U.S. patent application number 17/118314 was filed with the patent office on 2021-03-25 for nucleotide hemi-sulfate salt for the treatment of hepatitis c virus.
This patent application is currently assigned to Atea Pharmaceuticals, Inc.. The applicant listed for this patent is Atea Pharmaceuticals, Inc.. Invention is credited to Adel Moussa, Jean-Pierre Sommadossi.
Application Number | 20210087217 17/118314 |
Document ID | / |
Family ID | 1000005264310 |
Filed Date | 2021-03-25 |
View All Diagrams
United States Patent
Application |
20210087217 |
Kind Code |
A1 |
Moussa; Adel ; et
al. |
March 25, 2021 |
NUCLEOTIDE HEMI-SULFATE SALT FOR THE TREATMENT OF HEPATITIS C
VIRUS
Abstract
A hemi-sulfate salt of the structure: ##STR00001## to treat a
host infected with hepatitis C, as well as pharmaceutical
compositions and dosage forms, including solid dosage forms,
thereof.
Inventors: |
Moussa; Adel; (Burlington,
MA) ; Sommadossi; Jean-Pierre; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Atea Pharmaceuticals, Inc. |
Boston |
MA |
US |
|
|
Assignee: |
Atea Pharmaceuticals, Inc.
Boston
MA
|
Family ID: |
1000005264310 |
Appl. No.: |
17/118314 |
Filed: |
December 10, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16918918 |
Jul 1, 2020 |
10894804 |
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17118314 |
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16687136 |
Nov 18, 2019 |
10906928 |
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16918918 |
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15885630 |
Jan 31, 2018 |
10519186 |
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16687136 |
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62575248 |
Oct 20, 2017 |
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62488366 |
Apr 21, 2017 |
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62469912 |
Mar 10, 2017 |
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62453437 |
Feb 1, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/708 20130101; A61K 9/20 20130101; A61K 31/7076 20130101;
A61P 31/14 20180101; C07H 19/20 20130101; C07B 2200/13 20130101;
A61K 9/2054 20130101 |
International
Class: |
C07H 19/20 20060101
C07H019/20; A61K 9/20 20060101 A61K009/20; A61K 31/708 20060101
A61K031/708; A61K 45/06 20060101 A61K045/06; A61P 31/14 20060101
A61P031/14; A61K 31/7076 20060101 A61K031/7076 |
Claims
1. A compound of the formula: ##STR00013##
2. The compound of claim 1 of the formula: ##STR00014##
3. The compound of claim 2 wherein the compound is at least 90%
free of the opposite phosphorus S-enantiomer.
4. The compound of claim 3 wherein the compound is at least 98%
free of the opposite phosphorus S-enantiomer.
5. The compound of claim 1 of the formula: ##STR00015##
6. The compound of claim 5 wherein the compound is at least 90%
free of the opposite phosphorus R-enantiomer.
7. The compound of claim 6 wherein the compound is at least 98%
free of the opposite phosphorus R-enantiomer.
8. A pharmaceutical composition comprising a compound of the
formula: ##STR00016## in a pharmaceutically acceptable carrier.
9. The pharmaceutical composition of claim 8, comprising a compound
of the formula: ##STR00017## in a pharmaceutically acceptable
carrier.
10. The pharmaceutical composition of claim 9, wherein the compound
is at least 90% free of the opposite phosphorus S-enantiomer.
11. The pharmaceutical composition of claim 10, wherein the
compound is at least 98% free of the opposite phosphorus
S-enantiomer.
12. The pharmaceutical composition of claim 8, comprising a
compound of the formula: ##STR00018## in a pharmaceutically
acceptable carrier.
13. The pharmaceutical composition of claim 12, wherein the
compound is at least 90% free of the opposite phosphorus
R-enantiomer.
14. The pharmaceutical composition of claim 13, wherein the
compound is at least 98% free of the opposite phosphorus
R-enantiomer.
15. The pharmaceutical composition of claim 8, wherein the
pharmaceutically acceptable carrier is suitable for oral
delivery.
16. The pharmaceutical composition of claim 15, wherein the
pharmaceutically acceptable dosage form suitable for oral delivery
is in a solid dosage form.
17. The pharmaceutical composition of claim 16, wherein the
pharmaceutically acceptable carrier is in the form of a tablet or a
capsule.
18. The pharmaceutical composition of claim 15, wherein the
pharmaceutically acceptable dosage form suitable for oral delivery
is in a liquid dosage form.
19. The pharmaceutical composition of claim 18, wherein the
pharmaceutically acceptable carrier is in the form of a suspension
or solution.
20. The pharmaceutical composition of claim 15, in an intravenous
formulation.
21. The pharmaceutical composition of claim 15, in a parenteral
formulation.
22. A method to treat hepatitis C in a human in need thereof
comprising an effective amount of a compound of the formula:
##STR00019## optionally in a pharmaceutically acceptable
carrier.
23. The method of claim 22 comprising a compound of the formula:
##STR00020## optionally in a pharmaceutically acceptable
carrier.
24. The method of claim 22 comprising a compound of the formula:
##STR00021## optionally in a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/918,918, filed Jul. 1, 2020, which is a continuation of U.S.
application Ser. No. 16/687,136, filed Nov. 18, 2019, which is a
continuation of U.S. application Ser. No. 15/885,630, filed Jan.
31, 2018, now U.S. Pat. No. 10,519,186, issued Dec. 21, 2019, which
claims the benefit of provisional U.S. Application Nos. 62/453,437
filed Feb. 1, 2017; 62/469,912 filed Mar. 10, 2017; 62/488,366
filed Apr. 21, 2017; and, 62/575,248 filed Oct. 20, 2017. The
entirety of these applications are incorporated by reference herein
for all purposes.
FIELD OF THE INVENTION
[0002] The present invention is the hemi-sulfate salt of a selected
nucleotide compound that has unexpected therapeutic properties to
treat a host infected with hepatitis C, as well as pharmaceutical
compositions and dosage forms thereof.
BACKGROUND OF THE INVENTION
[0003] Hepatitis C (HCV) is an RNA single-stranded virus and member
of the Hepacivirus genus. It is estimated that 75% of all cases of
liver disease are caused by HCV. HCV infection can lead to
cirrhosis and liver cancer, and if left to progress, liver failure
that may require a liver transplant.
[0004] Approximately 71 million people worldwide are living with
chronic HCV infections and approximately 399,000 people die each
year from HCV, mostly from cirrhosis and hepatocellular
carcinoma.
[0005] RNA polymerase is a key target for drug development against
RNA single stranded viruses. The HCV non-structural protein NS5B
RNA-dependent RNA polymerase is a key enzyme responsible for
initiating and catalyzing viral RNA synthesis. There are two major
subclasses of NS5B inhibitors: nucleoside analogs and
non-nucleoside inhibitors (NNIs). Nucleoside analogs are anabolized
to active triphosphates that act as alternative substrates for the
polymerase and non-nucleoside inhibitors (NNIs) bind to allosteric
regions on the protein. Nucleoside or nucleotide inhibitors mimic
natural polymerase substrates and act as chain terminators. They
inhibit the initiation of RNA transcription and elongation of a
nascent RNA chain.
[0006] In addition to targeting RNA polymerase, other RNA viral
proteins may also be targeted in combination therapies. For
example, HCV proteins that are additional targets for therapeutic
approaches are NS3/4A (a serine protease) and NS5A (a
non-structural protein that is an essential component of HCV
replicase and exerts a range of effects on cellular pathways).
[0007] In December 2013, the first nucleoside NS5B polymerase
inhibitor sofosbuvir (Sovaldi.RTM., Gilead Sciences) was approved.
Sovaldi.RTM. is a uridine phosphoramidate prodrug that is taken up
by hepatocytes and undergoes intracellular activation to afford the
active metabolite,
2'-deoxy-2'-.alpha.-fluoro-.beta.-C-methyluridine-5'-triphosphate.
##STR00002##
[0008] Sovaldi.RTM. is the first drug that has demonstrated safety
and efficacy to treat certain types of HCV infection without the
need for co-administration of interferon. Sovaldi.RTM. is the third
drug with breakthrough therapy designation to receive FDA
approval.
[0009] In 2014, the U.S. FDA approved Harvoni.RTM. (ledispasvir, a
NS5A inhibitor, and sofosbuvir) to treat chronic hepatitis C virus
Genotype 1 infection. Harvoni.RTM. is the first combination pill
approved to treat chronic HCV Genotype 1 infection. It is also the
first approved regimen that does not require administration with
interferon or ribavirin. In addition, the FDA approved simeprevir
(Olysio.TM.) in combination with sofosbuvir (Sovaldi.RTM.) as a
once-daily, all oral, interferon and ribavirin-free treatment for
adults with Genotype 1 HCV infection.
[0010] The U.S. FDA also approved AbbVie's VIEKIRA Pak.TM. in 2014,
a multi-pill pack containing dasabuvir (a non-nucleoside NS5B
polymerase inhibitor), ombitasvir (a NS5A inhibitor), paritaprevir
(a NS3/4A inhibitor), and ritonavir. The VIEKIRA Pak.TM. can be
used with or without the ribavirin to treat Genotype 1 HCV infected
patients including patients with compensated cirrhosis. VIEKIRA
Pak.TM. does not require interferon co-therapy.
[0011] In July 2015, the U.S. FDA approved Technivie.TM. and
Daklinza.TM. for the treatment of HCV genotype 4 and HCV Genotype
3, respectively. Technivie.TM. (Ombitasvir/paritaprevir/ritonavir)
was approved for use in combination with ribavirin for the
treatment of HCV genotype 4 in patients without scarring and
cirrhosis and is the first option for HCV-4 infected patients who
do not require co-administration with interferon. Daklinza.TM. was
approved for use with Sovaldi.RTM. to treat HCV genotype 3
infections. Daklinza.TM. is the first drug that has demonstrated
safety and efficacy in treating HCV Genotype 3 without the need for
co-administration of interferon or ribavirin.
[0012] In October 2015, the U.S. FDA warned that HCV treatments
Viekira Pak and Technivie can cause serious liver injury primarily
in patients with underlying advanced liver disease and required
that additional information about safety be added to the label.
[0013] Other current approved therapies for HCV include interferon
alpha-2b or pegylated interferon alpha-2b (Pegintron.RTM.), which
can be administered with ribavirin (Rebetol.RTM.), NS3/4A
telaprevir (Incivek.RTM., Vertex and Johnson & Johnson),
boceprevir (Victrelis.TM., Merck), simeprevir (Olysio.TM., Johnson
& Johnson), paritaprevir (AbbVie), Ombitasvir (AbbVie), the NNI
Dasabuvir (ABT-333) and Merck's Zepatier.TM. (a single-tablet
combination of the two drugs grazoprevir and elbasvir).
[0014] Additional NS5B polymerase inhibitors are currently under
development. Merck is developing the uridine nucleotide prodrug
MK-3682 (formerly Idenix IDX21437) and the drug is currently in
Phase II combination trials.
[0015] United States patents and WO applications that describe
nucleoside polymerase inhibitors for the treatment of Flaviviridae,
including HCV, include those filed by Idenix Pharmaceuticals (U.S.
Pat. Nos. 6,812,219; 6,914,054; 7,105,493; 7,138,376; 7,148,206;
7,157,441; 7,163,929; 7,169,766; 7,192,936; 7,365,057; 7,384,924;
7,456,155; 7,547,704; 7,582,618; 7,608,597; 7,608,600; 7,625,875;
7,635,689; 7,662,798; 7,824,851; 7,902,202; 7,932,240; 7,951,789;
8,193,372; 8,299,038; 8,343,937; 8,362,068; 8,507,460; 8,637,475;
8,674,085; 8,680,071; 8,691,788, 8,742,101, 8,951,985; 9,109,001;
9,243,025; US2016/0002281; US2013/0064794; WO/2015/095305;
WO/2015/081133; WO/2015/061683; WO/2013/177219; WO/2013/039920;
WO/2014/137930; WO/2014/052638; WO/2012/154321); Merck (U.S. Pat.
Nos. 6,777,395; 7,105,499; 7,125,855; 7,202,224; 7,323,449;
7,339,054; 7,534,767; 7,632,821; 7,879,815; 8,071,568; 8,148,349;
8,470,834; 8,481,712; 8,541,434; 8,697,694; 8,715,638, 9,061,041;
9,156,872 and WO/2013/009737); Emory University (U.S. Pat. Nos.
6,348,587; 6,911,424; 7,307,065; 7,495,006; 7,662,938; 7,772,208;
8,114,994; 8,168,583; 8,609,627; US 2014/0212382; and
WO2014/1244430); Gilead Sciences/Pharmasset Inc. (U.S. Pat. Nos.
7,842,672; 7,973,013; 8,008,264; 8,012,941; 8,012,942; 8,318,682;
8,324,179; 8,415,308; 8,455,451; 8,563,530; 8,841,275; 8,853,171;
8,871,785; 8,877,733; 8,889,159; 8,906,880; 8,912,321; 8,957,045;
8,957,046; 9,045,520; 9,085,573; 9,090,642; and 9,139,604) and
(U.S. Pat. Nos. 6,908,924; 6,949,522; 7,094,770; 7,211,570;
7,429,572; 7,601,820; 7,638,502; 7,718,790; 7,772,208; RE42,015;
7,919,247; 7,964,580; 8,093,380; 8,114,997; 8,173,621; 8,334,270;
8,415,322; 8,481,713; 8,492,539; 8,551,973; 8,580,765; 8,618,076;
8,629,263; 8,633,309; 8,642,756; 8,716,262; 8,716,263; 8,735,345;
8,735,372; 8,735,569; 8,759,510 and 8,765,710); Hoffman La-Roche
(U.S. Pat. No. 6,660,721), Roche (U.S. Pat. Nos. 6,784,166;
7,608,599, 7,608,601 and 8,071,567); Alios BioPharma Inc. (U.S.
Pat. Nos. 8,895,723; 8,877,731; 8,871,737, 8,846,896, 8,772,474;
8,980,865; 9,012,427; US 2015/0105341; US 2015/0011497; US
2010/0249068; US2012/0070411; WO 2015/054465; WO 2014/209979; WO
2014/100505; WO 2014/100498; WO 2013/142159; WO 2013/142157; WO
2013/096680; WO 2013/088155; WO 2010/108135), Enanta
Pharmaceuticals (U.S. Pat. Nos. 8,575,119; 8,846,638; 9,085,599; WO
2013/044030; WO 2012/125900), Biota (U.S. Pat. Nos. 7,268,119;
7,285,658; 7,713,941; 8,119,607; 8,415,309; 8,501,699 and
8,802,840), Biocryst Pharmaceuticals (U.S. Pat. Nos. 7,388,002;
7,429,571; 7,514,410; 7,560,434; 7,994,139; 8,133,870; 8,163,703;
8,242,085 and 8,440,813), Alla Chem, LLC (U.S. Pat. No. 8,889,701
and WO 2015/053662), Inhibitex (U.S. Pat. No. 8,759,318 and
WO/2012/092484), Janssen Products (U.S. Pat. Nos. 8,399,429;
8,431,588, 8,481,510, 8,552,021, 8,933,052; 9,006,29 and 9,012,428)
the University of Georgia Foundation (U.S. Pat. Nos. 6,348,587;
7,307,065; 7,662,938; 8,168,583; 8,673,926, 8,816,074; 8,921,384
and 8,946,244), RFS Pharma, LLC (U.S. Pat. Nos. 8,895,531;
8,859,595; 8,815,829; 8,609,627; 7,560,550; US 2014/0066395; US
2014/0235566; US 2010/0279969; WO/2010/091386 and WO 2012/158811)
University College Cardiff Consultants Limited (WO/2014/076490, WO
2010/081082; WO/2008/062206), Achillion Pharmaceuticals, Inc.
(WO/2014/169278 and WO 2014/169280), Cocrystal Pharma, Inc. (U.S.
Pat. No. 9,173,893), Katholieke Universiteit Leuven (WO
2015/158913), Catabasis (WO 2013/090420) and the Regents of the
University of Minnesota (WO 2006/004637).
[0016] Atea Pharmaceuticals, Inc. has disclosed
.beta.-D-2'-deoxy-2'-.alpha.-fluoro-2'-.beta.-C-substituted-2-modified-N.-
sup.6-(mono- and di-methyl) purine nucleotides for the treatment of
HCV in U.S. Pat. No. 9,828,410 and PCT Application No. WO
2016/144918. Atea has also disclosed
.beta.-D-2'-deoxy-2'-substituted-4'-substituted-2-N-substituted-6-aminopu-
rine nucleotides for the treatment of paramyxovirus and
orthomyxovirus infections in US 2018/0009836 and WO
2018/009623.
[0017] There remains a strong medical need to develop anti-HCV
therapies that are safe, effective and well-tolerated. The need is
accentuated by the expectation of drug resistance. More potent
direct-acting antivirals could significantly shorten treatment
duration and improve compliance and SVR (sustained viral response)
rates for patients infected with all HCV genotypes.
[0018] It is therefore an object of the present invention to
provide compounds, pharmaceutical compositions, methods, and dosage
forms to treat and/or prevent infections of HCV.
SUMMARY OF THE INVENTION
[0019] It has been surprisingly discovered that the hemisulfate
salt of Compound 1, which is provided below as Compound 2, exhibits
unexpected advantageous therapeutic properties, including enhanced
bioavailability and target organ selectivity, over its free base
(Compound 1).
[0020] These unexpected advantages could not have been predicted in
advance. Compound 2 is thus a therapeutically superior composition
of matter to administer in an effective amount to a host in need
thereof, typically a human, for the treatment of hepatitis C.
Compound 2 is referred to as the hemi-sulfate salt of
isopropyl((S)-(((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. Compound 1 is disclosed in U.S. Pat. No.
9,828,410.
##STR00003##
[0021] Compound 2, as Compound 1, is converted to its corresponding
triphosphate nucleotide (Compound 1-6) in the cell, which is the
active metabolite and inhibitor of RNA polymerase (see Scheme 1
below). Since Compound 1-6 is produced in the cell and does not
leave the cell, it is not measurable in the plasma. However, the
5'-OH metabolite Compound 1-7 (see Scheme 1) is exported from the
cell, and therefore is measurable in plasma and acts as a surrogate
of the concentration of intracellular active metabolite Compound
1-6.
[0022] It has been discovered that the plasma concentration in vivo
of surrogate Compound 1-7, and thus intracellular Compound 1-6, is
substantially higher when Compound 2 is administered in vivo than
when Compound 1 is administered in vivo. In a head-to-head
comparison of dogs dosed with Compound 1 and Compound 2 (Example
19, Table 28), dosing with Compound 2 achieved an AUC.sub.(0-4hrs)
of the ultimate guanine 5'-OH nucleoside metabolite (1-7) that is
twice as high as the AUC following Compound 1 dosing. It is
unexpected that a non-covalent salt has such an effect on plasma
concentration of the parent drug (Compound 1).
[0023] Additionally, Compound 2 selectively partitions in vivo to
the liver over the heart (Example 19, Table 29), which is
beneficial since the liver is the diseased organ in hosts infected
with HCV. Dogs were dosed with Compound 1 or Compound 2 and the
concentration of the active triphosphate (1-6) in the liver and
heart was measured. The liver to heart ratio of the active
triphosphate concentration was higher after dosing with Compound 2
compared to Compound 1 as shown in Table 29. Specifically, the
liver/heart partitioning ratio for Compound 2 is 20 compared to a
liver/heart partitioning ratio of 3.1 for Compound 1. This data
indicates, unexpectedly, that the administration of Compound 2
results in the preferential distribution of the active guanine
triphosphate (Compound 1-6) in the liver over the heart when
compared to Compound 1, which reduces potential off-target effects.
It was unexpected that administration of Compound 2 would
significantly reduce undesired off-target partitioning. This allows
for the administration of Compound 2 at a higher dose than Compound
1, if desired by the healthcare practitioner.
[0024] In addition, liver and heart tissue levels of the active
guanine triphosphate derivative of Compound 2 (metabolite 1-6) were
measured after oral doses of Compound 2 in rats and monkeys
(Example 20). High levels of the active guanine triphosphate (1-6)
were measured in the liver of all species tested. Importantly,
unquantifiable levels of the guanine triphosphate (1-6) were
measured in monkey hearts, and this is indicative of liver-specific
formation of the active triphosphate. It was thus discovered that
compared to Compound 1 dosing, Compound 2 dosing improves guanine
triphosphate (1-6) distribution.
[0025] When administered to healthy and hepatitis C infected
patients, Compound 2 was well tolerated after a single oral dose
and C.sub.max, T.sub.max and AUC.sub.tot pharmacokinetic parameters
were comparable in both groups (Tables 34 and 35). As described in
Example 24, a single dose of Compound 2 in HCV-infected patients
resulted in a significant antiviral activity. Plasma exposure of
metabolite 1-7 was mostly dose-proportional over the studied
range.
[0026] Individual pharmacokinetic/pharmacodynamic analyses of
patients dosed with Compound 2 showed that the viral response
correlated with plasma exposure of metabolite 1-7 of Compound 2
(Example 24, FIGS. 23A-23F), indicating that profound vial
responses are achievable with robust doses of Compound 2.
[0027] Example 24 confirms that, as non-limiting embodiments,
single oral doses of 300 mg, 400 mg, and 600 mg result in
significant antiviral activity in humans. The C.sub.24 trough
plasma concentration of metabolite 1-7 following a 600 mg dose of
Compound 2 doubled from the C.sub.24 trough plasma concentration of
metabolite 1-7 following a 300 mg dose of Compound 2.
[0028] FIG. 24 and Example 25 highlight the striking invention
provided by Compound 2 for the treatment of hepatitis C. As shown
in FIG. 24, the steady-state trough plasma levels (C.sub.24,ss) of
metabolite 1-7 following Compound 2 dosing in humans (600 mg QD
(550 mg free base equivalent) and 450 mg QD (400 mg free base
equivalent)) was predicted and compared to the EC.sub.95 of
Compound 1 in vitro across a range of HCV clinical isolates to
determine if the steady state plasma concentration is consistently
higher than the EC.sub.95, which would result in high efficacy
against multiple clinical isolates in vivo. The EC.sub.95 for
Compound 1 is the same as the EC.sub.95 of Compound 2. For Compound
2 to be effective, the steady-state trough plasma level of
metabolite 1-7 should exceed the EC.sub.95.
[0029] As shown in FIG. 24, the EC.sub.95 of Compound 2 against all
tesed clinical isolates ranged from approximately 18 nM to 24
nM.
