U.S. patent application number 15/238553 was filed with the patent office on 2017-02-02 for uracyl spirooxetane nucleosides.
The applicant listed for this patent is Janssen Sciences Ireland UC. Invention is credited to Ioannis Nicolaos Houpis, Tim Hugo Maria Jonckers, Pierre Jean-Marie Bernard Raboisson, Abdellah Tahri.
Application Number | 20170029455 15/238553 |
Document ID | / |
Family ID | 48576965 |
Filed Date | 2017-02-02 |
United States Patent
Application |
20170029455 |
Kind Code |
A1 |
Houpis; Ioannis Nicolaos ;
et al. |
February 2, 2017 |
URACYL SPIROOXETANE NUCLEOSIDES
Abstract
The present invention relates to compounds of the formula I:
##STR00001## including any possible stereoisomers thereof, wherein
R.sup.9 has the meaning as defined herein,or a pharmaceutically
acceptable salt or solvate thereof. The present invention also
relates to processes for preparing said compounds, pharmaceutical
compositions containing them and their use, alone or in combination
with other HCV inhibitors, in HCV therapy.
Inventors: |
Houpis; Ioannis Nicolaos;
(Antwerp, BE) ; Jonckers; Tim Hugo Maria;
(Heist-op-den-Berg, BE) ; Raboisson; Pierre Jean-Marie
Bernard; (Rosieres, BE) ; Tahri; Abdellah;
(Anderlecht, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen Sciences Ireland UC |
Little Island |
|
IE |
|
|
Family ID: |
48576965 |
Appl. No.: |
15/238553 |
Filed: |
August 16, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14403587 |
Nov 25, 2014 |
9422323 |
|
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PCT/EP2013/060704 |
May 24, 2013 |
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15238553 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/7072 20130101;
C07H 19/06 20130101; A61P 43/00 20180101; C07H 19/10 20130101; A61P
31/14 20180101; C07H 1/00 20130101; C07H 19/11 20130101; C07H 19/24
20130101 |
International
Class: |
C07H 19/10 20060101
C07H019/10 |
Foreign Application Data
Date |
Code |
Application Number |
May 25, 2012 |
EP |
12169425.1 |
Claims
1. A compound of formula I: ##STR00011## including any possible
stereoisomer thereof, wherein: R.sup.9 is C.sub.1-C.sub.6alkyl,
phenyl, C.sub.3-C.sub.7cycloalkyl or C.sub.1-C.sub.3alkyl
substituted with 1, 2 or 3 substituents each independently selected
from phenyl, naphtyl, C.sub.3-C.sub.6cycloalkyl, hydroxy, or
C.sub.1-C.sub.6alkoxy; or a pharmaceutically acceptable salt or
solvate thereof.
2. A compound according to claim 1 which is of formula Ia:
##STR00012##
3. A compound according to claim 1, wherein R.sup.9 is
C.sub.1-C.sub.6alkyl or C.sub.1-C.sub.2alkyl substituted with
phenyl, C.sub.1-C.sub.2alkoxy or C.sub.3-C.sub.6cycloalkyl.
4. A compound according to claim 1, wherein R.sup.9 is
C.sub.2-C.sub.4alkyl.
5. A compound according to claim 1, wherein R.sup.9 is
i-propyl.
6. A compound according to claim 1, which is of formula Ib:
##STR00013##
7. (canceled)
8. (canceled)
9. A compound of formula VI: ##STR00014## including any
stereochemical form, pharmaceutically acceptable salt or solvate
thereof.
10. A pharmaceutical composition comprising a compound according to
claim 1, and a pharmaceutically acceptable carrier.
11. (canceled)
12. A compound according to claim 1 for use in the prevention or
treatment of an HCV infection in a mammal.
13. A product containing (a) a compound of formula I as defined in
claim 1, and (b) another HCV inhibitor, as a combined preparation
for simultaneous, separate or sequential use in the treatment of
HCV infections.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to spirooxetane nucleosides and
nucleotides that are inhibitors of the hepatitis C virus (HCV).
[0002] HCV is a single stranded, positive-sense RNA virus belonging
to the Flaviviridae family of viruses in the hepacivirus genus. The
NS5B region of the RNA polygene encodes a RNA dependent RNA
polymerase (RdRp), which is essential to viral replication.
Following the initial acute infection, a majority of infected
individuals develop chronic hepatitis because HCV replicates
preferentially in hepatocytes but is not directly cytopathic. In
particular, the lack of a vigorous T-lymphocyte response and the
high propensity of the virus to mutate appear to promote a high
rate of chronic infection. Chronic hepatitis can progress to liver
fibrosis, leading to cirrhosis, end-stage liver disease, and HCC
(hepatocellular carcinoma), making it the leading cause of liver
transplantations. There are six major HCV genotypes and more than
50 subtypes, which are differently distributed geographically. HCV
genotype 1 is the predominant genotype in Europe and in the US. The
extensive genetic heterogeneity of HCV has important diagnostic and
clinical implications, perhaps explaining difficulties in vaccine
development and the lack of response to current therapy.
[0003] Transmission of HCV can occur through contact with
contaminated blood or blood products, for example following blood
transfusion or intravenous drug use. The introduction of diagnostic
tests used in blood screening has led to a downward trend in
post-transfusion HCV incidence. However, given the slow progression
to the end-stage liver disease, the existing infections will
continue to present a serious medical and economic burden for
decades.
[0004] Current HCV therapy is based on (pegylated) interferon-alpha
(IFN-.alpha.) in combination with ribavirin. This combination
therapy yields a sustained virologic response in more than 40% of
patients infected by genotype 1 HCV and about 80% of those infected
by genotypes 2 and 3. Beside the limited efficacy against HCV
genotype 1, this combination therapy has significant side effects
and is poorly tolerated in many patients. Major side effects
include influenza-like symptoms, hematologic abnormalities, and
neuropsychiatric symptoms. Hence there is a need for more
effective, convenient and better-tolerated treatments.
[0005] Recently, therapy possibilities have extended towards the
combination of a HCV protease inhibitor (e.g. Telaprevir or
boceprevir) and (pegylated) interferon-alpha
(IFN-.alpha.)/ribavirin.
[0006] Experience with HIV drugs, in particular with HIV protease
inhibitors, has taught that sub-optimal pharmacokinetics and
complex dosing regimes quickly result in inadvertent compliance
failures. This in turn means that the 24 hour trough concentration
(minimum plasma concentration) for the respective drugs in an HIV
regime frequently falls below the IC.sub.90 or ED.sub.90 threshold
for large parts of the day. It is considered that a 24 hour trough
level of at least the IC.sub.50, and more realistically, the
IC.sub.90 or ED.sub.90, is essential to slow down the development
of drug escape mutants. Achieving the necessary pharmacokinetics
and drug metabolism to allow such trough levels provides a
stringent challenge to drug design.
