Note: Descriptions are shown in the official language in which they were submitted.
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2'-FLUORO SUBSTITUTED CARBA-NUCLEOSIDE ANALOGS FOR
ANTIVIRAL TREATMENT
FIELD OF THE INVENTION
The invention relates generally to compounds with antiviral activity, more
particularly nucleosides active against Flaviviridae infections and most
particularly to
inhibitors of hepatitis C virus RNA-dependent RNA polymerase.
BACKGROUND OF THE INVENTION
Viruses comprising the Flaviviridae family comprise at least three
distinguishable genera including pestiviruses,.flaviviruses, and hepaciviruses
(Calisher, et al., J. Gen. Virol., 1993, 70, 37-43). While pest/viruses cause
many
economically important animal diseases such as bovine viral diarrhea virus
(BVDV),
classical swine fever virus (CSFV, hog cholera) and border disease of sheep
(BDV),
their importance in human disease is less well characterized (Mocnnig, V., et
al., Adv.
Vir. Res. 1992, 48, 53-98). Flaviviruses arc responsible for important human
diseases
such as dengue fever and yellow fever while hepaciviruses cause hepatitis C
virus
infections in humans. Other important viral infections caused by the
Flaviviridae
family include West Nile virus (WNV) Japanese encephalitis virus (JEV), tick-
borne
encephalitis virus, Junjin virus, Murray Valley encephalitis, St Louis
encephalitis,
Omsk hemorrhagic fever virus and Zika virus. Combined, infections from the
Flaviviridae virus family cause significant mortality, morbidity and economic
losses
throughout the world. Therefore, there is a need to develop effective
treatments for
Flaviviridue virus infections.
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The hepatitis C virus (HCV) is the leading cause of chronic liver disease
worldwide (Boyer, N. et al. Hepatol. 32:98-112, 2000) so a significant focus
of
current antiviral research is directed toward the development of improved
methods of
treatment of chronic HCV infections in humans (Di Besceglie, A.M. and Bacon,
B.
R., Scientific American, Oct.: 80-85, (1999); Gordon, C. P., et al., J. Med.
Chem.
2005, 48, 1-20; Maradpour, D.; et al., Nat. Rev. Micro. 2007, 5(6), 453-463).
A
number of HCV treatments are reviewed by Bymock et al. in Antiviral Chemistry
&
Chemotherapy, 11:2; 79-95 (2000).
RNA-dependent RNA polymerase (RdRp) is one of the best studied targets for
the development of novel HCV therapeutic agents. The NS5B polymerase is a
target
for inhibitors in early human clinical trials (Sommadossi, J., WO 01/90121 A2,
US
2004/0006002 Al). These enzymes have been extensively characterized at the
biochemical and structural level, with screening assays for identifying
selective
inhibitors (De Clercq, E. (2001) J. Pharmacol. Exp.Ther. 297:1-10; De Clercq,
E.
(2001) J. Clin. Virol. 22:73-89). Biochemical targets such as NS5B are
important in
developing HCV therapies since HCV does not replicate in the laboratory and
there
are difficulties in developing cell-based assays and preclinical animal
systems.
Currently, there are primarily two antiviral compounds, ribavirin, a
nucleoside
analog, and interferon-alpha (a) (IFN), that are used for the treatment of
chronic HCV
infections in humans. Ribavirin alone is not effective in reducing viral RNA
levels,
has significant toxicity, and is known to induce anemia. The combination of
1FN and
ribavirin has been reported to be effective in the management of chronic
hepatitis C
(Scott, L. J., ct al. Drugs 2002, 62, 507-556) but less than half the patients
infected
with some genotypes show a persistent benefit when given this treatment. Other
patent applications disclosing the use of nucleoside analogs to treat
hepatitis C virus
include WO 01/32153, WO 01/60315, WO 02/057425, WO 02/057287, WO
02/032920, WO 02/18404, WO 04/046331, W02008/089105 and W02008/141079
but additional treatments for HCV infections have not yet become available for
patients.
2
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Virologic cures of patients with chronic HCV infection are difficult to
achieve
because of the prodigious amount of daily virus production in chronically
infected
patients and the high spontaneous mutability of HCV virus (Neumann, et al.,
Science
1998, 282, 103-7; Fukimoto, et al., Hepatology, 1996, 24, 1351-4; Domingo, et
al.,
Gene, 1985, 40, 1-8; Martell, et al., J. Virol, 1992, 66, 3225-9. Experimental
anti-
viral nucleoside analogs have been shown to induce viable mutations in the HCV
virus both in vivo and in vitro (Migliaccio, et al., J. Biol. Chem. 2003, 926;
Carroll, et
al., Antimicrobial Agents Chemotherapy 2009, 926; Brown, A. B., Expert Opin.
Investig. Drugs 2009, 18, 709-725). Therefore, drugs having improved antiviral
properties, particularly enhanced activity against resistant strains of virus;
improved
oral bioavailability; fewer undesirable side effects and extended effective
half-life in
vivo (De Francesco, R. et al. (2003) Antiviral Research 58:1-16) are urgently
needed.
Certain ribosidcs of the nueleobascs pyrrolo[1,2-f][1,2,4]triazine,
imidazo[1,5-
f][1,2,4]triazine, imidazo[1,2-f][1,2,4]triazine, and [1,2,4]triazo1o[4,3-
1][1,2,4]triazine
have been disclosed in Carbohydrate Research 2001, 331(1), 77-82; Nucleosides
&
Nucleotides (1996), 15(1-3), 793-807; Tetrahedron Letters (1994), 35(30), 5339-
42;
Heterocycles (1992), 34(3), 569-74; J. Chem. Soc. Perkin Trans. 11985, 3, 621-
30;
J. Chem. Soc. Perkin Trans. I 1984, 2, 229-38; WO 2000056734; Organic Letters
(2001), 3(6), 839-842; J. Chem, Soc. Perkin Trans. 1 1999, 20, 2929-2936; and
J.
Med. Chem. 1986, 29(11), 2231-5. However, these compounds have not been
disclosed as useful for the treatment of HCV.
Ribosides of pytTolo[1,2-f][1,2,4]triazinyl, imidazo[1,5-f][1,2,4]triazinyl,
imidazo[1,2-f][1,2,41triazinyl, and [1,2,4]triazolo[4,3-f][1,2,41triazinyl
nucleobases
with antiviral, anti-HCV, and anti-RdRp activity have been disclosed by Babu,
Y. S.,
W02008/089105 and W02008/141079; Cho, et al., W02009/132123 and Francom,
et al. W02010/002877. Butler, et al., W02009/132135, has disclosed anti-viral
pyrrolo[1,2-f][1,2,4]triazinyl, imidazo[1,5-f][1,2,4]triazinyl, imidazo[1,2-
f][1,2,4]triazinyl, and [1,2,4]triazolo[4,3-f][1,2,4]triazinyl nucleosides
wherein the l'
position of the nucleoside sugar is substituted.
3
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SUMMARY OF THE INVENTION
Provided are compounds that inhibit viruses of the Flaviviridae family. The
invention also comprises compounds of Formula I or Formula IV-VI that inhibit
viral
nucleic acid polyrnerases, particularly HCV RNA-dependent RNA polymerase
(RdRp), rather than cellular nucleic acid polymerases. The compounds of
Formula I
or Formula IV-VI have been discovered to be efficacious against both wild type
and
S282T mutant strains of HCV virus. Therefore, a compound of Formula I or
Formula
IV-VI are useful for treating Flaviviridae infections in humans and other
animals.
In one embodiment, provided are compounds of Foimula I:
R8
Xi
R7
X2
0 _________________________ CH2
0
R5
R3 Ri
R4 R2
Formula
or a pharmaceutically acceptable salt, thereof;
wherein:
R1 is (C1¨C8)alkyl, (C4¨C8)carbocyclylalkyl, (C1¨C8)substituted alkyl,
(C2¨C8)alkenyl, (C2¨C8)substituted alkcnyl, (C2¨C8)alkynyl, (C2¨C8)substituted
alkynyl, or aryl(Ci-C8)alkyl;
R2 is halogen;
each R3, R4, or R5 is independently H, ORB, N(Ra)1, N3, CN, NO2, S(0)õRa,
halogen, (C1¨C8)alkyl, (C4¨C8)earboeyelylalkyl, (C1¨C8)substituted alkyl,
(C2¨C8)alkenyl, (C2¨C8)substitutcd alkcnyl, (C7¨C8)alkynyl, (C2¨C8)substituted
alkynyl, or aryl(Ci-C8)alkyl;
4
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802.CIPF2
or any two of R3, R4 or R5 on adjacent carbon atoms when taken together are -
0(C0)0- or when taken together with the ring carbon atoms to which they are
attached form a double bond;
R6 is H, ORa, N(Ra)2, N3, CN, NO2, S(0)11Ra, -C(--0)R11, -C(=0)0R11, -
C(=0)NR11R12, -C(=0)SR11, -S(0)R11, -S(0)2R11, -S(0)(0R11). -S(0)2(0R11),
-SO2NRI1R12, halogen, (C1-Cs)alkyl, (C4-C8)carbocyclylalkyl, (C1-
C8)substituted
alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl,
(C2-C8)substituted alkynyl, or aryl(C1-C8)alkyl;
each n is independently 0; 1, or 2;
each Ra is independently H, (Ci-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl,
aryl (Ci-C8)alkyl, (C4-C8)carbocyclylalkyl, -C(=0)R11, -C(=0)0R11, -
C(=0)NRI1R125
-C(=0)SR11, -S(0)R11, -S(0)2R11, -S(0)(0R11), -S(0)2(0R11), or -SO2NRI IR12;
R7 is H, -C(--0)R11, -C(=0)0R11, -C(=0)NR11R12, -C(=0)SR11, -S(0)R", -
S(0)2R", -S(0)(0R11), -S(0)2(0R11), -SO2NR11R12, or
(I
W17P i
w2
= ,
each Y or Y1 is, independently, 0, S. NR,+N(0)(R), N(OR), +N(0)(0R), or
N-NR2;
WI and W2, when taken together, are -Y3(C(RY)2)3Y3-; or one of WI or W2
together with either R3 or R4 is -Y3- and the other of WI or W2 is Formula Ia;
or WI
and W2 are each, independently, a group of the Formula Ia:
(1
Y
1
Rx Y2 P y2 __
y2 i-
Rx /
M2
5
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802.CIPF2
Formula Ia
wherein:
each Y2 is independently a bond, 0, CR2, NR, +N(0)(R), N(OR), +N(0)(0R),
N-NR2, S, S-S, S(0), or S(0)2;
each Y3 is independently 0, S, or NR;
M2 is 0, 1 or 2;
each Rx is independently IV or the formula:
y 1
RY\ IRY
V RY
y2'
Mid
_
Ml2c\
Mid
Mia
wherein;
each Mla, Mle, and Mid is independently 0 or 1;
M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;
each RY is independently H, F, Cl, Br, I, 011, R, -C(=Y1)R, -C(=Y1)0R, -
C(Y1)N(R)2, -N(R)2, -+N(R)3, -SR, -S(0)R, -S(0)2R, -S(0)(0R), -S(0)2(0R),
-0C(=Y1)0R, -0C(=Y1)(N(R)2), -SC(=Y1)R, -SC(=Y1)0R, -
SC(=Y1)(NR)2), -N(R)C(=Y1)R, -N(R)C(=Y1)0R, -N(R)C(=Y1)N(R)2, -SO2NR2,
-CN, -N3, -NO2, -OR, or W3; or when taken together, two RY on the same carbon
atom form a earbocyclie ring of 3 to 7 carbon atoms;
each R is independently H, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-
C8)alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl, (C2-C8) substituted
alkynyl,
C6-C20 aryl, C6-C20 substituted aryl, C2-C20 heterocyclyl, C2-C20 substituted
heterocyclyl, arylalkyl or substituted arylalkyl;
W3 is W4 or W5; W4 is R, -C(Y1)RY, -C(Y1)W5, -SO2RY, or -S02W5; and W' is
carbocycle or a heterocycle wherein W5 is independently substituted with 0 to
3 RY
groups;
each X1 or X2 is independently C-R1 or N;
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each R8 is halogen, NRI1R12, N(RH)OR11, NRHNR11R12, N3, NO, NO2, CHO,
CN, .-CH(-NR"), -CH-NNHRH, -CH=N(OR11), -CH(OR11)2, -C(=0)NR11RI2,
-C(=S)NRHR12, -C(=0)0R11, (Ci-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl,
(C4-C8)carbocyclylalkyl, optionally substituted aryl, optionally substituted
heteroaryl,
-C8)alkyl, -S(0)(Ci-C8)alkyl, aryl(Ci-C8)alkyl, OR" or SR";
each R9 or R16 is independently H, halogen, NRI1R12, N(R11)0R11,
NRHNRI1R12, N -35
NO, NO2, CHO, CN, -CH(=NR11), -CH=NTINR11, -CH=N(OR11),
-CH(OR11)2, -C(=0)NR I I'sK 12,
C(=S)NRIIR12, C(=0)0R11, RH, ORI I or SR";
each RH or R12 is independently H, (CI -C8)alkyl, (C2-C8)alkenyl, (C2-
C8)alkynyl, (C4-C8)earbocyclylalkyl, optionally substituted aryl, optionally
substituted heteroaryl, -C(=0)(C1-C8)alkyl, -S(0),,(Ci-C8)alkyl or aryl(Ci-
C8)alkyl; or
RH and R12 taken together with a nitrogen to which they are both attached form
a 3 to
7 membered heterocyclic ring wherein any one carbon atom of said heterocyclic
ring
can optionally be replaced with -0-, -S- or -Nle-;
wherein each (CI-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or aryl(CI-C8)alkyl
of each R1, R3, R4, R5, R6, RH or R12 is, independently, optionally
substituted with one
or more halo, hydroxy, CN, N3, N(Ra)2 or Ole; and wherein one or more of the
non-
terminal carbon atoms of each said (CI-C8)alkyl may be optionally replaced
with -0-,
-S- or -NRa-.
In another embodiment, provided are compounds of Formula I or Formula IV-
.. VI and pharmaceutically acceptable salts thereof and all racemates,
enantiomers,
diastereomers, tautomers, polymorphs, pseudopolymorphs and amorphous forms
thereof.
In another embodiment, provided are novel compounds of Formula I or
Formula IV-VI with activity against infectious Flaviviridae viruses. Without
wishing
to be bound by theory, the compounds of the invention may inhibit viral RNA-
dependent RNA polymerase and thus inhibit the replication of the virus. They
are
useful for treating human patients infected with a human virus such as
hepatitis C.
7
,
,
In another embodiment, provided are pharmaceutical compositions comprising
an effective amount of a Formula I or a Formula IV-VI compound, or a
pharmaceutically acceptable salt thereof, in combination with a
pharmaceutically
acceptable diluent or carrier.
In one embodiment, the present application provides a compound which is:
NH2
NH2 z
N
-z-
=.,,,,0 = 0
='::r---µ.
;7/ 41-P-0 0 \ N, j 0 HN-P-0
3
O N O N
6
11
,J
,N.
NH2 NH2
N
0
N
(g 121N-A-o 0 \ N, .... j
oI N
oI N
40 .
6 -.
II 6- P-
-Ao
NH2
NH2
0
N
'II ________________ HN-P-0 0 \ NI, . j ''())/ 9
I N 0 HN-P-0 0 N, .......j
0
O N
C5- :F = :
0- F
, 5
or
NH2
0
) H
0 HN-P-0-v0 N,
oI N
II ,,,,.,..,\ 6
0
,
or a pharmaceutically acceptable salt, thereof.
8
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In another embodiment, the present application provides for combination
pharmaceutical agent comprising:
a) a first pharmaceutical composition comprising a compound of
Formula
I or Formula IV-VI; or a pharmaceutically acceptable salt, solvate, or ester
thereof;
and
b) a second pharmaceutical composition comprising at least one additional
therapeutic agent selected from the group consisting of interferons, ribavirin
or its
analogs, HCV NS3 protease inhibitors, NS5a inhibitors, alpha-glucosidase 1
inhibitors, hepatoprotectants, mevalonate decarboxylase antagonists,
antagonists of
the renin-angiotensin system, other anti-fibrotic agents, endothelin
antagonists,
nucleoside or nucleotide inhibitors of HCV NS5B polymerase, non-nucleoside
inhibitors of HCV NS5B polymerase, HCV NS5A inhibitors, TLR-7 agonists,
cyclophillin inhibitors, HCV IRES inhibitors, pharmacokinetic enhancers and
other
drugs for treating HCV; or mixtures thereof.
In another embodiment, the present application provides for a method of
inhibiting HCV polymerase, comprising contacting a cell infected with HCV with
an
effective amount of a compound of Formula I or Formula IV-VI; or a
pharmaceutically acceptable salts, solvate, and/or ester thereof.
In another embodiment, the present application provides for a method of
inhibiting HCV polymerase, comprising contacting a cell infected with HCV with
an
effective amount of a compound of Formula I or Formula IV-VI; or a
pharmaceutically acceptable salts, solvate, and/or ester thereof; and at least
one
additional therapeutic agent.
In another embodiment, the present application provides for a method of
treating and/or preventing a disease caused by a viral infection wherein the
viral
infection is caused by a virus selected from the group consisting of dengue
virus,
8a
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yellow fever virus, West Nile virus, Japanese encephalitis virus, tick-borne
encephalitis virus, Junjin virus, Murray Valley encephalitis virus, St Louis
encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus,
Zika
virus and Hepatitis C virus; by administering to a subject in need thereof a
therapeutically effective amount of a compound of Formula I or Formula IV-VI,
or a
pharmaceutically acceptable salt thereof
In another embodiment, the present application provides for a method of
treating HCV in a patient, comprising administering to said patient a
therapeutically
effective amount of a compound of Formula I or Formula IV-VI; or a
pharmaceutically acceptable salt, solvate, and/or ester thereof.
In another embodiment, the present application provides for a method of
treating HCV in a patient, comprising administering to said patient a
therapeutically
effective amount of a compound of Formula I or Formula IV-VI; or a
pharmaceutically acceptable salt, solvate, and/or ester thereof; and at least
one
additional therapeutic agent.
Another aspect of the invention provides a method for the treatment or
prevention of the symptoms or effects of an HCV infection in an infected
animal
which comprises administering to, i.e. treating, said animal with a
pharmaceutical
combination composition or formulation comprising an effective amount of a
Formula
I compound or Formula IV-VI, and a second compound having anti-HCV properties.
In another aspect, the invention also provides a method of inhibiting HCV,
comprising administering to a mammal infected with HCV an amount of a Formula
I
or Formula IV-VI compound, effective to inhibit the replication of HCV in
infected
cells in said mammal.
In another aspect, provided is the use of a compound of Formula I or Formula
IV-VI for the manufacture of a medicament for the treatment of F/aviviridae
viral
infections. In another aspect, provided is a compound of Formula I or Formula
IV-VI
for use in treating a Flaviviridae viral infection. In one embodiment, the
Flaviviridae
viral infection is acute or chronic HCV infection. In one embodiment of each
aspect
9
of use and compound, the treatment results in the reduction of one or more of
the viral
loads or clearance of RNA in the patient.
In one embodiment, there is provided a pharmaceutical composition,
comprising a compound as defined herein or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable carrier.
In another embodiment, there is provided the use of a compound defined
herein or a pharmaceutically acceptable salt thereof, for treating or
preventing a
Flaviviridae virus infection.
In another embodiment, there is provided the use of a compound as defined
herein or a pharmaceutically acceptable salt thereof, for treating a
Flaviviridae virus
.. infection.
In another embodiment, there is provided the use of a compound as defined
herein or a pharmaceutically acceptable salt thereof, for treating or
preventing a
Hepatitis C virus infection.
In another embodiment, there is provided the use of acompound as defined
herein or a pharmaceutically acceptable salt thereof, for treating a Hepatitis
C virus
infection.
In another embodiment, there is provided the use of a compound as defined
herein or a pharmaceutically acceptable salt thereof, for the manufacture of a
medicament for treating or preventing a Flaviviridae virus infection.
In another embodiment, there is provided the use of a compound as defined
herein or a pharmaceutically acceptable salt thereof, for the manufacture of a
medicament for treating a Flaviviridae virus infection.
In another embodiment, there is provided the use of a compound as defined
herein or a pharmaceutically acceptable salt thereof, for the manufacture of a
medicament for treating or preventing a Hepatitis C virus infection.
In another embodiment, there is provided the use of a compound as defined
herein or a pharmaceutically acceptable salt thereof, for the manufacture of a
medicament for treating a Hepatitis C virus infection.
In another embodiment, there is provided the use of a pharmaceutical
composition as defined herein, for treating or preventing a Flaviviridae virus
infection.
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In another embodiment, there is provided the use of a pharmaceutical
composition as defined herein, for treating a Flaviviridae virus infection.
In another embodiment, there is provided the use of a pharmaceutical
composition as defined herein, for treating or preventing a Hepatitis C virus
infection.
In another embodiment, there is provided the use of a pharmaceutical
composition as defined herein, for treating a Hepatitis C virus infection.
In another aspect, the invention also provides processes and novel
intermediates disclosed herein which are useful for preparing Formula I or
Formula
IV-VI compounds of the invention.
In other aspects, novel methods for synthesis, analysis, separation,
isolation,
purification, characterization, and testing of the compounds of this invention
are
provided.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
Reference will now be made in detail to certain embodiments of the invention,
examples of which are illustrated in the accompanying description, structures
and
formulas. While the invention will be described in conjunction with the
enumerated
embodiments, it will be understood that they are not intended to limit the
invention to
those embodiments. On the contrary, the invention is intended to cover all
alternatives,
modifications, and equivalents, which may be included within the scope of the
present
invention.
In another aspect, compounds of Formula I are represented by Formula II:
R8
R7 N
X2\
0 _______________________
0 R9
R3 ______________________________ R1
R4
10a
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Formula II
or a pharmaceutically acceptable salt, thereof;
wherein:
RI is (Ci-C8)alkyl, (C4-C8)carbocyclylalkyl, (Ci-C8)substituted alkyl,
(C2-C8)alkenyl, (C2-C8)substitute,d alkenyl, (C2-Cg)alkynyl, (C2-
Cs)substituted
alkynyl, or aryl(CI-C8)alkyl;
each R3, R4, or R5 is independently H, ORa, N(Ra)2, N3, CN, NO2, S(0)R11,
halogen, (Ci-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl,
(C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-COalkynyl, (C2-C8)substituted
alkynyl, or aryl(Ci-C8)a1ky1;
or any two of R3, R4 or R5 on adjacent carbon atoms when taken together are -
0(C0)0- or when taken together with the ring carbon atoms to which they are
attached form a double bond;
R6 is H, ORE, N(Ra)2, N3, CN, NO2, S(0)R', -C(-0)R11, -C(-0)0R11, -
C(-0)NRIIR12, -C(-0)SR11, -S(0)R11, -S(0)2R11, -S(0)(0R11), -S(0)2(0R11),
-SO2NR11R12, halogen, (Ci-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-
C8)substituted
alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl,
(C2-C8)substituted alkynyl, or aryl(C1-C8)alkyl;
each n is independently 0, 1, or 2;
each Ra is independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl,
aryl(C1-C8)alkyl, (C4-C8)carbocyclyla1kyl, -C(-0)R11, -C(=0)0R11, -
C(=0)NRI1R12,
-C(=0)SR11, -S(0)R11, -5(0)2R11, -S(0)(OR11), -S(0)2(0R11), or -SO2NR11R12;
R7 is H, -C(=0)R11, -C(=0)0R11, -C(=0)NR11R12, -C(=0)SR11, -S(0)R11, -
S(0)2R11, -S(0)(OR11), -S(0)2(OR11), -SO2NR11R12, or
W2
=
each Y or Y1 is, independently, 0, S, NR, +N(0)(R), N(OR), 1N(0)(OR), or
N-NR2;
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802.CIPF2
W1 and W2, when taken together, are -Y3(C(RY)2)3Y3-; or one of WI or W2
together with either R3 or R4 is -Y3- and the other of W1 or W2 is Formula la;
or W1
and W2 are each, independently, a group of the Formula Ia:
yl
________________________________ Y2 -P ______ y2 ___
y2
Rx
M2
Formula fa
wherein:
each Y2 is independently a bond, 0, CR2, NR, 'N(0)(R), N(OR), EN(0)(0R),
N-NR2, S, S-S, S(0), or S(0)2;
each Y3 is independently 0, S, or NR;
M2 is 0. 1 or 2;
each Rx is independently R or the formula:
_ yl _ yl
RY\ IRY
V
y2 y _
_ -M 1 2c\ / Mid
Mic
Mla
wherein:
each Mla, Mle, and Mid is independently 0 or 1;
Ml2c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;
each RY is independently H, F, Cl, Br, I, OH, R, -C(=Y1)R, -C(=-Y1)0R, -
C(=Y1)N(R)2, -N(R)2, -4N(R)3, -SR, -S(0)R, -S(0)2R, -S(0)(0R), -S(0)2(OR), -
OC(=Y1)R, -0C(=Y1)0R, -0C(=Y1)(N(R)2), -SC(=Y1)R, -SC(=Y1)0R, -
SC(=Y1)(N(R)2), -N(R)C(=Y1)R, -N(R)C(=Y1)0R, -N(R)C(----Y1)N(R)2, -SO2NR2,
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-CN, -N3, -NO2, -OR, or W3; or when taken together, two RY on the same carbon
atom form a carbocyclic ring of 3 to 7 carbon atoms;
each R is independently H, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-
C8)alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl, (C2-C8) substituted
alkynyl,
C6-C20 aryl, C6-C20 substituted aryl, C2-C20 heterocyclyl, C2-C20 substituted
heterocyclyl, arylalkyl or substituted arylalkyl;
W3 is W4 or W5; W4 is R, -C(Y1)R, -C(Y1)W5, -SO2RY, or -S02W5; and W5 is
a carbocycle or a heterocycle wherein W5 is independently substituted with 0
to 3 RY
groups;
each X1 or X2 is independently C-R1 or N;
each R8 is halogen, NR' R'2, N(R11)0RH, NR11NR11,-. 12,
K N3, NO, NO2, CHO,
CN, -CH(=NR11), -CH=NNHR11, -CH-N(0R1 1), -CH(OR11)2, -C(=0)NRI IR12,
-C( Ri2, =S)NR11 -C(0)OR' 1, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-
C8)alkynyl,
(C4-C8)carbocyclylalkyl, optionally substituted aryl, optionally substituted
heteroaryl,
-C(=0)(Ci-C8)alkyl, -S(0)n(CI-C8)a1kyl, aryl(Ci-C8)alkyl, OR11 or SR11;
each R9 or R16 is independently H, halogen, NR11-K 12,
N(R11)0RI I ,
NRI INRIIR12, N3, NO, NO2, CHO, CN, -CH(=NR11), -CH=NHNR11, -CH=N(ORI
-CH(OR11)2, -c(=.0)NR11-R 125
C(=S)NR11R12, -C(=--0)0R11, R", OR" or SR";
each R11 or R12 is independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-
C8)alkynyl, (C4-C8)carbocyclylalkyl, optionally substituted aryl, optionally
substituted heteroaryl, -C(=0)(Ci-C8)alky1, -S(0)(C1-C8)a1ky1 or aryl(Ci-
C8)alkyl; or
RH and R12 taken together with a nitrogen to which they are both attached form
a 3 to
7 membered heterocyclic ring wherein any one carbon atom of said heterocyclic
ring
can optionally be replaced with -0-, -S- or
wherein each (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or aryl(Ci-C8)alkyl
of each R1, R1, R4, R5, R6, R11 or R12 is, independently, optionally
substituted with one
or more halo, hydroxy, CN, N3, N(Ra), or Ole; and wherein one or more of the
non-
terminal carbon atoms of each said (C1-C8)alkyl may be optionally replaced
with -0-,
-S- or -NRa-.
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In one embodiment of the invention of Formula II, RI is (Ci¨C8)alkyl, (C2¨C8)
alkenyl or (C2¨C8)alkynyl. In another aspect of this embodiment, RI is
(C1¨Cg)alkyl.
In another aspect of this embodiment. RI is methyl, CH2F, or ethynyl. In
another
aspect of this embodiment, R1 is methyl. In another aspect of this embodiment,
RI is
(Ci¨C8)alkyl and R6 is H. In another aspect of this embodiment, RI is
(C1¨C8)alkyl
and at least one of XI or X2is N. In another aspect of this embodiment, RI is
(C1¨C8)alkyl and R6 is CN, OH, or CH3.
In one embodiment of Formula II, R3 is H, ORa, N(Ra),, N3, CN, SR', halogen,
(CI¨C8)alkyl, (C2¨C8)alkenyl or (C2¨C8)alkynyl. In one aspect of this
embodiment,
R3 is H. In another aspect of this embodiment, R3 is H and RI is (C1¨C8)alkyl,
(C7¨C8) alkenyl or (C2¨C8)alkynyl. In another aspect of this embodiment, R3 is
H
and RI is (Ci¨C8)alkyl. In another aspect of this embodiment, R3 is H and RI
methyl,
CH2F, or ethynyl. In another aspect of this embodiment, R3 is H and RI is
methyl. In
another aspect of this embodiment, R3 is H, RI is (Ci¨C8)alkyl and at least
one of X1
or X2 is N. In another aspect of this embodiment, R3 is H, RI is methyl and at
least
one of X1 or X2 is N. In another aspect of this embodiment, R3 is FI, RI is
(C1¨C8)alkyl and R6 is CN, OH, or CH3. In another aspect of this embodiment,
R3 is
H, RI is methyl and R6 is CN, OH, or CH3. In another aspect of this
embodiment, R3
is H, RI is methyl and R6 is H.
In one embodiment of Fonnula II, R4 is H, ORa, N(Ra),, N3, CN, SRa, halogen,
(Ci¨C8)alkyl, (C7¨C8)alkenyl or (C2¨C8)alkynyl. In another aspect of this
embodiment, R4 is H or ORE. hi another aspect of this embodiment, R4 is OR.
another aspect of this embodiment, R4 is ORa and III is (Ci¨C8)alkyl, (C2¨C8)
alkenyl
or (C2¨C8)alkynyl. In another aspect of this embodiment, R4 is ORa and RI is
(Ci¨C8)alkyl, (C2¨C8) alkenyl or (C2¨C8)alkynyl. In another aspect of this
embodiment, R4 is ORa and 111 is (C1¨C8)alkyl. In another aspect of this
embodiment,
R4 is Ole and RI is methyl. In another aspect of this embodiment, R4 is Ole,
RI is
(Ci¨C8)alkyl and at least one of XI or X2 is N. In another aspect of this
embodiment,
R4 is OR, RI is methyl and at least one of X1 or X2is N. In another aspect of
this
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embodiment, R4 is Ole, RI is (Ci¨C8)alkyl and R6 is CN, OH, or CH3 In another
aspect of this embodiment. R4 is ORa, R1 is methyl and R6 is CN, OH, or CH3.