[0030] As shown in FIG. 24, Compound 2 at a dose of 450 mg QD (400
mg free base equivalent) in humans provides a predicted steady
state trough plasma concentration (C.sub.24,ss) of approximately 40
ng/mL. Compound 2 at a dose of 600 mg QD (550 mg free base
equivalent) in humans provides a predicted steady state trough
plasma concentration (C.sub.24,ss) of approximately 50 ng/mL.
[0031] Therefore, the predicted steady state plasma concentration
of surrogate metabolite 1-7 is almost double the EC.sub.95 against
all tested clinical isolates (even the hard to treat GT3a), which
indicates superior performance.
[0032] In contrast, the EC.sub.95 of the standard of care
nucleotide sofosbuvir (Sovaldi) ranges from 50 nM to 265 nM across
all tested HCV clinical isolates, with an EC.sub.95 less than the
predicted steady state concentration at the commercial dosage of
400 mg for only two isolates, GT2a and GT2b. The EC.sub.95 for the
commercial dosage of 400 mg of sofosbuvir is greater than the
predicted steady state concentration for other clinical isolates,
GT1a, GT1b, GT3a, GT4a, and GT4d.
[0033] The data comparing the efficacy and pharmacokinetic steady
state parameters in FIG. 24 clearly demonstrates the unexpected
therapeutic importance of Compound 2 for the treatment of hepatitis
C. In fact, the predicted steady-state (C.sub.24,ss) plasma level
after administration of Compound 2 is predicted to be at least
2-fold higher than the EC.sub.95 for all genotypes tested, and is
3- to 5-fold more potent against GT2. This data indicates that
Compound 2 has potent pan-genotypic antiviral activity in humans.
As shown in FIG. 24, the EC.sub.95 of sofosbuvir against GT1, GT3,
and GT4 is greater than 100 ng/mL. Thus surprisingly, Compound 2 is
active against HCV at a dosage form that delivers a lower
steady-state trough concentration (40-50 ng/mL) than the
steady-state tough concentration (approximately 100 ng/mL) achieved
by the equivalent dosage form of sofosbuvir.
[0034] In one embodiment, therefore, the invention includes a
dosage form of Compound 2 that provides a metabolite 1-7
steady-state plasma trough concentration (C.sub.24,ss) between
approximately 15-75 ng/mL, for example, 20-60 ng/mL, 25-50 ng/mL,
40-60 ng/mL, or even 40-50 ng/mL. This is unexpected in light of
the fact that the steady state concentration of the equivalent
metabolite of sofosbuvir is approximately 100 ng/mL.
[0035] Additionally, it has been discovered that Compound 2 is an
unusually stable, highly soluble, non-hygroscopic salt with
activity against HCV. This is surprising because a number of salts
of Compound 1 other than the hemi-sulfate salt (Compound 2),
including the mono-sulfate salt (Compound 3), are not physically
stable, but instead deliquesce or become gummy solids (Example 4),
and thus are not suitable for stable solid pharmaceutical dosage
forms. Surprisingly, while Compound 2 does not become gummy, it is
up to 43 times more soluble in water compared to Compound 1 and is
over 6 times more soluble than Compound 1 under simulated gastric
fluid (SGF) conditions (Example 15).
[0036] As discussed in Example 16, Compound 2 remains a white solid
with an IR that corresponds to the reference standard for 6 months
under accelerated stability conditions (40.degree. C./75% RH).
Compound 2 is stable for 9 months at ambient conditions (25.degree.
C./60% RH) and refrigerator conditions (5.degree. C.).
[0037] Solid dosage forms (50 mg and 100 mg tablets) of Compound 2
are also chemically stable under accelerated (40.degree. C./75% RH)
and refrigeration conditions (5.degree. C.) for 6 months (Example
26). Compound 2 is stable under ambient conditions (25.degree.
C./60% RH) in a solid dosage form for at least 9 months.
[0038] Scheme 1 provides the metabolic pathway of Compound 1 and
Compound 2, which involves the initial de-esterification of the
phosphoramidate (metabolite 1-1) to form metabolite 1-2. Metabolite
1-2 is then converted to the
N.sup.6-methyl-2,6-diaminopurine-5'-monophosphate derivative
(metabolite 1-3), which is in turn metabolized to the free
5'-hydroxyl-N.sup.6-methyl-2,6-diaminopurine nucleoside (metabolite
1-8) and
((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-
-hydroxy-4-methyltetrahydrofuran-2-yl)methyl dihydrogen phosphate
as the 5'-monophosphate (metabolite 1-4). Metabolite 1-4 is
anabolized to the corresponding diphosphate (metabolite 1-5) and
then the active triphosphate derivative (metabolite 1-6). The
5'-triphosphate can be further metabolized to generate
2-amino-9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltet-
rahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (1-7). Metabolite 1-7
is measurable in plasma and is therefore a surrogate for the active
triphosphate (1-6), which is not measurable in plasma.
##STR00004##
[0039] In one embodiment, the invention is Compound 2 and its use
to treat hepatitis C (HCV) in a host in need thereof, optionally in
a pharmaceutically acceptable carrier. In one aspect, Compound 2 is
used as an amorphous solid. In another aspect, Compound 2 is used
as a crystalline solid.
[0040] The present invention further includes an exemplary
on-limiting process for the preparation of Compound 2 that includes
[0041] (i) a first step of dissolving Compound 1 in an organic
solvent, for example, acetone, ethyl acetate, methanol,
acetonitrile, or ether, or the like, in a flask or container;
[0042] (ii) charging a second flask or container with a second
organic solvent, which may be the same as or different from the
organic solvent in step (i), optionally cooling the second solvent
to 0-10 degrees C., and adding dropwise H.sub.2SO.sub.4 to the
second organic solvent to create a H.sub.2SO.sub.4/organic solvent
mixture; and wherein the solvent for example may be methanol;
[0043] (iii) adding dropwise the H.sub.2SO.sub.4/solvent mixture at
a molar ratio of 0.5/1.0 from step [0044] (ii) to the solution of
Compound 1 of step (i) at ambient or slightly increased or
decreased temperature (for example 23-35 degrees C.); [0045] (iv)
stirring the reaction of step (iii) until precipitate of Compound 2
is formed, for example at ambient or slightly increased or
decreased temperature; [0046] (v) optionally filtering the
resulting precipitate from step (iv) and washing with an organic
solvent; and [0047] (vi) optionally drying the resulting Compound 2
in a vacuum, optionally at elevated a temperature, for example, 55,
56, 57, 58, 59, or 60.degree. C.
[0048] In one embodiment, the organic solvent in step (i) is
3-methyl-2-pentanone. In one embodiment, the organic solvent in
step (i) is ethyl isopropyl ketone. In one embodiment, the organic
solvent in step (i) is methyl propionate. In one embodiment, the
organic solvent in step (i) is ethyl butyrate.
[0049] Despite the volume of antiviral nucleoside literature and
patent filings, Compound 2 has not been specifically disclosed.
Accordingly, the present invention includes Compound 2, or a
pharmaceutically acceptable composition or dosage form thereof, as
described herein.
[0050] Compounds, methods, dosage forms, and compositions are
provided for the treatment of a host infected with a HCV virus via
administration of an effective amount of Compound 2. In certain
embodiments, Compound 2 is administered at a dose of at least about
100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750,
800, 850, 900, 950, or 1000 mg. In certain embodiments, Compound 2
is administered for up to 12 weeks, for up to 10 weeks, for up to 8
weeks, for up to 6 weeks, or for up to 4 weeks. In alternative
embodiments, Compound 2 is administered for at least 4 weeks, for
at least 6 weeks, for at least 8 weeks, for at least 10 weeks, or
for at least 12 weeks. In certain embodiments, Compound 2 is
administered at least once a day or every other day. In certain
embodiments, Compound 2 is administered in a dosage form that
achieves a steady-state trough plasma level (C.sub.24,ss) of
metabolite 1-7 between approximately 15-75 ng/mL. In one
embodiment, Compound 2 is administered in a dosage form that
achieves a steady-state trough plasma level (C.sub.24,ss) of
metabolite 1-7 between approximately 20-60 ng/mL. In certain
embodiments, Compound 2 is administered in a dosage form that
achieves an AUC of metabolite 1-7 between approximately 1,200
ng*h/mL and 3,000 ng*h/mL. In one embodiment, Compound 2 is
administered in a dosage form that achieves an AUC of metabolite
1-7 between approximately 1,500 and 2,100 ng*h/mL.
[0051] The compounds, compositions, and dosage forms can also be
used to treat related conditions such as anti-HCV antibody positive
and antigen positive conditions, viral-based chronic liver
inflammation, liver cancer resulting from advanced hepatitis C
(hepatocellular carcinoma (HCC)), cirrhosis, chronic or acute
hepatitis C, fulminant hepatitis C, chronic persistent hepatitis C
and anti-HCV-based fatigue. The compound or formulations that
include the compounds can also be used prophylactically to prevent
or restrict the progression of clinical illness in individuals who
are anti-HCV antibody- or antigen-positive or who have been exposed
to hepatitis C.
[0052] The present invention thus includes the following features:
[0053] (a) Compound 2 as described herein; [0054] (b) Prodrugs of
Compound 2 [0055] (c) Use of Compound 2 in the manufacture of a
medicament for treatment of a hepatitis C virus infection; [0056]
(d) Compound 2 for use to treat hepatitis C, optionally in a
pharmaceutically acceptable carrier; [0057] (e) A method for
manufacturing a medicament intended for the therapeutic use for
treating a hepatitis C virus infection, characterized in that
Compound 2, or a pharmaceutically acceptable salt, as described
herein is used in the manufacture; [0058] (e) A pharmaceutical
formulation comprising an effective host-treating amount of
Compound 2 with a pharmaceutically acceptable carrier or diluent;
[0059] (f) Processes for the preparation of therapeutic products
that contain an effective amount of Compound 2; [0060] (g) Solid
dosage forms, including those that provide an advantageous
pharmacokinetic profile; and [0061] (h) Processes for the
manufacture of Compound 2, as described herein.
BRIEF DESCRIPTION OF THE FIGURES
[0062] FIG. 1A is an overlay of XRPD diffractograms of samples 1-1
(amorphous Compound 1), 1-2 (crystalline Compound 1), and 1-3
(amorphous Compound 2) prior to stability studies for
characterization purposes as described in Example 2 and Example 5.
The x-axis is 2Theta measured in degrees and the y-axis is
intensity measured in counts.
[0063] FIG. 1B is the HPLC chromatograph of amorphous Compound 1
(sample 1-1) to determine purity as described in Example 2. The
purity of the sample was 98.7%. The x-axis is time measured in
minutes and the y-axis is intensity measured in counts.
[0064] FIG. 2A is the HPLC chromatograph of crystalline Compound 1
(sample 1-2) to determine purity as described in Example 2. The
purity of the sample was 99.11%. The x-axis is time measured in
minutes and the y-axis is intensity measured in counts.
[0065] FIG. 2B is a DSC and TGA graph of crystalline Compound 1
(sample 1-2) prior to any stability studies for characterization
purposes as described in Example 2. The x-axis is temperature
measured in .degree. C., the left y-axis heat flow measured in
(W/g), and the right y-axis is weight measured in percent.
[0066] FIG. 3 is an X-ray crystallography image of Compound 1
showing the absolute stereochemistry as described in Example 2.
[0067] FIG. 4A is an overlay of XRPD diffractograms of samples 1-1
(amorphous Compound 1), 1-2 (crystalline Compound 1), and 1-3
(amorphous Compound 2) after storing at 25.degree. C. and 60%
relative humidity for 14 days as described in Example 2. The x-axis
is 2Theta measured in degrees and the y-axis is intensity measured
in counts.
[0068] FIG. 4B is an overlay of XRPD diffractograms of samples 1-4,
1-5, 1-6, 1-7, and 1-9 after storing at 25.degree. C. and 60%
relative humidity for 7 days as described in Example 4. The x-axis
is 2Theta measured in degrees and the y-axis is intensity measured
in counts.
[0069] FIG. 5A is an overlay of XRPD diffractograms of samples 1-4,
1-6, 1-7, and 1-9 after storing at 25.degree. C. and 60% relative
humidity for 14 days as described in Example 4. The x-axis is
2Theta measured in degrees and the y-axis is intensity measured in
counts.
[0070] FIG. 5B is the XRPD pattern of amorphous Compound 2 (sample
1-3) as described in Example 5. The x-axis is 2Theta measured in
degrees and the y-axis is intensity measured in counts.
[0071] FIG. 6A is the HPLC chromatograph of amorphous Compound 2
(sample 1-3) to determine purity as described in Example 5. The
purity of the sample was 99.6%. The x-axis is time measured in
minutes and the y-axis is intensity measured in counts.
[0072] FIG. 6B is a DSC and TGA graph for amorphous Compound 2
(sample 1-3) prior to any stability studies for characterization
purposes as described in Example 5. The x-axis is temperature
measured in .degree. C., the left y-axis heat flow measured in
(W/g), and the right y-axis is weight measured in percent.
[0073] FIG. 7A is an overlay of XRPD diffractograms of crystalline
samples (samples 2-2, 2-6, and 2-7) and poorly crystalline samples
(samples 2-3, 2-4, 2-5, and 2-8) identified from the
crystallizations of Compound 2 (Example 6). The x-axis is 2Theta
measured in degrees and the y-axis intensity measured in
counts.
[0074] FIG. 7B is an overlay of XRPD diffractograms of amorphous
samples (samples 2-9, 2-10, and 2-11) identified from the
crystallizations of Compound 2 (Example 6). The x-axis is 2Theta
measured in degrees and the y-axis intensity measured in
counts.
[0075] FIG. 8A is an overlay of XRPD diffractograms of samples
(samples 2-2, 2-3, 2-4, 2-5, 2-6, 2-7 and 2-8) after 6 days storage
at 25.degree. C. and 60% relative humidity (Example 6). The x-axis
is 2Theta measured in degrees and the y-axis intensity measured in
counts.
[0076] FIG. 8B is a DSC and TGA graph for sample 2-2 (Example 6).
The x-axis is temperature measured in .degree. C., the left y-axis
heat flow measured in (W/g), and the right y-axis is weight
measured in percent. Experimental procedures for DSC and TGA
collection are given in Example 2.
[0077] FIG. 9A is a DSC and TGA graph for sample 2-3 (Example 6).
The x-axis is temperature measured in .degree. C., the left y-axis
heat flow measured in (W/g), and the right y-axis is weight
measured in percent. Experimental procedures for DSC and TGA
collection are given in Example 2.
[0078] FIG. 9B is a DSC and TGA graph for sample 2-4 (Example 6).
The x-axis is temperature measured in .degree. C., the left y-axis
heat flow measured in (W/g), and the right y-axis is weight
measured in percent. Experimental procedures for DSC and TGA
collection are given in Example 2.
[0079] FIG. 10A is a DSC and TGA graph for sample 2-5 (Example 6).
The x-axis is temperature measured in .degree. C., the left y-axis
heat flow measured in (W/g), and the right y-axis is weight
measured in percent. Experimental procedures for DSC and TGA
collection are given in Example 2.
[0080] FIG. 10B is a DSC and TGA graph for sample 2-6 (Example 6).
The x-axis is temperature measured in .degree. C., the left y-axis
heat flow measured in (W/g), and the right y-axis is weight
measured in percent. Experimental procedures for DSC and TGA
collection are given in Example 2.
[0081] FIG. 11A is a DSC and TGA graph for sample 2-7 (Example 6).
The x-axis is temperature measured in .degree. C., the left y-axis
heat flow measured in (W/g), and the right y-axis is weight
measured in percent. Experimental procedures for DSC and TGA
collection are given in Example 2.
[0082] FIG. 11B is a DSC and TGA graph for sample 2-8 (Example 6).
The x-axis is temperature measured in .degree. C., the left y-axis
heat flow measured in (W/g), and the right y-axis is weight
measured in percent. Experimental procedures for DSC and TGA
collection are given in Example 2.
[0083] FIG. 12A is the XRPD pattern of amorphous Compound 4 (sample
3-12) as discussed in Example 7. The x-axis is 2Theta measured in
degrees and the y-axis is intensity measured in counts. No
crystallization of a malonate salt was observed regardless of the
solvent used.
[0084] FIG. 12B is an overlay of XRPD diffractograms of amorphous
samples (samples 3-6, 3-10, 3-11, and 3-12) identified from the
attempted crystallization of compound 1 with malonate salt (Example
7). The x-axis is 2Theta measured in degrees and the y-axis is
intensity measured in counts.
[0085] FIG. 13A is the HPLC chromatogram of sample 3-12 from the
attempted crystallizations of compound 1 with malonate salt as
described in Example 7. The sample was 99.2% pure. The x-axis is
time measured in minutes and the y-axis is intensity measured in
mAu.
[0086] FIG. 13B is an overlay of XRPD diffractograms of solid
samples obtained from the crystallization using LAG (samples 4-13,
4-12, 4-9, 4-3, and 4-1) compared to Compound 1 (sample 1-2) as
described in Example 8. All the XRDP match the patterns of the
crystalline acid counter ion with no additional peaks. The x-axis
is 2Theta measured in degrees and the y-axis is 10 intensity
measured in counts.
[0087] FIG. 14A is an overlay of XRPD diffractograms of samples
obtained from utilizing ethyl acetate as a crystallization solvent
(samples 6-13, 6-12, 6-11, 6-10, 6-8, 6-7, 6-6, 6-5, 6-4, and 6-2)
compared to crystalline Compound 1 (sample 1-2) as described in
Example 10. The XRPD patterns were generally found to match the
Compound 1 pattern with the exception of samples 6-2, 6-4, and 6-5
that exhibit slight differences. The x-axis is 2Theta measured in
degrees and the y-axis is intensity measured in counts.
[0088] FIG. 14B is an overlay of XRPD diffractogram of sample 5-1
following a second dissolution in MEK and the addition of the
antisolvent cyclohexane and pamioc acid as described in Example 9.
Sample 5-1, crystallized in pamioc acid, was a solid following
maturation, but the XRPD pattern matched the pattern of pamioc
acid.
[0089] FIG. 15A is an overlay of XRPD diffractograms of samples
obtained from utilizing ethyl acetate as a crystallization solvent
(samples 6-5, 6-4, and 6-2) compared to crystalline Compound 1
(sample 1-2) as described in Example 10. The XRPD patterns were
generally found to match the Compound 1 pattern with the exception
of samples 6-2, 6-4, and 6-5 that exhibit slight differences. The
x-axis is 2Theta measured in degrees and the y-axis is intensity
measured in counts and labeled with the acid used in
crystallization.
[0090] FIG. 15B is the XRPD pattern for Compound 2 as described in
Example 14. The x-axis is 2Theta measured in degrees and the y-axis
is intensity measured in counts.
[0091] FIG. 16A is a graph of the active TP (metabolite 1-6)
concentration levels in the livers and hearts of rats, dogs, and
monkeys (Example 18). The x-axis is the dosage measured in mg/kg
for each species and the y-axis is the active TP concentration
measured in ng/g.
[0092] FIG. 16B is a graph of the active TP (metabolite 1-6)
concentration levels in the liver and heart of dogs (n=2) measured
4 hours after a single oral dose of Compound 1 or Compound 2
(Example 19). The x-axis is the dosage of each compound measured in
mg/kg and the y-axis is the active TP concentration measured in
ng/g.
[0093] FIG. 17 is the plasma profile of Compound 1 and metabolite
1-7 in rats given a single 500 mg/kg oral dose of Compound 2
(Example 20) measured 72 hours post-dose. The x-axis is time
measured in hours and the y-axis is plasma concentration measured
in ng/mL.
[0094] FIG. 18 is the plasma profile of Compound 1 and metabolite
1-7 in monkeys given single oral doses of 30 mg, 100 mg, or 300 mg
of Compound 2 (Example 20) measured 72 hours post-dose. The x-axis
is time measured in hours and the y-axis is plasma concentration
measured in ng/mL.
[0095] FIG. 19 is a graph of EC.sub.95 measured in nM of sofosbuvir
and Compound 1 against HCV clinical isolates. EC.sub.95 values for
Compound 1 are 7-33 times lower than sofosbuvir (Example 22). The
x-axis is labeled with the genotype and the y-axis is EC.sub.95
measured in nM.
[0096] FIG. 20 is a graph of EC.sub.50 measured in nM of sofosbuvir
and Compound 1 against laboratory strains of HCV Genotypes 1a, 1b,
2a, 3a, 4a, and 5a. Compound 1 is approximately 6-11 times more
potent than sofosbuvir in Genotypes 1-5 (Example 22). The x-axis is
labeled with the genotype and the y-axis is EC.sub.50 measured in
nM.
[0097] FIG. 21 is a graph of the mean plasma concentration-time
profile of Compound 1 following the administration of a single dose
of Compound 2 in all cohorts of Part B of the study as described in
Example 24. Compound 1 was quickly absorbed and rapidly metabolized
within approximately 8 hours in all cohorts from Part B. The x-axis
is the time measured in hours and the y-axis is the geometric mean
plasma concentration measured in ng/mL.
[0098] FIG. 22 is a graph of the mean plasma concentration-time
profile of metabolite 1-7 following the administration of a single
dose of Compound 2 in all cohorts of Part B of the study as
described in Example 24. Metabolite 1-7 exhibited sustained plasma
concentration in all cohorts from Part B. The x-axis is the time
measured in hours and the y-axis is the geometric mean plasma
concentration measured in ng/mL.
[0099] FIG. 23A is an individual pharmacokinetic/pharmacodynamic
analysis of a subject enrolled in the 1b cohort as described in
Example 24. The graph shows plasma metabolite 1-7 exposure and HCV
RNA reduction levels. The dashed line represents the minimum
concentration of metabolite 1-7 required to sustain a viral
response greater than the EC.sub.95 value against GT1b. The x-axis
is time measured in hours. The left y-axis is metabolite 1-7 plasma
concentration measured in ng/mL and the right y-axis is the HCV RNA
reduction measured in log.sub.10 IU/mL.
[0100] FIG. 23B is an individual pharmacokinetic/pharmacodynamic
analysis of a subject enrolled in the 1b cohort as described in
Example 24. The graph shows plasma metabolite 1-7 exposure and HCV
RNA reduction levels. The dashed line represents the minimum
concentration of metabolite 1-7 required to sustain a viral
response greater than the EC.sub.95 value against GT1b. The x-axis
is time measured in hours. The left y-axis is metabolite 1-7 plasma
concentration measured in ng/mL and the right y-axis is the HCV RNA
reduction measured in log.sub.10 IU/mL.