[0007] The NS5B RdRp is essential for replication of the
single-stranded, positive sense, HCV RNA genome. This enzyme has
elicited significant interest among medicinal chemists. Both
nucleoside and non-nucleoside inhibitors of NS5B are known.
Nucleoside inhibitors can act as a chain terminator or as a
competitive inhibitor, or as both. In order to be active,
nucleoside inhibitors have to be taken up by the cell and converted
in vivo to a triphosphate. This conversion to the triphosphate is
commonly mediated by cellular kinases, which imparts additional
structural requirements on a potential nucleoside polymerase
inhibitor. In addition this limits the direct evaluation of
nucleosides as inhibitors of HCV replication to cell-based assays
capable of in situ phosphorylation.
[0008] Several attempts have been made to develop nucleosides as
inhibitors of HCV RdRp, but while a handful of compounds have
progressed into clinical development, none have proceeded to
registration. Amongst the problems which HCV-targeted nucleosides
have encountered to date are toxicity, mutagenicity, lack of
selectivity, poor efficacy, poor bioavailability, sub-optimal
dosage regimes and ensuing high pill burden and cost of goods.
[0009] Spirooxetane nucleosides, in particular
1-(8-hydroxy-7-(hydroxy-methyl)-1,6-dioxaspiro[3.41]octan-5-yl)pyrimidine-
-2,4-dione derivatives and their use as HCV inhibitors are known
from WO2010/130726, and WO2012/062869, including
CAS-1375074-52-4.
[0010] There is a need for HCV inhibitors that may overcome at
least one of the disadvantages of current HCV therapy such as side
effects, limited efficacy, the emerging of resistance, and
compliance failures, or improve the sustained viral response.
[0011] The present invention concerns a group of HCV-inhibiting
uracyl spirooxetane derivatives with useful properties regarding
one or more of the following parameters: antiviral efficacy towards
at least one of the following genotypes 1a, 1b, 2a, 2b, 3,4 and 6,
favorable profile of resistance development, lack of toxicity and
genotoxicity, favorable pharmacokinetics and pharmacodynamics and
ease of formulation and administration.
DESCRIPTION OF THE INVENTION
[0012] In one aspect the present invention provides compounds that
can be represented by the formula I:
##STR00002##
including any possible stereoisomer thereof, wherein:
[0013] R.sup.9 is C.sub.1-C.sub.6alkyl, phenyl,
C.sub.3-C.sub.7cycloalkyl or C.sub.1-C.sub.3alkyl substituted with
1, 2 or 3 substituents each independently selected from phenyl,
naphtyl, C.sub.3-C.sub.6cycloalkyl, hydroxy, or
C.sub.1-C.sub.6alkoxy;
[0014] or a pharmaceutically acceptable salt or solvate
thereof.
[0015] Of particular interest are compounds of formula I or
subgroups thereof as defined herein, that have a structure
according to formula Ia:
##STR00003##
[0016] In one embodiment of the present invention, R.sup.9 is
C.sub.1-C.sub.6alkyl, phenyl, C.sub.3-C.sub.7cycloalkyl or
C.sub.1-C.sub.3alkyl substituted with 1 substituent selected from
phenyl, C.sub.3-C.sub.6cycloalkyl, hydroxy, or
C.sub.1-C.sub.6alkoxy. In another embodiment of the present
invention, R.sup.9 in Formula I or Ia is C.sub.1-C.sub.6alkyl or
C.sub.1-C.sub.2alkyl substituted with phenyl C.sub.1-C.sub.2alkoxy
or C.sub.3-C.sub.6cycloalkyl. In a more preferred embodiment,
R.sup.9 is C.sub.2-C.sub.4alkyl and in a most preferred embodiment,
R.sup.9 is i-propyl.
[0017] A preferred embodiment according to the invention is a
compound according to formula Ib:
##STR00004##
including any pharmaceutically acceptable salt or solvate thereof
and the use of compound (V) in the synthesis of a compound
according to Formula I, Ia or Ib.
[0018] The invention further relates to a compound of formula
V:
##STR00005##
including any pharmaceutically acceptable salt or solvate thereof
and the use of compound (V) in the synthesis of a compound
according to Formula I, Ia or Ib.
[0019] In addition, the invention relates to a compound of formula
VI:
##STR00006##
including any stereochemical form and/or pharmaceutically
acceptable salt or solvate thereof
[0020] Additionally, the invention relates to a pharmaceutical
composition comprising a compound according to Formula I, Ia or Ib,
and a pharmaceutically acceptable carrier. The invention also
relates to a product containing (a) a compound of formula I, Ia or
Ib a, and (b) another HCV inhibitor, as a combined preparation for
simultaneous, separate or sequential use in the treatment of HCV
infections
[0021] Yet another aspect of the invention relates to a compound
according to Formula I, Ia or Ib or a pharmaceutical composition
according to the present invention for use as a medicament,
preferably for use in the prevention or treatment of an HCV
infection in a mammal.
[0022] In a further aspect, the invention provides a compound of
formula I Ia or Ib or a pharmaceutically acceptable salt, hydrate,
or solvate thereof, for use in the treatment or prophylaxis (or the
manufacture of a medicament for the treatment or prophylaxis) of
HCV infection. Representative HCV genotypes in the context of
treatment or prophylaxis in accordance with the invention include
genotype 1b (prevalent in Europe) or 1a (prevalent in North
America). The invention also provides a method for the treatment or
prophylaxis of HCV infection, in particular of the genotype 1a or
1b.
[0023] Of particular interest is compound 8a mentioned in the
section "Examples" as well as the pharmaceutically acceptable acid
addition salts of this compound.
[0024] The compounds of formula I have several centers of
chirality, in particular at the carbon atoms 1', 2', 3', and 4'.
Although the stereochemistry at these carbon atoms is fixed, the
compounds may display at least 75%, preferably at least 90%, such
as in excess of 95%, or of 98%, enantiomeric purity at each of the
chiral centers.
[0025] The phosphorus center can be present as R.sub.p or S.sub.p,
or a mixture of such stereoisomers, including racemates.
Diastereoisomers resulting from the chiral phosphorus center and a
chiral carbon atom may exist as well.
