U.S. patent application number 16/383382 was filed with the patent office on 2019-10-03 for fused indazole pyridone compounds as antivirals.
The applicant listed for this patent is Novartis AG. Invention is credited to Jiping FU, Wooseok HAN, Subramanian KARUR, Peichao LU, Keith Bruce PFISTER, Joseph Michael YOUNG.
Application Number | 20190300530 16/383382 |
Document ID | / |
Family ID | 62148437 |
Filed Date | 2019-10-03 |
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United States Patent
Application |
20190300530 |
Kind Code |
A1 |
FU; Jiping ; et al. |
October 3, 2019 |
FUSED INDAZOLE PYRIDONE COMPOUNDS AS ANTIVIRALS
Abstract
The invention provides compounds of Formula (I) ##STR00001## as
described herein, along with pharmaceutically acceptable salts,
pharmaceutical compositions containing such compounds, and methods
to use these compounds, salts and compositions for treating viral
infections, particularly infections caused by hepatitis B virus,
and reducing the occurrence of serious conditions associated with
HBV.
Inventors: |
FU; Jiping; (Danville,
CA) ; HAN; Wooseok; (Cambridge, MA) ; KARUR;
Subramanian; (Dublin, CA) ; LU; Peichao;
(Pleasant Hill, CA) ; PFISTER; Keith Bruce; (San
Ramon, CA) ; YOUNG; Joseph Michael; (Pleasant Hill,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG |
Basel |
|
CH |
|
|
Family ID: |
62148437 |
Appl. No.: |
16/383382 |
Filed: |
April 12, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15965172 |
Apr 27, 2018 |
10301312 |
|
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16383382 |
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62490890 |
Apr 27, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4985 20130101;
C07D 471/14 20130101; C07D 401/12 20130101; C07D 405/12 20130101;
A61K 45/06 20130101; A61P 31/12 20180101; C07D 471/22 20130101;
C07D 491/16 20130101 |
International
Class: |
C07D 471/14 20060101
C07D471/14; C07D 401/12 20060101 C07D401/12; C07D 491/16 20060101
C07D491/16; A61P 31/12 20060101 A61P031/12; A61K 45/06 20060101
A61K045/06; C07D 471/22 20060101 C07D471/22; A61K 31/4985 20060101
A61K031/4985; C07D 405/12 20060101 C07D405/12 |
Claims
1. A compound of formula (I): ##STR00270## wherein: R.sup.1 is H or
halo; R.sup.2 is independently selected at each occurrence from
halo, CN, C.sub.1-C.sub.3 haloalkyl, C.sub.1-C.sub.3 haloalkoxy,
--OR.sup.3, --N(R)R.sup.3, and C.sub.1-C.sub.3 alkyl optionally
substituted with up to three groups selected from
R.sup.3,--N(R)R.sup.3, CN, --OH, --CONR.sub.2, --COOR, and
--OR.sup.3; R.sup.3 is a C.sub.1-C.sub.4 alkyl that is optionally
substituted with one to three groups selected from halo,
C.sub.1-C.sub.3 haloalkyl, --OH, C.sub.1-C.sub.3 alkoxy,
C.sub.1-C.sub.3 haloalkoxy, oxo, CN, --NH.sub.2,
--NH(C.sub.1-C.sub.3 alkyl), --N(C.sub.1-C.sub.3 alkyl).sub.2,
--COOR, --CONR.sub.2, C.sub.3-C.sub.5 cycloalkyl, and a 4-6
membered cyclic ether, wherein the C.sub.3-C.sub.5 cycloalkyl and
4-6 membered cyclic ether are each optionally substituted with one
or two groups selected from halo, --OH, C.sub.1-C.sub.3 alkoxy, and
C.sub.1-C.sub.3 alkyl; or R.sup.3 is a C.sub.3-C.sub.5 cycloalkyl
ring or 4-6 membered cyclic ether, wherein the C.sub.3-C.sub.5
cycloalkyl ring or 4-6 membered cyclic ether are each optionally
substituted with one to three groups selected from halo,
C.sub.1-C.sub.3 alkyl, --OH, C.sub.1-C.sub.3 alkoxy,
C.sub.1-C.sub.3 haloalkoxy, C.sub.1-C.sub.3 haloalkyl, --NH.sub.2,
--NH(C.sub.1-C.sub.3 alkyl), and --N(C.sub.1-C.sub.3 alkyl).sub.2;
n is 0, 1, or 2; W is --COOR.sup.4, --C(O)NH--SO.sub.2R.sup.5,
--C(O)NH--SO.sub.2NR.sub.2, 5-tetrazolyl, or 1,2,4-oxadiazol-3-yl-5
(4H)-one; R.sup.4 is H or C.sub.1-C.sub.6 alkyl that is optionally
substituted with one to three groups selected from halo, --OR, oxo,
CN, and --NR.sub.2; R.sup.5 is C.sub.1-C.sub.6 alkyl that is
optionally substituted with one to three groups selected from halo,
--OR, oxo, CN, and --NR.sub.2; R.sup.6 is H or C.sub.1-C.sub.6
alkyl; R.sup.7 is H or C.sub.1-C.sub.6 alkyl, or R.sup.7 taken
together with R.sup.9 and the atoms connecting R.sup.7 with R.sup.9
forms a ring as described below; R.sup.8 is H or C.sub.1-C.sub.6
alkyl; R.sup.9 is selected from: (1) H; (2) C.sub.1-C.sub.6 alkyl
optionally substituted with up to three groups selected from
C.sub.3-C.sub.6 cycloalkyl, --OR, --NR.sub.2, halo, CN, COOR,
CONR.sub.2, and oxo; and (3) a ring selected from (a)
C.sub.3-C.sub.6 cycloalkyl, (b) phenyl, (c) 5-6 membered
heterocyclyl containing one or two heteroatoms selected from N, O
and S as ring members, and (d) 5-6 membered heteroaryl containing
one or two heteroatoms selected from N, O and S as ring members,
wherein each of rings (a) through (d) is optionally substituted
with up to three groups selected from C.sub.1-C.sub.2 alkyl,
(CH.sub.2).sub.0-2--OR, --NR.sub.2, halo, CN, COOR, and CONR.sub.2;
or R.sup.9 taken together with R.sup.7 and the atoms connecting
R.sup.9 with R.sup.7 forms a 3-7 membered cycloalkyl ring, or a 3-7
membered heterocyclic ring containing N, O or S as a ring member;
wherein the cycloalkyl or heterocyclic ring is optionally
substituted with up to three groups selected from R, --OR,
--NR.sub.2, halo, CN, COOR, CONR.sub.2, and oxo; R is independently
selected at each occurrence from H and C.sub.1-C.sub.3 alkyl
optionally substituted with one to three groups selected from halo,
--OH, C.sub.1-C.sub.3 alkoxy, oxo, CN, --NH.sub.2,
--NH(C.sub.1-C.sub.3 alkyl), --N(C.sub.1-C.sub.3 alkyl).sub.2, and
cyclopropyl; and two R groups directly attached to the same atom
can optionally be taken together with the atom to which both are
attached to form a 3-6 membered ring that can optionally contain a
heteroatom selected from N, O and S as a ring member, and can be
substituted by up to two groups selected from --OH, oxo,
C.sub.1-C.sub.3 alkyl, and C.sub.1-C.sub.3 alkoxy; or a
pharmaceutically acceptable salt thereof.
2. A compound according to claim 1, wherein the compound is of the
formula: ##STR00271## or a pharmaceutically acceptable salt
thereof.
3. A compound according to claim 1, which is of the formula:
##STR00272## or a pharmaceutically acceptable salt thereof.
4. The compound of claim 1, which is selected from: ##STR00273##
##STR00274## ##STR00275## ##STR00276## ##STR00277## ##STR00278##
##STR00279## ##STR00280## ##STR00281## ##STR00282## ##STR00283##
and the individual enantiomers of any one of these; or a
pharmaceutically acceptable salt thereof.
5. A pharmaceutical composition, comprising a compound of claim 1
admixed with at least one pharmaceutically acceptable carrier.
6. A method to treat a subject having a hepatitis B infection,
which comprises administering to the subject a compound of claim
1.
7. A method to inhibit replication of hepatitis B virus, which
comprises contacting the hepatitis B virus, either in vitro or in
vivo, with a compound according to claim 1.
8. A pharmaceutical combination, comprising a compound of claim 1
and at least one additional therapeutic agent.
9. A compound according to claim 1 for use in therapy.
10. Use of a compound according claim 1 in the manufacture of a
medicament.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
15/965,172 filed 27 Apr. 2018, which claims the benefit of priority
to U.S. Ser. No. 62/490,890, filed 27 Apr. 2017, the contents of
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to novel fused tetracyclic
pyridone compounds that are inhibitors of hepatitis virus
replication, and are thus useful to treat viral infections, and
particularly hepatitis B virus (HBV). The invention provides novel
tetracyclic pyridone compounds as disclosed herein, pharmaceutical
compositions containing such compounds, and methods of using these
compounds and compositions in the treatment and prevention of HBV
infections.
BACKGROUND
[0003] Globally, over 400 million people are chronically infected
with hepatitis B virus (HBV), and more than 12 million reside in
the United States alone. Of those chronically infected patients, up
to 40 percent will eventually develop complications of liver
failure from cirrhosis or development of hepatocellular carcinoma
(HCC). HBV belongs to the family of Hepadnaviridae, a group of
small hepatotropic DNA viruses that replicate through the reverse
transcription of an RNA intermediate. The 3.2-kb HBV genome in
viral particles is in a circular, partially double-stranded DNA
conformation (relaxed circular DNA or rcDNA). The HBV genome
consists of four overlapping open reading frames (ORFs), which
encode for the core, polymerase (Pol), envelope, and X proteins.
rcDNA is transcriptionally inert and must be converted into
covalently closed circular DNA (cccDNA) in the nucleus of infected
cells before viral RNAs can be transcribed. cccDNA is the only
template for HBV transcription and, because HBV RNA templates
genomic reverse transcription, its persistence is required for
persistent infection.
[0004] The envelope of HBV comprises a mixture of surface antigen
proteins (HBsAg). The HBsAg coat is a mixture of three overlapping
proteins: all three share a common region, which corresponds to the
smallest of the three proteins (SHBsAg). The mixture consists
mostly of SHBsAg, but also includes Medium HBsAg, which comprises
SHBsAg plus an additional polypeptide segment, and Large HBsAg,
which comprises M HBsAg plus another added polypeptide segment. In
addition to forming the infectious virion particle, the S, M and L
HBsAg proteins also assemble into a subviral particle knows as the
22-nm particle, which is not infectious but contains the same
proteins that envelope the infectious virus particles. Indeed,
these subviral, non-infectious particles have been used as a
vaccine, since they contain the same antigenic surface proteins as
the infectious HBV virion, and thus elicit antibodies that
recognize the infectious agent. Interestingly, these subviral
particles greatly outnumber infectious virions, and are believed to
protect the infectious virions from the immune system of the
infected host. By sheer numbers, they may act as decoys,
distracting immune responses from the infectious virus particles,
but in addition they are reported to suppress the function of
immune cells (monocytes, dendritic cells and natural killer cells)
and may thus impair the immune response to HBV. Because these
subviral particles protect infectious HBV from the host immune
system, reducing the level of subviral particles has been
recognized as a viable therapeutic approach. See, e.g.,
WO2015/113990.
[0005] One of the key diagnostic symptoms of chronic HBV is the
high serum levels of the hepatitis B surface antigen (HBsAg).
Clinical data in recent years suggest that sustained virologic
response is often associated with on-treatment HBsAg decline during
the early phase of the treatment as early as week 8, while
sustained exposure to HBsAg and other viral antigens may lead to
HBV-specific immune-tolerance. Chronic HB patients who experienced
larger and faster decreases in serum HBsAg levels achieved
significantly higher rate (.about.40%) of sustained virologic
response as defined by sustained viral control post treatment.
[0006] Current treatment options for HBV include interferon
therapies and nucleoside/nucleotide inhibitors of the viral DNA
polymerase, such as entecavir and tenofovir. These focus on
reduction in the level of viremia and toleration of hepatic
dysfunction, and may have adverse side-effects and also select for
drug-resistant virus variants during long term therapy. More
importantly, these therapies cannot eradicate the intrahepatic HBV
cccDNA pool in chronic hepatitis B patients or limit the
transcription of HBsAg from the pre-existing cccDNA, nor do they
affect the secretion of synthesized HBsAg into patients' blood to
counteract the host innate immune response. As a result, these HBV
treatments are in most cases life-long therapies, and
discontinuation often leads to virological relapse.
[0007] Accordingly, there remains a need for more effective
treatments for HBV, especially for treating chronic HBV infections
(cHBV). The invention provides compounds that are believed to
operate by suppression of the secretion of the 22 nm subviral
particles containing HBsAg. These compounds are useful to treat HBV
infections and to reduce the incidence of serious liver disorders
caused by HBV infections.
SUMMARY
[0008] The present invention provides novel compounds that inhibit
secretion of HBsAg from cells infected with hepatitis B virus and
thereby reduce viral load and viral replication in patients having
chronic HBV infection. Thus the compounds of the invention are
suitable for treatment of patients with HBV, including chronic
HBV.
[0009] In one aspect, the invention provides compounds of Formula
(I):
##STR00002##
[0010] wherein:
[0011] R.sup.1 is H or halo;
[0012] R.sup.2 is independently selected at each occurrence from
halo, CN, C.sub.1-C.sub.3 haloalkyl, C.sub.1-C.sub.3 haloalkoxy,
--OR.sup.3, --N(R)R.sup.3, and C.sub.1-C.sub.3 alkyl optionally
substituted with up to three groups selected from R.sup.3,
--N(R)R.sup.3, CN, --OH, --CONR.sub.2, --COOR, and --OR.sup.3;
[0013] R.sup.3 is a C.sub.1-C.sub.4 alkyl that is optionally
substituted with one to three groups selected from halo,
C.sub.1-C.sub.3 haloalkyl, --OH, C.sub.1-C.sub.3 alkoxy,
C.sub.1-C.sub.3 haloalkoxy, oxo, CN, --NH.sub.2,
--NH(C.sub.1-C.sub.3 alkyl), --N(C.sub.1-C.sub.3 alkyl).sub.2,
--COOR, --CONR.sub.2, C.sub.3-C.sub.5 cycloalkyl, and a 4-6
membered cyclic ether, wherein the C.sub.3-C.sub.5 cycloalkyl and
4-6 membered cyclic ether are each optionally substituted with one
or two groups selected from halo, --OH, C.sub.1-C.sub.3 alkoxy, and
C.sub.1-C.sub.3 alkyl;
[0014] or R.sup.3 is a C.sub.3-C.sub.5 cycloalkyl ring or 4-6
membered cyclic ether, wherein the C.sub.3-C.sub.5 cycloalkyl ring
or 4-6 membered cyclic ether are each optionally substituted with
one to three groups selected from halo, C.sub.1-C.sub.3 alkyl,
--OH, C.sub.1-C.sub.3 alkoxy, C.sub.1-C.sub.3 haloalkoxy,
C.sub.1-C.sub.3 haloalkyl, --NH.sub.2, --NH(C.sub.1-C.sub.3 alkyl),
and --N(C.sub.1-C.sub.3 alkyl).sub.2;
[0015] n is 0, 1, or 2;
[0016] W is --COOR.sup.4, --C(O)NH--SO.sub.2R.sup.5,
--C(O)NH--SO.sub.2NR.sub.2, 5-tetrazolyl, or
1,2,4-oxadiazol-3-yl-5(4H)-one;
[0017] R.sup.4 is H or C.sub.1-C.sub.6 alkyl that is optionally
substituted with one to three groups selected from halo, --OR, oxo,
CN, and --NR.sub.2;
[0018] R.sup.5 is C.sub.1-C.sub.6 alkyl that is optionally
substituted with one to three groups selected from halo, --OR, oxo,
CN, and --NR.sub.2;
[0019] R.sup.6 is H or C.sub.1-C.sub.6 alkyl;
[0020] R.sup.7 is H or C.sub.1-C.sub.6 alkyl,
[0021] or R.sup.7 taken together with R.sup.9 and the atoms
connecting R.sup.7 with R.sup.9 forms a ring as described
below;
[0022] R.sup.8 is H or C.sub.1-C.sub.6 alkyl;
[0023] R.sup.9 is selected from: H;
[0024] C.sub.1-C.sub.6 alkyl optionally substituted with up to
three groups selected from C.sub.3-C.sub.6 cycloalkyl, --OR,
--NR.sub.2, halo, CN, COOR, CONR.sub.2, and oxo; and
[0025] a ring selected from (a) C.sub.3-C.sub.6 cycloalkyl, (b)
phenyl, (c) 5-6 membered heterocyclyl containing one or two
heteroatoms selected from N, O and S as ring members, and (d) 5-6
membered heteroaryl containing one or two heteroatoms selected from
N, O and S as ring members, wherein each of rings (a) through (d)
is optionally substituted with up to three groups selected from
C.sub.1-C.sub.2 alkyl, (CH.sub.2).sub.0-2--OR, --NR.sub.2, halo,
CN, COOR, and CONR.sub.2;
[0026] or R.sup.9 taken together with R.sup.7 and the atoms
connecting R.sup.9 with R.sup.7 forms a 3-7 membered cycloalkyl
ring, or a 3-7 membered heterocyclic ring containing N, O or S as a
ring member; wherein the cycloalkyl or heterocyclic ring is
optionally substituted with up to three groups selected from R,
--OR, --NR.sub.2, halo, CN, COOR, CONR.sub.2, and oxo;
[0027] R is independently selected at each occurrence from H and
C.sub.1-C.sub.3 alkyl optionally substituted with one to three
groups selected from halo, --OH, C.sub.1-C.sub.3 alkoxy, oxo, CN,
--NH.sub.2, --NH(C.sub.1-C.sub.3 alkyl), --N(C.sub.1-C.sub.3
alkyl).sub.2, and cyclopropyl;
[0028] and two R groups directly attached to the same atom can
optionally be taken together with the atom to which both are
attached to form a 3-6 membered ring that can optionally contain a
heteroatom selected from N, O and S as a ring member, and can be
substituted by up to two groups selected from --OH, oxo,
C.sub.1-C.sub.3 alkyl, and C.sub.1-C.sub.3 alkoxy;
[0029] or a pharmaceutically acceptable salt thereof.
[0030] The invention also includes pharmaceutical compositions
containing these compounds, methods to use these compounds and
compositions to treat viral infections, pharmaceutical combinations
comprising these compounds, and methods to use the compounds in the
manufacture of a medicament.
DETAILED DESCRIPTION
[0031] For purposes of interpreting this specification, the
following definitions will apply, and whenever appropriate, terms
used in the singular will also include the plural.
[0032] Terms used in the specification have the following meanings
unless the context clearly indicates otherwise:
[0033] As used herein, the term "subject" refers to an animal. In
certain aspects, the animal is a mammal. A subject also refers to
for example, primates (e.g., humans), cows, sheep, goats, horses,
dogs, cats, rabbits, rats, mice, fish, birds and the like. In
certain embodiments, the subject is a human. A "patient" as used
herein refers to a human subject.
[0034] As used herein, the term "inhibition" or "inhibiting" refers
to the reduction or suppression of a given condition, symptom, or
disorder, or disease, or a significant decrease in the baseline
activity of a biological activity or process.
[0035] As used herein, the term "treating" or "treatment" of any
disease or disorder refers in one embodiment, to ameliorating the
disease or disorder (i.e., slowing or arresting or reducing the
development of the disease or at least one of the clinical symptoms
thereof). In another embodiment "treating" or "treatment" refers to
alleviating or ameliorating at least one physical parameter
including those which may not be discernible by the patient. In yet
another embodiment, "treating" or "treatment" refers to modulating
the disease or disorder, either physically, (e.g., stabilization of
a discernible symptom), physiologically, (e.g., stabilization of a
physical parameter), or both. In yet another embodiment, "treating"
or "treatment" refers to preventing or delaying the onset or
development or progression of the disease or disorder.
[0036] As used herein, the term "a," "an," "the" and similar terms
used in the context of the present invention (especially in the
context of the claims) are to be construed to cover both the
singular and plural unless otherwise indicated herein or clearly
contradicted by the context.
[0037] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g. "such as") provided herein is intended
merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed.
[0038] "Optionally substituted" means the group referred to can be
substituted at one or more positions by any one or any combination
of the radicals listed thereafter. The number, placement and
selection of substituents is understood to encompass only those
substitutions that a skilled chemist would expect to be reasonably
stable; thus `oxo` would not be a substituent on an aryl or
heteroaryl ring, for example, and a single carbon atom would not
have three hydroxy or amino substituents. Unless otherwise
specified, optional substituents are typically up to four groups
selected from halo, oxo, CN, amino, hydroxy, --C.sub.1-3 alkyl,
--OR*, --NR*.sub.2, --SR*, --SO.sub.2R*, --COOR*, and
--CONR*.sub.2, where each R* is independently H or C.sub.1-3
alkyl.
[0039] "Aryl" as used herein refers to a phenyl or naphthyl group
unless otherwise specified. Aryl groups unless otherwise specified
may be optionally substituted with up to four groups selected from
halo, CN, amino, hydroxy, C.sub.1-3 alkyl, --OR*, --NR*.sub.2,
--SR*, --SO.sub.2R*, --COOR*, and --CONR*.sub.2, where each R* is
independently H or C.sub.1-3 alkyl.
[0040] "Halo" or "halogen", as used herein, may be fluorine,
chlorine, bromine or iodine.
[0041] "C.sub.1-6 alkyl" or "C.sub.1-C.sub.6 alkyl", as used
herein, denotes straight chain or branched alkyl having 1-6 carbon
atoms. If a different number of carbon atoms is specified, such as
C.sub.4 or C.sub.3, then the definition is to be amended
accordingly, such as "C.sub.1-4 alkyl" will represent methyl,
ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and
tert-butyl.
[0042] "C.sub.1-6 alkylene" or "C.sub.1-C.sub.6 alkylene", as used
herein, denotes straight chain or branched alkyl having 1-6 carbon
atoms and two open valences for connection to two other groups. If
a different number of carbon atoms is specified, such as C.sub.4 or
C.sub.3, then the definition is to be amended accordingly, such as
"C.sub.1-4 alkylene" will represent methylene (--CH.sub.2--),
ethylene (--CH.sub.2CH.sub.2--), straight chain or branched
propylene (--CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2--CHMe-CH.sub.2--), and the like.
[0043] "C.sub.1-6 alkoxy", as used herein, denotes straight chain
or branched alkoxy (--O-Alkyl) having 1-6 carbon atoms. If a
different number of carbon atoms is specified, such as C.sub.4 or
C.sub.3, then the definition is to be amended accordingly, such as
"C.sub.1-4 alkoxy" will represent methoxy, ethoxy, propoxy,
isopropoxy, butoxy, isobutoxy, sec-butoxy and tert-butoxy.
[0044] "C.sub.1-4 Haloalkyl" or "C.sub.1-C.sub.4 haloalkyl" as used
herein, denotes straight chain or branched alkyl having 1-4 carbon
atoms wherein at least one hydrogen has been replaced with a
halogen. The number of halogen replacements can be from one up to
the number of hydrogen atoms on the unsubstituted alkyl group. If a
different number of carbon atoms is specified, such as C.sub.6 or
C.sub.3, then the definition is to be amended accordingly. Thus
"C.sub.1-4 haloalkyl" will represent methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, sec-butyl and tert-butyl that have at
least one hydrogen substituted with halogen, such as where the
halogen is fluorine: CF.sub.3CF.sub.2--, (CF.sub.3).sub.2CH--,
CH.sub.3--CF.sub.2--, CF.sub.3CF.sub.2--, CF.sub.3, CF.sub.2H--,
CF.sub.3CF.sub.2CH(CF.sub.3)-- or
CF.sub.3CF.sub.2CF.sub.2CF.sub.2--.
[0045] "C.sub.3-8 cycloalkyl" as used herein refers to a saturated
monocyclic hydrocarbon ring of 3 to 8 carbon atoms. Examples of
such groups include cyclopropyl, cyclobutyl, cyclopentyl and
cyclohexyl. If a different number of carbon atoms is specified,
such as C.sub.3-C.sub.6, then the definition is to be amended
accordingly.
[0046] "4- to 8-Membered heterocyclyl", "5- to 6-membered
heterocyclyl", "3- to 10-membered heterocyclyl", "3- to 14-membered
heterocyclyl", "4- to 14-membered heterocyclyl" and "5- to
14-membered heterocyclyl", refers, respectively, to 4- to
8-membered, 5- to 6-membered, 3- to 10-membered, 3- to 14-membered,
4- to 14-membered and 5- to 14-membered heterocyclic rings; unless
otherwise specified, such rings contain 1 to 7, 1 to 5, or 1 to 3
heteroatoms selected from the group consisting of nitrogen, oxygen
and sulfur as ring members, and the rings may be saturated, or
partially saturated but not aromatic. The heterocyclic group can be
attached to another group at a nitrogen or a carbon atom. The term
"heterocyclyl" includes single ring groups, fused ring groups and
bridged groups. Examples of such heterocyclyl include, but are not
limited to pyrrolidine, piperidine, piperazine, pyrrolidinone,
morpholine, tetrahydrofuran, tetrahydrothiophene,
tetrahydrothiopyran, tetrahydropyran, 1,4-dioxane, 1,4-oxathiane,
8-aza-bicyclo[3.2.1]octane, 3,8-diazabicyclo[3.2.1]octane,
3-Oxa-8-aza-bicyclo[3.2.1]octane, 8-Oxa-3-aza-bicyclo[3.2.1]octane,
2-Oxa-5-aza-bicyclo[2.2.1]heptane, 2,5-Diaza-bicyclo[2.2.1]heptane,
azetidine, ethylenedioxo, oxetane or thiazole. In certain
embodiments, if not otherwise specified, heterocyclic groups have
1-2 heteroatoms selected from N, O and S as ring members, and 4-7
ring atoms, and are optionally substituted with up to four groups
selected from halo, oxo, CN, amino, hydroxy, C.sub.1-3 alkyl,
--OR*, --NR*.sub.2, --SR*, --SO.sub.2R*, --COOR*, and
--CONR*.sub.2, where each R* is independently H or C.sub.1-3 alkyl.
In particular, heterocyclic groups containing a sulfur atom are
optionally substituted with one or two oxo groups on the
sulfur.
[0047] "4-6 membered cyclic ether" as used herein refers to a 4 to
6 membered ring comprising one oxygen atom as a ring member.
Examples include oxetane, tetrahydrofuran and tetrahydropyran.
[0048] "Heteroaryl" is a completely unsaturated (aromatic) ring.
The term "heteroaryl" refers to a 5-14 membered monocyclic- or
bicyclic- or tricyclic-aromatic ring system, having 1 to 8
heteroatoms selected from N, O or S. Typically, the heteroaryl is a
5-10 membered ring or ring system (e.g., 5-7 membered monocyclic
group or an 8-10 membered bicyclic group), often a 5-6 membered
ring containing up to four heteroatoms selected from N, O and S,
though often a heteroaryl ring contains no more than one divalent O
or S in the ring. Typical heteroaryl groups include furan,
isothiazole, thiadiazole, oxadiazole, indazole, indole, quinoline,
2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4-, or
5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-,
4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or
5-isoxazolyl, 3- or 5-(1,2,4-triazolyl), 4- or 5-(1,2,
3-triazolyl), tetrazolyl, triazine, pyrimidine, 2-, 3-, or
4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl,
2-pyrazinyl, and 2-, 4-, or 5-pyrimidinyl. Heteroaryl groups are
optionally substituted with up to four groups selected from halo,
CN, amino, hydroxy, C.sub.1-3 alkyl, --OR*, --NR*.sub.2, --SR*,
--SO.sub.2R*, --COOR*, and --CONR*.sub.2, where each R* is
independently H or C.sub.1-3 alkyl.
[0049] The term "hydroxy" or "hydroxyl" refers to the group
--OH.
[0050] Various embodiments of the invention are described herein.
It will be recognized that features specified in each embodiment
may be combined with other specified features to provide further
embodiments.
[0051] The following enumerated embodiments are representative of
the invention:
[0052] A compound of formula (I):
##STR00003##
[0053] wherein:
[0054] R.sup.1 is H or halo;
[0055] R.sup.2 is independently selected at each occurrence from
halo, CN, C.sub.1-C.sub.3 haloalkyl, C.sub.1-C.sub.3 haloalkoxy,
--OR.sup.3, --N(R)R.sup.3, and C.sub.1-C.sub.3 alkyl optionally
substituted with up to three groups selected from R.sup.3,
--N(R)R.sup.3, CN, --OH, --CONR.sub.2, --COOR, and --OR.sup.3;
[0056] R.sup.3 is a C.sub.1-C.sub.4 alkyl that is optionally
substituted with one to three groups selected from halo,
C.sub.1-C.sub.3 haloalkyl, --OH, C.sub.1-C.sub.3 alkoxy,
C.sub.1-C.sub.3 haloalkoxy, oxo, CN, --NH.sub.2,
--NH(C.sub.1-C.sub.3 alkyl), --N(C.sub.1-C.sub.3 alkyl).sub.2,
--COOR, --CONR.sub.2, C.sub.3-C.sub.5 cycloalkyl, and a 4-6
membered cyclic ether, wherein the C.sub.3-C.sub.5 cycloalkyl and
4-6 membered cyclic ether are each optionally substituted with one
or two groups selected from halo, --OH, C.sub.1-C.sub.3 alkoxy, and
C.sub.1-C.sub.3 alkyl;
[0057] or R.sup.3 is a C.sub.3-C.sub.5 cycloalkyl ring or 4-6
membered cyclic ether, wherein the C.sub.3-C.sub.5 cycloalkyl ring
or 4-6 membered cyclic ether are each optionally substituted with
one to three groups selected from halo, C.sub.1-C.sub.3 alkyl,
--OH, C.sub.1-C.sub.3 alkoxy, C.sub.1-C.sub.3 haloalkoxy,
C.sub.1-C.sub.3 haloalkyl, --NH.sub.2, --NH(C.sub.1-C.sub.3 alkyl),
and --N(C.sub.1-C.sub.3 alkyl).sub.2;
[0058] n is 0, 1, or 2;
[0059] W is --COOR.sup.4, --C(O)NH--SO.sub.2R.sup.5,
--C(O)NH--SO.sub.2NR.sub.2, 5-tetrazolyl, or
1,2,4-oxadiazol-3-yl-5(4H)-one;
[0060] R.sup.4 is H or C.sub.1-C.sub.6 alkyl that is optionally
substituted with one to three groups selected from halo, --OR, oxo,
CN, and --NR.sub.2;
[0061] R.sup.5 is C.sub.1-C.sub.6 alkyl that is optionally
substituted with one to three groups selected from halo, --OR, oxo,
CN, and --NR.sub.2;
[0062] R.sup.6 is H or C.sub.1-C.sub.6 alkyl;
[0063] R.sup.7 is H or C.sub.1-C.sub.6 alkyl, or R.sup.7 taken
together with R.sup.9 and the atoms connecting R.sup.7 with R.sup.9
forms a ring as described below;
[0064] R.sup.8 is H or C.sub.1-C.sub.6 alkyl;
[0065] R.sup.9 is selected from:
[0066] H;
[0067] C.sub.1-C.sub.6 alkyl optionally substituted with up to
three groups selected from C.sub.3-C.sub.6 cycloalkyl, --OR,
--NR.sub.2, halo, CN, COOR, CONR.sub.2, and oxo; and
[0068] a ring selected from (a) C.sub.3-C.sub.6 cycloalkyl, (b)
phenyl, (c) 5-6 membered heterocyclyl containing one or two
heteroatoms selected from N, O and S as ring members, and (d) 5-6
membered heteroaryl containing one or two heteroatoms selected from
N, O and S as ring members, wherein each of rings (a) through (d)
is optionally substituted with up to three groups selected from
C.sub.1-C.sub.2 alkyl, (CH.sub.2).sub.0-2--OR, --NR.sub.2, halo,
CN, COOR, and CONR.sub.2;
[0069] or R.sup.9 taken together with R.sup.7 and the atoms
connecting R.sup.9 with R.sup.7 forms a 3-7 membered cycloalkyl
ring, or a 3-7 membered heterocyclic ring containing N, O or S as a
ring member; wherein the cycloalkyl or heterocyclic ring is
optionally substituted with up to three groups selected from R,
--OR, --NR.sub.2, halo, CN, COOR, CONR.sub.2, and oxo;
[0070] R is independently selected at each occurrence from H and
C.sub.1-C.sub.3 alkyl optionally substituted with one to three
groups selected from halo, --OH, C.sub.1-C.sub.3 alkoxy, oxo, CN,
--NH.sub.2, --NH(C.sub.1-C.sub.3 alkyl), --N(C.sub.1-C.sub.3
alkyl).sub.2, and cyclopropyl;
[0071] and two R groups directly attached to the same atom can
optionally be taken together with the atom to which both are
attached to form a 3-6 membered ring that can optionally contain a
heteroatom selected from N, O and S as a ring member, and can be
substituted by up to two groups selected from --OH, oxo,
C.sub.1-C.sub.3 alkyl, and C.sub.1-C.sub.3 alkoxy;
[0072] or a pharmaceutically acceptable salt thereof.
[0073] When R.sup.3 is a cyclic ether in this embodiment, it is
typically selected from oxetanyl, tetrahydrofuranyl, and
tetrahydropyranyl.
[0074] 2. The compound of embodiment 1, or a pharmaceutically
acceptable salt thereof, wherein R.sup.1 is H or F.
[0075] 3. The compound according to any one of the preceding
embodiments, or a pharmaceutically acceptable salt thereof, wherein
R.sup.2 is --OR.sup.3.
[0076] 4. The compound according to any one of embodiments 1 to 3,
wherein W is --COOR.sup.4; or a pharmaceutically acceptable salt
thereof. In preferred compounds of this embodiment, W is
--COOH.
[0077] 5. The compound of any of embodiments 1-4, wherein R.sup.6
is H and R.sup.8 is H; or a pharmaceutically acceptable salt
thereof.
[0078] 6. The compound of any of embodiments 1-5, wherein R.sup.9
is C.sub.1-C.sub.6 alkyl optionally substituted with up to two
groups selected from C.sub.3-C.sub.6 cycloalkyl, --OR, --NR.sub.2,
halo, CN, COOR, CONR.sub.2, and oxo; or R.sup.9 is cyclopropyl,
cyclobutyl, or oxetanyl and is optionally substituted by one or two
groups selected from methyl, halo, and (CH.sub.2).sub.0-2--OR;
[0079] or a pharmaceutically acceptable salt thereof.
[0080] 7. The compound of any of embodiments 1-5, wherein R.sup.9
is taken together with R.sup.7 and the atoms connecting R.sup.9 to
R.sup.7 to form a 5-6 membered cycloalkyl ring or a 5-6 membered
heterocyclic ring containing N, O or S as a ring member; wherein
the cycloalkyl or heterocyclic ring is optionally substituted with
up to two groups selected from R, --OR, --NR.sub.2, halo, CN, COOR,
CONR.sub.2, and oxo;
[0081] or a pharmaceutically acceptable salt thereof.
[0082] 8. The compound of any of embodiments 1-7, wherein n is 1;
or a pharmaceutically acceptable salt thereof.
[0083] 9. The compound of any one of embodiments 7-8, which is of
the formula:
##STR00004##
[0084] wherein:
[0085] R.sup.9 taken together with R.sup.7 and the atoms connecting
R.sup.9 with R.sup.7 forms a 5 or 6 membered cycloalkyl ring or a 5
or 6 membered heterocyclic ring containing O as a ring member;
wherein the cycloalkyl or heterocyclic ring is optionally
substituted with one or two groups selected from R, --OR,
--NR.sub.2, halo, and CN; and
[0086] R.sup.4 is H or C.sub.1-C.sub.4 alkyl;
[0087] or a pharmaceutically acceptable salt thereof.
[0088] 10. The compound of any of embodiments 1-6, which is of the
formula:
##STR00005##
[0089] wherein R.sup.4 is H or C.sub.1-C.sub.4 alkyl; and
[0090] R.sup.9 is C.sub.1-C.sub.4 alkyl optionally substituted with
hydroxy or methoxy, or R.sup.9 is C.sub.3-C.sub.4 cycloalkyl
optionally substituted with methyl or (CH.sub.2).sub.0-2--OR;
[0091] or a pharmaceutically acceptable salt thereof.
[0092] 11. A compound according to embodiment 10, which is of the
formula:
##STR00006##
[0093] or a pharmaceutically acceptable salt thereof.
[0094] 12. A compound according to embodiment 8, wherein the
compound is of the formula:
##STR00007##
[0095] or a pharmaceutically acceptable salt thereof.
[0096] 13. A compound according to embodiment 8, which is of the
formula:
##STR00008##
[0097] or a pharmaceutically acceptable salt thereof. In compounds
of this embodiment, R is preferably methyl, or two R groups on the
same carbon taken together form a ring selected from cyclopropyl,
cyclobutyl and oxetanyl.
[0098] 14. The compound of any one of embodiments 1-13, wherein
R.sup.1 is H; or a pharmaceutically acceptable salt thereof.
[0099] 15. The compound of any one of embodiments 1-13, wherein
R.sup.1 is F; or a pharmaceutically acceptable salt thereof.
[0100] 16. The compound of any one of embodiments 1-15, wherein
R.sup.4 is H; or a pharmaceutically acceptable salt thereof.
[0101] 17. The compound of embodiment 1, which is selected
from:
##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013##
##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018##
##STR00019##
and the individual enantiomers of any one of these; or a
pharmaceutically acceptable salt thereof.
[0102] In particular, each of the compounds of the Examples,
including each compound listed in Table I, is a specific embodiment
of the compounds of the invention.
[0103] 18. A pharmaceutical composition, comprising a compound of
any of the preceding embodiments admixed with at least one
pharmaceutically acceptable carrier.
[0104] 19. A method to treat a subject having a hepatitis B
infection, which comprises administering to the subject a compound
of any of embodiments 1-17 or a pharmaceutical composition of claim
17.