In
another aspect of this embodiment, R4 is ORa, R1 is methyl and R6 is H. In
another
aspect of this embodiment, R4 is OH and R1 is methyl. In another aspect of
this
embodiment, R4 is Oli, R1 is (Ci¨C8)alkyl and at least one of X1 or X2 is N.
In
another aspect of this embodiment, R4 is OH, R1 is methyl and at least one of
X1 or X2
is N. In another aspect of this embodiment, R4 is OH, R1 is (Ci¨C8)alkyl and
R6 is
CN, OH, or CH3. In another aspect of this embodiment, R4 is OH, R1 is methyl
and R6
is CN, OH, or CH3. In another aspect of this embodiment, R4 is OH, R1 is
methyl and
R6 is H.
In one embodiment of Foimula II, R5 is H, ORa, N(Ra)7, N3, CN, Slaa, halogen,
(Ci¨C8)alkyl, (C2¨C8)alkenyl or (C2¨C8)alkynyl. In another aspect of this
embodiment, R4 is 1-1 or ORa. In another aspect of this embodiment, R4 is ORa.
In
another aspect of this embodiment, R4 is ORa and R1 is (C1¨C8)alkyl, (C2¨C8)
alkenyl
or (C2¨C8)alkynyl. In another aspect of this embodiment, R4 is ORa and R1 is
(Ci¨C8)alkyl, (C2¨C8) alkenyl or (C2¨C8)alkynyl. In another aspect of this
embodiment, R4 is ORa and RI is (C1¨C8)alkyl. In another aspect of this
embodiment,
R4 is ORa and R1 is methyl. In another aspect of this embodiment, R4 is ORa,
R1 is
(Ci¨C8)alkyl and at least one of X1 or X2 is N. In another aspect of this
embodiment,
R4 is Ole, R1 is methyl and at least one of X' or X2is N. In another aspect of
this
embodiment, R4 is Ole, R1 is (CI¨C8)alkyl and R6 is CN, OH, or CH3. In another
aspect of this embodiment. R4 is ORa, R1 is methyl and R6 is CN, OH, or CH3.
In
another aspect of this embodiment, R4 is ORa, R1 is methyl and R6 is H. In
another
aspect of this embodiment, R4 is OH and R1 is methyl. In another aspect of'
this
embodiment, R4 is OH, RI is (Ci¨C8)alkyl and at least one of X1 or X2 is N. In
another aspect of this embodiment, R4 is OH, R1 is methyl and at least one of
X1 or X2
is N. In another aspect of this embodiment, R4 is OH, R1 is (Ci¨C8)alkyl and
R6 is
CN, OH, or CH3 In another aspect of this embodiment, R4 is OH, R1 is methyl
and R6
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is CN, OH, or CH3. In another aspect of this embodiment, R4 is OH, RI is
methyl and
R6 is H. In another aspect of this embodiment, R5 is N3.
In another embodiment of Formula II, R5 is H. In another aspect of this
embodiment, R4 is H or Ole. In another aspect of this embodiment, R4 is ORa.
In
another aspect of this embodiment, R4 is ORa and RI is (Ci¨C8)alkyl, (C2¨C8)
alkenyl
or (C2¨C8)alkynyl. In another aspect of this embodiment, R4 is ORa and RI is
(C1¨C8)alkyl, (C2¨C8) alkenyl or (C2¨C8)alkynyl. In another aspect of this
embodiment, R4 is OR and RI is (Ci¨C8)alkyl. In another aspect of this
embodiment,
R4 is ORa and RI is methyl. In another aspect of this embodiment, R4 is ORa,
RI is
(C1¨C8)alkyl and at least one of XI or X2 is N. In another aspect of this
embodiment,
R4 is Ole, RI is methyl and at least one of XI or X2 is N. In another aspect
of this
embodiment, R4 is ORa, RI is (Ci¨C8)alkyl and R6 is CN, OH, or CH3 In another
aspect of this embodiment, R4 is ORE, RI is methyl and R6 is CN, OH, or CH3.
In
another aspect of this embodiment, R4 is ORa, RI is methyl and R6 is H. In
another
aspect of this embodiment, R4 is OH and RI is methyl. In another aspect of
this
embodiment, R4 is OH, RI is (CI¨C8)alkyl and at least one of Xi or X2 is N. In
another aspect of this embodiment, R4 is OH, RI is methyl and at least one of
XI or X2
is N. In another aspect of this embodiment, R4 is OH, R is (CI¨C8)alkyl and R6
is
CN, OH, or CH3. In another aspect of this embodiment, R4 is OH, RI is methyl
and R6
is CN. OH, or CH3. In another aspect of this embodiment, R4 is OH, RI is
methyl and
R6 is H.
In another embodiment of Formula II, R6 is H, CN, ORa or CH3. In another
aspect of this embodiment R6 is H. In another aspect of this embodiment R6 is
CN. In
another aspect of this embodiment R6 is ORa. In another aspect of this
embodiment le
is OH. In another aspect of this embodiment R6 is CH3. In another aspect of
this
embodiment, R4 is H or ORa. In another aspect of this embodiment, R4 is ORa.
In
another aspect of this embodiment, R4 is ORa and RI is (Ci¨C8)alkyl, (C2¨Cg)
alkenyl
or (C2¨C8)alkynyl. In another aspect of this embodiment, R4 is ORa and RI is
(C1¨C8)alkyl, (C2¨C8) alkenyl or (C2¨C8)alkynyl. In another aspect of this
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embodiment, R4 is ORa and RI is (CI-C8)alkyl. In another aspect of this
embodiment,
R4 is ORa and RI is methyl. In another aspect of this embodiment, R4 is ORa,
RI is
(CI-C8)alkyl and at least one of XI or X2is N. In another aspect of this
embodiment,
R4 is ORa, RI is methyl and at least one of XI or X2is N. In another aspect of
this
embodiment, R4 is Ole, RI is (C1-C8)alky1 and R6 is CN, OH, or CH3 In another
aspect of this embodiment, R4 is Ole, RI is methyl and R6 is CN, OH, or CH3.
In
another aspect of this embodiment, R4 is ORa, RI is methyl and R6 is H. In
another
aspect of this embodiment, R4 is 01-1 and RI is methyl. In another aspect of
this
embodiment, R4 is OH, RI is (CI-C8)alkyl and at least one of X1 or X2 is N. In
another aspect of this embodiment, R4 is OH, RI is methyl and at least one of
X1 or X2
is N. In another aspect of this embodiment, R4 is OH, RI is (C1--C8)alkyl and
R6 is
CN, OH, or CH3 In another aspect of this embodiment, R4 is OH, RI is methyl
and R6
is CN, OH, or CH3. In another aspect of this embodiment, R4 is OH, RI is
methyl and
R6 is H.
In another embodiment of Formula II, R6 is CN, ORa or CH3. In another
aspect of this embodiment R6 is CN. In another aspect of this embodiment R6 is
ORE.
In another aspect of this embodiment R6 is OH. In another aspect of this
embodiment
R6 is CH3. In another aspect of this embodiment, R4 is H or ORa. In another
aspect of
this embodiment, R4 is ORa. In another aspect of this embodiment, R4 is ORa
and R1
is (C1-C8)alkyl, (C2--C8) alkenyl or (G2-C8)alkynyl. In another aspect of this
embodiment, R4 is ORa and RI is (C1-C8)alkyl, (CT-Cs) alkcnyl or (C2-
C8)alkynyl. In
another aspect of this embodiment, R4 is ORa and RI is (Ci-C8)alkyl. In
another
aspect of this embodiment, R4 is ORa and RI is methyl. In another aspect of
this
embodiment, R4 is ORa, RI is (C1-C8)alkyl and at least one of X1 or X2 is N.
In
another aspect of this embodiment, R4 is ORa, RI is methyl and at least one of
XI or
X2is N. In another aspect of this embodiment, R4 is OR, RI is (CI-C8)alkyl and
R6 is
CN, OH, or CH3 In another aspect of this embodiment, R4 is ORa, RI is methyl
and le
is CN, OH, or CH3. In another aspect of this embodiment, R4 is OH and RI is
methyl.
In another aspect of this embodiment, R4 is OH, RI is (Ci-C8)alkyl and at
least one of
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X1 or X2 is N. In another aspect of this embodiment, R4 is OH, 121 is methyl
and at
least one of Xi or X2 is N. In another aspect of this embodiment, R4 is OH, R1
is
(C1¨C8)alkyl and R6 is CN, OH, or CH3 In another aspect of this embodiment, R4
is
OH, R1 is methyl and R6 is CN, OH, or CH3.
In one embodiment of Formula II, R7 is H, -C(=0)R11, -C(=0)0R11, -
C(-0)SR11 or
0
W2
. In a aspect of this embodiment, R7 is H. In another aspect of this
embodiment, R7 is -C(=0)R11. In another aspect of this embodiment, R7 is -
C(=0)RI I
wherein R11 is (C1-C8)alkyl. In another aspect of this embodiment, R7 is
0
wi
W2 6 i . In another aspect of
this embodiment R s H. In another aspect of
this embodiment R6 is CN. In another aspect of this embodiment R6 is ORa. In
another aspect of this embodiment R6 is OH. In another aspect of this
embodiment R6
is CH3. In another aspect of this embodiment, R4 is H or ORa. In another
aspect of
this embodiment, R4 is ORa. In another aspect of this embodiment, R4 is ORa
and R1
is (Ci¨C8)alkyl, (C2¨C8) alkenyl or (C2¨C8)alkynyl. hi another aspect of this
embodiment, R4 is ORa and R1 is (C1¨C8)alkyl. In another aspect of this
embodiment,
R4 is ORa and R1 is methyl. In another aspect of this embodiment, R4 is ORa,
R1 is
(Cr¨C8)alkyl and at least one of X1 or X2 is N. In another aspect of this
embodiment,
R4 is ORB, R1 is methyl and at least one of X1 or X2 is N. In another aspect
of this
embodiment, R4 is ORa, R1 is (C1¨C8)alkyl and R6 is CN, OH, or CH3 In another
aspect of this embodiment, R4 is ORa, R1 is methyl and R6 is CN, OH, or CH3.
In
another aspect of this embodiment, R4 is ORa, R1 is methyl and R6 is H. In
another
aspect of this embodiment, R4 is OH and R1 is methyl. In another aspect of
this
embodiment, R4 is OH, R1 is (Ci¨C8)allcyl and at least one of Xi or X2 is N.
In
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another aspect of this embodiment, R4 is OH, RI is methyl and at least one of
XI or X2
is N. In another aspect of this embodiment, R4 is OH, RI is (C1¨C8)alkyl and
R6 is
CN, OH, or CH3 In another aspect of this embodiment, R4 is OH, RI is methyl
and R6
is CN, OH, or CH3. In another aspect of this embodiment, R4 is OH, RI is
methyl and
R6 is H.
In one embodiment of Formula II, XI is N or C-121 . In another aspect of this
embodiment, Xl is N. In another aspect of this embodiment, XI is C-RIO. In
another
aspect of this embodiment, X2 is C-H. In another aspect of this embodiment, XI
is N
and X2 is C-11 In another aspect of this embodiment, XI is C-R' and X2 is CH.
In
another aspect of this embodiment R6 is H. In another aspect of this
embodiment R6 is
CN. In another aspect of this embodiment R6 is OR.a. In another aspect of this
embodiment R6 is OH. In another aspect of this embodiment R6 is CH3. In
another
aspect of this embodiment, R4 is H or OR". In another aspect of this
embodiment, R4
is OR". In another aspect of this embodiment, R4 is OR" and RI is
(CI¨C8)alkyl,
(C2¨C8) alkenyl or (C2¨C8)alkynyl. In another aspect of this embodiment, R4 is
OR"
and RI is (C1¨C8)alkyl. In another aspect of this embodiment, R4 is Ole and RI
is
methyl. In another aspect of this embodiment, R4 is ORa, RI is (Ca¨C8)alkyl
and at
least one of XI or X2 is N. In another aspect of this embodiment, R4 is Ole,
RI is
methyl and at least one of XI or X2 is N. In another aspect of this
embodiment, R4 is
OR", RI is (C1¨C8)alkyl and R6 is CN, OH, or CH3 In another aspect of this
embodiment, R4 is OR", RI is methyl and R6 is CN, OH, or CH3. In another
aspect of
this embodiment, R4 is OH and RI is methyl. In another aspect of this
embodiment,
R4 is OH, RI is (CI¨C8)alkyl and at least one of XI or X2 is N. In another
aspect of
this embodiment, R4 is OH, RI is methyl and at least one of X' or X2 is N. In
another
aspect of this embodiment, R4 is OH, RI is (C1¨C8)alkyl and R6 is CN, OH, or
CH3.
In another aspect of this embodiment, R4 is OH, RI is methyl and R6 is CN, OH,
or
CH3. In another aspect of this embodiment, R4 is OH. RI is methyl and R6 is H.
In another embodiment of Formula II, each Rg is independently halogen,
NR"R'2,
N(R11)0R11, NRI INRI 1R12, OR"
or Se. In another aspect of this
embodiment, RI is methyl, CH?F or ethynyl. In another aspect of this
embodiment,
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R1 is methyl. In another aspect of this embodiment, R9 is H, halogen, or
NR11R12. in
another aspect of this embodiment, R9 is H, halogen, or NR' 'R'2 and R1 is
methyl,
CH2F, or ethynyl. In another aspect of this embodiment, R9 is H, halogen, or
NR11R12
and R1 is methyl. In another aspect of this embodiment, R8 is NH2 and R9 is H
or
halogen. In another aspect of this embodiment, R8 is NH2 and R9 is H or
halogen and
R1 is methyl, CH2F, or ethynyl. In another aspect of this embodiment, R8 is
NH2 and
R9 is H or halogen and R1 is methyl. In another aspect of this embodiment, R8
and R9
are each NH2. In another aspect of this embodiment, R8 and R9 are each NH, and
R1
is methyl. In another aspect of this embodiment, R8 and R9 are each NH2 and R1
is
methyl, CH2F or ethynyl. In another aspect of this embodiment, R8 is OH and R9
is
NFL. In another aspect of this embodiment, R8 is OH, R9 is NH2 and R1 is
methyl. In
another aspect of this embodiment, R8 is OH, R9 is NH2 and RI is methyl, CH2F,
or
ethynyl. In another aspect of this embodiment R6 is H. In another aspect of
this
embodiment R6 is CN. In another aspect of this embodiment R6 is ORa. In
another
aspect of this embodiment R6 is OH. In another aspect of this embodiment R6 is
CH3.
In another aspect of this embodiment, R4 is H or ORa. In another aspect of
this
embodiment, R4 is ORa. In another aspect of this embodiment, R4 is ORa and R1
is
(C1¨C8)alkyl, (C2¨C8) alkenyl or (C2¨C8)alkynyl. In another aspect of this
embodiment, R4 is OR and R1 is (Ci¨C8)alkyl. In another aspect of this
embodiment,
R4 is ORa and R1 is methyl. In another aspect of this embodiment, R4 is ORa,
R1 is
(Ci¨C8)alkyl and at least one of X1 or X2 is N. In another aspect of this
embodiment,
R4 is ORa, R1 is methyl and at least one of X1 or X2 is N. In another aspect
of this
embodiment, R4 is ORa, R1 is (CI¨C8)alkyl and R6 is CN, OH, or CH3. In another
aspect of this embodiment, R4 is ORa, R1 is methyl and R6 is CN, OH, or CH3.
In
another aspect of this embodiment, R4 is ORa, R1 is methyl and R6 is H. In
another
aspect of this embodiment, R4 is OH and R1 is methyl. In another aspect of
this
embodiment, R4 is OH, R1 is (C1¨Cg)alkyl and at least one of X1 or X2 is N. In
another aspect of this embodiment, R4 is OH, R1 is methyl and at least one of
X1 or X2
is N. In another aspect of this embodiment, R4 is OH. R1 is (Ci¨C8)alkyl and
R6 is
CN, OH, or CH3. In another aspect of this embodiment, R4 is OH, R1 is methyl
and R6
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is CN, OH, or CH3. In another aspect of this embodiment, R4 is OH, RI is
methyl and
R6 is H.
In another embodiment of Formula II, each RI is, independently, H, halogen,
CN or optionally substituted heteroaryl. In another aspect of this embodiment,
RI is
methyl. In another aspect of this embodiment, RI is methyl, CH2F or ethynyl.
In
another aspect of this embodiment, R9 is H, halogen, or NR' 'R12. In another
aspect of
this embodiment, R9 is H, halogen, or NR' 1R'2 and RI is methyl. In another
aspect of
this embodiment, R9 is H, halogen, or NR' IRI2 and RI is methyl, CH2F, or
ethynyl. In
another aspect of this embodiment, R8 is NH2 and R9 is H or halogen. In
another
aspect of this embodiment, R8 is NH2 and R9 is H or halogen and RI is methyl.
In
another aspect of this embodiment, le is NH, and R9 is H or halogen and RI is
methyl,
CH2F, or ethynyl. In another aspect of this embodiment, R8 and R9 are each
NH2. In
another aspect of this embodiment, R8 and R9 are each NH2 and RI is methyl. In
another aspect of this embodiment, R8 and R9 are each NH, and RI is methyl,
CH2F or
ethynyl. In another aspect of this embodiment, Rs is OH and R9 is NH,. In
another
aspect of this embodiment, R8 is OH, R9 is NH2 and RI is methyl. In another
aspect of
this embodiment, R8 is OH, R9 is NH2 and RI is methyl, CH2F, or ethynyl. In
another
aspect of this embodiment R6 is H. In another aspect of this embodiment R6 is
CN. In
another aspect of this embodiment R6 is ORa. In another aspect of this
embodiment R6
is OH. In another aspect of this embodiment R6 is CH3. In another aspect of
this
embodiment, R4 is I-I or OR'. In another aspect of this embodiment, R4 is ORa.
In
another aspect of this embodiment, R4 is OR and R1 is (Ci¨C8)alkyl, (C/¨C8)
alkenyl
or (C2¨C8)alkynyl. In another aspect of this embodiment, R4 is ORa and RI is
(Ci¨C8)alkyl. In another aspect of this embodiment, R4 is ORa and R1 is
methyl. In
another aspect of this embodiment, R4 is Ole, RI is (Ca ¨C8)alkyl and at least
one of
XI or X2 is N. In another aspect of this embodiment, R4 is ORa, RI is methyl
and at
least one of XI or X2 is N. In another aspect of this embodiment, R4 is ORa,
R1 is
(Ci¨C8)alkyl and R6 is CN, OH, or CH3. In another aspect of this embodiment,
R4 is
OR, RI is methyl and R6 is CN, OH, or CH3. In another aspect of this
embodiment,
R4 is ORa, RI is methyl and R6 is H. In another aspect of this embodiment, R4
is OH
21
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802.CIPF2
and RI is methyl. In another aspect of this embodiment, R4 is OH, RI is
(CI¨C8)alkyl
and at least one of XI or X2 is N. In another aspect of this embodiment, R4 is
OH, RI
is methyl and at least one of XI or X2 is N. In another aspect of this
embodiment, R4 is
OH, RI is (Ci¨C8)alkyl and R6 is CN, OH, or C1-13. In another aspect of this
embodiment, R4 is OH, RI is methyl and R6 is CN, OH, or CH3. In another aspect
of
this embodiment, R4 is OH, RI is methyl and R6 is H.
In another embodiment, compounds of Formula I or Formula II are
represented by Formula
R8
R7 / N
.X2\
0 N NR9
0
R1
H
R4 E.
Formula III
or a pharmaceutically acceptable salt, thereof;
wherein:
R1 is CH3, CH2F, or ethynyl and all remaining variables are defined as for
Formula I.
In one embodiment of Formula III, R4 is H, ORa, N(Ra)2, N3, CN, SRa,
halogen, (C1¨C8)alkyl, (C2¨C8)alkenyl or (C2¨C8)alkynyl. In another aspect of
this
embodiment, R4 is H or ORa. In another aspect of this embodiment, R4 is ORa.
In
another aspect of this embodiment, R4 is OR and RI is CH3, CH2F, or ethynyl.
In
another aspect of this embodiment, R4 is Ole and RI is methyl. In another
aspect of
this embodiment, R4 is ORa, RI is methyl and at least one of XI or X2is N. In
another
aspect of this embodiment, R4 is ORa, RI is methyl and R6 is CN, OH, or CH3.
In
another aspect of this embodiment, R4 is ORa, RI is methyl and R6 is H. In
another
aspect of this embodiment, R4 is OH and RI is methyl. In another aspect of
this
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embodiment, R4 is OH, RI is methyl and at least one of XI or X2 is N. In
another
aspect of this embodiment, R4 is OH, RI is methyl and R6 is CN, OH, or CH3. In
another aspect of this embodiment, R4 is OH, RI is methyl and R6 is H.
In another embodiment of Formula III, R6 is H, CN, ORa or CH3. In another
aspect of this embodiment R6 is H. In another aspect of this embodiment R6 is
CN. In
another aspect of this embodiment R6 is ORa. In another aspect of this
embodiment R6
is OH. In another aspect of this embodiment R6 is CH3. In another aspect of
this
embodiment, R4 is H or ORa. In another aspect of this embodiment, R4 is ORa.
In
another aspect of this embodiment, R4 is ORa and RI is methyl. In another
aspect of
this embodiment, R4 is ORa, RI is methyl and at least one of XI or X2 is N. In
another
aspect of this embodiment, R4 is ORa, RI is methyl and R6 is CN, OH, or CH3.
In
another aspect of this embodiment, R4 is ORa, RI is methyl and R6 is H. In
another
aspect of this embodiment, R4 is OH and RI is methyl. In another aspect of
this
embodiment, R4 is OH, Rl is methyl and at least one of XI or X2 is N. In
another
aspect of this embodiment, R4 is OH, RI is methyl and R6 is CN, OH, or CH3. In
another aspect of this embodiment, R4 is OH, RI is methyl and R6 is H.
In another embodiment of Formula III, R6 is CN, ORa or CH3. In another
aspect of this embodiment R6 is CN. In another aspect of this embodiment R6 is
ORa.
In another aspect of this embodiment R6 is OH. In another aspect of this
embodiment
R6 is CH3. In another aspect of this embodiment, R4 is H or Ole. In another
aspect of
this embodiment, R4 is ORB. In another aspect of this embodiment, R4 is OR and
RI
is methyl. In another aspect of this embodiment, R4 is Ole, RI is methyl and
at least
one of XI or X2 is N. In another aspect of this embodiment, R4 is ORa, RI is
methyl
and R6 is CN, OH, or CH3. In another aspect of this embodiment, R4 is OH and
RI is
methyl. In another aspect of this embodiment, R4 is OH, RI is methyl and at
least one
of XI or X2is N. In another aspect of this embodiment, R4 is OH, RI is methyl
and R6
is CN, OH, or CH3.
In one embodiment of Formula III, R7 is H, -C(-0)RII, -C(-0)0R11, -
C(=0)SRI or
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0
p _____________
w2
. In a aspect of this embodiment, R7 is H. In another aspect of this
embodiment, R7 is -C(=0)R11. In another aspect of this embodiment, R7 is -
C(=0)RI I
wherein R" is (CI-C8)alkyl. In another aspect of this embodiment, R7 is
0
I
W2
. In another aspect of this embodiment R6 is H. In another aspect of
this embodiment R6 is CN. In another aspect of this embodiment R6 is ORa. In
another aspect of this embodiment R6 is OH. In another aspect of this
embodiment R6
is CH3. In another aspect of this embodiment, R4 is H or OR In another aspect
of
this embodiment, R4 is ORa and RI is methyl. In another aspect of this
embodiment,
R4 is ORa, RI is methyl and at least one of X1 or X2is N. In another aspect of
this
embodiment, R4 is ORa, RI is methyl and R6 is CN, OH, or CH3. In another
aspect of
this embodiment, R4 is Ole, R1 is methyl and R6 is H. In another aspect of
this
embodiment, R4 is OH and RI is methyl. In another aspect of this embodiment,
R4 is
OH, RI is methyl and at least one of X1 or X2 is N. In another aspect of this
embodiment, R4 is OH, R1 is methyl and R6 is CN, OH, or CH3. In another aspect
of
this embodiment, R4 is OH, RI is methyl and R6 is H.
90 In one embodiment of Formula III, X1 is N or C-R' . In another aspect of
this
embodiment, X1 is N. In another aspect of this embodiment, X1 is C-R10. In
another
aspect of this embodiment, X2 is C-H. In another aspect of this embodiment, X1
is N
and X2 is C-H. In another aspect of this embodiment, X1 is C-R1 and X2 is CH.
In
another aspect of this embodiment R6 is H. In another aspect of this
embodiment R6 is
CN. In another aspect of this embodiment R6 is ORa. In another aspect of this
embodiment R6 is OH. In another aspect of this embodiment R6 is CH3. In
another
aspect of this embodiment, R4 is H or ORa. In another aspect of this
embodiment, R4
is OR. In another aspect of this embodiment, R4 is ORa and R1 is methyl. In
another
aspect of this embodiment, R4 is OW, RI is methyl and at least one of X.1 or
X2 is N.
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.. In another aspect of this embodiment, R4 is ORa, RI is methyl and R6 is CN,
OH, or
CH3. In another aspect of this embodiment, R4 is OH and RI is methyl. In
another
aspect of this embodiment, R4 is OH, RI is methyl and at least one of XI or
X2is N. In
another aspect of this embodiment, R4 is OH, RI is methyl and R6 is CN, OH, or
CH3.
In another aspect of this embodiment. R4 is OH, RI is methyl and R6 is H.
In another embodiment of Fonnula III, each R8 is independently halogen,
NR1IR12, N(R11)0R1', NRIINR11R12, ORI I or SR''. In another aspect of this
embodiment, RI is methyl, CH2F or ethynyl. In another aspect of this
embodiment,
R1 is methyl. In another aspect of this embodiment, R9 is H, halogen, or
NRI1R12. In
another aspect of this embodiment, R9 is H, halogen, or NR' IR12 and RI is
methyl,
.. CH2F, or ethynyl. In another aspect of this embodiment, R9 is H, halogen,
or NRH iR 2
and R1 is methyl. In another aspect of this embodiment, R8 is NH2 and R9 is H
or
halogen. In another aspect of this embodiment, R8 is NH? and R9 is H or
halogen and
R1 is methyl, CH2F, or ethynyl. In another aspect of this embodiment, R8 is
NH? and
R9 is H or halogen and R1 is methyl. In another aspect of this embodiment, R8
and R9
.. are each NH?. In another aspect of this embodiment, R8 and R9 are each NH?
and RI
is methyl, CH2F or ethynyl. In another aspect of this embodiment, R8 and R9
are each
NH2 and R1 is methyl. In another aspect of this embodiment, R8 is OH and R9 is
NH2.
In another aspect of this embodiment. R8 is OH, R9 is NH? and RI is methyl,
CH2F, or
ethynyl. In another aspect of this embodiment, R8 is OH, R9 is NH2 and RI is
methyl.
In another aspect of this embodiment R6 is H. In another aspect of this
embodiment
R6 is CN. In another aspect of this embodiment R6 is ORa. In another aspect of
this
embodiment R6 is OH. In another aspect of this embodiment R6 is CH3. In
another
aspect of this embodiment, R4 is H or OR'. In another aspect of this
embodiment, R4
is ORa and RI is methyl. In another aspect of this embodiment, R4 is ORa, RI
is
.. methyl and at least one of X1 or X2 is N. In another aspect of this
embodiment, R4 is
Ole, RI is methyl and R6 is CN, OH, or CH3. In another aspect of this
embodiment,
R4 is ORa, RI is methyl and R6 is H. In another aspect of this embodiment, R4
is OH
and RI is methyl. In another aspect of this embodiment, R4 is OH, RI is methyl
and at
least one of XI or X2 is N. In another aspect of this embodiment, R4 is OH, RI
is
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methyl and R6 is CN, OH, or CH3. In another aspect of this embodiment, R4 is
OH, RI
is methyl and R6 is H.
In another embodiment of Formula Ill, each R1 is, independently, H, halogen,
CN or optionally substituted heteroaryl. In another aspect of this embodiment,
RI is
methyl. In another aspect of this embodiment, R9 is H, halogen, or NRI1Ri 2.
In
another aspect of this embodiment, R9 is H, halogen, or NR' 'R'2 and RI is
methyl. In
another aspect of this embodiment, R8 is NH-, and R9 is H or halogen. In
another
aspect of this embodiment, R8 is NH2 and R9 is H or halogen and RI is methyl.
In
another aspect of this embodiment, R8 and R9 are each NH,. In another aspect
of this
embodiment, R8 and R9 are each NH, and R1 is methyl. In another aspect of this
embodiment, R8 is OH and R9 is NH2. In another aspect of this embodiment, R8
is
OH, R9 is NEll and RI is methyl. In another aspect of this embodiment R6 is H.
In
another aspect of this embodiment R6 is CN. In another aspect of this
embodiment R6
is ORa. In another aspect of this embodiment R6 is OH. In another aspect of
this
embodiment R6 is CH3. In another aspect of this embodiment, R4 is H or Ole. In
another aspect of this embodiment, R4 is ORa and RI is methyl. In another
aspect of
this embodiment, R4 is Ole, R1 is methyl and at least one of XI or X2 is N. In
another
aspect of this embodiment, R4 is ORa, R' is methyl and R6 is CN, OH, or CH3.
In
another aspect of this embodiment, R4 is ORa, RI is methyl and R6 is H. In
another
aspect of this embodiment, R4 is OH and R1 is methyl. In another aspect of
this
.. embodiment, R4 is OH, RI is methyl and at least one of X1 or X2 is N. In
another
aspect of this embodiment, R4 is OH, RI is methyl and R6 is CN, OH, or CH3. In
another aspect of this embodiment, R4 is OH, RI is methyl and R6 is H.