[0026] The compounds of formula I are represented as a defined
stereoisomer, except for the stereoisomerism at the phosphorous
atom. The absolute configuration of such compounds can be
determined using art-known methods such as, for example, X-ray
diffraction or NMR and/or implication from starting materials of
known stereochemistry. Pharmaceutical compositions in accordance
with the invention will preferably comprise stereoisomerically pure
forms of the indicated stereoisomer of the particular compound of
formula I.
[0027] Pure stereoisomeric forms of the compounds and intermediates
as mentioned herein are defined as isomers substantially free of
other enantiomeric or diastereomeric forms of the same basic
molecular structure of said compounds or intermediates. In
particular, the term "stereoisomerically pure" concerns compounds
or intermediates having a stereoisomeric excess of at least 80%
(i.e. minimum 90% of one isomer and maximum 10% of the other
possible isomers) up to a stereoisomeric excess of 100% (i.e. 100%
of one isomer and none of the other), more in particular, compounds
or intermediates having a stereoisomeric excess of 90% up to 100%,
even more in particular having a stereoisomeric excess of 94% up to
100% and most in particular having a stereoisomeric excess of 97%
up to 100%, or of 98% up to 100%. The terms "enantiomerically pure"
and "diastereomerically pure" should be understood in a similar
way, but then having regard to the enantiomeric excess, and the
diastereomeric excess, respectively, of the mixture in
question.
[0028] Pure stereoisomeric forms of the compounds and intermediates
of this invention may be obtained by the application of art-known
procedures. For instance, enantiomers may be separated from each
other by the selective crystallization of their diastereomeric
salts with optically active acids or bases. Examples thereof are
tartaric acid, dibenzoyl-tartaric acid, ditoluoyltartaric acid and
camphorsulfonic acid. Alternatively, enantiomers may be separated
by chromatographic techniques using chiral stationary layers. Said
pure stereochemically isomeric forms may also be derived from the
corresponding pure stereochemically isomeric forms of the
appropriate starting materials, provided that the reaction occurs
stereospecifically. Preferably, if a specific stereoisomer is
desired, said compound is synthesized by stereospecific methods of
preparation. These methods will advantageously employ
enantiomerically pure starting materials.
[0029] The diastereomeric racemates of the compounds of formula I
can be obtained separately by conventional methods. Appropriate
physical separation methods that may advantageously be employed
are, for example, selective crystallization and chromatography,
e.g. column chromatography.
[0030] The pharmaceutically acceptable addition salts comprise the
therapeutically active non-toxic acid and base addition salt forms
of the compounds of formula I. Of interest are the free, i.e.
non-salt forms of the compounds of formula I, or of any subgroup of
compounds of formula I specified herein.
[0031] The pharmaceutically acceptable acid addition salts can
conveniently be obtained by treating the base form with such
appropriate acid. Appropriate acids comprise, for example,
inorganic acids such as hydrohalic acids, e.g. hydrochloric or
hydrobromic acid, sulfuric, nitric, phosphoric and the like acids;
or organic acids such as, for example, acetic, propionic,
hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic,
succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e.
hydroxyl-butanedioic acid), tartaric, citric, methanesulfonic,
ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic,
salicylic, p-aminosalicylic, palmoic and the like acids. Conversely
said salt forms can be converted by treatment with an appropriate
base into the free base form.
[0032] The compounds of formula I containing an acidic proton may
also be converted into their non-toxic metal or amine addition salt
forms by treatment with appropriate organic and inorganic bases.
Appropriate base salt forms comprise, for example, the ammonium
salts, the alkali and earth alkaline metal salts, e.g. the lithium,
sodium, potassium, magnesium, calcium salts and the like, salts
with organic bases, e.g. the benzathine, N-methyl-D-glucamine,
hydrabamine salts, and salts with amino acids such as, for example,
arginine, lysine and the like.
[0033] The term "solvates" covers any pharmaceutically acceptable
solvates that the compounds of formula I as well as the salts
thereof, are able to form. Such solvates are for example hydrates,
alcoholates, e.g. ethanolates, propanolates, and the like.
[0034] Some of the compounds of formula I may also exist in their
tautomeric form. For example, tautomeric forms of amide
(--C(.dbd.O)--NH--) groups are iminoalcohols (--C(OH).dbd.N--),
which can become stabilized in rings with aromatic character. The
uridine base is an example of such a form. Such forms, although not
explicitly indicated in the structural formulae represented herein,
are intended to be included within the scope of the present
invention.
SHORT DESCRIPTION OF THE FIGURE
[0035] FIG. 1: In vivo efficacy of compound 8a and CAS-1375074-52-4
as determined in a humanized hepatocyte mouse model.
DEFINITIONS
[0036] As used herein "C.sub.1-C.sub.nalkyl" as a group or part of
a group defines saturated straight or branched chain hydrocarbon
radicals having from 1 to n carbon atoms. Accordingly,
"C.sub.1-C.sub.4alkyl" as a group or part of a group defines
saturated straight or branched chain hydrocarbon radicals having
from 1 to 4 carbon atoms such as for example methyl, ethyl,
1-propyl, 2-propyl, 1-butyl, 2-butyl, 2-methyl-1-propyl,
2-methyl-2-propyl. "C.sub.1-C.sub.6alkyl" encompasses
C.sub.1-C.sub.4alkyl radicals and the higher homologues thereof
having 5 or 6 carbon atoms such as, for example, 1-pentyl,
2-pentyl, 3-pentyl, 1-hexyl, 2-hexyl, 2-methyl-1-butyl,
2-methyl-1-pentyl, 2-ethyl-1-butyl, 3-methyl-2-pentyl, and the
like. Of interest amongst C.sub.1-C.sub.6alkyl is
C.sub.1-C.sub.4alkyl.
[0037] `C.sub.1-C.sub.nalkoxy` means a radical
--O--C.sub.1-C.sub.11alkyl wherein C.sub.1-C.sub.nalkyl is as
defined above. Accordingly, `C.sub.1-C.sub.6alkoxy` means a radical
--O--C.sub.1-C.sub.6alkyl wherein C.sub.1-C.sub.6alkyl is as
defined above. Examples of C.sub.1-C.sub.6alkoxy are methoxy,
ethoxy, n-propoxy, or isopropoxy. Of interest is
`C.sub.1-C.sub.2alkoxy`, encompassing methoxy and ethoxy.
[0038] "C.sub.3-C.sub.6cycloalkyl" includes cyclopropyl,
cyclobutyl, cyclopentyl, and cyclohexyl.
[0039] In one embodiment, the term "phenyl-C.sub.1-C.sub.6alkyl" is
benzyl.