[0105] 20. The method of embodiment 19, wherein the compound of any
one of embodiments 1-17 or the pharmaceutical composition of
embodiment 18 is used in combination with an additional therapeutic
agent selected from an interferon or peginterferon, an HBV
polymerase inhibitor, a viral entry inhibitor, a viral maturation
inhibitor, a capsid assembly inhibitor, an HBV core modulator, a
reverse transcriptase inhibitor, a TLR-agonist, or an
immunomodulator.
[0106] 21. A method to inhibit replication of hepatitis B virus,
which comprises contacting the hepatitis B virus, either in vitro
or in vivo, with a compound according to any one of embodiments
1-17.
[0107] 22. A pharmaceutical combination, comprising a compound of
any of embodiments 1-17 and at least one additional therapeutic
agent.
[0108] 23. A compound according to any of embodiments 1-17 for use
in therapy.
[0109] 24. The compound according to embodiment 23 wherein the
therapy is treatment of a bacterial infection.
[0110] 25. Use of a compound according to any one of embodiments
1-17 in the manufacture of a medicament.
[0111] Another embodiment of the invention provides a compound as
described above, or a pharmaceutically acceptable salt thereof, for
use as a medicament. In one aspect, the medicament is for treatment
of a subject having an HBV infection. In a particular embodiment,
the subject is a human diagnosed with chronic HBV.
[0112] Also within the scope of this invention is the use of a
compound of formula (I), or a pharmaceutically acceptable salt
thereof, for the manufacture of a medicament; in some embodiments,
this medicament is for the treatment or prevention of a viral
disease and/or infection in a human being, particularly where the
virus involved is HBV.
[0113] Included within the scope of this invention is a
pharmaceutical composition, comprising a compound of Formula (I),
or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier or excipient. Optionally, the
composition comprises at least two pharmaceutically acceptable
carriers and/or excipients.
[0114] According to a further aspect of this embodiment the
pharmaceutical composition according to this invention further
comprises a therapeutically effective amount of at least one other
antiviral agent.
[0115] The invention also provides the use of a pharmaceutical
composition as described hereinabove for the treatment of a HBV
infection in a human being having or at risk of having the
infection.
[0116] The invention also provides the use of a pharmaceutical
composition as described hereinabove for the treatment of HBV
infection in a human being having or at risk of having the
disease.
[0117] Another aspect of the invention involves a method of
treating or preventing a hepatitis B viral disease and/or infection
in a human being by administering to the human being an antivirally
effective amount of a compound of the invention, a pharmaceutically
acceptable salt thereof, or a composition as described above, alone
or in combination with at least one other antiviral agent,
administered together or separately.
[0118] An additional aspect of this invention refers to an article
of manufacture comprising a composition of the invention that is
effective to treat a hepatitis B viral disease and/or infection;
and packaging material comprising a label which indicates that the
composition can be used to treat disease and/or infection by a
hepatitis B virus; wherein the composition comprises a compound of
formula (I) according to this invention or a pharmaceutically
acceptable salt thereof.
[0119] Still another aspect of this invention relates to a method
of inhibiting the replication of HBV, comprising exposing the virus
to an effective amount of the compound of formula (I), or a salt
thereof, under conditions where replication of the virus is
inhibited. This method can be practiced in vitro or in vivo.
[0120] Further included in the scope of the invention is the use of
a compound of formula (I), or a salt thereof, to inhibit the
replication of HBV.
[0121] In all of the embodiment referring to a compound of Formula
(I), the compound of Formula (I) can be a compound according to any
of embodiments 1-17 described above.
[0122] In some embodiments, the compound of Formula (I) is
co-administered with or used in combination with at least one
additional therapeutic agent selected from: an interferon or
peginterferon, an HBV polymerase inhibitor, a viral entry
inhibitor, a viral maturation inhibitor, a capsid assembly
inhibitor, an HBV core modulator, a reverse transcriptase
inhibitor, a TLR-agonist, or an immunomodulator. Optionally, the
compound of Formula (I) may be prepared for simultaneous or
sequential use in combination with an additional therapeutic agent;
or the compound of Formula (I) may be combined into a
pharmaceutical combination comprising a compound of Formula (I) and
at least one additional therapeutic agent. Some particular
therapeutic agents that may be used in combination with the
compounds of the invention include immunomodulators described
herein, interferon alfa 2a, interferon alfa-2b, pegylated
interferon alfa-2a, pegylated interferon alfa-2b, TLR-7 and TLR-9
agonists, entecavir, tenofovir, cidofovir, telbivudine, didanosine,
zalcitabine, stavudine, lamivudine, abacavir, emtricitabine,
apricitabine, atevirapine, ribavirin, acyclovir, famciclovir,
valacyclovir, ganciclovir, adefovir, efavirenz, nevirapine,
delavirdine, and etravirine. Suitable core modulators are disclosed
in WO2013/096744; suitable HBV capsid inhibitors are described in
US2015/0252057.
[0123] These additional agents may be combined with the compounds
of this invention to create a single pharmaceutical dosage form.
Alternatively these additional agents may be separately
administered to the patient as part of a multiple dosage form, for
example, using a kit. Such additional agents may be administered to
the patient prior to, concurrently with, or following the
administration of a compound of the invention, or a
pharmaceutically acceptable salt thereof. Alternatively, these
additional therapeutic agents may be administered separately from
and optionally by different routes of administration and on
different dosing schedules from the compound of the invention,
provided the compound of the invention and the additional
therapeutic agent are used concurrently for treatment of an HBV
infection or a disorder caused or complicated by an HBV
infection.
[0124] The dose range of the compounds of the invention applicable
per day is usually from 0.01 to 100 mg/kg of body weight,
preferably from 0.1 to 50 mg/kg of body weight. Each dosage unit
may conveniently contain from 5% to 95% active compound (w/w).
Preferably such preparations contain from 20% to 80% active
compound.
[0125] The actual pharmaceutically effective amount or therapeutic
dosage will of course depend on factors known by those skilled in
the art such as age and weight of the patient, route of
administration and severity of disease. In any case the combination
will be administered at dosages and in a manner which allows a
pharmaceutically effective amount to be delivered based upon
patient's unique condition.
[0126] When the composition of this invention comprises a
combination of a compound of the invention and one or more
additional therapeutic or prophylactic agent, both the compound and
the additional agent should be present at dosage levels of between
about 10 to 100%, and more preferably between about 10 and 80% of
the dosage normally administered in a monotherapy regimen.
[0127] Antiviral agents contemplated for use in such combination
therapy include agents (compounds or biologicals) that are
effective to inhibit the formation and/or replication of a virus in
a human being, including but not limited to agents that interfere
with either host or viral mechanisms necessary for the formation
and/or replication of a virus in a human being. Such agents can be
selected from entecavir, tenofovir, cidofovir, telbivudine,
didanosine, zalcitabine, stavudine, lamivudine, abacavir,
emtricitabine, apricitabine, atevirapine, ribavirin, acyclovir,
famciclovir, valacyclovir, ganciclovir, adefovir, efavirenz,
nevirapine, delavirdine, and etravirine, and immunomodulators
described herein including interferons and pegylated interferons,
TLR-7 agonists, and TLR-9 agonists.
[0128] Many compounds of the invention contain one or more chiral
centers. These compounds may be made and used as single isomers or
as mixtures of isomers. Methods for separating the isomers,
including diastereomers and enantiomers, are known in the art, and
examples of suitable methods are described herein. In certain
embodiments, the compounds of the invention are used as a single
substantially pure isomer, meaning at least 90% of a sample of the
compound is the specified isomer and less than 10% of the sample is
any other isomer or mixture of isomers. Preferably, at least 95% of
the sample is a single isomer. Selection of a suitable isomer is
within the ordinary level of skill, as one isomer will typically be
more active in the in vivo or in vitro assay described herein for
measuring HBV activity, and will be the preferred isomer. Where in
vitro activity differences between isomers are relatively small,
e.g. less than about a factor of 4, a preferred isomer may be
selected based on activity level against viral replication in cell
culture, using methods such as those described herein: the isomer
having a lower MIC (minimum inhibitory concentration) or EC-50 is
preferred.
[0129] The compounds of the invention may be synthesized by the
general synthetic routes illustrated below, specific examples of
which are described in more detail in the Examples. Additional
guidance for synthesis of the compounds of Formula (I) and
synthetic intermediates useful for these syntheses are disclosed in
published PCT applications WO2015/113990 and WO2015/173164.
[0130] Scheme 1 illustrates a general method useful to make
compounds of the invention, as demonstrated in the Examples herein.
A variety of indazole-3-carboxylate starting materials are known in
the art. The carboxylate can be reduced to an alcohol using methods
known in the art, and the alcohol can be protected with a
protecting group such as known silyl ethers (e.g., TBS). The N-2
nitrogen can be alkylated with a suitable alph-haloketone to
introduce the group containing R.sup.9. Reductive amination is one
method to introduce nitrogen at the carbonyl center. Once the
primary amine is in place, the protected alcohol at C.sub.3 of the
indazole can be deprotected and oxidized to an aldehyde oxidation
state, at which point it cyclizes with the primary amine to form
the new 6-membered ring substituted with R.sup.9.
[0131] The imine of the new ring is then annulated to form an
additional fused ring by a method known in the art, using (Z)-ethyl
2-(ethoxymethylene)-3-oxobutanoate. The new ring is then oxidized
to provide the pyridone ring shown in Formula (I). Methods useful
in preparing these compounds are disclosed in published PCT
applications WO2015/113990 and WO2015/173164.
##STR00020##
[0132] Using this general method, other known starting materials,
and the examples herein, a person of ordinary skill can synthesize
compounds of Formula (I). Enantiomers of these compounds can be
separated by chiral HPLC and similar known methods. Alternatively,
enantiomers may be synthesized by the general synthetic routes
illustrated in Scheme 2, specific examples of which are described
in more detail in the Examples.
##STR00021##
[0133] While one enantiomer of compounds of this formula is
typically more active than the other enantiomer, both isomers
exhibit activity on HBsAg as demonstrated herein.
[0134] The term "an optical isomer" or "a stereoisomer" refers to
any of the various stereoisomeric configurations which may exist
for a given compound of the present invention and includes
geometric isomers. It is understood that a substituent may be
attached at a chiral center of a carbon atom. The term "chiral"
refers to molecules which have the property of
non-superimposability on their mirror image partner, while the term
"achiral" refers to molecules which are superimposable on their
mirror image partner. Therefore, the invention includes
enantiomers, diastereomers or racemates of the compound.
"Enantiomers" are a pair of stereoisomers that are
non-superimposable mirror images of each other. A 1:1 mixture of a
pair of enantiomers is a "racemic" mixture. The term is used to
designate a racemic mixture where appropriate. "Diastereoisomers"
are stereoisomers that have at least two asymmetric atoms, but
which are not mirror-images of each other. The absolute
stereochemistry is specified according to the Cahn-lngold- Prelog
R-S system. When a compound is a pure enantiomer the
stereochemistry at each chiral carbon may be specified by either R
or S. Resolved compounds whose absolute configuration is unknown
can be designated (+) or (-) depending on the direction (dextro- or
levorotatory) which they rotate plane polarized light at the
wavelength of the sodium D line. Certain compounds described herein
contain one or more asymmetric centers or axes and may thus give
rise to enantiomers, diastereomers, and other stereoisomeric forms
that may be defined, in terms of absolute stereochemistry, as (R)-
or (S)-.
[0135] Depending on the choice of the starting materials and
procedures, the compounds can be present in the form of one of the
possible isomers or as mixtures thereof, for example as pure
optical isomers, or as isomer mixtures, such as racemates and
diastereoisomer mixtures, depending on the number of asymmetric
carbon atoms. The present invention is meant to include all such
possible stereoisomers, including racemic mixtures, diasteriomeric
mixtures and optically pure forms. Optically active (R)- and
(S)-isomers may be prepared using chiral synthons or chiral
reagents, or resolved using conventional techniques. If the
compound contains a double bond, the substituent may be E or Z
configuration. If the compound contains a disubstituted cycloalkyl,
the cycloalkyl substituent may have a cis- or trans-configuration.
All tautomeric forms are also intended to be included.
[0136] Any resulting mixtures of isomers can be separated on the
basis of the physicochemical differences of the constituents, into
the pure or substantially pure geometric or optical isomers or
diastereomers, for example, by chromatography and/or fractional
crystallization.
[0137] Any resulting racemates of final products or intermediates
can be resolved into the optical antipodes by known methods, e.g.,
by separation of the diastereomeric salts thereof, obtained with an
optically active acid or base, and liberating the optically active
acidic or basic compound. In particular, a basic moiety may thus be
employed to resolve the compounds of the present invention into
their optical antipodes, e.g., by fractional crystallization of a
salt formed with an optically active acid, e.g., tartaric acid,
dibenzoyl tartaric acid, diacetyl tartaric acid, di-O,O'-p-toluoyl
tartaric acid, mandelic acid, malic acid or camphor-10-sulfonic
acid. Racemic products can also be resolved by chiral
chromatography, e.g., high pressure liquid chromatography (HPLC)
using a chiral adsorbent.
[0138] Furthermore, the compounds of the present invention,
including their salts, can also be obtained in the form of their
hydrates, or include other solvents used for their crystallization.
The compounds of the present invention may inherently or by design
form solvates with pharmaceutically acceptable solvents (including
water); therefore, it is intended that the invention embrace both
solvated and unsolvated forms. The term "solvate" refers to a
molecular complex of a compound of the present invention (including
pharmaceutically acceptable salts thereof) with one or more solvent
molecules. Such solvent molecules are those commonly used in the
pharmaceutical art, which are known to be innocuous to the
recipient, e.g., water, ethanol, and the like. The term "hydrate"
refers to the complex where the solvent molecule is water.
[0139] The compounds of the present invention, including salts,
hydrates and solvates thereof, may inherently or by design form
polymorphs.
[0140] As used herein, the terms "salt" or "salts" refers to an
acid addition or base addition salt of a compound of the present
invention. "Salts" include in particular "pharmaceutically
acceptable salts". The term "pharmaceutically acceptable salts"
refers to salts that retain the biological effectiveness and
properties of the compounds of this invention and, which typically
are not biologically or otherwise undesirable. In many cases, the
compounds of the present invention are capable of forming acid
and/or base salts by virtue of the presence of amino and/or
carboxyl groups or groups similar thereto.
[0141] Pharmaceutically acceptable acid addition salts can be
formed with inorganic acids and organic acids, e.g., acetate,
aspartate, benzoate, besylate, bromide/hydrobromide,
bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate,
chloride/hydrochloride, chlortheophyllonate, citrate,
ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate,
hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate,
laurylsulfate, malate, maleate, malonate, mandelate, mesylate,
methylsulphate, naphthoate, napsylate, nicotinate, nitrate,
octadecanoate, oleate, oxalate, palmitate, pamoate,
phosphate/hydrogen phosphate/dihydrogen phosphate,
polygalacturonate, propionate, stearate, succinate,
sulfosalicylate, tartrate, tosylate and trifluoroacetate salts.
[0142] Inorganic acids from which salts can be derived include, for
example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid, and the like.
[0143] Organic acids from which salts can be derived include, for
example, acetic acid, propionic acid, glycolic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic
acid, and the like. Pharmaceutically acceptable base addition salts
can be formed with inorganic and organic bases.
[0144] Inorganic bases from which salts can be derived include, for
example, ammonium salts and metals from columns I to XII of the
periodic table. In certain embodiments, the salts are derived from
sodium, potassium, ammonium, calcium, magnesium, iron, silver,
zinc, and copper; particularly suitable salts include ammonium,
potassium, sodium, calcium and magnesium salts.
[0145] Organic bases from which salts can be derived include, for
example, primary, secondary, and tertiary amines, substituted
amines including naturally occurring substituted amines, cyclic
amines, basic ion exchange resins, and the like. Certain organic
amines include isopropylamine, benzathine, cholinate,
diethanolamine, diethylamine, lysine, meglumine, piperazine and
tromethamine.
[0146] The pharmaceutically acceptable salts of the present
invention can be synthesized from a basic or acidic moiety, by
conventional chemical methods. Generally, such salts can be
prepared by reacting free acid forms of these compounds with a
stoichiometric amount of the appropriate base (such as Na, Ca, Mg,
or K hydroxide, carbonate, bicarbonate or the like), or by reacting
free base forms of these compounds with a stoichiometric amount of
the appropriate acid. Such reactions are typically carried out in
water or in an organic solvent, or in a mixture of the two.
Generally, use of non-aqueous media like ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile is desirable, where
practicable. Lists of additional suitable salts can be found, e.g.,
in "Remington's Pharmaceutical Sciences", 20th ed., Mack Publishing
Company, Easton, Pa., (1985); and in "Handbook of Pharmaceutical
Salts: Properties, Selection, and Use" by Stahl and Wermuth
(Wiley-VCH, Weinheim, Germany, 2002).
[0147] Any formula given herein is intended to represent unlabeled
forms as well as isotopically labeled forms of the compounds of the
present invention having up to three atoms with non-natural isotope
distributions, e.g., sites that are enriched in deuterium or
.sup.13C or .sup.15N. Isotopically labeled compounds have
structures depicted by the formulas given herein except that one or
more atoms are replaced by an atom having a selected atomic mass or
mass number other than the natural-abundance mass distribution.
Examples of isotopes that can be usefully over-incorporated into
compounds of the invention include isotopes of hydrogen, carbon,
nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as
.sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C, .sup.15N, .sup.18F
.sup.31P, .sup.32P, .sup.35S, .sup.36Cl, .sup.125I respectively.
The invention includes various isotopically labeled compounds of
the present invention, for example those into which radioactive
isotopes, such as .sup.3H and .sup.14C, or those in which
non-radioactive isotopes, such as .sup.2H and .sup.13C are present
at levels substantially above normal isotope distribution. Such
isotopically labelled compounds are useful in metabolic studies
(with .sup.14C, for example), reaction kinetic studies (with, for
example .sup.2H or .sup.3H), detection or imaging techniques, such
as positron emission tomography (PET) or single-photon emission
computed tomography (SPECT) including drug or substrate tissue
distribution assays, or in radioactive treatment of patients. In
particular, an .sup.18F labeled compound of the present invention
may be particularly desirable for PET or SPECT studies.
Isotopically-labeled compounds of the present invention can
generally be prepared by conventional techniques known to those
skilled in the art or by processes analogous to those described in
the accompanying Examples and Preparations using an appropriate
isotopically-labeled reagent in place of the non-labeled reagent
typically employed. Labeled samples may be useful with quite low
isotope incorporation, such as where a radiolabel is used to detect
trace amounts of the compound.
[0148] Further, site-specific substitution with heavier isotopes,
particularly deuterium (i.e., .sup.2H or D), may afford certain
therapeutic advantages resulting from greater metabolic stability,
for example increased in vivo half-life or reduced dosage
requirements or an improvement in therapeutic index. It is
understood that deuterium in this context is regarded as a
substituent of a compound of the present invention, and typically a
sample of a compound having deuterium as a substituent has at least
50% deuterium incorporation at the labeled position(s). The
concentration of such a heavier isotope, specifically deuterium,
may be defined by the isotopic enrichment factor. The term
"isotopic enrichment factor" as used herein means the ratio between
the isotopic abundance and the natural abundance of a specified
isotope. If a substituent in a compound of this invention is
denoted deuterium, such compound has an isotopic enrichment factor
for each designated deuterium atom of at least 3500 (52.5%
deuterium incorporation at each designated deuterium atom), at
least 4000 (60% deuterium incorporation), at least 4500 (67.5%
deuterium incorporation), at least 5000 (75% deuterium
incorporation), at least 5500 (82.5% deuterium incorporation), at
least 6000 (90% deuterium incorporation), at least 6333.3 (95%
deuterium incorporation), at least 6466.7 (97% deuterium
incorporation), at least 6600 (99% deuterium incorporation), or at
least 6633.3 (99.5% deuterium incorporation).
[0149] Pharmaceutically acceptable solvates in accordance with the
invention include those wherein the solvent of crystallization may
be isotopically substituted, e.g. D.sub.2O, d.sup.6-acetone,
d.sup.6-DMSO.
[0150] Compounds of the present invention that contain groups
capable of acting as donors and/or acceptors for hydrogen bonds may
be capable of forming co-crystals with suitable co-crystal formers.
These co-crystals may be prepared from compounds of the present
invention by known co-crystal forming procedures. Such procedures
include grinding, heating, co-subliming, co-melting, or contacting
in solution compounds of the present invention with the co-crystal
former under crystallization conditions and isolating co-crystals
thereby formed. Suitable co-crystal formers include those described
in WO 2004/078163. Hence the invention further provides co-crystals
comprising a compound of the present invention.
Methods of Use
[0151] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g. "such as") provided herein is intended
merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed.
[0152] The compounds of the invention can be administered by known
methods, including oral, parenteral, inhalation, and the like. In
certain embodiments, the compound of the invention is administered
orally, as a pill, lozenge, troche, capsule, solution, or
suspension. In other embodiments, a compound of the invention is
administered by injection or infusion. Infusion is typically
performed intravenously, often over a period of time between about
15 minutes and 4 hours. In other embodiments, a compound of the
invention is administered intranasally or by inhalation; inhalation
methods are particularly useful for treatment of respiratory
infections. Compounds of the present invention exhibit oral
bioavailability, so oral administration is sometimes preferred.
[0153] In certain embodiments of the present invention, a compound
of the present invention is used in combination with a second
antiviral agent, such as those named herein.
[0154] By the term "combination", is meant either a fixed
combination in one dosage unit form, as separate dosage forms
suitable for use together either simultaneously or sequentially, or
as a kit of parts for the combined administration where a compound
of the present invention and a combination partner may be
administered independently at the same time or separately within
time intervals that especially allow that the combination partners
show a cooperative, e.g., synergistic, effect, or any combination
thereof.
[0155] The second antiviral agent may be administered in
combination with the compounds of the present inventions wherein
the second antiviral agent is administered prior to,
simultaneously, or after the compound or compounds of the present
invention. When simultaneous administration of a compound of the
invention with a second agent is desired and the route of
administration is the same, then a compound of the invention may be
formulated with a second agent into the same dosage form. An
example of a dosage form containing a compound of the invention and
a second agent is a tablet or a capsule.
[0156] In some embodiments, a combination of a compound of the
invention and a second antiviral agent may provide synergistic
activity. The compound of the invention and second antiviral agent
may be administered together, separate but simultaneously, or
sequentially.
[0157] An "effective amount" of a compound is that amount necessary
or sufficient to treat or prevent a viral infection and/or a
disease or condition described herein. In an example, an effective
amount of a compound of Formula I is an amount sufficient to treat
viral infection in a subject. In another example, an effective
amount is an amount sufficient to treat HBV in a subject in need of
such treatment. The effective amount can vary depending on such
factors as the size and weight of the subject, the type of illness,
or the particular compound of the invention. For example, the
choice of the compound of the invention can affect what constitutes
an "effective amount." One of ordinary skill in the art would be
able to study the factors contained herein and make the
determination regarding the effective amount of the compounds of
the invention without undue experimentation.
[0158] The regimen of administration can affect what constitutes an
effective amount. The compound of the invention can be administered
to the subject either prior to or after the onset of a viral
infection. Further, several divided dosages, as well as staggered
dosages, can be administered daily or sequentially, or the dose can
be continuously infused, or can be a bolus injection. Further, the
dosages of the compound(s) of the invention can be proportionally
increased or decreased as indicated by the exigencies of the
therapeutic or prophylactic situation.
[0159] Compounds of the invention may be used in the treatment of
states, disorders or diseases as described herein, or for the
manufacture of pharmaceutical compositions for use in the treatment
of these diseases. The invention provides methods of use of
compounds of the present invention in the treatment of these
diseases or for preparation of pharmaceutical compositions having
compounds of the present invention for the treatment of these
diseases.
[0160] The language "pharmaceutical composition" includes
preparations suitable for administration to mammals, e.g., humans.
When the compounds of the present invention are administered as
pharmaceuticals to mammals, e.g., humans, they can be given per se
or as a pharmaceutical composition containing, for example, 0.1 to
99.5% (more preferably, 0.5 to 90%) of at least one compound of
Formula (I) or any subgenus thereof as active ingredient in
combination with a pharmaceutically acceptable carrier, or
optionally two or more pharmaceutically acceptable carriers.
[0161] The phrase "pharmaceutically acceptable carrier" is art
recognized and includes a pharmaceutically acceptable material,
composition or vehicle, suitable for administering compounds of the
present invention to mammals. The carriers include liquid or solid
filler, diluent, excipient, solvent or encapsulating material,
involved in carrying or transporting the subject agent from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically acceptable carriers include: sugars, such
as lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations. Typically, pharmaceutically acceptable
carriers are sterilized and/or substantially pyrogen-free.
[0162] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0163] Examples of pharmaceutically acceptable antioxidants
include: water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, .alpha.-tocopherol,
and the like; and metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0164] Formulations of the present invention include those suitable
for oral, nasal, inhalation, topical, transdermal, buccal,
sublingual, rectal, vaginal and/or parenteral administration. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any methods well known in the art of pharmacy.
The amount of active ingredient that can be combined with a carrier
material to produce a single dosage form will generally be that
amount of the compound that produces a therapeutic effect.
Generally, out of one hundred percent, this amount will range from
about 1 percent to about ninety-nine percent of active ingredient,
preferably from about 5 percent to about 70 percent, most
preferably from about 10 percent to about 30 percent.
[0165] Methods of preparing these formulations or compositions
include the step of bringing into association a compound of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a compound of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0166] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored base, for example, usually
sucrose and acacia or tragacanth), powders, granules, or as a
solution or a suspension in an aqueous or non-aqueous liquid, or as
an oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. A compound of the
present invention may also be administered as a bolus, electuary or
paste.
[0167] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; humectants, such as glycerol; disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate;
solution retarding agents, such as paraffin; absorption
accelerators, such as quaternary ammonium compounds; wetting
agents, such as, for example, cetyl alcohol and glycerol
monostearate; absorbents, such as kaolin and bentonite clay;
lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and coloring agents. In the case of capsules, tablets and
pills, the pharmaceutical compositions may also comprise buffering
agents. Solid compositions of a similar type may also be employed
as fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugars, as well as high molecular
weight polyethylene glycols and the like.
[0168] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0169] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions that
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions that can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0170] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluent commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0171] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0172] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0173] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active compound.
[0174] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0175] Dosage forms for the topical or transdermal administration
of a compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants that may be required.
[0176] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0177] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0178] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
compound in the proper medium. Absorption enhancers can also be
used to increase the flux of the compound across the skin. The rate
of such flux can be controlled by either providing a rate
controlling membrane or dispersing the active compound in a polymer
matrix or gel.
[0179] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0180] Pharmaceutical compositions of this invention suitable for
parenteral administration may comprise one or more compounds of the
invention in combination with one or more pharmaceutically
acceptable carriers such as sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents.
[0181] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, glycol ethers, polyols (such as glycerol,
propylene glycol, polyethylene glycol, and the like), and suitable
mixtures thereof, vegetable oils, such as olive oil, and injectable
organic esters, such as ethyl oleate. Proper fluidity can be
maintained, for example, by the use of coating materials, such as
lecithin, by the maintenance of the required particle size in the
case of dispersions, and by the use of surfactants.
[0182] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents that delay
absorption such as aluminum monostearate and gelatin.
[0183] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0184] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions that are
compatible with body tissue.
[0185] The preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are of course
given by forms suitable for each administration route. For example,
they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, etc., administration
by injection, infusion or inhalation; topical by lotion or
ointment; and rectal by suppositories.
[0186] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and infusion.
Intravenous infusion is sometimes a preferred method of delivery
for compounds of the invention. Infusion may be used to deliver a
single daily dose or multiple doses. In some embodiments, a
compound of the invention is administered by infusion over an
interval between 15 minutes and 4 hours, typically between 0.5 and
3 hours. Such infusion may be used once per day, twice per day or
up to three times per day.
[0187] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0188] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0189] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0190] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0191] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular compound employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0192] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved.
[0193] In general, a suitable daily dose of a compound of the
invention will be that amount of the compound that is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above.
Generally, intravenous and subcutaneous doses of the compounds of
this invention for a patient, when used for the indicated effects,
will range from about 0.0001 to about 100 mg per kilogram of body
weight per day, more preferably from about 0.01 to about 50 mg per
kg per day, and still more preferably from about 0.1 to about 20 mg
per kg per day. An effective amount is that amount which prevents
or treats a viral infection, such as HBV.
[0194] Treatment with a compound or composition described herein
may be repeated daily for a period sufficient to reduce or
substantially eliminate an HBV infection or viral load. For
example, treatment may be continued for a week, or two weeks, or
3-4 weeks, or 4-8 weeks, or 8-12 weeks, 2-6 months, or longer,
e.g., until viral load or other measure of infection shows a
substantial reduction in viral load or viral activity or other
signs or symptoms of HBV infection. The skilled treating physician
can readily determine a suitable duration of treatment.
[0195] If desired, the effective daily dose of the active compound
may be administered as a single dose per day, or as two, three,
four, five, six or more sub-doses administered separately at
appropriate intervals throughout the day, optionally, in unit
dosage forms. Compounds delivered orally or by inhalation, are
commonly administered in one to four doses per day. Compounds
delivered by injection are typically administered once per day, or
once every other day. Compounds delivered by infusion are typically
administered in one to three doses per day. When multiple doses are
administered within a day, the doses may be administered at
intervals of about 4 hours, about 6 hours, about 8 hours or about
12 hours.
[0196] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical composition such as those described
herein. Thus methods of using the compounds of the invention
include administering the compound as a pharmaceutical composition,
wherein at least one compound of the invention is admixed with a
pharmaceutically acceptable carrier prior to administration.
Use of Compounds of the Invention in Combination with
Immunomodulators
[0197] The compounds and compositions described herein can be used
or administered in combination with one or more therapeutic agents
that act as immunomodulators, e.g., an activator of a costimulatory
molecule, or an inhibitor of an immune-inhibitory molecule, or a
vaccine. The Programmed Death 1 (PD-1) protein is an inhibitory
member of the extended CD28/CTLA4 family of T cell regulators
(Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et
al. (2003) J. Immunol. 170:711-8). PD-1 is expressed on activated B
cells, T cells, and monocytes. PD-1 is an immune-inhibitory protein
that negatively regulates TCR signals (Ishida, Y. et al. (1992)
EMBO J. 11:3887-3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol.
Immunother. 56(5):739-745), and is up-regulated in chronic
infections. The interaction between PD-1 and PD-L1 can act as an
immune checkpoint, which can lead to, e.g., a decrease in
infiltrating lymphocytes, a decrease in T-cell receptor mediated
proliferation, and/or immune evasion by cancerous or infected cells
(Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005)
Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin.
Cancer Res. 10:5094-100). Immune suppression can be reversed by
inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the
effect is additive when the interaction of PD-1 with PD-L2 is
blocked as well (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA
99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).
Immunomodulation can be achieved by binding to either the
immune-inhibitory protein (e.g., PD-1) or to binding proteins that
modulate the inhibitory protein (e.g., PD-L1, PD-L2).
[0198] In one embodiment, the combination therapies of the
invention include an immunomodulator that is an inhibitor or
antagonist of an inhibitory molecule of an immune checkpoint
molecule. In another embodiment the immunomodulator binds to a
protein that naturally inhibits the immuno-inhibitory checkpoint
molecule. When used in combination with antiviral compounds, these
immunomodulators can enhance the antiviral response, and thus
enhance efficacy relative to treatment with the antiviral compound
alone.
[0199] The term "immune checkpoints" refers to a group of molecules
on the cell surface of CD4 and CD8 T cells. These molecules can
effectively serve as "brakes" to down-modulate or inhibit an
adaptive immune response. Immune checkpoint molecules include, but
are not limited to, Programmed Death 1 (PD-1), Cytotoxic
T-Lymphocyte Antigen 4 (CTLA-4), B7H1, B7H4, OX-40, CD137, CD40,
and LAG3, which directly inhibit immune cells. Immunotherapeutic
agents which can act as immune checkpoint inhibitors useful in the
methods of the present invention, include, but are not limited to,
inhibitors of PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT,
LAIR1, CD160, 2B4 and/or TGFR beta. Inhibition of an inhibitory
molecule can be performed by inhibition at the DNA, RNA or protein
level. In some embodiments, an inhibitory nucleic acid (e.g., a
dsRNA, siRNA or shRNA), can be used to inhibit expression of an
inhibitory molecule. In other embodiments, the inhibitor of an
inhibitory signal is a polypeptide, e.g., a soluble ligand, or an
antibody or antigen-binding fragment thereof, that binds to the
inhibitory molecule.
[0200] By "in combination with," it is not intended to imply that
the therapy or the therapeutic agents must be administered at the
same time and/or formulated for delivery together, although these
methods of delivery are within the scope described herein. The
immunomodulator can be administered concurrently with, prior to, or
subsequent to, one or more compounds of the invention, and
optionally one or more additional therapies or therapeutic agents.
The therapeutic agents in the combination can be administered in
any order. In general, each agent will be administered at a dose
and/or on a time schedule determined for that agent. It will
further be appreciated that the therapeutic agents utilized in this
combination may be administered together in a single composition or
administered separately in different compositions. In general, it
is expected that each of the therapeutic agents utilized in
combination be utilized at levels that do not exceed the levels at
which they are utilized individually. In some embodiments, the
levels utilized in combination will be lower than those utilized
individually.
[0201] In certain embodiments, the antiviral compounds described
herein are administered in combination with one or more
immunomodulators that are inhibitors of PD-1, PD-L1 and/or PD-L2.
Each such inhibitor may be an antibody, an antigen binding fragment
thereof, an immunoadhesin, a fusion protein, or an oligopeptide.
Examples of such immunomodulators are known in the art.
[0202] In some embodiments, the immunomodulator is an anti-PD-1
antibody chosen from MDX-1106, Merck 3475 or CT-011.
[0203] In some embodiments, the immunomodulator is an immunoadhesin
(e.g., an immunoadhesin comprising an extracellular or PD-1 binding
portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc
region of an immunoglobulin sequence).
[0204] In some embodiments, the immunomodulator is a PD-1 inhibitor
such as AMP-224.
[0205] In some embodiments, the immunomodulator is a PD-L1
inhibitor such as anti-PD-L1 antibody.
[0206] In some embodiments, the immunomodulator is an anti-PD-L1
binding antagonist chosen from YW243.55.S70, MPDL3280A, MEDI-4736,
MSB-0010718C, or MDX-1105. MDX-1105, also known as BMS-936559, is
an anti-PD-L1 antibody described in WO2007/005874. Antibody
YW243.55.S70 is an anti-PD-L1 described in WO 2010/077634.
[0207] In some embodiments, the immunomodulator is nivolumab (CAS
Registry Number: 946414-94-4). Alternative names for nivolumab
include MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558. Nivolumab
is a fully human IgG4 monoclonal antibody which specifically blocks
PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies
that specifically bind to PD-1 are disclosed in U.S. Pat. No.
8,008,449, EP2161336 and WO2006/121168.
[0208] In some embodiments, the immunomodulator is an anti-PD-1
antibody Pembrolizumab. Pembrolizumab (also referred to as
Lambrolizumab, MK-3475, MK03475, SCH-900475 or KEYTRUDA.RTM.;
Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1.
Pembrolizumab and other humanized anti-PD-1 antibodies are
disclosed in Hamid, O. et al. (2013) New England Journal of
Medicine 369 (2): 134-44, U.S. Pat. No. 8,354,509, WO2009/114335,
and WO2013/079174.
[0209] In some embodiments, the immunomodulator is Pidilizumab
(CT-011; Cure Tech), a humanized IgG1k monoclonal antibody that
binds to PD1. Pidilizumab and other humanized anti-PD-1 monoclonal
antibodies are disclosed in WO2009/101611.
[0210] Other anti-PD1 antibodies useful as immunomodulators for use
in the methods disclosed herein include AMP 514 (Amplimmune), and
anti-PD1 antibodies disclosed in U.S. Pat. No. 8,609,089, US
2010028330, and/or US 20120114649. In some embodiments, the
anti-PD-L1 antibody is MSB0010718C. MSB0010718C (also referred to
as A09-246-2; Merck Serono) is a monoclonal antibody that binds to
PD-L1.
[0211] In some embodiments, the immunomodulator is MDPL3280A
(Genentech/Roche), a human Fc optimized IgG1 monoclonal antibody
that binds to PD-L1. MDPL3280A and other human monoclonal
antibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743 and U.
S Publication No.: 20120039906. Other anti-PD-L1 binding agents
useful as immunomodulators for methods of the invention include
YW243.55.S70 (see WO2010/077634), MDX-1105 (also referred to as
BMS-936559), and anti-PD-L1 binding agents disclosed in
WO2007/005874.
[0212] In some embodiments, the immunomodulator is AMP-224
(B7-DCIg; Amplimmune; e.g., disclosed in WO2010/027827 and
WO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocks
the interaction between PD1 and B7-H1.
[0213] In some embodiments, the immunomodulator is an anti-LAG-3
antibody such as BMS-986016. BMS-986016 (also referred to as
BMS986016) is a monoclonal antibody that binds to LAG-3. BMS-986016
and other humanized anti-LAG-3 antibodies are disclosed in US
2011/0150892, WO2010/019570, and WO2014/008218
[0214] In certain embodiments, the combination therapies disclosed
herein include a modulator of a costimulatory molecule or an
inhibitory molecule, e.g., a co-inhibitory ligand or receptor.
[0215] In one embodiment, the costimulatory modulator, e.g.,
agonist, of a costimulatory molecule is chosen from an agonist
(e.g., an agonistic antibody or antigen-binding fragment thereof,
or soluble fusion) of OX40, CD2, CD27, CDS, ICAM-1, LFA-1
(CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR,
HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83
ligand.
[0216] In another embodiment, the combination therapies disclosed
herein include an immunomodulator that is a costimulatory molecule,
e.g., an agonist associated with a positive signal that includes a
costimulatory domain of CD28, CD27, ICOS and/or GITR.
