, a compound of Formula II, Formula IIA, or Formula IIB is used in the treatment of an infection of DENV1, DENV2, DENV3, or DENV4.
In certain embodiments, R4a is selected to be the side chain of a naturally occurring amino acid, for example:
In certain alternative embodiments, variable R4a of Formula II, IIA, or IIB is C7-C15alkyl, variable R4b is hydrogen, and all other variables are as defined herein. In alternative embodiments, the compound is selected from
Treatment, as used herein, refers to the administration of Compound 1 or Compound 2 to a host, for example a human that is or may become infected with Dengue virus.
The term “prophylactic” or preventative, when used, refers to the administration of Compound 1 or 2 to prevent or reduce the likelihood of an occurrence of the viral disorder. The present invention includes both treatment and prophylactic or preventative therapies. In one embodiment, Compound 1 or 2 is administered to a host who has been exposed to and thus is at risk of infection by a Dengue virus infection.
The invention is directed to a method of treatment or prophylaxis of a Dengue virus, including drug resistant and multidrug resistant forms of DENV and related disease states, conditions, or complications. The method comprises administering to a host in need thereof, typically a human, with an effective amount of Compound 1 or 2 as described herein, optionally in combination with at least one additional bioactive agent, for example, an additional anti-DENV agent, further in combination with a pharmaceutically acceptable carrier additive and/or excipient.
In yet another aspect, the present invention is a method for prevention or prophylaxis of a Dengue infection or a disease state or related or follow-on disease state, condition or complication of a Dengue infection.
In an alternative embodiment, Compound 2 is provided as the hemisulfate salt of a phosphoramidate of Compound 1 other than the specific phosphoramidate described in the compound illustration such as in Formula I or II. A wide range of phosphoramidates are known to those skilled in the art that include various esters and phospho-esters, any combination of which can be used to provide an active compound as described herein in the form of a hemisulfate salt.
It is well recognized that drug-resistant variants of viruses can emerge after prolonged treatment with an antiviral agent. Drug resistance sometimes occurs by mutation of a gene that encodes for an enzyme used in viral replication. The efficacy of a drug against DENV infection, can be prolonged, augmented, or restored by administering the compound in combination or alternation with another, and perhaps even two or three other, antiviral compounds that induce a different mutation or act through a different pathway, from that of the principle drug.
Alternatively, the pharmacokinetic, bio distribution, half-life, or other parameter of the drug can be altered by such combination therapy (which may include alternation therapy if considered concerted). Since the disclosed Compound 2 and Compound 1 is an NS5 polymerase inhibitor, it may be useful to administer the compound to a host in combination with, for example a
The cell lines, viruses and compounds described herein were used to generate the data presented in Example 2, 3, 4, 5, 6, 7, and 8. Huh-7 (human liver carcinoma; AcceGen Biotechnology, Fairfield, NJ) cells were maintained in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 μg/mL penicillin and 100 μg/mL streptomycin (Lonza, Walkersville, MD). Vero 76 cells (American Type Culture Collection (ATCC), Manassas, VA) used for the virus yield reduction assay were maintained similarly. BHK-21 (baby hamster kidney; ATCC, Manassas, VA) cells were maintained in Minimum Essential Medium with Earle's salts (EMEM) containing 1 mM sodium pyruvate and 25 μg/mL kanamycin, supplemented with 10% FBS. All cell cultures were maintained at 37° C. in an atmosphere of 5% CO2 and ≥95% humidity. Infections were performed in EMEM supplemented with 5% FBS and 50 μg/mL gentamicin.
The Dengue viruses (DENV-2 NGC and DENV-3 H87) were obtained from ATCC (Manassas, VA), and DENV-2 used in the human PBMC and BHK-21 assays was a clinical isolate. Japanese encephalitis (JEV SA-14), West Nile (WNO2 Kern 515), Yellow fever (YFV 17D) and Zika (ZIKV MR766) viruses were obtained from the University of Texas Medical Branch (Galveston, TX) while the Powassan (POWV Spooner and LB strains) and Usutu (USUV TC-508) viruses were from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch. For studies using fresh human PBMCs, cells from a single male donor (Lot LS-88-45477C) were obtained from BioIVT (Westbury, NY) or were isolated from blood collected from healthy donors using Ficoll-Paque (Pham et al., 2008. Hepatitis C virus replicates in the same immune cell subsets in chronic hepatitis C and occult infection. Gastroenterology. 134:812-22, Pham et al., 2004. Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C. J Virol 78:5867-74). The DENV-2 studies in PBMCs and BHK21 cells were completed at University degli Studi di Cagliari (Monserrato, Italy). The other virus studies for efficacy and specificity were conducted at Utah State University (Logan, UT) and ImQuest BioSciences (Frederick, MD). Protocols at the latter laboratory for the different RNA and DNA viruses—HCV, HIV-1, Influenza A & B, RSV, Rhinovirus, Herpes simplex-1 and Adenovirus—have been previously described (Good et al., 2020. Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus. PLos One 15:30227104). Compound 2 (AT-752) and its free base Compound 1 (AT-281) were synthesized by a stereospecific process and, along with its plasma metabolite Compound 6 (AT-273), were prepared for Atea Pharmaceuticals by Topharman Shanghai Co., Ltd., Shanghai, China. (Compound 5 (AT-9010) and the TP internal standards used to quantify Compound 5 (AT-9010) were synthesized by NuBlocks (Oceanside, CA). Stock solutions were prepared in DMSO and stored at −20° C. Frozen plasma, human and from SD rat (BioreclamationlVT, Westbury, NY) and from Cynomolgus monkey (Suzhou Xishan, China) was purchased to test the stability of Compound 1 (AT-281) at 37° C.
The antiviral activity of Compound 1 (AT-281) was evaluated against flaviviruses DENV-2 NGC, DENV-3, JEV, POWV, USUV, WNV, YFV and ZIKV using a neutral red dye uptake assay (described in Example 3) to determine inhibition of virus-induced and compound-induced cytopathic effect (CPE) and using a virus yield reduction (VYR) assay (described in Example 4) as a second, independent determination of the inhibition of viral replication. Human hepatocarcinoma (Huh-7), baby hamster kidney (BHK-21) or human peripheral blood mononuclear cells (PBMCs) were acutely infected with the different viruses and exposed to serial dilutions of the drug (described in Example 5). The activity of Compound 1 (AT-281) was measured in infected cells using the neutral red assay and/or the virus yield reduction (VYR) assay, to determine the effective concentration required to achieve 50% inhibition (EC50) of the virus-induced cytopathic effect (CPE), the concentration to reduce virus yield by 1 log10 (EC90) and the cytotoxic concentration of the drug to cause death to 50% of viable cells incubated without virus (CC50) (see Table 1). After a 3- to 6-day incubation, the effective concentration of Compound 1 (AT-281), a 2′-fluoro-2′-C-methyl guanosine nucleotide prodrug, required to achieve 50% inhibition (EC50) of the virus-induced cytopathic effect (CPE) ranged from 0.19 to 1.41 μM (see tables on next page).
aneutral red assay
bVYR assay
chighest concentration tested
aneutral red assay
While the growth kinetics of different flaviviruses can differ in cell culture, Compound 1 (AT-281) showed similar potent effect in reducing viral titer against all 7 flaviviruses including 11 different strains tested in vitro (Table 1A) the antiviral activity was also maintained across Dengue serotype 1, 2, 3, and 4 across all tested strains (Table 1). The inhibition curves for DENV, WNV and YFV (
To assess the antiviral specificity of Compound 1 (AT-281), serial dilutions were incubated with various host cell lines infected with a panel of DNA and RNA viruses other than flaviviruses. The activity of Compound 1 (AT-281) was measured in infected cells using the neutral red assay to determine the effective concentration required to inhibit viral replication 50% (EC50) (described in Example 3) and the threshold concentration required to obtain a perceptible effect on 50% of the cells incubated without virus (CC50). Compound 1 (AT-281) had no activity against the DNA viruses tested and was only weakly or not active against several RNA viruses (Table 2). However, it had a high selectivity (>2,000) towards HCV, inhibiting the virus at nanomolar levels (Table 2). The potential cytotoxicity of Compound 1 (AT-281) was assessed in multiple cell lines with resulting CC50 values greater than 100 μM (Table 2, below).
A further lack of cytotoxicity was demonstrated in human induced pluripotent stem cell (iPS) cardiomyocytes (described in Example 6) and in granulocyte macrophage (GM) and erythroid (E) human bone marrow progenitor cells (described in Example 7). When incubated with Compound 1 (AT-281), these cells also had CC50 values greater than 100 μM, whereas the positive control compounds had the expected cytotoxic effects (CC50 values of 7 μM for doxazosin with cardiomyocytes and 2 and 3 μM for AZT in the GM and E cell assays, respectively). Additionally, it has been previously shown that the active TP, Compound 5 (AT-9010), does not inhibit the in vitro enzyme activities of human cellular DNA-dependent DNA polymerases α, β or γ with estimated IC50 values>100 μM, nor is it likely to affect mitochondrial integrity or inhibit human mitochondrial DNA-directed RNA polymerase (POLRMT; (Good et al., Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus. PLos One 15:e0227104).
The neutral red assay procedure herein was used to generate the data marked “neutral red assay” or “NR” in Tables 1A and 1B of Example 2. AT-281 was dissolved in DMSO at a concentration of 10 mg/mL and serially diluted using eight half-log dilutions so that the highest test concentration was 100 μg/mL (172 μM). Each dilution was added to 5 wells of a 96-well plate with 80-100% confluent Huh-7 cells. Three wells of each dilution were infected with virus, and two wells remained uninfected as toxicity controls. Six untreated wells were infected as virus controls and six untreated wells were left uninfected to use as virus controls. Viruses were diluted to achieve MOIs of approximately 0.001 CCID50 (50% cell culture infectious dose) per cell (0.037 and 0.028 CCID50 for POWV LB strain and USUV, respectively). Plates were incubated at 37° C. in a humidified atmosphere containing 5% CO2. On day 3 (ZIKV, EEEV and POWV Spooner), day 4 (CHIKV), day 5 (YFV, RVFV, MERS, POWV LB and USUV) or day 6 (WNV, JEV, DENV-2 NGC and DENV-3) post-infection, when untreated virus control wells reached maximum CPE, the plates were stained with neutral red dye for approximately 2 hours (±15 minutes). Supernatant dye was removed, wells were rinsed with PBS, and the incorporated dye was extracted in 50:50 Sorensen citrate buffer/ethanol for >30 minutes. The optical density was read on a spectrophotometer at 540 nm and converted to percent of controls. The concentrations of test compound required to prevent virus-induced CPE by 50% (EC50) and to cause 50% cell death in the absence of virus (CC50) were calculated (Smee et al., 2017. Evaluation of cell viability dyes in antiviral assays with RNA viruses that exhibit different cytopathogenic properties. J Virol Methods 246:51-57; Repetto et al., 2008. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature Protoc 3:1125-1131). The selective index is the CC50 divided by EC50 except where indicated. Selectivity index (SI) values between 0-3.9 indicate inactive compounds; SI of 4-9.9 indicates minimally active compounds, SI of 10-49.9 indicates moderately active compounds and SI values>50 indicate highly active compounds. Compounds with SI values>100 are indistinguishable from one another.