[0040] As used herein, the term `(.dbd.O)` or `oxo` forms a
carbonyl moiety when attached to a carbon atom. It should be noted
that an atom can only be substituted with an oxo group when the
valency of that atom so permits.
[0041] The term "monophosphate, diphosphate or triphosphate ester"
refers to groups:
##STR00007##
[0042] Where the position of a radical on a molecular moiety is not
specified (for example a sunstituent on phenyl) or is represented
by a floating bond, such radical may be positioned on any atom of
such a moiety, as long as the resulting structure is chemically
stable. When any variable is present more than once in the
molecule, each definition is independent.
[0043] Whenever used herein, the term `compounds of formula I`, or
`the present compounds` or similar terms, it is meant to include
the compounds of Formula I, Ia and Ib, including the possible
stereochemically isomeric forms, and their pharmaceutically
acceptable salts and solvates.
[0044] The present invention also includes isotope-labeled
compounds of formula I or any subgroup of formula I, wherein one or
more of the atoms is replaced by an isotope that differs from the
one(s) typically found in nature. Examples of such isotopes include
isotopes of hydrogen, such as .sup.2H and .sup.3H; carbon, such as
.sup.11C, .sup.13C and .sup.14C nitrogen, such as .sup.13N and
.sup.15N; oxygen, such as .sup.15O, .sup.17O and .sup.18O;
phosphorus, such as .sup.31P and .sup.32P, sulphur, such as
.sup.35S; fluorine, such as .sup.18F; chlorine, such as .sup.36Cl;
bromine such as .sup.75Br, .sup.76Br, .sup.77Br and .sup.82Br; and
iodine, such as .sup.123I, .sup.124I and .sup.131I. Isotope-labeled
compounds of the invention can be prepared by processes analogous
to those described herein by using the appropriate isotope-labeled
reagents or starting materials, or by art-known techniques. The
choice of the isotope included in an isotope-labeled compound
depends on the specific application of that compound. For example,
for tissue distribution assays, a radioactive isotope such as
.sup.3H or .sup.14C is incorporated. For radio-imaging
applications, a positron emitting isotope such as .sup.11C,
.sup.18F, .sup.13N or .sup.15O will be useful. The incorporation of
deuterium may provide greater metabolic stability, resulting in,
e.g. an increased in vivo half life of the compound or reduced
dosage requirements.
[0045] General Synthetic Procedures
[0046] The following schemes are just meant to be illustrative and
are by no means limiting the scope.
[0047] The starting material
1-[(4R,5R,7R,8R)-8-hydroxy-7-(hydroxymethyl)-1,6-dioxa-spiro[3.41]octan-5-
-yl]pyrimidine-2,4(1H,3H )-dione (1) can be prepared as exemplified
in WO2010/130726. Compound (1) is converted into compounds of the
present invention via a p-methoxybenzyl protected derivative (4) as
exemplified in the
##STR00008##
[0048] In Scheme 1, R.sup.9 can be C.sub.1-C.sub.6alkyl, phenyl,
naphtyl, C.sub.3-C.sub.7cycloalkyl or C.sub.1-C.sub.3alkyl
substituted with 1,2 or 3 substituents each independently selected
from phenyl, C.sub.3-C.sub.6cycloalkyl, hydroxy, or
C.sub.1-C.sub.6alkoxy, preferably R.sup.9 is C.sub.1-C.sub.6alkyl
or C.sub.1-C.sub.2alkyl substituted with phenyl,
C.sub.1-C.sub.2alkoxy or C.sub.3-C.sub.6cycloalkyl, even more
preferably R.sup.9 is C.sub.2-C.sub.4alkyl and most preferably
R.sup.9 is i-propyl.
[0049] In a further aspect, the present invention concerns a
pharmaceutical composition comprising a therapeutically effective
amount of a compound of formula I as specified herein, and a
pharmaceutically acceptable carrier. Said composition may contain
from 1% to 50%, or from 10% to 40% of a compound of formula I and
the remainder of the composition is the said carrier. A
therapeutically effective amount in this context is an amount
sufficient to act in a prophylactic way against HCV infection, to
inhibit HCV, to stabilize or to reduce HCV infection, in infected
subjects or subjects being at risk of becoming infected. In still a
further aspect, this invention relates to a process of preparing a
pharmaceutical composition as specified herein, which comprises
intimately mixing a pharmaceutically acceptable carrier with a
therapeutically effective amount of a compound of formula I, as
specified herein.
[0050] The compounds of formula I or of any subgroup thereof may be
formulated into various pharmaceutical forms for administration
purposes. As appropriate compositions there may be cited all
compositions usually employed for systemically administering drugs.
To prepare the pharmaceutical compositions of this invention, an
effective amount of the particular compound, optionally in addition
salt form or metal complex, as the active ingredient is combined in
intimate admixture with a pharmaceutically acceptable carrier,
which carrier may take a wide variety of forms depending on the
form of preparation desired for administration. These
pharmaceutical compositions are desirable in unitary dosage form
suitable, particularly, for administration orally, rectally,
percutaneously, or by parenteral injection. For example, in
preparing the compositions in oral dosage form, any of the usual
pharmaceutical media may be employed such as, for example, water,
glycols, oils, alcohols and the like in the case of oral liquid
preparations such as suspensions, syrups, elixirs, emulsions and
solutions; or solid carriers such as starches, sugars, kaolin,
lubricants, binders, disintegrating agents and the like in the case
of powders, pills, capsules, and tablets. Because of their ease in
administration, tablets and capsules represent the most
advantageous oral dosage unit forms, in which case solid
pharmaceutical carriers are obviously employed. For parenteral
compositions, the carrier will usually comprise sterile water, at
least in large part, though other ingredients, for example, to aid
solubility, may be included. Injectable solutions, for example, may
be prepared in which the carrier comprises saline solution, glucose
solution or a mixture of saline and glucose solution. Injectable
suspensions may also be prepared in which case appropriate liquid
carriers, suspending agents and the like may be employed. Also
included are solid form preparations intended to be converted,
shortly before use, to liquid form preparations. In the
compositions suitable for percutaneous administration, the carrier
optionally comprises a penetration enhancing agent and/or a
suitable wetting agent, optionally combined with suitable additives
of any nature in minor proportions, which additives do not
introduce a significant deleterious effect on the skin. The
compounds of the present invention may also be administered via
oral inhalation or insufflation in the form of a solution, a
suspension or a dry powder using any art-known delivery system.