[0217] Exemplary GITR agonists include, e.g., GITR fusion proteins
and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies),
such as, a GITR fusion protein described in U.S. Pat. No.
6,111,090, European Patent No.: 090505B1, U.S. Pat. No. 8,586,023,
PCT Publication Nos.: WO 2010/003118 and 2011/090754, or an
anti-GITR antibody described, e.g., in U.S. Pat. No. 7,025,962,
European Patent No.: 1947183B1, U.S. Pat. Nos. 7,812,135,
8,388,967, 8,591,886, European Patent No.: EP 1866339, PCT
Publication No.: WO 2011/028683, PCT Publication No.: WO
2013/039954, PCT Publication No.: WO2005/007190, PCT Publication
No.: WO 2007/133822, PCT Publication No.: WO2005/055808, PCT
Publication No.: WO 99/40196, PCT Publication No.: WO 2001/03720,
PCT Publication No.: WO99/20758, PCT Publication No.:
WO2006/083289, PCT Publication No.: WO 2005/115451, U.S. Pat. No.
7,618,632, and PCT Publication No.: WO 2011/051726.
[0218] In one embodiment, the immunomodulator used is a soluble
ligand (e.g., a CTLA-4-Ig), or an antibody or antibody fragment
that binds to PD-L1, PD-L2 or CTLA4. For example, the anti-PD-1
antibody molecule can be administered in combination with an
anti-CTLA-4 antibody, e.g., ipilimumab, for example. Exemplary
anti-CTLA4 antibodies include Tremelimumab (IgG2 monoclonal
antibody available from Pfizer, formerly known as ticilimumab,
CP-675,206); and Ipilimumab (CTLA-4 antibody, also known as
MDX-010, CAS No. 477202-00-9).
[0219] In one embodiment, an anti-PD-1 antibody molecule is
administered after treatment with a compound of the invention as
described herein.
[0220] In another embodiment, an anti-PD-1 or PD-L1 antibody
molecule is administered in combination with an anti-LAG-3 antibody
or an antigen-binding fragment thereof. In another embodiment, the
anti-PD-1 or PD-L1 antibody molecule is administered in combination
with an anti-TIM-3 antibody or antigen-binding fragment thereof. In
yet other embodiments, the anti-PD-1 or PD-L1 antibody molecule is
administered in combination with an anti-LAG-3 antibody and an
anti-TIM-3 antibody, or antigen-binding fragments thereof. The
combination of antibodies recited herein can be administered
separately, e.g., as separate antibodies, or linked, e.g., as a
bispecific or trispecific antibody molecule. In one embodiment, a
bispecific antibody that includes an anti-PD-1 or PD-L1 antibody
molecule and an anti-TIM-3 or anti-LAG-3 antibody, or
antigen-binding fragment thereof, is administered. In certain
embodiments, the combination of antibodies recited herein is used
to treat a cancer, e.g., a cancer as described herein (e.g., a
solid tumor). The efficacy of the aforesaid combinations can be
tested in animal models known in the art. For example, the animal
models to test the synergistic effect of anti-PD-1 and anti-LAG-3
are described, e.g., in Woo et al. (2012) Cancer Res.
72(4):917-27).
[0221] Exemplary immunomodulators that can be used in the
combination therapies include, but are not limited to, e.g.,
afutuzumab (available from Roche.RTM.); pegfilgrastim
(Neulasta.RTM.); lenalidomide (CC-5013, Revlimid.RTM.); thalidomide
(Thalomid.RTM.), actimid (CC4047); and cytokines, e.g., IL-21 or
IRX-2 (mixture of human cytokines including interleukin 1,
interleukin 2, and interferon .gamma., CAS 951209-71-5, available
from IRX Therapeutics).
[0222] Exemplary doses of such immunomodulators that can be used in
combination with the antiviral compounds of the invention include a
dose of anti-PD-1 antibody molecule of about 1 to 10 mg/kg, e.g., 3
mg/kg, and a dose of an anti-CTLA-4 antibody, e.g., ipilimumab, of
about 3 mg/kg.
[0223] Examples of embodiments of the methods of using the
antiviral compounds of the invention in combination with an
immunomodulator include these, which may be used along with a
compound of Formula I or any subgenus or species thereof that is
disclosed herein:
[0224] i. A method to treat a viral infection in a subject,
comprising administering to the subject a compound of Formula (I)
as described herein, and an immunomodulator.
[0225] ii. The method of embodiment i, wherein the immunomodulator
is an activator of a costimulatory molecule or an inhibitor of an
immune checkpoint molecule.
[0226] iii. The method of either of embodiments i and ii, wherein
the activator of the costimulatory molecule is an agonist of one or
more of OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS
(CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT,
NKG2C, SLAMF7, NKp80, CD160, B7-H3 and CD83 ligand.
[0227] iv. The method of any of embodiments i-iii above, wherein
the inhibitor of the immune checkpoint molecule is chosen from
PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1,
CD160, 2B4 and TGFR beta.
[0228] v. The method of any of embodiments i-iii, wherein the
inhibitor of the immune checkpoint molecule is chosen from an
inhibitor of PD-1, PD-L1, LAG-3, TIM-3 or CTLA4, or any combination
thereof.
[0229] vi. The method of any of embodiments i-v, wherein the
inhibitor of the immune checkpoint molecule is a soluble ligand or
an antibody or antigen-binding fragment thereof, that binds to the
immune checkpoint molecule.
[0230] vii. The method of any of embodiments i-vi, wherein the
antibody or antigen-binding fragment thereof is from an IgG1 or
IgG4 (e.g., human IgG1 or IgG4).
[0231] viii. The method of any of embodiments i-vii, wherein the
antibody or antigen-binding fragment thereof is altered, e.g.,
mutated, to increase or decrease one or more of: Fc receptor
binding, antibody glycosylation, the number of cysteine residues,
effector cell function, or complement function.
[0232] ix. The method of any of embodiments i-viii, wherein the
antibody molecule is a bispecific or multispecific antibody
molecule that has a first binding specificity to PD-1 or PD-L1 and
a second binding specificity to TIM-3, LAG-3, or PD-L2.
[0233] x. The method of any of embodiments i-ix, wherein the
immunomodulator is an anti-PD-1 antibody chosen from Nivolumab,
Pembrolizumab or Pidilizumab.
[0234] xi. The method of any of embodiments i-x, wherein the
immunomodulator is an anti-PD-L1 antibody chosen from YW243.55.S70,
MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.
[0235] xii. The method of any of embodiments i-x, wherein the
immunomodulator is an anti-LAG-3 antibody molecule.
[0236] xiii. The method of embodiment xii, wherein the anti-LAG-3
antibody molecule is BMS-986016.
[0237] xiv. The method of any of embodiments i-x, wherein the
immunomodulator is an anti-PD-1 antibody molecule administered by
injection (e.g., subcutaneously or intravenously) at a dose of
about 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20
mg/kg, about 1 to 5 mg/kg, or about 3 mg/kg., e.g., once a week to
once every 2, 3, or 4 weeks.
[0238] xv. The method of embodiment xiv, wherein the anti-PD-1
antibody molecule is administered at a dose from about 10 to 20
mg/kg every other week.
[0239] xvi. The method of embodiment xv, wherein the anti-PD-1
antibody molecule, e.g., nivolumab, is administered intravenously
at a dose from about 1 mg/kg to 3 mg/kg, e.g., about 1 mg/kg, 2
mg/kg or 3 mg/kg, every two weeks.
[0240] xvii. The method of embodiment xv, wherein the anti-PD-1
antibody molecule, e.g., nivolumab, is administered intravenously
at a dose of about 2 mg/kg at 3-week intervals.
[0241] The compounds as described herein may be synthesized by the
general synthetic routes below, specific examples of which are
described in more detail in the Examples.
General Synthetic Procedures
[0242] All starting materials, building blocks, reagents, acids,
bases, dehydrating agents, solvents, and catalysts utilized to
synthesize the compounds of the invention are either commercially
available or can be produced by organic synthesis methods known to
one of ordinary skill in the art (Houben-Weyl 4th Ed. 1952, Methods
of Organic Synthesis, Thieme, Volume 21). General methods for
synthesis of compounds of the invention are illustrated by the
Examples below, the general method in Scheme 1, and by methods
disclosed in published PCT applications WO2015/113990 and
WO2015/173164.
LIST OF ABBREVIATIONS
[0243] Ac acetyl
ACN Acetonitrile
[0244] AcOEt/EtOAc Ethyl acetate AcOH acetic acid aq aqueous Bn
benzyl Bu butyl (nBu=n-butyl, tBu=tert-butyl)
CDI Carbonyldiimidazole
[0245] DBU 1,8-Diazabicyclo[5.4.0]-undec-7-ene Boc20 di-tert-butyl
dicarbonate
DCE 1,2-Dichloroethane
DCM Dichloromethane
[0246] DIAD Diisopropyl azodicarboxylate
DiBAl-H Diisobutylaluminum Hydride
DIPEA N-Ethyldiisopropylamine
DMA N,N-dimethylacetamide
DMAP Dimethylaminopyridine
DMF N,N'-Dimethylformamide
DMSO Dimethylsulfoxide
[0247] EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EI
Electrospray ionisation
Et.sub.2O Diethylether
Et.sub.3N Triethylamine
Ether Diethylether
[0248] EtOAc Ethyl acetate
EtOH Ethanol
[0249] FA Formic acid
FC Flash Chromatography
[0250] h hour(s) HCl Hydrochloric acid
HOBt 1-Hydroxybenzotriazole
HPLC High Performance Liquid Chromatography
H.sub.2O Water
[0251] IPA isopropanol L liter(s)
LC-MS Liquid Chromatography Mass Spectrometry
[0252] LiHMDS Lithium bis(trimethylsilyl)amide Me methyl
MeI Iodomethane
MeOH Methanol
[0253] mg milligram min minute(s) mL milliliter
MS Mass Spectrometry
[0254] Pd/C palladium on charcoal PG protecting group Ph phenyl
Ph.sub.3P triphenyl phosphine
Prep Preparative
[0255] Rf ratio of fronts RP reverse phase Rt Retention time rt
Room temperature
SFC Supercritical Fluid Chromatography
SiO.sub.2 Silica gel
[0256] T3P.RTM. Propylphosphonic acid anhydride TBAF
Tetrabutylammonium fluoride TBDMS t-Butyldimethylsilyl
TEA Triethylamine
[0257] TFA Trifluoroacetic acid
THF Tetrahydrofuran
TLC Thin Layer Chromatography
[0258] TsCl toluene sulfonyl chloride
[0259] Within the scope of this text, a readily removable group
that is not a constituent of the particular desired end product of
the compounds of the present invention is designated a "protecting
group," unless the context indicates otherwise. The protection of
functional groups by such protecting groups, the protecting groups
themselves, and their cleavage reactions are described for example
in standard reference works, such as e.g., Science of Synthesis:
Houben-Weyl Methods of Molecular Transformation. Georg Thieme
Verlag, Stuttgart, Germany. 2005. 41627 pp. (URL:
http://www.science-of-synthesis.com (Electronic Version, 48
Volumes)); J. F. W. McOmie, "Protective Groups in Organic
Chemistry", Plenum Press, London and New York 1973, in T. W. Greene
and P. G. M. Wuts, "Protective Groups in Organic Synthesis", Third
edition, Wiley, New York 1999, in "The Peptides"; Volume 3
(editors: E. Gross and J. Meienhofer), Academic Press, London and
New York 1981, in "Methoden der organischen Chemie" (Methods of
Organic Chemistry), Houben Weyl, 4th edition, Volume 15/I, Georg
Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jeschkeit,
"Aminosauren, Peptide, Proteine" (Amino acids, Peptides, Proteins),
Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and in
Jochen Lehmann, "Chemie der Kohlenhydrate: Monosaccharide und
Derivate" (Chemistry of Carbohydrates: Monosaccharides and
Derivatives), Georg Thieme Verlag, Stuttgart 1974. A characteristic
of protecting groups is that they can be removed readily (i.e.,
without the occurrence of undesired secondary reactions) for
example by solvolysis, reduction, photolysis or alternatively under
physiological conditions (e.g., by enzymatic cleavage).
[0260] Salts of compounds of the present invention having at least
one salt-forming group may be prepared in a manner known per se.
For example, salts of compounds of the present invention having
acid groups may be formed, for example, by treating the compounds
with metal compounds, such as alkali metal salts of suitable
organic carboxylic acids, e.g., the sodium salt of 2-ethyl hexanoic
acid, with organic alkali metal or alkaline earth metal compounds,
such as the corresponding hydroxides, carbonates or hydrogen
carbonates, such as sodium or potassium hydroxide, carbonate or
hydrogen carbonate, with corresponding calcium compounds or with
ammonia or a suitable organic amine, stoichiometric amounts or only
a small excess of the salt-forming agent preferably being used.
Acid addition salts of compounds of the present invention are
obtained in customary manner, e.g., by treating the compounds with
an acid or a suitable anion exchange reagent. Internal salts of
compounds of the present invention containing acid and basic
salt-forming groups, e.g., a free carboxy group and a free amino
group, may be formed, e.g., by the neutralization of salts, such as
acid addition salts, to the isoelectric point, e.g., with weak
bases, or by treatment with ion exchangers.
[0261] Salts can be converted in customary manner into the free
compounds; metal and ammonium salts can be converted, for example,
by treatment with suitable acids, and acid addition salts, for
example, by treatment with a suitable basic agent.
[0262] Mixtures of isomers obtainable according to the invention
can be separated in a manner known per se into the individual
isomers; diastereoisomers can be separated, for example, by
partitioning between polyphasic solvent mixtures, recrystallization
and/or chromatographic separation, for example over silica gel or
by, e.g., medium pressure liquid chromatography over a reversed
phase column, and racemates can be separated, for example, by the
formation of salts with optically pure salt-forming reagents and
separation of the mixture of diastereoisomers so obtainable, for
example by means of fractional crystallization, or by
chromatography over optically active column materials.
[0263] Intermediates and final products can be worked up and/or
purified according to standard methods, e.g., using chromatographic
methods, distribution methods, (re-) crystallization, and the
like.
EXAMPLES
[0264] The invention is illustrated by the following examples,
which should not be construed as limiting. The assays used to
demonstrate the efficacy of compounds of Formula (I) in these
assays are generally regarded as predictive of efficacy in
subjects.
General Conditions:
[0265] Mass spectra were run on LC-MS systems using electrospray
ionization. These were WATERS Acquity Single Quard Detector.
[M+H].sup.+ refers to mono-isotopic molecular weights.
[0266] NMR spectra were run on open access Varian 400 or Varian 500
NMR spectrometers. Spectra were measured at 298K and were
referenced using the solvent peak. Chemical shifts for .sup.1H NMR
are reported in parts per million (ppm).
[0267] Mass spectra were run on LC-MS systems with one of the
following conditions:
[0268] Waters Acquity UPLC-H class system equipped with SQD
detector.
[0269] Column: ACQUITY UPLC HSS C.sub.18 (50*2.1) mm, 1.8 u.
[0270] Column temperature: Ambient.
[0271] Mobile Phase: A) 0.1% FA+5 mM Ammonium Acetate in Water.
[0272] B) 0.1% FA in Acetonitrile.
[0273] Gradient: 5-5% solvent B in 0.40 min, 5-35% solvent B in
0.80 min, 35-55% solvent B in 1.2 min,
[0274] 55-100% solvent B in 2.5 min.
[0275] Flow rate: 0.55 mL/min.
[0276] Compounds were detected by a Waters Photodiode Array
Detector.
[0277] Waters LCMS system equipped with ZQ 2000 detector.
[0278] Column: X-BRIDGE C.sub.18 (50*4.6) mm, 3.5 u.
[0279] Column temperature: Ambient.
[0280] Mobile Phase: A) 0.1% NH.sub.3 in Water. [0281] B) 0.1%
NH.sub.3 in Acetonitrile.
[0282] Gradient: 5-95% solvent B in 5.00 min.
[0283] Flow rate: 1.0 mL/min.
[0284] Compounds were detected by a Waters Photodiode Array
Detector.
[0285] Waters ACQUITY UPLC system and equipped with a ZQ 2000 MS
system.
[0286] Column: Kinetex by Phenomenex, 2.6 um, 2.1.times.50 mm
[0287] Column temperature: 50.degree. C.
[0288] Gradient: 2-88% (or 00-45%, or 65-95%) solvent B over a 1.29
min period
[0289] Flow rate: 1.2 mL/min.
[0290] Compounds were detected by a Waters Photodiode Array
Detector.
[0291] Chiral separations were done with the following columns:
[0292] AD: ChiralPak AD-H, SFC 21.times.250 mm
[0293] OD: ChiralPak OD-H, SFC 21.times.250 mm
Example 1.1
6-(tert-butyl)-10-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1-
,2-b]indazole-3-carboxylic acid [1.1-I] and [1.1-II]
##STR00022##
[0294] Step 1: (7-methoxy-2H-indazol-3-yl)methanol [1.1a]
##STR00023##
[0296] To methyl 7-methoxy-2H-indazole-3-carboxylate (950 mg, 4.61
mmol) was added THF (Volume: 25 mL), cooled to 0.degree. C. then
LAH (2M in THF; 3.46 mL, 6.91 mmol) was added. The reaction was
allowed to warm to room temperature and stirred for 2 hours or
until done by LCMS. The reaction was cooled in ice bath, then
quenched carefully by adding excess water dropwise (0.8 ml) and as
salts formed magnesium sulfate and then sodium sulfate was added.
The reaction was removed from ice bath and stirred for 1 hour,
filtered through celite, concentrated to residue to give the
desired 1.1a assume in quantitative yield, used as is LC-MS (m/z):
179.1 [M+H]+, 0.48 min.
Step 2: 3-(((tert-butyldimethyl
silyl)oxy)methyl)-7-methoxy-2H-indazole [1.1 b]
##STR00024##
[0298] To 1.1a (810 mg, 4.55 mmol) was added DCM (Volume: 25 mL)
and imidazole (990 mg, 14.55 mmol) and the mixture was stirred at
room temperature for 5 minutes. Then TBDMSCl (2055 mg, 13.64 mmol)
was added. The reaction was stirred at room temperature for 2 hours
or until done by LCMS. To the reaction was added 10 ml of methanol,
stirred for 2-3 minutes and concentrated most of solvent off. Then
was added 250 ml of ethyl acetate, and the organic layer was washed
with saturated sodium bicarbonate, water, saturated salt solution,
dried with anhydrous sodium sulfate, filtered and concentrated to
residue to give desired product 1.1b, assumed to be in quantitative
yield, which was used as is. LC-MS (m/z): 293.3 [M+H]+, 1.15
min.
Step 3:
1-(3-(((tert-butyldimethylsilyl)oxy)methyl)-7-methoxy-2H-indazol-2-
-yl)-3,3-dimethylbutan-2-one [1.1c]
##STR00025##
[0300] To 1.1b (1300 mg, 4.45 mmol) was added DMF (Volume: 20 mL),
and Lithium Carbonate (1314 mg, 17.78 mmol) and the mixture was
stirred for 10 minutes at 75-80.degree. C. Then
1-bromo-3,3-dimethylbutan-2-one (1990 mg, 11.11 mmol) was added
with the reaction at 75-80.degree. C., and the mixture was stirred
at 75-80.degree. C. for 15 hours or until done by LCMS. To the
reaction was added 150 ml of ethyl acetate, which was washed with
saturated sodium bicarbonate, water, saturated salt solution, dried
over sodium sulfate, filtered and concentrated to residue. The
crude material was purified by silica gel column chromatography,
using 0 to 30% EtOAc/heptanes. The desired regio-isomer eluted
second and was concentrated to constant mass to give 520 mg of the
desired product 1.1c in 30% yield. LC-MS (m/z): 391.3 [M+H]+, 1.31
min.
Step 4:
(2-(2-amino-3,3-dimethylbutyl)-7-methoxy-2H-indazol-3-yl)methanol
[1.1d]
##STR00026##
[0302] To 1.1c (520 mg, 1.331 mmol) was added MeOH (Volume: 8 mL),
ammonium acetate (1539 mg, 19.97 mmol) and sodium cyanoborohydride
(251 mg, 3.99 mmol). The reaction was then stirred at 55.degree. C.
for 40 hours or until done by LCMS. To the crude reaction was added
350 ml of DCM and 25 ml of methanol, extracted with 1:1 solution of
(6 M NaOH, saturated salt solution). The aqueous layer was back
extracted with DCM. The organics were combined washed with
saturated salt solution, dried with sodium sulfate, filtered
through 1 cm.times.2 cm celite filter plug, washed with DCM with
10% methanol, concentrated to residue to give the desired product
1.1d assume in quantitative yield, used as is. LC-MS (m/z): 278.3
[M+H]+, 0.51 min.
Step 5: 3-(tert-butyl)-7-methoxy-3,4-dihydropyrazino[1,2-b]indazole
[1.1e]
##STR00027##
[0304] To 1.1d (360 mg, 1.298 mmol) was added DCM (Volume: 12 mL),
and manganese dioxide (1128 mg, 12.98 mmol). The reaction was then
stirred room temperature for 2 hours. Then additional manganese
dioxide (564 mg, 6.49 mmol) was added and stirred overnight for a
total of 20 hours or until done by LCMS. Additional manganese
dioxide can be added if needed. To the crude was added 30 ml of DCM
stirred for 30 minutes then filtered through a 1 cm.times.2 cm
celite filter plug, flushed with DCM and concentrated to residue.
The residue was dissolved in 5 ml of DCM and excess TFA (0.300 mL,
3.89 mmol) was added stirred for 5-10 minutes at room temperature
then concentrated to residue give the desired product 1.1e assume
in quantitative yield, used as is. LC-MS (m/z): 258.3 [M+H]+, 0.59
min.
Step 6: ethyl
6-(tert-butyl)-10-methoxy-2-oxo-2,6,7,13c-tetrahydro-1H-pyrido[2',1':3,4]-
pyrazino[1,2-b]indazole-3-carboxylate [1.1f]
##STR00028##
[0306] To 1.1e (250 mg, 0.972 mmol) was added Ethanol (Volume: 8
mL), and (Z)-ethyl 2-(ethoxymethylene)-3-oxobutanoate (543 mg, 2.91
mmol). The reaction was then stirred at 85-90.degree. C. for 16
hours or until done by LCMS. The reaction was concentrated to
residue to give the desired product 1.1f, assumed to be in
quantitative yield, which was used as is in the next step. LC-MS
(m/z): 398.3 [M+H]+, 0.77 min.
Step 7: ethyl
6-(tert-butyl)-10-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[-
1,2-b]indazole-3-carboxylate [1.1g-I] and [1.1g-II]
##STR00029##
[0308] To 1.1f (384 mg, 0.966 mmol) was added DME (Volume: 7 mL),
and then chloranil (238 mg, 0.966 mmol). The reaction was stirred
at 90-95.degree. C. for 90 minutes or until done by LCMS. The crude
reaction was concentrated to residue and purified by silica gel
chromatography, using 0-100% EtOAc (with 25% ethanol)/heptane. The
desired fractions were concentrated to constant mass to give 140 mg
of the desired racemic product 1.1g in 37% yield. LC-MS (m/z):
396.4 [M+H]+, 0.77 min.
[0309] The above racemic material 128 mg was separated by chiral
chromatography using (OD column, SFC=100 ml/min,
CO.sub.2/MeOH=75/25, 276 bar) to give products 1.1g-I (peak 1, tR
4.40 min.) at 58 mg and product 1.1g-II (peak 2, tR 6.69 min.) at
55 mg.
Step 8:
6-(tert-butyl)-10-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]py-
razino[1,2-b]indazole-3-carboxylic acid [1.1-I] and [1.1-II]
##STR00030##
[0311] To 1.1g-I (58 mg, 0.147 mmol) was added THF (Volume: 1.2 ml,
Ratio: 12.00), MeOH (Volume: 0.1 ml, Ratio: 1.000), Water (Volume:
0.8 ml, Ratio: 8.00) and then NaOH 3M (0.147 ml, 0.440 mmol). The
reaction was stirred at room temperature for 2 hours or until done
by LCMS. The solvent was concentrated off, the residue was
dissolved in 4 ml of DMSO with 5% water, purified by reverse phase
prep LC, and lyophilized. The product was re-dissolved in 1:1
ACN/water and re-lyophilized to give 10.0 mg of the desired product
1.1-I as TFA salt in 14% yield. LC-MS (m/z): 368.3 [M+H]+, 0.80
min. .sup.1H NMR (DMSO-d6) .delta.: 8.95 (s, 1H), 7.66 (d, J=8.5
Hz, 1H), 7.20-7.32 (m, 2H), 6.84 (d, J=7.6 Hz, 1H), 5.12 (br s,
2H), 4.97 (br s, 1H), 3.96 (s, 3H), 0.70 (s, 9H)
[0312] To 1.1g-II (55 mg, 0.139 mmol) was added THF (Volume: 1.2
ml, Ratio: 12.00), MeOH (Volume: 0.1 ml, Ratio: 1.000), Water
(Volume: 0.8 ml, Ratio: 8.00) and then 3M NaOH (0.139 ml, 0.417
mmol). The reaction was stirred at room temperature for 2 hours or
until done by LCMS. The solvent was concentrated off, the residue
was dissolved in 4 ml of DMSO with 5% water, purified by reverse
phase prep LC, and lyophilized. The product was re-dissolved in 1:1
ACN/water and re-lyophilized to give 23.8 mg of the desired product
1.1-II as TFA salt in 35% yield. LC-MS (m/z): 368.3 [M+H]+, 0.80
min. 1H NMR (DMSO-d6) .delta.: 8.95 (s, 1H), 7.65 (d, J=8.3 Hz,
1H), 7.21-7.33 (m, 2H), 6.84 (d, J=7.6 Hz, 1H), 5.12 (br s, 2H),
4.96 (br s, 1H), 3.96 (s, 3H), 0.70 (s, 9H)
Example 1.2
6-(tert-butyl)-11-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1-
,2-b]indazole-3-carboxylic acid [1.2-I] and [1.2-II]
##STR00031##
[0313] Step 1 to 7: ethyl
6-(tert-butyl)-11-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[-
1,2-b]indazole-3-carboxylate [1.2g-I] and [1.2g-II]
##STR00032##
[0315] Compound 1.2g was synthesized from the starting material;
methyl 6-methoxy-2H-indazole-3-carboxylate by the process of
Example 1.1 following steps 1 through 7 resulting in the desired
product 1.2g as a racemate. LC-MS (m/z): 396.3 [M+H]+, 0.79
min.
[0316] The above racemic material (65 mg) was separated by chiral
chromatography using AD column, (SFC=100 ml/min,
CO.sub.2/EtOH=70/30, 266 bar) to give products 1.2g-I (peak 1, tR
2.11 min.) at 32 mg and product 1.2g-II (peak 2, tR 5.42 min.) at
30 mg.
Step 8:
6-(tert-butyl)-11-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]py-
razino[1,2-b]indazole-3-carboxylic acid [1.2-I] and [1.2-II]
##STR00033##
[0318] To 1.2g-I (32 mg, 0.081 mmol) was added THF (Volume: 1.2 ml,
Ratio: 12.00), MeOH (Volume: 0.1 ml, Ratio: 1.000), Water (Volume:
0.8 ml, Ratio: 8.00) and then 3M NaOH (0.081 ml, 0.243 mmol). The
reaction was stirred at room temperature for 2 hours or until done
by LCMS. The solvent was concentrated off, the residue was
dissolved in 4 ml of DMSO with 5% water, purified by reverse phase
prep LC, and lyophilized. The product was re-dissolved in 1:1
ACN/water and re-lyophilized to give 8.8 mg of the desired product
1.2-I as TFA salt in 22% yield. LC-MS (m/z): 368.3 [M+H]+, 0.81
min. 1H NMR (DMSO-d6) .delta.: 8.95 (s, 1H), 8.06 (d, J=9.2 Hz,
1H), 7.32 (s, 1H), 7.16 (s, 1H), 6.95-7.03 (m, 1H), 5.01-5.15 (m,
2H), 4.93 (d, J=4.5 Hz, 1H), 3.85 (s, 3H), 0.70 (s, 9H)
[0319] To 1.2g-II (30 mg, 0.076 mmol) was added THF (Volume: 1.2
ml, Ratio: 12.00), MeOH (Volume: 0.1 ml, Ratio: 1.000), Water
(Volume: 0.8 ml, Ratio: 8.00) and then 3M NaOH (0.076 ml, 0.228
mmol). The reaction was stirred at room temperature for 2 hours or
until done by LCMS. The solvent was concentrated, the residue was
dissolved in 4 ml of DMSO with 5% water, purified by reverse phase
prep LC, and lyophilized. The product was re-dissolved in 1:1
ACN/water and re-lyophilized to give 7.4 mg of the desired product
1.2-II as TFA salt in 20% yield. LC-MS (m/z): 368.3 [M+H]+, 0.81
min. .sup.1H NMR (DMSO-d6) .delta.: 8.95 (s, 1H), 8.06 (d, J=9.2
Hz, 1H), 7.32 (s, 1H), 7.16 (s, 1H), 6.99 (br d, J=9.2 Hz, 1H),
5.00-5.18 (m, 2H), 4.93 (d, J=4.5 Hz, 1H), 3.85 (s, 3H), 0.70 (s,
9H)
Example 1.3
6-(tert-butyl)-12-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1-
,2-b]indazole-3-carboxylic acid [1.3-] and [1.3-II]
##STR00034##
[0320] Step 1 to 7: ethyl
6-(tert-butyl)-12-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[-
1,2-b]indazole-3-carboxylate [1.3g-I] and [1.3g-II]
##STR00035##
[0322] Compound 1.3g was synthesized from the starting material;
methyl 5-methoxy-2H-indazole-3-carboxylate by the process of
Example 1.1 following steps 1 through 7 resulting in the desired
product 1.3g as a racemate. LC-MS (m/z): 396.3 [M+H]+, 0.81
min.
[0323] The above racemic material (148 mg) was separated by chiral
chromatography using AD column, (SFC=100 ml/min,
CO.sub.2/EtOH=85/15, 236 bar) to give products 1.3g-I (peak 1, tR
10.41 min.) at 56 mg and product 1.3g-II (peak 2, tR 12.55 min.) at
68 mg.
Step 8:
6-(tert-butyl)-12-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]py-
razino[1,2-b]indazole-3-carboxylic acid [1.3-I] and [1.3-II]
##STR00036##
[0325] To 1.3g-I (55 mg, 0.139 mmol) was added THF (Volume: 1.2 ml,
Ratio: 12.00), MeOH (Volume: 0.1 ml, Ratio: 1.000), Water (Volume:
0.8 ml, Ratio: 8.00) and then 3M NaOH (0.139 ml, 0.417 mmol). The
reaction was stirred at room temperature for 2 hours or until done
by LCMS. The solvent was concentrated, the residue was dissolved in
4 ml of DMSO with 5% water, purified by reverse phase prep LC, and
lyophilized. The product was re-dissolved in 1:1 ACN/water and
re-lyophilized to give 18.7 mg of the desired product 1.3-I as TFA
salt in 27% yield. LC-MS (m/z): 368.3 [M+H]+, 0.83 min. .sup.1H NMR
(DMSO-d6) .delta.: 8.94 (s, 1H), 7.75 (d, J=9.2 Hz, 1H), 7.42 (s,
1H), 7.36 (s, 1H), 7.10 (dd, J=9.5, 1.9 Hz, 1H), 5.02-5.17 (m, 2H),
4.94 (d, J=4.5 Hz, 1H), 3.93 (s, 3H), 0.70 (s, 9H). To 1.3g-II (67
mg, 0.169 mmol) was added THF (Volume: 1.2 ml, Ratio: 12.00), MeOH
(Volume: 0.1 ml, Ratio: 1.000), Water (Volume: 0.8 ml, Ratio: 8.00)
and then 3M NaOH (0.169 ml, 0.508 mmol). (30 mg, 0.076 mmol) was
added THF (Volume: 1.2 ml, Ratio: 12.00), MeOH (Volume: 0.1 ml,
Ratio: 1.000), Water (Volume: 0.8 ml, Ratio: 8.00) and then 3M NaOH
(0.076 ml, 0.228 mmol). The reaction was stirred at room
temperature for 2 hours or until done by LCMS. The solvent was
concentrated, the residue was dissolved in 4 ml of DMSO with 5%
water, purified by reverse phase prep LC, and lyophilized. The
product was re-dissolved in 1:1 ACN/water and re-lyophilized to
give 29.8 mg of the desired product 1.3-II as TFA salt in 36%
yield. LC-MS (m/z): 368.3 [M+H]+, 0.82 min. .sup.1H NMR (DMSO-d6)
.delta.: 8.94 (s, 1H), 7.75 (d, J=9.2 Hz, 1H), 7.42 (s, 1H), 7.36
(s, 1H), 7.10 (dd, J=9.2, 1.9 Hz, 1H), 5.02-5.18 (m, 2H), 4.94 (d,
J=4.5 Hz, 1H), 3.93 (s, 3H), 0.70 (s, 9H)
Example 1.4
6-(tert-butyl)-13-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1-
,2-b]indazole-3-carboxylic acid [1.4-I] and [1.4-II]
##STR00037##
[0326] Step 1 to 7: ethyl
6-(tert-butyl)-13-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[-
1,2-b]indazole-3-carboxylate [1.4g-I] and [1.4g-II]
##STR00038##
[0328] Compound 1.4g was synthesized from the starting material;
methyl 4-methoxy-2H-indazole-3-carboxylate by the process of
Example 1.1 following steps 1 through 7 resulting in the desired
product 1.4g as a racemate. LC-MS (m/z): 396.3 [M+H]+, 0.81 min.
The above racemic material (60 mg) was purified by chiral
chromatography using OD column, (SFC=100 ml/min,
CO.sub.2/MeOH=88/12, 246 bar) to give enriched racemate 1.4g (peak
1, tR 8.74 min., 25% and peak 2, tR 11.37 min., 75%) at 30 mg.
Step 8:
6-(tert-butyl)-13-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]py-
razino[1,2-b]indazole-3-carboxylic acid [1.4-I] and [1.4-II]
##STR00039##
[0330] To the above enriched racemate 1.4g (30 mg, 0.076 mmol) was
added THF (Volume: 0.5 ml, Ratio: 5.00), MeOH (Volume: 0.1 ml,
Ratio: 1.000), Water (Volume: 0.3 ml, Ratio: 3.00) and then 3M NaOH
(0.076 ml, 0.228 mmol). The reaction was stirred at room
temperature for 2 hours or until done by LCMS. The solvent was
concentrated, the residue was dissolved in 2 ml of DMSO with 5%
water, purified by reverse phase prep LC, and lyophilized to give
18 mg of enriched racemic product 1.4 as TFA salt in 49% yield.
LC-MS (m/z): 368.3 [M+H]+, 0.77 min.
[0331] The above enriched material (18 mg) was further purified by
chiral chromatography using (AS column, SFC=100 ml/min,
CO.sub.2/EtOH=80/20, 226 bar) to give products 1.4-I (peak 1, tR
4.76 min.) at 2.6 mg in 9% yield and product 1.4-II (peak 2, tR
9.41 min.) at 7.7 mg in 27% yield both as free base.
[0332] 1.4-I LC-MS (m/z): 368.3 [M+H]+, 0.82 min. .sup.1H NMR
(DMSO-d6) .delta.: 8.90 (br s, 1H), 7.89 (br s, 1H), 7.30-7.43 (m,
2H), 6.77 (br d, J=5.7 Hz, .sup.1H), 4.99-5.18 (m, 2H), 4.93 (br s,
1H), 3.99 (s, 3H), 0.71 (s, 9H)
[0333] 1.4-II LC-MS (m/z): 368.3 [M+H]+, 0.81 min. .sup.1H NMR
(DMSO-d6) .delta.: 8.91 (br s, 1H), 7.90 (br s, 1H), 7.29-7.42 (m,
2H), 6.77 (br d, J=6.1 Hz, .sup.1H), 5.02-5.17 (m, 2H), 4.93 (br s,
1H), 3.99 (s, 3H), 0.71 (s, 9H)
Example 1.5
6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazo-
le-3-carboxylic acid [1.5-I] and [1.5-II]
##STR00040##
[0334] Step 1 to 7: ethyl
6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indaz-
ole-3-carboxylate [1.5g-I] and [1.5g-II]
##STR00041##
[0336] Compound 1.5g was synthesized from the starting material;
methyl 2H-indazole-3-carboxylate by the process of Example 1.1
following steps 1 through 7 resulting in the desired product, 1.5g,
as a racemate. LC-MS (m/z): 366.3 [M+H]+, 0.68 min. The above
racemic material (60 mg) was purified by chiral chromatography
using AD column, (SFC=100 ml/min, CO.sub.2/EtOH=70/30, 256 bar) to
give products 1.5g-I (peak 1, tR 2.30 min.) at 13 mg and product
1.5g-II (peak 2, tR 3.97 min.) at 13 mg.