[0101] FIG. 23C is an individual pharmacokinetic/pharmacodynamic
analysis of a subject enrolled in the 1b cohort as described in
Example 24. The graph shows plasma metabolite 1-7 exposure and HCV
RNA reduction levels. The dashed line represents the minimum
concentration of metabolite 1-7 required to sustain a viral
response greater than the EC.sub.95 value against GT1b. The x-axis
is time measured in hours. The left y-axis is metabolite 1-7 plasma
concentration measured in ng/mL and the right y-axis is the HCV RNA
reduction measured in log.sub.10 IU/mL.
[0102] FIG. 23D is an individual pharmacokinetic/pharmacodynamic
analysis of a subject enrolled in the 3b cohort as described in
Example 24. Each graph shows plasma metabolite 1-7 exposure and HCV
RNA reduction levels. The dashed line represents the minimum
concentration of metabolite 1-7 required to sustain a viral
response greater than the EC.sub.95 value against GT1b. The x-axis
is time measured in hours. The left y-axis is metabolite 1-7 plasma
concentration measured in ng/mL and the right y-axis is the HCV RNA
reduction measured in log.sub.10 IU/mL.
[0103] FIG. 23E is an individual pharmacokinetic/pharmacodynamic
analysis of a subject enrolled in the 3b cohort as described in
Example 24. Each graph shows plasma metabolite 1-7 exposure and HCV
RNA reduction levels. The dashed line represents the minimum
concentration of metabolite 1-7 required to sustain a viral
response greater than the EC.sub.95 value against GT1b. The x-axis
is time measured in hours. The left y-axis is metabolite 1-7 plasma
concentration measured in ng/mL and the right y-axis is the HCV RNA
reduction measured in log.sub.10 IU/mL.
[0104] FIG. 23F is an individual pharmacokinetic/pharmacodynamic
analysis of a subject enrolled in the 3b cohort as described in
Example 24. Each graph shows plasma metabolite 1-7 exposure and HCV
RNA reduction levels. The dashed line represents the minimum
concentration of metabolite 1-7 required to sustain a viral
response greater than the EC.sub.95 value against GT1b. The x-axis
is time measured in hours. The left y-axis is metabolite 1-7 plasma
concentration measured in ng/mL and the right y-axis is the HCV RNA
reduction measured in log.sub.10 IU/mL.
[0105] FIG. 24 is a graph of the EC.sub.95 values of Compound 1 and
sofosbuvir against clinical isolates of GT1, GT2, GT3, and GT4
HCV-infected patients. The dashed horizontal line () represents the
steady-state trough concentration (C.sub.24,ss) of sofosbuvir
nucleoside following a dose of 400 mg QD of sofosbuvir. The full
horizontal line () represents the steady-state trough concentration
(C.sub.24,ss) of metabolite 1-7 following 600 mg of Compound 2
(equivalent to 550 mg of Compound 1). The dotted horizontal line ()
represents the steady-state trough concentration (C.sub.24,ss) of
metabolite 1-7 following 450 mg of Compound 2 (equivalent to 400 mg
of Compound 1). As discussed in Example 25, the predicted
steady-state trough plasma level (C.sub.24,ss) of metabolite 1-7
following 600 mg and 450 mg of Compound 2 exceeds the in vitro
EC.sub.95 of Compound 1 against all tested clinical isolates. The
steady state trough plasma level (C.sub.24,ss) of sofosbuvir only
exceeds the EC.sub.95 at GT2 clinical isolates. The x-axis is
labeled with the clinical isolates and the table under the x-axis
lists the EC.sub.95 values for Compound 1 and sofosbuvir. The
y-axis is the EC.sub.95 against the clinical isolates measured in
ng/mL. EC.sub.95 is expressed as nucleoside equivalent. Sofosbuvir
and Compound 2 were administered daily (QD).
[0106] FIG. 25 is a flow diagram showing the manufacturing process
of 50 mg and 100 mg tablets of Compound 2 as described in Example
26. In step 1, microcrystalline cellulose, Compound 2, lactose
monohydrate, and croscarmellose sodium are filtered through a 600 M
screen. In step 2, the contents from step 1 are loaded into a
V-blender and mixed for 5 minutes at 25 rpm. In step 3, magnesium
stearate is filtered through a 600 M screen. In step 4, magnesium
stearate is loaded into the V-blender containing the contents from
step 2 (microcrystalline cellulose, Compound 2, lactose
monohydrate, and croscarmellose sodium) and mixed for 2 minutes at
25 rpm. The common blend is then divided for the production of 50
mg tablets and 100 mg tablets. To produce 50 mg tablets, the blend
from step 4 is compressed with 6 mm round standard concave tooling.
To produce 100 mg tablets, the blend from step 4 is compressed with
8 mm round standard concave tooling. The tablets are then packaged
into HDPE bottles induction-sealed with PP caps with desiccant.
[0107] FIG. 26 is the hemi-sulfate salt that exhibits advantageous
pharmacological properties over its corresponding free base for the
treatment of an HCV virus.
DETAILED DESCRIPTION OF THE INVENTION
[0108] The invention disclosed herein is a compound, method,
composition, and solid dosage form for the treatment of infections
in or exposure to humans and other host animals of the HCV virus
that includes the administration of an effective amount of the
hemi-sulfate salt of
isopropyl((S)-(((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 (Compound 2) as described herein, optionally in a
pharmaceutically acceptable carrier. In one embodiment, Compound 2
is an amorphous solid. In yet another embodiment, Compound 2 is a
crystalline solid.
##STR00005##
[0109] The compound, compositions, and dosage forms can also be
used to treat conditions related to or occurring as a result of an
HCV viral exposure. For example, the active compound can be used to
treat HCV antibody positive- and HCV antigen-positive conditions,
viral-based chronic liver inflammation, liver cancer resulting from
advanced hepatitis C (e.g, hepatocellular carcinoma), cirrhosis,
acute hepatitis C, fulminant hepatitis C, chronic persistent
hepatitis C, and anti-HCV-based fatigue.
[0110] The active compounds and compositions can also be used to
treat the range of HCV genotypes. At least six distinct genotypes
of HCV, each of which have multiple subtypes, have been identified
globally. Genotypes 1-3 are prevalent worldwide, and Genotypes 4,
5, and 6 are more limited geographically. Genotype 4 is common in
the Middle East and Africa. Genotype 5 is mostly found in South
Africa. Genotype 6 predominately exists in Southeast Asia. Although
the most common genotype in the United States is Genotype 1,
defining the genotype and subtype can assist in treatment type and
duration. For example, different genotypes respond differently to
different medications and optimal treatment times vary depending on
the genotype infection.
[0111] Within genotypes, subtypes, such as Genotype 1a and Genotype
1b, respond differently to treatment as well. Infection with one
type of genotype does not preclude a later infection with a
different genotype.
[0112] As described in Example 22, Compound 2 is active against the
range of HCV genotypes, including Genotypes 1-5. In one embodiment,
Compound 2 is used to treat HCV Genotype 1, HCV Genotype 2, HCV
Genotype 3, HCV Genotype 4, HCV Genotype 5, or HCV Genotype 6. In
one embodiment, Compound 2 is used to treat HCV Genotype 1a. In one
embodiment, Compound 2 is used to treat HCV Genotype 1b. In one
embodiment, Compound 2 is used to treat HCV Genotype 2a. In one
embodiment, Compound 2 is used to treat HCV Genotype 2b. In one
embodiment, Compound 2 is used to treat HCV Genotype 3a. In one
embodiment, Compound 2 is used to treat HCV Genotype 4a. In one
embodiment, Compound 2 is used to treat HCV Genotype 4d.
[0113] In one embodiment, Compound 1 or Compound 2 is used to treat
HCV Genotype 5a. In one embodiment, Compound 1 or Compound 2 is
used to treat HCV Genotype 6a. In one embodiment, Compound 1 or
Compound 2 is used to treat HCV Genotype 6b, 6c, 6d, 6e, 6f, 6g,
6h, 6i, 6j, 6k, 6l, 6m, 6n, 6o, 6p, 6q, 6r, 6s, 6t, or 6u.
[0114] As discussed in Example 25 and shown in FIG. 24, the
predicted steady-state trough concentration (C.sub.24,ss) of
metabolite 1-7 following a dose of 450 mg (400 mg free base) and a
dose of 600 mg (550 mg free base) of Compound 2 is approximately 40
ng/mL to 50 ng/mL. This C.sub.24,ss level exceeded the EC.sub.95 of
Compound 1 at HCV Genotypes 1a, 1b, 2a, 2b, 3a, 4a, and 4d. This
data confirms that Compound 2 has potent-pan genotypic activity.
This is surprising because Compound 2 achieves a smaller
steady-state trough concentration (C.sub.24,ss) than the
steady-state trough concentration (C.sub.24,ss) of the nucleoside
metabolite of sofosbuvir following equivalent sofosbuvir dosing.
The steady-state trough concentration (C.sub.24,ss) of the
corresponding nucleoside metabolite of sofosbuvir is approximately
100 ng/mL, but this level only exceeds the EC.sub.95 of sofosbuvir
against GT2 clinical isolates (FIG. 24). Compound 2 is more potent
than sofosbuvir against GT1, GT2, GT3, and GT4, and therefore
allows a dosage form that delivers a smaller steady-state trough
concentration of its metabolite which is nonetheless efficacious
against all tested genotypes of HCV. In one embodiment, a dosage
form of Compound 2 is delivered that achieves a metabolite 1-7
steady-state trough concentration (C.sub.24,ss) between
approximately 15-75 ng/mL. In one embodiment, a dosage form of
Compound 2 is delivered that achieves a metabolite 1-7 steady-state
trough concentration (C.sub.24,ss) between approximately 20-60
ng/mL, 20-50 ng/mL, or 20-40 ng/mL.
[0115] In one embodiment, the compound, formulations, or solid
dosage forms that include the compound can also be used
prophylactically to prevent or retard the progression of clinical
illness in individuals who are HCV antibody- or HCV
antigen-positive or who have been exposed to hepatitis C.
[0116] In particular, it has been discovered that Compound 2 is
active against HCV and exhibits superior drug-like and
pharmacological properties compared to its free base (Compound
1).
[0117] Surprisingly, Compound 2 is more bioavailable and achieves a
higher AUC than Compound 1 (Example 19) and Compound 2 is more
selective for the target organ, the liver, than Compound 1 (Example
19).
[0118] Compound 2 is also advantageous over Compound 1 in terms of
solubility and chemical stability. This is surprising because the
mono-sulfate salt of
isopropyl((S)-(((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 (Compound 3) is unstable and exhibits the appearance
of a sticky gum, while Compound 2, the hemi-sulfate salt, is a
stable white solid. The hemisulfate salt, both as a solid and in a
solid dosage form, is very stable over 9 months and is not
hydroscopic.
##STR00006##
[0119] Despite the volume of antiviral nucleoside literature and
patent filings, Compound 2 has not been specifically disclosed.
[0120] Compound 2 has S-stereochemistry at the phosphorus atom
which has been confirmed with X-ray crystallography (FIG. 3,
Example 2). In alternative embodiments, Compound 2 can be used in
the form of any desired ratio of phosphorus R- and S-enantiomers,
including up to pure enantiomers. In some embodiments, Compound 2
is used in a form that is at least 90% free of the opposite
enantiomer, and can be at least 98%, 99%, or even 100% free of the
opposite enantiomer. Unless described otherwise, an
enantiomerically enriched Compound 2 is at least 90% free of the
opposite enantiomer. In addition, in an alternative embodiment, the
amino acid of the phosphoramidate can be in the D- or
L-configuration, or a mixture thereof, including a racemic
mixture.
[0121] Unless otherwise specified, the compounds described herein
are provided in the .beta.-D-configuration. In an alternative
embodiment, the compounds can be provided in a
.beta.-L-configuration. Likewise, any substituent group that
exhibits chirality can be provided in racemic, enantiomeric,
diastereomeric form, or any mixture thereof. Where a
phosphoramidate exhibits chirality, it can be provided as an R or S
chiral phosphorus derivative or a mixture thereof, including a
racemic mixture. All of the combinations of these stereo
configurations are alternative embodiments in the invention
described herein. In another embodiment, at least one of the
hydrogens of Compound 2 (the nucleotide or the hemi-sulfate salt)
can be replaced with deuterium. These alternative configurations
include, but are not limited to,
##STR00007##
I. Hemi-sulfate salt of
isopropyl((S)-(((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 (Compound 2)
[0122] The active compound of the invention is Compound 2, which
can be provided in a pharmaceutically acceptable composition or
solid dosage form thereof. In one embodiment, Compound 2 is an
amorphous solid. In yet a further embodiment, Compound 2 is a
crystalline solid.
Synthesis of Compound 2
[0123] The present invention further includes a non-limiting
illustrative process for the preparation of Compound 2 that
includes [0124] (i) a first step of dissolving Compound 1 in an
organic solvent, for example, acetone, ethyl acetate, methanol,
acetonitrile, or ether, or the like, in a flask or container;
[0125] (ii) charging a second flask or container with a second
organic solvent, which may be the same as or different from the
organic solvent in step (i), optionally cooling the second solvent
to 0-10 degrees C., and adding dropwise H.sub.2SO.sub.4 to the
second organic solvent to create a H.sub.2SO.sub.4/organic solvent
mixture; and wherein the solvent for example may be methanol;
[0126] (iii) adding dropwise the H.sub.2SO.sub.4/solvent mixture at
a molar ratio of 0.5/1.0 from step (ii) to the solution of Compound
1 of step (i) at ambient or slightly increased or decreased
temperature (for example 23-35 degrees C.); [0127] (iv) stirring
the reaction of step (iii) until precipitate of Compound 2 is
formed, for example at ambient or slightly increased or decreased
temperature; [0128] (v) optionally filtering the resulting
precipitate from step (iv) and washing with an organic solvent; and
[0129] (vi) optionally drying the resulting Compound 2 in a vacuum,
optionally at elevated a temperature, for example, 55, 56, 57, 58,
59, or 60.degree. C.
[0130] In certain embodiments, step (i) above is carried out in
acetone. Further, the second organic solvent in step (ii) may be
for example methanol and the mixture of organic solvents in step
(v) is methanol/acetone.
[0131] In one embodiment, Compound 1 is dissolved in ethyl acetate
in step (i). In one embodiment, Compound 1 is dissolved in
tetrahydrofuran in step (i). In one embodiment, Compound 1 is
dissolved in acetonitrile in step (i). In an additional embodiment,
Compound 1 is dissolved in dimethylformamide in step (i).
[0132] In one embodiment, the second organic solvent in step (ii)
is ethanol. In one embodiment, the second organic solvent in step
(ii) is isopropanol. In one embodiment, the second organic solvent
in step (ii) is n-butanol.
[0133] In one embodiment, a mixture of solvents are used for
washing in step (v), for example, ethanol/acetone. In one
embodiment, the mixture of solvent for washing in step (v) is
isopropanol/acetone. In one embodiment, the mixture of solvent for
washing in step (v) is n-butanol/acetone. In one embodiment, the
mixture of solvent for washing in step (v) is ethanol/ethyl
acetate. In one embodiment, the mixture of solvent for washing in
step (v) is isopropanol/ethyl acetate. In one embodiment, the
mixture of solvent for washing in step (v) is n-butanol/ethyl
acetate. In one embodiment, the mixture of solvent for washing in
step (v) is ethanol/tetrahydrofuran. In one embodiment, the mixture
of solvent for washing in step (v) is isopropanol/tetrahydrofuran.
In one embodiment, the mixture of solvent for washing in step (v)
is n-butanol/tetrahydrofuran. In one embodiment, the mixture of
solvent for washing in step (v) is ethanol/acetonitrile. In one
embodiment, the mixture of solvent for washing in step (v) is
isopropanol/acetonitrile. In one embodiment, the mixture of solvent
for washing in step (v) is n-butanol/acetonitrile. In one
embodiment, the mixture of solvent for washing in step (v) is
ethanol/dimethylformamide. In one embodiment, the mixture of
solvent for washing in step (v) is isopropanol/dimethylformamide.
In one embodiment, the mixture of solvent for washing in step (v)
is n-butanol/dimethylformamide.
II. Metabolism of
Isopropyl((S)-(((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 (Compound 2)
[0134] The metabolism of Compound 1 and Compound 2 involves the
production of a 5'-monophosphate and the subsequent anabolism of
the N.sup.6-methyl-2,6-diaminopurine base (1-3) to generate
((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-fluoro-3-hyd-
roxy-4-methyltetrahydrofuran-2-yl)methyl dihydrogen phosphate (1-4)
as the 5'-monophosphate. The monophosphate is then further
anabolized to the active triphosphate species: the 5'-triphosphate
(1-6). The 5'-triphosphate can be further metabolized to generate
2-amino-9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltet-
rahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (1-7). Alternatively,
5'-monophophate 1-2 can be metabolized to generate the purine base
1-8. The metabolic pathway for
isopropyl((S)-(((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 is illustrated in Scheme 1 (shown above).
III. Additional Salts of Compound 1
[0135] In alternative embodiments, the present invention provides
Compound 1 as an oxalate salt (Compound 4) or an HCl salt (Compound
5).
##STR00008##
[0136] Both the 1:1 oxalate salt and the 1:1 HC salt form solids
with reasonable properties for solid dosage forms for the treatment
of a host such as a human with hepatitis C. However, the oxalate
salt may be less desired, and perhaps not suitable, if the patient
is susceptible to kidney stones. The HCl salt is more hydroscopic
than the hemisulfate salt. Thus, the hemisulfate salt remains the
most desired salt form of Compound 1 with unexpected
properties.
IV. Definitions
[0137] The term "D-configuration" as used in the context of the
present invention refers to the principle configuration which
mimics the natural configuration of sugar moieties as opposed to
the unnatural occurring nucleosides or "L" configuration. The term
"0" or "anomer" is used with reference to nucleoside analogs in
which the nucleoside base is configured (disposed) above the plane
of the furanose moiety in the nucleoside analog.
[0138] The terms "coadminister" and "coadministration" or
combination therapy are used to describe the administration of
Compound 2 according to the present invention in combination with
at least one other active agent, for example where appropriate at
least one additional anti-HCV agent. The timing of the
coadministration is best determined by the medical specialist
treating the patient. It is sometimes preferred that the agents be
administered at the same time. Alternatively, the drugs selected
for combination therapy may be 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.
[0139] The term "host", as used herein, refers to a unicellular or
multicellular organism in which a HCV virus can replicate,
including cell lines and animals, and typically a human. The term
host specifically refers to infected cells, cells transfected with
all or part of a HCV genome, and animals, in particular, primates
(including chimpanzees) and humans. In most animal applications of
the present invention, the host is a human patient. Veterinary
applications, in certain indications, however, are clearly
anticipated by the present invention (such as chimpanzees). The
host can be for example, bovine, equine, avian, canine, feline,
etc.
Isotopic Substitution
[0140] The present invention includes compounds and the use of
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 (.sup.2H) and tritium
(.sup.3H) may be used anywhere in described structures.
Alternatively or in addition, isotopes of carbon, e.g., .sup.13C
and .sup.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 .alpha.-deuterium
kinetic isotope effect) or next to or near the site of bond
breakage (a .beta.-deuterium kinetic isotope effect). Achillion
Pharmaceuticals, Inc. (WO/2014/169278 and WO/2014/169280) describes
deuteration of nucleotides to improve their pharmacokinetic or
pharmacodynamic, including at the 5-position of the molecule.
[0141] 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 (H), deuterium (2H)
and tritium (3H). Thus, reference herein to a compound encompasses
all potential isotopic forms unless the context clearly dictates
otherwise.
[0142] The term "isotopically-labeled" analog refers to an analog
that is a "deuterated analog", a ".sup.13C-labeled analog," or a
"deuterated/.sup.13C-labeled analog." The term "deuterated analog"
means a compound described herein, whereby a H-isotope, i.e.,
hydrogen/protium (H), is substituted by a H-isotope, i.e.,
deuterium (.sup.2H). 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.
V. Methods of Treatment or Prophylaxis
[0143] Treatment, as used herein, refers to the administration of
Compound 2 to a host, for example a human that is or may become
infected with a HCV virus.
[0144] The term "prophylactic" or preventative, when used, refers
to the administration of Compound 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 2 is administered to a host
who has been exposed to and thus is at risk of infection by a
hepatitis C virus infection.
[0145] The invention is directed to a method of treatment or
prophylaxis of a hepatitis C virus, including drug resistant and
multidrug resistant forms of HCV and related disease states,
conditions, or complications of an HCV infection, including
cirrhosis and related hepatotoxicities, as well as other conditions
that are secondary to a HCV infection, such as weakness, loss of
appetite, weight loss, breast enlargement (especially in men), rash
(especially on the palms), difficulty with clotting of blood,
spider-like blood vessels on the skin, confusion, coma
(encephalopathy), buildup of fluid in the abdominal cavity
(ascites), esophageal varices, portal hypertension, kidney failure,
enlarged spleen, decrease in blood cells, anemia, thrombocytopenia,
jaundice, and hepatocellular cancer, among others. The method
comprises administering to a host in need thereof, typically a
human, with an effective amount of Compound 2 as described herein,
optionally in combination with at least one additional bioactive
agent, for example, an additional anti-HCV agent, further in
combination with a pharmaceutically acceptable carrier additive
and/or excipient.
[0146] In yet another aspect, the present invention is a method for
prevention or prophylaxis of an HCV infection or a disease state or
related or follow-on disease state, condition or complication of an
HCV infection, including cirrhosis and related hepatotoxicities,
weakness, loss of appetite, weight loss, breast enlargement
(especially in men), rash (especially on the palms), difficulty
with clotting of blood, spider-like blood vessels on the skin,
confusion, coma (encephalopathy), buildup of fluid in the abdominal
cavity (ascites), esophageal varices, portal hypertension, kidney
failure, enlarged spleen, decrease in blood cells, anemia,
thrombocytopenia, jaundice, and hepatocellular (liver) cancer,
among others, said method comprising administering to a patient at
risk with an effective amount Compound 2 as described above in
combination with a pharmaceutically acceptable carrier, additive,
or excipient, optionally in combination with another anti-HCV
agent. In another embodiment, the active compounds of the invention
can be administered to a patient after a hepatitis-related liver
transplantation to protect the new organ.