[0051] It is especially advantageous to formulate the
aforementioned pharmaceutical compositions in unit dosage form for
ease of administration and uniformity of dosage. Unit dosage form
as used herein refers to physically discrete units suitable as
unitary dosages, each unit containing a predetermined quantity of
active ingredient calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
Examples of such unit dosage forms are tablets (including scored or
coated tablets), capsules, pills, suppositories, powder packets,
wafers, injectable solutions or suspensions and the like, and
segregated multiples thereof
[0052] The compounds of formula I show activity against HCV and can
be used in the treatment and/or prophylaxis of HCV infection or
diseases associated with HCV. The latter include progressive liver
fibrosis, inflammation and necrosis leading to cirrhosis, end-stage
liver disease, and HCC. The compounds of this invention moreover
are believed to be active against mutated strains of HCV and show a
favorable pharmacokinetic profile and have attractive properties in
terms of bioavailability, including an acceptable half-life, AUC
(area under the curve) and peak values and lacking unfavorable
phenomena such as insufficient quick onset and tissue
retention.
[0053] The in vitro antiviral activity against HCV of the compounds
of formula I can be tested in a cellular HCV replicon system based
on Lohmann et al. (1999) Science 285:110-113, with the further
modifications described by Krieger et al. (2001) Journal of
Virology 75: 4614-4624 (incorporated herein by reference), which is
further exemplified in the examples section. This model, while not
a complete infection model for HCV, is widely accepted as the most
robust and efficient model of autonomous HCV RNA replication
currently available. It will be appreciated that it is important to
distinguish between compounds that specifically interfere with HCV
functions from those that exert cytotoxic or cytostatic effects in
the HCV replicon model, and as a consequence cause a decrease in
HCV RNA or linked reporter enzyme concentration. Assays are known
in the field for the evaluation of cellular cytotoxicity based for
example on the activity of mitochondrial enzymes using fluorogenic
redox dyes such as resazurin. Furthermore, cellular counter screens
exist for the evaluation of non-selective inhibition of linked
reporter gene activity, such as firefly luciferase. Appropriate
cell types can be equipped by stable transfection with a luciferase
reporter gene whose expression is dependent on a constitutively
active gene promoter, and such cells can be used as a
counter-screen to eliminate non-selective inhibitors.
[0054] Due to their anti-HCV properties, the compounds of formula
I, including any possible stereoisomers, the pharmaceutically
acceptable addition salts or solvates thereof, are useful in the
treatment of warm-blooded animals, in particular humans, infected
with HCV, and in the prophylaxis of HCV infections. The compounds
of the present invention may therefore be used as a medicine, in
particular as an anti-HCV or a HCV-inhibiting medicine. The present
invention also relates to the use of the present compounds in the
manufacture of a medicament for the treatment or the prevention of
HCV infection. In a further aspect, the present invention relates
to a method of treating a warm-blooded animal, in particular human,
infected by HCV, or being at risk of becoming infected by HCV, said
method comprising the administration of an anti-HCV effective
amount of a compound of formula I, as specified herein. Said use as
a medicine or method of treatment comprises the systemic
administration to HCV-infected subjects or to subjects susceptible
to HCV infection of an amount effective to combat the conditions
associated with HCV infection.
[0055] In general it is contemplated that an antiviral effective
daily amount would be from about 1 to about 30 mg/kg, or about 2 to
about 25 mg/kg, or about 5 to about 15 mg/kg, or about 8 to about
12 mg/kg body weight. Average daily doses can be obtained by
multiplying these daily amounts by about 70. It may be appropriate
to administer the required dose as two, three, four or more
sub-doses at appropriate intervals throughout the day. Said
sub-doses may be formulated as unit dosage forms, for example,
containing about 1 to about 2000 mg, or about 50 to about 1500 mg,
or about 100 to about 1000 mg, or about 150 to about 600 mg, or
about 100 to about 400 mg of active ingredient per unit dosage
form.
[0056] As used herein the term "about" has the meaning known to the
person skilled in the art. In certain embodiments the term "about"
may be left out and the exact amount is meant. In other embodiments
the term "about" means that the numerical following the term
"about" is in the range of .+-.15%, or of .+-.10%, or of .+-.5%, or
of .+-.1%, of said numerical value.
EXAMPLES
[0057] Synthesis of Compound (8a)
##STR00009##
[0058] Synthesis of Compound (2)
[0059] Compound (2) can be prepared by dissolving compound (1) in
pyridine and adding 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane.
The reaction is stirred at room temperature until complete. The
solvent is removed and the product redissolved in CH.sub.2Cl.sub.2
and washed with saturated NaHCO.sub.3 solution. Drying on
MgSO.sub.4 and removal of the solvent gives compound (2).
[0060] Synthesis of Compound (3)
[0061] Compound (3) is prepared by reacting compound (2) with
p-methoxybenzylchloride in the presence of DBU as the base in
CH.sub.3CN.
[0062] Synthesis of Compound (4)
[0063] Compound (4) is prepared by cleavage of the bis-silyl
protecting group in compound (3) using TBAF as the fluoride
source.
[0064] Synthesis of Compound (6a)
[0065] A solution of isopropyl alcohol (3.86 mL,0.05mol) and
triethylamine (6.983 mL, 0.05 mol) in dichloromethane (50 mL) was
added to a stirred solution of POCl.sub.3 (5) (5.0 mL, 0.0551mol)
in DCM (50 mL) dropwise over a period of 25 min at -5.degree. C.
After the mixture stirred for 1 h, the solvent was evaporated, and
the residue was suspended in ether (100 mL). The triethylamine
hydrochloride salt was filtered and washed with ether (20 mL). The
filtrate was concentrated, and the residue was distilled to give
the (6) as a colorless liquid (6.1g, 69% yield).
[0066] Synthesis of Compound (7a)
[0067] To a stirred suspension of (4) (2.0 g, 5.13 mmol) in
dichloromethane (50 mL) was added triethylamine (2.07 g, 20.46
mmol) at room temperature. The reaction mixture was cooled to
-20.degree. C., and then (6a) (1.2 g, 6.78 mmol) was added dropwise
over a period of 10 min. The mixture was stirred at this
temperature for 15 min and then NMI was added (0.84 g, 10.23 mmol),
dropwise over a period of 15 min. The mixture was stirred at
-15.degree. C. for 1 h and then slowly warmed to room temperature
in 20 h. The solvent was evaporated, the mixture was concentrated
and purified by column chromatography using petroleum ether/EtOAc
(10:1 to 5:1 as a gradient) to give (7a) as white solid (0.8 g, 32%
yield).