Step 8:
6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2--
b]indazole-3-carboxylic acid [1.5-I] and [1.5-II]
##STR00042##
[0338] To 1.5g-I (12 mg, 0.033 mmol) was added THF (Volume: 0.2 ml,
Ratio: 1.000), MeOH (Volume: 0.2 ml, Ratio: 1.000) and then 3M NaOH
(0.033 ml, 0.099 mmol). The reaction was stirred at room
temperature for 2 hours or until done by LCMS. The solvent was
concentrated, the residue was dissolved in 1.2 ml of DMSO, purified
by reverse phase prep LC, and lyophilized. The product was
re-dissolved in 1:1 ACN/water and re-lyophilized to give 6.8 mg of
the desired product 1.5-I as TFA salt in 45% yield. LC-MS (m/z):
338.3 [M+H]+, 0.68 min. .sup.1H NMR (DMSO-d6) .delta.: 8.99 (s,
1H), 8.20 (d, J=8.5 Hz, 1H), 7.85 (d, J=8.5 Hz, 1H), 7.43-7.52 (m,
1H), 7.33-7.41 (m, 2H), 5.10-5.25 (m, 2H), 4.99 (d, J=4.7 Hz, 1H),
0.71 (s, 9H). To 1.5g-II (12 mg, 0.033 mmol) was added THF (Volume:
0.2 ml, Ratio: 1.000), MeOH (Volume: 0.2 ml, Ratio: 1.000) and then
3M NaOH (0.033 ml, 0.099 mmol). The reaction was stirred at room
temperature for 2 hours or until done by LCMS. The solvent was
concentrated off, the residue was dissolved in 1.2 ml of DMSO,
purified by reverse phase prep LC, and lyophilized. The product was
re-dissolved in 1:1 ACN/water and re-lyophilized to give 6.8 mg of
the desired product 1.5-II as TFA salt in 45% yield. LC-MS (m/z):
338.3 [M+H]+, 0.68 min. .sup.1H NMR (DMSO-d6) .delta.: 8.99 (s,
1H), 8.20 (d, J=8.5 Hz, 1H), 7.85 (d, J=8.5 Hz, 1H), 7.43-7.50 (m,
1H), 7.31-7.41 (m, 2H), 5.10-5.21 (m, 2H), 4.99 (d, J=4.4 Hz, 1H),
0.71 (s, 9H)
Example 2.1
6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4-
]pyrazino[1,2-b]indazole-3-carboxylic acid [2.1-I] and [2.1-II]
##STR00043##
[0339] Step 1: (7-(benzyloxy)-2H-indazol-3-yl)methanol [2.1a]
##STR00044##
[0341] To methyl 7-(benzyloxy)-2H-indazole-3-carboxylate (6000 mg,
21.25 mmol) was added THF (Volume: 100 mL), and stirred to dissolve
at room temperature. Then the reaction placed in room temperature
water bath and 2M LAH in THF (15.94 mL, 31.9 mmol) was added
carefully. The reaction was stirred for 2 hours at room temperature
or until done by LCMS. The reaction was cooled in ice bath, then
quenched carefully by adding additional THF (Volume: 100 mL), then
excess water dropwise (3.0 ml). As the salts formed magnesium
sulfate was added followed by sodium sulfate. The reaction was
removed from ice bath and stirred for 55 minutes, filtered through
celite, concentrated to residue to give 4850 mg of the desired
product 2.1a in 90% yield, used as is. LC-MS (m/z): 255.2 [M+H]+,
0.71 min.
Step 2:
7-(benzyloxy)-3-(((tert-butyldimethylsilyl)oxy)methyl)-2H-indazole
[2.1b]
##STR00045##
[0343] To 2.1a (4850 mg, 19.07 mmol) was added DCM (Volume: 100 mL)
and imidazole (4155 mg, 61.0 mmol) stirred at room temperature for
5 minutes. Then TBDMSCl (8624 mg, 57.2 mmol) was added. The
reaction was stirred at room temperature for 15 hours or until done
by LCMS. To the reaction was added 10 ml of methanol stirred for
2-3 minutes and concentrated most of solvent off. Then was added
300 ml of ethyl acetate, which was washed with saturated sodium
bicarbonate, water, saturated salt solution, dried sodium sulfate,
filtered and concentrated to residue to give desired product 2.1b
assume in quantitative yield, used as is. LC-MS (m/z): 369.5
[M+H]+, 1.16 min.
Step 3:
1-(7-(benzyloxy)-3-(((tert-butyldimethylsilyl)oxy)methyl)-2H-indaz-
ol-2-yl)-3,3-dimethylbutan-2-one [2.1c]
##STR00046##
[0345] To 2.1b (6985, 18.95 mmol) was added DMF (Volume: 60 mL) and
lithium carbonate (5602 mg, 76 mmol) and the mixture was stirred
for 10 minutes at 90.degree. C. Then
1-bromo-3,3-dimethylbutan-2-one (8484 mg, 47.4 mmol) was added with
the reaction at 90.degree. C., and then stirred at 90.degree. C.
for 15 hours or until done by LCMS. To the cooled reaction was
added 450 ml of ethyl acetate, which was washed with saturated
sodium bicarbonate, water, saturated salt solution, dried sodium
sulfate, filtered and concentrated to residue. The crude material
was purified by silica gel column chromatography, using 0 to 25%
EtOAc/heptanes. The desired regio-isomer eluted second and was
concentrated to constant mass to give 3100 mg of the desired
product 2.1c in 35% yield. LC-MS (m/z): 467.4 [M+H]+, 1.30 min.
Step 4:
(2-(2-amino-3,3-dimethylbutyl)-7-(benzyloxy)-2H-indazol-3-yl)metha-
nol [2.1d]
##STR00047##
[0347] To 2.1c (3100 mg, 6.64 mmol) was added MeOH (Volume: 29 mL),
ammonium acetate (7680 mg, 100 mmol) and sodium cyanoborohydride
(1252 mg, 19.93 mmol). The reaction was then stirred at 55.degree.
C. for 40 hours or until done by LCMS. To the crude reaction was
added 350 ml of DCM and 25 ml of methanol, extracted with 1:1
solution of (6 M NaOH, saturated salt solution). The aqueous layer
was back extracted with DCM. The organics were combined, washed
with saturated salt solution, dried with sodium sulfate, filtered
through 1 cm.times.2 cm celite filter plug, washed with DCM with
10% methanol, concentrated to residue to give the desired product
2.1d (assume in quantitative yield), which was used as is. LC-MS
(m/z): 354.3 [M+H]+, 0.73 min.
Step 5:
7-(benzyloxy)-3-(tert-butyl)-3,4-dihydropyrazino[1,2-b]indazole
[2.1e]
##STR00048##
[0349] To 2.1d (2340 mg, 6.62 mmol) was added DCM (Volume: 45 mL),
and manganese dioxide (5755 mg, 66.2 mmol). The reaction was then
stirred room temperature for 2 hours. Then additional manganese
dioxide (2878 mg, 33.1 mmol) was added and the mixture was stirred
overnight for a total of 20 hours or until done by LCMS. Additional
manganese dioxide can be added if needed. To the crude was added 30
ml of DCM stirred for 30 minutes then filtered through a 1
cm.times.2 cm celite filter plug, flushed with DCM and concentrated
to residue. The residue was dissolved in 20 ml of DCM and excess
TFA (1.530 mL, 19.86 mmol) was added, and the mixture was stirred
for 5-10 minutes at room temperature then concentrated to residue
to give the desired product 2.1e (assume quantitative yield), which
was used as is. LC-MS (m/z): 334.3 [M+H]+, 0.82 min.
Step 6: ethyl
10-(benzyloxy)-6-(tert-butyl)-2-oxo-2,6,7,13c-tetrahydro-1H-pyrido[2',1':-
3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.1f]
##STR00049##
[0351] To 2.1e (2200 mg, 6.60 mmol) was added Ethanol (Volume: 35
mL), and (Z)-ethyl 2-(ethoxymethylene)-3-oxobutanoate (3686 mg,
19.79 mmol). The reaction was then stirred at 85-90.degree. C. for
16 hours or until done by LCMS. The reaction was concentrated to
residue to give the desired product 2.1f assume in quantitative
yield, used as is. LC-MS (m/z): 474.6 [M+H]+, 0.92 min.
Step 7: ethyl
10-(benzyloxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyraz-
ino[1,2-b]indazole-3-carboxylate [2.1g]
##STR00050##
[0353] To 2.1f (3120 mg, 6.59 mmol) was added DME (Volume: 40 mL),
and then chloranil (1620 mg, 6.59 mmol). The reaction was stirred
at 90-95.degree. C. for 90 minutes or until done by LCMS. The
reaction cooled, 2 ml of water was added; stirred for 5 minutes
then diluted with 350 ml of DCM with 2% ethanol. Saturated sodium
bicarbonate (100 ml) and 150 ml of water were added and the mixture
was stirred for 5 minutes. The reaction was filtered and the layers
were separated. The organic layer was washed with water 2.times.,
saturated salt solution, dried with sodium sulfate, filtered and
concentrated to residue. The crude material was purified by silica
gel chromatography, using 0-75% EtOAc (with 25% ethanol)/heptane.
The desired fractions were concentrated to constant mass to give
1145 mg of the desired product 2.1g in 37% yield. LC-MS (m/z):
472.5 [M+H]+, 0.98 min. 1H NMR (DMSO-d6) .delta.: 8.51 (s, 1H),
7.48-7.57 (m, 3H), 7.30-7.45 (m, 3H), 7.19 (t, J=8.0 Hz, 1H), 6.90
(d, J=7.6 Hz, 1H), 6.81 (s, 1H), 5.27 (s, 2H), 5.00-5.08 (m, 2H),
4.69 (br d, J=3.0 Hz, 1H), 4.22 (q, J=7.0 Hz, 2H), 1.26 (t, J=7.1
Hz, 3H), 0.68 (s, 9H).
Step 8: ethyl
6-(tert-butyl)-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[-
1,2-b]indazole-3-carboxylate [2.1h]
##STR00051##
[0355] To 2.1g (1120 mg, 2.375 mmol) was flushed with argon then
Pd--C(758 mg, 0.713 mmol) was added. Then under argon with a
syringe Ethanol (Volume: 40 mL) was added. To the reaction was
added a hydrogen filled balloon. The reaction was evacuated and
refilled 5.times. with hydrogen and then stirred at room
temperature for 2 hours or until done by LCMS. The reaction was
stopped by removing the hydrogen balloon and flushing the reaction
with argon. Then 100 ml of THF was added and the mixture stirred
for 30 minutes. The reaction was filtered through 1.times.2 cm
celite plug, flushed with THF and concentrated to residue to give
900 mg of the desired product 2.1h in 99% yield, which was used as
is. LC-MS (m/z): 382.5 [M+H]+, 0.68 min.
Step 9: ethyl
6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,-
4]pyrazino[1,2-b]indazole-3-carboxylate [2.1i-I] and [2.1i-II]
##STR00052##
[0357] To 2.1h (425 mg, 1.114 mmol) was added DMF (Volume: 10 mL)
and cesium carbonate (1089 mg, 3.34 mmol). The reaction was stirred
at room temperature for 10 minutes, then 1-bromo-3-methoxypropane
(341 mg, 2.228 mmol) was added and the reaction was heated to
50-55.degree. C. and stirred for 2 hours or until done by LCMS. The
reaction was cooled, diluted with 225 ml of DCM with 2% ethanol,
washed with saturated sodium bicarbonate, water, saturated salt
solution, dried with sodium sulfate, filtered and concentrated to
residue. The crude material was purified by silica gel
chromatography, using 0-100% EtOAc (with 25% ethanol)/heptane. The
desired fractions were concentrated to constant mass to give 434 mg
of the desired racemic product 2.1i in 86% yield. LC-MS (m/z):
454.5 [M+H]+, 0.82 min. 1H NMR (<dmso>) .delta.: 8.52 (s,
1H), 7.50 (d, J=8.5 Hz, 1H), 7.17 (t, J=8.0 Hz, 1H), 6.74-6.83 (m,
2H), 5.05-5.13 (m, 1H), 4.98-5.05 (m, 1H), 4.69 (d, J=4.5 Hz, 1H),
4.11-4.28 (m, 4H), 3.51 (t, J=6.3 Hz, 2H), 3.25 (s, 3H), 2.04
(quin, J=6.3 Hz, 2H), 1.26 (t, J=7.1 Hz, 3H), 0.68 (s, 9H).
[0358] The above racemic material 434 mg was separated by chiral
chromatography using OD column, (SFC=80 ml/min,
CO.sub.2/MeOH=70/30, 266 bar) to give products 2.1i-I (peak 1, tR
3.93 min.) at 170 mg and product 2.1i-II (peak 2, tR 6.98 min.) at
172 mg.
Step 10:
6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.1-I] and
[2.1-II]
##STR00053##
[0360] To 2.1i-I (170 mg, 0.375 mmol) was added acetonitrile
(Volume: 5 ml, Ratio: 1.000), Water (Volume: 5 ml, Ratio: 1.000)
and then LiOH (1.125 ml, 1.125 mmol). The reaction was stirred at
room temperature for 2 hours or until done by LCMS. The reaction
was diluted with 20 ml of water, acidified with 1M HCl to pH of
2-3, then 100 ml of DCM with 5% ethanol was added and 15 ml of
saturated sodium chloride solution. The organic layer was
separated, the aqueous layer was extracted again, 50 ml of DCM with
5% ethanol. The organic layers were combined, washed with water,
saturated sodium chloride solution, dried sodium sulfate, filtered
through sodium sulfate plug 1 cm.times.1.5 cm and concentrated to
residue. The residue was dissolved in 1:1 ACN/water and lyophilized
to give 154 mg of the desired product 2.1-I as free base in 96%
yield. LC-MS (m/z): 426.4 [M+H]+, 0.85 min. 1H NMR (DMSO-d6)
.delta.: 8.95 (s, 1H), 7.63 (d, J=8.5 Hz, 1H), 7.18-7.31 (m, 2H),
6.82 (d, J=7.6 Hz, 1H), 5.03-5.26 (m, 2H), 4.95 (d, J=4.8 Hz, 1H),
4.20 (t, J=6.4 Hz, 2H), 3.51 (t, J=6.3 Hz, 2H), 3.25 (s, 3H), 2.04
(quin, J=6.3 Hz, 2H), 0.69 (s, 9H). To 2.1i-II (172 mg, 0.379 mmol)
was added acetonitrile (Volume: 5 ml, Ratio: 1.000), Water (Volume:
5 ml, Ratio: 1.000) and then LiOH (1.138 ml, 1.138 mmol). The
reaction was stirred at room temperature for 2 hours or until done
by LCMS. The reaction was diluted with 20 ml of water, acidified
with 1M HCl to pH of 2-3, then 100 ml of DCM with 5% ethanol was
added and 15 ml of saturated sodium chloride solution. The organic
layer was separated, the aqueous layer was extracted again, 50 ml
of DCM with 5% ethanol. The organic layers were combined, washed
with water, saturated sodium chloride solution, dried sodium
sulfate, filtered through sodium sulfate plug 1 cm.times.1.5 cm and
concentrated to residue. The residue was dissolved in 1:1 ACN/water
and lyophilized to give 154 mg of the desired product 2.1-II as
free base in 94% yield. LC-MS (m/z): 426.4 [M+H]+, 0.85 min.
.sup.1H NMR (DMSO-d6) .delta.: 8.95 (s, 1H), 7.63 (d, J=8.5 Hz,
1H), 7.17-7.28 (m, 2H), 6.82 (d, J=7.6 Hz, 1H), 5.04-5.23 (m, 2H),
4.95 (d, J=4.7 Hz, 1H), 4.20 (t, J=6.4 Hz, 2H), 3.51 (t, J=6.2 Hz,
2H), 3.25 (s, 3H), 2.04 (quin, J=6.3 Hz, 2H), 0.69 (s, 9H)
Example 2.2
6-(tert-butyl)-10-(2-methoxyethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]-
pyrazino[1,2-b]indazole-3-carboxylic acid [2.2-I] and [2.2-II]
##STR00054##
[0361] Step 1: ethyl
6-(tert-butyl)-10-(2-methoxyethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4-
]pyrazino[1,2-b]indazole-3-carboxylate [2.2a-I] and [2.2a-II]
##STR00055##
[0363] To 2.1h (425 mg, 1.114 mmol) was added DMF (Volume: 10 mL)
and cesium carbonate (1089 mg, 3.34 mmol). The reaction was stirred
at room temperature for 10 minutes then 1-bromo-2-methoxyethane
(310 mg, 2.228 mmol) was added and the reaction was heated to
50-55.degree. C. and stirred for 2 hours or until done by LCMS. The
reaction was cooled, diluted with 225 ml of DCM with 2% ethanol,
washed with saturated sodium bicarbonate, water, saturated salt
solution, dried with sodium sulfate, filtered and concentrated to
residue. The crude material was purified by silica gel
chromatography, using 0-100% EtOAc (with 25% ethanol)/heptane. The
desired fractions were concentrated to constant mass to give 414 mg
of the desired racemic product 2.2a in 85% yield. LC-MS (m/z):
440.5 [M+H]+, 0.78 min. The above racemic material 414 mg was
separated by chiral chromatography using AD Column, (SFC=100
ml/min, CO.sub.2/EtOH=75/25, 226 bar) to give products 2.2a-I (peak
1, tR 3.55 min.) at 134 mg and product 2.2a-II (peak 2, tR 4.97
min.) at 104 mg.
Step 2:
6-(tert-butyl)-10-(2-methoxyethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2'-
,1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.2-I] and
[2.2-II]
##STR00056##
[0365] To 2.2a-I (134 mg, 0.305 mmol) was added acetonitrile
(Volume: 5 ml, Ratio: 1.000), Water (Volume: 5 ml, Ratio: 1.000)
and then LiOH (0.915 ml, 0.915 mmol). The reaction was stirred at
room temperature for 2 hours or until done by LCMS. The reaction
was diluted with 20 ml of water acidified with 1M HCl to pH of 2-3,
then 100 ml of DCM with 5% ethanol was added and 15 ml of saturated
sodium chloride solution. The organic layer was separated, the
aqueous layer was extracted again, 50 ml of DCM with 5% ethanol.
The organic layers were combined, washed with water, saturated
sodium chloride solution, dried sodium sulfate, filtered through
sodium sulfate plug 1 cm.times.1.5 cm and concentrated to residue.
The residue was dissolved in 1:1 ACN/water and lyophilized to give
117 mg of the desired product 2.2-I as free base in 93% yield.
LC-MS (m/z): 412.4 [M+H]+, 0.80 min. .sup.1H NMR (DMSO-d6) .delta.:
8.94 (s, 1H), 7.64 (d, J=8.5 Hz, 1H), 7.15-7.30 (m, 2H), 6.83 (d,
J=7.6 Hz, 1H), 5.05-5.22 (m, 2H), 4.96 (d, J=4.6 Hz, 1H), 4.24-4.34
(m, 2H), 3.70-3.78 (m, 2H), 3.33 (s, 3H), 0.69 (s, 9H). To 2.2a-II
(104 mg, 0.237 mmol) was added acetonitrile (Volume: 4 ml, Ratio:
1.000), Water (Volume: 4 ml, Ratio: 1.000) and then LiOH (0.710 ml,
0.710 mmol). The reaction was stirred at room temperature for 2
hours or until done by LCMS. The reaction was diluted with 20 ml of
water, acidified with 1M HCl to pH of 2-3, then 100 ml of DCM with
5% ethanol was added and 15 ml of saturated sodium chloride
solution. The organic layer was separated, the aqueous layer was
extracted again, 50 ml of DCM with 5% ethanol. The organic layers
were combined, washed with water, saturated sodium chloride
solution, dried sodium sulfate, filtered through sodium sulfate
plug 1 cm.times.1.5 cm and concentrated to residue. The residue was
dissolved in 1:1 ACN/water and lyophilized to give 93 mg of the
desired product 2.2-II as free base in 95% yield. LC-MS (m/z):
412.4 [M+H]+, 0.80 min. .sup.1H NMR (DMSO-d6) .delta.: 8.94 (s,
1H), 7.64 (d, J=8.5 Hz, 1H), 7.18-7.32 (m, 2H), 6.83 (d, J=7.6 Hz,
1H), 5.03-5.29 (m, 2H), 4.95 (d, J=4.6 Hz, 1H), 4.23-4.32 (m, 2H),
3.68-3.79 (m, 2H), 3.33 (s, 3H), 0.69 (s, 9H).
Illustrative Example for Synthesis of Compounds Wherein
R.sup.1.dbd.F
Step 1:
5-(4-methoxy-3-(3-methoxypropoxy)phenyl)-2,2-dimethylcyclopentanon-
e [1a]
##STR00057##
[0367] A mixture of Pd(OAc).sub.2 (8.16 mg, 0.036 mmol), sodium
tert-butoxide (0.454 g, 4.72 mmol),
dicyclohexyl(2'-methyl-[1,1'-biphenyl]-2-yl)phosphane (32 mg),
2,2-dimethylcyclopentanone (0.547 ml, 4.36 mmol) and
4-bromo-1-methoxy-2-(3-methoxypropoxy)benzene (1 g, 3.63 mmol) in
toluene (4.0 mL) was heated in a sealed vial at 50.degree. C. for
18 hours. The mixture was diluted with EtOAc and filtered. The
filtrate was concentrated and the remaining oil was purified by
silica gel column chromatography, EtOAc/heptane 5 to 50%, to give
product (500 mg, 45% yield). LC-MS (m/z): 307.2 [M+H].sup.+.
.sup.1H NMR (400 MHz, CDCl.sub.3): 7.72-7.28 (m, 1H), 6.94-6.61 (m,
2H), 4.10 (m, 2H), 3.91-3.77 (m, 4H), 3.65-3.49 (m, 2H), 3.41-3.27
(m, 3H), 2.36 (d, J=4.2 Hz, 1H), 2.19-1.88 (m, 4H), 1.88-1.71 (m,
1H), 1.21-1.11 (m, 3H), 1.07 (s, 3H).
Step 2:
5-(4-methoxy-3-(3-methoxypropoxy)phenyl)-2,2-dimethylcyclopentanam-
ine [1b]
##STR00058##
[0369] To the mixture of
5-(4-methoxy-3-(3-methoxypropoxy)phenyl)-2,2-dimethylcyclopentanone
(320 mg, 1.0 mmol) in MeOH (3 mL) was added acetic acid ammonia
salt (1.6 g, 20.9 mmol) and sodium cyanoborohydride (656 mg, 10.4
mmol). The mixture was stirred at 80.degree. C. for 8 hours and
then was concentrated under reduced pressure. The remaining
material was diluted with EtOAc, washed with water and brine, dried
over Na.sub.2SO.sub.4 and concentrated. The crude material was used
in the next step with no further purification. LCMS (m/z): 308.0
[M+H].sup.+.
Step 3: N-(5-(4-methoxy-3-(3-methoxypropoxy)phenyl)-2,2
dimethylcyclopentyl)formamide [1c]
##STR00059##
[0371] To the mixture of
5-(4-methoxy-3-(3-methoxypropoxy)phenyl)-2,2-dimethylcyclopentanamine
(320 mg, 1.041 mmol) in dioxane (3 ml) was added formic acid (0.160
mL, 4.16 mmol). The mixture was stirred at 100.degree. C. for 6
hours. The mixture was concentrated to afford the crude product
which was used in the next step with no further purification. LCMS
(m/z): 336.2 [M+H].sup.+.
Step 4:
7-methoxy-8-(3-methoxypropoxy)-3,3-dimethyl-2,3,3a,9b-tetrahydro-1-
H-cyclopenta[c]isoquinoline [1d-I] and [1d-II]
##STR00060##
[0373] To a mixture of
N-(5-(4-methoxy-3-(3-methoxypropoxy)phenyl)-2,2-dimethylcyclopentyl)forma-
mide (349 mg, 1.04 mmol) in acetonitrile (1.8 ml) was added
POCl.sub.3 (140 .mu.l, 1.50 mmol). The mixture was stirred at
85.degree. C. for 2 hours and then concentrated. The residue was
dissolved in EtOAc and basified by adding ammonium hydroxide
solution. The phases were separated and the organic layer was
washed with brine, dried over Na.sub.2SO.sub.4 and concentrated.
The remaining material was purified by silica gel chromatography,
acetone/heptane 5 to 50% to give product rac-1d-I and
rac-1d-II.
[0374] Trans isomer rac-1d-II: (70 mg, 21% yield). LCMS (m/z):
318.3 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.22 (s, 1H),
6.93-6.83 (m, 1H), 6.70 (s, 1H), 4.16 (t, J=6.4 Hz, 2H), 3.94-3.83
(m, 3H), 3.57 (q, J=4.7 Hz, 2H), 3.35 (d, J=1.5 Hz, 4H), 2.84 (s,
2H), 2.21-1.98 (m, 4H), 1.84-1.70 (m, 3H), 1.68-1.53 (m, 3H), 1.22
(s, 4H), 1.06 (s, 4H).
[0375] Cis isomer rac-1d-I: (54 mg, 16% yield). LCMS (m/z): 318.3
[M+H].sup.+. 1H NMR (400 MHz, CDCl.sub.3): 8.17 (s, 1H), 6.75 (s,
1H), 6.67 (s, 1H), 4.14 (t, J=6.2 Hz, 3H), 3.92-3.83 (m, 3H), 3.77
(d, J=10.3 Hz, 1H), 3.57 (t, J=5.4 Hz, 3H), 3.35 (d, J=1.6 Hz, 3H),
3.25 (q, J=9.4 Hz, 1H), 2.42-2.21 (m, 2H), 2.19-2.01 (m, 3H), 1.64
(dd, J=20.6, 7.8 Hz, 4H), 1.45 (t, J=8.0 Hz, 2H), 1.24 (d, J=6.1
Hz, 4H), 0.89 (s, 4H). The relative configuration of rac-1d-I and
rac-1d-II were confirmed by nuclear Overhauser effect (nOe)
experiments.
Step 5. Ethyl
8-fluoro-10-methoxy-11-(3-methoxypropoxy)-3,3-dimethyl-7-oxo-1,2,3,3a,7,1-
2b-hexahydrocyclopenta[c]pyrido[2,1-a]isoquinoline-6-carboxylate
[1e-I] and [1e-II]
##STR00061##
[0376] ethyl
(Z)-2-(ethoxymethyiene)-4,4-difluoro-3-((trimethylsilyl)oxy)but-3-enoate
[0377] Under an argon atmosphere, a mixture of Mg (3.69 g, 152
mmol) and TMSCl (19.43 mL, 152 mmol) was treated with ultrasound
irradiation for 15 min. To the mixture was added DMF (30 mL)),
ethyl (Z)-2-(ethoxymethylene)-4,4,4-trifluoro-3-oxobutanoate (4.56
g, 19 mmol) was added dropwise at 50.degree. C. under an argon
atmosphere. The reaction mixture was stirred for additional 3 min.
at 50.degree. C. After removal of excess TMSCl in vacuo, the crude
mixture was filtered and the filtrate (containing DMF) was used in
the next step without further purification.
##STR00062##
[0378] To a suspension of ZnI.sub.2 (920 mg, 2.88 mmol)) and 1d-I
(915 mg, 2.88 mmol) in dry MeCN (10 mL), a solution of crude ethyl
(Z)-2-(ethoxymethylene)-4,4-difluoro-3-((trimethylsilyl)oxy)but-3-enoate
(5091 mg, 17.30 mmol) in dry DMF (30 mL) was added dropwise at
50.degree. C., and the reaction mixture was stirred overnight. The
reaction mixture was poured into 10% HCl and extracted with DCM.
The organic layer was washed with brine, and dried over MgSO.sub.4.
After filtration, the organic layer was concentrated and the crude
oil purified by silica gel chromatography (0-10% MeOH/EtOAc) to
afford rac-1e (1.2 g, 2.53 mmol, 88% yield)) as a pale yellow
solid. The material was then purified by chiral SFC (AD column,
flow rate 100 ml/min, CO.sub.2/EtOH=70/30, 256 bar) to provide to
provide products 1e-1 (tR 2.4 min) and 1e-2 (tR 4.4 min, 280 mg).
LC-MS (m/z): 474.2 [M+H].sup.+.
Step 6:
(3aS,12bR)-8-fluoro-10-methoxy-11-(3-methoxypropoxy)-3,3-dimethyl--
7-oxo-1,2,3,3a,7,12b-hexahydrocyclopenta[c]pyrido[2,1-a]isoquinoline-6-car-
boxylic acid [3.1]
##STR00063##
[0380] To the solution of 1e-2 (330 mg, 0.697 mmol) in THF (1 mL)
was added NaOH (1.394 mL, 1.394 mmol). The reaction mixture was
stirred for 2 h, then the reaction was acidified with 1.5 ml 1N HCl
and extracted with dichloromethane. The organic layer was washed
with brine, and dried over MgSO.sub.4. After filtration and
concentration, the resultant solid was recrystallized from hot
EtOH/Water (5 ml; 5 ml) and the solids collected by vacuum
filtration. The material was further lyophilized from MeCN and
water to give product (230 mg, 0.511 mmol, 73.3%) as a tan solid.
LC-MS (m/z): 446.4 [M+H].sup.+. .sup.1H NMR (500 MHz, DMSO-d.sup.6)
.delta. 8.82 (s, 1H), 7.56 (s, 1H), 7.20 (s, 1H), 4.52 (d, J=5.5
Hz, 1H), 4.20-4.06 (m, 2H), 3.82 (s, 3H), 3.31 (dd, J=15.9, 4.8 Hz,
2H), 3.26 (s, 3H), 3.18 (d, J=15.9 Hz, 1H), 2.01 (p, J=6.3 Hz, 2H),
1.57-1.50 (m, 1H), 0.87 (d, J=6.6 Hz, 3H), 0.73 (d, J=6.7 Hz, 3H).
For each compound of Formula (I), the enantiomer having greater
potency in the HBsAg assay is preferred. By analogy to other
compounds believed to bind in the same target site, the more potent
isomer in the HBsAg assay in each case is believed to have an
absolute stereochemical configuration corresponding to these
structures:
##STR00064##
[0381] where the dashed arc indicates R.sup.7 and R.sup.9 taken
together form a ring as described for certain compounds of Formula
(I) and substructures thereof that are shown herein.
Example 2.3
6-(tert-butyl)-2-oxo-10-(2-(2,2,2-trifluoroethoxy)ethoxy)-6,7-dihydro-2H-p-
yrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.3]
##STR00065##
[0382] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-(2-(2,2,2-trifluoroethoxy)ethoxy)-6,7-dihydro-2H--
pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.3a]
##STR00066##
[0384] To 2.1h (16 mg, 0.042 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (47.8 mg, 0.147 mmol). The reaction was
stirred at room temperature for 5 minutes then
2-(2-bromoethoxy)-1,1,1-trifluoroethane (21.71 mg, 0.105 mmol) was
added. The reaction was heated to 70.degree. C. and stirred for 3
hours or until done by LCMS. The reaction was cooled, 0.5 ml of DMF
was added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.3a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 508.5 [M+H].sup.+,
0.88 min.
Step 2:
6-(tert-butyl)-2-oxo-10-(2-(2,2,2-trifluoroethoxy)ethoxy)-6,7-dihy-
dro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.3]
##STR00067##
[0386] To 2.3a (21.32 mg, 0.042 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (168 .mu.l, 0.168 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 8.2
mg of the desired racemic product 2.3 as TFA salt in 32% yield.
LC-MS (m/z): 480.4 [M+H].sup.+, 0.89 min. .sup.1H NMR (DMSO-d6)
.delta.: 8.94 (s, 1H), 7.66 (d, J=8.5 Hz, 1H), 7.19-7.30 (m, 2H),
6.84 (d, J=7.6 Hz, 1H), 5.06-5.22 (m, 2H), 4.95 (d, J=4.5 Hz, 1H),
4.30-4.38 (m, 2H), 4.20 (q, J=9.4 Hz, 2H), 3.99-4.06 (m, 2H), 0.69
(s, 9H)
Example 2.4
6-(tert-butyl)-10-((1-(methoxymethyl)cyclobutyl)methoxy)-2-oxo-6,7-dihydro-
-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.4]
##STR00068##
[0387] Step 1: ethyl
6-(tert-butyl)-10-((1-(methoxymethyl)cyclobutyl)methoxy)-2-oxo-6,7-dihydr-
o-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate
[2.4a]
##STR00069##
[0389] To 2.1h (16 mg, 0.042 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (47.8 mg, 0.147 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-(bromomethyl)-1-(methoxymethyl)cyclobutane (20.25 mg, 0.105 mmol)
was added. The reaction was heated to 70.degree. C. and stirred for
3 hours or until done by LCMS. The reaction was cooled, 0.5 ml of
DMF was added, then filtered through a 0.45 nM in line filter. The
DMF solution with the desired product 2.4a was used as is for the
next step, assume quantitative yield. LC-MS (m/z): 494.5
[M+H].sup.+, 0.96 min.
Step 2:
6-(tert-butyl)-10-((1-(methoxymethyl)cyclobutyl)methoxy)-2-oxo-6,7-
-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic
acid [2.4]
##STR00070##
[0391] To 2.4a (20.73 mg, 0.042 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq. (168 .mu.l, 0.168 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 4.0
mg of the desired racemic product 2.4 as TFA salt in 16% yield.
LC-MS (m/z): 466.5 [M+H].sup.+, 1.00 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.63 (d, J=8.6 Hz, 1H),
7.19-7.27 (m, 2H), 6.87 (d, J=7.6 Hz, 1H), 5.17-5.25 (m, 1H),
5.05-5.13 (m, 1H), 4.94 (d, J=4.9 Hz, 1H), 4.05-4.18 (m, 2H), 3.47
(s, 2H), 3.25 (s, 3H), 1.85-1.97 (m, 6H), 0.69 (s, 9H)
Example 2.5
6-(tert-butyl)-10-(difluoromethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]-
pyrazino[1,2-b]indazole-3-carboxylic acid [2.5]
##STR00071##
[0392] Step 1: ethyl
6-(tert-butyl)-10-(difluoromethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4-
]pyrazino[1,2-b]indazole-3-carboxylate [2.5a]
##STR00072##
[0394] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (77 mg, 0.236 mmol). The reaction was stirred
at room temperature for 5 minutes then ethyl
2-bromo-3,3-difluoropropanoate (41.0 mg, 0.189 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 20 hours, or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.5a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 432.5 [M+H].sup.+,
0.91 min.
Step 2:
6-(tert-butyl)-10-(difluoromethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2'-
,1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.5]
##STR00073##
[0396] To 2.5a (20.28 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 2 hours or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 0.8
mg of the desired racemic product 2.5 as TFA salt in 3% yield.
LC-MS (m/z): 404.4 [M+H].sup.+, 0.88 min. .sup.1H NMR
(<cdcl3>) .delta.: 8.58 (s, 1H), 7.74 (d, J=8.5 Hz, 1H), 7.35
(t, J=8.0 Hz, 1H), 7.31 (s, 1H), 7.18 (d, J=1.4 Hz, 1H), 6.81-7.17
(m, 1H), 5.22 (d, J=14.9 Hz, 1H), 4.96 (dd, J=14.9, 5.4 Hz, 1H),
4.26 (d, J=5.2 Hz, 1H), 0.85 (s, 9H)
Example 2.6
6-(tert-butyl)-10-((S)-2-methylbutoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3-
,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.6]
##STR00074##
[0397] Step 1: ethyl
6-(tert-butyl)-10-((S)-2-methylbutoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':-
3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.6a]
##STR00075##
[0399] To 2.1h (16 mg, 0.042 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (47.8 mg, 0.147 mmol). The reaction was
stirred at room temperature for 5 minutes then
(S)-1-bromo-2-methylbutane (15.84 mg, 0.105 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.6a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 452.5 [M+H].sup.+,
1.01 min.
Step 2:
6-(tert-butyl)-10-((S)-2-methylbutoxy)-2-oxo-6,7-dihydro-2H-pyrido-
[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.6]
##STR00076##
[0401] To 2.6a (18.97 mg, 0.042 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq. (168 .mu.l, 0.168 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 6.5
mg of the desired racemic product 2.6 as TFA salt in 26% yield.
LC-MS (m/z): 424.5 [M+H].sup.+, 1.05 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.62 (d, J=8.5 Hz, 1H),
7.18-7.28 (m, 2H), 6.82 (d, J=7.6 Hz, 1H), 5.16-5.24 (m, 1H),
5.05-5.14 (m, 1H), 4.94 (d, J=4.8 Hz, 1H), 3.88-4.10 (m, 2H),
1.83-1.98 (m, 1H), 1.50-1.66 (m, 1H), 1.20-1.33 (m, 1H), 1.01 (t,
J=6.6 Hz, 3H), 0.92 (t, J=7.4 Hz, 3H), 0.69 (s, 9H)
Example 2.7
10-(sec-butoxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyraz-
ino[1,2-b]indazole-3-carboxylic acid [2.7]
##STR00077##
[0402] Step 1: ethyl
10-(sec-butoxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyra-
zino[1,2-b]indazole-3-carboxylate [2.7a]
##STR00078##
[0404] To 2.1h (16 mg, 0.042 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (47.8 mg, 0.147 mmol). The reaction was
stirred at room temperature for 5 minutes then 2-bromobutane (14.37
mg, 0.105 mmol) was added. The reaction was heated to 70.degree. C.
and stirred for 3 hours or until done by LCMS. The reaction was
cooled, 0.5 ml of DMF was added, then filtered through a 0.45 nM in
line filter. The DMF solution with the desired product 2.7a was
used as is for the next step, assume quantitative yield. LC-MS
(m/z): 438.5 [M+H].sup.+, 0.92 min.
Step 2:
10-(sec-butoxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3-
,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.7]
##STR00079##
[0406] To 2.7a (18.97 mg, 0.042 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq. (168 .mu.l, 0.168 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 6.5
mg of the desired racemic product 2.7 as TFA salt in 27% yield.
LC-MS (m/z): 410.5 [M+H].sup.+, 0.95 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.60 (dd, J=8.5, 1.8 Hz, 1H),
7.19-7.26 (m, 2H), 6.82 (dd, J=7.7, 3.0 Hz, 1H), 5.14-5.23 (m, 1H),
5.04-5.13 (m, 1H), 4.95 (d, J=4.8 Hz, 1H), 4.66 (dt, J=12.5, 6.2
Hz, 1H), 1.58-1.83 (m, 2H), 1.31 (dd, J=10.9, 6.0 Hz, 3H),
0.89-0.99 (m, 3H), 0.69 (s, 9H)
Example 2.8
6-(tert-butyl)-2-oxo-10-((tetrahydrofuran-3-yl)methoxy)-6,7-dihydro-2H-pyr-
ido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.8]
##STR00080##
[0407] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-((tetrahydrofuran-3-yl)methoxy)-6,7-dihydro-2H-py-
rido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.8a]
##STR00081##
[0409] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
(tetrahydrofuran-3-yl)methyl 4-methylbenzenesulfonate (24.19 mg,
0.094 mmol) was added. The reaction was heated to 70.degree. C. and
stirred for 3 hours or until done by LCMS. The reaction was cooled,
0.5 ml of DMF was added, then filtered through a 0.45 nM in line
filter. The DMF solution with the desired product 2.8a was used as
is for the next step, assume quantitative yield. LC-MS (m/z): 466.5
[M+H].sup.+, 0.79 min.