In another embodiment, provided arc compounds of Formula IV:
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R8
R7--0 N N<%j\
0 R-
R3 R1
= =
R4 R2
Formula IV
or a pharmaceutically acceptable salt, thereof;
wherein:
R1 is (C1-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl,
(C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl, (C2-C8)substituted
alkynyl, or aryl(C -C8)alkyl;
R2 is halogen;
R3, R4, and R5 are each independently H, halogen, ORa, N(Ra)2, N3, CN, NO2,
S(0)õRa, (Ci-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl,
(C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl, (C2-C8)substituted
alkynyl, or aryl(C1-C8)alkyl;
or any two of R3, R4 or R5 on adjacent carbon atoms when taken together are -
0(C0)0- or when taken together with the ring carbon atoms to which they are
attached form a double bond;
each n is independently 0, 1, or 2;
each Ra is independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl,
aryl(Ci-C8)alkyl, (C4-C8)carbocyclylalkyl, -C(=0)R11, -C(0)OR", -C(-0)NR1IR12,
-8(0)R11, -S(0)2R11, -8(0)(0R11 ), -8(0)2(0R11), or -SO2NR11R12;
R7 is H, -C(=0)R11, -C(=0)0R11, -C(=0)NRI IR12, -C(=0)SRI I , -8(0)Ril -
S(0)2R11, -S(0)(0R11), -S(0)2(ORI1), -SO2NRI 1R12, or
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)
vv1----7
vv2
'
,
Y is 0, S. NR, 'N(0)(R), N(OR), +N(0)(0R), or N¨NR2;
W1 and W2, when taken together, are ¨Y3(C(RY)2)3Y3-; or
one of WI or W2 together with either R3 or R4 is ¨Y3- and the other of WI or
W2 is Formula Ia; or
W1 and W2 are each, independently, a group of Foimula IVa:
_ _
Rx (y2 _______________________________ )1 \
y2 i
Rx / y2 _____________________________________________
M2
Formula IVa
wherein:
each Y1 is, independently, 0, S, NR, +N(0)(R), N(OR), +N(0)(0R), or
N¨NR2;
each Y2 is independently a bond, 0, CR2, NR, +N(0)(R), N(OR), +N(0)(0R),
N¨NR2, S, S¨S, S(0), or S(0)2;
each Y3 is independently 0, S, or NR;
M2 is 0, 1 or 2;
each Rx is a group of Formula IVb:
28
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802.CIPF2
yl _ yl
RY RY
y2 y2 y
Mid
_ M12c
1M1c
M1 a
Formula IVb
wherein:
each Ml a, M1 c, and Mid is independently 0 or 1;
M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;
each RY is independently H, F, Cl, Br, I, OH, -C(=Y1)R, -C(=Y1)R13, -
C(=Y1)0R, -C(-Y1)N(R)2, -N(R)2, -+N(R)3, -SR, -S(0)R, -S(0)2R, -S(0)2R13, -
S(0)(0R), -S(0)2(0R), -0C(=Y1)R, -0C(=Y1)0R, -0C(=Y1)(N(R)2), -SC(=Y1)R, -
SC(=Y1)0R, -SC(----Y1)(N(R)2), -N(R)C(=Y1)R, -N(R)C(-=Y1)0R, -N(R)C(=Y1)N(R)2,
-SO2NR2, -CN, -NO2, -OR, (CI-CO alkyl, (C2-C8)alkenyl. (C2-C8) alkynyl,
C6-C20 aryl, C3-C20 carbocyclyl, C2-C20 heterocyclyl, arylalkyl,
heteroarylalkyl;
wherein each (C1-C8) alkyl, (C2-C8)alkenyl, (C2-C8) alkynyl, C6-C20 aryl,
C3-C20 carbocyclyl, C2-C20 heterocyclyl, arylalkyl, or heteroarylalkyl
is optionally substituted with 1-3 R2 groups;
or when taken together, two RY on the same carbon atom form a carbocyclic
ring of 3 to 7 carbon atoms;
each R is independently H, (C1-C8) alkyl, (C2-C8)alkenyl, (C2-C8) alkynyl,
C6-C20 aryl, C3-C20 carbocyclyl, C2-C20 heterocyclyl, or arylalkyl;
each R8 is halogen, NR' 'R'2, N(R11)OR", NR11NR11R12, N3, NO, NO2, OR11
or S(0)R";
each R9 is independently H, halogen, NRI1R12, N(R11)0R11, NRIINR11R12, N3,
NO, NO2, CHO, CN, I), -CI-1=NHNRI I, -CH=N(OR11), -CH(OR1 1)2,
-C(=0)NRIIR12, -C(=S)NR11R12, -C(=0)0R11, RI 1, OR11 or
each RI 1 or R12 is independently (C1-C8)alkyl, (C2-C8)alkenyl, (C2-
C8)alkynyl, (C4-C8)carbocyclylalkyl, optionally substituted aryl, optionally
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substituted heteroaryl, -C(=0)(C1-C8)alkyl, -S(0)õ(CI-C8)alky1 or aryl(C1-
C8)alkyl; or
R11 and R12 taken together with a nitrogen to which they are both attached
form a 3 to
7 membered heterocyclic ring wherein any one carbon atom of said heterocyclic
ring
can optionally be replaced with -0-, -S- or -NRb-;
each R13 is independently a carbocycle or heterocycle optionally substituted
with 1-3 R2 groups;
each R2 is independently, halogen, CN, N3, N(R)2, OR, -SR, -S(0)R, -S(0)7R,
-S(0)(0R), -S(0)2(0R), -C(=Y1)R, -C(=Y1)0R, or C(=Y1)N(R)2;
wherein each (Ci-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or aryl(Ci-C8)alkyl
; ; ; ;
R3 R4 R5 R6 RI or R12 , is
of each R1, independently, optionally substituted with
one
or more halo, hydroxy, CN, N3, N(Rb)2 or ORb; and wherein one or more of the
non-
terminal carbon atoms of each said (CI-C8)alkyl may be optionally replaced
with -0-,
-S- or -Nle;
each Rb is independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl,
aryl(C -C8)alkyl, (C4-C8)carbocyclylalkyl, -C(=0)R21, -C(-0)0R21, -C(-0)NR21
R22,
-C(=0)SR21, -S(0)R21, -S(0)2R21, -S(0)(0R21);
S(0)2(0R21), or -SO2NR21R22;
each R21 or R22 is independently H, (CI-C8)alkyl, (C2-C8)alkenyl, (C2-
C8)alkynyl. (C4-C8)carbocyclylalkyl, -C(=0)(Ci-C8)alkyl, -S(0)(Ci-C8)alkyl or
aryl(C1-C8)alkyl;
with the optional proviso that compounds 1, Id, le, 2, TP-1, A-1, 8, and 21
are
excluded.
In another aspect of this embodiment Y and Y1 is 0. In another aspect of this
embodiment R8 is halogen, NR11,-.K 12,
N(R11)0R11, NRI INRI1R12, U-11
K or S(0)õRi 1.
In another aspect of this embodiment R9 is IL halogen, S(0)R" or NR1 1R 12. In
another aspect of this embodiment R4 is ORE. In another aspect of this
embodiment
R1 is CH:3. In another aspect of this embodiment R2 is F. In another aspect of
this
embodiment R7 is
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802.CIPF2
w2
=
wherein Y is ¨0-; WI is Formula la and W2 together with R4 is ¨0-.In another
embodiment, compounds of Foimula IV are represented by Formula V:
R8
0 R9
H R1
-
z.
4 -T.
R F
Formula V
wherein R1 is methyl or ethynyl, and R4 is ORa. In another aspect of this
embodiment R7 is H or
0
w2
In another aspect of this embodiment, compound of Formula V are represented
the following structures:
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N H2
R N
RY'0'=1 _________ ."< 9
11 R
HN-/(11:1';0-\\za)52N,
0
N
Ar
HO F ,
NH2
R R N
RY.0 -,/, 0
/I
0 HN-P-0 \ r\I
Ar/6
o
IR--.0 ,
NH2
NH2
),---
/.._0---NI NN
R, R 0 0-0 N
1
RY-0-P----__ -' ''
0 H 8 "F
0
. , NH2
NH2
RR
0 FOR 0-P-0 0 \ N, _J
0
RY, I . . , ,
N-P--_, ,:- -, R R õ
R, II 0 F ¨
0 HO F
NH2
R p
RY.Ø, \< ' s 0 N.-";i----
0 R/F1R HN-P-0-1\r0
/6 N
Ar
Ha -F .
In another embodiment, provided are compounds of Formula VI:
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NH2
0
"rEz T
Formula VI
or a pharmaceutically acceptable salt, thereof;
wherein:
R4 is Gle;
each n is independently 0, I, or 2;
each Ra is independently H, (CI -C8)alkyl, (C2-Cg)alkenyl, (C2-C8)alkynyl,
aryl (C -C8)alkyl, (C4-C8)carbocyclylalkyl, -C(=0)R11, -C(=0)0R11, -C(-
0)NRIIR12,
-C(=-0)SR11, -S(0)R11, -S(0)2R11, -S(0)(0R11), -S(0)2(0R11), or -SO2NRI1R12;
R7 is H, -C(=0)R11, -C(=0)0R _c(=o)NRIIR 11, 12. -C(=0)SR11, -S(0)R11, -
S(0)2R11, -S(0)(0R11), -S(0)2(0R11), -SO2NRI 'R12, or
W11 H
vv2 =
Y is 0, S, NR, +N(0)(R), N(OR), +N(0)(0R), or N-NR2;
WI and W2, when taken together, are -Y3(C(RY)2)3Y3-; or
one of WI or W2 together with R4 is -Y3- and the other of WI or W2 is Formula
Ia; or
WI and W2 are each, independently, a group of Formula Via:
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( YI
Rx Y2 IP ______________________________________ Y2 __
2
- 1' /
----
_
M2
Formula Via
wherein:
each Y1 is, independently, 0, S, NR, 4N(0)(R), N(OR), N(0)(0R), or
N-NR2
each Y2 is independently a bond, 0, CR2, NR, +N(0)(R), N(04 +N(0)(014
N-NR, S, S-S, S(0), or S(0)21
each Y3 is independently 0, S, or NR;
M2 is 0,1 or 2;
each lel is a group of Fonnula VIb:
- -
y1
_ y1 _ Ry Ry
y2 R
----r--_ Y
1.õ _ M12c /
Mid
\
M1 a M1c
Formula VII,
wherein:
each Mla, Mle, and Mid is independently 0 or 1;
M1 2c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;
each RY is independently H, F, Cl, Br, I, OH, -C(=Y1)R, -C(Y1)R'3, -
C(=Y1)0R, -C(=Y1)N(R)2, -N(R)2, -+N(R)3, -SR, -S(0)R, -S(0)2R, -S(0)2R13, -
S(0)(0R), -S(0)2(0R), -0C(=Y1)R, -0C(=Y1)0R, -0C(=Y1)(N(R)2), -SC(=Y1)R, -
SC(=Y1)0R, -SC(=Y1)(N(R)2), -N(R)C(=Y1)R, -N(R)C(=Y1)0R, -N(R)C(=Y1)N(R)2,
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-SO2NR2. -CN, -N3, -NO2, -OR, (C1-C8) alkyl, (C2-C8)alkenyl, (C2-C8) alkynyl,
C6-C20 aryl, C3-C20 carbocyclyl, C2-C20 heterocyclyl, arylalkyl,
heteroarylalkyl;
wherein each (C1-C8) alkyl, (C2-C8)alkenyl, (C2-C8) alkynyl, C6-C20 aryl,
C3-C20 carbocyclyl, C2-C20 heterocyclyl, arylalkyl, or heteroarylalkyl
is optionally substituted with 1-3 R2 groups:
each R is independently H, (C1-C8) alkyl, (C2-C8)alkenyl, (C2-C8) alkynyl,
C6-C20 aryl, C3-C20 carbocyclyl, C2-C20 heterocyclyl, or arylalkyl;
each R11 or R12 is independently H, (CI-C8)alkyl, (C2-C8)alkenyl, (C2-
C8)alkynyl, (C4-C8)earbocyclylalkyl, optionally substituted aryl, optionally
substituted heteroaryl, -C(=0)(Ci-C8)alkyl, -S(0)õ(Ci-C8)alkyl or aryl(CI-
C8)alkyl;
each R13 is independently a carbocycle or heterocycle optionally substituted
with 1-3 RN groups;
each R2 is independently, halogen, CN, N3, N(R)2, OR, -SR, -S(0)R, -S(0)2R,
-S(0)(0R), -S(0)2(0R), -C(=Y1)R, -C(Y1)OR, or C(=Y1)N(R)2;
wherein each (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or aryl(CI-C8)alkyl
of each R4, R'' or R12 is, independently, optionally substituted with one or
more halo,
hydroxy, CN, N3, N(Rh)2 or OR"; and wherein one or more of the non-terminal
carbon
atoms of each said (Ci-C8)alkyl may be optionally replaced with -0-, -S- or
each Rb is independently H, (C1-CR)alkyl, (C2-CR)a1kenyl, (C2-C8)alkynyl,
aryl(Ci-C8)alkyl, (C4-C8)carbocyclylalkyl, -C(=0)R21, -C(=0)0R21,
_c(=o)NR21R22,
-C(=0)SR21, -S(0)R21, -S(0)2R21, -S(0)(0R21), -S(0)2(0R21), or -S02NR21R22;
each R21 or R22 is independently H, (Ci-C8)alkYl, (C2-C8)alkenyl, (C2-
C8)alkynyl, (C4-C8)carbocyclylalkyl, -C(=0)(Ci-C8)alkyl, -S(0),1(Ci-C8)alkyl
or
aryl(CI-C8)alkyl; and
with the optional proviso that compounds 1, lc, 1d, le, 2, TP-1, A-1, 8, and
21
are excluded.
In another aspect of this embodiment Ra is H, (Ci-C8)alkyl, or
C6)alkyl; R7 or R7 together with R4 is
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_ -
o RR
II RY
H __________ 0 __ P ___
-
0 HN-P--
1
OH _ ,0
- 1-35 , Ar5
, T b I
RYa0 N-P-0/ RY-0-P,õ ,b RYNN_pi
,b
H li II 0 R= II 0
0 0 0 0
RY,S R R RY 0 RR
>1 \K 9 _____________________________________________ ,,,-- > \< 0
il s
0 R R 0-P-0- 0 R R HN-P--
1 1
/N,FR 0
R
,or Ar/ =
,
wherein
a is the point of attachment to R7;
b is the point of attachment to R4;
Ar is phenyl or naphthyl, wherein the phenyl and naphthyl are optionally
substituted with 1-3 R2 groups;
each RY is independently (C1-C8) alkyl or C5-C6 carbocyclyl, wherein the
alkyl and carbocycly1 are optionally substituted with 1-3 R2 groups;
each R is independently H. (C1-C6) alkyl, or arylalkyl; and
each R2 is independently halogen, CN, N(R)2, OR, -SR, -S(0)R, -S(0)2R, -
S(0)(0R), -S(0)2(0R), -C(0)R, -C(=0)0R, or C(=0)N(R)2.
In another embodiment, compounds of Formula IV-VI are represented by
compounds having a structure:
NH2
NH2 :-
N
0 0
ti it It
O I \sN f \
HO-1?-0-P-O-P-0¨vo Ns j o HN¨P-0--yN
N
OH OH OH \ I N
,
\ .
Ho . H6 ''
9 5
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NH2 "-- NH2
0 Ny--- ..-- 0
ii \ L
, ,2.,_.
HN''''P
0 A -0 0 N, ,,__J 0
N
O 'N--
0
11 HO -P. 41, HO
NH )õ..,..____. NH2
z-
N 0--, ____ ' 0 N
/I \ ti ___riN\N
crOcriN -rj4-() \ -,,--7---N.N, j.,,N
/ -\70 ---1 0 HN-P-0 0
O N
0
4. Ho -P
. .:
HO P
, 5
NH2
0 N _,,,_(.. NH2 .
0 HN-P-0-y---N,
I N 0 HN-P-0 0
I N
0
Ho P
It HO --P
, ,
NH2
NH2 -.,,..,.0 F 0
0 HN-P-0
C) N
%_}
/=_(... 0
6
-,õ
NH2 NH2
9 IN1._____ _
_,2_,:[ N-N
0 HN-P-0-v -2N 0 HN-P-0 0 , _J N,
I N I N
0 0
lik a -E.
-----o -----Lo , ,
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NH2 > NH2
0 \ ---r--,C 0 __ .; 0 N,,____-
0 ______________________________ HN-P-0-y \ -N, _JN 71 1\-iN-P-0
I N
I N
0
_
(J . __ .
6
0 F
0
AO
, ,
NH2 ---,
NH2
N
\\// riii \ 'T--N '--"--,-0-, pi
6 HN-P-0 0 N,
6
01 N 0 N
11 6
lik ¨
HO F
-0
CI / /
NH2 NH2
5N,ri,
-N,N../Ni
0N _F,... 6 , ,E,
[I H 11
0 0 , 0 0 ,
NH2 NH2
N,,,..7___
0S-1\1=N--/N
-f
if N-P-0'
H
0 8 0 H8 , ,
NH2 NH2
N.-.T.-HN NN
N,N--,)
N
0444"=_11- 0
)-0-11
0 611 ___________ F 1 !
-'-'-'-11.-----d. F. --
, ,
38
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HO
NH2
N,
0
H
0
C31--"O
NH
HO t
,or
NH2
\ 9
0 HN-F1)--0 0 \ N, J
0
Hd
=
or a pharmaceutically acceptable salt, thereof.
In one embodiment of Formulas and Formulas IV-VI, R11 or R12 is
independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl,
(C4¨C8)carbocyclylalkyl, optionally substituted aryl, optionally substituted
heteroaryl,
-C(=0)(CI-C8)alkyl, -S(0)(Ci-C8)alkyl or aryl(C1-C8)alkyl. In another
embodiment,
R" and R12 taken together with a nitrogen to which they are both attached,
form a 3 to
I 5 7 membered heterocyclic ring wherein any one carbon atom of said
heterocyclic ring
can optionally be replaced with -0-, -S- or Therefore, by way of example
and
not limitation, the moiety NRLRl2 can be represented by the heterocycles:
¨N/ / \ / \ / \ f--N)
N 0 __ N S ¨N N Ra _____
\ _______________________ / \ __ / \ __ /
and the like.
In another embodiment of Formulas I-III and Formulas IV-VI, each R3, R4, R5,
R6, R11 or R12 is, independently, (CI-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl
or
aryl(CI-C8)alkyl, wherein said (Ci-C8)alkyl, (C2-C8)aikenyl, (C2-C8)alkynyl or
aryl(Ci-C8)alkyl are, independently, optionally substituted with one or more
halo,
hydroxy, CN, N3, N(Ra)2 or ORa. Therefore, by way of example and not
limitation,
R3, R4, R5, R6, R" or R12 could represent moieties such as -CH(NI-17)CH3, -
39
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CH(OH)CH2CH3, -CH(Nt2)CH(CH3)2, -CH2CF3, -(CH2)2CH(N3)CH3, -(CH2)6NH2
and the like.
In another embodiment of Formula I-III and Formula IV-V1, R3, R4, R5, R6,
R11 or R12 is (CI-C8)alkyl wherein one or more of the non-terminal carbon
atoms of
each said (C1-C8)alkyl may be optionally replaced with -0-, -S- or ¨Nle-.
Therefore,
by way of example and not limitation, R3, R4, R5, R6, -ii
K. or R12 could represent
moieties such as -C1420CH3, -C1-120CH2CH3, -CH2OCH(CH3)2, -CH2SCH3, -
(CH2)60CH3, -(CH2)6N(CH3)2 and the like.
In another embodiment, Formulas I-III is a compound selected from the group
consisting of
S' S'
N N
\,,,i,--
Bzi - 'N\s¨
OH S
Bz , Bz ,
NH2 NH2 NH2
HO
0 N,
.-S---- NH2
HC3 HO F HO F
0 0
0 0 0
\ NH \ Y(NH
HO HO-P-O-P-O-P-0 0 N, i
OH
H2 OH OH N¨\NH2
N
HC3 -F Ho 'F
NH2 NH2
S --------., 0
Ho .F NN
N___NNH2 / HO--NO NµN__:_iN
S---\,--C) '
0 . .
Ho -F
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NH NH2
NH2
N N
\ \ N \ \
N _IN \
0
Bz7 N, ,_____/ 0 \m
A Bz/ ---y) '1\1=-- HO 0 \ Ns ____ õJ-
______________ OH N CN ______________________________ N
OH
6 /6
Bz/ , Bz ,
. HO F ,
NH2 NH2 NH2
HO 0 N, N _ j HO 0 N,
õ NNI--- ., N----
."0-- HO F HO F , Ho -P
NH2
,
H n
________________________ 0 \ \ .0 - \-\\NH2
N
0 HN-p-O 0 N, , , ___J.
0 . N
"CN NI77-----/N
II HO P IIP Hd -F
, CI ,
NH2
z-
0
N
0 HN-P-0A0
. N
IIO :-
HO F
,
NH2
\
0 HN-P-0 0 Nsj
(!) , N
"CN
HO
:-_ :-
F
,
NH2
:-. NH2 \_0\ j 0
0
,,,
0 H O H I
411 IF
-F
- = '--N Hd 'F 4
, ,
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"S
NH2
I
NH2
0 \ N
NisN-
0 H ' 0 H '
--- _______________________ - ' N
HO' -F HO' -F.
, ,
HN NH2 NH2
0
0 '
,,...z,.,
= N H 0
100
HO 6 -F. 110 H6 -F.
p
\
õs.
..------0 NH2 0 NH2
\
0
, NN Ct
0 H 6 N
H6 -F HO -F
0 NH2
NH2 )..._
0 \
II
HO¨P-0___,J p .,
OH CN N 0
'CN N
HO F >-04
0 HO
NH2
NH2
---- '- N
\N, 0 \ N,N.-)
,,.........z0 N 0
\ '''ON 0,), . ., "ON
O. I
Hc5 u
_ F
,.= .,.. ____c/7-------(5 -F
,
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NH2
NH
---- N
N 0
) ___ 0\ 'ON
,-, : ' P--____.-: ---
//. 'N---P-6-- :": -..õ----,,,,,,-,11.--.1\i/ u F
0 H H
0 , 0 ,
NH2
N NH2 NH2
Bz/ NI' ---'( \ ______NN
s. Bz0---%\70 Ns ii HO 0 Ns
OH N N---
.-.:'ION
/0
Bz Bz0 F HO F
NH2
N
..--,-õT----
NH2 \
/-N
N
HO,? 0, j /..--C___.'"--C-N N
HO/IDO 7, N` 7 0
ION N--------
HO-P-0.
õ n
Ho. ..F 0
NH2 NHDMTr
N
N ------. .. ''= N
------\ ,O) . "CNN
0 0
0-----.õ µ -- --. ' "CN
0 , Fid' -F ,
TrO NHDMTr
S
_______________ \ ti \
0 0-P-0
FI-I ON N
.: 1.
Ho -F ,
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HO
NH2
S
N., II \ N
0 ______________ 0-P-0
NH
HO -F
,
0
...
: Ny(
NH
>-C) O 1\11\1*-OAO \ N =N---(
NH2
(--
. __________________________
HO:-. :-
F
,
NH2
N NH2
N \
1
/0 0 N, ,__/N
\ \
Bz N HO 0 N, ,,,,_,JN
N
. .
Bz,c5
HO F
, ,
NH2
NH2 \ -... N
0 = 9 \ N
\ _____ 0 .-'1- 0 \ -,. N
N )r- N-P-
0 H
N
0
r---N-Ig-C) 0
N
0 H 6 HO -F
H6 -F.
NH2 NH2 NH2
HO 0 N
INI( N 4 N
N
,,y2/ ;____N/ \'N
HOE, / HO 0 N 0 NA,N,J , vj
V¨
'', OH _______________________________________ . . ',0----.
. .
HO 'P , Ho F Ho -P.
NH2
0 0 0 N \
11 li It \
HO-P- O-P -0-P- 0 0
OH OH OH . N
CN
HO -P ,
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0
NH2
Nz------rliNH
O 0 0
II li II
0
OH OH OH , N
''H OH
",-
Ho , HO. F ,
0 0 NH2
N
N------?L'NH
HO \ 2
0
..-,4A-
""CN
0_/0
HO
.: :-
F
H6 F 0 , ,
0 NH2 0
N N
V ____,,,_)-1(NH
0 HN-H0 0 - ' Ns ___ J\
'"1-1 N- NH2
0¨/
)-04
0 HO F
HO "F
,
NH2 NH2
\N
--.."---7--)\\
N
0 N . N
'ICN
\¨\
(.,//' -1\1-1D--d ": \ ,= :.
HO-P---6 ..
,., H ii ii
0 , 0 , ,
NH2 NH2
NN N____-__?:-. N
HO \ N,N .-----1-.
0
OH ,,
NH2 HO
0 \,ONN'N-----NH2
. ,
H6 'F. HO' F , ,
HO---y___
NH2
S N_____(
\ 0
\ i N
0
NH "'ON N- NNH2
HO =F
,
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NH2 _________________________________________________________ NH2
0
N
HN-P-0 0 N, 0
0
0
'H H
HO
NH2
N
0
H
OH
=
and HO
or a pharmaceutically acceptable salt or ester thereof.
In another embodiment, provided is a compound useful for the synthesis of the
compounds of Formula I selected from the group consisting of
0 " 0
N-P-
" NO NO2 NO2
-
H '
0
ON HN
\-0 0 \-0 0
)nN-vo
0 H 6 No2 0 H 6 NO2
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/--O)rr_ 9
N¨P-0
0 H
0
NO2
S(0)2
N¨P-0 ir¨O)H
N¨P-0
0 H
0 0 H
0
NO2 NO2
and ; or salts or esters
thereof.
DEFINITIONS
Unless stated otherwise, the following Willis and phrases as used herein are
intended to have the following meanings:
When trade names are used herein, applicants intend to independently include
the trade name product and the active pharmaceutical ingredient(s) of the
trade name
product.
1 5 As used herein, "a compound of the invention" or "a compound of
Formula I"
means a compound of Formula I or a pharmaceutically acceptable salt, thereof.
Similarly, with respect to isolatable intermediates, the phrase "a compound of
Formula (number)" means a compound of that formula and pharmaceutically
acceptable salts, thereof.
"Alkyl" is hydrocarbon containing normal, secondary, tertiary or cyclic carbon
atoms. For example, an alkyl group can have 1 to 20 carbon atoms (i.e., C1-C2o
alkyl), 1 to 8 carbon atoms (i.e., CI-C8 alkyl), or 1 to 6 carbon atoms (i.e.,
Ci-C6
alkyl). Examples of suitable alkyl groups include, but are not limited to,
methyl (Me, -
CH3), ethyl (Et, -CH2CH3), 1-propyl (11-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-
Pr,
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I-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-l-
propyl
" (i.-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH1)CH2CH3),
2-methyl-
2-propyl (I-Bu, I-butyl, -C(CH3)3), -pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-
pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl
(-C(CH3)2CH2CH3), 3-methy1-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-I-butyl
(-CH2CH2CH(CH3)2), 2-methyl-I -butyl (-CH2CH(CH3)CH2CH3), 1-hexyl
(-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-
CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methy1-2-
pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2),
3-methy1-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-
CH(CH2CH3)CH(CH3)2), 2,3-dimethy1-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethy1-2-
butyl (-CH(CH3)C(CH3)3, and octyl (-(CI-12)7C113),
"Alkoxy" means a group having the formula -0-alkyl, in which an alkyl
group, as defined above, is attached to the parent molecule via an oxygen
atom. The
alkyl portion of an alkoxy group can have 1 to 20 carbon atoms (i.e., Ci-C20
alkoxy),
1 to 12 carbon atoms(i.e., C1-C12 alkoxy), or 1 to 6 carbon atoms( i.e., C1-C6
alkoxy).
Examples of suitable alkoxy groups include, but are not limited to, methoxy (-
0-CH3
or -0Me), ethoxy (-0CH2CH3 or -0Et), t-butoxy (-0-C(CH3)3 or -0tBu) and thc
like.
"Haloalkyl" is an alkyl group, as defined above, in which one or more
hydrogen atoms of the alkyl group is replaced with a halogen atom. The alkyl
portion
of a haloalkyl group can have 1 to 20 carbon atoms (i.e., C1-C20 haloalkyl), 1
to 12
carbon atoms(i. e. , Cl-C!2 haloalkyl), or 1 to 6 carbon atoms(i.e. , C1-C6
alkyl).
Examples of suitable haloalkyl groups include, but are not limited to, -CF3, -
CHF2,
-CH2CF3, and the like.
"Alkenyr is a hydrocarbon containing normal, secondary, tertiary or cyclic
carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon, sp2
double
bond. For example, an alkenyl group can have 2 to 20 carbon atoms (i.e., C2-
C20
alkenyl), 2 to 8 carbon atoms (i.e., C2-CR alkenyl), or 2 to 6 carbon atoms
(i.e., C2-C6
alkenyl). Examples of suitable alkenyl groups include, but are not limited to.
ethylene
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802.C1PF2
or vinyl (-CH=CH2), ally' (-CH2CH=CH2), cyclopentenyl (-05H7), and 5-hexenyl
(-CH2CH2CH2CH2CH=CH2).
"Alkynyl" is a hydrocarbon containing normal, secondary, tertiary or cyclic
carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon, .sp
triple
bond. For example, an alkynyl group can have 2 to 20 carbon atoms (i.e., C2-
C20
alkynyl), 2 to 8 carbon atoms (i.e., C2-C8 alkyne,), or 2 to 6 carbon atoms
(i.e., C2-C6
alkynyl). Examples of suitable alkynyl groups include, but are not limited to,
acetylenic propargyl (-CH2CEECH), and the like.
"Alkylene" refers to a saturated, branched or straight chain or cyclic
hydrocarbon
radical having two monovalent radical centers derived by the removal of two
hydrogen
atoms from the same or two different carbon atoms of a parent alkane. For
example, an
alkylene group can have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or I to 6
carbon
atoms. Typical alkylene radicals include, but are not limited to, methylene (-
CH2-),
1,1-ethyl (-CH(CH3)-), 1,2-ethyl (-CH2CH2-), 1,1-propyl (-CH(CH2C13)-), 1,2-
propyl
(-CH7CH(CH3)-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH7CH2Cl2CH7-), and the
like.
"Alkenylene" refers to an unsaturated, branched or straight chain or cyclic
hydrocarbon radical having two monovalent radical centers derived by the
removal of
two hydrogen atoms from the same or two different carbon atoms of a parent
alkene.
For example, and alkenylene group can have 1 to 20 carbon atoms, 1 to 10
carbon atoms,
or 1 to 6 carbon atoms. Typical alkenylene radicals include, but are not
limited to, 1,2-
ethylene (-CH=CH-).
"Alkynylene" refers to an unsaturated, branched or straight chain or cyclic
hydrocarbon radical having two monovalent radical centers derived by the
removal of
two hydrogen atoms from the same or two different carbon atoms of a parent
alkyne.