[0068] Synthesis of Compound (8a)
[0069] To a solution of (7a) in CH.sub.3CN (30 mL) and H.sub.2O (7
mL) was add CAN portion wise below 20.degree. C. The mixture was
stirred at 15-20.degree. C. for 5 h under N.sub.2. Na.sub.2SO.sub.3
(370 mL) was added dropwise into the reaction mixture below
15.degree. C., and then Na.sub.2CO.sub.3 (370 mL) was added. The
mixture was filtered and the filtrate was extracted with
CH.sub.2C1.sub.2 (100 mL*3). The organic layer was dried and
concentrated to give the residue. The residue was purified by
column chromatography to give the target compound (8a) as white
solid. (Yield: 55%)
[0070] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.45 (dd,
J=7.53, 6.27 Hz, 6H), 2.65 -2.84 (m, 2H), 3.98 (td, J=10.29, 4.77
Hz, 1H), 4.27 (t, J=9.66 Hz, 1H), 4.43 (ddd, J=8.91, 5.77, 5.65 Hz,
1H), 4.49-4.61 (m, 1H), 4.65 (td, J=7.78, 5.77 Hz, 1H), 4.73 (d,
J=7.78 Hz, 1H), 4.87 (dq, J=12.74, 6.30 Hz, 1H), 5.55 (br. s., 1H),
5.82 (d, J=8.03 Hz, 1H), 7.20 (d, J=8.03 Hz, 1H), 8.78 (br. s.,
1H); .sup.31P NMR (CHLOROFORM-d) .delta. ppm -7.13; LC-MS: 375
(M+1)+
[0071] Synthesis of Compound (VI)
##STR00010##
[0072] Step 1: Synthesis of Compound (9)
[0073] Compound (1), CAS 1255860-33-3 (1200 mg, 4.33 mmol) and
1,8-bis(dimethyl-amino)naphihalene (3707 mg. 17.3 mmol) were
dissolved in 24.3 mL of trimethylphosphate. The solution was cooled
to 0.degree. C. Compound (5) (1.21 mL, 12.98 mmol) was added, and
the mixture was stirred well maintaining the temperature at
0.degree. C. for 5 hours. The reaction was quenched by addition of
120 mL of tetraethyl-ammonium bromide solution (1M) and extracted
with CH.sub.2Cl.sub.2 (2.times.80 mL). Purification was done by
preparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-10
.mu.m, 30.times.150 mm, mobile phase: 0.25% NH.sub.4HCO.sub.3
solution in water, CH.sub.3CN), yielding two fractions. The purest
fraction was dissolved in water (15 mL) and passed through a
manually packed Dowex (H.sup.+) column by elution with water. The
end of the elution was determined by checking UV absorbance of
eluting fractions. Combined fractions were frozen at -78.degree. C.
and lyophilized. Compound (9) was obtained as a white fluffy solid
(303 mg, (0.86 mmol, 20% yield), which was used immediately in the
following reaction.
[0074] Step 2: Preparation of Compound (VI)
[0075] Compound (9) (303 mg, 0.86 mmol) was dissolved in 8 mL water
and to this solution was added NN-Dicyclohexyl-4-morpholine
carboxamidine (253.8 mg, 0.86 mmol) dissolved in pyridine (8.4 mL).
The mixture was kept for 5 minutes and then evaporated to dryness,
dried overnight in vacuo overnight at 37.degree. C. The residu was
dissolved in pyridine (80 mL). This solution was added dropwise to
vigorously stirred DCC (892.6 mg, 4.326 mmol) in pyridine (80 mL)
at reflux temperature. The solution was kept refluxing for 1.5 h
during which some turbidity was observed in the solution. The
reaction mixture was cooled and evaporated to dryness. Diethylether
(50 mL) and water (50 mL) were added to the solid residu.
N'N-dicyclohexylurea was filtered off, and the aqueous fraction was
purified by preparative HPLC (Stationary phase: RP XBridge Prep C18
OBD-10 .mu.m, 30.times.150 mm, mobile phase: 0.25%
NH.sub.4HCO.sub.3 solution in water, CH.sub.3CN), yielding a white
solid which was dried overnight in vacuo at 38.degree. C. (185 mg,
0.56 mmol, 65% yield). LC-MS: (M+H).sup.+: 333. .sup.1H NMR (400
MHz, DMSO-d.sub.6) d ppm 2.44-2.59 (m, 2H) signal falls under DMSO
signal, 3.51 (td, J=9.90, 5.50 Hz, 1H), 3.95-4.11 (m, 2H), 4.16 (d,
J=10.34 Hz, 1H), 4.25-4.40 (m, 2H), 5.65 (d, J=8.14 Hz, 1H), 5.93
(br. s., 1H), 7.46 (d, J=7.92 Hz, 1H), 2H's not observed
BIOLOGICAL EXAMPLES
[0076] Replicon Assays
[0077] The compounds of formula I were examined for activity in the
inhibition of HCV-RNA replication in a cellular assay. The assay
was used to demonstrate that the compounds of formula I inhibited a
HCV functional cellular replicating cell line, also known as HCV
replicons. The cellular assay was based on a bicistronic expression
construct, as described by Lohmann et al. (1999) Science vol. 285
pp. 110-113 with modifications described by Krieger et al. (2001)
Journal of Virology 75: 4614-4624, in a multi-target screening
strategy.
[0078] Replicon Assay (A)
[0079] In essence, the method was as follows. The assay utilized
the stably transfected cell line Huh-7 luc/neo (hereafter referred
to as Huh-Luc). This cell line harbors an RNA encoding a
bicistronic expression construct comprising the wild type NS3-NS5B
regions of HCV type 1b translated from an internal ribosome entry
site (IRES) from encephalomyocarditis virus (EMCV), preceded by a
reporter portion (FfL-luciferase), and a selectable marker portion
(neo.sup.R, neomycine phosphotransferase). The construct is
bordered by 5' and 3' NTRs (non-translated regions) from HCV
genotype 1b.
[0080] Continued culture of the replicon cells in the presence of
G418 (neo.sup.R) is dependent on the replication of the HCV-RNA.
The stably transfected replicon cells that express HCV-RNA, which
replicates autonomously and to high levels, encoding inter alia
luciferase, were used for screening the antiviral compounds.
[0081] The replicon cells were plated in 384-well plates in the
presence of the test and control compounds which were added in
various concentrations. Following an incubation of three days, HCV
replication was measured by assaying luciferase activity (using
standard luciferase assay substrates and reagents and a Perkin
Elmer ViewLux.TM. ultraHTS microplate imager). Replicon cells in
the control cultures have high luciferase expression in the absence
of any inhibitor. The inhibitory activity of the compound on
luciferase activity was monitored on the Huh-Luc cells, enabling a
dose-response curve for each test compound. EC.sub.50 values were
then calculated, which value represents the amount of the compound
required to decrease the level of detected luciferase activity by
50%, or more specifically, the ability of the genetically linked
HCV replicon RNA to replicate.