Step 2:
6-(tert-butyl)-2-oxo-10-((tetrahydrofuran-3-yl)methoxy)-6,7-dihydr-
o-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.8]
##STR00082##
[0411] To 2.8a (21.88 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 3.7
mg of the desired racemic product 2.8 as TFA salt in 13% yield.
LC-MS (m/z): 438.4 [M+H].sup.+, 0.83 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.64 (d, J=8.5 Hz, 1H),
7.18-7.30 (m, 2H), 6.85 (d, J=7.5 Hz, 1H), 5.04-5.24 (m, 2H), 4.95
(d, J=4.8 Hz, 1H), 4.00-4.21 (m, 2H), 3.72-3.87 (m, 2H), 3.57-3.71
(m, 2H), 2.69-2.84 (m, 1H), 1.99-2.13 (m, 1H), 1.65-1.77 (m, 1H),
0.69 (s, 9H)
Example 2.9
6-(tert-butyl)-2-oxo-10-(2-(trifluoromethoxy)ethoxy)-6,7-dihydro-2H-pyrido-
[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.9]
##STR00083##
[0412] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-(2-(trifluoromethoxy)ethoxy)-6,7-dihydro-2H-pyrid-
o[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.9a]
##STR00084##
[0414] To 2.1h (15 mg, 0.039 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (44.8 mg, 0.138 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-bromo-2-(trifluoromethoxy)ethane (18.97 mg, 0.098 mmol) was
added. The reaction was heated to 50.degree. C. and stirred for 15
minutes or until done by LCMS. The reaction was cooled, 0.5 ml of
DMF was added, then filtered through a 0.45 nM in line filter. The
DMF solution with the desired product 2.9a was used as is for the
next step, assume quantitative yield. LC-MS (m/z): 494.6
[M+H].sup.+, 0.99 min.
Step 2:
6-(tert-butyl)-2-oxo-10-(2-(trifluoromethoxy)ethoxy)-6,7-dihydro-2-
H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.9]
##STR00085##
[0416] To 2.9a (19.25 mg, 0.039 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (156 .mu.l, 0.156 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 5.3
mg of the desired racemic product 2.9 as TFA salt in 23% yield.
LC-MS (m/z): 466.4 [M+H].sup.+, 0.92 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.69 (d, J=8.5 Hz, 1H),
7.21-7.31 (m, 2H), 6.87 (d, J=7.6 Hz, 1H), 5.05-5.28 (m, 2H), 4.96
(d, J=4.7 Hz, 1H), 4.48 (br dd, J=18.4, 4.9 Hz, 4H), 0.69 (s,
9H)
Example 2.10
6-(tert-butyl)-10-(2,2-difluoroethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3-
,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.10]
##STR00086##
[0417] Step 1: ethyl
6-(tert-butyl)-10-(2,2-difluoroethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':-
3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.10a]
##STR00087##
[0419] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then 2,2-difluoroethyl
trifluoromethanesulfonate (25.3 mg, 0.118 mmol) was added. The
reaction was heated to 50.degree. C. and stirred for 15 minutes or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.10a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 446.4 [M+H].sup.+,
0.81 min.
Step 2:
6-(tert-butyl)-10-(2,2-difluoroethoxy)-2-oxo-6,7-dihydro-2H-pyrido-
[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.10]
##STR00088##
[0421] To 2.10a (20.94 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 3.6
mg of the desired racemic product 2.10 as TFA salt in 14% yield.
LC-MS (m/z): 418.4 [M+H].sup.+, 0.84 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.72 (d, J=8.5 Hz, 1H),
7.21-7.32 (m, 2H), 6.92 (d, J=7.6 Hz, 1H), 6.35-6.65 (m, 1H),
5.05-5.27 (m, 2H), 4.95 (d, J=4.7 Hz, 1H), 4.51 (td, J=14.5, 3.4
Hz, 2H), 0.69 (s, 9H).
Example 2.11
[0422]
6-(tert-butyl)-2-oxo-10-propoxy-6,7-dihydro-2H-pyrido[2',1':3,4]pyr-
azino[1,2-b]indazole-3-carboxylic acid [2.11]
##STR00089##
Step 1: ethyl
6-(tert-butyl)-2-oxo-10-propoxy-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[-
1,2-b]indazole-3-carboxylate [2.11a]
##STR00090##
[0424] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then 1-bromopropane
(14.51 mg, 0.118 mmol) was added. The reaction was heated to
70.degree. C. and stirred for 3 hours or until done by LCMS. The
reaction was cooled, 0.5 ml of DMF was added, then filtered through
a 0.45 nM in line filter. The DMF solution with the desired product
2.11a was used as is for the next step, assume quantitative yield.
LC-MS (m/z): 424.5 [M+H].sup.+, 0.89 min.
Step 2:
6-(tert-butyl)-2-oxo-10-propoxy-6,7-dihydro-2H-pyrido[2',1':3,4]py-
razino[1,2-b]indazole-3-carboxylic acid [2.11]
##STR00091##
[0426] To 2.11a (19.90 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 5.4
mg of the desired racemic product 2.11 as TFA salt in 22% yield.
LC-MS (m/z): 396.4 [M+H].sup.+, 0.92 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.62 (d, J=8.5 Hz, 1H),
7.18-7.29 (m, 2H), 6.81 (d, J=7.6 Hz, 1H), 5.04-5.22 (m, 2H), 4.94
(d, J=4.8 Hz, 1H), 4.02-4.16 (m, 2H), 1.75-1.88 (m, 2H), 1.02 (t,
J=7.4 Hz, 3H), 0.69 (s, 9H)
Example 2.12
6-(tert-butyl)-10-(2-(2-methoxyethoxy)ethoxy)-2-oxo-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.12]
##STR00092##
[0427] Step 1: ethyl
6-(tert-butyl)-10-(2-(2-methoxyethoxy)ethoxy)-2-oxo-6,7-dihydro-2H-pyrido-
[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.12a]
##STR00093##
[0429] To 2.1h (18 mg, 0.047 mmol) was added DMF (volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-bromo-2-(2-methoxyethoxy)ethane (21.60 mg, 0.118 mmol) was added.
The reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.12a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 484.5 [M+H].sup.+,
0.77 min.
Step 2:
6-(tert-butyl)-10-(2-(2-methoxyethoxy)ethoxy)-2-oxo-6,7-dihydro-2H-
-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.12]
##STR00094##
[0431] To 2.12a (22.73 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 6.6
mg of the desired racemic product 2.12 as TFA salt in 24% yield.
LC-MS (m/z): 456.5 [M+H].sup.+, 0.81 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.65 (d, J=8.5 Hz, 1H),
7.19-7.30 (m, 2H), 6.83 (d, J=7.6 Hz, 1H), 5.04-5.25 (m, 2H), 4.95
(d, J=4.7 Hz, 1H), 4.23-4.34 (m, 2H), 3.79-3.88 (m, 2H), 3.61 (dd,
J=5.6, 3.8 Hz, 2H), 3.47 (dd, J=5.6, 3.8 Hz, 2H), 3.23 (s, 3H),
0.69 (s, 9H)
Example 2.13
6-(tert-butyl)-10-(2,3-dimethoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1'-
:3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.13]
##STR00095##
[0432] Step 1: ethyl
6-(tert-butyl)-10-(2,3-dimethoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1-
':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.13a]
##STR00096##
[0434] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-bromo-2,3-dimethoxypropane (21.60 mg, 0.118 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.13a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 484.5 [M+H].sup.+,
0.78 min.
Step 2:
6-(tert-butyl)-10-(2,3-dimethoxypropoxy)-2-oxo-6,7-dihydro-2H-pyri-
do[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.13]
##STR00097##
[0436] To 2.13a (20.94 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 5.6
mg of the desired racemic product 2.13 as TFA salt in 21% yield.
LC-MS (m/z): 456.5 [M+H].sup.+, 0.81 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.65 (d, J=8.5 Hz, 1H),
7.14-7.29 (m, 2H), 6.84 (dd, J=7.6, 4.0 Hz, 1H), 5.03-5.25 (m, 2H),
4.95 (d, J=4.7 Hz, 1H), 4.11-4.31 (m, 2H), 3.76 (br t, J=4.5 Hz,
1H), 3.47-3.62 (m, 2H), 3.40 (d, J=3.6 Hz, 3H), 3.28 (s, 3H), 0.69
(s, 9H).
Example 2.14
6-(tert-butyl)-10-(3-ethoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]-
pyrazino[1,2-b]indazole-3-carboxylic acid [2.14]
##STR00098##
[0437] Step 1: ethyl
6-(tert-butyl)-10-(3-ethoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4-
]pyrazino[1,2-b]indazole-3-carboxylate [2.14a]
##STR00099##
[0439] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-bromo-3-ethoxypropane (19.71 mg, 0.118 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.14a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 468.5 [M+H].sup.+,
0.86 min.
Step 2:
6-(tert-butyl)-10-(3-ethoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2'-
,1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.14]
##STR00100##
[0441] To 2.14a (21.98 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 6.2
mg of the desired racemic product 2.14 as TFA salt in 23% yield.
LC-MS (m/z): 440.5 [M+H].sup.+, 0.90 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.63 (d, J=8.5 Hz, 1H),
7.19-7.29 (m, 2H), 6.82 (d, J=7.6 Hz, 1H), 5.04-5.23 (m, 2H), 4.95
(d, J=4.7 Hz, 1H), 4.20 (t, J=6.4 Hz, 2H), 3.55 (t, J=6.3 Hz, 2H),
3.42 (q, J=7.0 Hz, 2H), 2.04 (quin, J=6.3 Hz, 2H), 1.09 (t, J=7.0
Hz, 3H), 0.69 (s, 9H)
Example 2.15
6-(tert-butyl)-10-(2-isopropoxyethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3-
,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.15]
##STR00101##
[0442] Step 1: ethyl
6-(tert-butyl)-10-(2-isopropoxyethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':-
3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.15a]
##STR00102##
[0444] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reacting was
stirred at room temperature for 5 minutes then
2-(2-bromoethoxy)propane (19.71 mg, 0.118 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.15a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 468.5 [M+H].sup.+,
0.87 min.
Step 2:
6-(tert-butyl)-10-(2-isopropoxyethoxy)-2-oxo-6,7-dihydro-2H-pyrido-
[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.15]
##STR00103##
[0446] To 2.15a (20.94 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 4.5
mg of the desired racemic product 2.15 as TFA salt in 17% yield.
LC-MS (m/z): 440.5 [M+H].sup.+, 0.91 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.64 (d, J=8.5 Hz, 1H),
7.19-7.28 (m, 2H), 6.83 (d, J=7.6 Hz, 1H), 5.06-5.23 (m, 2H), 4.95
(d, J=4.5 Hz, 1H), 4.21-4.30 (m, 2H), 3.74-3.82 (m, 2H), 3.66 (dt,
J=12.2, 6.1 Hz, 1H), 1.11 (d, J=6.1 Hz, 6H), 0.69 (s, 9H).
Example 2.16
6-(tert-butyl)-10-isobutoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino-
[1,2-b]indazole-3-carboxylic acid [2.16]
##STR00104##
[0447] Step 1: ethyl
6-(tert-butyl)-10-isobutoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazin-
o[1,2-b]indazole-3-carboxylate [2.16a]
##STR00105##
[0449] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-bromo-2-methylpropane (16.17 mg, 0.118 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.16a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 438.5 [M+H].sup.+,
0.95 min.
Step 2:
6-(tert-butyl)-10-isobutoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]-
pyrazino[1,2-b]indazole-3-carboxylic acid [2.16]
##STR00106##
[0451] To 2.16a (20.56 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 5.2
mg of the desired racemic product 2.16 as TFA salt in 21% yield.
LC-MS (m/z): 410.5 [M+H].sup.+, 1.00 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.62 (d, J=8.5 Hz, 1H),
7.14-7.29 (m, 2H), 6.81 (d, J=7.6 Hz, 1H), 5.05-5.29 (m, 2H), 4.95
(d, J=4.8 Hz, 1H), 3.77-4.04 (m, 2H), 2.12 (dt, J=13.3, 6.6 Hz,
1H), 1.02 (dd, J=6.5, 4.9 Hz, 6H), 0.69 (s, 9H).
Example 2.17
6-(tert-butyl)-10-(3-hydroxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4-
]pyrazino[1,2-b]indazole-3-carboxylic acid [2.17]
##STR00107##
[0452] Step 1: ethyl
6-(tert-butyl)-10-(3-hydroxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,-
4]pyrazino[1,2-b]indazole-3-carboxylate [2.17a]
##STR00108##
[0454] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then 3-bromopropan-1-ol
(16.40 mg, 0.118 mmol) was added. The reaction was heated to
70.degree. C. and stirred for 3 hours or until done by LCMS. The
reaction was cooled, 0.5 ml of DMF was added. The crude reaction
was purified by reverse phase prep LC, the desired fractions were
collected, 0.04 ml of 6M HCl was added and lyophilized to give 9.4
mg of the desired racemic product 2.17a as HCl salt in 42% yield
used as is. LC-MS (m/z): 440.5 [M+H].sup.+, 0.70 min.
Step 2:
6-(tert-butyl)-10-(3-hydroxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2-
',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.17]
##STR00109##
[0456] To 2.17a (9.4 mg, 0.021 mmol) was dissolved in 1 ml of DMF,
then was added LiOH 1M aq (86 .mu.l, 0.086 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude was
purified by reverse phase prep LC, the desired fractions were
collected, 0.04 ml of 6M HCl was added and lyophilized. The product
was re-dissolved in 1:1 ACN/water, additional 0.02 ml of 6M HCl was
added and lyophilized again. Then the sample was again redissolved
in 1:1 ACN/water and lyophilized to give 3.2 mg of the desired
racemic product 2.17 as HCl salt in 31% yield. LC-MS (m/z): 412.4
[M+H].sup.+, 0.71 min. .sup.1H NMR (DMSO-d.sub.6) .delta.: 8.94 (s,
1H), 7.63 (d, J=8.5 Hz, 1H), 7.19-7.28 (m, 2H), 6.82 (d, J=7.6 Hz,
1H), 5.04-5.24 (m, 2H), 4.95 (d, J=4.8 Hz, 1H), 4.22 (t, J=6.3 Hz,
2H), 3.60 (t, J=6.2 Hz, 2H), 1.95 (quin, J=6.2 Hz, 2H), 0.69 (s,
9H).
Example 2.18
6-(tert-butyl)-10-(2-hydroxyethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]-
pyrazino[1,2-b]indazole-3-carboxylic acid [2.18]
##STR00110##
[0457] Step 1: ethyl
6-(tert-butyl)-10-(2-hydroxyethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4-
]pyrazino[1,2-b]indazole-3-carboxylate [2.18a]
##STR00111##
[0459] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then 2-bromoethanol
(14.74 mg, 0.118 mmol) was added. The reaction was heated to
70.degree. C. and stirred for 3 hours or until done by LCMS. The
reaction was cooled, 0.5 ml of DMF was added. The crude reaction
was purified by reverse phase prep LC, the desired fractions were
collected, 0.04 ml of 6M HCl was added and lyophilized to give 7.0
mg of the desired racemic product 2.18a as HCl salt in 32% yield
used as is. LC-MS (m/z): 426.4 [M+H].sup.+, 0.68 min.
Step 2:
6-(tert-butyl)-10-(2-hydroxyethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2'-
,1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.18]
##STR00112##
[0461] To 2.18a (7.0 mg, 0.016 mmol) was dissolved in 1 ml of DMF,
then was added LiOH 1M aq (0.066 mL, 0.066 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude was
purified by reverse phase prep LC, the desired fractions were
collected, 0.04 ml of 6M HCl was added and lyophilized. The product
was re-dissolved in 1:1 ACN/water, additional 0.02 ml of 6M HCl was
added and lyophilized again. Then the sample was again redissolved
in 1:1 ACN/water and lyophilized to give 2.3 mg of the desired
racemic product 2.18 as HCl salt in 29% yield. LC-MS (m/z): 398.4
[M+H].sup.+, 0.69 min. .sup.1H NMR (DMSO-d.sub.6) .delta.: 8.94 (s,
1H), 7.63 (d, J=8.5 Hz, 1H), 7.16-7.29 (m, 2H), 6.82 (d, J=7.6 Hz,
1H), 5.07-5.20 (m, 2H), 4.96 (br d, J=3.8 Hz, 1H), 4.17 (t, J=4.9
Hz, 2H), 3.81 (br t, J=4.9 Hz, 2H), 0.69 (s, 9H)
Example 2.19
6-(tert-butyl)-2-oxo-10-(3-(trifluoromethoxy)propoxy)-6,7-dihydro-2H-pyrid-
o[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.19]
##STR00113##
[0462] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-(3-(trifluoromethoxy)propoxy)-6,7-dihydro-2H-pyri-
do[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.19a]
##STR00114##
[0464] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-bromo-3-(trifluoromethoxy)propane (24.42 mg, 0.118 mmol) was
added. The reaction was heated to 70.degree. C. and stirred for 3
hours or until done by LCMS. The reaction was cooled, 0.5 ml of DMF
was added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.19a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 508.6 [M+H].sup.+,
1.02 min.
Step 2:
6-(tert-butyl)-2-oxo-10-(3-(trifluoromethoxy)propoxy)-6,7-dihydro--
2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.19]
##STR00115##
[0466] To 2.19a (23.85 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 10.6
mg of the desired racemic product 2.19 as TFA salt in 37% yield.
LC-MS (m/z): 480.4 [M+H].sup.+, 0.97 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.66 (d, J=8.5 Hz, 1H),
7.17-7.31 (m, 2H), 6.86 (d, J=7.6 Hz, 1H), 5.04-5.23 (m, 2H), 4.95
(d, J=4.6 Hz, 1H), 4.21-4.35 (m, 4H), 2.22 (br t, J=6.2 Hz, 2H),
0.69 (s, 9H).
Example 2.20
6-(tert-butyl)-2-oxo-10-(2-(2-oxopyrrolidin-1-yl)ethoxy)-6,7-dihydro-2H-py-
rido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.20]
##STR00116##
[0467] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-(2-(2-oxopyrrolidin-1-yl)ethoxy)-6,7-dihydro-2H-p-
yrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.20a]
##STR00117##
[0469] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-(2-bromoethyl)pyrrolidin-2-one (22.66 mg, 0.118 mmol) was added.
The reaction was heated to 70.degree. C. and stirred for 7 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.20a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 493.5 [M+H].sup.+,
0.93 min.
Step 2:
6-(tert-butyl)-2-oxo-10-(2-(2-oxopyrrolidin-1-yl)ethoxy)-6,7-dihyd-
ro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.20]
##STR00118##
[0471] To 2.20a (23.15 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 7.4
mg of the desired racemic product 2.20 as TFA salt in 27% yield.
LC-MS (m/z): 465.4 [M+H].sup.+, 0.73 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.66 (d, J=8.5 Hz, 1H),
7.18-7.29 (m, 2H), 6.86 (d, J=7.6 Hz, 1H), 5.07-5.24 (m, 2H), 4.95
(d, J=4.2 Hz, 1H), 4.28 (t, J=5.6 Hz, 2H), 3.58-3.68 (m, 2H),
3.47-3.55 (m, 2H), 2.13-2.26 (m, 2H), 1.90 (quin, J=7.5 Hz, 2H),
0.69 (s, 9H).
Example 2.21
6-(tert-butyl)-10-(3-(2-methoxyethoxy)propoxy)-2-oxo-6,7-dihydro-2H-pyrido-
[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.21]
##STR00119##
[0472] Step 1: ethyl
6-(tert-butyl)-10-(3-(2-methoxyethoxy)propoxy)-2-oxo-6,7-dihydro-2H-pyrid-
o[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.21a]
##STR00120##
[0474] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-bromo-3-(2-methoxyethoxy)propane (23.25 mg, 0.118 mmol) was
added. The reaction was heated to 70.degree. C. and stirred for 7
hours or until done by LCMS. The reaction was cooled, 0.5 ml of DMF
was added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.21a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 498.6 [M+H].sup.+,
0.88 min.
Step 2:
6-(tert-butyl)-10-(3-(2-methoxyethoxy)propoxy)-2-oxo-6,7-dihydro-2-
H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.21]
##STR00121##
[0476] To 2.21a (23.39 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 12.5
mg of the desired racemic product 2.21 as TFA salt in 44% yield.
LC-MS (m/z): 470.5 [M+H].sup.+, 0.82 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.63 (d, J=8.5 Hz, 1H),
7.17-7.28 (m, 2H), 6.82 (d, J=7.6 Hz, 1H), 5.05-5.25 (m, 2H), 4.95
(d, J=4.7 Hz, 1H), 4.20 (t, J=6.3 Hz, 2H), 3.59 (t, J=6.3 Hz, 2H),
3.49-3.54 (m, 2H), 3.40-3.44 (m, 2H), 3.21 (s, 3H), 2.04 (quin,
J=6.2 Hz, 2H), 0.69 (s, 9H).
Example 2.22
10-((1,4-dioxan-2-yl)methoxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2-
',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.22]
##STR00122##
[0477] Step 1: ethyl
10-((1,4-dioxan-2-yl)methoxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.22a]
##STR00123##
[0479] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
(1,4-dioxan-2-yl)methyl methanesulfonate (23.15 mg, 0.118 mmol) was
added. The reaction was heated to 70.degree. C. and stirred for 3
hours or until done by LCMS. The reaction was cooled, 0.5 ml of DMF
was added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.22a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 482.4 [M+H].sup.+,
0.76 min.
Step 2:
10-((1,4-dioxan-2-yl)methoxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H--
pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.22]
##STR00124##
[0481] To 2.22a (22.63 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized. The crude
product was re-dissolved in 1 ml of DMF and re-purified by reverse
phase prep LC, and lyophilized to give 6.2 mg of the desired
racemic product 2.22 as TFA salt in 22% yield. LC-MS (m/z): 454.6
[M+H].sup.+, 0.86 min. .sup.1H NMR (DMSO-d.sub.6) .delta.: 8.95 (s,
1H), 7.66 (d, J=8.5 Hz, 1H), 7.19-7.29 (m, 2H), 6.83 (d, J=6.7 Hz,
1H), 5.16-5.25 (m, 1H), 5.05-5.15 (m, 1H), 4.95 (d, J=4.7 Hz, 1H),
4.11-4.18 (m, 2H), 3.91-4.04 (m, 1H), 3.82-3.91 (m, 1H), 3.75-3.82
(m, 1H), 3.61-3.71 (m, 2H), 0.69 (s, 9H).
Example 2.23
6-(tert-butyl)-10-(carboxymethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]p-
yrazino[1,2-b]indazole-3-carboxylic acid [2.23]
##STR00125##
[0482] Step 1: ethyl
6-(tert-butyl)-10-(2-methoxy-2-oxoethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',-
1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.23a]
##STR00126##
[0484] To 2.1h (16 mg, 0.042 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (47.8 mg, 0.147 mmol). The reaction was
stirred at room temperature for 5 minutes then methyl
2-bromoacetate (16.04 mg, 0.105 mmol) was added. The reaction was
heated to 50.degree. C. and stirred for 1 hour or until done by
LCMS. The reaction was cooled, 0.5 ml of DMF was added, then
filtered through a 0.45 nM in line filter. The DMF solution with
the desired product 2.23a was used as is for the next step, assume
quantitative yield. LC-MS (m/z): 454.4 [M+H].sup.+, 0.76 min.
Step 2:
6-(tert-butyl)-10-(carboxymethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',-
1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.23]
##STR00127##
[0486] To 2.23a (19.05 mg, 0.042 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (168 .mu.l, 0.168 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 8.0
mg of the desired racemic product 2.23 as TFA salt in 36% yield.
LC-MS (m/z): 412.4 [M+H].sup.+, 0.68 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 13.10 (br s, 1H), 8.95 (s, 1H), 7.68 (d,
J=8.5 Hz, 1H), 7.28 (s, 1H), 7.22 (t, J=8.0 Hz, 1H), 6.74 (d, J=7.6
Hz, 1H), 5.13 (d, J=2.3 Hz, 2H), 4.96 (br s, 1H), 4.89 (s, 2H),
0.69 (s, 9H).
Example 2.24
6-(tert-butyl)-10-((1-((methylsulfonyl)methyl)cyclopropyl)methoxy)-2-oxo-6-
,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic
acid [2.24]
##STR00128##
[0487] Step 1: ethyl
6-(tert-butyl)-10-((1-((methylsulfonyl)methyl)cyclopropyl)methoxy)-2-oxo--
6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate
[2.24a]
##STR00129##
[0489] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-(bromomethyl)-1-((methylsulfonyl)methyl)cyclopropane (26.8 mg,
0.118 mmol) was added. The reaction was heated to 70.degree. C. and
stirred for 3 hours or until done by LCMS. The reaction was cooled,
0.5 ml of DMF was added, then filtered through a 0.45 nM in line
filter. The DMF solution with the desired product 2.24a was used as
is for the next step, assume quantitative yield. LC-MS (m/z): 528.5
[M+H].sup.+, 0.85 min.
Step 2:
6-(tert-butyl)-10-((1-((methylsulfonyl)methyl)cyclopropyl)methoxy)-
-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxyli-
c acid [2.24]
##STR00130##
[0491] To 2.24a (24.80 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 2.4
mg of the desired racemic product 2.24 as TFA salt in 8% yield.
LC-MS (m/z): 500.4 [M+H].sup.+, 0.78 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.65 (d, J=8.6 Hz, 1H),
7.17-7.29 (m, 2H), 6.75 (d, J=7.6 Hz, 1H), 5.17-5.28 (m, 1H),
5.05-5.15 (m, 1H), 4.96 (br d, J=4.8 Hz, 1H), 4.26 (d, J=10.0 Hz,
1H), 4.07 (d, J=9.9 Hz, 1H), 3.01 (s, 3H), 0.78-0.93 (m, 4H), 0.70
(s, 9H).
Example 2.25
6-(tert-butyl)-2-oxo-10-((tetrahydrofuran-2-yl)methoxy)-6,7-dihydro-2H-pyr-
ido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.25]
##STR00131##
[0492] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-((tetrahydrofuran-2-yl)methoxy)-6,7-dihydro-2H-py-
rido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.25a]
##STR00132##
[0494] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
2-(bromomethyl)tetrahydrofuran (19.47 mg, 0.118 mmol) was added.
The reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.25a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 466.5 [M+H].sup.+,
0.90 min.
Step 2:
6-(tert-butyl)-2-oxo-10-((tetrahydrofuran-2-yl)methoxy)-6,7-dihydr-
o-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.25]
##STR00133##
[0496] To 2.25a (21.88 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 7.6
mg of the desired racemic product 2.25 as TFA salt in 29% yield.
LC-MS (m/z): 438.4 [M+H].sup.+, 0.84 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.64 (d, J=8.5 Hz, 1H),
7.14-7.30 (m, 2H), 6.83 (d, J=7.6 Hz, 1H), 5.03-5.23 (m, 2H), 4.95
(d, J=4.5 Hz, 1H), 4.20-4.33 (m, 1H), 4.05-4.19 (m, 2H), 3.80 (qd,
J=7.1, 3.5 Hz, 1H), 3.64-3.74 (m, 1H), 1.98-2.11 (m, 1H), 1.78-1.97
(m, 2H), 1.66-1.75 (m, 1H), 0.69 (s, 9H).
Example 2.26
6-(tert-butyl)-10-(oxetan-3-yloxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]p-
yrazino[1,2-b]indazole-3-carboxylic acid [2.26]
##STR00134##
[0497] Step 1: ethyl
6-(tert-butyl)-10-(oxetan-3-yloxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]-
pyrazino[1,2-b]indazole-3-carboxylate [2.26a]
##STR00135##
[0499] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then 3-bromooxetane
(16.16 mg, 0.118 mmol) was added. The reaction was heated to
70.degree. C. and stirred for 3 hours or until done by LCMS. The
reaction was cooled, 0.5 ml of DMF was added, then filtered through
a 0.45 nM in line filter. The DMF solution with the desired product
2.26a was used as is for the next step, assume quantitative yield.
LC-MS (m/z): 438.5 [M+H].sup.+, 0.81 min.
Step 2:
6-(tert-butyl)-10-(oxetan-3-yloxy)-2-oxo-6,7-dihydro-2H-pyrido[2',-
1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.26]
##STR00136##
[0501] To 2.26a (20.56 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 5.4
mg of the desired racemic product 2.26 as TFA salt in 21% yield.
LC-MS (m/z): 410.4 [M+H].sup.+, 0.74 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.70 (d, J=8.5 Hz, 1H), 7.28
(s, 1H), 7.20 (t, J=8.0 Hz, 1H), 6.50 (d, J=7.5 Hz, 1H), 5.48 (br
t, J=5.4 Hz, 1H), 5.06-5.24 (m, 2H), 4.99 (dt, J=11.9, 5.9 Hz, 3H),
4.64 (ddd, J=11.6, 7.0, 5.1 Hz, 2H), 0.69 (s, 9H).
Example 2.27
6-(tert-butyl)-2-oxo-10-(piperidin-4-yloxy)-6,7-dihydro-2H-pyrido[2',1':3,-
4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.27]
##STR00137##
[0502] Step 1: ethyl
10-((1-(tert-butoxycarbonyl)piperidin-4-yl)oxy)-6-(tert-butyl)-2-oxo-6,7--
dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate
[2.27a]
##STR00138##
[0504] To 2.1h (16 mg, 0.042 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (47.8 mg, 0.147 mmol). The reaction was
stirred at room temperature for 5 minutes then tert-butyl
4-bromopiperidine-1-carboxylate (27.7 mg, 0.105 mmol) was added.
The reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 10 ml of ethyl acetate
was added, washed with water 2.times., filtered and concentrated to
residue to give the desired product 2.27a used as is for the next
step, assume quantitative yield. LC-MS (m/z): 565.6 [M+H].sup.+,
0.98 min.
Step 2: ethyl
6-(tert-butyl)-2-oxo-10-(piperidin-4-yloxy)-6,7-dihydro-2H-pyrido[2',1':3-
,4]pyrazino[1,2-b]indazole-3-carboxylate [2.27b]
##STR00139##
[0506] To 2.27a (23.69 mg, 0.042 mmol) was added DCM (Volume: 1.8
mL, Ratio: 3.60), TFA (0.355 mL, 4.61 mmol) and stirred at room
temperature for 1 hour, or until done by LCMS. The reaction was
concentrated to residue to give the desired product 2.27b used as
is for the next step, assume quantitative yield. LC-MS (m/z): 465.5
[M+H].sup.+, 0.61 min.
Step 3:
6-(tert-butyl)-2-oxo-10-(piperidin-4-yloxy)-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.27]
##STR00140##
[0508] To 2.27b (19.51 mg, 0.042 mmol) that was added 1 ml of DMF
and LiOH 1M aq (0.294 mL, 0.294 mmol). The reaction was stirred at
room temperature for 30 minutes or until done by LCMS. Additional
LiOH 1M aq. can be added as needed. The crude reaction was purified
by reverse phase prep LC and lyophilized to give 3.4 mg of the
desired racemic product 2.27 as TFA salt in 14% yield. LC-MS (m/z):
437.5 [M+H].sup.+, 0.64 min. .sup.1H NMR (DMSO-d.sub.6) .delta.:
8.96 (s, 1H), 8.50 (br s, 2H), 7.69 (d, J=8.5 Hz, 1H), 7.15-7.31
(m, 2H), 6.97 (d, J=7.7 Hz, 1H), 5.12 (br s, 2H), 4.95 (br s, 1H),
4.89-4.93 (m, 1H), 3.12 (br s, 2H), 2.17 (br s, 2H), 1.83-1.97 (m,
2H), 0.69 (s, 9H).
Example 2.28
6-(tert-butyl)-10-(2,3-dihydroxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1'-
:3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.28]
##STR00141##
[0509] Step 1: ethyl
6-(tert-butyl)-10-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-oxo-6,7-dih-
ydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate
[2.28a]
##STR00142##
[0511] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
4-(bromomethyl)-2,2-dimethyl-1,3-dioxolane (23.01 mg, 0.118 mmol)
added. The reaction was heated to 70.degree. C. and stirred for 3
hours or until done by LCMS. The reaction was cooled, 0.5 ml of DMF
was added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.28a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 496.4 [M+H].sup.+,
0.84 min.
Step 2:
6-(tert-butyl)-10-(2,3-dihydroxypropoxy)-2-oxo-6,7-dihydro-2H-pyri-
do[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.28]
##STR00143##
[0513] To 2.28a (23.29 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
LC-MS (m/z): 468.4 [M+H].sup.+, 0.89 min. Additional LiOH 1M aq.
can be added as needed. The crude reaction was purified by reverse
phase prep LC using ACN/water with 0.1% TFA. The desired fractions
(in ACN/water with 0.1% TFA) were combined and let sit at room
temperature for 20 hours to give the desired deprotected product,
followed by LCMS. Then 0.2 ml of 3M HCl solution was added and
lyophilized. The product was re-dissolved in 1:1 ACN/water and
re-lyophilized to give 7.0 mg of the desired racemic product 2.28
as HCl salt in 31% yield. LC-MS (m/z): 428.4 [M+H].sup.+, 0.63 min.
.sup.1H NMR (DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.63 (d, J=8.5
Hz, 1H), 7.18-7.29 (m, 2H), 6.82 (d, J=7.6 Hz, 1H), 5.05-5.24 (m,
2H), 4.96 (br d, J=4.5 Hz, 1H), 4.17 (dt, J=9.8, 3.6 Hz, 1H),
3.99-4.09 (m, 1H), 3.90 (br s, 1H), 3.49 (br d, J=5.6 Hz, 2H), 0.69
(s, 9H).
Example 2.29
6-(tert-butyl)-10-cyclobutoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazi-
no[1,2-b]indazole-3-carboxylic acid [2.29]
##STR00144##
[0514] Step 1: ethyl
6-(tert-butyl)-10-cyclobutoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyraz-
ino[1,2-b]indazole-3-carboxylate [2.29a]
##STR00145##
[0516] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then bromocyclobutane
(15.93 mg, 0.118 mmol) was added. The reaction was heated to
70.degree. C. and stirred for 3 hours or until done by LCMS. The
reaction was cooled, 0.5 ml of DMF was added, then filtered through
a 0.45 nM in line filter. The DMF solution with the desired product
2.29a was used as is for the next step, assume quantitative yield.
LC-MS (m/z): 436.4 [M+H].sup.+, 0.91 min.
Step 2:
6-(tert-butyl)-10-cyclobutoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,-
4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.29]
##STR00146##
[0518] To 2.29a (20.47 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 2.0
mg of the desired racemic product 2.29 as TFA salt in 8% yield.
LC-MS (m/z): 408.4 [M+H].sup.+, 0.94 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.62 (d, J=8.5 Hz, 1H), 7.25
(s, 1H), 7.20 (t, J=8.0 Hz, 1H), 6.66 (d, J=7.5 Hz, 1H), 5.04-5.20
(m, 2H), 4.95 (d, J=4.5 Hz, 1H), 4.89 (quin, J=7.1 Hz, 1H),
2.07-2.20 (m, 2H), 1.83 (q, J=10.2 Hz, 1H), 1.61-1.75 (m, 1H), 0.69
(s, 9H).
Example 2.30
6-(tert-butyl)-10-(3-(methylsulfonyl)propoxy)-2-oxo-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.30]
##STR00147##
[0519] Step 1: ethyl
6-(tert-butyl)-10-(3-(methylsulfonyl)propoxy)-2-oxo-6,7-dihydro-2H-pyrido-
[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.30a]
##STR00148##
[0521] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-bromo-3-(methylsulfonyl)propane (23.72 mg, 0.118 mmol) was added.
The reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.30a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 502.4 [M+H].sup.+,
0.71 min.
Step 2:
6-(tert-butyl)-10-(3-(methylsulfonyl)propoxy)-2-oxo-6,7-dihydro-2H-
-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.30]
##STR00149##
[0523] To 2.30a (23.57 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 11.3
mg of the desired racemic product 2.30 as TFA salt in 40% yield.
LC-MS (m/z): 474.3 [M+H].sup.+, 0.72 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.66 (d, J=8.5 Hz, 1H),
7.18-7.30 (m, 2H), 6.83 (d, J=7.6 Hz, 1H), 5.05-5.25 (m, 2H), 4.95
(d, J=4.7 Hz, 1H), 4.24-4.36 (m, 2H), 3.30-3.35 (m, 2H), 3.04 (s,
3H), 2.18-2.32 (m, 2H), 0.69 (s, 9H).
Example 2.31
6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4-
]pyrazino[1,2-b]indazole-3-carboxylic acid [2.31]
##STR00150##
[0524] Step 1: ethyl
6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,-
4]pyrazino[1,2-b]indazole-3-carboxylate [2.31a]
##STR00151##
[0526] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-bromo-3-methoxypropane (18.05 mg, 0.118 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.31a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 454.5 [M+H].sup.+,
0.82 min.
Step 2:
6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2-
',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.31]
##STR00152##
[0528] To 2.31a (21.32 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 9.3
mg of the desired racemic product 2.31 as TFA salt in 36% yield.
LC-MS (m/z): 426.4 [M+H].sup.+, 0.83 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.63 (d, J=8.5 Hz, 1H),
7.13-7.30 (m, 2H), 6.82 (d, J=7.6 Hz, 1H), 5.04-5.26 (m, 2H), 4.95
(d, J=4.8 Hz, 1H), 4.20 (t, J=6.4 Hz, 2H), 3.51 (t, J=6.3 Hz, 2H),
3.25 (s, 3H), 2.04 (t, J=6.3 Hz, 2H), 0.69 (s, 9H).