For example, an alkynylene group can have 1 to 20 carbon atoms, 1 to 10 carbon
atoms,
or 1 to 6 carbon atoms. Typical alkynylenc radicals include, but arc not
limited to,
acetylene (-CF---C-), propargyl (-CH2C&--C-), and 4-pentynyl (-CH2CH2CH2C--).
"Amino" refers generally to a nitrogen radical which can be considered a
derivative of ammonia, having the formula ¨N(X)2, where each "X" is
independently H,
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802.C1PF2
substituted or unsubstituted alkyl, substituted or unsubstituted carbocyclyl,
substituted or
unsubstituted heterocyclyi, etc. The hybridization of the nitrogen is
approximately sp3.
Nonlimiting types of amino include -N112, -N(alkyl)2, -NH(alkyl), -
N(carbocycly1)2, -
NH(carbocycly1), -N(heterocycly1)2, -NII(heterocycly1), -N(aryl)2, -NH(ary1), -
N(alkyl)(ary1), -N(alkyl)(heterocycly1), -N(carbocycly1)(heterocycly1), -
N(ary1)(heteroary1), -N(alkyl)(heteroary1), etc. The term "alkylamino" refers
to an
amino group substituted with at least one alkyl group. Nonlimiting examples of
amino
groups include -NH2, -NH(CH3), -N(CH3)2, -NH(CH2CH3), - N(CH2CH3)2, -
NH(phenyl), -N(phenyl)z, -NH(benzyl), -N(benzy1)2, etc. Substituted alkylamino
refers
generally to alkylamino groups, as defined above, in which at least one
substituted alkyl,
as defined herein, is attached to the amino nitrogen atom. Non-limiting
examples of
substituted alkylamino includes -NH(alkylene-C(0)-0H), -NH(alkylene-C(0)-0-
alkyl),
-N(alkylene-C(0)-0H)2, -N(alkylene-C(0)-0-alkyl)2, etc.
"Aryl" means an aromatic hydrocarbon radical derived by the removal of one
hydrogen atom from a single carbon atom of a parent aromatic ring system. For
example, an aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or
6 to 10
carbon atoms. Typical aryl groups include, but are not limited to, radicals
derived from
benzene (e.g., phenyl), substituted benzene, naphthalene, anthracene,
biphenyl, and the
like.
"Arylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen
atoms bonded to a carbon atom, typically a terminal or sp3 carbon atom, is
replaced
with an aryl radical. Typical arylalkyl groups include, but are not limited
to, henzyl,
2-phenylethan-1-yl, naphthylmethyl, 2-naphthylethan- 1 -yl, naphihobenzyl,
2-naphthophenylethan-1-y1 and the like. The arylalkyl group can comprise 7 to
20
carbon atoms, e.g., the alkyl moiety is 1 to 6 carbon atOms and the aryl
moiety is 6 to
14 carbon atoms.
"Arylalkenyl" refers to an acyclic alkenyl radical in which one of the
hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon
atom, but
also an sp2 carbon atom, is replaced with an aryl radical. The aryl portion of
the
arylalkenyl can include, for example, any of the aryl groups disclosed herein,
and the
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alkenyl portion of the arylalkenyl can include, for example, any of the
alkenyl groups
disclosed herein. The arylalkenyl group can comprise 8 to 20 carbon atoms,
e.g., the
alkenyl moiety is 2 to 6 carbon atoms and the aryl moiety is 6 to 14 carbon
atoms.
"Arylalkynyl" refers to an acyclic alkynyl radical in which one of the
hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon
atom, but
also an sp carbon atom, is replaced with an aryl radical. The aryl portion of
the
arylalkynyl can include, for example, any of the aryl groups disclosed herein,
and the
alkynyl portion of the arylalkynyl can include, for example, any of the
alkynyl groups
disclosed herein. The arylalkynyl group can comprise 8 to 20 carbon atoms,
e.g., the
alkynyl moiety is 2 to 6 carbon atoms and the aryl moiety is 6 to 14 carbon
atoms.
The term "substituted" in reference to alkyl, alkylene, aryl, arylalkyl,
alkoxy,
heterocyclyl, heteroaryl, carboeyelyl, etc. . for example, "substituted
alkyl",
"substituted alkylene", "substituted aryl", "substituted arylalkyl'',
"substituted
heterocyclyl", and "substituted carbocyclyl", unless otherwise indicated,
means alkyl,
alkylene, aryl, arylalkyl, heterocyclyl, carbocycly1 respectively, in which
one or more
hydrogen atoms are each independently replaced with a non-hydrogen
substituent.
Typical substituents include, but are not limited to, -X, -Rb, -0-, =0, -01e, -
SRb,
- -1\1+Rb3, =NW), -CX3, -CN, -OCN, -SCN, -N-=C=0, -NCS, -NO, -NO2, =N2,
-N3, -NHC(=0)Rb, -0C(=0)Rb, -NHC(=0)NRb2, -S(=0)2-, -S(=0)20H, -S(=0)2Rb,
-0S(=0)20Rb, -S(--0)9NRh2, -S(=-0)Rb, -0P(=0)(0Rb)2, -P(=0)(0Rh)2, -P(=0)(0-
)2,
-P(-----0)(OH)2, -P(0)(0Rb)(0), -C(=0)Rb, -C(=0)X, _C(S)Rh, -C(0)OR', -C(0)0-,
-C(S)0Rh, -C(0)SR", -C(S)SRb, -C(0)NRb2, -C(S)NRh2, -C(=NRh)NRh2, where each
X is independently a halogen: F, Cl, Br, or I; and each Rb is independently H,
alkyl,
aryl, arylalkyl, a heterocycle, or a protecting group or prodrug moiety.
Alkylene,
alkenylene, and alkynylene groups may also be similarly substituted. Unless
otherwise
indicated, when the term "substituted" is used in conjunction with groups such
as
arylalkyl, which have two or more moieties capable of substitution, the
substituents can
be attached to the aryl moiety, the alkyl moiety, or both.
The term "prodrug" as used herein refers to any compound that when
administered to a biological system generates the drug substance, i.e., active
ingredient,
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as a result of spontaneous chemical reaction(s), enzyme catalyzed chemical
reaction(s),
photolysis, and/or metabolic chemical reaction(s). A prodrug is thus a
covalently
modified analog or latent form of a therapeutically active compound.
One skilled in the art will recognize that substituents and other moieties of
the
compounds of Formula 1-Ill and Formula IV-VI should be selected in order to
provide a
compound which is sufficiently stable to provide a pharmaceutically useful
compound
which can be formulated into an acceptably stable pharmaceutical composition.
The
definitions and substituents for various genus and subgenus of the present
compounds
are described and illustrated herein. It should be understood by one skilled
in the art
that any combination of the definitions and substituents described above
should not
result in an inoperable species or compound. "Inoperable species or compounds"
means compound structures that violates relevant scientific principles (such
as, for
example, a carbon atom connecting to more than four covalent bonds) or
compounds
too unstable to permit isolation and formulation into pharmaceutically
acceptable
dosage forms.
"Heteroalkyl" refers to an alkyl group where one or more carbon atoms have
been replaced with a heteroatom, such as, 0, N, or S. For example, if the
carbon atom of
the alkyl group which is attached to the parent molecule is replaced with a
heteroatom
(e.g., 0, N, or S) the resulting heteroalkyl groups are, respectively, an
alkoxy group (e.g.,
-OCH3, etc.), an amine (e.g., -NHCH3, -N(CH3)2, etc.), or a thioalkyl group
(e.g.,
-SCH3). if a non-terniinal carbon atom of the alkyl group which is not
attached to the
parent molecule is replaced with a heteroatom (e.g., 0, N, or S) the resulting
heteroalkyl
groups are, respectively, an alkyl ether (e.g., -CH2CH2-0-CH3, etc.), an alkyl
amine
(e.g., -CH2NHC113, -CH3N(CH3)2, etc.), or a thioalkyl ether (e.g.,-CH2-S-CH3).
If a
terminal carbon atom of the alkyl group is replaced with a heteroatom (e.g.,
0, N, or S),
the resulting heteroalkyl groups are, respectively, a hydroxyalkyl group
(e.g.,
-CH2CH2-OH), an aminoalkyl group (e.g., -CH3NH2), or an alkyl thiol group
(e.g.,
-CH2CI-13-SH). A heteroalkyl group can have, for example, 1 to 20 carbon
atoms, 1 to
10 carbon atoms, or 1 to 6 carbon atoms. A C1-C6 heteroalkyl group means a
heteroalkyl
group having 1 to 6 carbon atoms.
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"Heterocycle" or "heterocyclyr as used herein includes by way of example
and not limitation those heterocycles described in Paquette, Leo A.;
Principles of
Modern Heterocyclic Chemistry (W.A. Benjamin, New York, 1968), particularly
Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A
Series of
Monographs" (John Wiley & Sons, New York, 1950 to present), in particular
Volumes 13, 14, 16, 19, and 28; and]. Am. Chem. Soc. (1960) 82:5566. In one
specific embodiment of the invention "heterocycle" includes a "carbocycle" as
defined herein, wherein one or more (e.g. 1, 2, 3, or 4) carbon atoms have
been
replaced with a heteroatom (e.g. 0, N, or S). The terms "heterocycle" or
"heterocycly1" includes saturated rings, partially unsaturated rings, and
aromatic rings
(i.e., heteroaromatic rings). Substituted heterocyelyls include, for example,
heterocyclic rings substituted with any of the substituents disclosed herein
including
carbonyl groups. A non-limiting example of a carbonyl substituted heterocyclyl
is:
N NH
0
Examples of heterocycles include by way of example and not limitation
pyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,
tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,
furanyl,
thienyl, pyToly1, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl,
thianaphthalenyl,
indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-
piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, prTolinyl, tetrahydrofuranyl,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,
octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-
1,5,2-
dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl,
xanthenyl,
phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl,
indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly, purinyl, 4H-quinolizinyl,
phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl,
pteridinyl, 4aH-
carbazolyl, carbazolyl, B-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl,
isochromanyl,
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chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,
piperazinyl,
indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl,
benzotriazolyl,
benzisoxazolyl, oxindolyl, benzoxazolinyl, isatinoyl, and bis-
tetrahydrofuranyl:
V.
By way of example and not limitation, carbon bonded heterocycles are bonded
at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a
pyridazine, position
2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position
2, 3, 4, or 5
of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or
tetrahydropyrrole,
position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4, or 5
of an
isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position
2, 3, or 4 of
an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3,
4, 5, 6, 7, or 8
of an isoquinoline. Still more typically, carbon bonded heterocycles include 2-
pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-
pyridazinyl, 5-
pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-
pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl,
4-
thiazolyl, or 5-thiazolyl.
By way of example and not limitation, nitrogen bonded heterocycles are
bonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-
pyrroline, 3-
pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole,
pyrazoline, 2-pyrazolinc, 3-pyrazoline, piperidine, piperazine, indole,
indoline, 1H-
indazole, position 2 of a isoindole, or isoindoline, position 4 of a
morpholine, and
position 9 of a carbazole, or B-carboline. Still more typically, nitrogen
bonded
heterocycles include 1-aziridyl, 1-azetedyl, 1-imidazolyl, 1-pyrazolyl, and
1-piperidinyl.
"Heterocyclylalkyl" refers to an acyclic alkyl radical in which one of the
hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon
atom, is
replaced with a heterocyclyi radical (i.e., a heterocyclyl-alkylene- moiety).
Typical
heterocyclyl alkyl groups include, but are not limited to heterocyclyl-CH2-, 2-
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(heterocyclyl)ethan-l-yl, and the like, wherein the "heterocyclyl" portion
includes any
of the heterocyclyl groups described above, including those described in
Principles of
Modern Heterocyclic Chemistry. One skilled in the art will also understand
that the
heterocyclyl group can be attached to the alkyl portion of the heterocyclyl
alkyl by
means of a carbon-carbon bond or a carbon-heteroatom bond, with the proviso
that
the resulting group is chemically stable. The heterocyclyl alkyl group
comprises 3 to
carbon atoms, e.g., the alkyl portion of the arylalkyl group is 1 to 6 carbon
atoms
and the heterocyclyl moiety is 2 to 14 carbon atoms. Examples of
heterocyclylalkyls
include by way of example and not limitation 5-membered sulfur, oxygen, and/or
nitrogen containing heterocycles such as thiazolylmethyl, 2-thiazolyiethan-l-
yl,
15 imidazolylmethyl, oxazolylmethyl, thiadiazolylmethyl, etc., 6-membered
sulfur,
oxygen, and/or nitrogen containing heterocycles such as piperidinylmethyl,
piperazinylmethyl, morpholinylmethyl, pyridinylmethyl, pyridizylmethyl,
pyrimidylmethyl, pyrazinylmethyl, etc.
"Heterocyclylalkenyl" refers to an acyclic alkenyl radical in which one of the
20 hydrogen atoms bonded to a carbon atom, typically a terminal or sp3
carbon atom, but
also a sp2 carbon atom, is replaced with a heterocyclyl radical (i.e., a
heterocyclyl-
alkenylene- moiety). The heterocyclyl portion of the heterocyclyl alkenyl
group
includes any of the heterocyclyl groups described herein, including those
described in
Principles of Modem Heterocyclic Chemistry, and the alkenyl portion of the
heterocyclyl alkenyl group includes any of the alkenyl groups disclosed
herein. One
skilled in the art will also understand that the heterocyclyl group can be
attached to
the alkenyl portion of the heterocyclyl alkenyl by means of a carbon-carbon
bond or a
carbon-heteroatom bond, with the proviso that the resulting group is
chemically
stable. The heterocyclyl alkenyl group comprises 4 to 20 carbon atoms, e.g.,
the
alkenyl portion of the heterocyclyl alkenyl group is 2 to 6 carbon atoms and
the
heterocyclyl moiety is 2 to 14 carbon atoms.
"Heterocyclylalkynyl" refers to an acyclic alkynyl radical in which one of the
hydrogen atoms bonded to a carbon atom, typically a terminal or sp3 carbon
atom, but
also an sp carbon atom, is replaced with a heterocyclyl radical (i.e., a
heteroeyelyl-
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alkynylene- moiety). The heterocyclyl portion of the heterocyclyl alkynyl
group
includes any of the heterocyclyl groups described herein, including those
described in
Principles of Modern Heterocyclic Chemistry, and the alkynyl portion of the
heterocyclyl alkynyl group includes any of the alkynyl groups disclosed
herein. One
skilled in the art will also understand that the heterocyclyl group can be
attached to
the alkynyl portion of the heterocyclyl alkynyl by means of a carbon-carbon
bond or a
carbon-heteroatom bond, with the proviso that the resulting group is
chemically
stable. The heterocyclyl alkynyl group comprises 4 to 20 carbon atoms, e.g.,
the
alkynyl portion of the heterocyclyl alkynyl group is 2 to 6 carbon atoms and
the
heterocyclyl moiety is 2 to 14 carbon atoms.
"Heteroaryl" refers to an aromatic heterocyclyl having at least one heteroatom
in the ring. Non-limiting examples of suitable heteroatoms which can be
included in
the aromatic ring include oxygen, sulfur, and nitrogen. Non-limiting examples
of
heteroaryl rings include all of those aromatic rings listed in the definition
of
"heterocyclyl", including pyridinyl, pyrrolyl, oxazolyl, indolyl, isoindolyl,
purinyl,
furanyl, thienyl, benzofuranyl, benzothiophenyl, carbazolyl, imidazolyl,
thiazolyl,
isoxazolyl, pyrazolyl, isothiazolyl, quinolyl, isoquinolyl, pyridazyl,
pyrimidyl,
pyrazyl, etc.
"Carbocycle" or "carbocycly1" refers to a saturated (i.e., cycloalkyl),
partially
unsaturated (e.g., cycloakenyl, cycloalkadienyl, etc.) or aromatic ring having
3 to 7
carbon atoms as a monocycle, 7 to 12 carbon atoms as a bicycle, and up to
about 20
carbon atoms as a polycycle. Monocyclic carbocycles have 3 to 7 ring atoms,
still
more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring
atoms, e.g.,
arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring
atoms arranged
as a bicyclo [5.6] or [6,6] system, or spiro-fused rings. Non-limiting
examples of
monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-
cyclopcnt-l-
enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohcxyl, I -cyclohex-l-enyl,
1-
cyclohex-2-enyl, 1-cyclohcx-3-cnyl, and phenyl. Non-limiting examples of
bicyclo
carbocycles includes naphthyl, tetrahydronapthalene, and decaline.
"Carbocyclylalkyl" refers to an acyclic alkyl radical in which one of the
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hydrogen atoms bonded to a carbon atom is replaced with a carbocyclyl radical
as
described herein. Typical, but non-limiting, examples of earbocyclylalkyl
groups
include cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl,
cyclopentylmethyl
and cyclohexylmethyl.
"Arylheteroalkyl" refers to a heteroalkyl as defined herein, in which a
hydrogen atom (which may be attached either to a carbon atom or a heteroatom)
has
been replaced with an aryl group as defined herein. The aryl groups may be
bonded
to a carbon atom of the heteroalkyl group, or to a heteroatom of the
heteroalkyl group,
provided that the resulting arylheteroalkyl group provides a chemically stable
moiety.
For example, an arylheteroalkyl group can have the general formulae -alkylene-
0-aryl, -alkylene-O-alkylene-aryl, -alkylene-NH-aryl, -alkylene-NH-alkylene-
aryl,
-alkylene-S-aryl, -alkylene-S-alkylene-aryl, etc. In addition, any of the
alkylene
moieties in the general formulae above can be further substituted with any of
the
substituents defined or exemplified herein.
"Heteroarylalkyl" refers to an alkyl group, as defined herein, in which a
hydrogen atom has been replaced with a heteroaryl group as defined herein. Non-
limiting examples of heteroaryl alkyl include -CI12-pyridinyl, -CH2-pyrrolyl,
-CH2-oxazolyl, -CH2-indolyl, -CH2-isoindolyl, -CH2-purinyl, -CH2-furanyl,
-CH2-thienyl, -CH2-benzofuranyl, -CH2-benzothiophenyl, -CH2-carbazolyl,
-CH2-thiazolyl, -CH2-isoxazolyl, -CH2-Pyrazolyl, -CH2-isothiazolyl,
-CH2-quinolyl, -CH2-isoquinolyl, -CH2-pyridazyl, -CH2-pyrimidyl, -CH2-pyrazyl,
-CH(CH3)-pyridinyl, -CH(CH3)-pyrrolyl, -CH(CH3)-oxazolyl, -CH(CH3)-indolyl,
-CH(CH3)-isoindolyl, -CH(CH3)-purinyl, -CH(CH3)-furanyl, -CH(CH3)-thienyl,
-CH(CH3)-benzofuranyl, -CH(CH3)-benzothiophenyl, -CH(CH3)-carbazolyl,
-CH(CH3)-imidazolyl, -CH(CH3)-thiazolyl, -CH(CH3)-isoxazolyl,
-CH(CH3)-pyrazolyl, -CH(CH3)-isothiazolyl, -CH(CI3)-quinolyl,
-CH(CH3)-isoquinolyl, -CH(CH3)-pyridazyl, -CH(CH3)-pyrimidyl.
-CH(CH3)-pyrazyl, etc.
The term "optionally substituted" in reference to a particular moiety of the
compound of Formula I-III and Formula IV-VI (e.g., an optionally substituted
aryl
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group) refers to a moiety wherein all substituents are hydrogen or wherein one
or
more of the hydrogens of the moiety may be replaced by substituents such as
those
listed under the definition of "substituted" or as otherwise indicated.
The term "optionally replaced" in reference to a particular moiety of the
compound of Formula 1-III and Formula PV-VI (e.g., the carbon atoms of said
(C1-
C8)alkyl may be optionally replaced by ¨0-, -S-, or ¨NRa-) means that one or
more of
the methylene groups of the (Ci-C8)alkyl may be replaced by 0, 1, 2, or more
of the
groups specified (e.g., ¨0-, -S-, or ¨NRa-).
The term "non-terminal carbon atom(s)" in reference to an alkyl, alkenyl,
alkynyl, alkylene, alkenylene, or alkynylene moiety refers to the carbon atoms
in the
moiety that intervene between the first carbon atom of the moiety and the last
carbon
atom in the moiety. Therefore, by way of example and not limitation, in the
alkyl
moiety -CH2(C)H2(C*)H2CH3 or alkylene moiety -CH2(C*)H2(C*)H2CH2- the C*
atoms would be considered to be the non-terminal carbon atoms.
Certain Y and Y1 alternatives are nitrogen oxides such as +N(0)(R) or
+N(0)(0R). These nitrogen oxides, as shown here attached to a carbon atom, can
also
0 0
N+ N+
be represented by charge separated groups such as R or OR
respectively, and are intended to be equivalent to the aforementioned
representations
for the purposes of describing this invention.
"Linker" or "link" means a chemical moiety comprising a covalent bond or a
chain of atoms. Linkers include repeating units of alkyloxy (e.g.
polyethyleneoxy,
PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino, Jeffaminem);
and
diacid ester and amides including succinate, suceinamide, diglyeolate,
malonate, and
caproamide.
The terms such as "oxygen-linked", "nitrogen-linked", "carbon-linked",
"sulfur-linked", or "phosphorous-linked" mean that if a bond between two
moieties
can be formed by using more than one type of atom in a moiety, then the bond
formed
between the moieties is through the atom specified. For example, a nitrogen-
linked
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amino acid would be bonded through a nitrogen atom of the amino acid rather
than
through an oxygen or carbon atom of the amino acid.
Unless otherwise specified, the carbon atoms of the compounds of Formula I-
III and Formula IV-VI are intended to have a valence of four. In some chemical
structure representations where carbon atoms do not have a sufficient number
of
variables attached to produce a valence of four, the remaining carbon
substituents
needed to provide a valence of four should be assumed to be hydrogen. For
example,
R8
R7 / N
X\
0 \ N
0 R9
'R6
R3
1:k4 has the same -meaning as
R8
R7 / N
X2\
0 _______ CH2 N
0
R3 CH3
F
"Protecting group" refers to a moiety of a compound that masks or alters the
properties of a functional group or the properties of the compound as a whole.
The
chemical substructure of a protecting group varies widely. One function of a
protecting group is to serve as an intermediate in the synthesis of the
parental drug
substance. Chemical protecting groups and strategies for
protection/deproteetion are
well known in the art. See: "Protective Groups in Organic Chemistry", Theodora
W.
Greene (John Wiley & Sons, Inc., New York, 1991. Protecting groups arc often
utilized to mask the reactivity of certain functional groups, to assist in the
efficiency
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of desired chemical reactions, e.g. making and breaking chemical bonds in an
ordered
and planned fashion. Protection of functional groups of a compound alters
other
physical properties besides the reactivity of the protected functional group,
such as the
polarity, lipophilicity (hydrophobicity), and other properties which can be
measured
by common analytical tools. Chemically protected intermediates may themselves
be
biologically active or inactive.
Protected compounds may also exhibit altered, and in some cases, optimized
properties in vitro and in vivo, such as passage through cellular membranes
and
resistance to enzymatic degradation or sequestration. In this role, protected
compounds with intended therapeutic effects may be referred to as prodrugs.
Another
function of a protecting group is to convert the parental drug into a prodrug,
whereby
the parental drug is released upon conversion of the prodrug in vivo. Because
active
prodrugs may be absorbed more effectively than the parental drug, prodrugs may
possess greater potency in vivo than the parental drug. Protecting groups are
removed
either in vitro, in the instance of chemical intermediates, or in vivo, in the
case of
prodrugs. With chemical intermediates, it is not particularly important that
the
resulting products after deprotection, e.g. alcohols, be physiologically
acceptable,
although in general it is more desirable if the products are pharmacologically
innocuous.
"Prodrug moiety" means a labile functional group which separates from the
active inhibitory compound during metabolism, systemically, inside a cell, by
hydrolysis, enzymatic cleavage, or by some other process (Bundgaard, Hans,
"Design
and Application of Prodrugs" in Textbook of Drug Design and Development
(1991), P.
Krogsgaard-Larsen and H. Bundgaard, Eds. Harwood Academic Publishers, pp. 113-
191). Enzymes which are capable of an enzymatic activation mechanism with the
phosphonate prodrug compounds of the invention include, but are not limited
to,
amidases, esterases, microbial enzymes, phospholipases, cholinesterases, and
phosphases. Prodrug moieties can serve to enhance solubility, absorption and
lipophilicity to optimize drug delivery, bioavailability and efficacy.
A prodrug moiety may include an active metabolite or drug itself.
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Exemplary prodrug moieties include the hydrolytically sensitive or labile
acyloxymethyl esters ¨CH20C(=0)R3 and acyloxymethyl carbonates
¨C1-120C(=0)0R3 where R3 is C1¨C6 alkyl, C1¨C6 substituted alkyl, C6¨C20
aryl or
C6¨C20 substituted aryl. The acyloxyalkyl ester was used as a prodrug strategy
for
carboxylic acids and then applied to phosphates and phosphonates by Farquhar
et al
(1983) J. Pharm. Sci. 72: 324; also US Patent Nos. 4816570, 4968788, 5663159
and
5792756. In certain compounds of the invention, a prodrug moiety is part of a
phosphate group. The acyloxyalkyl ester may be used to deliver phosphoric
acids
across cell membranes and to enhance oral bioavailability. A close variant of
the
acyloxyalkyl ester, the alkoxycarbonyloxyalkyl ester (carbonate), may also
enhance
oral bioavailability as a prodrug moiety in the compounds of the combinations
of the
invention. An exemplary acyloxymethyl ester is pivaloyloxymethoxy, (POM)
¨CI-120C(=0)C(CH3)3. An exemplary acyloxyrnethyl carbonate prodrug moiety is
pivaloyloxymethylcarbonate (POC) ¨CH20C(=0)0C(C113)3.
The phosphate group may be a phosphate prodrug moiety. The prodrug
moiety may be sensitive to hydrolysis, such as, but not limited to those
comprising a
pivaloyloxymethyl carbonate (POC) or POM group. Alternatively, the prodrug
moiety may be sensitive to enzymatic potentiated cleavage, such as a lactate
ester or a
phosphonamidate-ester group.
Aryl esters of phosphorus groups, especially phenyl esters, are reported to
enhance oral bioavailability (DeLambert et al (1994)J. Med. Chem. 37: 498).
Phenyl
esters containing a carboxylic ester ortho to the phosphate have also been
described
(Khamnei and Torrence, (1996).1. Med. Chem. 39:4109-4115). Benzyl esters are
reported to generate the parent phosphonie acid. In some cases, substituents
at the
ortho-orpara-position may accelerate the hydrolysis. Benzyl analogs with an
acylated phenol or an alkylated phenol may generate the phenolic compound
through
the action of enzymes, e.g. esterases, oxidases, etc., which in turn undergoes
cleavage
at the benzylic C-0 bond to generate the phosphoric acid and the quinone
methide
intermediate. Examples of this class of prodrugs are described by Mitchell et
al
(1992) J. Chem. Soc. Perkin Trans. 12345; Brook et al WO 91/19721. Still other
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benzylic prodrugs have been described containing a carboxylic ester-containing
group
attached to the benzylic methylene (Glazier et al WO 91/19721). Thio-
containing
prodrugs are reported to be useful for the intracellular delivery of
phosphonate drugs.
These proesters contain an ethylthio group in which the thiol group is either
esterified
with an acyl group or combined with another thiol group to form a disulfide.
Deesterification or reduction of the disulfide generates the free thio
intermediate
which subsequently breaks down to the phosphoric acid and episulfide (Puech et
al
(1993) Antiviral Res., 22: 155-174; Benzaria eta! (1996)1. Med. Chem. 39:
4958).
Cyclic phosphonate esters have also been described as prodrugs of phosphorus-
containing compounds (Erion et al, US Patent No. 6312662).
It is to be noted that all enantiomers, diastereomers, and racemic mixtures,
tautomers, polymorphs, pseudopolymorphs of compounds within the scope of
Formula I, Formula II, Formula III, Formula IV, Foimula V, or Formula VI and
phaimaceutically acceptable salts thereof are embraced by the present
invention. All
mixtures of such enantiomers and diastereomers are within the scope of the
present
invention.
A compound of Formula I-III and Formula IV-VI and its pharmaceutically
acceptable salts may exist as different polymorphs or pseudopolymorphs. As
used
herein, crystalline polymorphism means the ability of a crystalline compound
to exist
in different crystal structures. The crystalline polymorphism may result from
differences in crystal packing (packing polymorphism) or differences in
packing
between different conformers of the same molecule (conformational
polymorphism).
As used herein, crystalline pscudopolymorphism means the ability of a hydrate
or
solvate of a compound to exist in different crystal structures. The
pseudopolymorphs
of the instant invention may exist due to differences in crystal packing
(packing
pseudopolymorphism) or due to differences in packing between different
conformers
of the same molecule (conformational pseudopolymorphism). The instant
invention
comprises all polymorphs and pseudopolymorphs of the compounds of Formula I-
III
and Formula IV-VI and their pharmaceutically acceptable salts.
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A compound of Formula I-III and Formula IV-VI and its pharmaceutically
acceptable salts may also exist as an amorphous solid. As used herein, an
amorphous
solid is a solid in which there is no long-range order of the positions of the
atoms in
the solid. This definition applies as well when the crystal size is two
nanometers or
less. Additives, including solvents, may be used to create the amorphous forms
of the
instant invention. The instant invention comprises all amorphous forms of the
compounds of Formula I-III and Formula IV-VI and their pharmaceutically
acceptable salts.
Selected substituents comprising the compounds of Formula I-III and Formula
IV-VI are present to a recursive degree. In this context, "recursive
substituent" means
that a substituent may recite another instance of itself. Because of the
recursive
nature of such substituents, theoretically, a large number of compounds may be
present in any given embodiment. For example, Rx comprises a RY substituent.
RY
can be R. R can be W3. W3 can be W4 and W4 can be R or comprise substituents
comprising R. One of ordinary skill in the art of medicinal chemistry
understands
that the total number of such substituents is reasonably limited by the
desired
properties of the compound intended. Such properties include, by way of
example
and not limitation, physical properties such as molecular weight, solubility
or log P,
application properties such as activity against the intended target, and
practical
properties such as ease of synthesis.
By way of example and not limitation, W3 and RY are recursive substituents in
certain embodiments. Typically, each recursive substituent can independently
occur
20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0,
times in a given
embodiment. More typically, each recursive substituent can independently occur
12
or fewer times in a given embodiment. Even more typically, each recursive
substituent can independently occur 3 or fewer times in a given embodiment.