The virus yield reduction assay procedure herein was used to generate the data marked “VYR” or “VYR assay” in Tables 1A and 1B of Example 2. As previously published (Prichard et al., 1990. A microtiter virus yield reduction assay for the evaluation of antiviral compounds against human cytomegalovirus and herpes simplex virus. J Virol Methods 246:51-57), Vero 76 cells were seeded in 96-well plates and grown overnight (37° C.) to confluence. A sample of the supernatant fluid from each compound concentration was collected on day 3 (day 4 for POWV and day 7 for USUV) post infection (3 wells pooled) and tested for virus titer using a standard endpoint dilution CCID50 assay and titer calculations using the Reed-Muench equation (Reed et al., 1938. A simple method of estimating fifty percent endpoints. Am J Hygiene 27:493-497). The concentration of compound required to reduce virus yield by 90% (EC90) was determined by regression analysis.
The PBMC assay procedure herein was used to generate the data marked “PBMC” or “Human PBMC” in Tables 1A and 1B of Example 2. Compound 1 (AT-281) was dissolved in DMSO at 100 mM and then diluted in growth medium to final concentrations of 100, 20, 4 and 0.8 μM. PBMCs were resuspended in RPMI 1640 medium with 2 mM L-glutamine, 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (PBMC growth medium) and were subjected to a 3-day activation with PHA (5 μg/mL). Following a 1-h infection with DENV-2, the cells were seeded in 96-well plates (1×105 cells/well), in PBMC growth medium, either in the absence or presence of serial dilutions of Compound 1 (AT-281). Similarly, BHK-21 cells were grown to confluency in 96-well plates, then growth medium was replaced with fresh maintenance medium (growth medium with 1% inactivated FBS in place of 10% FBS) containing serially diluted Compound 1 (AT-281) and DENV-2 at a multiplicity of infection (MOI) of 0.01. Uninfected cells in the presence of serially diluted compound were used to assess the cytotoxicity of compounds. After a 3-day incubation at 37° C. in a humidified 5% CO2 atmosphere, cell viability was determined by the MTT method (Pauwels et al., 1988. Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J Virol Methods 20:309-21). The effective concentration of AT-281 required to prevent virus-induced cytopathic effect (CPE) by 50% (EC50) and to cause 50% cell death in the absence of virus (CC50) were calculated by regression analysis.
The cardiomyocyte cytotoxicity assay procedure herein was used to generate the CC50 data in Table 2 of Example 2. Cytotoxicity was evaluated in human iPS cardiomyocytes (Cellular Dynamics; Madison, WI) and in primary human bone marrow progenitor cells (Invitrogen, Grand Island, NY). Doxazosin mesylate and AZT, used as positive controls respectively, were purchased from Sigma Aldrich. The iPS cardiomyocytes, in medium from Cellular Dynamics, were seeded in 96-well plates pre-coated with 0.1% gelatin (Sigma) at 1.5×104 cells/well in a final volume of 100 μL, and incubated at 37° C. in 5% CO2 for 48 h. The cells were washed with DPBS, and AT-281 diluted in the same medium (100 μL) was added to the monolayer in triplicate and incubated at 37° C. in 5% CO2 for 3 days. Cell viability was measured by staining with CellTiter Glo®. The medium was removed from the test plates, and replaced with fresh medium (100 μL) and CellTiter Glo reagent (100 μL per well) before incubating at RT for 10 min. The well contents were transferred to a white 96-well plate and luminescence measured within 15 min on a Wallac 1450 Microbeta Trilux liquid scintillation counter.
The cardiomyocyte cytotoxicity assay procedure herein was used to confirm the CC50 data in Table 2 of Example 2. Bone marrow progenitor cells suspended in Iscove modified Dulbecco medium containing 15% heat-inactivated FBS, 10% giant cell tumor conditioned medium (Bone Marrow Plus, Sigma), 10 ng/mL recombinant human IL-6, 10 ng/mL recombinant human IL-3 and 25 ng/mL recombinant human granulocyte macrophage colony stimulating factor (GM-CSF, R&D systems), and a final concentration of 1% methylcellulose, were added to 6-well plates (1×105 cells/well) in a volume of 900 μL. AT-281 (100 μL) at 10 times high test concentration were added to each well in triplicate, incubated at 37° C. in 5% CO2 for 14 days, and colonies (greater than 30 cells) counted.
Since virus infection in the blood is an important component of Dengue fever, it was important to determine the stability of Compound 1 (AT-281) in blood, and the ability of PBMCs to phosphorylate Compound 1 (AT-281) to form the active triphosphate metabolite, Compound 5 (AT-9010). Compound 2 (AT-752) was found to be stable for up to 120 min in cynomolgus monkey and human plasma (98.2% and 93.5% of the respective starting 2 μM concentrations remained after 120 min of incubation), but was highly unstable in Sprague Dawley (SD) rat plasma with no drug remaining at the first time point (10 min of incubation). The half-life (T1/2) in rat plasma at 37° C. was estimated to be <3 minutes.
Fresh non-stimulated human PBMCs treated with 10 μM Compound 1 (AT-281) (as described in Example 9) for up to 6 h had a time-dependent increase in Compound 5 (AT-9010) concentrations over the entire exposure phase and continued to increase after drug washout, with a mean peak concentration of 1.88±0.01 pmol/106 cells at 10 h after initiation of exposure (
Upon receipt, fresh human PBMCs (single male donor, Lot LS-88-45477C) were centrifuged at 400×g for 5 min. The resulting cell pellet was suspended in 20 mL of warm PBMC growth medium. The cell density of the cell suspension was counted using an automated cell counter (Cellometer K2, Nexcelom) after staining with Trypan Blue and adjusted to 2×106 viable cells/mL. The human PBMC suspension was transferred to 24-well tissue culture treated plates at 0.5 mL/well (1×106 cells) and cultured in a humidified incubator maintained at 37° C. and 5% CO2 for 20-24 h. Then, Compound 1 (AT-281) stock solution was spiked into each well to achieve the final concentration of 10 μM, and the stimulated cells were incubated for 0, 2, 4 and 6 h at 37° C. and 5% CO2 atmosphere. At each time point, samples in triplicate were processed for analysis of Compound 5 (AT-9010). For the washout samples, the incubated PBMCs were collected and centrifuged at 400×g for 5 min. The supernatant was discarded and the cells washed with 0.5 mL medium, re-suspended in 0.5 mL medium and transferred to individual wells of 24-well plates. The human PBMCs were further incubated in a humidified incubator maintained at 37° C. and 5% CO2 for an additional 0, 2, 4, 16, 20, 24 and 28 h (i.e., 6, 8, 10, 22, 26, 30 and 34 h after the initiation of test article exposure). At each time point, triplicate samples were processed for Compound 5 (AT-9010) analysis as follows: Samples were transferred into 2 mL tubes and centrifuged at 800×g for 5 min. After aspirating the supernatant, each sample was vortexed after the addition of 0.1 mL water. They were then quenched with 0.3 mL ice-cold 60% MeOH, and stored at −70° C. until analyzed using LC/MS/MS (
To confirm the mechanism of action and antiviral target of Compound 5 (AT-9010), incorporation and elongation assays (described in Example 13) were run using the full-length DENV NS5 protein (described in Example 11) (serotype 2) and an annealed primer-template RNA pair mimicking the 3′ end of the DENV-2 genome (described in Example 12). In the absence of GTP, Compound 5 (AT-9010) was readily incorporated as a substitute at the +5 position of the RNA primer (
The DENV NS5 protein used in Example 10 was prepared by the procedure described herein. Full-length DENV NS5 (serotype 2, New Guinea C) was expressed and purified as previously described (Potisopon et al., 2017. Substrate selectivity of Dengue and Zika virus NS5 polymerase towards 2′-modified nucleotide analogues. Antiviral Res 140:25-36). Briefly, the gene coding for NS5 with a N-terminal 6-His tag was expressed from a pQE30 vector in E. coli NEB Express cells (New England Biolabs, Ipswich, MA) transformed with pRare2-LacI (Novagen, Madison, WI), and induced with 50 μM IPTG and 2% EtOH until the OD600 value reached 0.6. Cells were lysed with sonication, and the NS5 protein separated using TALON metal-affinity resin slurry (Clontech, Mountain View, CA) according to the manufacturer's instructions, washed and eluted. Size exclusion chromatography was carried out as a second purification step where the protein was loaded onto a Superdex 200 HR 16/20 column (GE Healthcare), and eluted in a buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol and 1 mM DTT.
The RNA oligonucleotides used in Example 10 was prepared as described herein. RNA oligonucleotides were purchased from Biomers.net (Ulm/Donau, Germany). A 20 nt template sequence (T20) corresponding to the 3′ end of the DENV-2 antigenome was annealed to a 10 nt complementary primer (P10) containing a fluorescent 6-FAM moiety at the 5′ end. Annealing was performed using a primer-template molar ratio of 1:1.5 in the presence of 110 mM KCl, incubated at 70° C. for 10 min and cooled slowly to room temperature. NTPs were purchased from GE Healthcare (Chicago, IL).
DENV NS5 was preincubated with the annealed P10/T20 RNA in an assembly buffer containing 20 mM HEPES pH 7.5, 10% glycerol, 5 mM MgCl2 and 5 mM DTT for 10 min at 30° C. to create an active RNA elongation complex. Reactions were started by adding AT-9010 with either all four NTPs, or UTP, ATP and CTP only. Final concentrations were 0.5 μM NS5, 0.25 μM P10/T20, 100 μM each NTP, and between 10-625 μM AT-9010, in a final buffer containing 20 mM HEPES pH 7.5, 15% glycerol, 5 mM MgCl2 and 5 mM DTT. Reactions were quenched at indicated timepoints in 2× volume FBD stop solution (formamide, 10 mM EDTA) and analysed on 20% acrylamide:bisacrylamide (19:1), 7 μM urea sequencing gels. RNA products were visualized using a Typhoon FluorImager and analyzed using ImageQuant software.