Example 2.32
10-(azetidin-3-ylmethoxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1'-
:3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.32]
##STR00153##
[0529] Step 1: ethyl
10-((1-(tert-butoxycarbonyl)azetidin-3-yl)methoxy)-6-(tert-butyl)-2-oxo-6-
,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate
[2.32a]
##STR00154##
[0531] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then tert-butyl
3-(bromomethyl)azetidine-1-carboxylate (29.5 mg, 0.118 mmol) was
added. The reaction was heated to 70.degree. C. and stirred for 3
hours or until done by LCMS. The reaction was cooled, 15 ml of
ethyl acetate was added, washed with water 2.times., saturated salt
solution, dried sodium sulfate, filtered and concentrated to
residue to give the desired product 2.32a used as is for the next
step, assume quantitative yield. LC-MS (m/z): 551.7 [M+H].sup.+,
1.01 min.
Step 2: ethyl
10-(azetidin-3-ylmethoxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyrido[2',1-
':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.32b]
##STR00155##
[0533] To 2.32a (26.0 mg, 0.047 mmol) was added DCM (Volume: 2 mL,
Ratio: 4.00) trifluoroacetic acid (0.4 mL, 5.19 mmol) and stirred
at room temperature for 30 minutes or until done by LCMS. The
reaction was concentrated to residue. The crude was dissolved in 1
ml of DMF and purified by reverse phase prep LC and lyophilized to
give 13.5 mg of the desired product 2.32b in 64% yield, used as is
for the next step. LC-MS (m/z): 451.4 [M+H].sup.+, 0.61 min.
Step 3:
10-(azetidin-3-ylmethoxy)-6-(tert-butyl)-2-oxo-6,7-dihydro-2H-pyri-
do[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.32]
##STR00156##
[0535] To 2.32b (13.5 mg, 0.030 mmol) that was added 1 ml of DMF
and LiOH 1M aq LiOH (0.120 mL, 0.120 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 6.6
mg of the desired racemic product 2.32 as TFA salt in 40% yield.
LC-MS (m/z): 423.4 [M+H].sup.+, 0.62 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.97 (s, 1H), 8.71 (br d, J=15.6 Hz, 2H),
7.70 (d, J=8.5 Hz, 1H), 7.21-7.33 (m, 2H), 6.88 (d, J=7.6 Hz, 1H),
5.12 (br s, 2H), 4.96 (br s, 1H), 4.37 (br d, J=6.6 Hz, 2H), 4.11
(br s, 2H), 3.91 (br d, J=5.9 Hz, 2H), 0.69 (s, 9H).
Example 2.33
6-(tert-butyl)-2-oxo-10-(((S)-5-oxopyrrolidin-2-yl)methoxy)-6,7-dihydro-2H-
-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.33-I] and [2.33-II]
##STR00157##
[0536] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-(((S)-5-oxopyrrolidin-2-yl)methoxy)-6,7-dihydro-2-
H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate
[2.33a]
##STR00158##
[0538] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
(S)-5-(bromomethyl)pyrrolidin-2-one (21.00 mg, 0.118 mmol) was
added. The reaction was heated to 70.degree. C. and stirred for 3
hours or until done by LCMS. The reaction was cooled, 0.5 ml of DMF
was added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.33a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 479.4 [M+H].sup.+,
0.71 min.
Step 2:
6-(tert-butyl)-2-oxo-10-(((S)-5-oxopyrrolidin-2-yl)methoxy)-6,7-di-
hydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.33-I] and [2.33-II]
##STR00159##
[0540] To 2.33a (22.49 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC, using a Sunfire C.sub.18 OBD
column, eluting with ACN/water with 0.1% TFA, resulting in desired
products 2.33-I (peak 1, tR 5.97 min.) at 3.3 mg in 12% yield and
product 2.33-II (peak 2, tR 6.16 min.) at 2.5 mg in 9% yield, both
as TFA salts. 2.33-I LC-MS (m/z): 451.4 [M+H]+, 0.70 min. .sup.1H
NMR (DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.83 (s, 1H), 7.67 (d,
J=8.5 Hz, 1H), 7.18-7.30 (m, 2H), 6.85 (d, J=7.6 Hz, 1H), 5.05-5.21
(m, 2H), 4.95 (br d, J=4.2 Hz, 1H), 4.12 (d, J=4.9 Hz, 2H), 3.97
(br dd, J=7.5, 4.5 Hz, 1H), 2.30-2.40 (m, 1H), 2.08-2.26 (m, 2H),
1.90-2.01 (m, 1H), 0.69 (s, 9H). 2.33-II LC-MS (m/z): 451.4 [M+H]+,
0.71 min. .sup.1HNMR (DMSO-d.sub.6) .delta.: 8.96 (s, 1H) 7.84 (s,
1H) 7.67 (d, J=8.51 Hz, 1H) 7.20-7.30 (m, 2H) 6.86 (d, J=7.58 Hz,
1H) 5.07-5.22 (m, 2H) 4.95 (br d, J=4.55 Hz, 1H) 4.06-4.18 (m, 2H)
3.97 (br dd, J=7.51, 4.77 Hz, 1H) 2.28-2.39 (m, 2H) 2.08-2.23 (m,
2H) 1.94 (dt, J=16.81, 4.90 Hz, 1H) 0.69 (s, 9H)
Example 2.34
6-(tert-butyl)-2-oxo-10-(((R)-5-oxopyrrolidin-2-yl)methoxy)-6,7-dihydro-2H-
-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.34-I] and [2.34-II]
##STR00160##
[0541] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-(((R)-5-oxopyrrolidin-2-yl)methoxy)-6,7-dihydro-2-
H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate
[2.34a]
##STR00161##
[0543] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
(R)-5-(bromomethyl)pyrrolidin-2-one (21.00 mg, 0.118 mmol) was
added. The reaction was heated to 70.degree. C. and stirred for 3
hours or until done by LCMS. The reaction was cooled, 0.5 ml of DMF
was added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.34a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 479.4 [M+H].sup.+,
0.70 min.
Step 2:
6-(tert-butyl)-2-oxo-10-(((R)-5-oxopyrrolidin-2-yl)methoxy)-6,7-di-
hydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.34-I] and [2.34-II]
##STR00162##
[0545] To 2.34a (22.49 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC, using a Sunfire C18 OBD
column, eluting with ACN/water with 0.1% TFA, resulting in desired
products 2.34-I (peak 1, tR 5.86 min.) at 4.0 mg in 15% yield and
product 2.34-II (peak 2, tR 6.09 min.) at 3.4 mg in 13% yield, both
as TFA salts. 2.34-I LC-MS (m/z): 451.4 [M+H]+, 0.70 min. .sup.1H
NMR (DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.83 (s, 1H), 7.67 (d,
J=8.5 Hz, 1H), 7.17-7.28 (m, 2H), 6.85 (d, J=7.6 Hz, 1H), 5.06-5.20
(m, 2H), 4.95 (br d, J=4.2 Hz, 1H), 4.12 (d, J=4.9 Hz, 2H), 3.97
(br dd, J=7.7, 4.5 Hz, 1H), 2.30-2.40 (m, 1H), 2.08-2.26 (m, 2H),
1.89-2.01 (m, 1H), 0.69 (s, 9H). 2.34-II LC-MS (m/z): 451.4 [M+H]+,
0.71 min. .sup.1HNMR (DMSO-d.sub.6) .delta.: 8.96 (s, 1H), 7.84 (s,
1H), 7.67 (d, J=8.5 Hz, 1H), 7.19-7.33 (m, 2H), 6.86 (d, J=7.6 Hz,
1H), 5.03-5.24 (m, 2H), 4.95 (d, J=4.5 Hz, 1H), 4.07-4.23 (m, 2H),
3.97 (br dd, J=7.3, 4.5 Hz, 1H), 2.29-2.39 (m, 1H), 2.08-2.24 (m,
2H), 1.86-2.00 (m, 1H), 0.69 (s, 9H)
Example 2.35
6-(tert-butyl)-10-((1-methoxypropan-2-yl)oxy)-2-oxo-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.35-I] and
[2.35-II]
##STR00163##
[0546] Step 1: ethyl
6-(tert-butyl)-10-((1-methoxypropan-2-yl)oxy)-2-oxo-6,7-dihydro-2H-pyrido-
[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.35a]
##STR00164##
[0548] To 2.1h (16 mg, 0.042 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (47.8 mg, 0.147 mmol). The reaction was
stirred at room temperature for 5 minutes then
2-bromo-1-methoxypropane (16.05 mg, 0.105 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.35a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 454.5 [M+H].sup.+,
0.81 min.
Step 2:
6-(tert-butyl)-10-((1-methoxypropan-2-yl)oxy)-2-oxo-6,7-dihydro-2H-
-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.35-I] and [2.35-II]
##STR00165##
[0550] To 2.35a (19.05 mg, 0.042 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (168 .mu.l, 0.168 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC, using a Sunfire C.sub.18 OBD
column, eluting with ACN/water with 0.1% TFA, resulting in desired
products 2.35-I (peak 1, tR 5.50 min.) at 3.0 mg in 13% yield and
product 2.35-II (peak 2, tR 5.67 min.) at 3.0 mg in 13% yield, both
as TFA salts. 2.35-I LC-MS (m/z): 426.4 [M+H]+, 0.82 min. .sup.1H
NMR (DMSO-d.sub.6) .delta.: 8.93 (s, 1H), 7.61 (d, J=8.5 Hz, 1H),
7.18-7.28 (m, 2H), 6.85-6.92 (m, 1H), 5.13-5.22 (m, 1H), 5.03-5.12
(m, 1H), 4.94 (br d, J=4.7 Hz, 1H), 4.88-4.93 (m, 1H), 3.49-3.61
(m, 2H), 3.29 (s, 3H), 1.28 (d, J=6.2 Hz, 3H), 0.69 (s, 9H).
2.35-II LC-MS (m/z): 426.4 [M+H]+, 0.85 min. .sup.1HNMR
(DMSO-d.sub.6) .delta.: 8.93 (s, 1H), 7.61 (d, J=8.5 Hz, 1H),
7.18-7.27 (m, 2H), 6.85-6.91 (m, 1H), 5.14-5.20 (m, 1H), 5.05-5.13
(m, 1H), 4.94 (d, J=4.6 Hz, 1H), 4.87-4.93 (m, 1H), 3.49-3.61 (m,
2H), 3.28 (s, 3H), 1.30 (d, J=6.2 Hz, 3H), 0.69 (s, 9H).
Example 2.36
6-(tert-butyl)-10-(2-(dimethylamino)-2-oxoethoxy)-2-oxo-6,7-dihydro-2H-pyr-
ido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.36]
##STR00166##
[0551] Step 1: ethyl
6-(tert-butyl)-10-(2-(dimethylamino)-2-oxoethoxy)-2-oxo-6,7-dihydro-2H-py-
rido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.36a]
##STR00167##
[0553] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
2-bromo-N,N-dimethylacetamide (19.59 mg, 0.118 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.36a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 467.5 [M+H].sup.+,
0.70 min.
Step 2:
6-(tert-butyl)-10-(2-(dimethylamino)-2-oxoethoxy)-2-oxo-6,7-dihydr-
o-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.36]
##STR00168##
[0555] To 2.36a (21.93 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq. (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 9.7
mg of the desired racemic product 2.36 as TFA salt in 36% yield.
LC-MS (m/z): 439.4 [M+H].sup.+, 0.70 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.65 (d, J=8.5 Hz, 1H), 7.27
(s, 1H), 7.21 (t, J=8.0 Hz, 1H), 6.74 (d, J=7.6 Hz, 1H), 5.08-5.19
(m, 2H), 5.00 (d, J=1.7 Hz, 2H), 4.96 (br d, J=2.2 Hz, 1H), 3.03
(s, 3H), 2.85 (s, 3H), 0.69 (s, 9H).
Example 2.37
6-(tert-butyl)-2-oxo-10-((tetrahydrofuran-3-yl)oxy)-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.37]
##STR00169##
[0557] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-((tetrahydrofuran-3-yl)oxy)-6,7-dihydro-2H-pyrido-
[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.37a]
##STR00170##
[0558] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
3-bromotetrahydrofuran (17.81 mg, 0.118 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.37a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 452.4 [M+H].sup.+,
0.75 min.
Step 2:
6-(tert-butyl)-2-oxo-10-((tetrahydrofuran-3-yl)oxy)-6,7-dihydro-2H-
-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.37]
##STR00171##
[0560] To 2.37a (21.22 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 7.5
mg of the desired racemic product 2.37 as TFA salt in 29% yield.
LC-MS (m/z): 424.4 [M+H].sup.+, 0.77 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.66 (d, J=8.5 Hz, 1H),
7.16-7.29 (m, 2H), 6.80 (dd, J=7.5, 4.8 Hz, 1H), 5.25 (br s, 1H),
5.05-5.22 (m, 2H), 4.95 (d, J=4.8 Hz, 1H), 3.83-4.00 (m, 3H), 3.78
(tt, J=8.4, 4.3 Hz, 1H), 2.23-2.37 (m, 1H), 1.95-2.16 (m, 1H), 0.69
(s, 9H)
Example 2.38
6-(tert-butyl)-2-oxo-10-((tetrahydro-2H-pyran-4-yl)oxy)-6,7-dihydro-2H-pyr-
ido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.38]
##STR00172##
[0561] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-((tetrahydro-2H-pyran-4-yl)oxy)-6,7-dihydro-2H-py-
rido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.38a]
##STR00173##
[0563] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
4-bromotetrahydro-2H-pyran (19.47 mg, 0.118 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 20 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.38a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 466.5 [M+H].sup.+,
0.79 min.
Step 2:
6-(tert-butyl)-2-oxo-10-((tetrahydro-2H-pyran-4-yl)oxy)-6,7-dihydr-
o-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.38]
##STR00174##
[0565] To 2.38a (21.88 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 8.1
mg of the desired racemic product 2.38 as TFA salt in 31% yield.
LC-MS (m/z): 438.4 [M+H].sup.+, 0.81 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.64 (d, J=8.5 Hz, 1H),
7.16-7.29 (m, 2H), 6.94 (d, J=7.7 Hz, 1H), 5.05-5.25 (m, 2H), 4.95
(d, J=4.7 Hz, 1H), 4.79-4.91 (m, 1H), 3.89 (ddt, J=11.7, 7.9, 4.0
Hz, 2H), 3.46-3.58 (m, 3H), 2.06 (br dd, J=9.9, 4.9 Hz, 2H), 1.67
(ddd, J=13.1, 9.1, 3.7 Hz, 2H), 0.69 (s, 9H).
Example 2.39
6-(tert-butyl)-10-((3,3-difluorocyclobutyl)methoxy)-2-oxo-6,7-dihydro-2H-p-
yrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.39]
##STR00175##
[0566] Step 1: ethyl
6-(tert-butyl)-10-((3,3-difluorocyclobutyl)methoxy)-2-oxo-6,7-dihydro-2H--
pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.39a]
##STR00176##
[0568] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
3-(bromomethyl)-1,1-difluorocyclobutane (21.83 mg, 0.118 mmol) was
added. The reaction was heated to 70.degree. C. and stirred for 3
hours or until done by LCMS. The reaction was cooled, 0.5 ml of DMF
was added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.39a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 486.4 [M+H].sup.+,
0.92 min.
Step 2:
6-(tert-butyl)-10-((3,3-difluorocyclobutyl)methoxy)-2-oxo-6,7-dihy-
dro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.39]
##STR00177##
[0570] To 2.39a (22.82 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 9.1
mg of the desired racemic product 2.39 as TFA salt in 33% yield.
LC-MS (m/z): 458.4 [M+H].sup.+, 0.94 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.66 (d, J=8.5 Hz, 1H),
7.19-7.32 (m, 2H), 6.85 (d, J=7.6 Hz, 1H), 5.05-5.29 (m, 2H), 4.95
(d, J=4.8 Hz, 1H), 4.14-4.32 (m, 2H), 2.61-2.86 (m, 3H), 0.69 (s,
9H).
Example 2.40
6-(tert-butyl)-2-oxo-10-(3,3,3-trifluoropropoxy)-6,7-dihydro-2H-pyrido[2',-
1'. 3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.40]
##STR00178##
[0571] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-(3,3,3-trifluoropropoxy)-6,7-dihydro-2H-pyrido[2'-
,1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.40a]
##STR00179##
[0573] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
3-bromo-1,1,1-trifluoropropane (50.1 mg, 0.283 mmol) was added. The
reaction was heated to 85.degree. C. and stirred for 40 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.40a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 478.4 [M+H]+, 0.89
min.
Step 2:
6-(tert-butyl)-2-oxo-10-(3,3,3-trifluoropropoxy)-6,7-dihydro-2H-py-
rido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.40]
##STR00180##
[0575] To 2.40a (22.44 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq. (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 2.0
mg of the desired racemic product 2.40 as TFA salt in 7% yield.
LC-MS (m/z): 450.3 [M+H].sup.+, 0.91 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.69 (d, J=8.5 Hz, 1H),
7.19-7.34 (m, 2H), 6.90 (d, J=7.6 Hz, 1H), 5.06-5.24 (m, 2H), 4.95
(br d, J=4.7 Hz, 1H), 4.41 (t, J=6.0 Hz, 2H), 2.90 (tt, J=11.5, 5.6
Hz, 2H), 0.69 (s, 9H).
Example 2.41
6-(tert-butyl)-10-(2-fluoroethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]p-
yrazino[1,2-b]indazole-3-carboxylic acid [2.41]
##STR00181##
[0576] Step 1: ethyl
6-(tert-butyl)-10-(2-fluoroethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]-
pyrazino[1,2-b]indazole-3-carboxylate [2.41a]
##STR00182##
[0578] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-bromo-2-fluoroethane (14.98 mg, 0.118 mmol) was added. The
reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.41a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 428.4 [M+H].sup.+,
0.77 min.
Step 2:
6-(tert-butyl)-10-(2-fluoroethoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',-
1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.41]
##STR00183##
[0580] To 2.41a (20.09 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 9.5
mg of the desired racemic product 2.41 as TFA salt in 39% yield.
LC-MS (m/z): 400.3 [M+H].sup.+, 0.79 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.68 (d, J=8.5 Hz, 1H),
7.12-7.34 (m, 2H), 6.85 (d, J=7.6 Hz, 1H), 5.06-5.25 (m, 2H), 4.96
(d, J=4.7 Hz, 1H), 4.87-4.93 (m, 1H), 4.72-4.81 (m, 1H), 4.43-4.50
(m, 1H), 4.36-4.42 (m, 1H), 0.69 (s, 9H)
Example 2.42
6-(tert-butyl)-10-ethoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1,-
2-b]indazole-3-carboxylic acid [2.42]
##STR00184##
[0581] Step 1: ethyl
6-(tert-butyl)-10-ethoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazino[1-
,2-b]indazole-3-carboxylate [2.42a]
##STR00185##
[0583] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then bromoethane (12.86
mg, 0.118 mmol) was added. The reaction was heated to 70.degree. C.
and stirred for 3 hours or until done by LCMS. The reaction was
cooled, 0.5 ml of DMF was added, then filtered through a 0.45 nM in
line filter. The DMF solution with the desired product 2.42a was
used as is for the next step, assume quantitative yield. LC-MS
(m/z): 410.4 [M+H].sup.+, 0.82 min.
Step 2:
6-(tert-butyl)-10-ethoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyr-
azino[1,2-b]indazole-3-carboxylic acid [2.42]
##STR00186##
[0585] To 2.42a (19.25 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 7.2
mg of the desired racemic product 2.42 as TFA salt in 29% yield.
LC-MS (m/z): 382.4 [M+H].sup.+, 0.87 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.94 (s, 1H), 7.62 (d, J=8.5 Hz, 1H),
7.16-7.30 (m, 2H), 6.80 (d, J=7.6 Hz, 1H), 5.04-5.23 (m, 2H), 4.95
(d, J=4.6 Hz, 1H), 4.20 (q, J=7.0 Hz, 2H), 1.42 (t, J=6.9 Hz, 3H),
0.69 (s, 9H)
Example 2.43
6-(tert-butyl)-10-(3-(dimethylamino)propoxy)-2-oxo-6,7-dihydro-2H-pyrido[2-
',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.43]
##STR00187##
[0586] Step 1: ethyl
6-(tert-butyl)-10-(3-(dimethylamino)propoxy)-2-oxo-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.43a]
##STR00188##
[0588] To 2.1h (22 mg, 0.058 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (56.4 mg, 0.173 mmol). The reaction was
stirred at room temperature for 5 minutes then
3-bromo-N,N-dimethylpropan-1-amine (14.37 mg, 0.087 mmol) was
added. The reaction was heated to 50.degree. C. and stirred for 3
hours or until done by LCMS. The reaction was cooled, 0.5 ml of DMF
was added, then filtered through a 0.45 nM in line filter. The
crude reaction was purified by reverse phase prep LC and
lyophilized to give 17 mg of the desired racemic product 2.43a as
TFA salt in 63% yield. LC-MS (m/z): 467.5 [M+H].sup.+, 0.62
min.
Step 2:
6-(tert-butyl)-10-(3-(dimethylamino)propoxy)-2-oxo-6,7-dihydro-2H--
pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.43]
##STR00189##
[0590] To 2.43a (17 mg, 0.036 mmol) was added 1 ml of DMF and LiOH
1M aq (0.146 mL, 0.146 mmol). The reaction was stirred at room
temperature for 30 minutes or until done by LCMS. Additional LiOH
1M aq. can be added as needed. The crude reaction was purified by
reverse phase prep LC and lyophilized to give 9.0 mg of the desired
racemic product 2.43 as TFA salt in 43% yield. LC-MS (m/z): 439.4
[M+H].sup.+, 0.62 min. .sup.1H NMR (DMSO-d.sub.6) .delta.: 9.47 (br
s, 1H), 8.97 (s, 1H), 7.68 (d, J=8.5 Hz, 1H), 7.20-7.32 (m, 2H),
6.85 (d, J=7.6 Hz, 1H), 5.11 (br s, 2H), 4.96 (br s, 1H), 4.25 (br
t, J=5.4 Hz, 2H), 3.24-3.30 (m, 2H), 2.84 (d, J=4.0 Hz, 6H), 2.21
(br dd, J=9.2, 6.1 Hz, 2H), 0.69 (s, 9H).
Example 2.44
6-(tert-butyl)-2-oxo-10-((tetrahydro-2H-pyran-4-yl)methoxy)-6,7-dihydro-2H-
-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.44]
##STR00190##
[0591] Step 1: ethyl
6-(tert-butyl)-2-oxo-10-((tetrahydro-2H-pyran-4-yl)methoxy)-6,7-dihydro-2-
H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate
[2.44a]
##STR00191##
[0593] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
4-(bromomethyl)tetrahydro-2H-pyran (21.12 mg, 0.118 mmol) was
added. The reaction was heated to 70.degree. C. and stirred for 3
hours or until done by LCMS. The reaction was cooled, 0.5 ml of DMF
was added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.44a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 480.4 [M+H]+, 0.84
min.
Step 2:
6-(tert-butyl)-2-oxo-10-((tetrahydro-2H-pyran-4-yl)methoxy)-6,7-di-
hydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.44]
##STR00192##
[0595] To 2.44a (22.54 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq. (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 8.6
mg of the desired racemic product 2.44 as TFA salt in 32% yield.
LC-MS (m/z): 452.4 [M+H].sup.+, 0.86 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H), 7.63 (d, J=8.5 Hz, 1H),
7.19-7.30 (m, 2H), 6.82 (d, J=7.6 Hz, 1H), 5.16-5.24 (m, 1H),
5.04-5.15 (m, 1H), 4.95 (d, J=4.8 Hz, 1H), 3.97-4.11 (m, 2H), 3.89
(br dd, J=11.2, 2.8 Hz, 2H), 2.07-2.16 (m, 1H), 1.72 (br t, J=14.6
Hz, 2H), 1.31-1.46 (m, 2H), 0.69 (s, 9H)
Example 2.45
6-(tert-butyl)-10-((3-methyloxetan-3-yl)methoxy)-2-oxo-6,7-dihydro-2H-pyri-
do[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.45]
##STR00193##
[0596] Step 1: ethyl
6-(tert-butyl)-10-((3-methyloxetan-3-yl)methoxy)-2-oxo-6,7-dihydro-2H-pyr-
ido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.45a]
##STR00194##
[0598] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
3-(bromomethyl)-3-methyloxetane (19.47 mg, 0.118 mmol) was added.
The reaction was heated to 70.degree. C. and stirred for 3 hours or
until done by LCMS. The reaction was cooled, 0.5 ml of DMF was
added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.45a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 466.4 [M+H].sup.+,
0.79 min.
Step 2:
6-(tert-butyl)-10-((3-methyloxetan-3-yl)methoxy)-2-oxo-6,7-dihydro-
-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.45]
##STR00195##
[0600] To 2.45a (21.88 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 1.0
mg of the desired racemic product 2.45 as TFA salt in 4% yield.
LC-MS (m/z): 438.4 [M+H].sup.+, 0.81 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.96 (s, 1H), 7.67 (d, J=8.5 Hz, 1H),
7.19-7.31 (m, 2H), 6.90 (d, J=7.6 Hz, 1H), 5.24 (br d, J=14.9 Hz,
1H), 5.09 (br dd, J=14.7, 5.2 Hz, 1H), 4.95 (br d, J=5.0 Hz, 1H),
4.54 (t, J=5.5 Hz, 2H), 4.35 (dd, J=5.8, 3.1 Hz, 2H), 4.17-4.31 (m,
2H), 1.42 (s, 3H), 0.70 (s, 9H).
Example 2.46
6-(tert-butyl)-10-((1-cyanocyclopropyl)methoxy)-2-oxo-6,7-dihydro-2H-pyrid-
o[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [2.46]
##STR00196##
[0601] Step 1: ethyl
6-(tert-butyl)-10-((1-cyanocyclopropyl)methoxy)-2-oxo-6,7-dihydro-2H-pyri-
do[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [2.46a]
##STR00197##
[0603] To 2.1h (18 mg, 0.047 mmol) was added DMF (Volume: 0.5 mL)
and cesium carbonate (53.8 mg, 0.165 mmol). The reaction was
stirred at room temperature for 5 minutes then
1-(bromomethyl)cyclopropanecarbonitrile (18.88 mg, 0.118 mmol) was
added. The reaction was heated to 70.degree. C. and stirred for 3
hours or until done by LCMS. The reaction was cooled, 0.5 ml of DMF
was added, then filtered through a 0.45 nM in line filter. The DMF
solution with the desired product 2.46a was used as is for the next
step, assume quantitative yield. LC-MS (m/z): 461.4 [M+H].sup.+,
0.80 min.
Step 2:
6-(tert-butyl)-10-((1-cyanocyclopropyl)methoxy)-2-oxo-6,7-dihydro--
2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[2.46]
##STR00198##
[0605] To 2.46a (21.64 mg, 0.047 mmol) that was already in 1 ml of
DMF was added LiOH 1M aq (188 .mu.l, 0.188 mmol). The reaction was
stirred at room temperature for 30 minutes or until done by LCMS.
Additional LiOH 1M aq. can be added as needed. The crude reaction
was purified by reverse phase prep LC and lyophilized to give 5.8
mg of the desired racemic product 2.46 as TFA salt in 22% yield.
LC-MS (m/z): 433.4 [M+H].sup.+, 0.82 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.95 (s, 1H) 7.70 (d, J=8.51 Hz, 1H)
7.17-7.31 (m, 2H) 6.79 (d, J=7.58 Hz, 1H) 5.21-5.32 (m, 1H)
5.06-5.18 (m, 1H) 4.97 (d, J=4.99 Hz, 1H) 4.10-4.33 (m, 2H)
1.36-1.49 (m, 2H) 1.13-1.29 (m, 2H) 0.70 (s, 9H).
Example 2.47
6-(tert-butyl)-10-isopropoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazin-
o[1,2-b]indazole-3-carboxylic acid [2.47-] and [2.47-II]
##STR00199##
[0606] Step 1: ethyl
6-(tert-butyl)-10-isopropoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]pyrazi-
no[1,2-b]indazole-3-carboxylate [2.47a-I] and [2.47a-II]
##STR00200##
[0608] To 2.1h (250 mg, 0.655 mmol) was added DMF (Volume: 5 mL)
and cesium carbonate (641 mg, 1.966 mmol). The reaction was stirred
at room temperature for 10 minutes then 2-bromopropane (161 mg,
1.311 mmol) was added and the reaction was heated to 70.degree. C.
and stirred for 3 hours or until done by LCMS. The reaction was
cooled, diluted with 100 ml of DCM with 3% ethanol, washed with
water 3.times., saturated salt solution, dried with sodium sulfate,
filtered and concentrated to residue. The crude material was
purified by silica gel chromatography, using 0-85% EtOAc (with 25%
ethanol)/heptane. The desired fractions were concentrated to
constant mass to give 234 mg of the desired racemic product 2.47a
in 84% yield. LC-MS (m/z): 424.6 [M+H]+, 0.94 min. .sup.1H NMR
(DMSO-d.sub.6) .delta.: 8.51 (s, 1H), 7.47 (d, J=8.5 Hz, 1H), 7.17
(t, J=8.0 Hz, 1H), 6.79 (t, J=3.7 Hz, 2H), 5.04-5.12 (m, 1H),
4.96-5.04 (m, 1H), 4.87 (dt, J=12.1, 6.0 Hz, 1H), 4.69 (d, J=4.4
Hz, 1H), 4.22 (q, J=7.1 Hz, 2H), 1.34 (t, J=6.4 Hz, 6H), 1.26 (t,
J=7.1 Hz, 3H), 0.69 (s, 9H). The racemic material 2.47a, 370 mg was
separated by chiral chromatography using (AD column, SFC=100
ml/min, CO.sub.2/MeOH=80/20, 266 bar) to give products 2.47a-I
(peak 1, tR 3.72 min.) at 157 mg and product 2.47a-II (peak 2, tR
5.63 min.) at 170 mg.
Step 2:
6-(tert-butyl)-10-isopropoxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4-
]pyrazino[1,2-b]indazole-3-carboxylic acid [2.47-I] and
[2.47-II]
##STR00201##
[0610] To 2.47a-I (157 mg, 0.371 mmol) was added Acetonitrile
(Volume: 5 ml, Ratio: 1.000), Water (Volume: 5 ml, Ratio: 1.000)
and then LiOH (1.112 ml, 1.112 mmol). The reaction was stirred at
room temperature for 16 hours or until done by LCMS. The reaction
was diluted with 15 ml of water, acidified with 1M HCl to pH of
2-3, then 150 ml of DCM with 2% ethanol was added and 10 ml of
saturated sodium chloride solution. The organic layer was
separated, the aqueous layer was extracted again 1.times.75 ml DCM
with 2% ethanol. The organic layers were combined, washed with
water 4.times.75 ml, filtered and concentrated to residue. The
residue was dissolved in 10 ml of 1:1 ACN/water, filtered and
lyophilized to give 136.8 mg of the desired product 2.47-I as free
base in 92% yield. LC-MS (m/z): 396.3 [M+H].sup.+, 0.91 min.
.sup.1H NMR (400 MHz, METHANOL-d4) .delta. ppm: 8.82 (s, 1H) 7.52
(d, J=8.46 Hz, 1H) 7.26-7.36 (m, 2H) 6.85 (d, J=7.58 Hz, 1H)
5.09-5.19 (m, 1H) 5.02-5.09 (m, 1H) 4.90-4.96 (m, 1H) 4.76 (d,
J=4.50 Hz, 1H) 1.44 (d, J=6.06 Hz, 6H) 0.81 (s, 9H). To 2.47a-II
(170 mg, 0.401 mmol) was added Acetonitrile (Volume: 5 ml, Ratio:
1.000), Water (Volume: 5 ml, Ratio: 1.000) and then LiOH (1.204 ml,
1.204 mmol). The reaction was stirred at room temperature for 16
hours or until done by LCMS. The reaction was diluted with 15 ml of
water, acidified with 1M HCl to pH of 2-3, then 150 ml of DCM with
2% ethanol was added and 10 ml of saturated sodium chloride
solution. The organic layer was separated, the aqueous layer was
extracted again 1.times.75 ml DCM with 2% ethanol. The organic
layers were combined, washed with water 4.times.75 ml, filtered and
concentrated to residue. The residue was dissolved in 10 ml of 1:1
ACN/water, filtered and lyophilized to give 143.3 mg of the desired
product 2.47-II as free base in 89% yield. LC-MS (m/z): 396.3
[M+H].sup.+, 0.90 min. .sup.1H NMR (400 MHz, METHANOL-d4) .delta.
ppm: 8.82 (s, 1H) 7.51 (d, J=8.46 Hz, 1H) 7.26-7.38 (m, 2H) 6.85
(d, J=7.63 Hz, 1H) 5.10-5.18 (m, 1H) 5.03-5.09 (m, 1H) 4.89-4.96
(m, 1H) 4.76 (d, J=4.45 Hz, 1H) 1.44 (d, J=6.06 Hz, 6H) 0.80 (s,
9H)
Example 3.1
6-(1-hydroxy-2-methylpropan-2-yl)-10-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2-
',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [3.1-I] and
[3.1-II]
##STR00202##
[0611] Step 1: 4-(benzyloxy)-1-bromo-3,3-dimethylbutan-2-one
[3.1a]
##STR00203##
[0613] To 4-(benzyloxy)-3,3-dimethylbutan-2-one (48.17 g, 234 mmol)
in MeOH (Volume: 700 mL) cooled to -78.degree. C., Bromine (13.23
mL, 257 mmol) was added and stirred to rt. Saturated Sodium
bicarbonate was added until the solution is basic. The organics
were extracted into DCM, dried with Sodium Sulfate and concentrated
to give the pdt. The crude was purified by silica gel
chromatography using Heptane-EtOAc (0-15% EtOAc, 30 min) to give
33.2g of 3.1a in 49.9% yield. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. ppm 7.25-7.38 (m, 5H) 4.47-4.51 (m, 2H) 4.23 (s, 2H)
3.41-3.46 (m, 2H) 1.23 (s, 6H)
Step 2:
4-(benzyloxy)-1-(3-(((tert-butyldimethylsilyl)oxy)methyl)-7-methox-
y-2H-indazol-2-yl)-3,3-dimethylbutan-2-one [3.1b]
##STR00204##
[0615] To 1.1b (3.4 g, 11.63 mmol) and 3.1a (13.26 g, 46.5 mmol) in
DMSO (Volume: 124 ml, Ratio: 1.000)-Water (Volume: 6.22 ml, Ratio:
0.05), cesium carbonate (22.73 g, 69.8 mmol) was added and stirred
at rt for 4 hrs. The crude was extracted into DCM and concentrated.
EtOAc was added and washed with brine to remove any DMSO and
purified by silica gel chromatography using Heptane-EtOAc (0-20%
EtOAc, 23 min gradient elution) to give 1.24g of 3.1b in 21.5%
yield. LC-MS (m/z): 497.6 [M+H]+,1.23 min.
Step 3:
(2-(2-amino-4-(benzyloxy)-3,3-dimethylbutyl)-7-methoxy-2H-indazol--
3-yl)methanol [3.1c]
##STR00205##
[0617] To 3.1b (1.6 g, 3.22 mmol) in Methanol (Volume: 30 ml),
Ammonium acetate (4.97 g, 64.4 mmol) and Sodium cyanoborohydride
(2.024 g, 32.2 mmol) were added and stirred at 70.degree. C. for 3
days. The crude was diluted with DCM and washed with saturated
sodium bicarbonate and the organics concentrated. The organics
containing 3.1c were taken to the next step without further
purification, assume quantitative yield. LC-MS (m/z): 384.4
[M+H]+,0.74 min.
Step 4:
3-(1-(benzyloxy)-2-methylpropan-2-yl)-7-methoxy-3,4-dihydropyrazin-
o[1,2-b]indazole [3.1d]
##STR00206##
[0619] To 3.1c (1.6 g, 4.17 mmol) in DCM (Volume: 40 mL), Manganese
dioxide (7.25 g, 83 mmol) was added and stirred at rt. The reaction
stalls after stirring at rt for 2 hrs. The crude was filtered and
the filtrate was again treated with Manganese dioxide (7.25 g, 83
mmol) and stirred overnight. The crude reaction was again filtered
and concentrated to give the crude product 3.1d which was taken to
the next step as is, assume quantitative yield. LC-MS (m/z): 364.4
[M+H].sup.+,0.82 min.
Step 5: Ethyl
6-(1-(benzyloxy)-2-methylpropan-2-yl)-10-methoxy-2-oxo-1,6,7,13c-tetrahyd-
ro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate
[3.1e]
##STR00207##
[0621] 3.1d (0.994 g, 2.73 mmol) and ethyl
(Z)-2-(ethoxymethylene)-3-oxobutanoate (1.528 g, 8.20 mmol) was
heated in EtOH (Volume: 17 mL) at 120.degree. C. overnight. The
crude containing 3.1e was concentrated and taken to the next step,
assume quantitative yield. LC-MS (m/z): 504.5 [M+H].sup.+,0.98
min.
Step 6: Ethyl
6-(1-(benzyloxy)-2-methylpropan-2-yl)-10-methoxy-2-oxo-6,7-dihydro-2H-pyr-
ido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [3.1f]
##STR00208##
[0623] To 3.1e (1375 mg, 2.73 mmol) in DME (Volume: 40 ml),
Chloramine-T (738 mg, 3.00 mmol) was added and heated at reflux for
2 hrs. The crude was concentrated and redissolved in DCM-Methanol
(minimum amount) and purified by silica gel chromatography first
using Heptane-EtOAc (0-100%) to remove all non-polar SM/By-products
and then purified with DCM-Methanol (0-20% MeOH) to give after
concentration 630 mg of 3.1f in 46% yield. LC-MS (m/z): 502.4
[M+H].sup.+,0.96 min.
Step 7: Ethyl
6-(1-hydroxy-2-methylpropan-2-yl)-10-methoxy-2-oxo-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [3.1 g-I] and [3.1
g-II]
##STR00209##
[0625] To 3.1f (630 mg, 1.256 mmol) in EtOH (Volume: 40 mL), 10%
palladium on carbon (800 mg, 7.52 mmol) was added and purged with
nitrogen followed by hydrogen. A hydrogen balloon was attached and
the crude stirred for 4 hrs. The reaction needs to be monitored
closely as by-products form when the debenzylation is 70-80% done.