For
example, W3 will occur 0 to 8 times, RY will occur 0 to 6 times in a given
embodiment. Even more typically, W3 will occur 0 to 6 times and RY will occur
0 to 4
times in a given embodiment.
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Recursive substituents are an intended aspect of the invention. One of
ordinary skill in the art of medicinal chemistry understands the versatility
of such
substituents. To the degree that recursive substituents are present in an
embodiment
of the invention, the total number will be determined as set forth above.
The modifier "about" used in connection with a quantity is inclusive of the
stated value and has the meaning dictated by the context (e.g., includes the
degree of
error associated with measurement of the particular quantity).
The compounds of the Formula I-III and Formula IV-VI may comprise a
I I
phosphate group as R7, which may w2 be a prodrug moiety
wherein
each Y or Y1 is, independently, 0, S, NR, +N(0)(R), N(OR), +N(0)(0R), or
N¨NR,;
WI and W2, when taken together, are ¨Y3(C(RY)2)3Y3-; or one of WI or W2
together
with either R3or R4 is ¨Y3- and the other of WI or tf\r2 is Formula Ia; or WI
and W2 are
each, independently, a group of Formula la:
Y1\
Rx ______________________________ Y2 __ P ____ Y2 ___
y2
Rx
M2
wherein:
each Y2 is independently a bond, 0, CR7, NR, 'N(0)(R), N(OR), 'N(0)(0R),
N¨NR,, S, S¨S, S(0), or S(0)2;
each Y3 is independently 0, S, or NR;
M2 is 0,1 or 2;
each RY is independently H, F, Cl, Br, I, OH, R, -C(=Y1)R, -C(=Y1)0R, -
C(=YI)N(R),, -N(R)2, -+N(R)3, -SR, -S(0)R, -S(0)2R, -S(0)(04 -S(0)2(04 -
OC(¨Y1)R, -0C(=Y1)0R, -0C(=Y1)(N(R)2), -SC(=YI)R, -SC(=Y1)0R, -
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SC(=Y1)(N(R)2), -N(R)C(Y1)R, -N(R)C(=Y1)0R, or -N(R)C(=YI)N(R)2, ¨SO2NR2,
¨CN, ¨N3, ¨NO2, ¨OR, a protecting group or W3; or when taken together, two RY
on
the same carbon atom form a carbocyclic ring of 3 to 7 carbon atoms;
each Rx is independently RY, a protecting group, or the formula:
Yi
RY RY yi
y2
_ Y2
\ /
Mla M1 2c Mic Mid
wherein:
Mla, Mle, and Mid are independently 0 or 1;
Ml2c is 0, I, 2,3, 4, 5, 6, 7, 8, 9, 10,11 or 12;
each R is H, halogen, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8)
alkenyl,
(C1-C8) substituted allcenyl, (C1-C8) alkynyl, (C2-C8) substituted alkynyl,
C6¨C20 aryl,
C6¨C20 substituted aryl, C2¨C20 heterocycle, C2¨C20 substituted heterocyclyl,
arylalkyl, substituted arylalkyl or a protecting group;
W3 is W4 or W5; W4 is R, -C(YI)RY, -C(Y1)W5, -SO2RY, or -S02W5; and W5 is
carbocycle or a heterocycle wherein W5 is independently substituted with 0 to
3 RY
groups.
W5 carbocycles and W5 heterocycles may be independently substituted with 0
to 3 RY groups. W5 may be a saturated, unsaturated or aromatic ring comprising
a
mono- or bicyclic carbocycle or heterocycle. W5 may have 3 to 10 ring atoms,
e.g., 3
to 7 ring atoms. The W5 rings are saturated when containing 3 ring atoms,
saturated
or mono-unsaturated when containing 4 ring atoms, saturated, or mono- or di-
unsaturated when containing 5 ring atoms, and saturated, mono- or di-
unsaturated, or
aromatic when containing 6 ring atoms.
A W5 heterocycle may be a monocycle having 3 to 7 ring members (2 to 6
carbon atoms and 1 to 3 heteroatoms selected from N, 0, P, and S) or a bicycle
having
7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from
N, 0,
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P, and S). W5 heterocyclic monocycles may have 3 to 6 ring atoms (2 to 5
carbon
atoms and 1 to 2 heteroatoms selected from N, 0, and S); or 5 or 6 ring atoms
(3 to 5
carbon atoms and 1 to 2 heteroatoms selected from N and S). W5 heterocyclic
bicycles have 7 to 10 ring atoms (6 to 9 carbon atoms and 1 to 2 heteroatoms
selected
from N, 0, and S) arranged as a bicyclo [4,5], [5,5], [5,6], or [6,6] system;
or 9 to 10
ring atoms (8 to 9 carbon atoms and Ito 2 hetero atoms selected from N and S)
arranged as a bicyclo [5,6] or [6,6] system. The W5 heterocycle may be bonded
to Y2
through a carbon, nitrogen, sulfur or other atom by a stable covalent bond.
W5 heterocycles include for example, pyridyl, dihydropyridyl isomers,
piperidine, pyridazinyl, pyrimidinyl, pyrazinyl, s-triazinyl, oxazolyl,
imidazolyl,
thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, furanyl, thiofuranyl, thienyl,
and
pyrrolyl. W5 also includes, but is not limited to, examples such as:
_1 N
N
,
rfN
N s,
(5-
S , and
W5 carbocycles and heterocycles may be independently substituted with 0 to 3
R groups, as defined above. For example, substituted W5 carbocycles include:
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OH
/
/ CI
N
\
\OH /
CI
N/ \0
\ _______________________________________ /
1 NH2
\ / \
NH / ¨N NH
/ \ ___ /
/ \ / \S / \
o / /¨
¨N N SO2
Examples of substituted phenyl earbocycles include:
HN-)./._ HN 0--\
NH NMe2 2 e NH2
111 0 0 0
1... -.4. N.
0--\ <0 0¨\
0 0---\
NH
NH2 ) __ NH2 lit ) __ NH2
0 0
14.
\
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Embodiments of R7 or R7 together with R4 include the structures
o R
0
H __________ 0¨P ______
RY ___________________________________
0 s
OH 0
-1-3 Ar/
R R a a
RY'0,11)-( T N-p-o,b 0 /b RY`N¨
pi ,b
H II "
0 0 0 0
RY,S RR a RY 0 R R
> 0
s
0 R R R
0 R
0
R R ,or Ar =
wherein
a is the point of attachment to R7;
b is the point of attachment to R4;
Ar is phenyl or naphthyl, wherein the phenyl and naphthyl are optionally
substituted with 1-3 R2 groups;
each RY is independently (C1-C8) alkyl or C5¨C6 earbocyclyl, wherein the
alkyl and carbocyclyl are optionally substituted with 1-3 R2 groups;
each R is independently I-I, (C1-C6) alkyl, or arylalkyl; and
each R2 is independently halogen, CN, N(R)2, OR, -SR, -S(0)R, -S(0)2R, -
S(0)(0R), -S(0)2(0R), -C(=0)R, -C(=0)0R, or C(=0)N(R)2.
2
Embodiments of W of Formula 1-Ill and Formula IV-VI
compounds include substructures such as:
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801CIPF2
0
\ y2
y2b
RX
wherein each Y2b is, independently, 0 or N(R). In another aspect of this
embodiment, each Y21' is 0 and each le is independently:
0
R R
-y2 y2
Ml 2C
wherein Ml 2c is 1, 2 or 3 and each Y2 is independently a bond, 0, CR,, or S.
In another aspect of this embodiment, one Y2b_Rx is NI-1(R) and the other Y2b-
le is 0-
Rx wherein re is:
R R 0
R3
Ml 2C
wherein 1\412c is 2. In another aspect of this embodiment, each Y2b is 0 and
each le is independently:
R R 0
3
Ml2c
wherein M12c is 2. In another aspect of this embodiment, each Y2b is 0 and
each le is independently:
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0
R R
y2
M12c
wherein M1 2c is I and Y2 is a bond, 0, or CR1.
w
2
Other embodiments of w of Foumulas I-111 and Formulas IV-VI
compounds include substructures such as:
RY
/3 _____________________________________
/ RY
____________________________________________ RY
\z,P\
RY
Y3
RY
RY
wherein each Y3 is, independently, 0 or N(R). In another aspect of this
embodiment, each Y3 is 0. In another aspect of this embodiment, the
substructure is:
0
\O
RY
wherein RY is W5 as defined herein.
W1
Another embodiment of vv2 of Formula I-III and Formula IV-VI
includes the substructures:
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v2
0
9
wherein each Y2' is, independently, 0, N(R) or S.
W1 I
I I ___________________________________
P
Another embodiment of vv2
of Formula I-III and Formula IV-VI
compounds includes the substructures wherein one of WI or W2 together with
either
R3 or R4 is ¨Y3- and the other of W1 or W2 is Formula Ia. Such an embodiment
is
represented by a compound of Formula lb selected from:
R8
/
X N
0 ¨CH2 x2\ N
0
R9
"P
6
p
R "
Y3
R4 R2
R8
/
X N
0¨CH2 x2\ N
/ ON
R9
R1 R6
Y3
R3 R2 9
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R8
/ N
x2
0 _________________________ CH2 \
0N R9
P
R5
R R"
Y
zY3
R3 R2 or
R8
/ N
0¨CH2 x2\ N
0 R9
P
R5 ow'
==õ,
Ri R
,y3
R4 R2
Formula lb
In another aspect of the embodiment of Formula lb, each Y and Y3 is 0. In
another aspect of the embodiment of Formula lb, WI or W2 is x
each Y, Y3 and
Y2b is 0 and It.' is:
0
R R
M1 2c
wherein Ml2c is 1, 2 or 3 and each Y2 is independentlya bo21R.71,x0; , CR2, or
S.
is y
In another aspect of the embodiment of Formula Ib, W1 or W2 each Y, Y3
and Y2b is 0 and le is:
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0
R R
3
M12c
wherein Ml2c is 2. In another aspect of the embodiment of Formula lb, W' or
W2 is y213tc :-.x; each Y, Y3 and Y2b is 0 and Rx is:
0
R R
0
M12c
wherein M12c is 1 and Y2 is a bond, 0, or CR2.
vv1
2
Another embodiment of w of Formula I-III and Formula IV-VI
compounds includes a substructure:
y2
y2,"
wherein W5 is a carbocycle such as phenyl or substituted phenyl. In another
aspect of this embodiment, the substructure is:
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______________________________________ (R)o-3
0
RY
y2b
OR
0
wherein Y2b is 0 or N(R) and the phenyl carbocycle is substituted with 0 to 3
R groups. In another aspect of this embodiment of the substructure, Rx is:
0
R R
y2
Ml 2c
wherein M1 2c is 1,2 or 3 and each Y2 is independently a bond, 0, CR2, or S.
vv1--
vv2
Another embodiment of of Formula I-III and Formula IV-VI
includes substructure:
_________________________________ (RY)0-3 __________ (RY)0-3
0 0
CH3 ..õ,0 CH3
OR OR
0 or 0
The chiral carbon of the amino acid and lactate moieties may be either the R
or
S configuration or the racemic mixture.
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Ii ____________________________________
Another embodiment of w2
of Formula I-III and Formula IV-VI
is substructure
0
_________________________________ Y2
- 2
wherein each Y2 is, independently, ¨0- or -NH-. In another aspect of this
embodiment, RY is (C1-C8) alkyl, (Ci-C8) substituted alkyl, (C2-C8) alkenyl,
(C2-C8)
substituted alkenyl, (C2-C8) alkynyl or (C2-C8) substituted alkynyl. In
another aspect
of this embodiment, RY is (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8)
alkenyl,
(C2-C8) substituted alkenyl, (C2-C8) alkynyl or (C2-C8) substituted alkynyl;
and R is
Cl-I3. In another aspect of this embodiment, RY is (CI-C8) alkyl, (C1 -C8)
substituted
alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl or (C2-
C8)
substituted alkynyl; R is CH; and each Y2 is -NH-. In a aspect of this
embodiment,
W1 and W2 are, independently, nitrogen-linked, naturally occurring amino acids
or
naturally occurring amino acid esters. In another aspect of this embodiment,
W1 and
W2 arc, independently, naturally-occurring 2-hydroxy carboxylic acids or
naturally-
occurring 2-hydroxy carboxylic acid esters wherein the acid or ester is linked
to P
through the 2-hydroxy group.
2
Another embodiment of wof Formula I, Formula II, Formula
III, Formula IV, Formula V, or Formula VI is substructure:
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0
II
aF2'`
0,
Rx .
In one aspect of this embodiment, each .R.' is, independently, (C1-C8) alkyl.
In
another aspect of this embodiment, each Rx is, independently, C6-G20 aryl or
C6-C20
substituted aryl.
In a preferred embodiment,
0
I I
W1-----1 1
w2
is selected from
0 0
_
,.¨NHR
R R 0
0 0 2
___________________ S __________________________ S\
R21
_R R. 2 \if C(R)3, R
0 = - 0 =
,
0 0
Fl H
\ CH3 põ---0--______
0...- ------ \
I0,,,,y-----
(R)n or W5 .
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W1 I
Another embodiment of w2
of Formulas I-III and Formula IV-
VI is substructure
0
w2
wherein W1 and W2 are independently selected from one of the formulas in
Tables 20.1-20.37 and Table 30.1 below. The variables used in Tables 20.1-
20.37
(e.g., W23, R21, etc.) pertain only to Tables 20.1-20.37, unless otherwise
indicated.
The variables used in Tables 20.1 to 20.37 have the following definitions:
each R21 is independently H or (CI-C8)alkyl;
each R22 is independently H, R21, R23 or R24 wherein each R24 is independently
substituted with 0 to 3 R23;
1 5 each R23 is independently R23a, R23h, R23c or R23d, provided that when
R23 is
bound to a heteroatom, then R23 is R23 or R23d;
each R2" is independently F, Cl, Br, I, -CN, N3 or -NO2;
each R23b is independently y21;
each R23c is independently ¨R2x, -N(z2x)(R2x), _sR2x, _s(0)R2x, -S(0)2R2', _
S(0)(0R2x), -S(0)2(0R2x), -0C(= y2i)R2x, _oc(=y21)0R2x,
_oc(=y21)(N(R2x)(R2x)),
-SC(=y21)R2x; _sc(_y21)0R2x; _sc(=y21)(N(R2x)(R2x)), _N(R2x)c(=y21)R2x, _
N(R2x)C(=Y21)0R2x, or -N(R2x)C(=y21)(N(R1x)(R2x)) ;
each R23d is independently -C(= y2i)R2x, _c(=y21)0R2x or _
C(=Y21 )(N(R2x)(R2x));
each R2x is independently H, (Ci-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl,
aryl, heteroaryl; or two R2x taken together with a nitrogen to which they are
both
attached form a 3 to 7 membered heterocyclic ring wherein any one carbon atom
of
said heterocyclic ring can optionally be replaced with -0-, -S- or ¨NR21-; and
wherein
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one or more of the non-terminal carbon atoms of each said (CI-C8)alkyl may be
optionally replaced with -0-, -S- or
each R24 is independently (Ci-C8)alkyl, (C2-C8)alkenyl, or (C2-C8)alkynyl;
each R25 is independently R24 wherein each R24 is substituted with 0 to 3 R23
groups;
each R25a is independently (C1-C8)alky1ene, (C2-C8)alkenylene, or (C2-
C8)alkynylene any one of which said (C1-C8)alkylene, (C2-C8)alkenylene, or (C2-
C8)alkynylene is substituted with 0-3 R23 groups;
23 is independently W24 w25;
each W
each W24 is independently R25, -c(=y2i)R25, _c(=y21)w25, _S02R25, Or -
S02W25;
each W25 is independently carbocycle or heterocycle wherein W25 is
independently substituted with 0 to 3 R22 groups; and
each Y21 is independently 0 or S.
Table 20.1
OW230R 0
0 0 0
1 2 3
?
R21 0 H 0 CH3
0 0 0
4 5 6
0
0 CH3
0 0
7 8
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Table 20.2
0 0
o
0 CH3
9 10
CH3
CH3
0
11
Table 20.3
CH3 CH3 CH3
4.4 w23
0 0 R25 0' R24
0 0 0
12 13 14
CH3 CH3 CH3
R21
0 CH3
16 17
CH3 CH3
H3 CH3
0 0
18 19
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Table 20.4
CH3 CH3
o 0 CH3
20 21
CH3 CH3
0
22
Table 20.5
H3Cõ,
,
-0 w23 R2j 'R24
0
23 24 25
0CH3
0
26 27 28
H3C,,
0 0 CH3
0
29 30
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Table 20.6
?OCH3
o 0 CH3
31 32
CI-13
CH3 0
0
33
Table 20.7
w23 w23 w23
w23 R25 R24
0
34 35 36
w23 R25 R25
R21
w23 R25
o 0 0
37 38 39
R25 R25
R24 ?\0 R21
0
0
40 41
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Table 20.8
R24 R24 R24
?,
R24
0
0
44
42 43
R24 R21 R21
o
R21 OW23 0- R25
0 0 0
45 46 47
R21 R21
R25 cy Rz
0 0
48 49
Table 20.9
N R25 R24
H 0 H 0 H 0
50 51 52
f)N
- -3
H 0 H 0 H 0
53 54 55
CH3
1
H 0 H 0
56 57
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Table 20.10
e)
H 0 H 0 CH3
58 59
CH3
.3
H 0
Table 20.11
CH3 CH3 CH3
N(IN W23 R25 R24
H 0 H 0 H 0
61 62 63
CH3 CH3 CH3
N" H CH3
H 0 H 0 H 0
64 65 66
CH3 CH3
N3 N CH3
111 0 H 0
67 68
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Table 20.12
CH3 CH3
H 0 H 0 CH3
69 70
CH3 CH3
CH3
H 0
71
Table 20.13
CH3 CH3 CH3
H3C
?H3C ?H30
R25
N R24
H 0 H 0 H 0
72 73 74
OH 3 CH3 CH3
H3C ,H3C ,H3C
R21 rNXICCH3
H 0 H 0 H 0
75 76 77
CH3 CH3
JH3C H3C
r
NC H3
H 0 H 0
78 79
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Table 20.14
CH3 CH3
H3C
0
H3 H3
1
H 0 H 0 CH3
80 81
CH3 CH3
r
CH3
H 0
82
Table 20.15
w23 w23 w23
ej\
N R2J R24
HI
H 0 H 0 0
83 84 85
w23 R25 R25
0
a
N' R21 NC)1/V23 ''R25
H 0 H 0 H 0
86 87 88
R25 R25
R24 R21
'
H 0 H 0
89 90
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Table 20.16
R24 R24 R24
? ?,_
-,,
NNI-W23 4-, N R25 NN '''''''',./ -=R24
I
HI 0 I
H 0 H 0
91 92 93
R24 R21 R21
--N----\---- '-w23 --- N ---- \...---' "-
R25
I H 0 Ili 0 HI 0
94 95 96
R21 Rzi
I
?.,.._
-.. N.------...õ--- 0-.. R24 elõ ,õ ,-0.,
N. ---" R21
I I
H 0 H 0
97 98
Table 20.17
Nw23 ?N -C)R25 /Cs'
N R24
1 1 1
R23 0 R23 0 R23 0
99 100 101
N.--^'=.,õ---0-H /\ N.----',,,-- ,CH3
1 1 1
R23 0 R23 0 R23 0
102 103 104
/- ,õ----O.0 H3 4-N, N õ ---- \.,_- -õõ---- \
N,...., 13
I 1
R23 0 R23 0
'
105 106
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Table 2018.
R23 0 R23 0 CH3
107 108
CH3
CH3
R23 0
109
Table 20.19
CH3 CH3 CH3 CH3
R23 0 R23 0 R23 0 R23 0
110 111 112 113
CH3 CH3 CH3 CH3
0õ.
H N CH3 N CH3
R23 0 R23 0 R23 0 R23 0
114 115 116 117
Table 20.20
CH3 CH3
NI
R23 0 R23 0 CH3
118 119
CH3 CH3
()\
.3
R23 0
120
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Table 20.21
H30 cH3 H3c cH3 H3c cH3 H3c cH3
e* N>0 w23 ?=,=,,,N>R-- NK0 , ?\ >C)R,__, 0A /\ N A)R21
I I I I
R23 0 R23 0 R23 0 R23 0
121 122 123 124
H3C\ /CH3 H3C CH3 H3C CH3 H3C\ /CH3
?
'N27'H ?N><(()CH3 ,NX,õ,-0,,,,_,,,.0 H3
CH3
I I I I
R23 0 R23 0 R23 0 R23 0
125 126 127 128
Table 20.22
H30 CH3 H30\ /CH3 H3C CH3 CH3
P\N>0CH3 41,õ >,-0.,C H3
N N CH3
I \ I
R23 0 Rõ¨ 0 CH3 R23 0
129 ' 130 131
Table 20.23
w23 w23 w23 w23
?
N------(:)'`R25 ?-N--N.-----0"-R24 ''--N -''''''------ `= R21
I I 1 I I
R23 0 R23 0 R23 0 R23 0
132 133 134 135
R25 R25 R25 R25
I I I I
R23 0 R23 0 R23 0 R23 0
136 137 138 139
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Table 20.24
R24 R24 R24 R24
l
N Cs'' W23 N .--(:)µ' R25 N R21
R24
I I I I
R23 0 R23 0 R23 0 R23 0
140 141 142 143
R21 R21 R21 R21
'O23 (1-\.N.-----' `,R25 ('-'---N------\..----"a"-R24
I I 1 I
R23 0 R23 0 R23 0 R23 0
144 145 146 147
Table 20.25
, ,, ,.R21 fo,
W23 R25 R24 H R23
148 149 150 151 152 153
,.. w23 ?, R25 eo,_ R24 e),,.. R21 o.),,, _ H
eo, _R23
154 155 156 157 158 159
Table 20.26
?,,,,,,, ..., R25 e),,...., ..., R24 ?.õ..., .õ, R21
N N N N N N
I I
HH HI
II
-1
H H
160 161 162 163 164 165
= õyv23 ?...,...õ. ,.., R21
N N N N N N
I I 1 I 1 I
R23 R23 R23 R23 R23 R23
166 167 168 169 170 171
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Table 20.27
0
? p25a to, p25a
o,- R25
172 173
0 0
p25a ?, R25a
`1R24 "-R21
174 175
0 0
,,R25a ,,R25a
0 H 0 CH3
176 177
0 0 CH3
? 025a
3
0 0
178 179
Table 20.28
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H,C
0 0
,,R25a H3
O 0 0
CH3
180 181
0
0 CH3
0
= R25a
R25a
CH3
CH3
182 183
O 0 4111
= R25a
0"0 410 0 0
184 185
Table 20.29
O 0
0 0 R2'g
186 187
O 0
R24 0 0 R21
188 189
O 0
O 0 H 0 0 CH3
190 191
O 0 CH3
3
O 0 0 0
192 193
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Table 20.30
0
0
0 0
0 0
OH3
194 195
0 0 CH3
CH3
O'CH,
CH3
CH3
196 197
0 0
0 0 0 0
199
198
Table 20.31
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0
(i\R25 ,-W23 0
0 0 0
R25
200
201
0
0
R25 R21
0 0 0
0 0 O-
202
203
0 0
0,
,R258 ?
0 ,,CH3
0 0 0
204
205 H C
0 3
o
R25a
(0, R25a 0 0 0
207
206
Table 20.32
'CH3 0 CH3
? R25a el R25a
/\.
0 0 0 0 0 0 CH3
208 209
0 CH 3_ 0
CH3
0' 0 0 CH3 0 0 0
211 CH3
210
0
0
R25a
? R258 0 0 0
0 0 0
212 213
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Table 20.33
0
\/\/23 0
O 0 0
0 0 0
214
215
0
0
O 0 0
R21
0 0 0
216
217
0
0
O 0 0
0 0 0
218
219 HO
0 3
0 0 0
O 0 0 CH3
221
220
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Table 20.34
0 CH3 0 CH3
0 0 0 0 0 0 CH3
222 223
0 CH3_
0
-/¨\.
0 0 0 CH3
0 0 0
224 225 CH3
0
0
ed=-=õ.
0 0 0 ISO
0 0 0
226 227
Table 20.35
R25a 0 R258 0
Th.25
2 2280 290
R25a 0 R25a 0
M24 M21
2 2300 310
R25a 0 R25a 0
-'1\1 '.W23 R25
H 2320 H 2330
4), R25a 0 R25a 0
R24 -N-'
H 234 H 2350
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Table 20.36
e), R25a 0 e R.?..-5a 0
1\1 =-=====.õ--" '-
'"-. vv23
I I
R23 0 R23 0
236 237
,d, R25
1\ R25a 0
"--.....--- '-= R24
-'r
'1\r '''--- 'R21
I 1
R23 0 R23 0
238 239
.----''''-= .-%--- ",-,
I
-,,, õ..---:=,õ,,...,...)\,-
0 R22 R25
240 241
/1
I
R23 0
242 243
Table 20.37
/ ?
R22 --F-R25
244 '''-..,x1 245
? ?
oMj '0
R23
246
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Table 30.1
CH3 CH3
0CH3 e',.,
? N--11---- N CH3
I I
H 0
67 H 068
CH3 CH3
N N
I I
H690 H 70 0 CH3
CH3 CH3 CH3
CH3
N 0
I I
H 071 H2580
? 0 CH 4._ ...,.....,...- 3 , N
0............õ...--.,
N CH3
I I
H2480 H 0 249
CH3 ------"'''.NCH3
?
0...,.......õ.....--...,.._õ,-CH3 (1,...... .õ..--
y0õ.............."....õ,,,,...-
N N
I I
H2500 H 251
0 CH3
o ti N
I I
H 0 252 H 0 253 254
CI
0 CF3 \ s 0
0 HN-i
255 256 257 .
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Embodiments of Rx include esters, carbamates, carbonates, thioesters, amides,
thioamides, and urea groups:
R R /R
Y1
1
RY
RY y2 y2'
M12a
and M12a
Any reference to the compounds of the invention described herein also
includes a reference to a physiologically acceptable salt thereof. Examples of
physiologically acceptable salts of the compounds of the invention include
salts
derived from an appropriate base, such as an alkali metal or an alkaline earth
(for
example, Na+, Li+, K+, Ca+2 and Mg+2), ammonium and NR4+ (wherein R is
defined herein). Physiologically acceptable salts of a nitrogen atom or an
amino
group include (a) acid addition salts formed with inorganic acids, for
example,
hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acids, phosphoric
acid,
nitric acid and the like; (b) salts formed with organic acids such as, for
example,
acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric
acid, gluconic
acid, citric acid, malic acid, ascorbic acid, benzoic acid, isethionic acid,
lactobionic
acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,
naphthalenesulfonic
acid, methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid,
naplithalenedisulfonic acid, polygalacturonic acid, malonic acid,
sulfosalicylic acid,
glycolic acid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid,
phthalic
acid, mandelic acid, lactic acid, ethanesulfonic acid, lysine, arginine,
glutamic acid,
glycine, serine, threonine, alanine, isoleucine, leucine and the like; and (c)
salts
foinied from elemental anions for example, chlorine, bromine, and iodine.
Physiologically acceptable salts of a compound of a hydroxy group include the
anion
of said compound in combination with a suitable cation such as Na- and NR4+.
For therapeutic use, salts of active ingredients of the compounds of the
invention will be physiologically acceptable, i.e. they will be salts derived
from a
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physiologically acceptable acid or base. However, salts of acids or bases
which are
not physiologically acceptable may also find use, for example, in the
preparation or
purification of a physiologically acceptable compound. All salts, whether or
not
derived form a physiologically acceptable acid or base, are within the scope
of the
present invention.
Finally, it is to be understood that the compositions herein comprise
compounds of the invention in their un-ionized, as well as zwitterionic form,
and
combinations with stoichiometric amounts of water as in hydrates.
The compounds of the invention, exemplified by Formula I-III and Formula
IV-VI may have chiral centers, e.g. chiral carbon or phosphorus atoms. The
compounds of the invention thus include raccmic mixtures of all stercoisomers,
including enantiomers, diastercomers, and atropisomcrs. In addition, the
compounds
of the invention include enriched or resolved optical isomers at any or all
asymmetric,
chiral atoms. In other words, the chiral centers apparent from the depictions
are
provided as the chiral isomers or racemic mixtures. Both racemie and
diastereomerie
mixtures, as well as the individual optical isomers isolated or synthesized,
substantially free of their enantiomeric or diastereomeric partners, are all
within the
scope of the invention. The racemie mixtures are separated into their
individual,
substantially optically pure isomers through well-known techniques such as,
for
example, the separation of diastereomeric salts formed with optically active
adjuncts,
e.g., acids or bases followed by conversion back to the optically active
substances. In
most instances, the desired optical isomer is synthesized by means of
stereospeeific
reactions, beginning with the appropriate stereoisomer of the desired starting
material.
The term "chiral" refers to molecules which have the property of non-
superimposability of the mirror image partner, while the term "achiral''
refers to
molecules which are superimposable on their mirror image partner.
The term. "stercoisomers" refers to compounds which have identical chemical
constitution, but differ with regard to the arrangement of the atoms or groups
in space.
"Diastereomer" refers to a stereoisomer with two or more centers of chirality
and whose molecules are not mirror images of one another. Diastereomers have
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different physical properties, e.g. melting points, boiling points, spectral
properties,
and reactivities. Mixtures of diastereomers may separate under high resolution
analytical procedures such as electrophoresis and chromatography.
"Enantiomers" refer to two stereoisomers of a compound which are non-
superimposable mirror images of one another.
Stereochemical definitions and conventions used herein generally follow S. P.
Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book
Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic
Compounds (1994) John Wiley Sz. Sons, Inc., New York. Many organic compounds
exist in optically active forms, i.e., they have the ability to rotate the
plane of plane-
polarized light. In describing an optically active compound, the prefixes D
and L or R
and S are used to denote the absolute configuration of the molecule about its
chiral
center(s). The prefixes d and 1, D and L, or (+) and (-) are employed to
designate the
sign of rotation of plane-polarized light by the compound, with S, (-), or 1
meaning
that the compound is levorotatory while a compound prefixed with R, (+), or d
is
dextrorotatory. For a given chemical structure, these stereoisomers are
identical
except that they are mirror images of one another. A specific stereoisomer may
also
be referred to as an enantiomer, and a mixture of such isomers is often called
an
enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a
racemic
mixture or a racemate, which may occur where there has been no stereoselection
or
stereospecificity in a chemical reaction or process. The terms "racemic
mixture" and
"racemate" refer to an equimolar mixture of two enantiomerie species, devoid
of
optical activity.