[0147] 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. 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.
VI. Pharmaceutical Compositions and Dosage Forms
[0148] In an aspect of the invention, pharmaceutical compositions
according to the present invention comprise an anti-HCV virus
effective amount of Compound 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.
[0149] In an aspect of the invention, pharmaceutical compositions
according to the present invention comprise an anti-HCV effective
amount of Compound 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 an anti-HCV agent.
[0150] The invention includes pharmaceutical compositions that
include an effective amount to treat a hepatitis C virus infection
of Compound 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 hepatitis C virus
infection of Compound 2 of the present invention or prodrug, in a
pharmaceutically acceptable carrier or excipient.
[0151] One of ordinary skill in the art will recognize that a
therapeutically effective 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.
[0152] Compound 2 according to the present invention can be
formulated in a mixture with a pharmaceutically acceptable carrier.
In general, it is preferable to administer the pharmaceutical
composition in orally-administrable form, an 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.
[0153] In certain pharmaceutical dosage forms, the prodrug form of
the compounds, especially including acylated (acetylated or other),
and ether (alkyl and related) derivatives, phosphate esters,
thiophosphoramidates, phosphoramidates, and various salt forms of
the present compounds, may be used to achieve the desired effect.
One of ordinary skill in the art will recognize how to readily
modify the present compounds to prodrug forms to facilitate
delivery of active compounds to a targeted site within the host
organism or patient. The person of ordinary skill in the art also
will take advantage of favorable pharmacokinetic parameters of the
prodrug forms, where applicable, in delivering the present
compounds to a targeted site within the host organism or patient to
maximize the intended effect of the compound.
[0154] The amount of Compound 2 included within the therapeutically
active formulation according to the present invention is an
effective amount to achieve the desired outcome according to the
present invention, for example, for treating the HCV infection,
reducing the likelihood of a HCV infection or the inhibition,
reduction, and/or abolition of HCV or its secondary effects,
including disease states, conditions, and/or complications which
occur secondary to HCV. In general, a therapeutically effective
amount of the present compound in a pharmaceutical dosage form may
range from about 0.001 mg/kg to about 100 mg/kg per day or more,
more often, slightly less than about 0.1 mg/kg to more than about
25 mg/kg per day of the patient or considerably more, depending
upon the compound used, the condition or infection treated and the
route of administration. Compound 2 is often administered in
amounts ranging from about 0.1 mg/kg to about 15 mg/kg per day of
the patient, depending upon the pharmacokinetic of the agent in the
patient. This dosage range generally produces effective blood level
concentrations of active compound which may range from about 0.001
to about 100, about 0.05 to about 100 micrograms/cc of blood in the
patient.
[0155] Often, to treat, prevent or delay the onset of these
infections and/or to reduce the likelihood of an HCV virus
infection, or a secondary disease state, condition or complication
of HCV, Compound 2 will be administered in a solid dosage form in
an amount ranging from about 250 micrograms up to about 800
milligrams or more at least once a day, for example, at least about
5, 10, 20,25, 50,75, 100, 150,200, 250, 300, 350,400, 450, 500,
550, 600, 650, 700,750, or 800 milligrams or more, once, twice,
three, or up to four times a day according to the direction of the
healthcare provider. Compound 2 often administered orally, but may
be administered parenterally, topically, or in suppository form, as
well as intranasally, as a nasal spray or as otherwise described
herein. More generally, Compound 2 can be administered in a tablet,
capsule, injection, intravenous formulation, suspension, liquid,
emulsion, implant, particle, sphere, cream, ointment, suppository,
inhalable form, transdermal form, buccal, sublingual, topical, gel,
mucosal, and the like.
[0156] When a dosage form herein refers to a milligram weight dose,
it refers to the amount of Compound 2 (i.e., the weight of the
hemi-sulfate salt) unless otherwise specified to the contrary.
[0157] In certain embodiments, the pharmaceutical composition is in
a dosage form that contains from about 1 mg to about 2000 mg, from
about 10 mg to about 1000 mg, from about 100 mg to about 800 mg,
from about 200 mg to about 600 mg, from about 300 mg to about 500
mg, or from about 400 mg to about 450 mg of Compound 2 in a unit
dosage form. In certain embodiments, the pharmaceutical composition
is in a dosage form, for example in a solid dosage form, that
contains up to about 10, about 50, about 100, about 125, about 150,
about 175, about 200, about 225, about 250, about 275, about 300,
about 325, about 350, about 375, about 400, about 425, about 450,
about 475, about 500, about 525, about 550, about 575, about 600,
about 625, about 650, about 675, about 700, about 725, about 750,
about 775, about 800, about 825, about 850, about 875, about 900,
about 925, about 950, about 975, or about 1000 mg or more of
Compound 2 in a unit dosage form. In one embodiment, Compound 2 is
administered in a dosage form that delivers at least about 300 mg.
In one embodiment, Compound 2 is administered in a dosage form that
delivers at least about 400 mg. In one embodiment, Compound 2 is
administered in a dosage form that delivers at least about 500 mg.
In one embodiment, Compound 2 is administered in a dosage form that
delivers at least about 600 mg. In one embodiment, Compound 2 is
administered in a dosage form that delivers at least about 700 mg.
In one embodiment, Compound 2 is administered in a dosage form that
delivers at least about 800 mg. In certain embodiments, Compound 2
is administered at least once a day for up to 12 weeks. In certain
embodiments, Compound 2 is administered at least once a day for up
to 10 weeks. In certain embodiments, Compound 2 is administered at
least once a day for up to 8 weeks. In certain embodiments,
Compound 2 is administered at least once a day for up to 6 weeks.
In certain embodiments, Compound 2 is administered at least once a
day for up to 4 weeks. In certain embodiments, Compound 2 is
administered at least once a day for at least 4 weeks. In certain
embodiments, Compound 2 is administered at least once a day for at
least 6 weeks. In certain embodiments, Compound 2 is administered
at least once a day for at least 8 weeks. In certain embodiments,
Compound 2 is administered at least once a day for at least 10
weeks. In certain embodiments, Compound 2 is administered at least
once a day for at least 12 weeks. In certain embodiments, Compound
2 is administered at least every other day for up to 12 weeks, up
to 10 weeks, up to 8 weeks, up to 6 weeks, or up to 4 weeks. In
certain embodiments, Compound 2 is administered at least every
other day for at least 4 weeks, at least 6 weeks, at least 8 weeks,
at least 10 weeks, or at least 12 weeks. In one embodiment, at
least about 600 mg of Compound 2 is administered at least once a
day for up to 6 weeks. In one embodiment, at least about 500 mg of
Compound 2 is administered at least once a day for up to 6 weeks.
In one embodiment, at least about 400 mg of Compound 2 is
administered at least once a day for up to 6 weeks. In one
embodiment, at least 300 mg of Compound 2 is administered at least
once a day for up to 6 weeks. In one embodiment, at least 200 mg of
Compound 2 is administered at least once a day for up to 6 weeks.
In one embodiment, at least 100 mg of Compound 2 is administered at
least once a day for up to 6 weeks.
[0158] Metabolite 1-6 is the active triphosphate of Compound 2, but
metabolite 1-6 is not measurable in plasma. A surrogate for
metabolite 1-6 is metabolite 1-7. Metabolite 1-7 is a nucleoside
metabolite measurable in plasma and is therefore an indication of
the intracellular concentrations of metabolite 1-6. For maximum HCV
antiviral activity, a dosage form of Compound 2 must achieve a
metabolite 1-7 steady-state trough concentration (C.sub.24,ss) that
exceeds the EC.sub.95 value of Compound 2. As shown in FIG. 24, the
EC.sub.95 of Compound 1 against clinical isolates of GT1, GT2, GT3,
and GT4 is less than 25 ng/mL (Compound 1 EC.sub.95 and Compound 2
EC.sub.95 values are the same). In one embodiment, a dosage form of
Compound 2 is delivered that achieves a steady-state trough
concentration (C.sub.24,ss) of metabolite 1-7 that is between
approximately 15 to 75 ng/mL. In one embodiment, a dosage form of
Compound 2 is delivered that achieves a steady-state trough
concentration (C.sub.24,ss) of metabolite 1-7 that is between
approximately 20 to 60 ng/mL. In one embodiment, a dosage form of
Compound 2 is delivered that achieves a steady-state trough
concentration (C.sub.24,ss) of metabolite 1-7 that is between
approximately 30 to 60 ng/mL. In one embodiment, a dosage form of
Compound 2 is delivered that achieves a steady-state trough
concentration (C.sub.24,ss) of metabolite 1-7 that is between
approximately 20 to 50 ng/mL. In one embodiment, a dosage form of
Compound 2 is delivered that achieves a steady-state trough
concentration (C.sub.24,ss) of metabolite 1-7 that is between
approximately 30 to 50 ng/mL. In one embodiment, a dosage form of
Compound 2 is delivered that achieves a steady-state trough
concentration (C.sub.24,ss) of metabolite 1-7 that is between
approximately 20 to 45 ng/mL. In one embodiment, a dosage form of
Compound 2 is delivered that achieves a steady-state trough
concentration (C.sub.24,ss) of metabolite 1-7 that is between
approximately 20 to 30 ng/mL. In one embodiment, a dosage form of
Compound 2 is delivered that achieves a steady-state trough
concentration (C.sub.24,ss) of metabolite 1-7 that is between
approximately 20 to 35 ng/mL. In one embodiment, a dosage form of
Compound 2 is delivered that achieves a steady-state trough
concentration (C.sub.24,ss) of metabolite 1-7 that is between
approximately 20 to 25 ng/mL. Approximate dosage forms are .+-.10%
of the steady-state trough concentration.
[0159] In one embodiment, Compound 2 is dosed at an amount that
achieves a metabolite 1-7 AUC (area under the curve) of between
approximately 1,200 and 3,000 ng/mL. In one embodiment, Compound 2
is dosed at an amount that achieves a metabolite 1-7 AUC of between
approximately 1,500 and 3,000 ng/mL. In one embodiment, Compound 2
is dosed at an amount that achieves a metabolite 1-7 AUC of between
approximately 1,800 and 3,000 ng/mL. In one embodiment, Compound 2
is dosed at an amount that achieves a metabolite 1-7 AUC of between
approximately 2,100 and 3,000 ng/mL. In a preferred embodiment,
Compound 2 is dosed at amount that achieves a metabolite 1-7 AUC of
approximately 2,200 ng*h/mL. Approximate dosage forms are .+-.10%
of the AUC.
[0160] In the case of the co-administration of Compound 2 in
combination with another anti-HCV compound as otherwise described
herein, the amount of Compound 2 according to the present invention
to be administered in ranges from about 0.01 mg/kg of the patient
to about 800 mg/kg or more of the patient or considerably more,
depending upon the second agent to be co-administered and its
potency against the virus, the condition of the patient and
severity of the disease or infection to be treated and the route of
administration. The other anti-HCV agent may for example be
administered in amounts ranging from about 0.01 mg/kg to about 800
mg/kg. Examples of dosage amounts of the second active agent are
amounts ranging from about 250 micrograms up to about 750 mg or
more at least once a day, for example, at least about 5, 10, 20,
25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700,
or 800 milligrams or more, up to four times a day. In certain
preferred embodiments, Compound 2 may be often administered in an
amount ranging from about 0.5 mg/kg to about 50 mg/kg or more
(usually up to about 100 mg/kg), generally depending upon the
pharmacokinetic of the two agents in the patient. These dosage
ranges generally produce effective blood level concentrations of
active compound in the patient.
[0161] For purposes of the present invention, a prophylactically or
preventive effective amount of the compositions according to the
present invention falls within the same concentration range as set
forth above for therapeutically effective amount and is usually the
same as a therapeutically effective amount.
[0162] Administration of Compound 2 may range from continuous
(intravenous drip) to several oral or intranasal administrations
per day (for example, Q.I.D.) 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.
[0163] To prepare the pharmaceutical compositions according to the
present invention, a therapeutically effective amount of Compound 2
according to the present invention is often 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 media 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.
[0164] 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.
[0165] 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.
[0166] In typical embodiments according to the present invention,
Compound 2 and the compositions described are used to treat,
prevent or delay a HCV infection or a secondary disease state,
condition or complication of HCV.
VII. Combination and Alternation Therapy
[0167] 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 an HCV 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 is an NS5B polymerase
inhibitor, it may be useful to administer the compound to a host in
combination with, for example a [0168] (1) Protease inhibitor, such
as an NS3/4A protease inhibitor; [0169] (2) NS5A inhibitor; [0170]
(3) Another NS5B polymerase inhibitor; [0171] (4) NS5B
non-substrate inhibitor; [0172] (5) Interferon alfa-2a, which may
be pegylated or otherwise modified, and/or ribavirin; [0173] (6)
Non-substrate-based inhibitor; [0174] (7) Helicase inhibitor;
[0175] (8) Antisense oligodeoxynucleotide (S-ODN); [0176] (9)
Aptamer; [0177] (10) Nuclease-resistant ribozyme; [0178] (11) iRNA,
including microRNA and SiRNA; [0179] (12) Antibody, partial
antibody or domain antibody to the virus, or [0180] (13) Viral
antigen or partial antigen that induces a host antibody response.
Non limiting examples of anti-HCV agents that can be administered
in combination with Compound 2 of the invention, alone or with
multiple drugs from this lists, are [0181] (i) protease inhibitors
such as telaprevir (Incivek.RTM.), boceprevir (Victrelis.TM.),
simeprevir (Olysio.TM.), paritaprevir (ABT-450), glecaprevir
(ABT-493), ritonavir (Norvir), ACH-2684, AZD-7295, BMS-791325,
danoprevir, Filibuvir, GS-9256, GS-9451, MK-5172, Setrobuvir,
Sovaprevir, Tegobuvir, VX-135, VX-222, and, ALS-220; [0182] (ii)
NS5A inhibitor such as ACH-2928, ACH-3102, IDX-719, daclatasvir,
ledispasvir, velpatasvir (Epclusa), elbasvir (MK-8742), grazoprevir
(MK-5172), and Ombitasvir (ABT-267); [0183] (iii) NS5B inhibitors
such as AZD-7295, Clemizole, dasabuvir (Exviera), ITX-5061,
PPI-461, PPI-688, sofosbuvir (Sovaldi.RTM.), MK-3682, and
mericitabine; [0184] (iv) NS5B inhibitors such as ABT-333, and
MBX-700; [0185] (v) Antibody such as GS-6624; [0186] (vi)
Combination drugs such as Harvoni (ledipasvir/sofosbuvir); Viekira
Pak (ombitasvir/paritaprevir/ritonavir/dasabuvir); Viekirax
(ombitasvir/paritaprevir/ritonavir); G/P (paritaprevir and
glecaprevir); Technivie (ombitasvir/paritaprevir/ritonavir) and
Epclusa (sofosbuvir/velpatasvir) and Zepatier (elbasvir and
grazoprevir).
[0187] If Compound 2 is administered to treat advanced hepatitis C
virus leading to liver cancer or cirrhosis, in one embodiment, the
compound can be administered in combination or alternation with
another drug that is typically used to treat hepatocellular
carcinoma (HCC), for example, as described by Andrew Zhu in "New
Agents on the Horizon in Hepatocellular Carcinoma" Therapeutic
Advances in Medical Oncology, V 5(1), January 2013, 41-50. Examples
of suitable compounds for combination therapy where the host has or
is at risk of HCC include anti-angiogenic agents, sunitinib,
brivanib, linifanib, ramucirumab, bevacizumab, cediranib,
pazopanib, TSU-68, lenvatinib, antibodies against EGFR, mTor
inhibitors, MEK inhibitors, and histone decetylace inhibitors.
EXAMPLES
General Methods
[0188] .sup.1H, .sup.19F and .sup.31P NMR spectra were recorded on
a 400 MHz Fourier transform Brucker spectrometer. Spectra were
obtained DMSO-d.sub.6 unless stated otherwise. The spin
multiplicities are indicated by the symbols s (singlet), d
(doublet), t (triplet), m (multiplet) and, br (broad).
[0189] Coupling constants (J) are reported in Hz. The reactions
were generally carried out under a dry nitrogen atmosphere using
Sigma-Aldrich anhydrous solvents. All common chemicals were
purchased from commercial sources.
[0190] The following abbreviations are used in the Examples: [0191]
AUC: Area under the Curve [0192] C.sub.24:Concentration of the drug
in plasma at 24 hours [0193] C.sub.24,ss: Concentration at 24 hours
after dosing at steady state [0194] C.sub.max: Maximum
concentration of the drug achieved in plasma [0195] DCM:
Dichloromethane [0196] EtOAc: Ethyl acetate [0197] EtOH: Ethanol
[0198] HPLC: High pressure liquid chromatography [0199] NaOH:
Sodium hydroxide [0200] Na.sub.2SO.sub.4: Sodium sulphate
(anhydrous) [0201] MeCN: Acetonitrile [0202] MeNH.sub.2:
Methylamine [0203] MeOH: Methanol [0204] Na.sub.2SO.sub.4: Sodium
sulfate [0205] NaHCO.sub.3: Sodium bicarbonate [0206] NH.sub.4Cl:
Ammonium chloride [0207] NH.sub.4OH: Ammonium hydroxide [0208] PE:
Petroleum ether [0209] Ph.sub.3P: Triphenylphosphine [0210] RH:
relative humidity [0211] Silica gel (230 to 400 mesh, Sorbent)
[0212] t-BuMgCl: t-Butyl magnesium chloride [0213] T.sub.max: Time
at which C.sub.max is achieved [0214] THF: Tetrahydrofuran (THF),
anhydrous [0215] TP: Triphosphate
Example 1. Synthesis of Compound 1
##STR00009##
[0216] Step 1: Synthesis of
(2R,3R,4R,5R)-5-(2-Amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-2-(hydro-
xymethyl)-4-methyltetrahydrofuran-3-ol (2-2)
[0217] A 50 L flask was charged with methanol (30 L) and stirred at
10.+-.5.degree. C. NH.sub.2CH.sub.3 (3.95 Kg) was slowly ventilated
into the reactor at 10.+-.5.degree. C. Compound 2-1 (3.77 kg) was
added in batches at 20.+-.5.degree. C. and stirred for 1 hour to
obtain a clear solution. The reaction was stirred for an additional
6-8 hours, at which point HPLC indicated that the intermediate was
less than 0.1% of the solution. The reactor was charged with solid
NaOH (254 g), stirred for 30 minutes and concentrated at
50.+-.5.degree. C. (vacuum degree: -0.095). The resulting residue
was charged with EtOH (40 L) and re-slurried for 1 hour at
60.degree. C. The mixture was then filtered through celite and the
filter cake was re-slurried with EtOH (15 L) for 1 hour at
60.degree. C. The filtrate was filtered once more, combined with
the filtrate from the previous filtration, and then concentrated at
50.+-.5.degree. C. (vacuum degree: -0.095). A large amount of solid
was precipitated. EtOAc (6 L) was added to the solid residue and
the mixture was concentrated at 50.+-.5.degree. C. (vacuum degree:
-0.095). DCM was then added to the residue and the mixture was
re-slurried at reflux for 1 hour, cooled to room temperature,
filtered, and dried at 50.+-.5.degree. C. in a vacuum oven to
afford compound 2-2 as an off-white solid (1.89 Kg, 95.3%, purity
of 99.2%).
[0218] Analytic Method for Compound 2-2: The purity of compound 2-2
(15 mg) was obtained using an Agilent 1100 HPLC system with a
Agilent Poroshell 120 EC-C18 4.6*150 mm 4-Micron column with the
following conditions: 1 mL/min flow rate, read at 254 nm,
30.degree. C. column temperature, 15 .mu.L injection volume, and a
31 minute run time. The sample was dissolved in acetonitrile-water
(20:80) (v/v). The gradient method is shown below.
TABLE-US-00001 Time (min) A % (0.05 TFA in water) B %
(Acetonitrile) 0 95 5 8 80 20 13 50 50 23 5 95 26 5 95 26.1 95 5 31
95 5
Step 2: Synthesis of
isopropyl((S)-(((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 (Compound 1)
[0219] Compound 2-2 and compound 2-3 (isopropyl
((perfluorophenoxy)(phenoxy)phosphoryl)-L-alaninate) were dissolved
in THE (1 L) and stirred under nitrogen. The suspension was then
cooled to a temperature below -5.degree. C. and a 1.7 M solution of
t-BuMgCl solution (384 mL) was slowly added over 1.5 hours while a
temperature of 5-10.degree. C. was maintained. A solution of
NH.sub.4Cl (2 L) and water (8 L) was added to the suspension at
room temperature followed by DCM. The mixture was stirred for 5
minutes before a 5% aqueous solution of K.sub.2CO.sub.3 (10 L) was
added and the mixture was stirred for 5 additional minutes before
filtering through diatomite (500 g). The diatomite was washed with
DCM and the filtrate was separated. The organic phase was washed
with a 5% aqueous K.sub.2CO.sub.3 solution (10 L.times.2), brine
(10 L.times.3), and dried over Na.sub.2SO.sub.4 (500 g) for
approximately 1 hour. Meanwhile, this entire process was repeated 7
times in parallel and the 8 batches were combined. The organic
phases were filtered and concentrated at 45.+-.5.degree. C. (vacuum
degree of 0.09 Mpa). EtOAc was added and the mixture was stirred
for 1 hour at 60.degree. C. and then at room temperature for 18
hours. The mixture was then filtered and washed with EtOAc (2 L) to
afford crude Compound 1. The crude material was dissolved in DCM
(12 L), heptane (18 L) was added at 10-20.degree. C., and the
mixture was allowed to stir for 30 minutes at this temperature. The
mixture was filtered, washed with heptane (5 L), and dried at
50.+-.5.degree. C. to afford pure Compound 1 (1650 g, 60%).
[0220] Analytic Method for Compound 1: The purity of Compound 1 (25
mg) was obtained using an Agilent 1100 HPLC system with a Waters
XTerra Phenyl 5 .mu.m 4.6*250 mm column with the following
conditions: 1 mL/min flow rate, read at 254 nm, 30.degree. C.
column temperature, 15 .mu.L injection volume, and a 25 minute run
time. The sample was dissolved in acetonitrile-water (50:50) (v/v).
The gradient method is shown below.