The plasma pharmacokinetics (PK) of Compound 1 (AT-281) and its metabolites Compound 3 (AT-551), the intermediate prodrug, and Compound 6 (AT-273), the plasma surrogate for intracellular levels of the active triphosphate metabolite Compound 5 (AT-9010), were determined in CD-1 mice, SD rats and cynomolgus monkeys after single oral doses of Compound 2 (AT-752) at 420, 300 and 300 mg/kg, respectively (Table 3).
In rodents, the parent prodrug Compound 1 (AT-281) was quickly metabolized with the rapid appearance in plasma of its metabolites, Compound 3 (AT-551) and Compound 6 (AT-273) (
Concentrations of Compound 5 (AT-9010) in PBMCs isolated from mice and rats administered single oral doses of Compound 1 (AT-281) at 50 and 300 mg/kg and monkeys after a single oral dose of Compound 2 (AT-752) equivalent to 55 mg/kg Compound 1 (AT-281) were measured at various time points post dose (
To test the tolerability of Compound 1 (AT-281) and determine steady state concentrations of the active TP in PBMCs, mice were given a loading dose of 1000 mg/kg and, starting 4 h later, dosed twice a day (12 h apart) at 500 mg/kg for 3 days. The concentrations of Compound 5 (AT-9010) in pooled (n=3) PBMCs 12 h after the first and sixth 500 mg/kg dose were 0.421 and 0.575 pmol/106 cells, respectively, while the concentrations of plasma Compound 6 (AT-273) were 0.67±0.12 and 1.35±0.86 nmol/mL, respectively, for the same time points. There were no adverse clinical signs observed in the mice so the multiple doses of the prodrug were well-tolerated.
The studies using CD-1 mice, SD rats and cynomolgus monkeys in Example 17 were conducted at WuXi AppTec (Suzhou, China) in strict compliance with AAALAC International, NIH guidelines and the People's Republic of China, Ministry of Science and Technology, “Regulations for the Administration of Affairs Concerning Experimental Animals”, 2017. Protocols were reviewed and approved by WuXi AppTec's IACUC prior to study initiation, and all animals assessed by the WuXi AppTec veterinary staff throughout the studies. All animals were housed in rooms with controlled temperature (18 to 26° C.), relative humidity (40 to 70%) and light cycle (12 h artificial light and 12 h dark), with 100% airflow. They were provided with manipulatives/enrichment toys. The AG129 mouse study described herein was conducted at IBT Bioservices (Rockville, MD) in strict compliance with USDA Animal Welfare Act and follow the PHS and NIH Policy of Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals, National Research Council—ILAR, Revised 2011. The study was conducted in full compliance with protocols that were reviewed and approved by IBT's Institutional Animal Care and Use Committee (IACUC) prior to study initiation, and mice were assessed and monitored throughout the study by members of the IBT veterinary staff in accordance with PHS Policy and the USDA.
Male naïve CD-1 mice (Shanghai Sippr BK Laboratory Animals Co. Ltd., China) were administered a dose of Compound 2 (AT-752) at 420 mg/kg in 40% PEG400, 10% solutol HS15, 50% 100 mM citrate buffer, pH 4.5 (v/v) by oral gavage. Blood samples (N=3) were collected at 0.5, 1, 2, 4, 8, 12 and 24 h post-dose, plasma separated in EDTA and 1 μL dichlorvos (2 mg/mL; stabilizing agent to prevent in vitro hydrolysis of the ester moiety of Compound 1 (AT-281) by blood esterases) and stored at −70° C. Concentrations of Compound 1 (AT-281), and metabolites Compound 3 (AT-551) and Compound 6 (AT-273) were determined in the plasma by LC/MS/MS. For PBMCs, six male naïve CD-1 mice were administered a dose of Compound 2 (AT-752) (at an Compound 1 (AT-281) equivalent dose of 50 mg/kg) in 60% PEG400 by oral gavage. Blood samples were collected, in groups of three, at 4 and 12 h post-dose, PBMCs isolated, and concentrations of Compound 5 (AT-9010) were measured by LC/MS/MS.
Male naïve SD rats (Beijing Vital River Laboratory Animals Co. Ltd., China) were administered a dose of AT-752 at 300 mg/kg in 40% PEG400, 10% solutol HS15, 50% 100 mM citrate buffer, pH 4.5 (v/v) by oral gavage. Blood samples (n=4) were collected at 1, 2, 4, 8, 12 and 24 h post-dose, plasma separated and stored at −70° C. Concentrations of Compound 1 (AT-281), and metabolites Compound 3 (AT-551) and Compound 6 (AT-273) were determined by LC/MS/MS. For PBMCs, nine male naïve SD rats were administered a dose of Compound 2 (AT-752) (at a Compound 1 (AT-281) equivalent dose of 300 mg/kg) by oral gavage. Blood samples were collected, in groups of three, at 24, 48 and 72 h post-dose, PBMCs isolated, and Compound 5 (AT-9010) concentrations determined by LC/MS/MS.
Male non-naïve cynomolgus monkeys (Hainan Jingang Laboratory Animal Co. Ltd., China), at least 2 kg body weight, were individually housed during the study in stainless steel mesh cages, provided ad libitum access to RO water, fed twice daily (except on the day of dosing when they were fed once) with ˜60 g Certified Monkey Diet each feed (Beijing Vital Keao Feed Co., Ltd., Beijing, China) and given daily treats of fresh fruit. They (n=5) were administered a dose of Compound 2 (AT-752) at 300 mg/kg in 40% PEG400, 10% solutol HS15, 50% 100 mM citrate buffer, pH 4.5 (v/v) by oral gavage, and blood samples (˜0.5 mL) were collected at 1, 2, 4, 8, 12 and 24 h post dose. Plasma was separated at each time point for pharmacokinetics, and stored at −70° C. Concentrations of Compound 1 (AT-281), and metabolites Compound 3 (AT-551) and Compound 6 (AT-273) were determined by LC/MS/MS. Separately, three monkeys were given an oral dose of Compound 2 (AT-752) as powder in capsules at 60 mg/kg (Compound 2 (AT-281) equivalent dose of 55 mg/kg), and blood samples collected at 12, 24 and 48 h post dose. PBMCs were isolated and concentrations of Compound 5 (AT-9010) measured by LC/MS/MS. All animals were observed for any unusual or adverse clinical signs just before and immediately after dosing and prior to each blood collection time point, with no such signs noted.
Separately, two mouse studies with multiple dosing were conducted. First, 9 naïve CD-1 mice were administered Compound 1 (AT-281) in 100 mM citrate buffer, pH 4.5 by oral gavage at 500 mg/kg for the first dose and, 4 h later, 250 mg/kg for the second dose. Blood samples were collected, 3 animals per time point, at 4 (before the second dosing), 8 and 16 h post the first dose, and PBMCs isolated. Secondly, 9 naïve CD-1 mice were administered Compound 1 (AT-281) by oral gavage at 1000 mg/kg for the first dose, and then 4 h later, they were given twice daily doses 12 hours apart for 3 consecutive days at 500 mg/kg. Blood samples were collected, 3 animals per time point, at 16 (received one 500 mg/kg dose), 40 (received three 500 mg/kg doses) and 76 h post first dose (received six 500 mg/kg doses), and PBMCs were isolated. Concentrations of Compound 1 (AT-281) and Compound 6 (AT-273) were determined in the plasma, and Compound 5 (AT-9010) in the PBMCs by LC/MS/MS (described in Example 21).
To test the efficacy of Compound 2 (AT-752) against DENV infection, AG129 mice were subcutaneously inoculated with DENV-2 D2Y98P, a non-mouse-adapted strain that follows a similar disease kinetic as described in humans (Tan et al., 2010. A non mouse-adapted Dengue virus strain as a new model of severe Dengue infection in AG129 mice. PLoS Negl Trop Dis 4:3672). The prodrug was orally administered as a loading dose (1000 mg/kg) 4 h prior to viral challenge, and afterwards as twice a day (BID) doses (500 mg/kg) for 7 consecutive days, beginning 1 h post infection (pi). Viremia and spleen viral load, the primary endpoints, were evaluated at Days 4, 6, 7, 8 and 10 pi. Viremia (
Mice were evaluated daily for weight change, appearance, mobility and alertness, and euthanized when 20% weight loss or a health score >5 was reached. There were notable differences in body weight between the treated and vehicle control groups at Days 4 to Day 8 pi (p<0.001 by t-test). No vehicle-treated mice survived beyond Day 8 pi. Animals treated with Compound 2 (AT-752) had significantly reduced weight loss compared to vehicle control mice on Days 5, 7 and 8 pi (
The tissue samples used in Example 20 were collected according to the protocol described herein. Fifty-five female AG129 mice (α-, β-, γ-interferon knockout), 6-8 weeks old, were divided into two groups, with 25 mice in Group 1 (vehicle) and 30 mice in Group 2. On Day 0, all mice were challenged with 1×105 PFU of DENV-2 D2Y98P by subcutaneous injection. Group 1 animals received vehicle [(PEG400 (40%, v/v)/Solutol HS15 (10%, v/v)/100 mM Citrate buffer pH 4.5±0.2 (50%, v/v)] by oral gavage BID for 7 consecutive days starting at 1 h pi. Group 2 received a single dose of 1000 mg/kg of Compound 2 (AT-752), 4 h prior to challenge, followed by a 500 mg/kg dose 1 h post challenge (pi). BID dosing of Compound 2 (AT-752) at 500 mg/kg was continued for 7 consecutive days. The protocol called for five mice per group to be sacrificed and terminal sera and spleens to be collected on Days 4, 6, 7, 8 and 10 pi. On Day 20 pi, any surviving mice in Group 2 were sacrificed for terminal sera and spleen collection. Serum samples were stored at −80° C. until further analysis. Harvested spleen samples were weighed and flash-frozen in a mix of ethanol/dry ice and stored immediately at −80° C. All samples were processed and assayed for viral load via plaque assay. All animals were monitored daily for weight loss, morbidity, mortality, and neurological decay. Mice displaying severe illness as determined by >20% weight loss, a health score of greater than 5 (rating coat appearance, mobility and alertness), extreme lethargy, and/or paralysis were euthanized and harvested for terminal sera and spleens. Any mouse that was found dead, was harvested for spleen collection only.