Whenever a compound described herein is substituted with more than one of
the same designated group, e.g., "R" or "R1", then it will be understood that
the
groups may be the same or different, i.e., each group is independently
selected. Wavy
lines, - , indicate the site of covalent bond attachments to the adjoining
substructures, groups, moieties, or atoms.
The compounds of the invention can also exist as tautomeric isomers in certain
cases. Although only one delocalized resonance structure may be depicted, all
such
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forms are contemplated within the scope of the invention. For example, ene-
amine
tautomers can exist for purine, pyrimidine, imidazole, guanidine, amidine, and
tetrazole systems and all their possible tautomeric fauns are within the scope
of the
invention.
One skilled in the art will recognize that the pyrrolo[1,241[1,2,4]triazine,
imidazo[1,5-f][1,2,4]triazine, imidazo[1,2-f][1,2,4]triazine, and
[1,2,4]triazolo[4,3-
f][1,2,4]triazine nucleosides can exist in tautomeric forms. For example, but
not by
way of limitation, structures (a) and (b) can have equivalent tautomeric forms
as
shown below:
OH 0
XN X1,,,,,,/\ NH
X2\ X2\
= Rs R9
R8 R8
XN N
X2\
= OH 0
NH2 NH
XN NH
X2\ X2\
= R9N R9
a b.
All possible tautomeric forms of the heterocycles in all of the embodiments
disclosed herein are within the scope of the invention.
Methods of Inhibition of HCV polymerase
Another aspect of the invention relates to methods of inhibiting the activity
of
HCV polymerase comprising the step of treating a sample suspected of
containing
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HCV with a composition of the invention.
Compositions of the invention may act as inhibitors of HCV polymerase, as
intermediates for such inhibitors or have other utilities as described below.
The
inhibitors will bind to locations on the surface or in a cavity of HCV
polymerase
having a geometry unique to HCV polymerase. Compositions binding HCV
polymerase may bind with varying degrees of reversibility. Those compounds
binding substantially irreversibly are ideal candidates for use in this method
of the
invention. Once labeled, the substantially irreversibly binding compositions
are
useful as probes for the detection of HCV polymerase. Accordingly, the
invention
relates to methods of detecting HCV polymerase in a sample suspected of
containing
HCV polymerase comprising the steps of: treating a sample suspected of
containing
HCV polymerase with a composition comprising a compound of the invention bound
to a label; and observing the effect of the sample on the activity of the
label. Suitable
labels are well known in the diagnostics field and include stable free
radicals,
fluorophores, radioisotopes, enzymes, chemiluminescent groups and chromogens.
The compounds herein are labeled in conventional fashion using functional
groups
such as hydroxyl, carboxyl, sulfhydryl or amino.
Within the context of the invention, samples suspected of containing HCV
polymerasc include natural or man-made materials such as living organisms;
tissue or
cell cultures; biological samples such as biological material samples (blood,
serum,
urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, and the
like);
laboratory samples; food, water, or air samples; bioproduct samples such as
extracts
of cells, particularly recombinant cells synthesizing a desired glycoprotein;
and the
like. Typically the sample will be suspected of containing an organism which
produces HCV polymerase, frequently a pathogenic organism such as HCV.
Samples can be contained in any medium including water and organic
solvenfwater
mixtures. Samples include living organisms such as humans, and man made
materials
such as cell cultures.
The treating step of the invention comprises adding the composition of the
invention to the sample or it comprises adding a precursor of the composition
to the
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sample. The addition step comprises any method of administration as described
above.
If desired, the activity of HCV polymerase after application of the
composition
can be observed by any method including direct and indirect methods of
detecting
HCV polymerase activity. Quantitative, qualitative, and semiquantitative
methods of
determining HCV polymerase activity are all contemplated. Typically one of the
screening methods described above are applied, however, any other method such
as
observation of the physiological properties of a living organism are also
applicable.
Organisms that contain HCV polymerase include the HCV virus. The
compounds of this invention arc useful in the treatment or prophylaxis of HCV
.. infections in animals or in man.
However, in screening compounds capable of inhibiting human
immunodeficiency viruses, it should be kept in mind that the results of enzyme
assays
may not correlate with cell culture assays. Thus, a cell based assay should be
the
primary screening tool.
Screens for HCV polymerase Inhibitors.
Compositions of the invention are screened for inhibitory activity against
HCV polymerase by any of the conventional techniques for evaluating enzyme
activity. Within the context of the invention, typically compositions are
first screened
for inhibition of HCV polymerase in vitro and compositions showing inhibitory
activity are then screened for activity in vivo. Compositions having in vitro
Ki
(inhibitory constants) of less then about 5 X 10-6 M, typically less than
about IX 10-
7 M and preferably less than about 5 X 10-8 M are preferred for in vivo use.
Useful in vitro screens have been described in detail and will not be
elaborated
here. However, the examples describe suitable in vitro assays.
Pharmaceutical Formulations
The compounds of this invention are formulated with conventional carriers
and excipients, which will be selected in accord with ordinary practice.
Tablets will
contain excipients, glidants, fillers, binders and the like. Aqueous
formulations are
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prepared in sterile form, and when intended for delivery by other than oral
administration generally will be isotonic. All formulations will optionally
contain
excipients such as those set forth in the "Handbook of Pharmaceutical Ex
cipients"
(1986). Excipients include ascorbic acid and other antioxidants, chelating
agents such
as EDTA, carbohydrates such as dextran, hydroxyalkylcellulose,
hydroxyalkylmethyleellulose, stearic acid and the like. The pH of the
foimulations
ranges from about 3 to about 11, but is ordinarily about 7 to 10.
While it is possible for the active ingredients to be administered alone it
may
be preferable to present them as pharmaceutical formulations. The
formulations, both
for veterinary and for human use, of the invention comprise at least one
active
ingredient, as above defined, together with one or more acceptable carriers
therefore
and optionally other therapeutic ingredients. The carrier(s) must be
"acceptable" in
the sense of being compatible with the other ingredients of the formulation
and
physiologically innocuous to the recipient thereof.
The formulations include those suitable for the foregoing administration
routes. The formulations may conveniently be presented in unit dosage form and
may
be prepared by any of the methods well known in the art of pharmacy.
Techniques
and formulations generally are found in Remington's Pharmaceutical Sciences
(Mack
Publishing Co., Easton, PA). Such methods include the step of bringing into
association the active ingredient with the carrier which constitutes one or
more
accessory ingredients. In general the foiniulations arc prepared by uniformly
and
intimately bringing into association the active ingredient with liquid
carriers or finely
divided solid carriers or both, and then, if necessary, shaping the product.
Formulations of the present invention suitable for oral administration may be
presented as discrete units such as capsules, cachets or tablets each
containing a
predetermined amount of the active ingredient; as a powder or granules; as a
solution
or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water
liquid
emulsion or a water-in-oil liquid emulsion. The active ingredient may also be
administered as a bolus, electuary or paste.
A tablet is made by compression or molding, optionally with one or more
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accessory ingredients. Compressed tablets may be prepared by compressing in a
suitable machine the active ingredient in a free-flowing form such as a powder
or
granules, optionally mixed with a binder, lubricant, inert diluent,
preservative, surface
active or dispersing agent. Molded tablets may be made by molding in a
suitable
machine a mixture of the powdered active ingredient moistened with an inert
liquid
diluent. The tablets may optionally be coated or scored and optionally are
formulated
so as to provide slow or controlled release of the active ingredient
therefrom.
For infections of the eye or other external tissues e.g. mouth and skin, the
formulations are preferably applied as a topical ointment or cream containing
the
active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including
active
ingredient(s) in a range between 0.1% and 20% in increments of 0.1% w/w such
as
0.6% w/w, 0.7% w/w, etc.), preferably 0.2 to 15% VW and most preferably 0.5 to
10% w/w. When formulated in an ointment, the active ingredients may be
employed
with either a paraffinic or a water-miscible ointment base. Alternatively, the
active
ingredients may be formulated in a cream with an oil-in-water cream base.
If desired, the aqueous phase of the cream base may include, for example, at
least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more
hydroxyl
groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol
and
polyethylene glycol (including PEG 400) and mixtures thereof. The topical
formulations may desirably include a compound which enhances absorption or
penetration of the active ingredient through the skin or other affected areas.
Examples
of such dermal penetration enhancers include dimethyl sulphoxide and related
analogs.
The oily phase of the emulsions of this invention may be constituted from
known ingredients in a known manner. While the phase may comprise merely an
emulsifier (otherwise known as an emulgent), it desirably comprises a mixture
of at
least one emulsifier with a fat or an oil or with both a fat and an oil.
Preferably, a
hydrophilic emulsifier is included together with a lipophilic emulsifier which
acts as a
stabilizer. It is also preferred to include both an oil and a fat. Together,
the
emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying
wax, and
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the wax together with the oil and fat make up the so-called emulsifying
ointment base
which forms the oily dispersed phase of the cream formulations.
Emulgents and emulsion stabilizers suitable for use in the formulation of the
invention include Tween 60, Span 80, cctostearyl alcohol, benzyl alcohol,
myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate.
The choice of suitable oils or fats for the formulation is based on achieving
the
desired cosmetic properties. The cream should preferably be a non-greasy, non-
staining and washable product with suitable consistency to avoid leakage from
tubes
or other containers. Straight or branched chain, mono- or dibasic alkyl esters
such as
di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty
acids,
.. isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-
ethylhexyl
palmitate or a blend of branched chain esters known as Crodamol CAP may be
used,
the last three being preferred esters. These may be used alone or in
combination
depending on the properties required. Alternatively, high melting point lipids
such as
white soft paraffin and/or liquid paraffin or other mineral oils are used.
Pharmaceutical formulations according to the present invention comprise a
combination according to the invention together with one or more
pharmaceutically
acceptable carriers or excipients and optionally other therapeutic agents.
Pharmaceutical formulations containing the active ingredient may be in any
form
suitable for the intended method of administration. When used for oral use for
example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible
powders
or granules, emulsions, hard or soft capsules, syrups or elixirs may be
prepared.
Compositions intended for oral use may be prepared according to any method
known
to the art for the manufacture of pharmaceutical compositions and such
compositions
may contain one or more agents including sweetening agents, flavoring agents,
coloring agents and preserving agents, in order to provide a palatable
preparation.
Tablets containing the active ingredient in admixture with non-toxic
pharmaceutically
acceptable excipient which are suitable for manufacture of tablets are
acceptable.
These excipients may be, for example, inert diluents, such as calcium or
sodium
carbonate, lactose, calcium or sodium phosphate; granulating and
disintegrating
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agents, such as maize starch, or alginic acid; binding agents, such as starch,
gelatin or
acacia; and lubricating agents, such as magnesium stearate, stearic acid or
talc.
Tablets may be uncoated or may be coated by known techniques including
microencapsulation to delay disintegration and adsorption in the
gastrointestinal tract
and thereby provide a sustained action over a longer period. For example, a
time
delay material such as glyceryl monostearate or glyeeryl distearate alone or
with a
wax may be employed.
Formulations for oral use may be also presented as hard gelatin capsules
where the active ingredient is mixed with an inert solid diluent, for example
calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient
is mixed
with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
Aqueous suspensions of the invention contain the active materials in
admixture with excipients suitable for the manufacture of aqueous suspensions.
Such
excipients include a suspending agent, such as sodium carboxymethyleellulose,
methylcellulose, hydroxypropyl methylcelluose, sodium alginate,
polyvinylpynolidone, gum tragacanth and gum acacia, and dispersing or wetting
agents such as a naturally-occurring phosphatide (e.g., lecithin), a
condensation
product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene
stearate), a
condensation product of ethylene oxide with a long chain aliphatic alcohol
(e.g.,
heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a
partial
ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene
sorbitan
monooleate). The aqueous suspension may also contain one or more preservatives
such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one
or
more flavoring agents and one or more sweetening agents, such as sucrose or
saccharin.
Oil suspensions may be formulated by suspending the active ingredient in a
vegetable oil, such as araehis oil, olive oil, sesame oil or coconut oil, or
in a mineral
oil such as liquid paraffin. The oral suspensions may contain a thickening
agent, such
as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those
set forth
above, and flavoring agents may be added to provide a palatable oral
preparation.
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These compositions may be preserved by the addition of an antioxidant such as
ascorbic acid.
Dispersible powders and granules of the invention suitable for preparation of
an aqueous suspension by the addition of water provide the active ingredient
in
admixture with a dispersing or wetting agent, a suspending agent, and one or
more
preservatives. Suitable dispersing or wetting agents and suspending agents are
exemplified by those disclosed above. Additional excipients, for example
sweetening,
flavoring and coloring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of
oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive
oil or
arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these.
Suitable
emulsifying agents include naturally-occurring gums, such as gum acacia and
gum
tragacanth, naturally-occurring phosphatides, such as soybean lecithin, esters
or
partial esters derived from fatty acids and hexitol anhydrides, such as
sorbitan
monooleate, and condensation products of these partial esters with ethylene
oxide,
such as polyoxyethylene sorbitan monooleate. The emulsion may also contain
sweetening and flavoring agents. Syrups and elixirs may be formulated with
sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations
may also
contain a demulcent, a preservative, a flavoring or a coloring agent.
The pharmaceutical compositions of the invention may be in the form of a
sterile injectable preparation, such as a sterile injectable aqueous or
oleaginous
suspension. This suspension may be formulated according to the known art using
those suitable dispersing or wetting agents and suspending agents which have
been
mentioned above. The sterile injectable preparation may also be a sterile
injectable
solution or suspension in a non-toxic parenterally acceptable diluent or
solvent, such
as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among
the
acceptable vehicles and solvents that may be employed arc water, Ringer's
solution
and isotonic sodium chloride solution. In addition, sterile fixed oils may
conventionally be employed as a solvent or suspending medium. For this purpose
any
bland fixed oil may be employed including synthetic mono- or diglycerides. In
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addition, fatty acids such as oleic acid may likewise be used in the
preparation of
injectables.
The amount of active ingredient that may be combined with the carrier
material to produce a single dosage form will vary depending upon the host
treated
and the particular mode of administration. For example, a time-release
formulation
intended for oral administration to humans may contain approximately 1 to 1000
mg
of active material compounded with an appropriate and convenient amount of
carrier
material which may vary from about 5 to about 95% of the total compositions
(weight:weight). The pharmaceutical composition can be prepared to provide
easily
measurable amounts for administration. For example, an aqueous solution
intended
for intravenous infusion may contain from about 3 to 5001.tg of the active
ingredient
per milliliter of solution in order that infusion of a suitable volume at a
rate of about
30 mL/hr can occur.
Formulations suitable for topical administration to the eye also include eye
drops wherein the active ingredient is dissolved or suspended in a suitable
carrier,
especially an aqueous solvent for the active ingredient. The active ingredient
is
preferably present in such foimulations in a concentration of 0.5 to 20%,
advantageously 0.5 to 10%, and particularly about 1.5% w/w.
Formulations suitable for topical administration in the mouth include lozenges
comprising the active ingredient in a flavored basis, usually sucrose and
acacia or
tragacanth; pastilles comprising the active ingredient in an inert basis such
as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the active
ingredient in a suitable liquid carrier.
Formulations for rectal administration may be presented as a suppository with
a suitable base comprising for example cocoa butter or a salicylate.
Formulations suitable for intrapulmonary or nasal administration have a
particle size for example in the range of 0.1 to 500 microns, such as 0.5, 1,
30,35 etc.,
which is administered by rapid inhalation through the nasal passage or by
inhalation
through the mouth so as to reach the alveolar sacs. Suitable formulations
include
aqueous or oily solutions of the active ingredient. Formulations suitable for
aerosol or
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dry powder administration may be prepared according to conventional methods
and
may be delivered with other therapeutic agents such as compounds heretofore
used in
the treatment or prophylaxis of HCV infections as described below.
Formulations suitable for vaginal administration may be presented as
pessaries, tampons, creams, gels, pastes, foams or spray formulations
containing in
addition to the active ingredient such carriers as are known in the art to be
appropriate.
Formulations suitable for parenteral administration include aqueous and non-
aqueous sterile injection solutions which may contain anti-oxidants, buffers,
baeteriostats and solutes which render the formulation isotonic with the blood
of the
intended recipient; and aqueous and non-aqueous sterile suspensions which may
include suspending agents and thickening agents.
The formulations are presented in unit-dose or multi-dose containers, for
example scaled ampoules and vials, and may be stored in a freeze-dried
(lyophilized)
condition requiring only the addition of the sterile liquid carrier, for
example water for
injection, immediately prior to use. Extemporaneous injection solutions and
suspensions are prepared from sterile powders, granules and tablets of the
kind
previously described. Preferred unit dosage formulations are those containing
a daily
dose or unit daily sub-dose, as herein above recited, or an appropriate
fraction thereof,
of the active ingredient.
It should be understood that in addition to the ingredients particularly
mentioned above the formulations of this invention may include other agents
conventional in the art having regard to the type of formulation in question,
for
example those suitable for oral administration may include flavoring agents.
The invention further provides veterinary compositions comprising at least
one active ingredient as above defined together with a veterinary carrier
therefore.
Veterinary carriers are materials useful for the purpose of administering the
composition and may be solid, liquid or gaseous materials which are otherwise
inert
or acceptable in the veterinary art and are compatible with the active
ingredient.
These veterinary compositions may be administered orally, parenterally or by
any
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other desired route.
Compounds of the invention are used to provide controlled release
pharmaceutical formulations containing as active ingredient one or more
compounds
of the invention ("controlled release formulations") in which the release of
the active
ingredient are controlled and regulated to allow less frequency dosing or to
improve
the pharmacokinetic or toxicity profile of a given active ingredient.
Effective dose of active ingredient depends at least on the nature of the
condition being treated, toxicity, whether the compound is being used
prophylactically (lower doses) or against an active viral infection, the
method of
delivery, and the pharmaceutical formulation, and will be determined by the
clinician
using conventional dose escalation studies. It can be expected to be from
about
0.0001 to about 100 mg/kg body weight per day; typically, from about 0.01 to
about
10 mg/kg body weight per day; more typically, from about .01 to about 5 mg/kg
body
weight per day; most typically, from about .05 to about 0.5 mg/kg body weight
per
day. For example, the daily candidate dose for an adult human of approximately
70
kg body weight will range from 1 mg to 1000 mg, preferably between 5 mg and
500
mg, and may take the form of single or multiple doses.
Routes of Administration
One or more compounds of the invention (herein referred to as the active
ingredients) are administered by any route appropriate to the condition to be
treated.
Suitable routes include oral, rectal, nasal, topical (including buccal and
sublingual),
vaginal and parenteral (including subcutaneous, intramuscular, intravenous,
intradermal, intrathecal and epidural), and the like. It will be appreciated
that the
preferred route may vary with for example the condition of the recipient. An
advantage of the compounds of this invention is that they are orally
bioavailable and
can be dosed orally.
Combination Therapy
Combinations of the compounds of Formula I-III and Formula IV-VI are
typically selected based on the condition to be treated, cross-reactivities of
ingredients
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and phannaco-properties of the combination. For example, when treating an
infection
(e.g., HCV), the compositions of the invention are combined with other active
therapeutic agents (such as those described herein).
Compositions of the invention are also used in combination with one or more
other active ingredients. Preferably, the other active therapeutic ingredients
or agents
are interferons, ribavirin or its analogs, HCV NS3 protease inhibitors, NS5a
inhibitors, alpha-glucosidase 1 inhibitors, hepatoprotectants, mevalonate
decarboxylase antagonists, antagonists of the renin-angiotensin system, other
anti-
fibrotic agents, endothelin antagonists, nucleoside or nucleotide inhibitors
of HCV
NS5B polymerase, non-nucleoside inhibitors of HCV NS5B polymerase, HCV NS5A
inhibitors, TLR-7 agonists, cyclophillin inhibitors, HCV TRES inhibitors,
pharmacokinetic enhancers or other drugs for treating HCV; or mixtures
thereof.
More specifically, one or more compounds of the present invention may be
combined with one or more compounds selected from the group consisting of
1) intcrfcrons, e.g., pcgylated rIFN-alpha 2b (PEG-Intron), pegylated rIFN-
alpha 2a (Pegasys), rIFN-alpha 2b (1ntron A), rIFN-alpha 2a (Rofcron-A),
interferon
alpha (MOR-22, OPC-18, Alfaferone, Alfanative, Multiferon, subalin),
interferon
alfacon-1 (Infergen), interferon alpha-nl (Wellferon), interferon alpha-n3
(Alferon),
interferon-beta (Avonex, DL-8234), interferon-omega (omega DUROS, Biomecl
510),
albinterferon alpha-2b (Albuferon), IFN alpha XL, BLX-883 (Locteron), DA-3021,
glycosylated interferon alpha-2h (AVI-005), PEG-Infergen, PEGylated interferon
lambda (PEGylated IL-29), and belerofon,
2) ribavirin and its analogs, e.g., ribavirin (Rebetol, Copegus), and
taribavirin
(Viramidine),
3) HCV NS3 protease inhibitors, e.g., boceprevir (SCH-503034 , SCH-7),
telaprevir (VX-950), VX-813, TMC-435 (TMC435350), ABT-450, BI-201335, BI-
1230, MK-7009, SCH-900518, VBY-376, VX-500, GS-9256, GS-9451, BMS-
790052, BMS-605339, PHX-1766, AS-101, YH-5258, YH5530, YH5531, and
ITMN-191 (R-7227),
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4) alpha-glucosidase 1 inhibitors, e.g., celgosivir (MX-3253), Miglitol, and
UT-231B,
5) hepatoprotectants, e.g., emericasan (IDN-6556), ME-3738, GS-9450 (LB-
84451), silibilin, and MitoQ,
6) nucleoside or nucleotide inhibitors of HCV NS5B polymerase, e.g., R1626,
.. R7128 (R4048), IDX184, IDX-102, PSI-7851, BCX-4678, valopicitabine (NM-
283),
and MK-0608,
7) non-nucleoside inhibitors of HCV NS5B polyrnerase, e.g., filibuvir (PF-
868554), ABT-333, ABT-072, BI-207127, VCH-759, VCH-916, JTK-652, MK-3281,
VBY-708, VCH-222, A848837, ANA-598, GL60667, GL59728, A-63890, A-48773,
A-48547, BC-2329, VCH-796 (nesbuvir), GSK625433, BILN-1941, XTL-2125, and
GS-9190,
8) HCV NS5A inhibitors, e.g., AZD-2836 (A-831), AZD-7295 (A-689), and
BMS-790052,
9) TLR-7 agonists, e.g., imiquimod, 852A, GS-9524, ANA-773, ANA-975,
.. AZD-8848 (DSP-3025), PF-04878691, and SM-360320,
10) cyclophillin inhibitors, e.g., DEB10-025, SCY-635, and NIM811,
11) HCV IRES inhibitors, e.g., MCI-067,
12) pharmacokinetic enhancers, e.g., BAS-100, SPI-452, PF-4194477, TMC-
41629, GS-9350, GS-9585, and roxythromycin,
13) other drugs for treating HCV, e.g., thymosin alpha 1 (Zadaxin),
nitazoxanide (Alinea, NTZ), BIVN-401 (virostat), PYN-17 (altirex),
KPE02003002,
actilon (CPG-10101), GS-9525, KRN-7000, civacir, GI-5005, XTL-6865, BIT225,
PTX-111, ITX2865, TT-033i, ANA 971, NOV-205, tarvacin, EHC-18, VGX-410C,
EMZ-702, AVI 4065, BMS-650032, BMS-791325, Bavituximab, MDX-1106 (ONO-
4538), Oglufanide, FK-788, and VX-497 (merimepodib)
14) mevalonate decarboxylase antagonists, e.g., statins, FIMGCoA synthase
inhibitors (e.g., hymeglusin), squalene synthesis inhibitors (e.g., zaragozic
acid);
15) angiotensin II receptor antagonists, e.g., losartan, irbesartan,
olmesartan,
candesartan, valsartan, telmisartan, eprosartan;
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16) angiotensin-converting enzyme inhibitors, e.g., captopril, zofenopril,
enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril,
fosinopril;
17) other anti-fibrotic agents, e.g., amiloride and
18) endothelin antagonists, e.g. bosentan and ambrisentan.
In yet another embodiment, the present application discloses pharmaceutical
.. compositions comprising a compound of the present invention, or a
pharmaceutically
acceptable salt, solvate, and/or ester thereof, in combination with at least
one
additional therapeutic agent, and a pharmaceutically acceptable carrier or
excipient.
According to the present invention, the therapeutic agent used in combination
with the compound or composition of the present invention can be any agent
having a
.. therapeutic effect when used in combination with the compound of the
present
invention. For example, the therapeutic agent used in combination with the
compound or composition of the present invention can be interferons, ribavirin
or its
analogs, HCV NS3 protease inhibitors, NS5a inhibitors, alpha-glucosidase 1
inhibitors, hepatoprotectants, mevalonate decarboxylase antagonists,
antagonists of
the renin-angiotensin system, other anti-fibrotic agents, endothelin
antagonists,
nucleoside or nucleotide inhibitors of HCV NS5B polymerase, non-nucleoside
inhibitors of HCV NS5B polymerase, HCV NS5A inhibitors, TLR-7 agonists,
cyclophillin inhibitors, HCV IRES inhibitors, pharmacokinetic enhancers or
other
drugs for treating HCV; or mixtures thereof.
More specifically, compositions of one or more compounds of the present
invention may be combined with one or more compounds selected from the group
consisting of
1) interferons, e.g., pegylated rIFN-alpha 2b (PEG-Intron), pegylatcd rIFN-
alpha 2a (Pegasys), r1FN-alpha 2b (1ntron A), rIFN-alpha 2a (Roferon-A),
interferon
alpha (MOR-22, OPC-18, Alfaferone, Alfanative, Multiferon, subalin),
interferon
alfacon-1 (Infergen), interferon alpha-nl (Wellferon), interferon alpha-n3
(Alferon),
interferon-beta (Avonex, DL-8234), interferon-omega (omega DUROS, Biomed 510),
albinterferon alpha-2b (Albuferon), IFN alpha XL, BLX-883 (Locteron), DA-3021,
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glycosylated interferon alpha-2b (AVI-005), PEG-Infergen, PEGylated interferon
lambda (PEGylated IL-29), and belerofon,
2) ribavirin and its analogs, e.g., ribavirin (Rebetol, Copegus), and
taribavirin
(Viramidine),
3) HCV NS3 protease inhibitors, e.g., boceprevir (SCH-503034 , SCH-7),
telaprevir (VX-950), VX-813, TMC-435 (TMC435350), ABT-450, BI-201335, BI-
1230, MK-7009, SCH-900518, VBY-376, VX-500, GS-9256, GS-9451, BMS-
790052, BMS-605339, PHX-1766, AS-101, YH-5258, YH5530, YH5531, and
1TMN-191 (R-7227),
4) alpha-glucosidase 1 inhibitors, e.g., celgosivir (MX-3253), Miglitol, and
UT-231B,
5) hepatoprotectants, e.g., emericasan (IDN-6556), ME-3738, GS-9450 (LB-
84451), silibilin, and MitoQ,
6) nucleoside or nucleotide inhibitors of HCV NS5B polymerase, e.g., R1626,
R7128 (R4048), IDX184, IDX-102, PSI-7851, BCX-4678, valopicitabine (NM-283),
and MK-0608,
7) non-nucleoside inhibitors of HCV NS5B polymerase, e.g., filibuvir (PF-
868554), ABT-333, ABT-072, BI-207127, VCH-759, VCH-916, JTK-652, MK-3281,
VBY-708, VCH-222, A848837, ANA-598, GL60667, GL59728, A-63890, A-48773,
A-48547, BC-2329, VCH-796 (nesbuvir), GSK625433, BILN-1941, XTL-2125, and
GS-9190,
8) HCV NS5A inhibitors, e.g., AZD-2836 (A-831), AZD-7295 (A-689), and
BMS-790052,
9) TLR-7 agonists, e.g., imiquimod, 852A, GS-9524, ANA-773, ANA-975,
AZD-8848 (DSP-3025), PF-04878691, and SM-360320,
10) cyclophillin inhibitors, e.g., DEB10-025, SCY-635, and NIM811,
11) HCV IRES inhibitors, e.g, MCI-067,
12) pharmacokinetic enhancers, e.g., BAS-100, SPI-452, PF-4194477, TMC-
41629, GS-9350, GS-9585, and roxythromycin,
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13) other drugs for treating HCV, e.g., thymosin alpha 1 (Zadaxin),
nitazoxanide (Alinea, NTZ), BIVN-401 (virostat), PYN-17 (altirex),
KPE02003002,
actilon (CPG-10101), GS-9525, KRN-7000, civacir, GI-5005, XTL-6865, BIT225,
PTX-111, ITX2865, TT-033i, ANA 971, NOV-205, tarvacin, EHC-18, VGX-410C,
EMZ-702, AVI 4065, BMS-650032, BMS-791325, Bavituximab, MDX-1106 (ONO-
4538), Oglufanide, FK-788, and VX-497 (merimepodib)
14) mevalonate decarboxylase antagonists, e.g., statins, HMGCoA synthase
inhibitors (e.g., hymeglusin), squalenc synthesis inhibitors (e.g., zaragozic
acid);
15) angiotensin 11 receptor antagonists, e.g., losartan, irbesartan,
olmesartan,
candesartan, valsartan, telmisartan, eprosartan;
16) angiotensin-converting enzyme inhibitors, e.g., captopril, zofenopril,
enalapril, ramipril, quinapri.1, perindopril, lisinopril, benazepril,
fosinopril;
17) other anti-fibrotic agents, e.g., amiloride and
18) endothelin antagonists, e.g. bosentan and ambrisentan.
In yet another embodiment, the present application provides a combination
pharmaceutical agent comprising:
a) a first pharmaceutical composition comprising a compound of the
present invention, or a pharmaceutically acceptable salt, solvate, or ester
thereof; and
b) a second pharmaceutical composition comprising at least one
additional therapeutic agent selected from the group consisting of HIV
protease
inhibiting compounds, HIV non-nucleoside inhibitors of reverse transeriptase,
HIV
nucleoside inhibitors of reverse transcriptase. HIV nucleotide inhibitors of
reverse
transcriptase, HIV intqu-ase inhibitors, gp41 inhibitors, CXCR4 inhibitors,
gp120
inhibitors. CCR5 inhibitors, intcrfcrons, ribavirin analogs, NS3 protease
inhibitors,
NS5a inhibitors, alpha-glucosidase 1 inhibitors, cyclophilin inhibitors,
hepatoprotectants, non-nucleoside inhibitors of HCV, and other drugs for
treating
HCV, and combinations thereof.