TABLE-US-00002 Time (min) A % (0.1% H.sub.3PO.sub.4 in water) B %
(Acetonitrile) 0 90 10 20 20 80 20.1 90 10 25 90 10
Example 2. Characterization of Amorphous and Crystalline Compound
1
[0221] Amorphous Compound 1 and crystalline Compound 1 were
initially analyzed by XRPD, .sup.1HNMR, and HPLC. The XRPD patterns
for both compounds are shown in FIG. 1A and the IPLC traces to
determine purity are shown in FIGS. 1B and 2A, respectively. Table
1 is a list of peaks from the XRPD of crystalline Compound 1 and
Table 2 is a list of relative retention times (RTT) from the IPLC
traces. Amorphous Compound 1 was 98.61% pure and crystalline
Compound 1 was 99.11% pure. Both compounds were a white solid. FIG.
2B is the TGA and DSC graphs of crystalline Compound 1. For
crystalline Compound 1, an endotherm was observed at 88.6.degree.
C. and there was a 7.8% mass loss from 80-110.degree. C.
[0222] A sample of Compound 1 was recrystallized from EtOAc/hexane
and drawn with ORTEP. The absolute structure of Compound 1 was
confirmed by the recrystallization of a single crystal. FIG. 3 is
the ORTEP drawing of Compound 1. Crystal data and measurement data
are shown in Table 3. The absolute stereochemistry of Compound 1
based on the X-ray crystallography is shown below:
##STR00010##
[0223] DSC data were collected on a TA Instruments Q2000 equipped
with a 50 position auto-sampler. The calibration for thermal
capacity was carried out using sapphire and the calibration for
energy and temperature was carried out using certified indium.
Typically approximately 3 mg of each sample, in a pin-holed
aluminum pan, was heated at 10.degree. C./min from 25.degree. C. to
200.degree. C. A purge of dry nitrogen at 50 ml/min was maintained
over the sample. The instrument control software was Advantage for
Q Series v2.8.0.394 and Thermal Advantage v5.5.3 and the data were
analyzed using Universal Analysis v4.5A.
[0224] TGA data were collected on a TA Instruments Q500 TGA,
equipped with a 16 position auto-sampler. The instrument was
temperature calibrated using certified Alumel and Nickel. Typically
5-10 mg of each sample was loaded onto a pre-tared aluminum DSC pan
and heated at 10.degree. C./min from ambient temperature to
350.degree. C. A nitrogen purge at 60 ml/min was maintained over
the sample. The instrument control software was Advantage for Q
Series v2.5.0.256 and Thermal Advantage v5.5.3 and the data were
analyzed using Universal Analysis v4.5.
[0225] Amorphous Compound 1 (1-1):
[0226] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.01-1.15
(m, 9H), 1.21 (d, J=7.20 Hz, 3H), 2.75-3.08 (m, 3H), 3.71-3.87 (m,
1H), 4.02-4.13 (m, 1H), 4.22-4.53 (m, 3H), 4.81 (s, 1H), 5.69-5.86
(m, 1H), 6.04 (br d, J=19.33 Hz, 4H), 7.12-7.27 (m, 3H), 7.27-7.44
(m, 3H), 7.81 (s, 1H)
[0227] Crystalline Compound 1 (1-2):
[0228] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.97-1.16
(m, 16H), 1.21 (d, J=7.07 Hz, 3H), 2.87 (br s, 3H), 3.08 (s, 2H),
3.79 (br d, J=7.07 Hz, 1H), 4.08 (br d, J=7.58 Hz, 1H), 4.17-4.55
(m, 3H), 4.81 (quin, J=6.25 Hz, 1H), 5.78 (br s, 1H), 5.91-6.15 (m,
4H), 7.10-7.26 (m, 3H), 7.26-7.44 (m, 3H), 7.81 (s, 1H)
TABLE-US-00003 TABLE 1 Peak list for crystalline Compound 1
Angle/.degree.2.theta. d spacing/.ANG. Intensity/Counts Intensity/%
6.03 14.64 1005 39.0 7.36 12.00 315 12.2 7.94 11.13 1724 66.9 9.34
9.47 2500 97.0 9.51 9.29 860 33.4 9.77 9.05 1591 61.8 11.08 7.98
2576 100.0 12.02 7.36 171 6.6 12.95 6.83 319 12.4 13.98 6.33 241
9.4 14.30 6.19 550 21.4 14.69 6.03 328 12.7 15.20 5.82 2176 84.5
15.94 5.56 1446 56.1 16.75 5.29 1009 39.2 17.29 5.13 700 27.2 17.72
5.00 1213 47.1 18.11 4.89 1565 60.8 18.46 4.80 302 11.7 18.89 4.69
385 14.9 19.63 4.52 636 24.7 20.37 4.36 1214 47.1 20.74 4.28 1198
46.5 21.24 4.18 640 24.8 22.31 3.98 961 37.3 22.88 3.88 806 31.3
23.43 3.79 355 13.8 24.08 3.69 573 22.2 24.49 3.63 159 6.2 25.00
3.56 351 13.6 25.36 3.51 293 11.4 26.09 3.41 235 9.1 26.26 3.39 301
11.7 26.83 3.32 696 27.0 27.35 3.26 436 16.9 27.46 3.25 363 14.1
28.07 3.18 200 7.8 28.30 3.15 195 7.6 28.82 3.10 599 23.3 29.85
2.99 217 8.4 30.26 2.95 186 7.2 30.75 2.91 333 12.9 31.12 2.87 149
5.8 31.85 2.81 238 9.2 33.28 2.69 261 10.1 34.77 2.58 171 6.6 35.18
2.55 175 6.8 36.83 2.44 327 12.7 37.41 2.40 172 6.7
TABLE-US-00004 TABLE 2 Relative Retention Times from HPLC
chromatographs of Amorphous Compound 1 and Crystalline Compound 1
Amorphous Compound 1 Crystalline Compound 1 RRT Area % RRT Area %
0.48 0.15 0.48 0.17 0.51 0.04 0.48 0.17 0.48 0.15 0.94 0.12 0.51
0.04 1.00 99.11 0.94 0.13 1.04 0.22 0.98 0.21 1.37 0.07 1.00 98.61
1.04 0.29 1.37 0.31
TABLE-US-00005 TABLE 3 Crystal and Data Measurement of Compound 1
Bond Precision C-C = 0.0297 A, Wavelength = 1.54184 Cell a =
10.1884(3) b = 28.6482(9) c = 12.9497(5) alpha = 90 beta =
113.184(4) gamma = 90 Temperature 150 K. Calculated Reported Volume
3474.5(2) 3474.5(2) Space Group P21 P 1 211 Hall Group P 2yb P 2yb
Moiety Formula C24 H34 F N7 O7 P 2(C24 H34 F N7 O7 P) Sum Formula
C24 H34 F N7 O7 P C48 H68 F2 N14 O14 P2 Mr 582.55 1165.10 Dx, g
cm.sup.-1 1.114 1.114 Z 4 2 Mu (mm.sup.-1) 1.139 1.139 F000 1228.0
1228.0 F000` 1233.21 h, k, l.sub.max 12, 34, 15 12, 34, 15
N.sub.ref 12742 [ 6510] 8259 T.sub.min, T.sub.max 0.790, 0.815
0.808, 1.000 T.sub.min` 0.716 Correction Method # Reported T
Limits: T.sub.min = 0.808 T.sub.max = 1.00 AbsCorr MULTI-SCAN Data
completeness 1.27/0.65 Theta (max) 68.244 R (reflections) 0.2091 (
7995) wR2 (reflections) 0.5338 ( 8259) S 2.875 Npar 716
[0229] This initial characterization was followed by storage at
25.degree. C./60% relative humidity (RH) for 14 days with analysis
by IPLC and XRPD after 7 and 14 days. FIG. 4A is the XRPD after 14
days at 25.degree. C./60% (RH). Amorphous Compound 1 (sample 1-1)
remained poorly crystalline, whereas crystalline Compound 1 (sample
1-2) retained its crystallinity, but both compounds were stable
after 14 days at 25.degree. C./60% (RH).
Example 3. Formation of Oxalate Salt Compound 4
[0230] Initially, the oxalate salt of Compound 1, Compound 4, was
formed by mixing the oxalic salt with solvent (5 vol, 100 .mu.L)
and allowing any solution to evaporate at room temperature. Any
suspension was matured (room temperature--50.degree. C.) for 3
hours and crystallinity was accessed.
##STR00011##
[0231] Table 4 shows the different solvents used in the production
of Compound 4. All solvents except for two (cyclohexane and
n-heptane) afforded crystalline products. Despite the high
crystallinity and solubility of Compound 4, oxalate salts are not
acceptable for clinical development due to the potential formation
of kidney stones and other salts of compound 1 were explored.
TABLE-US-00006 TABLE 4 Formation of Oxalate Compound 4 Observation
post acid addition at room Observation after Solvent temperature
maturation/evaporation EtOH Solution OXA-Form 1 IPA Solution
OXA-Form 1 Acetone Solution OXA-Form 1 MEK Solution OXA-Form 1
EtOAc Suspension OXA-Form 1 iPrOAc Suspension OXA-Form 1 THF
Solution OXA-Form 1 Toluene Solution OXA-Form 1 MeCN Solution
OXA-Form 1 IPA: 10% water Solution OXA-Form 1 TBME Suspension
OXA-Form 1 Cyclohexane Suspension Amorphous n-Heptane Suspension
Amorphous
Example 4. Salt Compounds of Amorphous Compound 1
[0232] Since the oxalate salt compound 4 (Example 3) could not be
carried forward in clinical trials due to its potential to form
kidney stones, amorphous salts of Compound were formed with the
counter ions listed in Table 5. Compound 1 was dissolved in
t-butanol (20 vol, 6 ml) and the solution was treated with the acid
counter-ions (1 equivalent for each sample except sample 1-9 which
had 0.5 equivalent of sulfate). The samples were then frozen with
the solvent removed by lyophilization. The residual solid in
samples 1-4, 1-5, 1-6, 1-7, 1-9 was initially analyzed by XRPD and
HPLC.
TABLE-US-00007 TABLE 5 Amorphous salt formation details Sample
Sample Stock solution ID details details Observation NMR 1-4 HCl
THF 1M White solid 3 fewer protons~0.3 (1:1) eq t-BuOH 1-5 Sulfuric
THF 1M White solid 3 fewer protons~0.3 (1:1) eq t-BuOH 1-6 Fumaric
MeOH:THF Glassy solid 1.05 eq fumaric acid (1:1) (1:1) 0.5M 0.84 eq
t-BuOH 1-7 Benzoic THF White solid 1.0 eq benzoic acid (1:1) 1M
0.34 eq t-BuOH 1-8 Succinic MeOH Sticky white ~1.1 eq succinic acid
(1:1) 1M solid 0.37 eq t-BuOH 1-9 Sulfuric THF White solid 3 fewer
protons~0.3 (0.5:1 1M eq t-BuOH acid:API)
[0233] .sup.1HNMR spectrum were taken for all samples.
[0234] Sample 1-4, HCl (1:1) salt:
[0235] .sup.1HNMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.93-1.39 (m,
16H), 2.97 (br s, 2H), 3.70-3.88 (in, 1H), 4.10 (br s, 1H),
4.18-4.49 (in, 3H), 4.70-4.88 (in, 1H), 5.71-5.94 (m, 1H), 6.07 (br
d, J=19.07 Hz, 2H), 7.14-7.27 (m, 3H), 7.29-7.44 (m, 2H), 7.83-8.19
(m, 1H)
[0236] Sample 1-5, Sulfuric (1:1) salt:
[0237] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.97-1.38
(m, 15H), 2.96 (br s, 2H), 4.06-4.18 (m, 1H), 4.19-4.49 (m, 3H),
4.66-4.91 (m, 1H), 5.70-5.95 (m, 1H), 5.96-6.16 (m, 2H), 7.10-7.27
(m, 3H), 7.30-7.43 (m, 2H), 7.88-8.19 (m, 1H)
[0238] Sample 1-6, Fumaric (1:1) salt:
[0239] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.95-1.31
(m, 21H), 2.87 (br s, 3H), 3.79 (br d, J=7.20 Hz, 1H), 4.01-4.13
(m, 1H), 4.16-4.23 (m, 1H), 4.16-4.24 (m, 1H), 4.20 (s, 1H),
4.18-4.23 (m, 1H), 4.24-4.52 (m, 1H), 4.24-4.52 (m, 1H), 4.24-4.49
(m, 1H), 4.72-4.88 (m, 1H), 5.68-5.86 (m, 1H), 6.04 (br d, J=19.33
Hz, 4H), 6.63 (s, 1H), 6.61-6.66 (m, 1H), 7.12-7.27 (m, 3H),
7.27-7.45 (m, 3H), 7.81 (s, 1H), 13.16 (br s, 2H)
[0240] Sample 1-7, Benzoic (1:1) salt:
[0241] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.96-1.30
(m, 15H), 2.87 (br s, 3H), 3.79 (br d, J=7.07 Hz, 1H), 4.07 (br s,
1H), 4.20 (s, 1H), 4.25-4.52 (m, 3H), 4.81 (s, 1H), 5.71-5.85 (m,
1H), 6.04 (br d, J=19.33 Hz, 4H), 7.08-7.27 (m, 3H), 7.27-7.43 (m,
3H), 7.45-7.57 (m, 2H), 7.63 (s, 1H), 7.81 (s, 1H), 7.95 (dd,
J=8.27, 1.33 Hz, 2H), 12.98 (br s, 1H)
[0242] Sample 1-8, Succinic (1:1) salt:
[0243] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.98-1.28
(m, 15H), 2.42 (s, 5H), 2.87 (br s, 3H), 3.57-3.62 (m, 1H),
3.70-3.86 (m, 1H), 4.02-4.14 (m, 1H), 4.20 (s, 1H), 4.24-4.51 (m,
3H), 4.70-4.88 (m, 1H), 5.69-5.86 (m, 1H), 6.04 (br d, J=19.33 Hz,
4H), 7.12-7.27 (m, 3H), 7.27-7.44 (m, 3H), 7.81 (s, 1H),
11.95-12.58 (m, 2H)
[0244] Sample 1-9, Sulfuric (0.5:1) salt:
[0245] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.02-1.31
(m, 15H), 2.94 (br s, 3H), 3.79 (br d, J=7.20 Hz, 2H), 4.09 (br s,
1H), 4.22-4.48 (m, 3H), 4.72-4.90 (m, 1H), 5.71-5.92 (m, 1H), 6.07
(br d, J=19.07 Hz, 2H), 7.12-7.28 (m, 3H), 7.31-7.44 (m, 2H),
7.75-8.19 (m, 1H).
[0246] The samples were then subjected to storage at 25.degree.
C./60% relative humidity (RH) for 14 days with analysis by HPLC and
XRPD after 7 (FIG. 4B) and 14 days (FIG. 5A). All prepared salts
remained amorphous and the observations are shown in Table 6. The
mono sulfate (sample 1-5) and succinate salts (sample 1-8) were
found to be physically unstable and deliquesced or became a gum
during the course of the study. Both the fumarate (sample 1-6) and
benzoate salts (sample 1-7) were found to be glassy solids. The HCl
salt (sample 1-4) was found to retain its physical appearance.
Surprisingly, the hemi-sulfate salt (sample 1-9) also retained its
physical appearance as a white solid in contrast to mono-sulfate
compound (sample 1-5), which was a sticky gum. Results are shown in
Table 6. The mono HCl salt (sample 1-4) and the hemi-sulfate salt
(sample 1-9) were found to be physically and chemically stable
after 2 weeks storage at 25.degree. C./60% relative humidity (RH).
Although both salts were stable over the two weeks, the
hemi-sulfate salt was superior to the HCl salt because the HCl salt
was hygroscopic, rendering it less useful compared to the
hemi-sulfate salt for long-term storage or use.
TABLE-US-00008 TABLE 6 Stability of samples after 7 and 14 days at
25.degree. C./60% RH Time exposed to 25.degree. C./60% RH (days)
Sample 0 7 14 ID HPLC Observation HPLC Observation HPLC Observation
1-1 98.6 White solid 98.7 White solid 98.5 White solid 1-2 99.1
White solid 99.2 White solid 99.0 White solid 1-3 99.7 White solid
99.6 White solid 99.4 White solid 1-4 98.7 White solid 98.8 White
solid 98.6 White solid 1-5 98.4 White solid 55.7 Sticky white --
Sticky gum solid 1-6 98.7 Glassy solid 98.6 Clear glassy 98.4 White
glassy solid solid 1-7 98.8 White solid 98.8 Clear glassy 98.7
Clear glassy solid solid 1-8 98.7 Sticky white -- Deliquesced/ --
Deliquesced solid sticky oil 1-9 98.7 White solid 98.1 White solid
96.4 White solid
Example 5. Characterization of Amorphous Compound 2
[0247] Amorphous Compound 2 was initially analyzed by XRPD,
.sup.1HNMR, DSC, TGA, and HPLC. The XRPD pattern for amorphous
Compound 2 overlaid with amorphous Compound 1 and crystalline
Compound 1 is shown in FIG. 1A and the XRPD pattern of amorphous
Compound 2 alone is shown in FIG. 5B. Table 7 is a peak list from
the XRPD pattern shown in FIG. 5B. The HPLC trace to determine
purity is shown in FIG. 6A. Table 8 is a list of relative retention
times (RTT) from the HPLC trace shown in FIG. 6A. Amorphous
Compound 2 was 99.6800 pure. FIG. 6B is a TGA and DSC graph of
amorphous Compound 2. Experimental details for the TGA and DSC
experiments are given in Example 2.
TABLE-US-00009 TABLE 7 Peak list for Amorphous Compound 2
Angle/.degree.2.theta. d spacing/.ANG. Intensity/Counts Intensity/%
4.20 21.03 486 81.8 4.67 18.91 482 81.0 5.16 17.10 595 100.0 9.13
9.68 547 92.0
TABLE-US-00010 TABLE 8 HPLC chromatogram of Amorphous Compound 2
Amorphous Compound 2 RRT Area % 0.48 0.02 0.48 0.02 0.67 0.01 0.94
0.13 1.00 99.68 1.04 0.06
[0248] Amorphous Compound 2:
[0249] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.93-1.29
(m, 13H), 2.94 (br s, 3H), 3.79 (td, J=10.04, 7.07 Hz, 2H),
4.05-4.19 (m, 1H), 4.19-4.50 (m, 3H), 4.81 (quin, J=6.25 Hz, 1H),
5.71-5.94 (m, 1H), 5.97-6.16 (m, 2H), 7.14-7.28 (m, 3H), 7.31-7.44
(m, 2H), 7.82-8.09 (m, 1H)
Example 6. Crystallization of Amorphous Compound 2
[0250] Since the hemi-sulfate salt was found to remain as a solid
after the 14 day stability study as shown in Table 6, preliminary
tests studying crystallization conditions using 11 different
solvents was conducted. Amorphous Compound 2 was suspended in 5
volumes of solvent at 25.degree. C. (sample 2-1, 2-2, 2-3, 2-4,
2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and 2-11). To those samples that
were not free flowing (2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, and
2-10), an additional 5 volumes of solvent was added. The samples
were then matured at 25-50.degree. C. (1.degree. C./min between
temperatures and 4 hour at each temperature) for 6 days except for
sample 2-1, which was observed to be a clear solution after 1 day
and was allowed to evaporate under ambient conditions. The results
are shown in Table 9. Crystalline patterns resulted from
crystallization with isobutanol (sample 2-1), acetone (sample 2-2),
EtOAc (sample 2-6), and iPrOAc (sample 2-7). Two poorly crystalline
samples were also identified from crystallization with MEK (sample
2-4) and MIBK (sample 2-5). The XRPD patterns are shown in FIG.
7A.
TABLE-US-00011 TABLE 9 Crystallization Conditions of Compound 2
Observation Observation Observation Sample after 5 after 10 after 1
day ID Solvent volumes volumes maturation XRPD 2-1 IPA Solid-not
Free flowing Solution, Gum free flowing suspension evaporated at RT
yielding a gum 2-2 Iso- Solid-not Free flowing Suspension
Crystalline- butanol free flowing suspension Pattern 2 2-3 Acetone
Solid-not Free flowing Suspension Crystalline- free flowing
suspension Pattern 3 2-4 MEK Solid-not Free flowing Suspension
Poorly free flowing suspension crystalline- Pattern 4 2-5 MIBK
Solid-not Free flowing Suspension Poorly free flowing suspension
crystalline- Pattern 4 2-6 EtOAc Solid-not Free flowing Suspension
Crystalline- free flowing suspension Pattern 1 2-7 iPrOAc Solid-not
Free flowing Suspension Crystalline- free flowing suspension
Pattern 1 2-8 THF Solid-not Free flowing Suspension Poorly free
flowing suspension crystalline 2-9 TBME Free flowing -- Suspension
Amorphous suspension 2-10 Toluene Solid-not Free flowing Suspension
Amorphous free flowing suspension 2-11 Heptane Free flowing --
Suspension Amorphous suspension
[0251] The seven samples (Samples 2-2, 2-3, 2-4, 2-5, 2-6, 2-7 and
2-8) were analyzed by DSC, TGA, .sup.1H-NMR and IC (Table 10, FIG.
8A, FIG. 8B, FIG. 9A, FIG. 9B, FIG. 10A, FIG. 10B, FIG. 11A, and
FIG. 11B) as well as by XRPD following 6 days storage at 25.degree.
C./60% relative humidity (RH) (all samples remained
crystalline/poorly crystalline following stability). All samples
retained roughly half an equivalent of sulfate, but contained a
relatively large amount of residual solvent. An overlay of the
X-ray diffractograms of amorphous samples 2-9, 2-10, and 2-11 is
shown in FIG. 7B.
TABLE-US-00012 TABLE 10 Characterization of crystalline Compound 2
samples IC Sample (corrected ID Solvent DSC TGA .sup.1HNMR for TGA)
2-2 Iso- Endo 113.8.degree. C. 8.3% 1.1 eq 0.45 eq butanol ambient-
iso- 140.degree. C. butanol 2-3 Acetone Endo 30-95.degree. C. 7.6%
0.5 eq 0.46 eq Endo 100-145.degree. C. ambient- acetone 140.degree.
C. 2-4 MEK Broad complex 8.5% 0.8 eq 0.45 eq endo 30-115.degree. C.
ambient- MEK Endo 115-145.degree. C. 140.degree. C. 2-5 MIBK Broad
endo 5.2% 0.2 eq 0.46 eq 30-105.degree. C. ambient- MIBK Endo
114.7.degree. C. 110.degree. C. 2-6 EtOAc Sharp endo 2.0% 0.9 eq
0.46 eq 113.6.degree. C. ambient- EtOAc 100.degree. C. 2-7 iPrOAc
Endo 30-90.degree. C. 1.6% 0.8 eq 0.45 eq ambient- iPrOAc
90.degree. C. 2-8 THF Endo 30-100.degree. C. 4.2% 0.7 eq 0.45 eq
Sharper endo ambient- THF 115.6.degree. C. 130.degree. C.