The reaction was filtered at this point and the crude was
concentrated. The concentrated crude was purified by silica gel
chromatography using Heptane-EtOAc with 25% EtOH (0-100%) to give
after concentration, 139 mg of 3.1g in 26.9% yield which was given
for chiral purification. LC-MS (m/z): 412.4 [M+H].sup.+,0.65 min.
The above racemic material 139 mg was separated by chiral
chromatography using (OD column, SFC=80 ml/min, CO2/MeOH=75/25, 266
bar) to give products 3.1g-I (peak 1, tR 5.17 min.) at 42 mg and
product 3.1g-II (peak 2, tR 9.86 min.) at 43 mg.
Step 8:
6-(1-hydroxy-2-methylpropan-2-yl)-10-methoxy-2-oxo-6,7-dihydro-2H--
pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [3.1-I]
and [3.1-II]
##STR00210##
[0627] To 3.1g-I (42 mg, 0.102 mmol) in Acetonitrile (Volume: 1,
Ratio: 1.000)-Water (Volume: 1, Ratio: 1.000), Lithium hydroxide
(15 mg, 0.357 mmol) was added and stirred at rt. After 1 hr, the
crude was acidified with 1M Sulfuric acid in 100 ml water (until
red on pH paper) and extracted into DCM and concentrated. The
organics were lyophilized to give 32.5 mg of 3.1-I in 79% yield.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.89 (s, 1H), 7.44 (d,
J=8.51 Hz, 1H), 7.23-7.33 (m, 2H), 6.74 (d, J=7.53 Hz, 1H), 5.17
(d, J=14.53 Hz, 1H), 4.85-5.02 (m, 2H), 4.07 (s, 3H), 3.27-3.42 (m,
2H), 1.03 (s, 3H), 0.36 (s, 3H). LC-MS (m/z): 384.3
[M+H].sup.+,0.69 min. To 3.1g-II (42 mg, 0.102 mmol) in
Acetonitrile (Volume: 1, Ratio: 1.000)-Water (Volume: 1, Ratio:
1.000), Lithium hydroxide (15 mg, 0.357 mmol) was added and stirred
at rt. After 1 hr, the crude was acidified with 1M Sulfuric acid in
100 ml water (until red on pH paper) and extracted into DCM and
concentrated. The organics were lyophilized to give 32.5 mg of
3.1-II in 79% yield. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.89
(s, 1H), 7.44 (d, J=8.46 Hz, 1H), 7.21-7.33 (m, 2H), 6.74 (d,
J=7.48 Hz, 1H), 5.17 (br d, J=14.48 Hz, 1H), 4.85-5.02 (m, 2H),
4.07 (s, 3H), 3.27-3.41 (m, 2H), 0.99-1.20 (m, 3H), 0.35 (s, 3H).
LC-MS (m/z): 384.3 [M+H].sup.+,0.69 min.
Example 4.1:
10-methoxy-6-(1-methoxy-2-methylpropan-2-yl)-2-oxo-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [4.1-I] and
[4.1-II]
##STR00211##
[0628] Step 1: 1-bromo-4-methoxy-3,3-dimethylbutan-2-one
##STR00212##
[0630] To 4-methoxy-3,3-dimethylbutan-2-one (9.81 g, 75 mmol) in
200 ml MeOH (Volume: 250 mL) cooled to -78.degree. C., Br2 (4.27
mL, 83 mmol) in 50 ml was added dropwise and stirred to rt
overnight. Saturated Sodium bicarbonate was added until the
solution is basic. The organics are extracted into DCM, dried with
Sodium Sulfate and concentrated to give 4.1a assume in quantitative
yield. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 4.24 (s, 2H)
3.35 (s, 2H) 3.31 (s, 3H) 1.21 (s, 6H)
Step 2:
1-(3-(((tert-butyldimethylsilyl)oxy)methyl)-7-methoxy-2H-indazol-2-
-yl)-4-methoxy-3,3-dimethylbutan-2-one [4.1b]
##STR00213##
[0632] To 1.1b (1.5 g, 5.13 mmol) and 4.1a (2.7 g, 12.91 mmol) in
DMSO (Volume: 47.9 ml, Ratio: 1.000))-Water (Volume: 3.36 ml,
Ratio: 0.07), cesium carbonate (6.68 g, 20.52 mmol) was added and
stirred at rt overnight. The crude was extracted into DCM and
concentrated. EtOAc was then added to the crude and washed with
brine to remove any DMSO and purified by silica gel chromatography
using Heptane-EtOAc (0-50% EtOAc) to give after concentration 572
mg of 4.1b (more polar on ISCO) in 26.5% yield. LC-MS (m/z): 421.4
[M+H].sup.+,1.26 min.
Step 3:
(2-(2-amino-4-methoxy-3,3-dimethylbutyl)-7-methoxy-2H-indazol-3-yl-
)methanol [4.1c]
##STR00214##
[0634] To 4.1b (572 mg, 1.360 mmol) in MeOH (Volume: 7), ammonium
acetate (1048 mg, 13.60 mmol) and sodium cyanoborohydride (0.9 g,
14.32 mmol) were added and stirred at 70.degree. C. for 3 days. The
crude was diluted with DCM and washed with saturated sodium
bicarbonate and the organics concentrated. The organics containing
4.1c were taken to the next step without further purification,
assume quantitative yield. LC-MS (m/z): 308.4 [M+H].sup.+,0.53
min.
Step 4:
7-methoxy-3-(1-methoxy-2-methylpropan-2-yl)-3,4-dihydropyrazino[1,-
2-b]indazole [4.1d]
##STR00215##
[0636] To 4.1c (0.418 g, 1.36 mmol) in DCM (Volume: 20 mL), MnO2
(4.3 g, 49.5 mmol) was added and stirred overnight. The crude was
filtered through celite and the celite washed with DCM. The organic
layers were concentrated and the crude containing 4.1 d was taken
to the next step, assume quantitative yield. LC-MS (m/z): 288.3
[M+H].sup.+,0.60 min.
Step 5: Ethyl
10-methoxy-6-(1-methoxy-2-methylpropan-2-yl)-2-oxo-1,6,7,13c-tetrahydro-2-
H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [4.1e]
##STR00216##
[0638] 4.1d (0.391 g, 1.36 mmol) and ethyl
(Z)-2-(ethoxymethylene)-3-oxobutanoate (0.760 g, 4.08 mmol) was
heated in EtOH (Volume: 6.80 ml) at 100.degree. C. overnight. The
crude containing 4.1e was concentrated and taken to the next step,
assume quantitative yield. LC-MS (m/z): 428.4 [M+H].sup.+,0.78
min.
Step 6: Ethyl
10-methoxy-6-(1-methoxy-2-methylpropan-2-yl)-2-oxo-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [4.1f-I] and
[4.1f-II]
##STR00217##
[0640] To 4.1e (1210 mg, 2.83 mmol) in DME (Volume: 20 mL),
Chloramine-T (834 mg, 3.40 mmol) was added and heated at reflux for
2 hrs. The crude was concentrated and redissolved in DCM-Methanol
(minimum amount) and purified by silica gel chromatography first
using Heptane-EtOAc (0-100%) to remove all non-polar SM/By-products
and then purified with DCM-Methanol (0-20% MeOH) to give after
concentration 130 mg of 4.1f in 10.8% yield. LC-MS (m/z): 426.4
[M+H].sup.+,0.77 min. The above racemic material 130 mg was
separated by chiral chromatography using (OD column, SFC=100
ml/min, CO.sub.2/MeOH=65/35, 266 bar) to give products 4.1f-I (peak
1, tR 2.04 min.) at 38 mg and product 4.1f-II (peak 2, tR 4.47
min.) at 37 mg.
Step 7:
10-methoxy-6-(1-methoxy-2-methylpropan-2-yl)-2-oxo-6,7-dihydro-2H--
pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [4.1-I]
and [4.1-II]
##STR00218##
[0642] To 4.1f-I (38 mg, 0.089 mmol) was added Acetonitrile
(Volume: 5 ml, Ratio: 1.000), Water (Volume: 5 ml, Ratio: 1.000)
and then LiOH (11.24 mg, 0.268 mmol). The reaction was stirred at
room temperature for 2 hours or until done by LCMS. The reaction
was diluted with 20 ml of water acidified with 1M HCl to pH of 2-3,
then 100 ml of DCM with 5% ethanol was added and 15 ml of saturated
sodium chloride solution. The organic layer was separated, the
aqueous layer was extracted again, 50 ml of DCM with 5% ethanol.
The organic layers were combined, washed with water, saturated
sodium chloride solution, dried sodium sulfate, filtered through
sodium sulfate plug 1 cm.times.1.5 cm and concentrated to residue.
The residue was dissolved in 1:1 ACN/water and lyophilized to give
29 mg of the desired product 4.1-I as free base in 78% yield. LC-MS
(m/z): 398.4 [M+H].sup.+, 0.79 min; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.69 (s, 1H), 7.45 (d, J=8.46 Hz, 1H),
7.17-7.36 (m, 2H), 6.74 (d, J=7.53 Hz, 1H), 5.12 (d, J=14.92 Hz,
1H), 4.91 (dd, J=5.45, 14.94 Hz, 1H), 4.73 (d, J=5.23 Hz, 1H), 4.07
(s, 3H), 3.37 (s, 3H), 3.02 (d, J=9.93 Hz, 1H), 2.93 (d, J=9.93 Hz,
1H), 1.03 (s, 3H), 0.36 (s, 3H). To 4.1f-II (37 mg, 0.087 mmol) was
added Acetonitrile (Volume: 5 ml, Ratio: 1.000), Water (Volume: 5
ml, Ratio: 1.000) and then LiOH (10.95 mg, 0.261 mmol). The
reaction was stirred at room temperature for 2 hours or until done
by LCMS. The reaction was diluted with 20 ml of water acidified
with 1M HCl to pH of 2-3, then 100 ml of DCM with 5% ethanol was
added and 15 ml of saturated sodium chloride solution. The organic
layer was separated, the aqueous layer was extracted again, 50 ml
of DCM with 5% ethanol. The organic layers were combined, washed
with water, saturated sodium chloride solution, dried sodium
sulfate, filtered through sodium sulfate plug 1 cm.times.1.5 cm and
concentrated to residue. The residue was dissolved in 1:1 ACN/water
and lyophilized to give 26 mg of the desired product 4.1-II as free
base in 71.5% yield. LC-MS (m/z): 398.4 [M+H].sup.+, 0.79 min;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.69 (s, 1H), 7.45 (d,
J=8.46 Hz, 1H), 7.17-7.36 (m, 2H), 6.74 (d, J=7.53 Hz, 1H), 5.12
(d, J=14.92 Hz, 1H), 4.92 (dd, J=5.43, 14.87 Hz, 1H), 4.73 (d,
J=5.18 Hz, 1H), 4.07 (s, 3H), 3.37 (s, 3H), 3.02 (d, J=9.98 Hz,
1H), 2.93 (d, J=9.93 Hz, 1H), 1.03 (s, 3H), 0.36 (s, 3H).
Example 5.1: 12-methoxy-3,3-dimethyl-7-oxo-2,3,3a,4a-tetrahydro-1H,
7H-cyclopenta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-6-carboxylic
acid [5.1-I] and [5.1-II]
##STR00219##
[0643] Step 1: 5-bromo-2,2-dimethylcyclopentan-1-one [5.1a]
##STR00220##
[0645] To 2,2-dimethylcyclopentan-1-one (25 g, 223 mmol) in MeOH
(Volume: 446 ml), Bromine (12.63 ml, 245 mmol) was added at crushed
ice temperature and stirred overnight. The crude was washed with
sat. Sodium bicarbonate and extracted into DCM, dried with
anhydrous sodium sulfate and concentrated to give 5.1a, assume in
quantitative yield. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm
4.30 (dd, J=6.92, 4.77 Hz, 1H) 2.30-2.52 (m, 1H) 2.05-2.24 (m, 2H)
1.74-1.88 (m, 1H) 1.15-1.28 (s, 3H) 1.07 (s, 3H)
Step 2:
5-(3-(((tert-butyldimethylsilyl)oxy)methyl)-7-methoxy-2H-indazol-2-
-yl)-2,2-dimethylcyclopentan-1-one [5.1b]
##STR00221##
[0647] To 5.1a (5.41 g, 28.3 mmol) and 1.1b (2.07 g, 7.08 mmol) in
DMSO (Volume: 25 ml, Ratio: 0.833)-Water (Volume: 1.5 mL, Ratio:
0.05), cesium carbonate (13.84 g, 42.5 mmol) was added and stirred
at rt overnight. The crude was extracted into DCM and concentrated.
EtOAc was added and washed with brine to remove any DMSO and
purified by silica gel chromatography using Heptane-EtOAc (0-50%
EtOAc) to give after concentration the desired product, 5.1b (1.14
g, 2.83 mmol, 40.0% yield) is the more polar product eluting around
40-50% EtOAc. LC-MS (m/z): 403.5 [M+H].sup.+,1.30 min.
Step 3:
(2-(2-amino-3,3-dimethylcyclopentyl)-7-methoxy-2H-indazol-3-yl)met-
hanol [5.1c]
##STR00222##
[0649] To 5.1b (1.14 g, 2.83 mmol) in MeOH (Volume: 15 ml),
ammonium acetate (2.183 g, 28.3 mmol) and sodium cyanoborohydride
(0.9 g, 14.32 mmol) were added and stirred at 70.degree. C. for 3
days. The crude was diluted with DCM and washed with saturated
sodium bicarbonate and the organics concentrated. The organics
containing 5.1c were taken to the next step without further
purification, assume quantitative yield. LC-MS (m/z): 290.4
[M+H].sup.+,0.61 min.
Step 4: 9-methoxy-3,3-dimethyl-2,3,3a,
11a-tetrahydro-1H-cyclopenta[5,6]pyrazino[1,2-b]indazole [5.1d]
##STR00223##
[0651] To 5.1c (0.819 g, 2.83 mmol) in DCM (Volume: 30 mL), MnO2
(5.5 g, 63.3 mmol) was added and stirred overnight. The crude was
filtered through celite and the celite washed with DCM. The organic
layers were concentrated and the crude containing 5.1d was taken to
the next step, assume quantitative yield. LC-MS (m/z): 270.3
[M+H].sup.+,0.61 min.
Step 5: Ethyl
12-methoxy-3,3-dimethyl-7-oxo-2,3,3a,8,8a,14a-hexahydro-1H,7H-cyclopenta[-
5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-6-carboxylate
[5.1e]
##STR00224##
[0653] 5.1d (0.762 g, 2.83 mmol) and ethyl
(Z)-2-(ethoxymethylene)-3-oxobutanoate (1.581 g, 8.49 mmol) was
heated in EtOH (Volume: 14.15 ml) at 120.degree. C. overnight. The
crude containing 5.1e was concentrated and taken to the next step,
assume quantitative yield. LC-MS (m/z): 410.4 [M+H].sup.+,0.78
min.
Step 6: Ethyl 12-methoxy-3,3-dimethyl-7-oxo-2,3,3a,
14a-tetrahydro-1H,7H-cyclopenta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indaz-
ole-6-carboxylate [5.1f-I] and [5.1f-II]
##STR00225##
[0655] To 5.1e (1159 mg, 2.83 mmol) in DME (Volume: 30 mL),
Chloramine-T (834 mg, 3.40 mmol) was added and heated at reflux for
2 hrs. The crude was concentrated and redissolved in DCM-Methanol
(minimum amount) and purified by silica gel chromatography first
using Heptane-EtOAc (0-100%) to remove all non-polar SM/By-products
and then purified with DCM-Methanol (0-20% MeOH) to give after
concentration 243 mg of 5.1f in 21% yield. LC-MS (m/z): 408.4
[M+H].sup.+,0.86 min. The above racemic material 243 mg was
separated by chiral chromatography using (SFC=100 ml/min,
CO.sub.2/MeOH=70/30, 246 bar) to give products 5.1f-I (peak 1, tR
3.58 min.) at 82 mg and product 5.1f-II (peak 2, tR 5.40 min.) at
66 mg.
Step 7:
12-methoxy-3,3-dimethyl-7-oxo-2,3,3a,14a-tetrahydro-1H,7H-cyclopen-
ta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-6-carboxylic acid
[5.1-I] and [5.1-II]
##STR00226##
[0657] To 5.1f-I (82 mg, 0.201 mmol) was added Acetonitrile
(Volume: 5 ml, Ratio: 1.000), Water (Volume: 5 ml, Ratio: 1.000)
and then LiOH (25.3 mg, 0.604 mmol). The reaction was stirred at
room temperature for 2 hours or until done by LCMS. The reaction
was diluted with 20 ml of water acidified with 1M HCl to pH of 2-3,
then 100 ml of DCM with 5% ethanol was added and 15 ml of saturated
sodium chloride solution. The organic layer was separated, the
aqueous layer was extracted again, 50 ml of DCM with 5% ethanol.
The organic layers were combined, washed with water, saturated
sodium chloride solution, dried sodium sulfate, filtered through
sodium sulfate plug 1 cm.times.1.5 cm and concentrated to residue.
The residue was dissolved in 1:1 ACN/water and lyophilized to give
51 mg of the desired product 5.1-I as free base in 63.5% yield.
LC-MS (m/z): 380.4 [M+H].sup.+, 0.83 min; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.51 (s, 1H), 7.50 (br d, J=8.41 Hz, 1H),
7.20-7.41 (m, 2H), 6.75 (br d, J=7.29 Hz, 1H), 5.24 (br s, 1H),
4.43 (br d, J=5.92 Hz, 1H), 4.08 (s, 3H), 3.23-3.44 (m, 1H), 2.53
(br dd, J=6.53, 14.60 Hz, 1H), 1.73-1.90 (m, 1H), 1.51-1.73 (m,
1H), 1.35 (s, 3H), 0.48 (s, 3H). To 5.1f-II (66 mg, 0.162 mmol) was
added Acetonitrile (Volume: 5 ml, Ratio: 1.000), Water (Volume: 5
ml, Ratio: 1.000) and then LiOH (20.39 mg, 0.486 mmol). The
reaction was stirred at room temperature for 2 hours or until done
by LCMS. The reaction was diluted with 20 ml of water acidified
with 1M HCl to pH of 2-3, then 100 ml of DCM with 5% ethanol was
added and 15 ml of saturated sodium chloride solution. The organic
layer was separated, the aqueous layer was extracted again, 50 ml
of DCM with 5% ethanol. The organic layers were combined, washed
with water, saturated sodium chloride solution, dried sodium
sulfate, filtered through sodium sulfate plug 1 cm.times.1.5 cm and
concentrated to residue. The residue was dissolved in 1:1 ACN/water
and lyophilized to give 53 mg of the desired product 5.1-II as free
base in 82% yield. LC-MS (m/z): 380.3 [M+H].sup.+, 0.82 min;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.51 (s, 1H), 7.49 (br d,
J=8.51 Hz, 1H), 7.21-7.41 (m, 2H), 6.75 (br d, J=7.48 Hz, 1H), 5.24
(br t, J=5.55 Hz, 1H), 4.43 (br d, J=6.36 Hz, 1H), 4.08 (s, 3H),
3.26-3.41 (m, 1H), 2.53 (br dd, J=6.55, 14.77 Hz, 1H), 1.81 (ddd,
J=5.40, 8.36, 13.33 Hz, 1H), 1.51-1.73 (m, 1H), 1.35 (s, 3H), 0.48
(s, 3H).
Example 5.2
12-fluoro-3,3-dimethyl-7-oxo-2,3,3a,14a-tetrahydro-1H,
7H-cyclopenta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-6-carboxylic
acid [5.2-1] and [5.2-II]
##STR00227##
[0658] Step 1 to 6: Ethyl 12-fluoro-3,3-dimethyl-7-oxo-2,3,3a,
14a-tetrahydro-1H,7H-cyclopenta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indaz-
ole-6-carboxylate [5.2f-I] and [5.2f-II]
##STR00228##
[0660] Compound 5.2f was synthesized from the starting material;
3-(((tert-butyldimethylsilyl)oxy)methyl)-7-fluoro-2H-indazole
(which is from methyl 7-fluoro-2H-indazole-3-carboxylate analogous
to method used for preparing 1.1b) by the process of Example 5.1
following steps 1 through 6 resulting in 310 mg of 5.2f as a
racemate in 27.7% yield. LC-MS (m/z): 396.5 [M+H].sup.+, 0.91 min.
The above racemic material (310 mg) was separated by chiral
chromatography using (OD column, SFC=100 ml/min,
CO.sub.2/MeOH80/20, 226 bar) to give products 5.2f-I (peak 1, tR
3.95 min.) at 110 mg and product 5.2f-II (peak 2, tR 6.11 min.) at
105 mg.
Step 7: 12-fluoro-3,3-dimethyl-7-oxo-2,3,3a,
14a-tetrahydro-1H,7H-cyclopenta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indaz-
ole-6-carboxylic acid [5.2-I] and [5.2-II]
##STR00229##
[0662] To 5.2f-I (105 mg, 0.266 mmol) was added Acetonitrile
(Volume: 4 ml, Ratio: 1.000), Water (Volume: 4 ml, Ratio: 1.000)
and then LiOH (33.4 mg, 0.797 mmol). The reaction was stirred at
room temperature for 2 hours or until done by LCMS. The reaction
was diluted with 20 ml of water acidified with 1M HCl to pH of 2-3,
then 100 ml of DCM with 5% ethanol was added and 15 ml of saturated
sodium chloride solution. The organic layer was separated, the
aqueous layer was extracted again, 50 ml of DCM with 5% ethanol.
The organic layers were combined, washed with water, saturated
sodium chloride solution, dried sodium sulfate, filtered through
sodium sulfate plug 1 cm.times.1.5 cm and concentrated to residue.
The residue was dissolved in 1:1 ACN/water and lyophilized to give
7 mg of the desired product 5.2-I as free base in 6.8% yield. LC-MS
(m/z): 368.3 [M+H].sup.+, 0.83 min; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. ppm 8.52 (s, 1H) 7.73 (d, J=8.51 Hz, 1H)
7.30-7.40 (m, 2H) 7.13 (dd, J=10.78, 7.56 Hz, 1H) 5.16-5.37 (m, 1H)
4.45 (d, J=6.50 Hz, 1H) 3.21-3.41 (m, 1H) 2.54 (dtd, J=14.80, 8.59,
8.59, 6.16 Hz, 1H) 1.84 (ddd, J=13.61, 8.72, 5.06 Hz, 1H) 1.64 (dt,
J=13.55, 8.27 Hz, 1H) 1.36 (s, 3H) 0.48 (s, 3H). To 5.2f-II (110
mg, 0.278 mmol) was added Acetonitrile (Volume: 4 ml, Ratio:
1.000), Water (Volume: 4 ml, Ratio: 1.000) and then LiOH (35 mg,
0.835 mmol). The reaction was stirred at room temperature for 2
hours or until done by LCMS. The reaction was diluted with 20 ml of
water acidified with 1M HCl to pH of 2-3, then 100 ml of DCM with
5% ethanol was added and 15 ml of saturated sodium chloride
solution. The organic layer was separated, the aqueous layer was
extracted again, 50 ml of DCM with 5% ethanol. The organic layers
were combined, washed with water, saturated sodium chloride
solution, dried sodium sulfate, filtered through sodium sulfate
plug 1 cm.times.1.5 cm and concentrated to residue. The residue was
dissolved in 1:1 ACN/water and lyophilized to give 7 mg of the
desired product 5.2-II as free base in 6.5% yield. LC-MS (m/z):
368.3 [M+H].sup.+, 0.83 min; .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. ppm 8.51 (s, 1H) 7.73 (d, J=8.51 Hz, 1H) 7.27-7.38 (m, 2H)
7.07-7.25 (m, 1H) 5.26 (t, J=5.75 Hz, 1H) 4.43 (d, J=6.50 Hz, 1H)
3.26-3.41 (m, 1H) 2.45-2.64 (m, 1H) 1.84 (ddd, J=13.58, 8.69, 5.06
Hz, 1H) 1.59-1.70 (m, 1H) 1.36 (s, 3H) 0.48 (s, 3H).
Example 6.1
12-(3-methoxypropoxy)-3,3-dimethyl-7-oxo-2,3,3a,14a-tetrahydro-1H,7H-cyclo-
penta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-6-carboxylic
acid [6.1-I] and [6.1-II]
##STR00230##
[0663] Step 1 to 6: Ethyl 12-(benzyloxy)-3,3-dimethyl-7-oxo-2,3,3a,
14a-tetrahydro-1H,7H-cyclopenta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indaz-
ole-6-carboxylate [6.1f]
##STR00231##
[0665] Compound 6.1f was synthesized from the starting material;
2.1b by the process of Example 5.1 following steps 1 through 6
resulting in the desired product 6.1f as a racemate. LC-MS (m/z):
484.4 [M+H].sup.+, 0.99 min.
Step 7: Ethyl 12-hydroxy-3,3-dimethyl-7-oxo-2,3,3a,
14a-tetrahydro-1H,7H-cyclopenta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indaz-
ole-6-carboxylate [6.1 g]
##STR00232##
[0667] To 6.1 f (315 mg, 0.651 mmol) in EtOH (Volume: 25 ml),
nitrogen was purged followed by addition of 10% Pd--C(350 mg, 0.329
mmol) and purged with nitrogen again. A hydrogen balloon was then
attached and the reaction mixture purged with hydrogen. The crude
was stirred for 1 hour and filtered on celite and purged with
DCM-Ethanol. The crude was concentrated to yield crude 262 mg of
6.1g in quantitative yield. LC-MS (m/z): 394.4 [M+H].sup.+, 0.69
min.
Step 8: Ethyl
12-(3-methoxypropoxy)-3,3-dimethyl-7-oxo-2,3,3a,14a-tetrahydro-1H,7H-cycl-
openta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-6-carboxylate
[6.1 h-I] and [6.1 h-II]
##STR00233##
[0669] To 6.1g (150 mg, 0.381 mmol) in DMF (Volume: 5 mL),
1-bromo-3-methoxypropane (0.083 mL, 0.763 mmol) and cesium
carbonate (373 mg, 1.144 mmol) were added and stirred. The crude
was filtered and rinsed with EtOAc and washed with sat. brine to
remove any DMF. The crude was purified by silica gel chromatography
using Heptane-(EtOAc:EtOH=75:25) (0-100%) to give after
concentration 99 mg of the desired product 6.1h in 55.8% yield,
which was given for chiral purification. LC-MS (m/z): 466.5
[M+H].sup.+, 0.84 min.
[0670] The above racemic material (99 mg) was separated by chiral
chromatography using (OD column, SFC=100 ml/min, CO2/MeOH=75/25,
246 bar) to give products 6.1h-I (peak 1, tR 5.24 min.) at 41 mg
and product 6.1h-II (peak 2, tR 7.61 min.) at 38 mg.
Step 9:
12-(3-methoxypropoxy)-3,3-dimethyl-7-oxo-2,3,3a,14a-tetrahydro-1H,-
7H-cyclopenta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-6-carboxylic
acid [6.1-I] and [6.1-II]
##STR00234##
[0672] To 6.1h-I (41 mg, 0.088 mmol) was added Acetonitrile
(Volume: 2 ml, Ratio: 1.000), Water (Volume: 1 ml, Ratio: 1.000)
and then LiOH (11.09 mg, 0.264 mmol). The reaction was stirred at
room temperature for 2 hours or until done by LCMS. The reaction
was diluted with 20 ml of water acidified with 1M HCl to pH of 2-3,
then 100 ml of DCM with 5% ethanol was added and 15 ml of saturated
sodium chloride solution. The organic layer was separated, the
aqueous layer was extracted again, 50 ml of DCM with 5% ethanol.
The organic layers were combined, washed with water, saturated
sodium chloride solution, dried sodium sulfate, filtered through
sodium sulfate plug 1 cm.times.1.5 cm and concentrated to residue.
The residue was dissolved in 1:1 ACN/water and lyophilized to give
19 mg of the desired product 6.1-I as free base in 46.8% yield.
LC-MS (m/z): 438.4 [M+H].sup.+, 0.88 min; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.48 (s, 1H), 7.40-7.57 (m, 1H), 7.25-7.34 (m,
2H), 6.78 (d, J=7.58 Hz, 1H), 5.23 (t, J=5.70 Hz, 1H), 4.29-4.44
(m, 3H), 3.55-3.71 (m, 2H), 3.29-3.40 (m, 4H), 2.38-2.64 (m, 1H),
2.24 (quin, J=6.32 Hz, 2H), 1.81 (ddd, J=5.14, 8.66, 13.60 Hz, 1H),
1.53-1.68 (m, 1H), 1.34 (s, 3H), 0.48 (s, 3H). To 6.1h-II (38 mg,
0.082 mmol) was added Acetonitrile (Volume: 2 ml, Ratio: 1.000),
Water (Volume: 1 ml, Ratio: 1.000) and then LiOH (10.28, 0.245
mmol). The reaction was stirred at room temperature for 2 hours or
until done by LCMS. The reaction was diluted with 20 ml of water
acidified with 1M HCl to pH of 2-3, then 100 ml of DCM with 5%
ethanol was added and 15 ml of saturated sodium chloride solution.
The organic layer was separated, the aqueous layer was extracted
again, 50 ml of DCM with 5% ethanol. The organic layers were
combined, washed with water, saturated sodium chloride solution,
dried sodium sulfate, filtered through sodium sulfate plug 1
cm.times.1.5 cm and concentrated to residue. The residue was
dissolved in 1:1 ACN/water and lyophilized to give 19 mg of the
desired product 6.1-II as free base in 50.5% yield. LC-MS (m/z):
438.4 [M+H].sup.+, 0.89 min; .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.49 (s, 1H), 7.47 (d, J=8.56 Hz, 1H), 7.24-7.34 (m, 2H),
6.78 (d, J=7.58 Hz, 1H), 5.23 (t, J=5.89 Hz, 1H), 4.28-4.44 (m,
3H), 3.53-3.74 (m, 2H), 3.29-3.40 (m, 4H), 2.38-2.65 (m, 1H), 2.24
(quin, J=6.32 Hz, 2H), 1.81 (ddd, J=5.23, 8.66, 13.60 Hz, 1H),
1.54-1.68 (m, 1H), 1.34 (s, 3H), 0.48 (s, 3H)
Example 6.2
12-(2-methoxyethoxy)-3,3-dimethyl-7-oxo-2,3,3a,14a-tetrahydro-1H,
7H-cyclopenta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-6-carboxylic
acid [6.2-1] and [6.2-II]
##STR00235##
[0673] Step 1: Ethyl
12-(2-methoxyethoxy)-3,3-dimethyl-7-oxo-2,3,3a,14a-tetrahydro-1H,7H-cyclo-
penta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-6-carboxylate
[6.2h-1] and [6.2h-II]
##STR00236##
[0675] To 6.1g (150 mg, 0.381 mmol) in DMF (Volume: 3 mL),
1-bromo-2-methoxyethane (0.076 mL, 0.763 mmol) and cesium carbonate
(373 mg, 1.144 mmol) were added and stirred. The crude was filtered
and rinsed with EtOAc and washed with sat. brine to remove any DMF.
The crude was purified by silica gel chromatography using
Heptane-(EtOAc:EtOH=75:25) (0-100%) to give after concentration 61
mg of 6.2h in 35.4% yield. LC-MS (m/z): 452.4 [M+H].sup.+, 0.80
min.
[0676] The above racemic material (61 mg) was separated by chiral
chromatography using (AD column, SFC=100 ml/min,
CO.sub.2/MeOH=70/30, 236 bar) to give products 6.2h-I (peak 1, tR
2.93 min.) at 28 mg and product 6.2h-II (peak 2, tR 4.91 min.) at
29 mg.
Step 2:
12-(2-methoxyethoxy)-3,3-dimethyl-7-oxo-2,3,3a,14a-tetrahydro-1H,7-
H-cyclopenta[5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-6-carboxylic
acid [6.2-I] and [6.2-II]
##STR00237##
[0678] To 6.2h-I (28 mg, 0.062 mmol) was added Acetonitrile
(Volume: 2 ml, Ratio: 1.000), Water (Volume: 1 ml, Ratio: 1.000)
and then LiOH (7.81 mg, 0.186 mmol). The reaction was stirred at
room temperature for 2 hours or until done by LCMS. The reaction
was diluted with 20 ml of water acidified with 1M HCl to pH of 2-3,
then 100 ml of DCM with 5% ethanol was added and 15 ml of saturated
sodium chloride solution. The organic layer was separated, the
aqueous layer was extracted again, 50 ml of DCM with 5% ethanol.
The organic layers were combined, washed with water, saturated
sodium chloride solution, dried sodium sulfate, filtered through
sodium sulfate plug 1 cm.times.1.5 cm and concentrated to residue.
The residue was dissolved in 1:1 ACN/water and lyophilized to give
15 mg of the desired product 6.2-I as free base in 54.3% yield.
LC-MS (m/z): 424.4 [M+H].sup.+, 0.82 min; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.48 (s, 1H), 7.42-7.55 (m, 1H), 7.25-7.33 (m,
2H), 6.77 (d, J=7.63 Hz, 1H), 5.23 (t, J=5.70 Hz, 1H), 4.30-4.48
(m, 3H), 3.86-3.99 (m, 2H), 3.49 (s, 3H), 3.21-3.43 (m, 1H), 2.51
(dt, J=8.46, 14.77 Hz, 1H), 1.80 (ddd, J=5.16, 8.68, 13.60 Hz, 1H),
1.49-1.68 (m, 1H), 1.34 (s, 3H), 0.47 (s, 3H). To 6.2h-II (29 mg,
0.064 mmol) was added Acetonitrile (Volume: 2 ml, Ratio: 1.000),
Water (Volume: 1 ml, Ratio: 1.000) and then LiOH (8.09 mg, 0.193
mmol). The reaction was stirred at room temperature for 2 hours or
until done by LCMS. The reaction was diluted with 20 ml of water
acidified with 1M HCl to pH of 2-3, then 100 ml of DCM with 5%
ethanol was added and 15 ml of saturated sodium chloride solution.
The organic layer was separated, the aqueous layer was extracted
again, 50 ml of DCM with 5% ethanol. The organic layers were
combined, washed with water, saturated sodium chloride solution,
dried sodium sulfate, filtered through sodium sulfate plug 1
cm.times.1.5 cm and concentrated to residue. The residue was
dissolved in 1:1 ACN/water and lyophilized to give 15 mg of the
desired product 6.2-II as free base in 52.4% yield. LC-MS (m/z):
424.4 [M+H].sup.+, 0.82 min; .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 8.48 (s, 1H), 7.48 (d, J=8.51 Hz, 1H), 7.25-7.34 (m, 2H),
6.76 (d, J=7.58 Hz, 1H), 5.23 (br t, J=5.77 Hz, 1H), 4.32-4.48 (m,
3H), 3.84-4.01 (m, 2H), 3.49 (s, 3H), 3.24-3.42 (m, 1H), 2.51 (br
dd, J=6.24, 14.79 Hz, 1H), 1.80 (ddd, J=5.14, 8.68, 13.57 Hz, 1H),
1.63-1.71 (m, 1H), 1.33 (s, 3H), 0.46 (s, 3H).
Example 7.1
6-(1-methoxy-2-methylpropan-2-yl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro--
2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid
[7.1-I] and [7.1-II]
##STR00238##
[0679] Step 1:
1-(7-(benzyloxy)-3-(((tert-butyldimethylsilyl)oxy)methyl)-2H-indazol-2-yl-
)-4-methoxy-3,3-dimethylbutan-2-one [7.1b]
##STR00239##
[0681] To 2.1b (7.5 g, 20.35 mmol) in DIPEA (35.5 ml, 204 mmol),
4.1a (14.44 g, 69.1 mmol) was added and heated with stirring for 4
hrs. at 110.degree. C. The crude was dissolved in 20 ml DCM and
washed with 0.723 ml Sulfuric Acid in water and the organics
concentrated. The organics were purified by silica gel
chromatography using Heptane-EtOAc (0-50%). The desired
regio-isomer eluted second and was concentrated to give 5.503 g of
the desired product 7.1b in 57.3% yield. LC-MS (m/z): 497.5
[M+H].sup.+, 1.32 min.
Step 2:
(2-(2-amino-4-methoxy-3,3-dimethylbutyl)-7-(benzyloxy)-2H-indazol--
3-yl)methanol [7.1c]
##STR00240##
[0683] To 7.1b (5.5 g, 11.07 mmol) in MeOH (Volume: 40 ml)ammonium
acetate (10.5 g, 136 mmol) and sodium cyanoborohydride (3.48 g,
55.4 mmol) were added and stirred at 70.degree. C. for 3 days. The
crude was diluted with DCM and washed with saturated sodium
bicarbonate and the organics concentrated. The organics containing
7.1c were taken to the next step without further purification,
assume quantitative yield. LC-MS (m/z): 384.5 [M+H].sup.+, 0.70
min.
Step 3:
7-(benzyloxy)-3-(1-methoxy-2-methylpropan-2-yl)-3,4-dihydropyrazin-
o[1,2-b]indazole [7.1d]
##STR00241##
[0685] To 7.1c (4.25 g, 11.07 mmol) in DCM (Volume: 50 ml), MnO2
(9.62 g, 111 mmol) was added and stirred at rt overnight. The crude
was filtered and the collected MnO2 rinsed with DCM and
concentrated to yield crude 7.1 d used as is, assume quantitative
yield. LC-MS (m/z): 364.5 [M+H].sup.+, 0.91 min.
Step 4: Ethyl
10-(benzyloxy)-6-(1-methoxy-2-methylpropan-2-yl)-2-oxo-1,6,7,13c-tetrahyd-
ro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate
[7.1e]
##STR00242##
[0687] 7.1d (4.02 g, 11.07 mmol) and ethyl
(Z)-2-(ethoxymethylene)-3-oxobutanoate (8.25 g, 44.3 mmol) was
heated in EtOH (Volume: 55 ml) at 120.degree. C. overnight. The
crude containing 7.1e was concentrated and taken to the next step
as is, assume quantitative yield. LC-MS (m/z): 504.5 [M+H].sup.+,
0.95 min.