[0082] Results (A)
[0083] Table 1 shows the replicon results (EC.sub.50, replicon) and
cytotoxicity results (CC.sub.50 (.mu.M) (Huh-7)) obtained for the
compound of the examples given above.
TABLE-US-00001 TABLE 1 Compound EC.sub.50 (.mu.M) CC.sub.50 (.mu.M)
number (HCV) (Huh-7) 8a 0.13 (n = 4) >100
[0084] Replicon Assays (B)
[0085] Further replicon assays were performed with compound 8a of
which the protocols and results are disclosed below.
[0086] Assay 1
[0087] The anti-HCV activity of compound 8a was tested in cell
culture with replicon cells generated using reagents from the
Bartenschlager laboratory (the HCV 1b bicistronic subgenomic
luciferase reporter replicon clone ET). The protocol included a
3-day incubation of 2500 replicon cells in a 384-well format in a
nine-point 1:4 dilution series of the compound. Dose response
curves were generated based on the firefly luciferase read-out. In
a variation of this assay, a 3 day incubation of 3000 cells in a
96-well format in a nine-point dilution series was followed by
qRT-PCR Taqman detection of HCV genome, and normalized to the
cellular transcript, RPL13 (of the ribosomal subunit RPL13 gene) as
a control for compound inhibition of cellular transcription.
[0088] Assay 2
[0089] The anti-HCV activity of compound 8a was tested in cell
culture with replicon cells generated using reagents from the
Bartenschlager laboratory (the HCV lb bicistronic subgenomic
luciferase reporter replicon clone ET or Huh-Luc-Neo). The protocol
included a 3-day incubation of 2.times.10.sup.4 replicon cells in a
96-well format in a six-point 1:5 dilution series of the compound.
Dose response curves were generated based on the luciferase
read-out.
[0090] Assay 3
[0091] The anti-HCV activity of compound 8a was tested in cell
culture with replicon cells generated using reagents from the
Bartenschlager laboratory (the HCV 1b bicistronic subgenomic
luciferase reporter replicon clone ET or Huh-Luc-Neo). The protocol
included either a 3-day incubation of 8.times.10.sup.3 cells or
2.times.10.sup.4 cells in a 96-well format in an eight-point 1:5
dilution series of the compound. Dose response curves were
generated based on the luciferase read-out.
[0092] Results
[0093] Table 2 shows the average replicon results (EC.sub.50,
replicon) obtained for compound 8a following assays as given
above.
TABLE-US-00002 TABLE 2 Assay Average EC.sub.50 value (8a): 1 57
.mu.M (n = 8) 2 17.5 .mu.M (n = 4) 3 >100 .mu.M (n = 1)
[0094] Primary Human Hepatocyte in Vitro Assay
[0095] The anti-HCV activity of compound 8a was determined in an in
vitro primary human hepatocyte assay. Protocols and results are
disclosed below.
[0096] Protocol
[0097] Hepatocyte Isolation and Culture
[0098] Primary human hepatocytes (PHH) were prepared from patients
undergoing partial hepatectomy for metastases or benign tumors.
Fresh human hepatocytes were isolated from encapsulated liver
fragments using a modification of the two-step collagenase
digestion method. Briefly, encapsulated liver tissue was placed in
a custom-made perfusion apparatus and hepatic vessels were
cannulated with tubing attached multichannel manifold. The liver
fragment was initially perfused for 20 min with a prewarmed
(37.degree. C.) calcium-free buffer supplemented with ethylene
glycol tetraacetic acid (EGTA) followed by perfusion with a
prewarmed (37.degree. C.) buffer containing calcium (CaCl.sub.2,
H.sub.2O.sub.2) and collagenase 0.05% for 10 min. Then, liver
fragment was gently shaken to free liver cells in Hepatocyte Wash
Medium. Cellular suspension was filtered through a gauze-lined
funnel. Cells were centrifuged at low speed centrifugation. The
supernatant, containing damaged or dead hepatocytes, non
parenchymal cells and debris was removed and pelleted hepatocytes
were re-suspended in Hepatocyte Wash Medium. Viability and cell
concentration were determined by trypan blue exclusion test.
[0099] Cells were resuspended in complete hepatocyte medium
consisting of William's medium (Invitrogen) supplemented with 100
IU/L insulin (Novo Nordisk, France), and 10% heat inactivated fetal
calf serum (Biowest, France), and seeded at a density 1.8.times.106
viable cells onto 6 well plates that had been precoated with a type
I collagen from calf skin (Sigma-Aldrich, France) The medium was
replaced 16-20 hours later with fresh complete hepatocyte medium
supplemented with hydrocortisone hemisuccinate (SERB, Paris,
France), and cells were left in this medium until HCV inoculation.
The cultures were maintained at 37.degree. C. in a humidified 5%
CO2 atmosphere.
[0100] The PHHs were inoculated 3 days after seeding. JFH1-HCVcc
stocks were used to inoculate PHHs for 12 hours, at a multiplicity
of infection (MOI) of 0.1 ffu per cell. After a 12-hours incubation
at 37.degree. C., the inoculum was removed, and monolayers were
washed 3 times with phosphate-buffered saline and incubated in
complete hepatocyte medium containing 0.1% dimethylsufoxide as
carrier control, 100 IU/ml of IFNalpha as negative control or else
increasing concentrations of compound 8a. The cultures then
[0101] Quantitation of HCV RNA
[0102] Total RNA was prepared from cultured cells or from filtered
culture supernatants using the RNeasy or Qiamp viral RNA minikit
respectively (Qiagen SA, Courtaboeuf, France) according to the
manufacturer's recommendations. HCV RNA was quantified in cells and
culture supernatants using a strand-specific reverse real-time PCR
technique described previously (Carriere M and al 2007):
[0103] Reverse transcription was performed using primers described
previously located in the 50 NCR region of HCV genome, tag-RC1
[0104] (5'-GGCCGTCATGGTGGCGAATAAGTCTAGCCATGGCGTTAGTA-3') and RC21
(5'-CTCCCGGGGCACTCGCAAGC-3') for the negative and positive strands,
respectively. After a denaturation step performed at 70.degree. C.
for 8 min, the RNA template was incubated at 4.degree. C. for 5 min
in the presence of 200 ng of tag-RC1 primer and 1.25 mM of each
deoxynucleoside triphosphate (dNTP) (Promega, Charbonnieres,
France) in a total volume of 12 .mu.l.