[0252] .sup.1HNMR spectrum were taken for all samples and listed
below.
[0253] Sample 2-2:
[0254] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.83 (d,
J=6.69 Hz, 7H), 0.99-1.26 (in, 14H), 1.61 (dt, J=13.26, 6.63 Hz,
1H), 3.73-3.87 (m, 2H), 4.03-4.18 (m, 1H), 4.18-4.51 (n, 4H),
4.66-4.92 (n, 1H), 4.70-4.90 (n, 1H), 4.72-4.88 (n, 1H), 5.81 (br
s, 1H), 5.93-6.11 (n, 2H), 7.10-7.26 (n, 3H), 7.14-7.26 (n, 1H),
7.30-7.41 (i, 2H), 7.94 (br s, 1H)
[0255] Sample 2-3:
[0256] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.00-1.26
(m, 13H), 2.09 (s, 3H), 3.74-3.87 (m, 2H), 4.10 (br d, J=7.70 Hz,
1H), 4.22-4.50 (m, 3H), 4.81 (quin, J=6.28 Hz, 1H), 5.71-5.90 (m,
1H), 5.96-6.15 (m, 2H), 7.12-7.26 (m, 3H), 7.31-7.41 (m, 2H),
7.79-8.07 (m, 1H)
[0257] Sample 2-4:
[0258] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.91 (t,
J=7.33 Hz, 3H), 1.01-1.28 (m, 13H), 2.08 (s, 2H), 3.72-3.89 (m,
2H), 4.10 (br d, J=8.08 Hz, 1H), 4.23-4.47 (m, 3H), 4.81 (quin,
J=6.25 Hz, 1H), 5.69-5.89 (m, 1H), 5.94-6.13 (m, 2H), 7.14-7.25 (m,
3H), 7.32-7.41 (m, 2H), 7.79-8.11 (m, 1H)
[0259] Sample 2-5:
[0260] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.86 (d,
J=6.69 Hz, 1H), 0.98-1.33 (m, 13H), 2.02-2.09 (m, 1H), 4.03-4.17
(m, 1H), 4.22-4.50 (m, 3H), 4.81 (quin, J=6.25 Hz, 1H), 5.81 (br s,
1H), 5.93-6.15 (m, 2H), 7.11-7.27 (m, 3H), 7.31-7.41 (m, 2H),
7.77-8.21 (m, 1H)
[0261] Sample 2-6:
[0262] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.98-1.28
(m, 15H), 2.00 (s, 3H), 3.99-4.14 (m, 3H), 4.21-4.49 (m, 3H), 4.81
(quin, J=6.22 Hz, 1H), 5.82 (br s, 1H), 5.93-6.14 (m, 2H),
7.11-7.26 (m, 3H), 7.29-7.42 (m, 2H), 7.79-8.17 (m, 1H)
[0263] Sample 2-7:
[0264] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.92-1.28
(m, 17H), 1.97 (s, 2H), 4.04-4.16 (m, 1H), 4.20-4.51 (m, 3H),
4.71-4.93 (m, 2H), 5.82 (br s, 1H), 5.95-6.14 (m, 2H), 7.11-7.28
(m, 3H), 7.31-7.43 (m, 2H), 7.75-8.21 (m, 1H)
[0265] Sample 2-8:
[0266] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.81-1.11
(m, 13H), 1.19 (s, 1H), 1.53-1.66 (m, 1H), 3.87-4.01 (m, 1H),
4.06-4.32 (m, 3H), 4.64 (quin, J=6.25 Hz, 1H), 5.55-5.75 (m, 1H),
5.77-5.97 (m, 2H), 6.94-7.10 (m, 3H), 7.13-7.26 (m, 2H), 7.66-7.96
(m, 1H)
Example 7. Failure to Crystallize Amorphous Malonate Salt (Compound
4)
[0267] As shown in Example 3, a crystalline oxalate salt was
identified when determining appropriate salts for Compound 1, but
oxalate salt Compound 4 could not be carried forward in clinical
trials due to its potential for causing kidney stones. Therefore,
crystallization of the chemically related malonate salt (Compound
5) was attempted using the same 11 solvents as for the hemi-sulfate
salt. Compound 1 (12.times.50 mg, samples 3-1, 3-2, 3-3, 3-4, 3-5,
3-6, 3-7, 3-8, 3-9, 3-10, 3-11, and 3-12) was dissolved in
t-butanol (20 vol) and the solutions were then treated with 1
equivalence of a malonic acid stock solution (1 M in THF). The
samples were then frozen with the solvent removed by
lyophilisation. To samples 3-1, 3-2, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8,
3-9, 3-10, and 3-11, relevant solvent (5 volumes) was added at room
temperature. Any resulting solutions were allowed to evaporate
under ambient conditions, while gums or solids were matured at
25-50.degree. C. (1.degree. C./min between temperatures and 4 hour
at each temperature) for 5 days. The solids were analyzed by XRPD
(FIG. 12B), but all samples were found to either form a gum or were
amorphous (FIG. 12B). Results are shown in Table 11. The one solid
(amorphous) sample (3-12) was analyzed by .sup.1H-NMR and HPLC, and
was found to contain around 1 equivalence of malonic acid (peaks
overlap) as well as 0.6 eq. t-BuOH. The compound was 99.2% pure
(FIG. 13A). FIG. 12A is an XRDP of sample 3-12 and FIG. 13A is the
HPLC chromatograph of sample 3-12.
[0268] Sample 3-12:
[0269] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.81-1.11
(m, 13H), 1.19 (s, 1H), 1.53-1.66 (m, 1H), 3.87-4.01 (m, 1H),
4.06-4.32 (m, 3H), 4.64 (quin, J=6.25 Hz, 1H), 5.55-5.75 (m, 1H),
5.77-5.97 (m, 2H), 6.94-7.10 (m, 3H), 7.13-7.26 (m, 2H), 7.66-7.96
(m, 1H)
TABLE-US-00013 TABLE 11 Crystallization Conditions of Amorphous
Malonate Salt Compound 4 Observation after 5 Observation days
Sample after 5 maturation/ ID Solvent volumes evaporation XRPD 3-1
IPA Clear solution* Clear gum -- 3-2 Isobutanol Clear solution*
Clear gum -- 3-3 Acetone Clear solution* Clear gum -- 3-4 MEK Clear
solution* Clear gum -- 3-5 MIBK Solution & Clear gum -- some
gum 3-6 EtOAc Clear solution* Clear gum A- & crystal- morphous
like appearance 3-7 iPrOAc Gum Clear gum -- 3-8 THF Clear solution*
Clear gum -- 3-9 TBME Thick Clear gum -- suspension 3-10 Toluene
White gum/ White gum A- solid morphous 3-11 Heptane White solid
White gum A- (static) morphous 3-12 -- (White solid- (Sticky white
A- no solvent) solid-ambient morphous conditions) *Evaporated at
room temperature
Example 8. Failure of Adequate Salt Formation Using Liquid Assisted
Grinding (LAG)
[0270] A liquid assisted grinding (LAG) study to determine
appropriate salts other than hemi-sulfate was performed using the
14 acidic counter ions in Table 12.
TABLE-US-00014 TABLE 12 Counter-ion stock solutions used in LAG
Crystallization Counter-ion Solvent (1M) Pamoic DMSO Malonic THF
D-Glucuronic Water DL-Mandelic THF D-Gluconic THF Glycolic THF
L-Lactic THF Oleic THF L-Ascorbic Water Adipic THF (heat) Caproic
THF Stearic THF Palmitic THF Methanesulfonic THF
[0271] Compound 1 (30 mg) was placed in HPLC vials with two 3 mm
ball bearings. The materials were wetted with solvent (15 .mu.l
ethanol, sample 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10,
4-11, 4-12, 4-13, and 4-14) and 1 equivalence of the acid
counter-ion was added. The samples were then ground for 2 hours at
650 rpm using a Fritsch milling system with an Automaxion adapter.
Most of the samples after grinding were found to be clear gums and
were not analyzed further (Table 13). Those that were observed to
contain solid were analyzed by XRPD and, in all cases, the patterns
obtained were found to match those of the crystalline acid counter
ion with no additional peaks (FIG. 13B).
TABLE-US-00015 TABLE 13 Observations and XRPD Results from LAG of
Compounds 1 Sample Observation ID Acid after grinding XRPD 4-1
Pamoic Yellow gum/solid Pamoic acid & amorphous halo 4-2
Malonic Clear gum -- 4-3 D-Glucuronic White gum/solid D-Glucuronic
acid & amorphous halo 4-4 DL-Mandelic Clear gum -- 4-5
D-Gluconic Clear gum -- 4-6 Glycolic Clear gum 4-7 L-Lactic Clear
gum -- 4-8 Oleic Clear gum -- 4-9 L-Ascorbic White gum/solid
L-Ascorbic acid & amorphous halo 4-10 Adipic Clear gum -- 4-11
Caproic Clear gum -- 4-12 Stearic White gum/solid Stearic acid
& amorphous halo 4-13 Palmitic White gum/solid Palmitic acid
& amorphous halo 4-4 Methanesulfonic Clear gum --
Example 9. Failure to Obtain Adequate Salt Formation using Methyl
Ethyl Ketone (MEK)
[0272] Methyl ethyl ketone (MEK) was next utilized as a solvent to
study appropriate salts other than the hemi-sulfate salt. Using the
14 acidic counter ions in Table 12, the study was performed by
dissolving Compound 1 (50 mg) in MEK (20 vol) at room temperature.
The solutions were treated with 1 equivalence of the selected
counter-ions (Table 12). The samples were then cooled down to
5.degree. C. at 0.1.degree. C./min and stirred at this temperature
overnight. All samples were allowed to evaporate under ambient
conditions and any solids observed were analyzed by XRPD. This
evaporation mainly produced gums, with the exception of the samples
with steric acid (sample 4-12) and palmitic acid (sample 5-13),
which afforded glassy solvents. These solids were amorphous by
XRPD, but no crystalline forms of the salt were obtained. Results
are shown in Table 14. (FIG. 13A).
TABLE-US-00016 TABLE 14 Results from dissolving Compound 1 in MEK
(20 volumes) Solvent Observation Observation Sample for acid upon
acid Observation upon ID Acid at 1M addition upon cooling
evaporation 5-1 Pamoic DMSO Yellow Yellow Yellow gum solution
solution 5-2 Malonic THF Solution Solution Clear gum 5-3 D- Water
Solution Solution Clear gum Glucuronic 5-4 DL- THF Solution
Solution Clear gum Mandelic 5-5 D- THF White Turbid Clear gum
Gluconic precipitate solution 5-6 Glycolic THF Solution Solution
Clear gum 5-7 L-Lactic THF Solution Solution Clear gum 5-8 Oleic
THF Solution Solution Clear gum 5-9 L-Ascorbic Water Solution
Solution Yellow gum 5-10 Adipic THF Solution Solution Clear gum
(heat) 5-11 Caproic THF Solution Solution Clear gum 5-12 Stearic
THF Solution Turbid Clear glassy solution solid* 5-13 Palmitic THF
Solution Solution Clear glassy solid* 5-14 Methane- THF Solution
Solution Clear gum sulfonic Stock solution prepared prior to acid
addition *Samples were analyzed by XRPD and gave amorphous patterns
plus peaks from the acid counter ion
[0273] Since all samples were amorphous, all samples were
redissolved in MEK (5 vol) and cyclohexane was added (20 vol
antisolvent) at room temperature followed by 1 hour of stirring at
25'C. The samples were then matured between 50-5.degree. C.
(1.degree. C./min between temperatures, 4 hours at each
temperature) for 2 days before the cycle was changed to
50-25.degree. C. for a further 4 days. The samples were observed by
eye following maturation. Results are shown in Table 15. Following
the maturation, all samples except 5-1 (with pamoic acid) were
found to begums. Sample 5-1, a yellow solid, was analyzed by XRPD,
and the pattern was found to match the known form of pamoic acid
(FIG. 14B),and therefore no crystalline forms of the salt were
obtained.
TABLE-US-00017 TABLE 15 Results from redissolving Compound 1 in MEK
(5 volumes) and antisolvent Observation Observation Observation
Sample Immediate after 10 after 60 after ID Observation minutes
minutes Maturation 5-1 Precipitate Gum Gum Yellow suspension** 5-2
Precipitate Gum Gum Gum 5-3 Precipitate/ Gum Gum Gum gum 5-4
Precipitate Gum Gum Gum 5-5 Precipitate/ Gum Gum Gum gum 5-6
Precipitate Gum Gum Gum 5-7 Precipitate Gum Gum Gum 5-8 Precipitate
Light Gum Gum suspension 5-9 Precipitate Gum Gum Gum 5-10
Precipitate Gum Gum Gum 5-11 Precipitate Light Gum Gum suspension
5-12 Precipitate Light Gum Gum suspension 5-13 Precipitate Light
Gum Gum suspension 5-14 Precipitate Gum Gum Gum **Sample analyzed
by XRPD with pattern matching known form of pamoic acid (no
additional peaks
Example 10. Failure to Obtain Adequate Salt Formation using Ethyl
Acetate
[0274] Ethyl acetate was next utilized to study appropriate salts
other than hemi-sulfate salt. Utilizing the 14 acidic counter ions
in Table 12, the study was performed by dissolving Compound 1 (50
mg) in ethyl acetate (20 vol) at 50'C. The solutions were treated
with 1 equivalent of the selected counter-ions (Table 12). The
samples were then cooled down to 5.degree. C. at 0.1.degree. C./min
and stirred at this temperature for 4 days. The solutions were
allowed to evaporate under ambient conditions while any solids were
analyzed by XRPD. The results from the crystallizations using ethyl
acetate are in Table 16. In contrast to Example 8 where MEK was the
solvent, the majority of samples were observed to be suspensions
following cooling of the acid:compound mixture (those that were
solutions were allowed to evaporate under ambient conditions).
However, the XRPD diffractograms were generally found to match
crystalline Compound 1. Samples 6-2, 6-4, and 6-5 have some slight
differences (FIG. 14A and FIG. 15A). No crystalline forms of the
salt were obtained.
TABLE-US-00018 TABLE 16 Results from dissolving Compound 1 in EtOAc
(20 volumes) Solvent for Observation Observation Observation Sample
acid at upon acid upon upon ID Acid 1 M addition Cooling XRPD
Evaporation 6-1 Pamoic DMSO Yellow Yellow -- Gum solution solution*
6-2 Malonic THF Solution White Slight -- suspension differences to
freebase 6-3 D-Glucuronic Water Solution Solution* -- Gum 6-4
DL-Mandelic THF Solution White Slight -- suspension differences to
freebase 6-5 D-Gluconic THF White Possible Slight -- precipitate
white gum differences to freebase 6-6 Glycolic THF Solution White
Freebase -- suspension 6-7 L-Lactic THF Solution White Freebase --
suspension 6-8 Oleic THF Solution White Freebase -- suspension 6-9
L-Ascorbic Water Solution Solution* -- White solid on side/ yellow
gum - amorphous 6-10 Adipic THF Solution White Freebase -- (heat)
suspension 6-11 Caproic THF Solution White Freebase -- suspension
6-12 Stearic THF Solution White Freebase -- suspension 6-13
Palmitic THF Solution White Freebase -- suspension 6-14
Methanesulfonic THF White Solution/ -- Clear gum precipitate clear
gum*
Example 11. Chemical Purity Determination by HPLC
[0275] Purity analysis in Example 2 and Example 4 was performed on
an Agilent HP1100 series system equipped with a diode array
detector and using ChemStation software vB.04.03 using the method
shown in Table 17.
TABLE-US-00019 TABLE 17 HPLC method for chemical purity
determinations Parameter Value Type of method Reverse phase with
gradient elution Sample Preparation 0.5 mg/ml in acetonitrile:water
1:1 Column Supelco Ascentis Express C18, 100 .times. 4.6 mm, 2.7
.mu.m Column Temperature (.degree. C.) 25 Injection (.quadrature.l)
5 Wavelength, Bandwidth (nm) 255, 90 Flow Rate (ml/min) 2 Phase A
0.1% TFA in water Phase B 0.085% TFA in acetonitrile Timetable Time
(min) % Phase A % Phase B 0 95 5 6 5 95 6.2 95 5 8 95 5
Example 12. X-Ray Powder Diffraction (XRPD) Techniques
[0276] The XRPD patterns in Examples 2, 3, 4, 5, 6, 7, 8, and 9
were collected on a PANalytical Empyrean diffractometer using Cu K
Q radiation (45 kV, 40 mA) in transmission geometry. A 0.5.degree.
slit, 4 mm mask and 0.4 rad Soller slits with a focusing mirror
were used on the incident beam. A PIXcel.sup.3D detector, placed on
the diffracted beam, was fitted with a receiving slit and 0.04 rad
Soller slits. The instrument is performance checked using silicon
powder on a weekly basis. The software used for data collection was
X'Pert Data Collector v. 5.3 and the data were analyzed and
presented using Diffrac Plus EVA v. 15.0.0.0 or Highscore Plus v.
4.5. Samples were prepared and analyzed in either a metal or
Millipore 96 well-plate in transmission mode. X-ray transparent
film was used between the metal sheets on the metal well-plate and
powders (approximately 1-2 mg) were used as received. The Millipore
plate was used to isolate and analyze solids from suspensions by
adding a small amount of suspension directly to the plate before
filtration under a light vacuum.
[0277] The scan mode for the metal plate used the gonio scan axis,
whereas a 20 scan was utilized for the Millipore plate. A
performance check was carried out using silicon powder (metal
well-plate). The details of the data collection were an angular
range of 2.5 to 32.0.degree. 2.theta., a step size of 0.0130
2.theta., and a total collection time of 2.07 minutes.
[0278] Samples were also collected on a Bruker D8 diffractometer
using Cu K.quadrature. radiation (40 kV, 40 mA), .theta.-2.theta.
goniometer, and divergence of V4 and receiving slits, a Ge
monochromator and a Lynxeye detector. The instrument is performance
checked using a certified Corundum standard (NIST 1976). The
software used for data collection was DiffracPlus XRD Commander
v2.6.1 and the data were analyzed and presented using Diffrac Plus
EVA v15.0.0.0.
[0279] Samples were run under ambient conditions as flat plate
specimens using powder as received. The sample was gently packed
into a cavity cut into polished, zero-background (510) silicon
wafer. The sample was rotated in its own plane during analysis. The
details of the data collection were an angular range of 2 to 420
2.theta., a step size of 0.05.degree. 2.theta., and collection time
of 0.5 s/step.
Example 13. Synthesis of Amorphous Compound 2
##STR00012##
[0281] A 250 mL flask was charged with MeOH (151 mL) and the
solution was cooled to 0-5.degree. C. A concentrated solution of
H.sub.2SO.sub.4 was added dropwise over 10 minutes. A separate
flask was charged with Compound 1 (151 g) and acetone (910 mL), and
the H.sub.2SO.sub.4/MeOH solution was added dropwise at
25-30.degree. C. over 2.5 hours. A large amount of solid was
precipitated. After the solution was stirred for 12-15 hours at
25-30.degree. C., the mixture was filtered, washed with
MeOH/acetone (25 mL/150 mL), and dried at 55-60.degree. C. in
vacuum to afford Compound 2 (121 g, 74%).
[0282] Analytic Method for Compound 2: The purity of Compound 2 was
obtained using an Agilent 1100 HPLC system with a Waters XTerra
Phenyl 5 .mu.m 4.6*250 mm column with the following conditions: 1
mL/min flow rate, read at 254 nm, 30.degree. C. column temperature,
10 L injection volume, and a 30 minute run time. The sample was
dissolved in ACN:water (90:10, v/v). The Gradient method for
separation is shown below. R.sub.t (min) of Compound 2 was
approximately 12.0 minutes.
TABLE-US-00020 Time 0.1% H.sub.3PO.sub.4 Acetonitrile (min) in
Water (A) % (B) % 0 90 10 20 20 80 20.1 90 10 30 90 10
[0283] .sup.1HNMR: (400 MHz, DMSO-d.sub.6): .delta. 8.41 (br, 1H),
7.97 (s, 1H), 7.36 (t, J=8.0 Hz, 2H), 7.22 (d, J=8.0 Hz, 2H), 7.17
(t, J=8.0 Hz, 1H), 6.73 (s, 2H), 6.07 (d, J=8.0 Hz, 1H), 6.00 (dd,
J=12.0, 8.0 Hz, 1H), 5.81 (br, 1H), 4.84-4.73 (m, 1H), 4.44-4.28
(m, 3H), 4.10 (t, J=8.0 Hz, 2H), 3.85-3.74 (m, 1H), 2.95 (s, 3H),
1.21 (s, J=4.0 Hz, 3H), 1.15-1.10 (m, 9H).
Example 14. Characterization of Compound 2
[0284] Compound 2 was further characterized by eye, .sup.1HNMR,
.sup.13CNMR, .sup.19FNMR, MS, HPLC, and XRPD (FIG. 15B). Residual
solvent was measured by GC. Water content was measured by Karl
Fischer Titration, and the water content was only 0.70%. Data is
summarized in Table 18.
TABLE-US-00021 TABLE 18 Summary of Additional Characterization Data
of Compound 2 Test Result Appearance White Solid NMR .sup.1HNMR
peaks are listed in Example 4 MS MS(ESI + ve) [M + H].sup.+ =
582.3-conforms to structure HPLC 99.8% by AUC at 254 nm (average of
two preparations) Residual Solvent Methanol-57 ppm by GC
Acetone-752 ppm Dichloromethane-50 ppm Ethyl Acetate-176 ppm Water
Content 0.70%
Example 15. Solubility of Compound 1 and Compound 2
[0285] Compound 1 and Compound 2 were both tested for solubility in
biorelevant test medias, including simulated gastric fluid (SGF),
fasted-state simulated gastric fluid (FaSSIF), and fed-state
gastric fluid (FeSSIF). Results for Compound 1 are shown in Table
19 and results for Compound 2 are shown in Table 20. Samples were
stirred at room temperature (20-25.degree. C.). Compound 2 was more
than 40-fold more soluble than Compound 1 in water at 2 hours and
more than 25-fold more soluble at 24 hours. In SGF conditions,
Compound 2 had a solubility of 84.2 mg/mL at 24 hours compared to
the solubility of 15.6 mg/mL of Compound 1 at the same time point.