Spleen samples collected according to Example 19 were homogenized in 250 μL DPBS using a TissueRuptor. Homogenates were then centrifuged and the supernatants collected and frozen immediately in two different aliquots until the plaque assay was performed. The results of the plaque assay are shown in
On the day prior to the assay, Vero cells were seeded at a density of 1×105 cells/well in 1 mL 1% Hi-FBS medium in 24-well tissue culture plates and incubated overnight at 37° C. in a 5% CO2 atmosphere. The following day, 10-fold serial dilutions (10−2 to 10−5) of the serum and spleen samples were prepared for titration in triplicate. Cell culture medium from the Vero cells was removed and 100 μL of MEM medium supplemented with 2 mM L-glutamine and 1× Pen/Strep was added along with 100 μL of the diluted samples. Cells were incubated for 1 h at 37° C. in a 5% CO2 atmosphere. Then, 1 mL 0.8% methylcellulose containing 2% FBS supplemented with 2 mM L-glutamine and 1× Pen/Strep was added to the cells without inoculum removal and incubated for an additional 3 days at 37° C. in a 5% CO2 atmosphere to allow plaque formation. Cells were fixed and permeabilized using a cold 80% ethanol/20% methanol mixture for 30 minutes at −20° C. 200 L anti-Dengue monoclonal antibody diluted 1:2000 in 5% nonfat milk was added to each well and incubated overnight at 4° C. Wells were washed 3 times with 1×DPBS, 200 μL of HRP-conjugated goat anti-mouse antibody at 1:2000 dilution in 5% non-fat milk was added, and the wells incubated for 1 h at room temperature. Viral plaques were resolved and counted using insoluble peroxidase substrate (TrueBlue) and a plaque counter. Data for the spleen viral load was normalized to the spleen weight for each individual mouse.
Pharmacokinetic measurements for
Data analysis of the pharmacokinetic data generated according to Example 21 and presented in
Healthy males and females 18-65 years of age were enrolled in single ascending dose (SAD) and multiple ascending dose (MAD) cohorts and randomized to receive oral Compound 2 or placebo according to Tables 4-7.
Safety assessments including adverse events, standard clinical laboratory tests, vital sign measurements, electrocardiograms, and physical examinations were performed.
Compound 2 was well tolerated and no serious adverse events or discontinuations due to adverse events were reported. Nonserious adverse events were mild or moderate in severity and resolved by the end of the study (Table 8). Sporadic cases of gastrointestinal related events including mild to moderate vomiting occurred mostly at higher doses.
Healthy males and females 18-65 years of age were enrolled in single ascending dose (SAD) and randomized to receive oral Compound 2 or placebo according to Tables 4-6.
Plasma and urine samples were collected at pre-determined timepoints and quantitated for Compound 1 and its metabolites Compounds 3, 6, and 7 using LC-MS/MS. Pharmacokinetic analyses were performed using non-compartmental approaches.
Upon oral dosing under fasting conditions, the parent prodrug Compound 1 was rapidly absorbed and cleared, followed by the appearance of Compound 3 which also exhibited a transient exposure (Tables 10-14, and
Plasma exposure of Compound 1 and its metabolites increased over the studied dose range from 250 to 1500 mg. Compound 7 increased in a dose-proportional manner. Compound 1 and Compound 3 increased in a greater than dose-proportional manner whereas Compound 6 was slightly less than dose-proportional.
A high-fat/high-calorie meal delayed and decreased peak level of Compound 1, Compound 3, and Compound 7, but had limited to no impact on their total exposure and slightly increased the plasma exposure of Compound 6.
Pharmacokinetic profiles were similar between the South/Southeast/East Asian and mostly Caucasian subject cohorts, suggesting absence of pharmacokinetic ethnic sensitivity. Therefore, Compound 2 does not require dose adjustment.
Following single oral administration of Compound 2 at 250 to 1500 mg, urine elimination was low for Compound 1 and Compound 3 and was modest for Compound 7 and Compound 6 (Table 9). Total urine recovery ranged from approximately 20% to approximately 30% across doses. Renal clearance of the nucleoside metabolites Compound 6 and Compound 7 exceeded estimated glomerular filtration rate suggesting involvement of active secretion in their renal environment.
Healthy males and females 18-65 years of age were enrolled in multiple ascending dose (MAD) and randomized to receive oral Compound 2 or placebo according to Tables 4 and 7.
Plasma and urine samples were collected at pre-determined timepoints and quantitated for Compound 1 and its metabolites Compounds 3, 6, and 7 using LC-MS/MS. Pharmacokinetic analyses were performed using non-compartmental approaches.
Plasma exposure of Compound 6 increased by approximately 25, 60 and 80% with QD, BID, and TID respectively due to its long plasma half-life. This is reflective of rapid accumulation and sustained intracellular exposure of the active metabolite Compound 5.
The 750 mg TID dose led to a rapid increase in plasma concentration of Compound 6, exceeding the 90% effective concentration (EC90) of the drug in inhibiting dengue virus replication in vitro (0.64 μM). These levels were maintained over the treatment period (
In tables 15-18, Cmax and AUCtau are represented by the mean±standard deviation. Tmax is represented as the median (minimum-maximum). AUCtau indicates the area under plasma concentration-time curve over the dosing interval.
Transcripts of the dengue viruses (DENV1 WP, DENV2 NGC, DENV3 VN32, DENV4 MY01) were modified with a nanoluciferase reporter between the 5′UTR and capsid gene as previously described (Baker et al., 2020b, 2020a) to create tagged viruses that are stable with cell culture passaging while producing a robust luciferase signal after inoculation for efficient screening of antiviral activity. Eight 2-fold serial dilutions of AT-281 were mixed with each reporter virus (MOIs of 0.1 for DENV-1, DENV-2 and DENV-3; MOI of 0.001 for DENV-4) and added to plates containing Huh-7 cells (RRID: CVCL 0336) that were seeded at 1×104 cells per well in a 96-well plate the previous day in Dulbecco's Modified Eagle Medium (Invitrogen, Carlsbad, CA) with 2% fetal bovine serum (Hyclone, Logan, UT). Cells were washed 48 h post infection three times with phosphate-buffered saline, followed by addition of NanoGlo® substrate diluted 1:100 in NanoGlo® Assay Buffer (Promega, Madison, WI). Luciferase activities were read by a BioTek Cytation 5 plate reader after 3 minutes. Data were analyzed with GraphPad Prism 9 software. Luciferase activities were normalized to DMSO treatment samples which were set to 100%. Error bars represent mean±SD. Results are representative of three independent experiments with each one analyzed in triplicate. The antiviral activity against the viruses tested can be found in
The proteins used in the thermal shift assay (Example 31) were prepared as described herein. Full length DENV NS5 and RdRp domains used in this study were expressed under the control of a T7-promoter in pET28a vectors in Escherichia coli (E. coli) NEB C2566 cells (New England Biolabs) carrying the pRARE2LacI (Novagen) plasmid. Proteins were expressed overnight at 17° C. in TB (with 25 μg/mL Kanamycin and 17 μg/mL Chloramphenicol), following induction with 100 μM IPTG and 2% EtOH (% v/v) at an OD600 of 0.8-1.
DENV MTases were expressed under the control of a T7-promoter in pDEST17 vectors in Escherichia coli (E. coli)) NEB C2566 cells (New England Biolabs) carrying the pRARE2LacI (Novagen) plasmid. Proteins were expressed overnight at 17° C. in TB (with 100 μg/mL Ampicillin and 17 μg/mL Chloramphenicol), following induction with 200 μM IPTG and 2% EtOH (% v/v) at an OD600 of 0.8-1.
The DENV NS5 used in the thermal shift assay (Example 31) were purified as described herein. The cells were disrupted by sonication on ice in a lysis buffer (50 mM NaP pH 8, 1 M NaCl, 20% glycerol) supplemented with, 1.0 mg/mL Lysozyme, 22 μg/mL DNase, 1.6% igepal, 0.5 mM TCEP and a complete protease inhibitor cocktail (COC) from Roche. Protein from the soluble fraction was loaded onto TALON® Superflow™ cobalt-based IMAC resin (Cytiva), washed 5 times with lysis buffer, 10 times with lysis buffer (igepal free) prior to elution with a lysis buffer supplemented with 250 mM imidazole and 250 mM glycine. Imidazole was removed by an overnight dialysis in a dialysis buffer (50 mM NaP pH 8, 20% glycerol, 150 mM NaCl, 250 mM glycine and 0.5 mM TCEP) before to perform a last step purification using a size exclusion chromatography (SEC) HiLoad® 16/600 Superdex® 200 μg column in a buffer (10 mM HEPES pH 8, 300 mM NaCl, 10% glycerol and 0.5 mM TCEP). Protein was then concentrated to 10 to 15 mg·mL-1 and stored at −80° C. after a last dialysis in the SEC buffer supplemented with 40% of glycerol.
The DENV RdRp used in the thermal shift assay (Example 31) were purified as described herein. The cells were disrupted by sonication on ice in a lysis buffer (50 mM NaP pH 8, 1 M NaCl, 20% glycerol) supplemented with, 1.0 mg/mL Lysozyme, 22 μg/mL DNase, 1.6% igepal, 0.5 mM TCEP and a complete protease inhibitor cocktail (COC) from Roche. Protein from the soluble fraction were loaded onto TALON® Superflow™ cobalt-based IMAC resin (Cytiva), washed 5 times with lysis buffer, 10 times with lysis buffer (igepal free) prior to elution with a lysis buffer supplemented with 250 mM imidazole and 250 mM glycine. Imidazole was removed by an overnight dialysis in a dialysis buffer (50 mM NaP pH 8, 20% glycerol, 150 mM NaCl, 250 mM glycine and 0.5 mM TCEP) before to perform a last step purification using a GE Hi-trap Heparine column. Protein was then concentrated to 10 to 15 mg·mL-1 and stored at −80° C. after a last dialysis in a final buffer containing 20 mM HEPES pH 8, 300 mM NaCl, 40% of glycerol and 0.5 mM TCEP.
The DENV MTase used in the thermal shift assay (Example 31) were purified as described herein. The cells were disrupted by sonication on ice in a lysis buffer (50 mM HEPES pH 7.5, 10% glycerol, 500 mM NaCl and 5 mM Imidazole) supplemented with 0.2 mM Benzamidine, 1 mg/mL Lysozyme, 22 μg/mL DNase and 0.5 mM TCEP. Protein from the soluble fraction were loaded onto TALON® Superflow™ cobalt-based IMAC resin (Cytiva) and washed with lysis buffer (1M NaCl) prior to elution with 250 mM imidazole. The protein was finally purified with a SEC (HiLoad® 16/600 Superdex® 200 μg) in a buffer of 50 mM HEPES pH 8, 300 mM NaCl and 0.5 mM TCEP. Protein was then concentrated to 10 to 15 mg·mL-1 and stored at −80° C. after a last dialysis in a final buffer containing 20 mM HEPES pH 8, 300 mM NaCl, 40% of glycerol and 0.5 mM TCEP.