Step 5: Ethyl
10-(benzyloxy)-6-(1-methoxy-2-methylpropan-2-yl)-2-oxo-6,7-dihydro-2H-pyr-
ido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [7.1f]
##STR00243##
[0689] To 7.1e (5.57 g, 11.07 mmol) was added DME (Volume: 80 mL)
and then iodine (8.43 g, 33.2 mmol). The reaction was stirred at
35-40.degree. C. overnight. The reaction was diluted with 400 ml of
DCM, treated with saturated sodium bicarbonate solution carefully
to avoid heat and CO2 evolution, saturated sodium bisulfite to
remove residual iodine/HI & saturated salt solution. The
organics were separated and dried with anhydrous sodium sulfate,
filtered and concentrated to residue. The residue was purified by
silica gel chromatography using 0-100% Heptane/EtOAc-EtOH (75:25),
concentrated to residue to give 1.51g of 7.1 f in 27.2% yield.
LC-MS (m/z): 502.5 [M+H].sup.+, 0.95 min.
Step 6: Ethyl
10-hydroxy-6-(1-methoxy-2-methylpropan-2-yl)-2-oxo-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [7.1 g]
##STR00244##
[0691] To 7.1f (1.51 g, 3.01 mmol) in MeOH (Volume: 30.1 ml),
nitrogen was purged followed by addition of degussa 10% Pd--C(1.5
g, 1.410 mmol) and purged with nitrogen again. A hydrogen balloon
was then attached and the reaction mixture purged with hydrogen.
The crude was stirred for 1 hour and filtered on celite and rinsed
with DCM-Ethanol. The crude was concentrated to give 1.04g of 7.1 g
in 84% yield and taken to the next step without further
purification. LC-MS (m/z): 412.4 [M+H].sup.+, 0.66 min.
Step 7: Ethyl
6-(1-methoxy-2-methylpropan-2-yl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-
-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [7.1
h-I] and [7.1 h-II]
##STR00245##
[0693] To 7.1g (900 mg, 2.187 mmol) in DMF (Volume: 8750 .mu.l),
1-bromo-3-methoxypropane (478 .mu.l, 4.37 mmol) and cesium
carbonate (2138 mg, 6.56 mmol) were added and stirred. The crude
was purified by silica gel chromatography using 0-100%
heptane-(EtOAc/EtOH:75/25) to give after concentration 667 mg of
7.1h in 63% yield. LC-MS (m/z): 484.5 [M+H].sup.+, 0.79 min.
[0694] The above racemic material (667 mg) was separated by chiral
chromatography using (AD column, SFC=100 ml/min,
CO.sub.2/MeOH=75/25, 236 bar) to give products 7.1h-I (peak 1, tR
2.71 min.) at 279 mg and product 7.1h-II (peak 2, tR 5.07 min.) at
279 mg.
Step 8:
6-(1-methoxy-2-methylpropan-2-yl)-10-(3-methoxypropoxy)-2-oxo-6,7--
dihydro-2H-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic
acid [7.1-I] and [7.1-II]
##STR00246##
[0696] To 7.1h-I (279 mg, 0.577 mmol) was added Acetonitrile
(Volume: 20 ml, Ratio: 1.000), Water (Volume: 20 ml, Ratio: 1.000)
and then LiOH (72.6 mg, 1.731 mmol). The reaction was stirred at
room temperature for 2 hours or until done by LCMS. The reaction
was diluted with 20 ml of water acidified with 1M HCl to pH of 2-3,
then 100 ml of DCM with 5% ethanol was added and 15 ml of saturated
sodium chloride solution. The organic layer was separated, the
aqueous layer was extracted again, 50 ml of DCM with 5% ethanol.
The organic layers were combined, washed with water, saturated
sodium chloride solution, dried sodium sulfate, filtered through
sodium sulfate plug 1 cm.times.1.5 cm and concentrated to residue.
The residue was dissolved in 1:1 ACN/water and lyophilized to give
234 mg of the desired product 7.1-I as free base in 85% yield.
LC-MS (m/z): 456.5 [M+H].sup.+, 0.82 min. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.76 (s, 1H) 7.63 (br d, J=8.41 Hz, 1H)
7.12-7.34 (m, 2H) 6.82 (br d, J=7.63 Hz, 1H) 5.12-5.24 (m, 1H)
4.95-5.12 (m, 2H) 4.20 (brt, J=6.38 Hz, 2H) 3.45-3.63 (m, 2H) 3.25
(s, 3H) 2.95-3.09 (m, 4H) 2.81 (br d, J=9.93 Hz, 1H) 1.87-2.13 (m,
2H) 0.88 (s, 3H) 0.36 (s, 3H). To 7.1h-II (279 mg, 0.577 mmol) was
added Acetonitrile (Volume: 20 ml, Ratio: 1.000), Water (Volume: 20
ml, Ratio: 1.000) and then LiOH (72.6 mg, 1.731 mmol). The reaction
was stirred at room temperature for 2 hours or until done by LCMS.
The reaction was diluted with 20 ml of water acidified with 1M HCl
to pH of 2-3, then 100 ml of DCM with 5% ethanol was added and 15
ml of saturated sodium chloride solution. The organic layer was
separated, the aqueous layer was extracted again, 50 ml of DCM with
5% ethanol. The organic layers were combined, washed with water,
saturated sodium chloride solution, dried sodium sulfate, filtered
through sodium sulfate plug 1 cm.times.1.5 cm and concentrated to
residue. The residue was dissolved in 1:1 ACN/water and lyophilized
to give 242 mg of the desired product 7.1-II as free base in 87%
yield. LC-MS (m/z): 456.5 [M+H].sup.+, 0.82 min; .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. ppm 8.69 (s, 1H) 7.43 (d, J=8.51 Hz, 1H)
7.27-7.33 (m, 2H) 6.77 (d, J=7.58 Hz, 1H) 5.15 (d, J=14.87 Hz, 1H)
4.90 (dd, J=14.89, 5.31 Hz, 1H) 4.72 (d, J=5.38 Hz, 1H) 4.33 (t,
J=6.65 Hz, 2H) 3.62 (t, J=5.82 Hz, 2H) 3.37 (s, 6H) 3.02 (d, J=9.93
Hz, 1H) 2.93 (d, J=9.98 Hz, 1H) 2.25 (quin, J=6.32 Hz, 2H) 1.04 (s,
3H) 0.37 (s, 3H).
Example 8.1
6-(tert-butyl)-1-fluoro-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[-
2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [8.1-I] and
[8.1-II]
##STR00247##
[0697] Step 1: (Z)-ethyl
2-(ethoxymethylene)-4,4-difluoro-3-((trimethylsilyl)oxy)but-3-enoate
[8.1a]
##STR00248##
[0699] Under an argon atmosphere, a mixture of magnesium powder
(1.214 g, 50 mmol) and trimethylsilyl chloride (6.34 mL, 50 mmol))
was sonicated prior to the reaction for 15-20 minutes. Then a
solution of ethyl
(Z)-2-(ethoxymethylene)-4,4,4-trifluoro-3-oxobutanoate (1.5 g, 6.25
mmol) in dry DMF (3 ml) was added dropwise over 5-6 minutes at
50.degree. C. under an argon atmosphere. The reaction mixture was
stirred for additional 30 min. at 50.degree. C. The crude mixture
was then filtered through a disposable filter funnel with
polyethylene frit. The resulting DMF solution with the desired
product 8.1a assume in quantitative yield, used as is.
Step 2: Ethyl
10-(benzyloxy)-6-(tert-butyl)-1-fluoro-2-oxo-6,7-dihydro-2H-pyrido[2',1':-
3,4]pyrazino[1,2-b]indazole-3-carboxylate [8.1 b]
##STR00249##
[0701] To 2.1e (420 mg, 1.260 mmol), crude 8.1a (1.513 g, 6.30
mmol) from above was added. Acetonitrile (Volume: 5 mL) and zinc
iodide (402 mg, 1.260 mmol) was then added and the crude refluxed
overnight at 50-60.degree. C. The crude was purified by silica gel
chromatography using 0 to 100% heptane-(EtOAc:EtOH=75:25) and the
desired fractions were concentrated to give 210 mg of the desired
product 8.1b in 34% yield. LC-MS (m/z): 490.4 [M+H].sup.+, 1.00
min.
Step 3: Ethyl
6-(tert-butyl)-1-fluoro-10-hydroxy-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,4]-
pyrazino[1,2-b]indazole-3-carboxylate [8.1c]
##STR00250##
[0703] To 8.1b (210 mg, 0.429 mmol) in Ethanol (Volume: 20 mL),
nitrogen was purged followed by addition of 10% Pd--C(210 mg, 1.973
mmol). Hydrogen gas was then bubbled through the solution with
venting followed by attaching a balloon of hydrogen and the
reaction stirred for 2 hrs. The crude was filtered on celite and
concentrated to yield crude 8.1c which was taken to the next step
without further purification, assume quantitative yield. LC-MS
(m/z): 400.4 [M+H].sup.+, 0.67 min.
Step 4: Ethyl
6-(tert-butyl)-1-fluoro-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido-
[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [8.1d-I] and [8.1
d-II]
##STR00251##
[0705] To 8.1c (103 mg, 0.258 mmol) in DMF (Volume: 1 mL),
1-bromo-3-methoxypropane (0.056 mL, 0.516 mmol) and cesium
carbonate (252 mg, 0.774 mmol) were added and stirred. The crude
was purified by silica gel chromatography using 0-100%
Heptane-(EtOAc/EtOH:75/25). The desired fractions were concentrated
to give 81 mg of the desired racemic product, 8.1d in 66% yield.
LC-MS (m/z): 472.5 [M+H].sup.+, 0.83 min. The above racemic
material (81 mg) was separated by chiral chromatography using (AD
column, SFC=100 ml/min, CO.sub.2/EtOH=70/30, 237 bar) to give
products 8.1d-I (peak 1, tR 2.77 min.) at 24 mg and product 8.1d-II
(peak 2, tR 5.52 min.) at 24 mg.
Step 5:
6-(tert-butyl)-1-fluoro-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-
-pyrido[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [8.1-I]
and [8.1-II]
##STR00252##
[0707] To 8.1d-I (24 mg, 0.051 mmol) was added Acetonitrile
(Volume: 3 ml, Ratio: 1.000), Water (Volume: 3 ml, Ratio: 1.000)
and then LiOH (6.41 mg, 0.153 mmol). The reaction was stirred at
room temperature for 2 hours or until done by LCMS. The reaction
was diluted with 20 ml of water acidified with 1M HCl to pH of 2-3,
then 100 ml of DCM with 5% ethanol was added and 15 ml of saturated
sodium chloride solution. The organic layer was separated, the
aqueous layer was extracted again, 50 ml of DCM with 5% ethanol.
The organic layers were combined, washed with water, saturated
sodium chloride solution, dried sodium sulfate, filtered through
sodium sulfate plug 1 cm.times.1.5 cm and concentrated to residue.
The residue was dissolved in 1:1 ACN/water and lyophilized to give
11 mg of the desired product 8.1-I as free base in 46.3% yield.
LC-MS (m/z): 444.4 [M+H].sup.+, 0.87 min. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.58 (s, 1H), 7.55 (dd, J=4.77, 8.63 Hz, 1H),
7.18-7.34 (m, 1H), 6.76 (d, J=7.58 Hz, 1H), 5.26 (d, J=14.87 Hz,
1H), 4.95 (dd, J=5.14, 14.87 Hz, 1H), 4.25-4.42 (m, 3H), 3.63 (t,
J=5.99 Hz, 2H), 3.38 (s, 3H), 2.25 (quin, J=6.27 Hz, 2H), 0.78-0.85
(m, 9H). To 8.1d-II (24 mg, 0.051 mmol) was added Acetonitrile
(Volume: 3 ml, Ratio: 1.000), Water (Volume: 3 ml, Ratio: 1.000)
and then LiOH (6.41 mg, 0.153 mmol). The reaction was stirred at
room temperature for 2 hours or until done by LCMS. The reaction
was diluted with 20 ml of water acidified with 1M HCl to pH of 2-3,
then 100 ml of DCM with 5% ethanol was added and 15 ml of saturated
sodium chloride solution. The organic layer was separated, the
aqueous layer was extracted again, 50 ml of DCM with 5% ethanol.
The organic layers were combined, washed with water, saturated
sodium chloride solution, dried sodium sulfate, filtered through
sodium sulfate plug 1 cm.times.1.5 cm and concentrated to residue.
The residue was dissolved in 1:1 ACN/water and lyophilized to give
11 mg of the desired product 8.1-II as free base in 46.3% yield.
LC-MS (m/z): 444.4 [M+H].sup.+, 0.87 min. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 8.59 (s, 1H), 7.55 (dd, J=4.77, 8.68 Hz, 1H),
7.18-7.34 (m, 1H), 6.76 (d, J=7.58 Hz, 1H), 5.25 (br d, J=14.77 Hz,
1H), 4.95 (br dd, J=3.99, 14.75 Hz, 1H), 4.27-4.40 (m, 3H), 3.63
(t, J=5.99 Hz, 2H), 3.38 (s, 3H), 2.25 (quin, J=6.27 Hz, 2H), 0.81
(s, 9H).
Example 9.1
(R)-6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1'-
:3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [9.1]
##STR00253##
[0708] Step 1: (R)-methyl
7-(benzyloxy)-2-(2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutyl)-2H-ind-
azole-3-carboxylate [9.1a]
##STR00254##
[0710] To methyl 7-(benzyloxy)-2H-indazole-3-carboxylate (18.5 g,
65.5 mmol) was added Triphenylphospine (20.63 g, 79 mmol),
(R)-tert-butyl (1-hydroxy-3,3-dimethylbutan-2-yl)carbamate (17.80
g, 82 mmol) and dissolved in THF (Volume: 185 mL). The reaction was
placed in a water bath and (E)-di-tert-butyl
diazene-1,2-dicarboxylate (18.11 g, 79 mmol) was added portion wise
to keep the internal temperature below 22.degree. C. The mixture
was stirred at room temperature for 3 hours or until done by LCMS.
The reaction was concentrated to residue. The crude residue was
dissolved in ethyl acetate with 20% heptane, washed with 0.5M HCl,
water, sodium bicarbonate, water, saturated salt solution, dried
with sodium sulfate, filtered and concentrated to residue. The
crude product was purified by silica gel chromatography using 0-25%
EtOAc in heptanes with the desired regio-isomer eluting first on
silica gel. The desired fractions were concentrated to constant
mass to give 27.3 grams of the desired product 9.1a in 87% yield.
LC-MS (m/z): 482.4 [M+H].sup.+, 1.15 min.
Step 2: (R)-methyl
2-(2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutyl)-7-hydroxy-2H-indazol-
e-3-carboxylate [9.1b]
##STR00255##
[0712] To 9.1a (27.3 g, 56.7 mmol) was added THF (Volume: 350 mL)
and flushed with nitrogen. Then Pd--C(3.02 g, 2.83 mmol) was added
carefully. Then to the reaction was added a hydrogen balloon. The
reaction was evacuated and refilled 7.times. with hydrogen and then
stirred at room temperature for 90 min, or until done by LCMS. To
the reaction was added 100 ml of DCM, flushed with nitrogen,
filtered through celite plug and flushed with THF. The solvent was
concentrated off to constant mass to give the desired product 9.1b
used as is for the next step, assume in quantitative yield. LC-MS
(m/z): 392.3 [M+H].sup.+, 0.91 min.
Step 3: (R)-methyl
2-(2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutyl)-7-(3-methoxypropoxy)-
-2H-indazole-3-carboxylate [9.1c]
##STR00256##
[0714] To 9.1b (22.2 g, 56.7 mmol) was added DMF (Volume: 3 mL) and
cesium carbonate (37.0 g, 113 mmol). The reaction was stirred at
room temperature for 5 minutes then 1-bromo-3-methoxypropane (13.02
g, 85 mmol) was added and the reaction was stirred at room
temperature for 3 hours or until done by LCMS. To the reaction was
added 800 ml of ethyl acetate with 20% heptane, washed with water
3.times., saturated salt solution, dried with sodium sulfate,
filtered and concentrated to residue. The crude material was
purified by silica gel chromatography using 0-45% ethyl acetate and
heptane. The desired fractions were concentrated to constant mass
to give 23.2 grams of the desired product 9.1c as a free base in
88% yield. LC-MS (m/z): 464.4 [M+H].sup.+, 1.03 min.
Step 4: (R)-tert-butyl
(1-(3-(hydroxymethyl)-7-(3-methoxypropoxy)-2H-indazol-2-yl)-3,3-dimethylb-
utan-2-yl)carbamate [9.1d]
##STR00257##
[0716] To 9.1c (23.2 g, 50.0 mmol) was added THF (Volume: 140 mL)
and then sodium borohydride (5.68 g, 150 mmol). The reaction was
heated to 60-65.degree. C. with stirring. Then methanol was added
slowly over 30-35 minutes and the reaction was stirred at
70-75.degree. C. for 3 hours or until done by LCMS. The reaction
was cooled to room temperature and placed in water bath for
cooling. Then carefully add 140 ml of saturated ammonium chloride
solution and then stirred for 45 minutes at room temperature. Then
750 ml of ethyl acetate was added and 70 ml of water, extracted,
the organic layer was washed with sat. bicarbonate solution, water,
saturated salt solution, dried with sodium sulfate, filtered and
concentrated to constant mass to give 21.4 grams of the desired
product 9.1d as a free base in 98% yield, used as is. LC-MS (m/z):
436.4 [M+H].sup.+, 0.83 min.
Step 5: (R)-tert-butyl
(1-(3-formyl-7-(3-methoxypropoxy)-2H-indazol-2-yl)-3,3-dimethylbutan-2-yl-
)carbamate [9.1e]
##STR00258##
[0718] To 9.1d (21.4 g, 49.1 mmol) was added DCM (Volume: 150 mL)
and then iodobenzene diacetate (22.16 g, 68.8 mmol). Then TEMPO
(0.768 g, 4.91 mmol) was added and the reaction was stirred at room
temperature for 3 hour or until done by LCMS. Then 700 ml of ethyl
acetate was added, washed with sodium thiosulfate, sodium carbonate
solution, water, saturated salt solution, dried sodium sulfate,
filtered and concentrated to residue. The crude material was
purified by silica gel chromatography using 0-50% ethyl acetate and
heptane. The desired fractions were concentrated to constant mass
to give 14.55 grams of the desired product 9.1e as a free base in
68% yield, used as is. LC-MS (m/z): 434.4 [M+H].sup.+, 0.96
min.
Step 6:
(R)-3-(tert-butyl)-7-(3-methoxypropoxy)-3,4-dihydropyrazino[1,2-b]-
indazole [9.1f]
##STR00259##
[0720] To 9.1e (14.55 g, 33.6 mmol) was added DCM (Volume: 180 mL)
and then TFA (38.8 mL, 503 mmol). The reaction was stirred at room
temperature for 1 hour or until done by LCMS. The reaction was
concentrated to residue to give the desired product 9.1f assume in
quantitative yield, used as is. LC-MS (m/z): 316.3 [M+H].sup.+,
0.66 min.
Step 7: (6R)-ethyl
6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-2,6,7,13c-tetrahydro-1H-pyrido-
[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylate [9.1 g]
##STR00260##
[0722] To 9.1f (10.59 g, 33.6 mmol) was added Ethanol (Volume: 110
mL, Ratio: 10.00), Water (Volume: 11 mL, Ratio: 1.000) and
(Z)-ethyl 2-(ethoxymethylene)-3-oxobutanoate (21.88 g, 118 mmol).
The reaction was then stirred at 90-95.degree. C. for 16 hours or
until done by LCMS. The reaction was concentrated to residue to
give the desired product 9.1g assume in quantitative yield, used as
is. LC-MS (m/z): 456.3 [M+H].sup.+, 0.78 min.
Step 8: (R)-ethyl
6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyrido[2',1':3,-
4]pyrazino[1,2-b]indazole-3-carboxylate [9.1 h]
##STR00261##
[0724] To 9.1g (15.3 g, 33.6 mmol) was added DME (Volume: 190 mL),
and then iodine (36.7 g, 144 mmol). The reaction was stirred at
35-40.degree. C. for 5 hours or until done by LCMS. The reaction
was diluted with 800 ml of DCM, washed with sodium bisulfite
solution 2.times., water, sodium carbonate solution, water,
saturated salt solution, dried with sodium sulfate, filtered and
concentrated to residue. The crude material was purified by silica
gel chromatography using 0-95% (ethyl acetate with 25% ethanol) and
heptane. The desired fractions were concentrated to constant mass
to give the desired crude product. To the residue was added 100 ml
of ethyl ether with 1% ethanol and heated to reflux with stirring
for 5 minutes, then was cooled to room temperature for 1 hour. The
solid was collected by filtration, washed with ethyl ether and
dried to constant mass. The crude product needed additional
purification by silica gel chromatography using 0-60% (DCM with 10%
ethanol) and DCM, the desired fractions were concentrated to
constant mass to give 7.71 grams of the desired product 9.1h as
free base in 51% yield, used as is. LC-MS (m/z): 454.3 [M+H].sup.+,
0.80 min. .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 8.52 (s, 1H)
7.50 (d, J=8.51 Hz, 1H) 7.17 (t, J=8.00 Hz, 1H) 6.72-6.85 (m, 2H)
5.06-5.12 (m, 1H) 4.98-5.05 (m, 1H) 4.69 (d, J=4.50 Hz, 1H)
4.14-4.28 (m, 4H) 3.51 (t, J=6.28 Hz, 2H) 3.25 (s, 3H) 2.04 (quin,
J=6.36 Hz, 2H) 1.26 (t, J=7.09 Hz, 3H) 0.68 (s, 9H).
Step 9:
(R)-6-(tert-butyl)-10-(3-methoxypropoxy)-2-oxo-6,7-dihydro-2H-pyri-
do[2',1':3,4]pyrazino[1,2-b]indazole-3-carboxylic acid [9.1]
##STR00262##
[0726] To 9.1h (7.71 g, 17.00 mmol) was added Acetonitrile (Volume:
120 ml, Ratio: 1.000), Water (Volume: 120 ml, Ratio: 1.000) and
then LiOH (51.0 ml, 51.0 mmol). The reaction was stirred at room
temperature for 2 hours or until done by LCMS. The reaction was
diluted with 400 ml of water acidified with 1M HCl to pH of 2-3,
then 800 ml of DCM with 3% ethanol was added and 200 ml of
saturated sodium chloride solution. The organic layer was
separated, the aqueous layer was extracted again 1.times.150 ml DCM
with 2% ethanol. The organic layers were combined, washed with
water 4.times.150 ml, filtered and concentrated to constant mass to
give 7.1 grams of the desired product 9.1 as free base in 99%
yield. The enantiomeric excess (e.e.) was determine by chiral
chromatography using AD column, SFC=5 mL/min, CO.sub.2/EtOH=75/25,
(with an enantiomer mixture separating at Rt 1.53 min. and Rt 2.21
min.) giving the desired product 9.1 at Rt 2.20 min., in 99% e.e.
LC-MS (m/z): 426.3 [M+H].sup.+, 0.81 min. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.95 (s, 1H) 7.63 (d, J=8.46 Hz, 1H)
7.15-7.32 (m, 2H) 6.82 (d, J=7.58 Hz, 1H) 5.14-5.24 (m, 1H)
5.04-5.13 (m, 1H) 4.95 (d, J=4.70 Hz, 1H) 4.20 (t, J=6.41 Hz, 2H)
3.51 (t, J=6.26 Hz, 2H) 3.25 (s, 3H) 2.01-2.08 (m, 2H) 0.69 (s,
9H). The product 9.1 can be converted to a
tris(hydroxymethyl)aminomethane (TRIS) salt by the following
procedure. To 9.1 (11.05 g, 26.0 mmol) was added Acetonitrile
(Volume: 185 ml, Ratio: 9.25) stirred at 50.degree. C. for 1 hour,
until full dissolved. Then
2-amino-2-(hydroxymethyl)propane-1,3-diol (3.15 g, 26.0 mmol) was
added and stirred for 3 hours at 50.degree. C. After 1 hour,
additional Acetonitrile (Volume: 20 ml, Ratio: 1.000) was added to
help stirring and continued at 50.degree. C. for a full 3 hours.
Then the solution was cooled to room temperature and stirred for 18
hours at room temperature. The solid was collected by filtration,
washed with ACN, dried under vacuum at 40.degree. C. for 24 hours
to give 12.82 grams of the desired product 9.1 as the TRIS salt in
89% yield. LC-MS (m/z): 426.3 [M+H].sup.+, 0.80 min. .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 8.93 (s, 1H), 7.63 (d, J=8.5
Hz, 1H), 7.25 (s, 1H), 7.22 (d, J=8.1 Hz, 1H), 6.82 (d, J=7.6 Hz,
1H), 5.15-5.22 (m, 1H), 5.05-5.13 (m, 1H), 4.94 (d, J=4.7 Hz, 1H),
4.34 (br s, 3H), 4.20 (t, J=6.4 Hz, 2H), 3.51 (t, J=6.3 Hz, 2H),
3.25 (s, 3H), 3.21 (s, 6H), 2.04 (quin, J=6.3 Hz, 2H), 0.69 (s,
9H).
Example 10.1
12-methoxy-3,3-dimethyl-7-oxo-1,3,3a,14a-tetrahydro-7H-furo[3',4':5,6]pyri-
do[2',1':3,4]pyrazino[1,2-b]indazole-6-carboxylic acid [10.1-I] and
[10.1-II]
##STR00263##
[0727] Step 1:
1-(3-(((tert-butyldimethylsilyl)oxy)methyl)-7-methoxy-2H-indazol-2-yl)-3--
hydroxy-3-methylbutan-2-one [10.1a]
##STR00264##
[0729] To a mixture of 1.1b (1.65 g, 5.64 mmol) and cesium
carbonate (7.35 g, 22.57 mmol) in DMSO (Volume: 19.89 ml, Ratio:
16) was added water (Volume: 1.243 ml, Ratio: 1.000). The mixture
was stirred 10 min, then 1-bromo-3-hydroxy-3-methylbutan-2-one
(2.043 g, 11.28 mmol) was added. After 30 min, 100 ml EtOAc was
added, and the organics washed with water, brine, dried over
Na.sub.2SO.sub.4, filtered, and concentrated. The material was
purified on SiO.sub.2 (0-60% EtOAc/Hep) to give after concentration
desired product 10.1a (0.8 g, 2.038 mmol, 36.1% yield) from the
more polar peak with the correct m/z by LC-MS. LC-MS (m/z): 393.4
[M+H].sup.+, 1.09 min.
Step 2:
4-(3-(((tert-butyldimethylsilyl)oxy)methyl)-7-methoxy-2H-indazol-2-
-yl)-2,2-dimethylfuran-3(2H)-one [10.1b]
##STR00265##
[0731] 10.1a (800 mg, 2.038 mmol) was dissolved in toluene (Volume:
2 mL). Bredereck's reagent (0.514 mL, 2.242 mmol) was added. The
flask was fitted with a reflux condenser, and heated to 100.degree.
C. At 1 h, the reaction was judged complete by LC-MS. The mixture
was cooled to rt and concentrated onto diatomaceous earth. The
crude material was purified on a 24 g SiO.sub.2 column (0->50%
EtOAc in heptanes) to provide after concentration desired product
10.1b (600 mg, 1.490 mmol, 73.1% yield) as a yellow oil from the
major peak. LC-MS (m/z): 403.5 [M+H].sup.+, 1.28 min.
Step 3:
4-(3-(((tert-butyldimethylsilyl)oxy)methyl)-7-methoxy-2H-indazol-2-
-yl)-2,2-dimethyldihydrofuran-3(2H)-one [10.1c]
##STR00266##
[0733] A 4 mL vial was charged with 10.1b (600 mg, 1.49 mmol) and
THF (Volume: 7450 .mu.l). The vial was cooled to -10.degree. C. in
an ice/brine bath. L-Selectride (1639 .mu.l, 1.639 mmol) was added
dropwise. The reaction was deemed complete by LC-MS at 20 min and
quenched with ammonium chloride, extracted with EtOAc, dried over
Na.sub.2SO.sub.4, filtered and concentrated. The crude material was
purified on SiO.sub.2 (0-60% EtOAc/Hep) to provide after
concentration desired product 10.1c (500 mg, 1.236 mmol, 83% yield)
as an oil. LC-MS (m/z): 405.4 [M+H].sup.+, 1.21 min.
Step 4:
9-methoxy-3,3-dimethyl-1,3,3a,11a-tetrahydrofuro[3',4':5,6]pyrazin-
o[1,2-b]indazole [10.1d]
##STR00267##
[0735] To 10.1c (230 mg, 0.568 mmol) in ethanol (Volume: 3 mL),
ammonium acetate (657 mg, 8.53 mmol) was added. The vial was sealed
and the mixture stirred at 60.degree. C. for 1 h. Sodium
cyanoborohydride (35.7 mg, 0.568 mmol) was added and stirring
continued for 1 h. Two more portions of sodium cyanoborohydride
were added over the next two hours. The crude mixture was diluted
with DCM and washed with 6 N NaOH to provide crude amine (420 mg,
50% pure). This material was taken up in THF (6 mL) and TBAF in THF
(1.2 mL, 1.2 mmol) was added. At 30 min, the reaction was diluted
with 1 N NaOH and extracted with DCM. The combined organics were
dried over Na.sub.2SO.sub.4, filtered and concentrated to provide
the hydroxyamine as a brown oil. LC-MS (m/z): 292.3 [M+H].sup.+,
0.66 min. The hydroxyamine was taken up in DCM (8.5 mL) and MnO2
(950 mg, 10.8 mmol) was added. The mixture was stirred for 20 h,
diluted with DCM, and passed through a plug of diatomaceous earth.
The solution was concentrated and the material was purified on 12 g
SiO.sub.2 (0->60% EtOAc/heptane). The peak starting at 30% EtOAc
was isolated as desired product 10.1d (120 mg, 51% yield) as a
white solid. LC-MS (m/z): 272.3 [M+H].sup.+, 0.68 min.
Step 5: ethyl 12-methoxy-3,3-dimethyl-7-oxo-1,3,3a,
14a-tetrahydro-7H-furo[3',4':5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-
-6-carboxylate [10.1e]
##STR00268##
[0737] A 4 mL vial was charged with 10.1d (120 mg, 0.44 mmol) in
water (1.8 ml). Ethyl (E)-2-(ethoxymethylene)-3-oxobutanoate (165
mg, 0.89 mmol) was added. The vial was sealed, purged with N2, and
heated to 80.degree. C. overnight. The mixture was extracted with
DCM, the combined organics dried over Na.sub.2SO.sub.4, filtered
and concentrated to provide an oil. The oil was taken up in DME
(1.8 mL) and p-chloroanil (217 mg) was added. The mixture was
heated at 100.degree. C. for 90 min. After cooling at rt, the
solvent was removed under streaming nitrogen to provide the desired
product 10.1e as a brown solid that was used without further
purification, assume quantitative yield. LC-MS (m/z): 410.5
[M+H].sup.+, 0.77 min.
Step 6: 12-methoxy-3,3-dimethyl-7-oxo-1,3,3a,
14a-tetrahydro-7H-furo[3',4':5,6]pyrido[2',1':3,4]pyrazino[1,2-b]indazole-
-6-carboxylic acid [10.1-I] and [10.1-II]
##STR00269##
[0739] To a solution of crude 10.1e (120 mg, 0.29 mmol) in THF (0.9
mL) was added aqueous LiOH (1 mL, 1.0 mmol) and stirred 1 hour. The
solution was acidified to pH 1 by adding 4.0 N HCl (aq.) and
extracted with DCM. The combined organic layers were dried over
Na.sub.2SO.sub.4 and concentrated to provide a brown solid, which
was purified first by achiral chromatography (Sunfire column
B-9m-1050, 0.1% TFA in H.sub.2O/MeCN, 75 mL/min), to give 59.9 mg
of racemic product 10.1 in 51% yield. 382.5 [M+H].sup.+, 0.88 min.
The above racemic product was purified by chiral chromatography (AS
column, SFC-UV15=5 mL/min, CO.sub.2/EtOH=75/25, crude in
MeOH+diethyl amine) to provide 10.1-I (17.7 mg, at 1.79 min
retention time) as a white solid and 10.1-II (13.6 mg, at 3.24 min.
retention time) as a white solid. .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. ppm 8.87 (s, 1H), 7.74 (d, J=8.5 Hz, 1H),
7.38 (s, 1H), 7.32 (dd, J=8.3, 7.6 Hz, 1H), 6.89 (d, J=7.6 Hz, 1H),
5.55 (dd, J=6.9, 3.3 Hz, 1H), 5.50 (d, J=6.6 Hz, 1H), 4.88 (d,
J=10.2 Hz, 1H), 4.42 (dd, J=10.9, 3.8 Hz), 3.98 (s, 3H), 1.47 (s,
3H), 0.56 (s, 3H). LC-MS (m/z): 382.5 [M+H].sup.+, 0.88 min.
Biological Examples
HBV Cell Line
[0740] HepG2-Clone42, a Tet-inducible HBV-expressing cell line with
a stably integrated 1.3mer copy of the HBV ayw strain, was
generated based on the Tet-inducible HepAD38 cell line with slight
modifications. Ladner S K, et al., AntimicrobialAgents and
Chemotherapy. 41(8):1715-1720 (1997). HepG2-Clone42 cells were
cultured in DMEM/F-12+Glutamax.TM. (Life Technologies, Carlsbad,
Calif., USA), supplemented with 10% fetal bovine serum (Life
Technologies), G-418 (Corning, Manassas, Va., USA) at a final
concentration of 0.5 mg/mL, and 5 .mu.g/mL Doxycycline (Sigma, St.
Louis, Mo., USA) and maintained in 5% CO2 at 37.degree. C.
HBsAg Assay
[0741] HepG2-Clone42 cells were seeded into black clear-bottom
96-well plates at a concentration of 6.0.times.10.sup.4 cells/well.
24 hours post-seeding, the cells were treated with 200 .mu.l/well
of media containing five-fold serial dilutions of compounds in
DMSO. DMSO alone was used as the no drug control. The final DMSO
concentration in all wells was 0.5%.
[0742] The HBsAg ELISA kit (Alpha Diagnostic International, San
Antonio, Tex., USE, Catalog #4110) was used to determine the level
(semi-quantitative) of secreted HBV sAg. The HBSAg ELISA assay was
performed following the manufacturer's protocol as described.
[0743] Step 1. Pipet 100 .mu.L each of compound or DMSO treated
samples into HBsAg ELISA plates. Seal plates and incubate at room
temp for 60 minutes.
[0744] Step 2. Aspirate samples and wash three times with Wash
Buffer. Dispense 100 .mu.L of antibody-HRP conjugate to each well.
Incubate at room temp for 30 minutes.
[0745] Step 3. Aspirate samples and wash three times with Wash
Buffer. Add 100 .mu.L of TMB Substrate to all wells and incubate 15
minutes at room temp.
[0746] Step 4. Dispense 100 .mu.L of Stop Solution to each well.
Measure absorbance of ELISA plate at 450 nm.
Dose Response Curves
[0747] Dose-response curves were generated and the EC.sub.50 value
was defined as the compound concentration at which HBsAg secretion
was reduced 50% compared to the DMSO control.
[0748] EC.sub.50 values were determined as follows:
[0749] Determine the percent of HBsAg secretion inhibition.
Calculate the percent inhibition on of HBsAg secretion inhibition
using the following equation:
100.times.(X.sub.C-M.sub.B)/(M.sub.D-M.sub.B)
[0750] where X.sub.C is the absorbance signal from compound-treated
well; M.sub.B is average absorbance signal (background signal) for
column 12 (no cells+HBsAg ELISA sample buffer) and M.sub.D is
average absorbance signal from DMSO-treated wells. Then calculate
EC.sub.50 values by non-linear regression using a four parameter
curve logistic equation.
[0751] The curve fit model employed is XLFit Dose Response One Site
Model 204: y=(A+((B-A)/(1+(10{circumflex over ( )}((C-x)*D)))))
where A is the minimum y value, B is the maximum y value, C is the
log EC50 value, and D is the slope factor. In the following
table+.gtoreq.100 nM; 100 nM>++.gtoreq.1 nM; 1 nM>+++
TABLE-US-00001 TABLE 1 In Vitro activity of selected compounds of
Formula (I). Compound HBsAg (nM) 1.1-I +++ 1.1-II + 1.2-I ++ 1.2-II
++ 1.3-I ++ 1.3-II ++ 1.4-I + 1.4-II ++ 1.5-I ++ 1.5-II + 2.1-I +++
2.1-II ++ 2.2-I + 2.2-II +++ 2.3 +++ 2.4 + 2.5 ++ 2.6 +++ 2.7 +++
2.8 +++ 2.9 +++ 2.10 +++ 2.11 +++ 2.12 ++ 2.13 ++ 2.14 +++ 2.15 +++
2.16 +++ 2.17 +++ 2.18 ++ 2.19 +++ 2.20 ++ 2.21 +++ 2.22 ++ 2.23 ++
2.24 + 2.25 ++ 2.26 ++ 2.27 + 2.28 + 2.29 +++ 2.30 ++ 2.31 +++ 2.32
+ 2.33-I + 2.33-II + 2.34-I + 2.34-II + 2.35-I ++ 2.35-II ++ 2.36
+++ 2.37 ++ 2.38 ++ 2.39 +++ 2.40 +++ 2.41 +++ 2.42 +++ 2.43 + 2.44
++ 2.45 ++ 2.46 ++ 2.47-I + 2.47-II +++ 3.1-I + 3.1-II ++ 4.1-I +++
4.1-II + 5.1-I +++ 5.1-II + 5.2-I ++ 5.2-II + 6.1-I + 6.1-II +++
6.2-I ++ 6.2-II +++ 7.1-I + 7.1-II +++ 8.1-I + 8.1-II ++ 9.1 +++
10.1-I + 10.1-II ++ 2.1i ++
* * * * *
References