Compound 2 was also more soluble at 2 hours in the SGF conditions
than Compound 1, and soluble enough to allow for testing even after
48 hours while testing at 48 hours was not done with Compound
1.
TABLE-US-00022 TABLE 19 Compound 1 solubility testing results Test
Solubility (in mg/mL) Descriptive Media 2 hours 24 hours Appearance
term Water 1.5 2.5 Clear Solution* Slightly Soluble SGF 13.8 15.6
Clear Solution Sparingly with gum at the Soluble bottom FaSSIF 1.7
1.7 Turbid Slightly Soluble FeSSIF 2.8 2.9 Turbid Slightly Soluble
*Sample appeared to be clear, yet a solubility of only 1.5 mg/mL
was achieved. Upon further investigation, it was noted that a gummy
film formed on the stir bar. The compound 1 active pharmaceutical
ingredient formed a gummy ball in diluent (90% water/10%
acetonitrile) during standard preparation which required a long
sonication time to dissolve completely.
TABLE-US-00023 TABLE 20 Compound 2 solubility testing results Test
Solubility (in mg/mL salt base) Descriptive Media 2 hours 24 hours
48 hours Appearance term Water 65.3 68.0 N/A Turbid Soluble SGF
89.0 84.2 81.3 Turbid Soluble FaSSIF 1.9 2.0 N/A Turbid Slightly
Soluble FeSSIF 3.3 3.4 N/A Turbid Slightly Soluble
Example 16. Chemical Stability of Compound 2
[0286] Compound 2 was tested for chemical stability at 25 and
40.degree. C. over a 6 month time period by monitoring organic
purity, water content, .sup.1HNMR, DSC, and Ramen IR. The container
closure system for the study was a combination medicinal valve bag
with a pharmaceutical laminated film over the pouch and desiccant
silica gel between the two layers. Compound 2 (1 g) was measured
into each container. Bags were then stored at 25.degree. C./60% RH
(relative humidity) and 40.degree. C./75% RH (relative humidity).
Organic purity, water content, .sup.1HNMR, DSC and Raman were
measured at Time 0, Month 1, Month 2, Month 3 and Month 6.
[0287] The purity of Compound 2 was obtained using a Shimadzu
LC-20AD system with a Waters XTerra Phenyl, 5 .mu.m, 4.6.times.250
mm column with the following conditions: 1 mL/min flow rate, read
at 254 nm, 35.degree. C. column temperature, and 10 L injection
volume. The sample was dissolved in acetonitrile-water (90:10)
(v/v). The gradient method is shown below.
TABLE-US-00024 Time (min) A % (ACN) B % (water) 0 90 10 20 20 80
20.1 90 10 30 90 10
[0288] The water content of Compound 2 (250 mg) was determined by a
water titration apparatus using the Karl Fischer titration
method.
[0289] Results are shown in Table 21 and Table 22. When Compound 2
was stored for 6 months at 25 and 40.degree. C., the rate of
degradation was minimal. At 3 months, Compound 2 was 99.75% percent
pure at the 25.degree. C. conditions and 99.58% pure at the
40.degree. C. conditions. At 6 months, Compound 2 was still 99.74%
pure at the 25.degree. C. conditions and 99.30% pure at the
40.degree. C. conditions. At 25.degree. C., the percent of
degradation product increased from 0.03% at Day 0 to 0.08% after 6
months. At 40.degree. C., the percent of degradation product
increased from 0.03% to 0.39%. Over the course of 6 months, the
percent of water increased approximately 0.6% at 25.degree. C. and
increased approximately 0.7% at 40.degree. C.
[0290] Characterization by .sup.1HNMR, Raman, and DSC of Compound 2
at 1, 2, 3, and 6 months was the same as the characterization of
Compound 2 on day 0 at both temperature conditions (Table 22),
highlighting the long-term stability of Compound 2.
TABLE-US-00025 TABLE 21 Compound 2 rate of degradation over 6
months at 25 and 40.degree. C. Percent of Maximum Time Percent
Percent Degradation Impurity Tested Water Purity Product Percent
25.degree. C. Day 0 1.2 99.82 0.03 0.12 Month 1 1.9 99.77 0.04 0.12
Month 2 1.8 99.75 0.06 0.12 Month 3 1.8 99.75 0.06 0.12 Month 6 1.8
99.74 0.08 0.13 40.degree. C. Day 0 1.2 99.82 0.03 0.12 Month 1 2.0
99.71 0.09 0.12 Month 2 1.9 99.63 0.15 0.12 Month 3 1.9 99.58 0.20
0.12 Month 6 1.9 99.30 0.39 0.14
TABLE-US-00026 TABLE 22 Characterization of Compound 2 during
degradation study Time Tested .sup.1HNMR Raman DSC 25.degree. C.
Day 0 Initial Test Initial Test Initial Test Month 1 The same as
The same as The same as Day 0 Day 0 Day 0 Month 2 The same as The
same as The same as Day 0 Day 0 Day 0 Month 3 The same as The same
as The same as Day 0 Day 0 Day 0 Month 6 The same as The same as
The same as Day 0 Day 0 Day 0 40.degree. C. Day 0 Initial Test
Initial Test Initial Test Month 1 The same as The same as The same
as Day 0 Day 0 Day 0 Month 2 The same as The same as The same as
Day 0 Day 0 Day 0 Month 3 The same as The same as The same as Day 0
Day 0 Day 0 Month 6 The same as The same as The same as Day 0 Day 0
Day 0
[0291] Additional chemical stability studies of Compound 2 were
measured to determine the impurity and water levels. Three
conditions were tested: accelerated stability (40 2.degree.
C./75.+-.5% RH) over a 6-month time period, ambient stability
(25.+-.2.degree. C./60.+-.5% RH) over a 9-month period, and
stability under refrigerator conditions (5 3.degree. C.) over a
9-month time period. The results for accelerated stability, ambient
stability, and refrigerator conditions are shown in Table 23, Table
24, and Table 25, respectively. Based on the results of these
studies, Compound 2 is very chemically stable.
[0292] In the accelerated stability study (Table 23), at each time
point (1.sup.st month, 3.sup.rd month, and 6.sup.th month) where
Compound 2 was measured, the appearance of Compound 2 was always a
white solid and the IR matched the reference standard. After six
months, the total related substance 1 impurities was only 0.08% and
there was no detection of related substance 2 and isomers.
TABLE-US-00027 TABLE 23 Accelerated Stability (40 .+-. 2.degree.
C./75 .+-. 5% RH) of Compound 2 Testing time point Items
Specification 0 month 1.sup.st month 3.sup.rd month 6.sup.th month
Appearance White or off- White White White White white solid solid
solid solid solid IR correspond correspond / correspond correspond
with with with with reference reference reference reference
standard standard standard standard Water .ltoreq.2.0% 0.45% 0.21%
0.36% 0.41% Related Impurity A .ltoreq.0.15% N.D. N.D. N.D. N.D.
Substance Impurity B .ltoreq.0.15% N.D. N.D. N.D. N.D. 1 Impurity F
.ltoreq.0.15% N.D. N.D. N.D. 0.01% Impurity H .ltoreq.0.15% N.D.
N.D. N.D. N.D. Any other .ltoreq.0.10% 0.01% 0.02% 0.01% 0.05%
single impurity Total .ltoreq.0.2% 0.01% 0.02% 0.02% 0.08%
Impurities Related Impurity G .ltoreq.0.15% N.D. N.D. N.D. N.D.
Substance 2 Isomer Impurity C .ltoreq.0.15% N.D. / N.D. N.D.
Impurity D .ltoreq.0.15% N.D. / N.D. N.D. Impurity E .ltoreq.0.15%
N.D. / N.D. N.D. Assay 98.0%~102.0% 98.8% 101.5% 99.6% 99.5%
Microbial TAMC .ltoreq.1000 cfu/g <1 cfu/g / / / Testing Mold
and Yeast .ltoreq.100 cfu/g <1 cfu/g / / / E. Coli Not Detected
N.D. / / / N.D.: Not Detected
[0293] In the ambient stability study where the appearance, IR,
water and impurity levels were measured for nine months, the
appearance of Compound 2 was always a white solid and the IR always
corresponded with the reference sample. The results (Table 24)
highlight how chemically stable Compound 2 is. After 9 months, the
percentage of water in the sample was only 0.20% and the total
related substance 1 impurities was only 0.02%. Similarly to the
accelerated stability studies, related substance 2 and any isomers
of Compound 2 were not detected.
TABLE-US-00028 TABLE 24 Ambient stability (25 .+-. 2.degree. C./60
.+-. 5% RH) of Compound 2 Testing time point 1.sup.st Item
Specification 0 month month 3.sup.rd month 6.sup.th month 9.sup.th
month Appearance White or off- White White White White solid
Off-white white solid solid solid solid solid IR correspond
correspond / correspond correspond correspond with with with with
with reference reference reference reference reference standard
standard standard standard standard Water .ltoreq.2.0% 0.45% 0.19%
0.29% 0.46% 0.20% Related Impurity .ltoreq.0.15% N.D. N.D. N.D.
N.D. N.D. Substance A 1 Impurity .ltoreq.0.15% N.D. N.D. 0.03% N.D.
N.D. B Impurity .ltoreq.0.15% N.D. N.D. 0.02% 0.01% N.D. F Impurity
.ltoreq.0.15% N.D. N.D. N.D. N.D. N.D. H Any other .ltoreq.0.10%
0.01% 0.01% 0.03% 0.02% 0.02% single impurity Total .ltoreq.0.2%
0.01% 0.02% 0.11% 0.05% 0.02% Impurities Related Impurity
.ltoreq.0.15% N.D. N.D. N.D. N.D. N.D. Substance G 2 Isomer
Impurity <0.15% N.D. / N.D. N.D. N.D. C Impurity .ltoreq.0.15%
N.D. / N.D. N.D. N.D. D Impurity .ltoreq.0.15% N.D. / N.D. N.D.
N.D. E Assay 98.0%~102.0% 98.8% 101.1% 99.6% 99.7% 100.9% Microbial
TAMC .ltoreq.1000 cfu/g <1 cfu/g / / / / Testing Mold and
.ltoreq.100 cfu/g <1 cfu/g / / / / Yeast E. Coli Not Detected
N.D. / / / / N.D.: Not Detected
[0294] The results of measuring the stability under refrigerator
conditions are shown in Table 25. The only impurities detected even
after 9 months were those from related substance 1 and water. The
water content after 9 months was 0.32% and the total impurities of
related substance 1 were only 0.01% of the sample. Compound 2 is
very chemically stable under refrigerator conditions.
TABLE-US-00029 TABLE 25 Stability under refrigerator conditions (5
.+-. 3.degree. C.) of Compound 2 Testing time point Item
Specification 0 month 1.sup.st month 3.sup.rd month 6.sup.th month
9.sup.th month Appearance White or off- White White White White
Off-white white solid solid solid solid solid solid IR correspond
correspond / correspond correspond correspond with with with with
with reference reference reference reference reference standard
standard standard standard standard Water .ltoreq.2.0% 0.45% 0.19%
0.32% 0.42% 0.32% Related Impurity .ltoreq.0.15% N.D. N.D. N.D.
N.D. N.D. Substance A 1 Impurity .ltoreq.0.15% N.D. N.D. 0.01% N.D.
N.D. B Impurity .ltoreq.0.15% N.D. N.D. N.D. N.D. N.D. F Impurity
.ltoreq.0.15% N.D. N.D. N.D. N.D. N.D. H Any other .ltoreq.0.10%
0.01% 0.01% 0.01% 0.01% 0.01% single impurity Total .ltoreq.0.2%
0.01% 0.01% 0.03% 0.03% 0.01% Impurities Related Impurity
.ltoreq.0.15% N.D. N.D. N.D. N.D. N.D. Substance G 2 Isomer
Impurity .ltoreq.0.15% N.D. / N.D. N.D. N.D. C Impurity
.ltoreq.0.15% N.D. / N.D. N.D. N.D. D Impurity .ltoreq.0.15% N.D. /
N.D. N.D. N.D. E Assay 98.0%~102.0% 98.8% 101.1% 100.2% 98.6%
101.4% Microbial TAMC .ltoreq.1000 cfu/g <1 cfu/g / / / /
Testing Mold and .ltoreq.100 cfu/g <1 cfu/g / / / / Yeast E.
Coli Not Detected N.D. / / / / N.D.: Not Detected
Example 17. Plasma Levels of Metabolites Following Single Oral
Doses of Compound 2
[0295] A single oral dose of Compound 2 was administered to rats,
dogs, and monkeys, and the plasma levels of certain metabolites
shown in Scheme 1 were measured.
[0296] The conversion of Compound 2 to Compound 1 and metabolite
1-7 are shown in Table 26 and the results for metabolite 1-8 and
metabolite 1-2 are shown in Table 27. In rats, low levels of
Compound 1 exposure were observed, but high levels of metabolite
1-7, the nucleoside metabolite of the active triphosphate
(metabolite 1-6), were observed. In monkeys, roughly
dose-proportional exposures of Compound 1 were measured. In dogs,
supra-proportional Compound 1 exposures, indicative of first-pass
metabolic clearance in the liver, were measured. Throughout the
study, significantly more vomiting in dogs (5/5 in high dose group)
than in monkeys (1/5 in high dose group) was observed.
TABLE-US-00030 TABLE 26 Plasma levels of Compound 1 and metabolite
1-7 after single oral doses of Compound 2 Compound 1 Metabolite 1-7
Dose* C.sub.max AUC.sub.0-last C.sub.max AUC.sub.0-last (mg/ (ng/
T.sub.max (hr*ng/ (ng/ (hr*ng/ Species kg) mL) (hr) mL) mL) mL)
Rat.sup.a 500 70.5 0.25 60.9 748 12000 Dog.sup.b 30 1530 0.25-1
1300 783 9270 100 8120 0.5-1 10200 2030 24200 300 21300 204 44300
4260 60800 Monkey.sup.b 30 63.5 0.5-2 176 42.5 1620 100 783 1-2
1100 131 3030 300 501 204 1600 93.6 3660 3 males per dose per
species; *dose formulations: .sup.a0.5% CMC, 0.5% Tween 80 in
water; .sup.bpowder in capsules
TABLE-US-00031 TABLE 27 Plasma levels of metabolites 1-8 and 1-2
after single oral dose of Compound 2 Metabolite 1-8 Metabolite 1-2
Dose* C.sub.max AUC.sub.0-last C.sub.max AUC.sub.0-last Species
(mg/kg) (ng/mL) (hr*ng/mL) (ng/mL) (hr*ng/mL) Rat.sup.a 500 5060
35100 9650 20300 Dog.sup.b 30 291 905 196 610 100 1230 4370 886
2830 300 5380 35300 2380 8710 Monkey.sup.b 30 209 5690 300 1730 100
406 12300 1350 8160 300 518 16800 1420 11400 3 males per dose per
species; *dose formulations: .sup.a0.5% CMC, 0.5% Tween 80 in
water; .sup.bpowder in capsules
Example 18. Tissue Exposure of Active Triphosphate Following
Compound 2 Oral Dose
[0297] Heart and liver tissue levels of the active triphosphate
(TP) of Compound 2 (metabolite 1-6) were measured 4 hours after
oral doses of Compound 2. Samples of liver and heart were obtained
at 4 hours after a single dose of Compound 2, flash-frozen,
homogenized and analyzed by LC-MS/MS for intracellular levels of
the active TP. Tissue levels were measured in rats, dogs, and
monkeys as shown in FIG. 16A. High levels of the active TP were
measured in the liver of all species tested. Relatively low levels
of the active TP were measured in the hearts of dogs due to
saturation of first-pass hepatic metabolism, and unquantifiable
levels of TP were measured in rat and monkey hearts, indicative of
liver-specific formation of the active TP. While not shown,
compared to Compound 1 dosing, Compound 2 dosing improved TP
distribution.
Example 19. Pharmacological Comparison of Compound 1 and Compound 2
in Dogs
[0298] A head-to-head comparison of dogs dosed with Compound 1 and
Compound 2 was conducted. The study measured plasma levels of
Compound 1 and metabolite 1-7 (from Scheme 1) out to 4 hours after
dosing with Compound 1 (25 mg/kg) and Compound 2 (30 mg/kg) (Table
28), and the AUC.sub.(0-4hr) of metabolite 1-7 was twice as great
with Compound 2 compared to Compound 1. Dose-normalized exposures
to Compound 1 and metabolite 1-7 are shown in Table 28. Values for
AUC.sub.(0-4hr) for Compound 1, metabolite 1-7, and the sum of
Compound 1+metabolite 1-7 were greater after dosing with Compound
2.
TABLE-US-00032 TABLE 28 Comparison of Plasma Levels following
dosing with Compound 1 and Compound 2 Mean Dose-normalized
AUC.sub.(0-4hr).sup.a (.mu.M*hr) for: Dosed Compound Metabolite
Compound 1 + Compound 1 1-7 Metabolite 1-7 Compound 1 0.2 1.9 2.1
(25 mg/kg) Compound 2 1.0 4.1 5.1 (30 mg/kg) .sup.aAUC.sub.(0-4hr)
values normalized to a dose of 25 mg/kg
[0299] Liver/heart ratio triphosphate concentrations indicate that
dosing with Compound 2, as compared to Compound 1, increases the
selective delivery of the triphosphate to the liver, as shown in
Table 29. The AUC.sub.(0-4hr) of the active guanine metabolite
(1-6) after administration of Compound 1 measured in the heart was
174 .mu.M*hr, while the AUC.sub.(0-4hr) of the active guanine
metabolite (1-6) after administration of Compound 2 measured in the
heart was 28 .mu.M*hr. The liver/heart ratio for Compound 2 was 20
compared to a liver/heart ratio of 3.1 for Compound 1.
TABLE-US-00033 TABLE 29 Comparison of Liver and Heart Exposure
following dosing with Compound 1 and Compound 2 Dosed Mean
Dose-normalized AUC.sub.(0-4hr)a (.mu.M*hr) for: Compound Liver
Heart Liver/Heart Compound 2 565 28.sup.b 20 Compound 1 537 174 3.1
.sup.aActive TP concentrations (1-6 Scheme 1) normalized to a dose
of 25 mg/kg .sup.bExtrapolated below the lower limit of
quantitation of the calibration curve
[0300] The effect of increased selectivity for the liver over the
heart when Compound 2 was administered compared to Compound 1 is
also shown in FIG. 16B. The heart and liver tissue levels of the
active triphosphate following a dosage of Compound 2 (30 mg/kg)
were compared to the tissue levels of the active triphosphate
following a dosage of Compound 1 (25 mg/kg). The concentration of
the active TP was higher in the liver than the heart for both
Compound 1 and Compound 2, but the active TP was more selective for
the liver over the heart when Compound 2 was dosed compared to
Compound 1.
Example 20. Plasma Profiles of Compound 2 Metabolites in Rats and
Monkeys
[0301] Male Sprague-Dawley rats and cynomolgus monkeys (3 animals
per dose group) were given single oral doses of Compound 2.
Aliquots of plasma prepared from blood samples treated with
Dichlorvos were analyzed by LC-MS/MS for concentrations of Compound
1 and metabolite 1-7 (the nucleoside metabolite of the active
triphosphate of Compound 2 shown in Scheme 1), and pharmacokinetic
parameters were determined using WinNonlin. The results for a
single 500 mg/kg dose in rats is shown in FIG. 17 and the results
for a single 30, 100, or 300 mg/kg dose in monkeys is shown in FIG.
18. The results are also summarized in Table 30.
[0302] High plasma levels of metabolite 1-7, the nucleoside
metabolite of the active triphosphate (TP) of Compound 2, are
indicative of formation of high levels of the TP, even in rats
where very low plasma levels of parent nucleotide prodrug are
observed due to the short half-life of Compound in rat blood (<2
min). Persistent plasma levels of metabolite 1-7 reflect the long
half-life of the TP.
[0303] In monkeys, plasma exposures (AUC) of Compound 1 were
roughly dose-proportional, while metabolite 1-7 exposures were
somewhat less than dose-proportional, although AUC values for both
parent drug and the nucleoside metabolite of the active TP continue
to increase up to the highest dose tested (300 mg/kg).
[0304] Oral administration of Compound in rats and monkeys produced
high and dose-dependent plasma exposures to metabolite 1-7 (the
nucleoside metabolite of the intracellular active triphosphate of
Compound 2); metabolite 1-7 exposures continued to increase up to
the highest dose tested, reflecting substantial formation of the
active TP in these species.
TABLE-US-00034 TABLE 30 Plasma levels of Compounds 1 and 1-7 after
single oral dose of Compound 2 Species Rat.sup.a Monkey.sup.b Dose
(mg/kg) 500 30 100 300 Compound 1 C.sub.max (ng/mL) 60.8 63.5 783
501 T.sub.max (hr) 0.25 0.5-2 1-2 204 AUC.sub.0-last 78.2 176 1100
1600 (hr*ng/mL) Metabolite 1-7 C.sub.max (ng/mL) 541 42.5 131 93.6
AUC.sub.0-last 9640 1620 3030 3660 (hr*ng/mL) T.sub.max (hr) 6-8
12-24 4 4-24 T.sub.1/2 (hr) 15.3 11.5 15.0 18.8 dose formulations:
.sup.a0.5% CMC, 0.5% Tween 80 in water; .sup.bpowder in
capsules
Example 21. The Effect of the Active Triphosphate of Compound 1 and
Compound 2 on Mitochondrial Integrity
[0305] The relative efficiency of incorporation of the active
triphosphate (TP) of Compound 1 and Compound 2, metabolite 1-6
(Scheme 1), by human mitochondrial RNA polymerase was compared to
the relative efficiency of the active TP of sofosbuvir and the
active TP of INX-189. Compound 1 and Compound 2 are not likely to
affect mitochondrial integrity since their active
triphosphateispoorlyincorporatedbyhumanmitochondrialRNApolymerasew-
ithanefficiency similar to that of the triphosphate of sofosbuvir;
the relative efficiency of incorporation of the triphosphate of
INX-189 was up to 55-fold greater. Results are shown in Table 31.