The thermal stability of the flavivirus proteins and their interaction with compounds were evaluated by using a thermal shift assay. Reactions were performed in white frame star PCR 96-well plates (ref 044705) with a final volume of 20 μL. Regarding the DENV NS5, Polymerase and MTase, the best conditions were found by using a buffer containing 20 mM Hepes pH7.5, 50 mM NaCl, 5 mM DTT, 1000 Glycerol and 1M Sorbitol. 0.4 μL of GTP, Sinefungin or AT-9010 were disposed in the plate (100 μM final concentration). Water was used as a control without compound. 2 μM of NS5, 2 μM of Polymerase or 6 μM of MTase were mixed in the buffer with 2.5×, 0.75× or 1.5× of dye respectively (final concentrations). The mix of protein and Protein thermal shift kit from ThermoFisher (ref 4461146) was prepared and 19.6 μl were dispensed into each well.
The plate was sealed with adhesive film and then submitted to a run on a CFX96 RT-PCR (Bio-Rad). The program started by a 1 min equilibration phase at 20° C., followed by a temperature gradient of 0.5° C./30 s, from 20° C. to 95° C., recording fluorescence every 0.5° C. Fluorescence was recorded using the FRET channel. Every experiment was performed in triplicate. Curves were plotted and analyzed to determine the melting temperature by using the Boltzmann sigmoidal equation with the GraphPad Prism software. Graphical representation of the data in the table below is shown in
Reactions were carried out in the presence of 2 mM MnCl2 or 5 mM MgCl2, the optimum concentrations of these catalytic ions for RNA synthesis by DENV2 RdRp (Selisko et al., 2006). Discrimination of AT-9010 against GTP was measured in four independent reactions, with similar results as those shown in
The transfer of tritiated methyl from [3H]-SAM onto RNA substrate was monitored by filter-binding assay (FBA), performed according to the method described previously (Paesen et al., 2015). The recombinant 2′-O-methyltransferase domain of the non-structural protein NS5 from DENV serotype 2 (DENV NS5 MTase) corresponding to residues 1-296 of NS5 was expressed and purified as described previously (Egloff et al., 2002). FBA was carried out in reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 2 μM SAM and 0.1 μM 3H-SAM (Perkin Elmer)] in the presence of 0.7 μM mGpppAC4 synthetic RNA and DENV2 NS5MTase protein (500 nM). The enzyme was first mixed with the compound (0.5 to 1000 μM) suspended in water before the addition of RNA substrate and SAM and then incubated at 30° C. Reaction mixtures were stopped after 30 min by their 10-fold dilution in ice-cold water. Samples were transferred to diethylaminoethyl (DEAE) filtermat (Perkin Elmer) using a Filtermat Harvester (Packard Instruments). The RNA-retaining mats were washed twice with 10 mM ammonium formate pH 8.0, twice with water and once with ethanol. They were soaked with scintillation fluid (Perkin Elmer), and 3H-methyl transfer to the RNA substrates was determined using a Wallac MicroBeta TriLux Liquid Scintillation Counter (Perkin Elmer). For IC50 measurements, values were normalized and fitted with Prism (GraphPad software) using the following equation: Y=100/(1+((X/IC50){circumflex over ( )}Hillslope)). IC50 is defined as the inhibitory compound concentration that causes 50% reduction in enzyme activity. Data curves can be found in
DENV NS5 was pre-incubated with the annealed P10/T20 RNA in an assembly buffer containing 20 mM HEPES pH 7.5, 10% glycerol, 5 mM MgCl2, and 5 mM DTT for 10 min at 30° C. to create an active RNA elongation complex.
Multiple nucleotide incorporation was tested as follows and the results shown in
Single nucleotide incorporation was tested as follows. Reactions were started by adding the corresponding NTP or analogue. Final concentrations were 1 μM NS5, 0.25 μM P10/T20, 100 μM NTP or analogue in a final buffer containing 20 mM HEPES pH 7.5, 15% glycerol, 5 mM MgCl2, and 5 mM DTT.
Reactions were quenched at the designated time points in 3× volume FBD stop solution (formamide, 10 mM EDTA) and analyzed on 20% acrylamide-bisacrylamide (19:1) 7 μM urea sequencing gels. RNA products were visualized using a Typhoon FluorImager then analyzed and quantified using ImageQuant software.
Crystallographic images produced by the protocol described herein are presented in
Crystallization conditions were adapted from (Lim et al., 2011) using the sitting-drop vapor diffusion method using crystallization buffer. All crystals were grown at 293.15 K, using a 1:2 ratio of protein (10 mg/mL) to precipitant solution (22% PEG 8000, 200 mM NaCl, 20 mM Tri-Sodium Citrate, 100 mM Tris pH 8.5). Crystals grew in 4 days and were soaked overnight with 1 mM of AT-9010 (final concentration). All crystals were cryo-protected with reservoir solution supplemented with 10% glycerol, and flash-frozen in liquid nitrogen at 100 K.
Data collection and diffraction data were collected at Soleil synchrotron. Original native data set was collected on Proxima2. Data set was processed and analysed with autoPROC toolbox (Vonrhein et al., 2011). Structure was solved by molecular replacement using PHASER (McCoy et al., 2007) and PDB 4CTK as a reference model. As previously observed the crystal Asymmetric Unit is composed of 2 chains, however the additional positive density blobs found in both chain is not equivalent. In one chain the extra density corresponds to a single molecule of SAM while in the other chain the positive density corresponds to one SAH and an AT-9010 molecules. The model was build using COOT (Emsley and Cowtan, 2004) and refined at 1.90 Å using PHENIX (Adams et al., 2010). The structure was checked and confirmed to have good stereochemistry according to MOLPROBITY (Chen et al., 2010). Data collection and refinement statistics are listed in Table 21. Structural analysis and figures was done using UCSF CHIMERA (Pettersen et al., 2004).
Compound 1 (the free base form of Compound 2) can be synthesized, for example, by reacting a compound of the formula Intermediate A with Intermediate B and an activator to form a compound of the formula Intermediate C, which is then reacted with phenol to form Compound 1:
Reaction of a compound of the formula Intermediate A, Intermediate B, an activator and a trialkylamine base preferentially forms the SP phosphoramidate (as described in WO 2022/040473A1 assigned to Atea Pharmaceuticals).
As used herein, an “activator” is a uronium-based peptide coupling reagent, including but not limited to 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) and 1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU).
As used herein, a “trialkylamine base” is an amine base with three C1-6 alkyl groups, including but not limited to triethylamine or diisopropylethylamine.
As used herein, a “pyridine base” is an optionally substituted pyridine molecule, including but not limited to pyridine, 2,6-dimethylpyridine (lutidine), or 2,4,6-trimethylpyridine (collidine).
If the SP phosphoramidate has electron withdrawing substituents on the aryl ester (Intermediate C), then the SP compound can be reacted further. The phosphorus stereochemistry can be inverted by reaction of the SP phosphoramidate (Intermediate C) with phenol and a base to form the RP phosphoramidate (Compound 1).
Intermediate A can be synthesized through a multistep displacement of phosphorus chloride (Intermediate E) followed by reductive deprotection of the benzyl protecting group.
In certain embodiments, the electron withdrawing group on the phenol of Intermediate A is an ortho-fluoro substituent (Intermediate A-1):
In certain embodiments, Intermediate A has one or more electron withdrawing substituents on the phenol. Electron withdrawing substituents include but are not limited to fluorine, chlorine, cyano, azido, nitro, trifluoromethyl, difluoromethyl, fluoromethyl, ester and amide. In certain embodiments, Intermediate A is used in the form of a salt. In certain embodiments, Intermediate A is used as the dihydroquinine salt.
The reaction of Intermediate C with phenol to form Compound 1 can be performed for example using a base such as an amine base, for example a guanidine base. Examples of guanidine bases are Bases A, B, C, D, E, F, G, and J. Bases A, C, D and J provided the highest yields of Compound 1. Intermediate C-1 was used as a model substrate for the reaction.
In certain embodiments, Compound 1 is prepared from a SP phosphoramidate Intermediate C, such as Intermediate C-1, C-2, C-3, C-4, C-5, C-6, C-7, and C-8. Intermediate C-1 through C-8 have different electron withdrawing groups on the phenol. These compounds can be reacted with phenol and a base to form Compound 1:
Procedure for synthesis of Intermediate A-1
Dichloromethane (2.0 L, 10 vol) was charged to a three-necked flask at 10° C. The flask was evacuated and refilled with nitrogen gas three times. Phosphorous oxychloride (200 g, 1.0 equiv) was charged to the flask, before the mixture was cooled to −55° C. Dichloromethane (200 mL, 1 vol), benzyl alcohol (141 g, 1 equiv), and triethylamine (139 g, 1.05 equiv) were added to a mixing flask at 10° C., before charging the resultant mixture to the phosphorous oxychloride solution over 40 minutes under nitrogen protection. The reaction mixture was stirred for 1 hour at −55° C., during which time white solids precipitated. The reaction appeared to be 99.6% completed, by HPLC analysis of a reaction mixture sample derivatized with benzylamine. L-Alanine isopropyl ester hydrochloride (219 g, 1 equiv) was charged to the reaction mixture, followed by triethylamine (271 g, 2.05 equiv) over 30 minutes.
The resultant reaction mixture was stirred for 1-2 hours, then analyzed for consumption of the first intermediate by HPLC analysis of a sample derivatized with benzylamine. The reaction appeared to be 97.7% complete.