[0105] Reverse transcription was carried out for 60 min at
60.degree. C. in the presence of 20 U RNaseOut.TM.
(Invitrogen,Cergy Pontoise, France) and 7.5 U Thermoscript.TM.
reverse transcriptase (Invitrogen), in the buffer recommended by
the manufacturer. An additional treatment was applied by adding 1
.mu.l (2U) RNaseH (Invitrogen) for 20 min at 37.degree. C.
[0106] The first round of nested PCR was performed with 2 .mu.l of
the cDNA obtained in a total volume of 50 .mu.l, containing 3 U Taq
polymerase (Promega), 0.5 mM dNTP, and 0.5 .mu.M RC1
(5'-GTCTAGCCATGGCGTTAGTA-3') and RC21 primers for positive-strand
amplification, or Tag (5'-GGCCGTCATGGTGGCGAATAA-3') and RC21
primers for negative strand amplification. The PCR protocol
consisted of 18 cycles of denaturation (94.degree. C. for 1 min),
annealing (55.degree. C. for 45 sec), and extension (72.degree. C.
for 2 min). The cDNA obtained was purified using the kit from
Qiagen, according to the manufacturer's instructions.
[0107] The purified product was then subjected to real-time PCR.
The reaction was carried out using the LightCycler 480 SYBR Green I
Master (2.times. con) Kit (Roche, Grenoble, France), with LC480
instruments and technology (Roche Diagnostics). PCR amplifications
were performed in a total volume of 10 .mu.l, containing 5 .mu.l of
Sybrgreen I Master Mix (2.times.), and 25 ng of the 197R
(5'-CTTTCGCGACCCAACACTAC-3') and 104 (5'-AGAGCCATAGTGGTCTGCGG-3')
primers. The PCR protocol consisted of one step of initial
denaturation for 10 min at 94.degree. C., followed by 40 cycles of
denaturation (95.degree. C. for 15 sec), annealing (57.degree. C.
for 5 sec), and extension (72.degree. C. for 8 sec).
[0108] The quantitation of 28Sr RNA by specific RT-PCR was used as
an internal standard to express the results of HCV positive or
negative strands per .mu.g of total hepatocyte RNA. Specific
primers for 28 S rRNA were designed using the Oligo6 software
5'-TTGAAAATCCGGGGGAGAG-3'(nt2717-2735) and
50-ACATTGTTCCAACATGCCAG-30 (nt 2816-2797). Reverse transcription
was performed using AMV reverse transcriptase (Promega), and the
PCR protocol consisted of one step of initial denaturation for 8
min at 95.degree. C., followed by 40 cycles of denaturation
(95.degree. C. for 15 sec), annealing (54.degree. C. for 5 sec),
and extension (72.degree. C. for 5 sec).
[0109] Results
[0110] Table 3 shows the anti-HCV activity of compound 8a as
determined in the in vitro primary human hepatocyte assay described
above. The numbers are expressed as 10.sup.6 HCV RNA copies/.mu.g
of total RNA. Results of two independent experiments (Exp 1 and Exp
2) are given. The data per experiment is the average of two
measurements.
[0111] Table 3: Effect of compound 8a on positive strand HCV-RNA
levels in primary human hepatocytes (expressed as 10.sup.6 HCV RNA
copies/.mu.g of total RNA).
TABLE-US-00003 TABLE 3 Exp. 1 Exp. 2 No HCV 0 0 HCV control 3.56
5.53 IFN.alpha. (100 IU/mL) 1.48 1.59 8a (0.195 .mu.M) 2.18 1.12 8a
(0.78 .mu.M) 2.25 1.3 8a (3.12 .mu.M) 1.09 0.94 8a (12.5 .mu.M)
2.17 1.3 8a(50 .mu.M) 0.94 1.33
[0112] In Vivo Efficacy Assay
[0113] The in vivo efficacy of compound 8a and CAS-1375074-52-4 was
determined in a humanized hepatocyte mouse model (PBX-mouse) as
previously described in Inoue et. al (Hepatology. 2007 April;
45(4):921-8) and Tenato et. al. (Am J Pathol 2004; 165-901-912)
with the following specification: Test animals: HCV G1a -infected
PXB-mice, male or female, >70% replacement index of human
hepatocytes. Dosing was performed p.o for 7 days at doses indicated
below wherein QD represents a single dose per day, BID represents
two doses per day.
[0114] Efficacy of compound 8a was compared to CAS-1375074-52-4.
Results are indicated in FIG. 1. The Figure shows the log drop HCV
viral RNA after dosing for a period of 7 days.
[0115] FIG. 1 clearly shows that a dosing of 100 mg/kg QD for CAS
1375074-52-4 (indicated as *, n=4) does not result in a significant
log drop in HCV viral RNA. This in strong contrast to each of the
indicated dose regimens for compound 8a, were a clear log drop is
observed for 100 mg/kg QD (indicated as .diamond-solid., n=3), 200
mg/kg QD (indicated as , n=4), 50 mg/kg BID (indicated as
.box-solid., n=4). The most pronounced log drop effect in viral RNA
is observed after a 7 day dosing of compound 8a at 100 mg/kg BID
(indicated as .tangle-solidup., n=4).
Sequence CWU 1
1
8141DNAHepatitis C virussource1..41/mol_type="unassigned DNA"
/organism="Hepatitis C virus" 1ggccgtcatg gtggcgaata agtctagcca
tggcgttagt a 41220DNAHepatitis C
virussource1..20/mol_type="unassigned DNA" /organism="Hepatitis C
virus" 2ctcccggggc actcgcaagc 20320DNAHepatitis C
virussource1..20/mol_type="unassigned DNA" /organism="Hepatitis C
virus" 3gtctagccat ggcgttagta 20421DNAHepatitis C
virussource1..21/mol_type="unassigned DNA" /organism="Hepatitis C
virus" 4ggccgtcatg gtggcgaata a 21520DNAHepatitis C
virussource1..20/mol_type="unassigned DNA" /organism="Hepatitis C
virus" 5ctttcgcgac ccaacactac 20620DNAHepatitis C
virussource1..20/mol_type="unassigned DNA" /organism="Hepatitis C
virus" 6agagccatag tggtctgcgg 20719DNAHepatitis C
virussource1..19/mol_type="unassigned DNA" /organism="Hepatitis C
virus" 7ttgaaaatcc gggggagag 19820DNAHepatitis C
virussource1..20/mol_type="unassigned DNA" /organism="Hepatitis C
virus" 8acattgttcc aacatgccag 20
* * * * *