The incorporation of these analogs by human mitochondrial
RNA-dependent RNA polymerase (POLRMT) were determined according to
Arnold et al. (Sensitivity of Mitochondrial Transcription and
Resistance of RNA Polymerase II Dependent Nuclear Transcription to
Antiviral Ribonucleotides. PLoS Pathog., 2012, 8, e1003030).
TABLE-US-00035 TABLE 31 Kinetic Parameters for Nucleotide Analogs
Evaluated with Human Mitochondrial RNA Polymerase Nucleotide
K.sub.pol K.sub.d,app K.sub.pol/Kd,app Relative Analog (s.sup.-1)
(.mu.M) (.mu.M.sup.-1s.sup.-1) Efficiency* 2`-deoxy-2`-F-2`-
0.00034 .+-. 590 .+-. 5.8 .times. 10.sup.-7 .+-. 1.0 .times.
10.sup.6 C-methyl UTP 0.00005 250 2.6 .times. 10.sup.-7 (active TP
of sofosbuvir) 2'-C-methyl GTP 0.051 .+-. 240 .+-. 2.1 .times.
10.sup.-4 .+-. 5.5 .times. 10.sup.-5 (active TP of INX- 0.002 26
0.2 .times. 10.sup.-4 189) Active TP of 0.0017 .+-. 204 .+-. 8.3
.times. 10.sup.-6 .+-. 2.2 .times. 10.sup.-6 Compound 1 and 0.0002
94 4.0 .times. 10.sup.-6 Compound 2 (metabolite 1-6) *Relative
efficiency = (K.sub.pol/K.sub.d,app).sub.analog
nucleotide/(K.sub.pol/K.sub.d,app).sub.natural nucleotide
Example 22. Activity of Compound 1 Against Replicons Containing the
NS5B Sequence
[0306] A panel of replicons containing the NS5B sequences from
various HCV genotypes derived from 6 laboratory reference strains
(GT1a, 1b, 2a, 3a, 4a and 5a) (FIG. 19) and from 8HCV patient
plasma samples (GT1a, 1b, 2a, 2b, 3a-1, 3a-2, 4a and 4d) (FIG. 20)
were used to determine the potency of Compound 1 and
sofosbuvir.
[0307] Compound 1 was more potent than sofosbuvir against clinical
and laboratory strains of HCV. Compound 1 showed potent
pan-genotypic antiviral activity in vitro against wild-type
clinical isolates with EC.sub.95<80 nM, which is 4- to 14-fold
more potent than sofosbuvir. As shown in FIG. 20, EC.sub.95 values
for Compound 1 were 7-33 times lower than sofosbuvir against
clinical isolates of all HCV genotypes tested. EC.sub.50 values for
Compound 1 were 6-11 times lower than sofosbuvir against laboratory
strains of HCV Genotypes 1-5 (FIG. 19).
Example 23. Single Ascending Dose (SAD) Study of Compound 2 in
Healthy Volunteers (Part A) and GT1-HCV Infected Patients (Part
B)
[0308] Compound 2 was tested in a single ascending dose (SAD) study
to measure its safety, tolerability, and pharmacokinetic in healthy
subjects (Part A). Part A was a randomized, double-blind,
placebo-controlled SAD study. Healthy subjects in Part A received a
single dose of Compound 2 or placebo in the fasting state. Subjects
were confined to the clinic from Day -1 to Day 6.
[0309] Dosing in each cohort was staggered such that 2 subjects (1
active:1 placebo) were evaluated for 48 hours after dosing before
the remainder of the cohort was dosed. Each cohort received
Compound 2 in ascending order. Dosing of sequential cohorts
occurred based on review of available safety data (through Day 5)
and plasma pharmacokinetic data (through 24 h) of the prior
cohort.
[0310] Dose escalation proceeded following satisfactory review of
these data. As pharmacokinetic and safety data emerged from prior
cohorts, doses evaluated in Cohorts 3a-4a were adjusted by
increments no more than 100 mg. The total maximum dose evaluated in
Part A did not exceed 800 mg. The dosing regimen for Part A is
shown in Table 32.
TABLE-US-00036 TABLE 32 Dosing Regimen for Compound 2
Administration Part A of Study N (active: Compound 2 Cohort
Population placebo) (Compound 1)* 1a Healthy 6:2 50 (45) mg .times.
1 day 2a Healthy 6:2 100 (90) mg .times. 1 day 3a Healthy 6:2 200
(180) mg .times. 1 day 4a Healthy 6:2 400 (360) mg .times. 1 day
*Clinical doses are expressed in terms of Compound 2, with the
approximate Compound 1 base equivalent in parenthesis
[0311] Healthy volunteers in the Part A portion of the study were
male and female subjects between the ages of 18 and 65. Active and
placebo recipients were pooled within each Part A cohort to
preserve the study blind.
[0312] Compound 2 was also tested in a single ascending dose (SAD)
study to measure its safety, tolerability, pharmacokinetic, and
antiviral activity in GT1-HCV infected patients (Part B). Subjects
in Part B received a single dose of Compound 2 in the fasting
state. Patients were confined to the clinic from Day -1 to Day
6.
[0313] Part B was initiated after the safety (through Day 5) and
plasma pharmacokinetic (through 24 h) data review from Cohort 3a in
Part A. Available safety data (through Day 5) and pharmacokinetic
data (through 24 h) was reviewed for the first cohort in Part B
(Cohort 1b) before enrolling subsequent Part B cohorts. Subsequent
Part B cohorts were only dosed following review of available safety
and pharmacokinetic data from the respective doses in Part A as
well as available safety (through Day 5) from the prior Part B
cohorts.
[0314] Dose escalation up to 600 mg in HCV-infected patients
proceeded following satisfactory review of these data. The dosing
regimen for Part B is shown in Table 33.
TABLE-US-00037 TABLE 33 Dosing Regimen for Compound 2 in Part B of
Study N Compound 2 Cohort Population (active) (Compound 1)* 1b GT1
HCV-Infected 3 100 (90) mg .times. 1 day 2b GT1 HCV-Infected 3 300
(270) mg .times. 1 day 3b GT1 HCV-Infected 3 400 (360) mg .times. 1
day 4b GT1 HCV-Infected 3 600 (540) mg .times. 1 day *Clinical
doses are expressed in terms of Compound 2, with the approximate
Compound 1 base equivalent in parenthesis.
[0315] Patients infected with HCV were treatment-naive,
non-cirrhotic GT1-infected subjects with a viral load of .gtoreq.5
log.sub.10 IU/mL.
[0316] No serious adverse events were recorded and no premature
discontinuations were required in either Part A or Part B. All
adverse effects were mild to moderate in intensity and no
dose-related patterns, including laboratory parameters, vital
signs, and ECGs were evident.
Example 24. Results of the Single Ascending Dose (SAD) Study of
Compound 2
[0317] Pharmacokinetic of Compound 1 and nucleoside metabolite 1-7
were measured following the single dose of Compound 2. The C.sub.24
trough plasma concentrations (C.sub.24h) of metabolite 1-7 in
HCV-infected patients following a 600 mg dose of Compound 2 was
25.8 ng/mL, which is more than double the plasma concentration dose
following 300 mg dose of Compound 2. Metabolite 1-7 (shown in
Scheme 1) can only be generated via dephosphorylation of the
intracellular phosphate metabolite 1-4, metabolite 1-5, and
metabolite 1-6, which is the active species. Therefore, metabolite
1-7 can be considered a surrogate of the active species. The
pharmacokinetic data for all cohorts is shown in Table 34 and Table
35. Values are reported as mean.+-.SD, except or T.sub.max where
median (range) is reported. Pharmacokinetic parameters were
comparable in healthy and HCV-infected patients.
TABLE-US-00038 TABLE 34 Human Pharmacokinetic of Compound 1 and
Metabolite 1-7 after Administration of a single dose of Compound 2
in Healthy Volunteers Dose C.sub.max AUC.sub.tot C.sub.24h (mg)
(ng/mL) T.sub.max (h) (ng*h/mL) T.sub.1/2 (h) (ng/mL) Part A,
Healthy Subjects Compd 1 50 46.4 .+-. 17.6 0.5 (0.5-0.5) 36.4 .+-.
12.3 0.32 .+-. 0.02 -- 100 156 .+-. 96.3 0.5 (0.5-1.0) 167 .+-. 110
0.53 .+-. 0.24 -- 200 818 .+-. 443 0.5 (0.5-3.0) 656 .+-. 255 0.71
.+-. 0.16 -- 400 1194 .+-. 401 0.5 (0.5-1.0) 1108 .+-. 326 0.86
.+-. 0.15 -- Metabolite 50 27.9 .+-. 5.62 3.5 (3.0-4.0) 285 .+-.
69.4 7.07 .+-. 4.59 2.28 .+-. 0.95 1-7 100 56.6 .+-. 14.0 4.0
(3.0-6.0) 663 .+-. 242 17.7 .+-. 14.7 4.45 .+-. 1.87 200 111 .+-.
38.8 5.0 (3.0-6.0) 1524 .+-. 497 15.9 .+-. 7.95 13.7 .+-. 5.09 400
153 .+-. 49.4 6.0 (4.0-8.0) 2342 .+-. 598 15.6 .+-. 6.37 23.5 .+-.
6.31 *Based on 24-hr profile.
TABLE-US-00039 TABLE 35 Human Pharmacokinetic of Compound 1 and
Metabolite 1-7 after Administration of Compound 2 in GT1-HCV
Infected Patients Dose C.sub.max AUC.sub.tot C.sub.24h (mg) (ng/mL)
T.sub.max (h) (ng*h/mL) T.sub.1/2 (h) (ng/mL) Compd 1 100 212 .+-.
32.0 0.5 (0.5-1.0) 179 .+-. 54.4 0.54 .+-. 0.12 -- 300 871 .+-. 590
0.5 (0.5-1.0) 818 .+-. 475 0.64 .+-. 0.20 -- 300 2277 .+-. 893 0.5
(0.5-1.0) 1856 .+-. 1025 0.84 .+-. 0.18 -- 400 2675 .+-. 2114 1.0
(1.0-2.0) 2408 .+-. 1013 0.86 .+-. 0.18 -- 600 3543 .+-. 1649 1.0
(0.5-1.0) 4132 .+-. 1127 0.70 .+-. 0.13 -- Metabolite 100 50.2 .+-.
15.4 6.0 (4.0-6.0) 538 .+-. 103* 8.4 .+-. 4.3* 3.60 .+-. 0.40 1-7
300 96.9 .+-. 38.9 6.0 (3.0-6.0) 1131 .+-. 273* 8.1 .+-. 2.4* 10.9
.+-. 3.51 300 123 .+-. 16.6 4.0 (3.0-6.0) 1420 .+-. 221 -- 18.0
.+-. 8.83 400 160 .+-. 36.7 4.0 (4.0-4.0) 2132 .+-. 120 11.6 .+-.
1.21 22.5 .+-. 3.29 600 198 .+-. 19.3 4.0 (4.0-6.0) 2176 .+-. 116
-- 25.8 .+-. 4.08 *Based on 24-hr profile.
[0318] The mean plasma concentration-time profiles of Compound 1
and metabolite 1-7 were also calculated for all cohorts of Part A
and Part B of the study. FIG. 21 is the mean plasma-concentration
of Compound 1 following a single dose of Compound 2 and FIG. 22 is
the mean plasma-concentration of metabolite 1-7 following a single
dose of Compound 2. As shown in FIG. 21, Compound 1 was quickly
absorbed and rapidly/extensively metabolized in all cohorts from
Part B. As shown in FIG. 22, metabolite 1-7 was a major metabolite
and exhibited sustained plasma concentrations. Plasma exposure of
Compound 1 was dose-related while exposure of metabolite 1-7 was
dose-proportional.
[0319] For the HCV-infected subjects of Part B, measurements of HCV
RNA quantitation were performed before, during, and after
administration of Compound 2. Plasma HCV RNA determinations were
performed through the use of a validated commercial assay. Baseline
was defined as the mean of Day -1 and Day 1 (pre-dose). A single
300 mg dose of Compound 2 (equivalent to 270 mg of Compound 1)
resulted insignificant antiviral activity in GT1b-HCV infected
subjects. The mean maximum HCV RNA reduction 24 hours post-dose
following a single 300 mg dose was 1.7 log.sub.10 IU/mL and this
compares to a-2 log.sub.10 IU/mL reduction after 1 day of 400 mg of
sofosbuvir monotherapy in GT1a HCV-infected subjects. The mean
maximum HCV RNA reduction 24 hours post-dose following a single 100
mg dose was 0.8 log.sub.10 IU/mL. The mean maximum HCV RNA
reduction was 2.2 log.sub.10 IU/mL following a single 400 mg dose.
Individual pharmacokinetic/pharmacodynamic analyses for the
individual subjects from Part B of the study are shown in FIGS.
23A-23F. Metabolite 1-7 concentration is plotted against HCV RNA
reduction concentration, and as shown in FIGS. 23A-23F, plasma HCV
RNA reduction correlates with plasma metabolite 1-7 exposure. Viral
response is sustained with metabolite 1-7 plasma concentrations
that are greater than the EC.sub.95 value against GT1b. The
correlation between plasma concentration and HCV RNA reduction
levels indicates that a more profound response will be achievable
with higher doses of Compound 2.
Example 25. Predicted Steady-State Trough Levels of Metabolite 1-7
Exceed Compound 1 EC.sub.95 Values Against Clinical Isolates of HCV
GT 1-4
[0320] As shown in FIG. 24, the steady-state trough plasma levels
(C.sub.24,ss) of metabolite 1-7 following Compound 2 dosing in
humans (600 mg QD (550 mg free base equivalent) and 450 mg QD (400
mg free base equivalent)) was predicted and compared to the
EC.sub.95 of Compound 1 in vitro across all tested clinical
isolates to determine if the steady state plasma concentration is
consistently higher than the EC.sub.95, which would result in high
efficacy against any or all tested clinical isolates in vivo. The
EC.sub.95 for Compound 1 is the same as the EC.sub.95 of Compound
2. For Compound 2 to be effective, the steady-state trough plasma
level of metabolite 1-7 should exceed the EC.sub.95.
[0321] As shown in FIG. 24, the EC.sub.95 of Compound 2 against all
tested clinical isolates ranged from approximately 18 to 24 nM.
[0322] As shown in FIG. 24, Compound 2 at a dose of 450 mg QD (400
mg free base equivalent) in humans of provides a predicted steady
state trough plasma concentration (C.sub.24,ss) of approximately 40
ng/mL. Compound 2 at a dose of 600 mg QD (550 mg free base
equivalent) in humans of provides a predicted steady state trough
plasma concentration (C.sub.24,ss) of approximately 50 ng/mL.
[0323] Therefore, the predicted steady state plasma concentration
of surrogate metabolite 1-7 is almost double the EC.sub.95 against
all tested clinical isolates (even the hard to treat GT3a), which
indicates superior performance.
[0324] In contrast, the EC.sub.95 of the standard of care
nucleotide sofosbuvir ranges from 50 to 265 nM across all tested
HCV clinical isolates, with an EC.sub.95 less than the predicted
steady state concentration at the commercial dosage of 400 mg for
only two isolates, GT2a and GT2b. The EC.sub.95 for the commercial
dosage of 400 mg of sofosbuvir is greater than the predicted steady
state concentration for other clinical isolates, GT1a, GT1b, GT3a,
GT4a, and GT4d.
[0325] The Compound 2 450 mg steady state trough plasma
concentration (C.sub.24,ss) was predicted using the 300 mg steady
state trough plasma concentration (C.sub.24,ss). The mean steady
state trough plasma concentration (C.sub.24,ss) at 300 mg was 26.4
ng/mL, and therefore the calculation was 26.4*450/300=39.6
ng/mL.
[0326] The 600 mg steady state trough plasma concentration
(C.sub.24,ss) was predicted using three approaches: 1) the 600 mg
Day 1 C.sub.24 mean was 25.8 ng/mL and a 60% increase was assumed
for reaching steady state. Therefore the calculation was
25.8*1.6=41.3 ng/mL; 2) the 400 mg day 1 C.sub.24 mean was 22.5
ng/mL and a 60% increase was assumed for reaching steady state.
Taking dose proportional PK into account, the calculation was
22.5*1.6*600/400=54 ng/mL; and 3) the 300 mg steady state trough
plasma concentration (C.sub.24,ss) was 26.4 ng/mL and a
proportional PK was assumed. Therefore the calculation was
26.4*2=52.8 ng/mL. The 600 mg steady state trough plasma
concentration (C.sub.24,ss) is the average of the 3 data points
((41.3+54+52.8)/3=49.3 ng/mL). There is generally about a 60%
increase in C.sub.24 at steady state compared to C.sub.24 following
a single dose.
[0327] The data comparing the efficacy and pharmacokinetic steady
state parameters in FIG. 24 clearly demonstrates the unexpected
therapeutic importance of Compound 2 for the treatment of hepatitis
C. In fact, the predicted steady-state plasma level after
administration of Compound 2 is predicted to be at least 2-fold
higher than the EC.sub.95 for all genotypes tested, and is 3- to
5-fold more potent against GT2. This data indicates that Compound 2
has potent pan-genotypic antiviral activity in humans. As shown in
FIG. 24, the EC.sub.95 of sofosbuvir at GT1, GT3, and GT4 is
greater than 100 ng/mL. Thus surprisingly, Compound 2 is active
against HCV at a dosage form that delivers a lower steady-state
trough concentration (40-50 ng/mL) than the steady-state tough
concentration (approximately 100 ng/mL) achieved by a similar
dosage form of sofosbuvir.
Example 26. Formulation Description and Manufacturing of Compound
2
[0328] A representative non-limiting batch formula for Compound 2
tablets (50 mg and 100 mg) is presented in Table 36. The tablets
were produced from a common blend using a direct compression
process as shown in FIG. 25. The active pharmaceutical ingredient
(API) is adjusted based on the as-is assay, with the adjustment
made in the percentage of microcrystalline cellulose.
[0329] The API and excipients (microcrystalline cellulose, lactose
monohydrate, and croscarmellose sodium) were screened, placed into
a V-blender (PK Blendmaster, 0.5 L bowl) and mixed for 5 minutes at
25 rpm. Magnesium Stearate was then screened, added and the blend
was mixed for an additional 2 minutes. The common blend was divided
for use in producing 50 mg and 100 mg tablets. The lubricated blend
was then compressed at a speed of 10 tablets/minutes using a single
punch research tablet press (Korsch XP1) and a gravity powder
feeder. The 50 tablets were produced using round standard concave 6
mm tooling and 3.5 kN forces. The 100 mg tablets were produced
using 8 mm round standard concave tooling and 3.9-4.2 kN
forces.
TABLE-US-00040 TABLE 36 Formulation of 50 mg and 100 mg Compound 2
Tablets Mg/unit Raw Material % w/w g/batch 50 mg Tablet 100 mg
Tablet Compound 2 50.0 180.0 50.0 100.0 Microcrystalline 20.0 72.0
20.0 40.0 Cellulose, USP/NF, EP Lactose Monohydrate, 24.0 86.4 24.0
48.0 USP/NF, BP, EP, JP Croscarmellose Sodium, 5.0 18.0 5.0 10.0
USP/NF, EP Magnesium Stearate, 1.0 3.6 1.0 2.0 USP/NF, BP, EP JP
Total 100.0 200.0
[0330] Compound 2 was adjusted based on the as-is assay, with the
adjustment made in the percentage of microcrystalline cellulose.
Compound 2 and excipients (microcrystalline cellulose, lactose
monohydrate, and croscarmellose sodium) were screened, placed into
a V-blender (PK Blendmaster, 0.5 L bowl) and mixed for 5 minutes at
25 rpm. Magnesium stearate was then screened, added and the blend
was mixed for an additional 2 minutes. The common blend was divided
for use in producing 50 mg and 100 mg tablets. The lubricated blend
was then compressed at a speed of 10 tablets/minutes using a single
punch research tablet press (Korsch XP1) and a gravity powder
feeder. The 50 mg tablets were produced using round standard
concave 6 mm tooling and 3.5 kN forces. The 100 mg tablets were
produced using 8 mm round standard concave tooling and 3.9-4.2 kN
forces. The specifications of the 50 mg and 100 mg tablets are
shown in Table 37.
TABLE-US-00041 TABLE 37 Specifications of 50 mg and 100 mg Tablets
of Compound 2 50 mg Tablets 100 mg Tablets Average Weight (n = 10)
100 .+-. 5 mg 200 .+-. 10 mg Individual Weight 100 .+-. 10 mg 200
.+-. 20 mg Hardness 5.3 kp 8.3 kp Disintegration <15 minutes
<15 minutes Friability NMT 0.5% NMT 0.5%
[0331] The 50 mg and 100 mg tablets produced as described above
were subjected to 6 month stability studies under three conditions:
5.degree. C. (refrigeration), 25.degree. C./60% RH (ambient), and
40.degree. C./75% RH (accelerated). Both the 50 mg and 100 mg
tablets were chemically stable under all three conditions
tested.
[0332] Under refrigeration conditions (5.degree. C.), both the 50
mg and 100 mg tablets remained white solids that did not change in
appearance from T=0 to T=6 months. Throughout the 6-month study, no
impurities were reported that were greater than 0.05% for either
the 50 mg tablets or the 100 mg tablets. The water content after 6
months was also less than 3.0% w/w for both tablets. Similar
results were reported when the tablets were subjected to ambient
conditions (25.degree. C./60% RH); no impurities that were greater
than 0.05% were reported throughout the 6 months for both tablets
and the water content did not exceed 3.0% w/w at the 6-month mark.
When the tablets were subjected to accelerated conditions
(40.degree. C./75% RH), the appearance of the 50 mg and 100 mg
tablets did not change from a white, round tablet. One impurity was
reported after 3 months, but the impurity was only 0.09%. A second
impurity was reported after 6 months, but the total impurity
percentage was only 0.21% for both the 50 mg and 100 mg tablets.
Water content was 3.4% w/w at 6 months for the 50 mg tablets and
3.2% w/w for the 100 mg tablets.
[0333] In a separate study, the stability of 50 mg and 100 mg
tablets of Compound 2 at ambient conditions (25.degree. C./60% RH)
was measured over 9 months. The appearance of the 50 mg and 100 mg
tablet did not change from a white round tablet over the course of
9 months. Impurities in the 50 mg tablet were less than 0.10% after
9 months and impurities in the 100 mg tablet were less than 0.05%.
The water content of the 50 mg tablet and the 100 mg tablet after 9
months was only 2.7% w/w and 2.6% w/w, respectively.
[0334] 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.
* * * * *