Dichloromethane (400 mL, 2 vol), 2-fluorophenol (146 g, 1 equiv), and triethylamine (139 g, 1.05 eq, added over 20 minutes) were combined in a mixing flask at 10° C., before addition to the reaction mixture over 43 minutes. The batch was warmed from −55° C. to 10° C. over a period of 3 hours, then stirred 2-4 hours. HPLC analysis of a sample derivatized with benzylamine showed 2.0% area of the second intermediate. The batch was filtered, and the filter cake was rinsed with dichloromethane (400 mL, 2 vol). Water (600 mL, 3 vol) was charged to the filtrate, and the mixture was stirred at 10° C. for 5 minutes. The mixture was allowed to settle, and the layers were separated. Hydrochloric acid in water (3.7% w/w, 1 L, 5 vol) was charged to the organics and the mixture was stirred for 5 minutes at 10° C. The mixture was allowed to settle, and the layers were separated. Sodium bicarbonate in water (5% w/w, 1 L, 5 vol) was charged to the organics and the mixture was stirred for 5 minutes at 5° C. The mixture was allowed to settle, and the layers were separated. Water (600 mL, 3 vol) was charged to the organics, and the mixture was stirred at 10° C. for 5 minutes. The mixture was allowed to settle, and the layers were separated. Charcoal (10 g, 5% w/w) was charged to the organics, and the resulting suspension was stirred for 2 hours at 10° C. The suspension was filtered to remove the charcoal, and the filter was rinsed with dichloromethane (100 mL). The filtrate was concentrated under vacuum at 45° C. to a volume of about 600 mL (3 vol). Isopropanol (600 mL, 3 vol) was charged to the mixture, which was again concentrated to about 600 mL (3 vol). Isopropanol (600 mL, 3 vol) was charged to the mixture, the mixture was adjusted to 25° C., and a sample was taken to quantify the amount of dichloromethane remaining (0.25% remained).
Isopropanol (1.9 L, 9.5 vol) was charged to the mixture, followed by dihydroquinine (DHQ, 387 g, 0.908 equiv). The mixture was stirred until a clear solution was formed. Palladium on carbon (10% w/w, KF=63%, 13.7 g) was charged to the mixture. The resulting suspension was degassed with nitrogen twice, then degassed with hydrogen three times. The mixture was stirred for 18-20 hours at 25° C. under 1 atm of hydrogen gas. Analysis of a sample by HPLC showed 0.05% of the third intermediate remaining.
The suspension was filtered and the cake was rinsed with isopropanol (100 mL, 0.5 vol). Solvent was distilled form the mixture under vacuum at 55° C. to a volume of approximately 1200 mL (6 vol), then acetonitrile (1.5 L, 7.5 vol) was charged to the mixture to provide a suspension. The distillation and acetonitrile addition were repeated two additional times. A sample of the resultant suspension was diluted with N-methyl pyrrolidone and analyzed to check for residual isopropanol (0.7% found). The suspension was stirred at 80-90° C. for 1-2 hours to provide a clear solution. The mixture was cooled to 5° C. with a cooling rate of 20° C. per hour, then stirred for 2-3 hours. The suspension was filtered and the cake was washed with cold acetonitrile (400 mL, 2 vol, at 5° C.). The cake was dried at 55° C. for 18-20 hours without vacuum to provide 591.7 g of Intermediate A-1 as a white powder (97.9% AUC purity, 71.8% yield over 4 steps).
1H NMR (400 MHz, DMSO) δ 12.50 (s, 1H), 8.75 (d, J=4.5 Hz, 1H), 7.96 (d, J=9.2 Hz, 1H), 7.70-7.48 (m, 3H), 7.41 (dd, J=9.2, 2.5 Hz, 1H), 7.12 (dd, J=11.0, 8.2 Hz, 1H), 7.00 (t, J=7.5 Hz, 1H), 6.97-6.86 (m, 1H), 6.59 (s, 1H), 6.05 (s, 1H), 4.77 (dq, J=12.5, 6.2 Hz, 1H), 3.97 (s, 4H), 3.87-3.66 (m, 2H), 3.57-3.34 (m, 2H), 3.07 (s, 1H), 2.84 (d, J=6.4 Hz, 1H), 1.97 (d, J=36.8 Hz, 3H), 1.87-1.61 (m, 2H), 1.38 (t, J=11.8 Hz, 1H), 1.33-1.18 (m, 2H), 1.16 (d, J=6.6 Hz, 3H), 1.09 (dd, J=6.2, 1.2 Hz, 6H), 0.75 (t, J=7.3 Hz, 3H).
13C NMR (101 MHz, DMSO) δ 174.69 (d, J=5.9 Hz), 158.27 (s), 154.97 (s), 152.58 (d, J=6.7 Hz), 147.78 (s), 146.04 (s), 144.19 (s), 142.55-142.06 (m), 131.72 (s), 126.16 (s), 124.38 (d, J=3.4 Hz), 122.73 (s), 122.67-122.04 (m), 119.39 (s), 116.16 (d, J=18.9 Hz), 102.09 (s), 67.71 (s), 66.45 (s), 9.32 (s), 56.87 (s), 55.27 (s), 50.96 (s), 43.12 (s), 35.28 (s), 26.04 (s), 24.89 (s), 21.83 (d, J=2.3 Hz), 21.21 (d, J=5.4 Hz), 17.74 (s), 11.86 (s).
31P NMR (162 MHz, DMSO) δ 1.04 (s).
19F NMR (377 MHz, DMSO) δ −132.65 (s).
Dichloromethane (1.33 L, 20 vol), Intermediate B (50.0 g, 1.0 equiv, 0.160 mol), dihydroquinine salt of Intermediate A (222 g, 2.2 equiv), and (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (82.3 g, 1.2 equiv) were under nitrogen charged to a 2 μL three-neck flask equipped with a reflux condenser and thermometer.
The resulting suspension was heated to 30-35° C. 2,6-Lutidine (20.6 g, 1.2 equiv) was charged to the reaction mixture over 2 hours and the suspension gradually dissolved during the addition. The mixture was stirred at 30-35° C. for 5-6 hours to provide a clear solution. The reaction was sampled and analysis by HPLC showed less than 5% of Intermediate B remaining. Solution yield calculated by assay of the sample was 64.2 g (66.9% yield). The reaction mixture was cooled to 0-10° C. over 1 hour.
Water (500 mL, 10 vol) was charged to the mixture and the batch was stirred for 2 minutes. Agitation was halted and the batch was allowed to settle into layers before removal of the aqueous layer. Aqueous hydrochloric acid (20% w/w, 133 g, 4.5 equiv) was charged over a period of 1 hour, over which time dihydroquinine salt precipitated. The solution was filtered, and the filter cake was rinsed with dichloromethane (50 mL, 1 vol) and the rinse was combined with the filtrate. Aqueous hydrochloric acid (7% w/w, 167 g, 2.0 equiv) was charged to the filtrate and the batch was agitated for 2 min. Agitation was stopped, the batch was allowed to settle into two layers, and the aqueous layer was removed. The organics were washed with aqueous sodium bicarbonate (5% w/w, 500 g, 1.9 equiv) three times, then washed with water (500 mL, 10 vol). Assay yield of the resultant organic layer was 59.4 g (61.9%). The organic layer was concentrated under vacuum at a temperature of 30-50° C. to a total volume of approximately 200 mL (4 vol). Toluene (500 mL, 10 vol) was charged to the mixture and the mixture was concentrated under vacuum at a temperature of 40-55° C. to a total volume of approximately 250 mL (5 vol). The toluene addition and concentration was repeated one additional time. Ethyl acetate (250 mL, 5 vol) was charged to the batch, and the mixture was heated to 65-75° C. While agitating, the mixture was cooled to 5° C. over 3 hours, during which time crystalline solid formed. The batch was held for 4 hours, then filtered to provide a yellow crystalline solid. Theis solid and ethyl acetate (250 mL, 5 vol) were charged to the flask and heated to 65-75° C. to provide a clear solution. The solution was cooled to 5° C. over 3 hours as crystalline solid formed. The batch was held at 5° C. for 4 hours, then filtered to provide a yellow solid. The yellow solid was dried at 45° C. for 15-16 hours to provide Intermediate C-1 (79 g, 56.2% corrected yield) as a yellow crystalline solid.
1H NMR (400 MHz, DMSO) δ 7.80 (s, 1H), 7.53-7.40 (m, 1H), 7.40-7.26 (m, 2H), 7.25-7.11 (m, 2H), 6.14 (dd, J=12.9, 10.4 Hz, 1H), 6.11-5.91 (m, 3H), 5.78 (d, J=6.4 Hz, 1H), 4.91-4.71 (m, H), 4.58-4.23 (m), 4.15-4.05 (m, 1H), 3.91-3.72 (m, 1H), 2.88 (s, 3H), 1.32-1.20 (m, 3H), 1.17-0.97 (m, 9H).
19F NMR (377 MHz, DMSO) δ −131.41 (s), −159.74 (s).
31P NMR (162 MHz, DMSO) δ 4.42 (s).
MS (ES+): Expected: 600.2; found: 600.3
Acetonitrile (255 mL, 8.5 vol), dimethyl sulfoxide (45 mL, 1.5 vol), Intermediate C-1 (30 g, 0.05 mol, 1.0 equiv), phenol (94.1 g, 20.0 equiv) 1,1,3,3-tetramethylguanidine (17.3 g, 3.0 equiv) were charged to a 350 mL flask under nitrogen equipped with a thermometer. The batch was heated to 30° C. for 22 hours while stirring.
The reaction was sampled and remaining Intermediate C-1 was found to be less than 2%. The reaction mixture was cooled to 0-10° C. over 1 hour, and the batch was poured into a reactor containing water (600 mL, 20 vol) while stirring. Methyl-tert-butyl ether (600 mL, 20 vol) was charged to the mixture. Aqueous hydrochloric acid (20% w/w, 90 g, 10 equiv) was charged over 0.5 hours to a pH value of 3-4 while maintaining the temperature of the batch at 0-10° C. The mixture was stirred for an additional 2 minutes, stirring was halted, the aqueous and organic layers were allowed to separate, and the aqueous layer was collected. Water (300 mL, 10 vol) was charged to the organic layer, followed by aqueous hydrochloric acid (20% w/w, 32 g, 3.5 equiv) over 0.5 hours while maintaining the temperature of the batch at 0-10° C.
The mixture was stirred for an additional 2 minutes, stirring was halted, the aqueous and organic layers were allowed to separate, and the aqueous layer was collected. Both aqueous layers were combined and cooled to 0-10° C. Aqueous sodium bicarbonate (5% w/w, 85 g, 1.0 equiv) was added over a period of 0.5 h to bring the pH to 8-9, followed by dichloromethane (300 mL, 10 vol) and water (300 mL, 10 vol). The mixture was stirred for 2 minutes, allowed to settle into two phases, and the aqueous phase was removed. The resultant organics were concentrated at 30-40° C. under vacuum to an approximate volume of 60 mL (2 vol).