Combinations of the compounds of Formula I-111 and Formula 1V-VI and
additional active therapeutic agents may be selected to treat patients
infected with
HCV and other conditions such as HIV infections. Accordingly, the compounds of
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Formula 1-ITT and Formula IV-VI may be combined with one or more compounds
useful in treating HIV, for example HIV protease inhibiting compounds, HIV non-
nucleoside inhibitors of reverse transcriptase, HIV nucleoside inhibitors of
reverse
transcriptase, HIV nucleotide inhibitors of reverse transcriptase, HIV
integrase
inhibitors, gp41 inhibitors, CXCR4 inhibitors, gp120 inhibitors, CCR5
inhibitors,
interferons, ribavirin analogs, NS3 protease inhibitors, NS5a inhibitors,
alpha-
glucosidase 1 inhibitors, cyclophilin inhibitors, hepatoprotectants, non-
nucleoside
inhibitors of HCV, and other drugs for treating HCV.
More specifically, one or more compounds of the present invention may be
combined with one or more compounds selected from the group consisting of 1)
HIV
protease inhibitors, e.g., amprenavir, atazanavir, fosamprenavir, indinavir,
lopinavir,
ritonavir, lopinavir + ritonavir, nelfinavir, saquinavir, tipranavir,
brecanavir,
darunavir, TMC-126, TMC-114, mozenavir (DMP-450), JE-2147 (AG1776),
AG1859, DG35, L-756423, R00334649, KNI-272, DPC-681, DPC-684, and
GW640385X, DG17, PPL-100, 2) a HIV non-nucleoside inhibitor of reverse
transcriptase, e.g., capravirine, emivirine, delaviridine, efavirenz,
nevirapine, (+)
calanolidc A, etravirinc, GW5634, DPC-083, DPC-961, DPC-963, MIV-150, and
TMC-120, TMC-278 (rilpivirine), cfavirenz, BILR 355 BS, VRX 840773, UK-
453,061, RDEA806, 3) a HIV nucleoside inhibitor of reverse transcriptasc,
e.g.,
zidovudine, emtricitabine, didanosine, stavudine, zalcitabine, lamivudine,
abacavir,
amdoxovir, elvucitabine, alovudine, MIV-210, raeivir ( -FTC), D-d4FC,
emtricitabine, phosphazide, fozivudine tidoxil, fosalvudine tidoxil,
apricitibine
(AVX754), amdoxovir, KP-1461, abacavir + lamivudine, abacavir + lamivudine +
zidovudine, zidovudine + lamivudine, 4) a HIV nucleotide inhibitor of reverse
transcriptase, e.g., tenofovir, tenofovir disoproxil fumarate + emtricitabine,
tenofovir
disoproxil futnarate + emtricitabine + efavirenz, and adefovir, 5) a HIV
integrase
inhibitor, e.g., eurcumin, derivatives of curcumin, chicoric acid, derivatives
of
chicoric acid, 3,5-dicaffcoylquinic acid, derivatives of 3,5-dicaffeoylquinic
acid,
aurintricarboxylic acid, derivatives of aurintricarboxylic acid, caffeic acid
phenethyl
ester, derivatives of caffeic acid phenethyl ester, tyrphostin, derivatives of
tyrphostin,
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quercetin, derivatives of quercetin, S-1360, zintevir (AR-177), L-870812, and
L-
870810, MK-0518 (raltegravir), BMS-707035, MK-2048, BA-011, BMS-538158,
GSK364735C, 6) a gp41 inhibitor, e.g., enfuvirtide, sifuvirtide, FB006M, TRI-
1144,
SPC3, DES6, Locus gp41, CovX, and REP 9, 7) a CXCR4 inhibitor, e.g., AMD-070,
8) an entry inhibitor, e.g., SPO1A, TNX-355, 9) a gp120 inhibitor, e.g., BMS-
488043
and BlockAide/CR, 10) a G6PD and NADH-oxidase inhibitor, e.g., immunitin, 10)
a
CCR5 inhibitor, e.g., aplaviroc, vicriviroc, INCB9471, PRO-140, INCB15050, PF-
232798, CCR5mAb004, and maraviroc, 11) an interferon, e.g., pegylated r1FN-
alpha
2b, pegylated r1FN-alpha 2a, r1FN-alpha 2b, IFN alpha-2b XL, rIFN-alpha 2a,
consensus IFN alpha, infergen, rebif, locteron, AVI-005, PEG-infergen,
pegylated
IFN-beta, oral interferon alpha, feron, reaferon, intermax alpha, r-IFN-beta,
infergen +
actimrnune, IFN-omega with DUROS, and albuferon, 12) ribavirin analogs, e.g.,
rebetol, copegus, VX-497, and viramidine (taribavirin) 13) NS5a inhibitors,
e.g., A-
831, A-689 and BMS-790052, 14) NS5b polymerase inhibitors, e.g., NM-283,
valopicitabine, R1626, PSI-6130 (R1656), IDX184, PSI-7851, HCV-796, BILB 1941,
MK-0608, NM-107, R7128, VCH-759, PF-868554, G5K625433, and XTL-2125, 15)
NS3 protease inhibitors, e.g., SCH-503034 (SCH-7), VX-950 (Telaprevir), ITMN-
191, and BILN-2065, 16) alpha-glucosidase I inhibitors, e.g., MX-3253
(celgosivir)
and UT-231B, 17) hepatoproteetants, e.g., IDN-6556, ME 3738. MitoQ, and LB-
84451, 18) non-nucleoside inhibitors of HCV, benzimidazole derivatives,
benzo-
1,2,4-thiadiazine derivatives, and phenylalanine derivatives, 19) other drugs
for
treating HCV, e.g-., zadaxin, nitazoxanide (alinea), BIVN-401 (virostat),
DEBIO-025,
VGX-410C, EMZ-702, AVI 4065, bavituximab, oglufanide, PYN-17, KPE02003002,
actilon (CPG-10101), KRN-7000, civacir, G1-5005, ANA-975, XTL-6865, ANA 971,
NOV-205, tarvacin, EHC-18, and NIM811, 19) phannacokinetic enhancers, e.g.,
BAS-100 and SPI452, 20)RNAse H inhibitors, e.g., ODN-93 and ODN-112, 21) other
anti-HIV agents, e.g., VGV-1, PA-457 (bevirimat), ampligen, HRG214, cytolin,
polymun, VGX-410, KD247, AMZ 0026, CYT 99007, A-221 HIV, BAY 50-4798,
MDX010 (iplimumab), PBS119, ALG889, and PA-1050040.
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It is also possible to combine any compound of the invention with one or more
other active therapeutic agents in a unitary dosage form for simultaneous or
sequential
administration to a patient. The combination therapy may be administered as a
simultaneous or sequential regimen. When administered sequentially, the
combination may be administered in two or more administrations.
Co-administration of a compound of the invention with one or more other
active therapeutic agents generally refers to simultaneous or sequential
administration
of a compound of the invention and one or more other active therapeutic
agents, such
that therapeutically effective amounts of the compound of the invention and
one or
more other active therapeutic agents are both present in the body of the
patient.
Co-administration includes administration of unit dosages of the compounds
of the invention before or after administration of unit dosages of one or more
other
active therapeutic agents, for example, administration of the compounds of the
invention within seconds, minutes, or hours of the administration of one or
more other
active therapeutic agents. For example, a unit dose of a compound of the
invention
can be administered first, followed within seconds or minutes by
administration of a
unit dose of one or more other active therapeutic agents. Alternatively, a
unit dose of
one or more other therapeutic agents can be administered first, followed by
administration of a unit dose of a compound of the invention within seconds or
minutes. In some cases, it may be desirable to administer a unit dose of a
compound
of the invention first, followed, after a period of hours (e.g., 1-12 hours),
by
administration of a unit dose of one or more other active therapeutic agents.
In other
cases, it may be desirable to administer a unit dose of one or more other
active
therapeutic agents first, followed, after a period of hours (e.g., 1-12
hours), by
administration of a unit dose of a compound of the invention.
The combination therapy may provide "synergy" and "synergistic", i.e. the
effect achieved when the active ingredients used together is greater than the
sum of
the effects that results from using the compounds separately. A synergistic
effect may
be attained when the active ingredients are: (1) co-formulated and
administered or
delivered simultaneously in a combined formulation; (2) delivered by
alternation or in
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parallel as separate formulations; or (3) by some other regimen. When
delivered in
alternation therapy, a synergistic effect may be attained when the compounds
are
administered or delivered sequentially, e.g. in separate tablets, pills or
capsules, or by
different injections in separate syringes. In general, during alternation
therapy, an
effective dosage of each active ingredient is administered sequentially, i.e.
serially,
whereas in combination therapy, effective dosages of two or more active
ingredients
are administered together. A synergistic anti-viral effect denotes an
antiviral effect
which is greater than the predicted purely additive effects of the individual
compounds of the combination.
In still yet another embodiment, the present application provides for methods
of inhibiting HCV polymerase in a cell, comprising: contacting a cell infected
with
HCV with an effective amount of a compound of Foiinula 1-Ill and Formula IV-
VI, or
a pharmaceutically acceptable salt, solvate, and/or ester thereof, whereby HCV
polymerase is inhibited.
In still yet another embodiment, the present application provides for methods
of inhibiting HCV polymerase in a cell, comprising: contacting a cell infected
with
HCV with an effective amount of a compound of Formula I-III and Formula IV-VI,
or
a pharmaceutically acceptable salt, solvate, and/or ester thereof, and at
least one
additional active therapeutic agent, whereby HCV polymerase is inhibited.
In still yet another embodiment, the present application provides for methods
of inhibiting HCV polymerase in a cell, comprising: contacting a cell infected
with
HCV with an effective amount of a compound of Formula 1-Ill and Formula IV-VI,
or
a pharmaceutically acceptable salt, solvate, and/or ester thereof, and at
least one
additional active therapeutic agent selected from the group consisting of one
or more
interferons, ribavirin or its analogs, HCV NS3 protease inhibitors, NS5a
inhibitors,
alpha-glucosidase 1 inhibitors, hepatoprotectants, mevalonate decarboxylase
antagonists, antagonists of the renin-angiotensin system, other anti-fibrotic
agents,
endothelin antagonists, nucleoside or nucleotide inhibitors of HCV NS5B
polymerase,
non-nucleoside inhibitors of HCV NS5B polymerase, HCV NS5A inhibitors, TLR-7
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agonists, cyclophillin inhibitors, HCV IRES inhibitors, pharmacokinetic
enhancers
and other drugs for treating HCV; or mixtures thereof.
In still yet another embodiment, the present application provides for methods
of treating HCV in a patient, comprising: administering to the patient a
therapeutically
effective amount of a compound of Formula 1411 and Formula IV-VI, or a
pharmaceutically acceptable salt, solvate, and/or ester thereof.
In still yet another embodiment, the present application provides for methods
of treating HCV in a patient, comprising: administering to the patient a
therapeutically
effective amount of a compound of Formula I-111 and Formula IV-VI, or a
pharmaceutically acceptable salt, solvate, and/or ester thereof, and at least
one
additional active therapeutic agent, whereby HCV polymerase is inhibited.
In still yet another embodiment, the present application provides for methods
of treating HCV in a patient, comprising: administering to the patient a
therapeutically
effective amount of a compound of Formula I-III and Formula IV-VI, or a
pharmaceutically acceptable salt, solvate, and/or ester thereof, and at least
one
additional active therapeutic agent selected from the group consisting of one
or more
interferons, ribavirin or its analogs, HCV NS3 protease inhibitors, NS5a
inhibitors,
alpha-glucosidase 1 inhibitors, hepatoprotectants, mevalonate decarboxylase
antagonists, antagonists of the renin-angiotensin system, other anti-fibrotic
agents,
endothelin antagonists, nucleoside or nucleotide inhibitors of HCV NS5B
polymerase,
.. non-nucleoside inhibitors of HCV NS5B polymerase, HCV NS5A inhibitors, TLR-
7
agonists, cyclophillin inhibitors, HCV IRES inhibitors, pharmacokinetic
enhancers
and other drugs for treating HCV; or mixtures thereof.
In still yet another embodiment, the present application provides for the use
of
a compound of the present invention, or a pharmaceutically acceptable salt,
solvate,
and/or ester thereof, for the preparation of a medicament for treating an HCV
infection in a patient.
Metabolites of the Compounds of the Invention
Also falling within the scope of this invention are the in vivo metabolic
products of the compounds described herein, to the extent such products are
novel and
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.. unobvious over the prior art. Such products may result for example from the
oxidation, reduction, hydrolysis, amidation, esterification and the like of
the
administered compound, primarily due to enzymatic processes. Accordingly, the
invention includes novel and unobvious compounds produced by a process
comprising contacting a compound of this invention with a mammal for a period
of
time sufficient to yield a metabolic product thereof. Such products typically
are
identified by preparing a radiolabelled (e.g. 14C or 3H) compound of the
invention,
administering it parenterally in a detectable dose (e.g. greater than about
0.5 mg/kg) to
an animal such as rat, mouse, guinea pig, monkey, or to man, allowing
sufficient time
for metabolism to occur (typically about 30 seconds to 30 hours) and isolating
its
conversion products from the urine, blood or other biological samples. These
products are easily isolated since they are labeled (others are isolated by
the use of
antibodies capable of binding epitopes surviving in the metabolite). The
metabolite
structures are determined in conventional fashion, e.g. by MS or NMR analysis.
In
general, analysis of metabolites is done in the same way as conventional drug
.. metabolism studies well-known to those skilled in the art. The conversion
products,
so long as they are not otherwise found in vivo, are useful in diagnostic
assays for
therapeutic dosing of the compounds of the invention even if they possess no
HCV
polym erase inhibitory activity of their own.
Recipes and methods for deteimining stability of compounds in surrogate
gastrointestinal secretions are known. Compounds are defined herein as stable
in the
gastrointestinal tract where less than about 50 mole percent of the protected
groups
are deprotected in surrogate intestinal or gastric juice upon incubation for 1
hour at
37 C. Simply because the compounds are stable to the gastrointestinal tract
does not
mean that they cannot be hydrolyzed in vivo. The prodrugs of the invention
typically
will be stable in the digestive system but may be substantially hydrolyzed to
the
parental drug in the digestive lumen, liver or other metabolic organ, or
within cells in
general.
Examples
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Certain abbreviations and acronyms are used in describing the experimental
details. Although most of these would be understood by one skilled in the art,
Table 1
contains a list of many of these abbreviations and acronyms.
Table 1. List of abbreviations and acronyms.
Abbreviation Meaning
Ac10 acetic anhydride
AIBN 2,2'-azobis(2-methylpropionitrile)
Bn benzyl
BnBr benzylbromide
BSA bis(trimethylsityl)acetamide
BzCl benzoyl chloride
CDI carbonyl diimidazole
DABCO 1,4-diazabicyclo[2.2.2]oetane
DBN 1,5-diazabicyclo[4.3.0]non-5-ene
DDQ 2,3 -dichloro-5,6-di cyano- 1,4-benzoquinone
DBU 1,5-diazabicyclo[5.4.0]undec-5-ene
DCA dichloroacetamide
DCC dicyclohexylcarbodiimide
DCM diehloromethane
DMAP 4-dimethylarninopyridine
DME 1 ,2-dim ethoxyethane
DMTCI dimethoxytrityl chloride
DMSO dimethylsulfoxide
DMTr 4, 4'-dimethoxytrityl
DMF dimethylformamide
Et0Ac ethyl acetate
ESI electrospray ionization
HMDS hexamethyldisilazane
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HPLC High pressure liquid chromatography
LDA lithium diisopropylamide
- -
LRMS low resolution mass spectrum
MCPBA meta-ehloroperbenzoic acid
MeCN acetonitrile
Me0H methanol
MMTC mono methoxytrityl chloride
m/z or mle mass to charge ratio
MEI+ mass plus 1
MW mass minus 1
Ms0H methanesulfonic acid
MS or ms mass spectrum
NBS N-bromosuccinimide
Ph phenyl
rt or r.t. room temperature
TBAF tetrabutylammonium fluoride
TMSCI chlorotrimethylsilane
TMSBr bromotrimethylsilane
TMSI iodotrimethylsilane
TMSOTf (trimethylsilyl)trifluoromethylsulfonate
TEA triethylamine
TBA tributylamine
TBAP tributylammonium pyrophosphate
TBSC1 t-butyldimethylsilyl chloride
TEAB triethylammonium bicarbonate
TFA trifluoroacetie acid
TLC or tic thin layer chromatography
Tr triphenylmethyl
Tol 4-methylbenzoyl
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Turbo Grignard 1:1 mixture of isopropylmagnesium chloride and lithium chloride
I
6 parts per million down field from tctramethylsilane
Preparation of Compounds
Compound 1
S'
sµ'N
s_
Bz
/OA 0
Bz
Br N---c /0 N
____________________________________________________ OH
Bu Li, BF3-Et20
/0 F /0 F
Bz THF Bz
la lb
To a suspension of 7-bromo-2,4-bis-methylsulfanyl-imidazo[2,1-
f][1,2,4]triazinc (prepared according to W02008116064, 500 mg, 1.72 mmol) in
anhydrous THF (5 mL) was dropwisc added BuLi (1.6 M in hexanes, 1.61 mL, 2.41
mmol) at -78 C. The suspension became red brown solution after 5 min, and then
a
mixture of la (prepared according to WO 200631725, 675 mg, 1.81 mmol) and
boron
trifluoride etherate (2.40 mL, 1.89 mmol) in THF (5 mL) was added dropwise to
the
mixture. After stirring for 2 h at -78 C, saturated NH4C1 was added to quench
the
reaction. The mixture was diluted with ethyl acetate; the organic layer was
washed
with brine and concentrated in vacua. The residue was purified by silica gel
column
chromatography (Et0Ac / hexanes), affording lb as a rich yellow foam (650 mg,
67%). 1H NMR (400 MHz, CDC13): 6 8.13 (d, 2H), 8.03 (d, 2H), 7.81 (d, 1H),
7.59
(t, 114), 7.45 (m, 3H), 7.36 (t, 2H), 6.40 (brs, 1H), 6.01 (dd, 1H), 4.78 (m,
2H), 4.60
(dd, 1H), 2.68 (s, 31-1), 2.45 (s, 311), 1.62 (d, 3H). 19F NMR (376 MHz,
CDC13): ö -
167.5. MS = 585.1 (M + H+).
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N
F"N
Bz Bz
Et3SiH
F BF3-Et20
Bz Bz
CH2Cl2
lb lc
To a solution of lb (820 mg, 1.40 mmol) in dichloromethane (20 mL) were
added boron trifluoride etherate (2 mL) and triethylsilane (2 mL), and stirred
at room
temperature for 16 h. Additional boron trifluoride etherate (1 mL) and
triethylsilane
(1 mL) were added, and stirred for 7 d. The mixture was diluted with
dichloromethane and saturated sodium bicarbonate. The organic layer was washed
sequentially with water, saturated ammonium chloride and brine, dried over
magnesium sulfate, and concentrated. The residue was purified by silica gel
column
chromatography (Et0Ac / hexanes), affording le (605 mg, 76%). ill NMR (400
MHz, CDCI3): 8 8.10 (d, J= 7.2Hz, 2H), 8.00 (d, J= 7.2 Hz, 2H), 7.66 (s, 1H),
7.61
(t, J= 7.2 Hz, 1H), 7.53 (t, J= 7.2 Hz, 1H), 7.46 (t, J= 7.2 Hz, 2H), 7.38 (t,
J= 7.2
Hz, 2H), 5.78 (m, 2H), 4.80 (dd, 1H), 4.68 (m, 1H), 4.60 (dd, 1H), 2.68 (s, 31-
1), 2.65
(s, 3H), 1.32 (d, 3H). 19F NMR (376 MHz, CDC13): 5 -149.9. MS = 569.1 (M +
H+).
S NH2
Bz/C)---y HCH"q0 NN7-4N,
1) NH3 N s,
,c5 2) Na0Et / THF HO F
Bz
1c Id
Compound le (635 mg, 1.12 mmol) was placed in a steel bomb reactor.
Liquid ammonia (-30 mL) was charged and the bomb reactor was tightly sealed.
The
mixture was stin-ed at 50 C for 16 h. After cooling to room temperature,
ammonia
was evaporated and the solid residue was dissolved in THF (10 mL) and Me0H (10
mL). Sodium ethoxide (25% wt. 0.63 mL) was added and stirred at 60 C for 40
min.
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The mixture was neutralized with AcOH and concentrated. The residue was
purified
by RP HPLC, affording the product id (175 mg, 48%). 'H NMR (400 MHz, DMSO-
d6): 6 8.21 (brs, 214), 7.60 (s, 1H), 5.45 (brs, 1H), 5.43 (d, 1H), 4.91 (t,
1H), 3.92 (m,
1H), 3.76 (m, 214), 3.57 (m, 1H), 2.44 (s, 3H), 1.09 (d, 3H). I9F NMR (376
MHz,
DMSO-d6): 5 -153.5. MS = 330.1 (M +
NH2 NH2
N
HO
N s, MCPBA
0' \\
0
HO CH2C12 HO =P
Id le
To a solution of id (175 mg, 0.53 mmol) in dichloromethane (11 mL) was
added MCPBA (370 mg, ¨ 1.5 mmol) and stirred at room temperature for 16 h. The
mixture was concentrated, affording crude 1 e which was used for the next
reaction
without purification. MS = 362.0 (M + H+).
NH2 NH2
HO
0 N.
0 INisN'N H2
NH3
\\
HO
0
Ho HO
1 e 1
Compound le (obtained from the previous reaction) was placed in a steel
bomb reactor. Liquid ammonia (-30 mL) was charged, and the bomb reactor was
tightly sealed. The mixture was stirred at 115 C for 3 d. After cooling to
room
temperature, ammonia was evaporated. The solid residue was purified by RP
HPLC,
affording compound 1(105 mg, 66% in two steps). 11-1 NMR (400 MHz, D20): 67.31
(s, 1H), 5.43 (d, J=.- 25.2 Hz, 1H), 4.07 (dd, J= 9.6, 23.2, 1H), 3.89 (m,
1H), 3.83 (dd,
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J= 2.4, 12.8 Hz, 1H), 3.67 (dd, J = 4.8, 12.8 Hz, 1H), 1.05 (d. J= 22.8 Hz,
3H). 19F
NMR (376 MHz, 1)20): 6 -153.5. MS = 299.2 (M + fl+).
Compound 2
NH2 0
'NH
HOA0 N, HOA0 Ns
adenosine deaminase
N NH2
HO "F water
HO F
1 2
To a solution of compound 1 (82 mg, 0.28 mmol) in water (340 mL) was
added adenosine deaminase (A5168 bovine spleen type IX from Sigma-Aldrich,
0.125
Unit per mL of water) and stirred at 37 C for 4 h. The mixture was
concentrated and
purified by RP HPLC, affording compound 2 (56 mg, 68%). 'H NMR (400 MHz,
D20): 67.35 (s, 1H), 5.46 (d, J= 25.2 Hz, 1H), 4.08 (dd, J= 9.6, 22.6, 1H),
3.93 (m,
1H), 3.87 (dd, J= 2.4, 12.8 Hz, 1H), 3.71 (dd, J= 4.8, 12.8 Hz, 1H), 1.12 (d,
J= 23.2
Hz, 3H). I9F NMR (376 MHz, 1)20): 8 -153.4. MS = 300.2 (M +
Compound 3
NH2
NH2
\ N
N,
Bz70-yro
Br N BzPAO
OH
BuLi, TMSCI
OF OF
Bz THF Bz
la 3b
To a suspension of 7-bromo-pyrrolo[2,1-f][1,2,4]triazin-4-ylamine (prepared
according to W02007056170, 2.13 g, 10 mmol) in THF (20 mL) was added TMSC1
(2.66 mL, 21 mmol) and stirred at room temperature for 16 h under argon. After
cooling to -78 C, a solution of BuLi (1.6 M, 21 mL, 33 mmol) in hcxancs was
added
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dropwise. The mixture was stirred for 1 h at the same temperature. A solution
of 1 a
(prepared according to WO 200631725, 4.46 g, 12 mmol) in THF (10 mL) was then
added. After stirring for 2 h at -78 C, saturated ammonium chloride was added
to
quench the reaction. The mixture was extracted with ethyl acetate. The organic
extract was concentrated in vacuo. The residue was purified by silica gel
chromatography (ethyl acetate / hexanes), affording 3b as a yellow solid (1.6
g, 32%).
MS = 507.1 (M + H+).
Alternative procedure for Compound 3b using 1,2-bis-
l(chlorodimethypsilanyllethane instead of chlorotrimethylsilane
To a suspension of 7-bromo-pyrrolo[2,1-f][1 .2,4]triazin-4-ylamine (500 mg,
2.35 mmol) in THF (6.5 mL) was added BuLi (1.6 M in hexanes, 1.6 mL) at -78 C.
After 30 mm., a solution of 1,2-bis-[(chlorodimethyl)silanyl]ethane (538 mg,
2.4
mmol) in THF (1.2 mL) was added. After 45 min., BuLi (1.6 mL) was added. After
an additional 30 min., BuLi (1.5 mL) was added. After 30 min., a solution of
la (610
mg, 1.64 mmol) in THF (2 mL) was then added dropwise. The resulting mixture
was
stirred at -78 C for 2 h under argon. Acetic acid (0.7 mL) was added dropwise
to
quench the reaction, followed by addition of saturated ammonium chloride. The
mixture was extracted with ethyl acetate. The organic extract was concentrated
in
vacuo. The residue was purified by silica gel chromatography (ethyl acetate /
hexanes), affording 3b (320 mg, 40%). The starting la was also recovered (350
mg)
from the chromatography.
NH2 NH2
N
TMSCN, Bz/0-0
___________________ OH TMSOTf CN
Bz F AcCN Bz F
3b 3c
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To a solution of compound 3b (50 mg, 0.1 mmol) and TMSCN (67 uL, 0.5
mmol) in acetonitrile (2.0 mL) at 0 C was added TMSOTf (91 uL, 0.5 mmol). The
reaction mixture was stirred at room temperature for 1 h, then at 65 C for 3
d. The
reaction was quenched with saturated NaHCO3 at room temperature, and diluted
with
CH3CO2Et. The organic phase was separated, washed with brine, dried over
Na2SO4,
filtered and concentrated. The residue was purified by RP-HPLC (acetonitrile /
water), to give the desired compound 3c (28 mg, 54%). MS = 516.1 (M +
NH2
NH2
Bz/0-0 ____________
28% NH3 in water HO 0 Ns /N
__________________ CN
/0- -F Me0H
Bz HO -F.
3c 3
To a solution of 3c (56 mg, 0.11 mmol) in methanol (1.2 mL) was added
ammonium hydroxide (28% in water, 0.8 mL) and stirred at room temperature for
16
h. The mixture was concentrated and the residue was purified by RP HPLC (water
/
acetonitrile), affording compound 3 (20 mg, 60%). 'H NMR (500 MHz, D20): 6
7.88
(s, 1H), 7.07 (d, 1H), 6.92 (d, 1H), 4.17 (m, 2H), 4.04 (dd, 1H), 3.87 (dd,
1H), 1.15 (d,
3H). MS = 308.1 (M + Fr).
Compound 4
NH2 NH
j0 0 Ns ___/N
HO-0 Ns /N
Bz N'
OH 28% NH3 in water OH
F HO
Bz CH3OH
3b 4
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To a solution of compound 3b (60 mg, 0.12 mmol) in methanol (0.5 mL) was
added ammonium hydroxide (28% in water, 0.5 mL) and stirred at room
temperature
for 16 h. The mixture was concentrated and the residue was purified by RP HPLC
(water / acetonitrile), affording compound 4 (25 mg, 70%). MS = 299.1 (M +
H+).
Compound 5
NH2 NH
0 0 N _N Et3SiH
Bz/ 'N1 BF3-Et20 Bz0tN
OH
CH20I2
/0 F /C5 -F
Bz Bz
3b 5a
28% NH3 in water
CH3OH
NH2
HO 0 N, /
N--""
Ho vr'
5
Compound 3b was converted to compound 5a by a procedure similar to
conversion of lb to lc. Compound 5a was then converted to compound 5 by a
procedure similar to conversion of 3c to 3. 1H NMR (300 MHz, D20): 6 7.68 (s,
1H), 6.75 (d, J= 4.5 Hz, 1H), 6.65 (d,J= 4.5 Hz, al), 5.65 (d, J= 25.2 Hz,
1H), 3.95
(m, 3H), 3.74 (dd, 1H), 0.98 (d, J= 22.8 Hz, 3H). 19F NMR (282 MHz, D70): 8 -
154.2. MS = 283.2 (M + H+).
General procedure for preparation of a nucleoside triphosphate:
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To a pear-shaped flask (5-15 mL) is charged with a nucleoside (-20 mg).
Trimethyl phosphate (0.5-1.0 mL) is added. The solution is cooled with ice-
water
bath. POCI3 (40-45 mg) is added and stirred at 0 C until the reaction is
complete (1 to
4 h; the reaction progress is monitored by ion-exchange HPLC; analytical
samples are
prepared by taking ¨3 IA of the reaction mixture and diluting it with 1.0 M
Et3N.H2CO3 (30-50 aL)). A solution of pyrophosphate-Bu3N (250 mg) and Bu3N (90-
105 mg) in acetonitrile or DMF (1-1.5 mL) is then added. The mixture is
stirred at
0 C for 0.3 to 2.5 h, and then the reaction is quenched with 1.0 M Et31\11-
12CO3 (-5
mL). The resulting mixture is stirred for additional 0.5-1 h while warming up
to room
temperature. The mixture is concentrated to dryness, re-dissolved in water (4
mL),
and purified by ion exchange HPLC. The fractions containing the desired
product is
concentrated to dryness, dissolved in water (-5 mL), concentrated to dryness,
and
again dissolved in water (-5 mL). NaHCO3 (30-50 mg) is added and concentrated
to
dryness. The residue is dissolved in water and concentrated to dryness again.
This
process is repeated 2-5 times. The residue is then subjected to C-18 HPLC
purification, affording the desired product as a sodium or salt.
Alternatively, the
crude reaction mixture is subjected to C-18 HPLC first and then ion exchange
HPLC
purification to afford the desired product as a triethylammonium salt.