Acetone (300 mL, 10 vol) was charged to the mixture. The batch was concentrated at 30-40° C. under vacuum to an approximate volume of 60 mL (2 vol). Acetone (180 mL, 6 vol) was charged to the mixture to provide a clear solution, which was cooled to 0-5° C. Aqueous hydrochloric acid (36.5% w/w, 10 g, 2.0 equiv) was charged in one portion and the mixture was stirred at 0-5° C. for 12 hours. The resultant suspension was filtered, and the recovered solid was dried to provide the bis-hydrochloride salt of Compound 1 (19.2 g, 59% yield) as an off-white solid.
1H NMR (400 MHz, DMSO) δ 9.68 (d, J=4.7 Hz, 1H), 8.14 (s, 3H), 7.36 (t, J=7.9 Hz, 2H), 7.18 (dd, J=17.8, 7.9 Hz, 3H), 6.11 (d, J=18.6 Hz, 2H), 4.84 (hept, J=6.2 Hz, 1H), 4.47 (dd, J=10.4, 5.9 Hz, 1H), 4.43-4.2, 1H), 3.77 (s, 1H), 3.15 (d, J=4.8 Hz, 3H), 1.28-1.07 (m, 12H).
13C NMR (101 MHz, DMSO) δ 173.14 (d, J=4.3 Hz), 153.46 (s), 151.11 (d, J=6.0 Hz), 149.58 (s), 130.03 (s), 124.98 (s), 120.62 (d, J=4.6 Hz), 112.08 (s), 102.03 (s), 100.24 (s), 80.59 (s), 72.00 (d, J=18.2 Hz), 68.43 (s), 65.85 (s), 50.42 (s), 40.49 (d, J=13.9 Hz), 40.35 (s), 40.14 (s), 39.93 (s), 39.72 (s), 39.51 (s), 39.30 (s), 31.16 (s), 30.07 (s), 21.84 (d, J=7.2 Hz), 20.08 (d, J=7.2 Hz), 17.15 (s), 16.91 (s).
19F NMR (377 MHz, DMSO) δ −160.09 (s).
31P NMR (162 MHz, DMSO) δ 3.66 (s).
MS (ES+): Expected: 582.22; found: 582.35
A mixture of benzyl alcohol (10.81 g, 0.1 mol) and TEA (10.62 g, 0.105 mol) was added dropwise to a solution of Phosphorus oxychloride (15.3 g, 0.1 mol) in DCM (150 mL) at −60° C. under nitrogen. After addition, the reaction was stirred at −60° C. for 1 h.
L-Alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) was added to the reaction and then TEA (20.74 g, 0.205 mol) was added dropwise at −60° C. The reaction mixture was stirred at −60° C. for 1 h. A substituted phenol and TEA (10.62 g, 0.105 mol) and DCM (20 mL) was added dropwise to the reaction at −60° C.
After addition, the reaction was stirred to room temperature overnight before being quenched with water. The organic layer was dried over Na2SO4, filtered and then the solvent was removed in vacuum. The residue was purified by silica gel column chromatography to give a colorless oil (benzyl protected Intermediates A-2, A-3, and A-7).
The colorless oil (0.1 mol) and isopropanol (200 mL, 5 vol) was charged into a 1000 mL 3-neck glass flask. Dihydroquinine (32.64 g, 0.1 mol) was added and stirred to give a clear solution. To this solution, 5% wet Pd/C (60% water by KF, 5% w/w dry basis) was charged, and hydrogenolysis was performed at 20-25° C. for 18-20 h using 1 atm of hydrogen. When the IPC showed the starting material was consumed completely, the mixture was filtered through a Buchner funnel, and the filtrate was stirred with 4.0 g of charcoal for 2 hours at 25-30° C. The mixture was filtered, and the cake was washed with isopropanol (40 mL). The filtrate was concentrated under vacuum less than 0.09 MPa at 50-60° C. to give a crude product.
Isopropyl acetate (200 mL, 5 vol) was added, and the mixture was concentrated again under vacuum less than 0.09 MPa at 50° C.-60° C. This step was repeated one more time with 5 vol of isopropyl acetate. To the residue, fresh isopropyl acetate (200 ml, 5 vol) was added, and the mixture was stirred at 80° C.-90° C. for 2-3 h to give a clear solution. Then it was cooled to 0° C.-10° C. with a cooling rate of 20° C./1 h and stirred at this temperature for 2-3 h. The solid was collected by filtration, rinsed with cold isopropyl acetate (40 mL, 1 vol) to give the wet cake, which was dried at 50° C. without vacuum for 18 h to give dihydroquinine salt of Intermediate A-2, A-3 or A-7 as an off-white solid.
1H NMR (400 MHz, DMSO) δ 12.30 (s, 1H), 8.74 (d, J=4.5 Hz, 1H), 7.96 (d, J=9.2 Hz, 1H), 7.64 (d, J=4.6 Hz, 1H), 7.62-7.56 (m, 1H), 7.52 (d, J=2.5 Hz, 1H), 7.41 (dd, J=9.2, 2.6 Hz, 1H), 7.25-7.03 (m, 1H), 6.97-6.76 (m, 1H), 6.57 (s, 1H), 6.02 (s, 1H), 4.77 (dt, J=12.5, 6.2 Hz, 1H), 3.97 (s, 4H), 3.75 (dd, J=12.1, 7.7 Hz, 2H), 3.59-3.28 (m, 3H), 3.09 (d, J=5.3 Hz, 1H), 2.86 (s, 1H), 2.01 (s, 2H), 1.92 (s, 1H), 1.88-1.64 (m, 2H), 1.38 (t, J=11.8 Hz, 1H), 1.33-1.18 (m, 2H), 1.14 (d, J=5.6 Hz, 3H), 1.09 (dd, J=6.2, 0.8 Hz, 6H), 0.75 (t, J=7.3 Hz, 3H).
MS (ES−): Expected: 322.07; found: 322.00
1H NMR (400 MHz, DMSO) δ 12.36 (s, 1H), 8.74 (d, J=4.5 Hz, 1H), 7.96 (d, J=9.2 Hz, 1H), 7.64 (d, J=4.5 Hz, 1H), 7.54 (d, J=2.5 Hz, 1H), 7.41 (dd, J=9.2, 2.6 Hz, 1H), 7.21 (dd, J=15.7, 8.1 Hz, 1H), 7.07 (dd, J=11.5, 2.0 Hz, 1H), 6.94 (d, J=8.2 Hz, 1H), 6.74 (td, J=8.5, 2.4 Hz, 1H), 6.57 (s, 1H), 6.04 (s, 1H), 4.77 (dt, J=12.5, 6.2 Hz, 1H), 3.97 (s, 4H), 3.76 (d, J=5.4 Hz, 2H), 3.63-3.25 (m, 2H), 3.09 (d, J=4.7 Hz, 1H), 2.85 (s, 1H), 1.97 (d, J=34.0 Hz, 3H), 1.88-1.62 (m, 2H), 1.38 (t, J=11.8 Hz, 1H), 1.33-1.18 (m, 2H), 1.18-1.03 (m, 9H), 0.75 (t, J=7.3 Hz, 3H).
MS (ES−) Expected: 304.08; found: 304.04
1H NMR (400 MHz, DMSO) δ 12.56 (s, 1H), 8.74 (d, J=4.5 Hz, 1H), 7.96 (d, J=9.2 Hz, 1H), 7.64 (d, J=4.5 Hz, 1H), 7.54 (d, J=2.6 Hz, 1H), 7.41 (dd, J=9.2, 2.6 Hz, 1H), 7.15 (dt, J=8.4, 4.3 Hz, 2H), 7.09-6.88 (m, 2H), 6.60 (s, 1H), 6.05 (s, 1H), 4.95-4.60 (m, 1H), 3.97 (s, 4H), 3.81-3.58 (m, 2H), 3.47 (d, J=7.4 Hz, 1H), 3.41 (s, 1H), 3.05 (s, 1H), 2.82 (s, 1H), 2.11-1.86 (m, 3H), 1.75 (d, J=36.1 Hz, 2H), 1.28 (ddt, J=20.8, 13.9, 9.0 Hz, 3H), 1.17-1.04 (m, 9H), 0.75 (t, J=7.3 Hz, 3H).
MS (ES−) Expected: 304.08; found: 304.18
A mixture of benzyl alcohol (10.81 g, 0.1 mol) and TEA (10.62 g, 0.105 mol) was added dropwise to a solution of Phosphorus oxychloride (15.3 g, 0.1 mol) in DCM (150 mL) at −60° C. under nitrogen. After addition, the reaction was stirred at −60° C. for 1 h. L-Alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) was added to the reaction and then TEA (20.74 g, 0.205 mol) was added dropwise at −60° C. The reaction mixture was stirred at −60° C. for 1 h. The substituted phenol corresponding to the phenyl substitution (0.1 mol) and TEA (10.62 g, 0.105 mol) and DCM (20 mL) was added dropwise to the reaction at −60° C., After addition, the reaction was stirred to room temperature overnight before being quenched with water. The organic layer was dried over Na2SO4, filtered and then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give a colorless oil.
The colorless oil (0.1 mol) and Isopropanol (200 mL, 5 vol) was charged into a 1000 mL 3-neck glass flask. Dihydroquinine (32.64 g, 0.1 mol) was added and stirred to give a clear solution. To this solution, 5% wet Pd/C (60% water by KF, 5% w/w dry basis) was charged, and hydrogenolysis was performed at 20° C.-25° C. for 18-20 h using 1 atm of hydrogen. When the IPC showed starting material was consumed completely, the mixture was filtered through a Buchner funnel, and the filtrate was stirred with 4.0 g of charcoal for 2 hours at 25° C.-30° C. The mixture was filtered, and the cake was washed with isopropanol (40 mL).
The filtrate was concentrated under vacuum less than 0.09 MPa at 50° C.-60° C. to give a crude product. Toluene (200 mL, 5 vol) was added, and the mixture was concentrated again under vacuum less than 0.09 MPa at 50° C.-60° C. This step was repeated two more time with 5 vol of Toluene give a residue as an oil. The residue was used the next step without purification.
A mixture of 3-chlorophenol (12.86 g, 0.1 mol) and TEA (10.62 g, 0.105 mol) was added dropwise to a solution of Phosphorus oxychloride (15.3 g, 0.1 mol) in DCM (150 mL) at −60° C. under nitrogen. After addition, the reaction was stirred at −60° C. for 1 h. L-Alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) was added to the reaction and then TEA (20.74 g, 0.205 mol) was added dropwise at −60° C. The reaction was stirred to room temperature overnight. Filtered and then the solvent was removed in vacuum, MTBE (128 mL, 10 v) was added, and the mixture was stirred for 1 h at 20° C., filtered, and the filtrate was stirred at 0° C. Then citric acid monohydrate (42 g, 0.2 mol) in H2O (200 mL) was added dropwise to the filtrate at 0° C. The reaction was stirred to room temperature overnight. The reaction was stood for layer separation. The organic layer was stirred with H2O (100 mL) and then stood for layer separation. DIPEA (12.9 g, 0.1 mol) was added to the organic phase. The mixture was concentrated under vacuum less than 0.09 MPa at 40° C. to give a crude product. The Purified product was obtained by pre-HPLC.
1H NMR (400 MHz, DMSO) δ 10.25 (s, 1H), 7.23 (dd, J=18.1, 10.0 Hz, 2H), 6.99 (td, J=8.0, 1.3 Hz, 2H), 4.80 (dt, J=12.5, 6.2 Hz, 1H), 3.81-3.62 (m, 2H), 3.60-3.43 (m, 2H), 3.04 (q, J=6.9 Hz, 2H), 2.17-1.95 (m, 1H), 1.32-1.19 (m, 13H), 1.18-1.08 (m, 9H).
MS (ES−): Expected: 320.05; found: 320.10
TEA (20.74 g, 0.205 mol) was added dropwise to a solution of 4-nitrophenyl dichlorophosphonate (25.6 g, 0.1 mol) and L-Alanine isopropyl ester hydrochloride (16.76 g, 0.1 mol) in DCM (256 mL, 10 v) at −10° C. under nitrogen. After addition, the reaction was stirred to room temperature overnight. The mixture was concentrated under vacuum less than 0.09 MPa at 40° C. MTBE (256 mL, 10 v) was added, and the mixture was stirred for 1 h at 20° C. Filtered and the filtrate was stirred at 0° C. Tetrabutylammonium Hydroxide (208 g, 0.2 mol, 25% w/w in H2O) was added dropwise to the filtrate at 0° C. After addition, the reaction was stirred to room temperature overnight. The reaction was stood for layer separation. The water layer was stirred with IPAC (200 mL) and then stood for layer separation. The IPAC layer was stirred with aq·HCl (200 mL, 1 mol/L) and then stood for layer separation. The organic layer was dried over Na2SO4 and filtered. Dihydroquinine (19.6 g, 0.06 mol) was added to the filtrate. The mixture was concentrated under vacuum less than 0.09 MPa at 50° C. to give a crude product, MTBE (256 mL, 10 v) was added, and the mixture was stirred for 1 h at 20° C. and filtered. The wet cake was dried for 16 h at 60° C. without vacuum. The product Intermediate A-8 was obtained.
1H NMR (400 MHz, DMSO) δ 11.89 (s, 1H), 8.74 (d, J=4.5 Hz, 1H), 8.15-8.04 (m, 2H), 7.96 (d, J=9.2 Hz, 1H), 7.64 (d, J=4.4 Hz, 1H), 7.49 (d, J=2.3 Hz, 1H), 7.45-7.32 (m, 3H), 6.59 (s, 1H), 6.00 (s, 1H), 4.75 (dt, J=12.5, 6.2 Hz, 1H), 4.07-3.86 (m, 5H), 3.85-3.68 (m, 1H), 3.62-3.22 (m, 4H), 3.10 (s, 1H), 2.86 (s, 1H), 1.97 (d, J=30.9 Hz, 3H), 1.76 (d, J=35.8 Hz, 2H), 1.40 (t, J=11.7 Hz, 1H), 1.34-1.18 (m, 2H), 1.18-1.00 (m, 9H), 0.75 (t, J=7.3 Hz, 3H).
MS (ES−): Expected: 331.07; found: 331.06
The salt of Intermediate A-1 to A-8 (1.7 eq.), Intermediate B (6.25 g, 20 mmol, 1.0 eq), diisopropyl ethylamine (2.6 g, 20 mmol, 1.0 eq.) and HATU (12.9 g, 34 mmol, 1.7 eq.) were added into 188 mL of dichloromethane (30 volumes). The mixture was heated to 40° C. and stirred for 18 hours under nitrogen. The reaction was monitored by TLC and HPLC.
After the reaction was complete, the reaction mixture was cooled to 0-10° C. and was washed with 2N HCl (100 mL) twice, 5% aqueous sodium bicarbonate (100 mL), the separated organic phase was dried over Na2SO4 and filtered. The filtrate was concentrated at 40-45° C. under vacuum to give a residue. The residue was purified by silica gel column chromatography to obtain the crude product. The crude product was recrystallized with EA (30 mL), the precipitated was dried without vacuum at 45-50° C. for 16 hours to afford Intermediates C-1 to C-5 or C-7.
1H NMR (400 MHz, DMSO) δ 7.79 (s, 1H), 7.54-7.35 (m, 2H), 7.28 (s, 1H), 7.06 (t, J=8.6 Hz, 1H), 6.27-5.87 (m, 4H), 5.75 (d, J=6.8 Hz, 1H), 4.81 (dt, J=12.5, 6.3 Hz, 1H), 4.37 (ddd, J=18.3, 14.3, 8.5 Hz, 3H), 4.16-3.96 (m, 1H), 3.79 (ddd, J=17.1, 7.2, 2.9 Hz, 1H), 2.88 (s, 3H), 1.24 (d, J=7.1 Hz, 3H), 1.20-1.01 (m, 9H).
MS (ES+): Expected: 618.20; found: 618.24
1H NMR (400 MHz, DMSO) δ 7.79 (s, 1H), 7.40 (dd, J=15.3, 8.0 Hz, 1H), 7.27 (s, 1H), 7.16-6.99 (m, 3H), 6.19-5.87 (m, 4H), 5.75 (d, J=6.6 Hz, 1H), 4.81 (dt, J=12.5, 6.2 Hz, 1H), 4.36 (ddd, J=18.4, 14.2, 8.3 Hz, 3H), 4.07 (dd, J=15.2, 8.0 Hz, 1H), 3.93-3.70 (m, 1H), 2.88 (s, 3H), 1.21 (d, J=7.0 Hz, 3H), 1.17-1.02 (m, 9H).
MS (ES+): Expected: 600.21; found: 600.24
1H NMR (400 MHz, DMSO) δ 7.79 (s, 1H), 7.28 (s, 1H), 7.15-6.90 (m, 3H), 6.20 (dd, J=12.6, 10.4 Hz, 1H), 6.13-5.88 (m, 3H), 5.77 (d, J=6.3 Hz, 1H), 4.80 (dq, J=12.5, 6.2 Hz, 1H), 4.54-4.24 (m, 3H), 4.07 (dd, J=17.6, 10.7 Hz, 1H), 3.92-3.69 (m, 1H), 2.87 (s, 3H), 1.22 (d, J=7.1 Hz, 3H), 1.15-1.03 (m, 9H).
MS (ES+): Expected: 618.20; found: 618.34
1H NMR (400 MHz, DMSO) δ 7.79 (s, 1H), 7.75-7.62 (m, 2H), 7.62-7.53 (m, 2H), 7.27 (s, 1H), 6.16 (dd, J=12.7, 10.2 Hz, 1H), 6.10-5.93 (m, 3H), 5.76 (d, J=6.8 Hz, 1H), 4.81 (dt, J=12.5, 6.2 Hz, 1H), 4.53-4.27 (m, 3H), 4.08 (dd, J=13.2, 6.3 Hz, 1H), 3.83 (ddd, J=17.1, 7.2, 2.9 Hz, 1H), 2.88 (s, 3H), 1.23 (d, J=7.1 Hz, 3H), 1.15-1.04 (m, 9H).
MS (ES+): Expected: 607.22; found: 607.19
1H NMR (400 MHz, DMSO) δ 7.80 (s, 1H), 7.22 (ddd, J=20.8, 13.3, 7.0 Hz, 5H), 6.23-5.86 (m, 4H), 5.73 (d, J=7.1 Hz, 1H), 4.81 (dt, J=12.5, 6.2 Hz, 1H), 4.54-4.20 (m, 3H), 4.06 (dd, J=12.8, 5.8 Hz, 1H), 3.90-3.67 (m, 1H), 2.89 (s, 3H), 1.21 (t, J=6.2 Hz, 3H), 1.15-1.03 (m, 9H).
MS (ES+): Expected: 600.21; found: 600.36
1H NMR (400 MHz, DMSO) δ 7.80 (s, 1H), 7.39 (t, J=8.1 Hz, 1H), 7.33 (s, 1H), 7.26 (d, J=7.9 Hz, 2H), 7.22-7.16 (m, 1H), 6.23-5.89 (m, 4H), 5.76 (d, J=6.8 Hz, 1H), 4.81 (dt, J=12.5, 6.2 Hz, 1H), 4.53-4.23 (m, 3H), 4.16-4.00 (m, 1H), 3.81 (ddd, J=17.1, 7.2, 2.9 Hz, 1H), 2.88 (s, 3H), 1.22 (d, J=7.1 Hz, 3H), 1.17-1.00 (m, 9H).
MS (ES+): Expected: 616.18; found: 616.15
1H NMR (400 MHz, DMSO) δ 8.31-8.18 (m, 2H), 7.79 (s, 1H), 7.44 (dd, J=14.3, 9.1 Hz, 2H), 7.18 (d, J=46.5 Hz, 1H), 6.35-6.15 (m, 1H), 6.12-5.88 (m, 3H), 5.75 (s, 1H), 4.88-4.73 (m, 1H), 4.45 (dd, J=14.1, 7.1 Hz, 3H), 4.18-4.01 (m, 1H), 3.81 (s, 1H), 2.89 (s, 3H), 1.24 (d, J=7.3 Hz, 3H), 1.10 (dd, J=14.0, 7.5 Hz, 9H).
MS (ES+): Expected: 627.21; found: 627.37
This specification has been described with reference to embodiments of the invention. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the invention as set forth in the claims below. Accordingly, the specification is to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of invention.
This application is a continuation of International Patent Application No. PCT/US2023/023739, filed in the U.S. Receiving Office on May 26, 2023, which claims the benefit of U.S. Provisional Application No. 63/346,274 filed May 26, 2022; U.S. Provisional Application No. 63/414,812 filed Oct. 10, 2022; and U.S. Provisional Application No. 63/427,359 filed Nov. 22, 2022. The entirety of each of these applications is hereby incorporated by reference for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63346274 | May 2022 | US | |
| 63414812 | Oct 2022 | US | |
| 63427359 | Nov 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2023/023739 | May 2023 | WO |
| Child | 18957362 | US |