Compound TP-1
0
0 0 0
it
HO-P-O-P-O-P-O-Aco
OH OH OH NH2
HO
TP-1
Compound TP-1 was prepared by the general method using Compound 2 as
starting material. 1H NMR (300 MHz, D20): 6 7.44 (s, 1H), 5.45 (d, J = 25.5
Hz,
1H), 4.0-4.4 (m, 4H), 3.05 (m, NC112CH3), 1.10 (in, NCH1CH3 and 2'-C-CH3). 31P
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NMR (121.4 MHz, D20): 6-9.5 (d, J = 22.1 Hz), -11.0 (d, 1= 19.9 Hz), -23.2 (t,
J
23.0 Hz). 19F NMR (282 MHz, D20): 6-153.9.
Compound TP-2
NH2
0 9 9
\
H0+0+- - 0F1)0-y N
Ns
OH OH OH N'
7====
Ha
TP-2
Compound TP-2 was prepared by the general method using Compound 3 as
starting material. IFINMR (300 MHz, D20): 6 7.82 (s, II-I), 7.03 (d, 1H), 6.90
(d,
1H), 4.1-4.4 (m, 4H), 3.05 (m, NC112CH3), 1.10 (m, NCH2CH3 and 2'-C-CH3). 31P
NMR (121.4 MHz, D20): 6 -10.7 (d, J = 19.5 Hz), -11.3 (d, J = 19.8 Hz), -23.1
(t, J =
19.8 Hz).
Compound TP-3
NH2
9 V 9
\
HOT)-0-H-0-Fi'-0 N N, /
OH OH OH
HO F
TP-3
Compound TP-3 was prepared by the general method using Compound 5 as
starting material. 11-1 NMR (300 MHz, D20): 6 7.73 (s, 1H), 6.87 (d, 1H), 6.82
(d,
1H), 5.71 (d, J = 24.6 Hz, I H) , 4.0-4.4 (m, 41-1), 3.05 (m, NCH2CH3), 1.14
(m,
NCH2CH3), 1.00 (d, J = 22.8 Hz, 3H, 2'-C-CH3). 31P NMR (121.4 MHz, D20): 6-8.1
(d, 1= 22.1 Hz), -11.1 (d, J = 19.9 Hz), -22.7 (t, J = 23.0 Hz). 19F NMR (282
MHz,
D20): 6 -155.6. MS = 520.9 (M - H+).
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Compound TP-8a
NH2
N
HO- P -0-P -0-P -0 N
OH OH OH
7-
HO F
TP-8a
Compound TP-8a was prepared by the general method using Compound 8
as starting material. 11-1 NMR (300 MHz, D20): 6 7.95 (s, 1H), 7.68 (s, 1H),
5.63 (d, J
= 25.5 Hz, 1H), 4.0-4.4 (m, 4H), 3.05 (m, NCH2CH3), 1.10 (m, NCH2CH3 and 2'-C-
CH3). 31P NMR (121.4 MHz, D20): 6 -9.20 (d, J = 22.1 Hz), -11.07 (d, 1= 19.9
Hz), -
23.82 (t, J = 23.0 Hz). 19F NMR (282 MHz, D20): 8 -155.9. MS = 521.6 (M -
General procedure for preparation of a nucleoside prodrug (Method A):
R8
X N
HOA0)¨N4N
R9
_
1H-tetrazole
0
2. 30% H202
R8
so
R4 F
0 X2.
\ 0
j:
7-R6 N R9
0 R1
A
To a solution of a nucleoside (0.1 mmol) in trimethylphosphite (1.0 mL) are
added /H-tetrazole (42 mg, 0.6 mmol) followed by addition of 2,2-dimethyl-
thiopropionic acid S-(2-{diisopropylamino-[2-(2,2-dimethyl-propionylsulfany1)-
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ethoxy]-phosphanyloxyl-ethyl) ester (prepared according to J. Med. Chem.,
1985, 38,
3941, 90 mg, 0.2 mmol) at 0 C. After stirring for 2 h, 30% hydrogen peroxide
in H20
(140 j_iL) was added to the mixture. The mixture was then allowed to warm up
to
room temperature. After 30 min stirring, 1 M Na2S203 in 1-120 (5 mL) was added
to
quench the reaction. The organic layer was washed with saturated aqueous
Na2CO3
(10 mL x 2), brine, concentrated in vacuo. The residue was purified by RP-HPLC
(MeCN¨H20 gradient) to afford a prodrug A.
Compound A-1
NH2
S 0 1\1:7---
0,11
ry-P-0 0 Nµ
z
HO F
A-1
Compound A-1 was prepared by Method A using compound 1 as starting
material. IFI NMR (400 MHz, CDC13): 6 7.42 (s, 1H), 5.47 (d, J= 26.4 Hz, 1H),
4.95
(brs, 2H), 4.59 (m, 2H), 4.35 (m, 1H, 4'-H), 4.18 (m, 2H, 5'-H), 4.10 (m, 4H),
3.13
(m, 4H), 1.24 (d, 3H), 1.22 (s, 9H), 1.19 (d, 9H). 31P NMR (161.9 MHz, CDC13):
6 -
1.26. MS = 667.1 (M Fr).
General procedure for preparation of a nucleoside prodrug (Method B):
Non-limiting examples of mono-phosphoramidate prodrugs comprising the
instant invention may be prepared according to general Scheme 1.
Scheme 1
135
0
0 RY 0 Ar0-7¨CI
ArO¨P1 ¨CI H2N,..,/ NH
HCI \O¨R
CI Rx
Rx
0
19a 19b 19c
R8 R8
X2 \
X2 0 Ar0 _N N
HO / 0
19c 0
= = 1R6 R9 = = 1R6
R9
RQ Z. R1
Os
R-4 k2 0 \R R4 ik2
19d
The general procedure comprises the reaction of an amino acid ester salt 19b,
e.g., HC1 salt, with an aryl dichlorophosphate 19a in the presence of about
two to ten
equivalents of a suitable base to give the phosphoramidate 19c. Suitable bases
include, but are not limited to, imidazoles, pyridines such as lutidine and
DMAP,
tertiary amines such as triethylamine and DABCO, and substituted amidines such
as
DBN and DBU. Tertiary amines are particularly preferred. Preferably, the
product of
each step is used directly in the subsequent steps without recrystallization
or
chromatography. Specific, but non-limiting, examples of 19a, 19b, and 19c can
be
found in WO 2006/121820. A nucleoside base 19d reacts with the phosphoramidate
19c in the presence of a suitable base. Suitable bases include, but are not
limited to,
imidazoles, pyridines such as lutidine and DMAP, tertiary amines such as
triethylamine and DABCO, and substituted amidines such as DBN and DBU. The
product B may be isolated by recrystallization and/or chromatography.
Compound B-1
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NH2
oI 9 N N
\
0 HN-P-0
N,
_
HO F
B-1
Phenyl ethoxyalaninyl phosphorochloridate (124 mg, 0.42 mmol; prepared
according to McGuigan et al, J. Med. Chem. 1993, 36, 1048-1052) was added to a
mixture of Compound 3 (20 mg, 0.065 mmol) and N-methylimidazole (42 1A,, 0.52
mmol) in anhydrous trimethyl phosphate (0.8 mL). The reaction mixture is
stirred for
3 h at room temperature, and then methanol was added to quench the reaction.
The
methanol solvent is removed under reduced pressure. The residue was purified
by
reverse-phase HPLC and then by silica gel column chromatography (100% ethyl
acetate), affording compound B-1 (10 mg, 27%). 31P NMR (121.4 MHz, CDC13): 5 -
3.42, 3.77. MS = 563.0 (M + Fe), 561.0 (M¨ H+).
Compound B-2
N /7"
N
0/
'CN
11,
CI
B-2
About 3.1 mmol of 4-chlorophenyl 2-propyloxyalaninyl phosphorochloridate
(prepared according to McGuigan et al, J. Med. Chem. 1993, 36, 1048-1052) is
added
to a mixture of about 0.5 mmol of Compound 3 and about 3.8 mmol of N-
methylimidazole in about 3 mL anhydrous trimethyl phosphate. The reaction
mixture
is stirred for about one hour to 24 hours at room temperature and methanol is
added to
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quench the reaction. The methanol solvent is removed under reduced pressure.
The
residue is purified by reverse-phase HPLC to give compound B-2.
Compound B-3
NH2
0
____________________________ \ it
0 HN-P-0 0 N.N
(1) = 'CN
HO F
B-3
Compound B-3 was obtained by a similar procedure used for compound B-1.
3/P NMR (121.4 MHz, CDC13): 6 -3.50, 3.76. MS = 577.2 (M +11+).
Compound B-4
NH2
V' 0
"
0
="CN N
HO F
B-4
Compound B-4 was obtained by a similar procedure used for compound B-1.
31P NMR (162 MHz, CD30D): 6 2.2. MS = 633.4 (M + Hi).
Compound B-5
NH2
\ ______________________ 0
C) 0 IN=KI-j
0 H 6
N
Hd
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B-5
Compound B-5 was obtained by a similar procedure used for compound B-1.
31P NMR (162 MHz, CDC13): 6 4.15, 4.27. MS = 549.3 (M +
Compound B-6
NH2
0 0 N
0 H 6
N
B-6
Compound B-6 was obtained by a similar procedure used for compound B-1.
31P NMR (162 MHz, CDC13): 6 3.50, 4.07. MS = 613.1 (M +
Compound B-7
NH2
0 0 N N
)r---N¨P---(3
0 H 6
Ho- 'F.
B-7
Compound B-7 was obtained by a similar procedure used for compound B-1,
using compound 5 as parent nucleoside. 31P NMR (162 MHz, CDC13): 6 3.37, 3.97.
MS =538.1 (M + Fr).
Compound B-8
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NH2
\-0 T o
N,
0 H 6
H -F
B-8
Compound B-8 was obtained by a similar procedure used for compound B-1,
using compound 5 as parent nucleoside. 31P NMR (162 MHz, CDC13): 6 3.69, 4.39.
MS = 588.1 (M + H+).
Alternative procedure for preparation of a nucleoside prodrug (Method C):
0
/
N-P-0
0 H 6
No2
C-1 a
Into a flask containing ethyl L-valine hydrochloride (2.5 g, 13.8 mmoL, 1
equiv.) was added CH2C12 (46 mL, 0.3 M) and phenyl dichlorophosphate (2.1 rnL,
13.8 mmoL, 1 equiv.) before being cooled to -10 C. After 10 minutes, TEA (3.8
mL,
13.8 mmoL, 1 equiv) was added slowly to the reaction mixture over five
minutes. The
reaction was allowed to proceed for an hour before p-nitrophenol (1.9 g, 13.8
mmoL,
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1 equiv.) was added to the reaction mixture followed by addition of more TEA
(3.8
mL, 13.8 mmoL, 1 equiv.) over five minutes. The reaction was allowed to warm
up
and proceed for another two hours. The reaction was concentrated in vacuo and
taken
up in diethyl ether (200 mL). The insoluble salts were filtered off and the
filtrate
concentrated in vacuo. Flash column chromatography was carried out using 4/1
Hex /
Et0Ac to furnish a clear oil as C-1a.
11-1 NMR (400 MHz, CDC13): d 8.21 (s, 2 H), 7.41 -7.20 (m, 7 H), 4.22 -4.05
(m, 3
H), 2.46 (s, 2 H), 1.99 (dd, J= 23.0, 20.1 Hz, 2 H), 1.68 (s, 1 H), 1.20 -1.05
(m, 8 H).
31P NMR (162 MHz, CDC13): d -2.79 (dd, J= 28.0, 4.2 Hz).
LC MS m/z 422.99 [M +
Compound C-1
NH2
N N
0 :7 0
)7N-P-C)-Ar0
0 H 6
HO ______________________________________ =- NF
C-1
Into a flask containing compound 3 (70 mg, 0.23 mmoL, 1 equiv.) was added
THF (1 mL, 0.2 M) and NMP (1 mL, 0.2 M) before cooling to 0 C. t-BuMgC1 (560
'IL, 2.5 equiv., 1M THF) was added slowly and allowed to stir for 5 minutes
before
the above phenolate C-1a (207 mg, 0.46 mmoL, 2 equiv. dissolved in 500 1t1.,
of THF)
was added. The reaction mixture was warmed to 50 C. The reaction was monitored
by LCMS. Once the reaction was complete, the mixture was then concentrated in
vacuo, and the residue was purified by HPLC, affording Compound C-.1.
1FINMR (400 MHz, CDCb) d 7.87 (s, 1 H), 7.24 - 7.10 (m, 4 H), 7.03 (t, J= 7.2
Hz,
1H), 6.81 (d, J= 4.6 Hz, I H), 6.52 (d, J= 4.7 Hz, 1 H), 5.61 (s, 2H), 4.46
(dd, J=
24.0, 11.4 Hz, 2 H), 4.33 -4.14 (m, 2 H), 4.06 (dt, .1= 7.2, 4.2 Hz, 2 H),
3.82- 3.70
(m, 1 H), 3.63 (t, J= 10.6 Hz, 2 H), 1.98 (s, 1 H), 1.17 (dd, 14.8, 7.6 Hz,
3 H),
.. 0.82 (dd, J= 22.8, 6.8 Hz, 6 H).
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.. 31P NMR (162 MHz, CDC13): d 5.11.
19F NMR (376 MHz, CDCI3): d -152.28.
LC MS ink 591.21 [M + 1-11.
\S
/0 0
0 H 0
1110NO2
C-2a
Compound C-2a was obtained in a procedure similar to that exemplified for
Compound C-la but using the methionine ester.
1H NMR (400 MHz, CDCI3) d 8.19 (s, 2 H), 7.44 ¨ 7.03 (m, 7 H), 4.11 (s, 2 H),
3.81
(d, J= 44.5 Hz, 1H), 2.04 (s, 3 H), 1.61 (s, 2 H), 1.21 (d, .1= 6.1 Hz, 2 H),
1.01 ¨0.65
(m, 4 H).
31P NMR (162 MHz, CDC13) d -2.00 (d, J¨ 12.9 Hz).
LC MS m/z 455.03 [M
Compound C-2
NH2
\ ______________________ 0 N
0 H 6
N
H0
C-2
Compound C-2 was obtained in a procedure similar to that exemplified for
Compound C-1 using Compound 3 and C-2a.
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1H NMR (400 MHz, CDC13) d 7.96 (d, J= 15.8 Hz, 1H), 7.40 - 7.06 (m, 13H), 6.93
(d, J= 6.7 Hz, 1H), 6.70 (s, 1H), 5.98 (s, 1H), 4.54 (dd, J= 21.6, 11.7 Hz,
2H), 4.32
(d, J= 12.0 Hz, 2H), 4.14 (dt, J= 13.0, 6.4 Hz, 4H), 2.44 (d, J= 7.5 Hz, 2H),
2.00 (d,
J= 16.2 Hz, 5H), 1.89 (s, 2H), 1.35 - 1.13 (m, 7H).
31P NMR (162 MHz, CDC13) d 4.12, 3.58.
19F NMR (376 MHz, CDC13) d -152.28 (s).
LC MS m/z 623.27 [M + H4].
HN
\-0 0
NO2
0 H 0
110
C-3a
Compound C-3a was obtained in a procedure similar to that exemplified for
Compound C-la but using a tryptophan ester.
1H NMR (400 MHz, CDC13) d 8.18 - 8.03 (m, 3 H), 7.29 - 7.08 (m, 8 H), 7.36-
6.98
(m, 3 H), 4.41 - 4.11 (m, I H), 4.15 - 3.95 (m 2 H), 3.68 - 3.80 (m, 1 H),
3.33 - 3.04
(in. 2 H), 1.06 -1.17 (m, 3 H).
31P NMR (162 MHz, CDC13) d -2.87, -2.99.
LC MS m/z 510.03 [M + H4].
Compound C-3
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HNTNH2
\--0
0
0 H 6
N
410 Hd
C-3
Compound C-3 was obtained in a procedure similar to that exemplified for
Compound C-1 using Compound 3 and C-3a.
1H NMR (400 MHz, CDC13) d 8.27 (s, 1H), 7.84 (s, 1H), 7.47 (s, 1H), 7.36- 6.77
(in,
11 H), 6.57 (s, 1 H), 4.40- 3.96 (m, 6 H), 3.20 (s, 4 H), 2.60 (s, 1H), 1.30-
1.04 (m,
6H).
31P NMR (162 MHz, CDC13) d 4.02, 3.75
19F NMR (376 MHz, CDC13) d -152.13.
LC MS m/z 678.32 [M + H+].
\-0 ;. 0
)nNk
NO2
0 H 0
C-4a
Compound C-4a was obtained in a procedure similar to that exemplified for
Compound C-la by substituting the phenylalanine ester.
1H NMR (400 MHz, CDC13) d 8.15 (t, J= 8.7 Hz, 2H), 7.43 - 7.11 (m, 10 H), 7.04
(ddd, J= 11.4, 6.7, 2.9 Hz, 2 H), 4.32 (ddd,J= 15.3, 11.3,6.1 Hz, 4 H), 4.15 -
3.99
(m, 7 H), 3.74 (td, J= 11.0, 5.0 Hz, 8 H), 3.01 (d, J= 5.7 Hz, 2 H), 1.17 (td,
J= 7.1,
5.2 Hz, 2 H).
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31P NMR (162 MHz, CDC13) d -2.97, -2.99.
LC MS m/z 471.03 [M +
Compound C-4
NH2
0 H 6
. N
C-4
Compound C-4 was obtained in a procedure similar to that exemplified for
Compound C-1 using Compound 3 and C-4a.
'H NMR (400 MHz, CDC13) d 7.92 (d, J= 13.2 H7, 1H), 7.46¨ 6.97 (m, 17H), 6.91
(s, 1H), 6.75 (s, 1H), 4.10 (dd, J= 29.6, 19.2 Hz, 8H), 2.97 (s, 3H), 1.32¨
1.05 (m,
7H).
31P NMR (162 MHz, CDC13) d 5.11.
19F NMR (376 MHz, CDC13) d -152.34 (s).
LC MS m/z 639.24 [M + ].
CN¨P No2
C-5a
Compound C-5a was obtained in a procedure similar to that exemplified for
Compound C-la but using the proline ester.
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11-i NMR (400 MHz, CDC13) d 8.20 (d, J = 7.8 Hz, 2 H), 7.45 - 7.08 (m, 7 H),
4.37
(td, J= 8.0, 3.8 Hz, 2 H), 4.17 - 3.98 (m, 2 H), 3.61 - 3.34 (m, 2 H), 2.21 -
1.77 (m, 3
H), 1.19 (td, J= 7.1, 3.8 Hz, 3 H).
31P NMR (162 MHz, CDC13) d-3,92, -3.96.
LC MS m/z 420.98 [M + H4].
Compound C-5
/----0 NH2
Ct 0 O
= = N
C-5
Compound C-5 was obtained in a procedure similar to that exemplified for
Compound C-1 using Compound 3 and C-5a.
1H NMR (400 MHz, CDC13) d 7.95 (d, J= 4.5 Hz, 1 H), 7.39 - 7.10 (m, 4 H), 6.92
(dd, J= 16.0, 4.6 Hz, 1 H), 6.69 (s, 1H), 6.03 (bs, 2 H), 4.46 -4.36 (m, 1 H),
4.36 -
3.96 (m, 4 H), 3.37 (d, J= 58.9 Hz, 2 H), 2.26 - 1.66 (m, 4 H), 1.39- 1.12 (m,
8 H).
31P NMR (162 MHz, CDCI3) d 3.47, 2.75.
19F NMR (376 MHz, CDC13) d -152.36.
LC MS m/z 589.14 [M + H4].
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0
NH2
\-0 0 N N
N
0 H 6
,\
HO F
Compound C-6
Compound C-6 was obtained in a procedure similar to that exemplified for
Compound C-1 using Compound 3 and the sulphonc analog of C-la.
1H NMR (400 MHz, CDC13) d 7.93 (s, 1 H), 7.89 (s, 1 H), 7.35 ¨ 7.01 (in, 5 H),
6.93
.. (d, J= 2.8 Hz, 1 H), 6.58 (d, J= 2.8 Hz, 1H), 5.79 (bs, 2 H), 4.30 (s, 6
H), 4.11 (d, J
¨ 7.0 Hz, 6H), 3.10 ¨ 2.84 (m, 3 H), 2.75 (s, 3 H), 2.54 (s, 6 H), 1.31 -1.15
(m, 6 H).
31P NMR (162 MHz, CDC13) d 3.39, 3.33.
I9F NMR (376 MHz, CDC13) d -152.40
LC MS m/z 655.24 IM +
Compound PD-A-8b
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NH2
o c:?
HN-P-0 HO 0 N,N,-.)
NO Ho
30d-1 Compound 8
NH2
z
0
t-BuMgCI
0 HN-P-0
0 Ns
NMP/THF 0
HO
PD-A-8b
To a solution of Compound 8 (200 mg, 0.71 mmol) in THF (1 mL) and NMP
(1 mL) under an atmosphere of argon at 0 C was added tert-butyl magnesium
chloride
(1.0 M in THF, 1.06mL, 1.06 mmol). After 15 minutes, compound 30d-1 (280 mg,
0.71 mmol) was added as a solution in THF. After 5 minutes, the reaction
mixture
was allowed to warm to room temperature and was stirred for 2 hours. The
reaction
mixture was cooled to 0 C, quenched with Me0H, and concentrated. The reaction
was purified by silica gel chromatography and then RP HPLC, affording PD-A-8b
(225 mg, 59%). IFI NMR (400 MHz, CDC13): d 8.09 (two s, 1H), 7.54 (two s, 1H),
7.31-7.12 (m, 5H), 5.66 (dd, 1H), 4.52-4.45 (m, 2H), 4.19-4.03 (m, 4H), 3.87-
3.69 (m,
1H), 1.35-1.15 (m, 9H). I'P NMR (161 MHz, CDC13): d4.14 (s), 3.55 (s).
LC/MS = 539 (M+ H+).
Retention time: 1.94 min
LC: Thermo Electron Surveyor HPLC
MS: Finnigan LCQ Advantage MAX Mass Spectrometer
Column: Phenomenex Polar RP 30 mm X 4.6 mm
Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid
Gradient: 0 min-0.1 min 5% ACN, 0.1 min-1.95 min 5%-100% ACN, 1.95 min-3.5
min 100% ACN, 3.5 min-3.55 min 100%-5% ACN, 3.55 min-4 min 5% ACN.
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Preparation of 30d-1
\()
'\ NH2 HCI
CI¨P-CI \
0 0 HN--CI
6
TEA
30a
30c-1
HO
NO2 o HN-P-0
TEA
NO2
30d-1
Compound 30d-1 was prepared from 30a in a matter similar to that of 30d-2
substituting alanine ethyl ester hydrochloride for alanine isopropyl ester
hydrochloride.
Compound (S)-PD-A-8c
NH2
0 HNP-1?-0 HO
NO2 Hos
(S)-30d-2 Compound 8
NH2
0
t-BuMgCI
NMP / THF 0
HO
(S)-PD-A-8c
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Compound (S)-PD-A-8c was prepared in a matter similar to that of PD-A-8b
substituting (S)-30d-2 for 30d-1. 1H NMR (400 MHz, CDC13): d 8.14 (s, 1H),
7.60
(s, 1H), 7.1-7.3 (m, 5H), 5.66 (did, 1H), 5.02 (in, 1H), 4.50 (m, 1H), 4.40
(m, 1H), 4.1-
4.3 (m, 2H), 3.98 (m, IH), 3.78 (m, 1H), 3.18 (brs, 1H), 1.15-1.4 (m, 12H).
31P NMR
(161 MHz, CDC13): d 3.70 (s).
LC/MS = 553 (M + H+).
Preparation of (S)-30d-2
CI¨P¨C1
) _______ 0
31a
HC ______________________________________________
-N-P-0
H '
0
0
p-Nitrophenol / IV'
TEA / CH2Cl2
30d-2 -0
Alanine isopropyl ester hydrochloride (7.95 g, 47.4 mmol) was suspended in
dichloromethane (100 mL). Compound 31a (10 g, 47.4 mmol) was added.
Triethyl amine (13.2 mL, 95 mmol) was then dropwise added over a period of 15
min.
(internal reaction temperature; -10 C ¨ -3 C). When the reaction was almost
complete (by phosphorous NMR), p-nitrophenol (6.29 g, 45.0 mmol) was added as
a
solid in one portion. To the resulting slurry was added triethylamine (6.28
mL, 45
mmol) over a period of 15 mm, The mixture was then wanned up to room
temperature. When the reaction was complete, MTBE (100 mL) was added. The
white precipitate was removed by filtration. The filter cake was washed with
MTBE
(3 x 50 mL). The filtrate and washings were combined and concentrated. The
residue
was purified by silica gel column chromatography (0 to 50% ethyl acetate /
hexanes),
affording compound 30d-2 as a 1:1 ratio of diastereomeric mixture (14.1 g,
77%). 1H
NMR (300 MHz, CDC13): 5 8.22 (2d, 2H), 7.2-7.4 (in, 7H), 5.0 (in, 1I-1), 4.09
(in,
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1H), 3.96 (m, 1H), 1.39 (2d, 3H), 1.22 (m, 6H). MS = 409.0 (M + H4), 407.2 (M -
H+).
Separation of two diatercomers of compound 30d-2
0
-N4-0
0 H =
0
N+ 0
o
-6
0 H (S)-30d-2
0
= 1\1+
-60 o
30d-2
0 H
0
diastereomeric mixture at phosphorous
1104 N+
-6
(R)-30d-2
The two di astereomers were separated by chiral column chromatography under
the
following conditions;
Column: Chiralpak IC, 2 x 25 cm
Solvent system: 70% heptane and 30% isopropanol (IPA)
Flow rate: 6 mL/min.
Loading volume per run: 1.0 mL
Concentration of loading sample: 150 mg / mL in 70% heptane and 30% IPA
(S)-compound 30d-2: retention time 43 mm. 31P NMR (162.1 MHz, CDC13): 6 -2.99
(s).
(R)-compound 30d-2: retention time 62 min. 31P NMR (162.1 MHz, CDC13): 6 -3.02
(s).
Alternatively, the two diastereomers were separated by crystallization under
the following procedure;
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Compound 30d-2 was dissolved in diethyl ether (-I 0 ml,/ gram). While
stirring, hexanes was then added until the solution became turbid. Seed
crystals (-10
mg / gram of compound 30d-2) were added to promote crystallization. The
resulting
suspension was gently stirred for 16 h, cooled to - 0 C, stirred for an
additional 2 h,
and filtered to collect the crystalline material (recovery yield of the
crystalline
material 35%-35%). The crystalline material contains -95% of (S)-compound 30d-
2
and -5% of (R)-compound 30d-2. Re-crystallization afforded 99%
diastereomerically pure (S)-isomer.
The following PD-A compounds as examples are made by the general
procedures:
Compound PD-A-8d
NH2
=
:= 0
\
0 HN¨PI-0 0 N,
0
Ho
Compound PD-A-8e
NH2
________________ 9 _\c_L;c_:.,,=N
=
HN¨P-0 0 N, J
0
-._
HO F
Compound PD-A-8f
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NH2
>0 _____________ 0
0 HN¨P-0 0 N, _J
01
111
HO F
Compound PD-A-8g
NH2
0
01
HO F
,and
Compound PD-A-8h
NH2
0
--- )
0 HN¨P-0 0 N,
01
HO F
General procedure for preparation of a nucleoside prodrug (Method D):
Non-limiting examples of 3'-0-acyalted mono-phosphoramidate prodrugs
comprising the instant invention may be prepared according to general Scheme
2.
Scheme 2
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R8
R8 0 x2 N N
0 X2 N N
Ar0.õ
yoi_N HNI
7 0 N -1R6 Rg
Ry, HN R3L1R1
Rx \ A be 2
0 0 R
2 OH --L
RHO R
0or Rz 0
Ft' 0 Rz 0
PD-A PD-B
The general procedure comprises the reaction of PD-A (R4 = OH) with a
carboxylic acid or an activated carboxylate such as an acyl chloride or an
acid
anhydride, which is generally known to those skilled in the art (Journal
ofMedicinal
Chemistry, 2006, 49, 6614 and Organic Letters, 2003, 6,807). When R8 = NH2,
protection of the amino group may be necessary. Briefly, to a solution of
compound
PD-A in acetonitrile (2 mL) is added N,N-dimethyforrnamide dimethyl acctal (¨
1.1
eq.) and stirred at room temperature for 1 h. After the protection of 6-amino
group is
complete, the mixture is then concentrated to dryness. To the residue are
added a
dehydrating agent such as DCC (¨ 4 eq.), acetonitrile and a carboxylic acid (¨
2 eq.).
The mixture is stirred at room temperature for 24 -48 h. Water (0.2 mL) and
trifluoroacetic acid (0.1 mL) are added at 0 C and stirred at room temperature
for 64
h. Sodium bicarbonate was added at 0 C. The mixture is stirred at room
temperature
for 0.5 h and filtered. The filtrate is concentrated and the residue was
purified by
silica gel column chromatography to afford compound PD-B. If an acyl chloride
or
an acid anhydride is used, a suitable base, such as triethylamine, is added
instead of a
dehydrating agent.
Compound PD-B-8i
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NH2
NH2 N
z 8 __ \
F 0
0
N, 0
0
0 F
Ho
PD-A-8b
PD-B-81
To a solution of PD-A-8b (100 mg, 0.19 mmol) in DCM (1.0 mL) under an
atmosphere of argon at room temperature was added N,N-dimethylformamide-
dimethylacetal (25 uL, 0.19 mmol). After 30 minutes, the reaction mixture was
concentrated. The reaction was taken up in DCM and concentrated. This process
was
repeated twice. The resulting residue was taken up in THF (1.0 mL) and cooled
to
0 C under an atmosphere of argon. To the solution was added triethylamine (79
L,
0.57 mmol) and DMAP (5mg, 0.04 mmol). After 5 minutes, isobutyryl chloride (60
uL, 0.57 mmol) was added. After 10 minutes, the reaction was allowed to warm
to
room temperature and was stirred for 3 hours. The mixture was cooled to 0 C,
quenched with a 5% TFA solution in water, and then allowed to stir at room
temperature for 4 hours. The resulting mixture was extracted with ethyl
acetate (3x).
The combined organic layers were dried with sodium sulfate, filtered and
concentrated. The residue was purified by RP HPLC (aeetonitrile / water),
affording
PD-B-8i (71 mg, 61%). 1H NMR (400 MHz, CDC13): d 8.17 (two s, 1H), 7.66 (two
s,
1H), 7.34-7.14 (m, 5H), 5.69 (dd, 1H), 5.56-5.43 (in, 1H), 4.55-4.01 (m, 5H),
3.79-
3.69 (m, 1H), 2.70-2.64 (m, 1H), 1.37-1.17 (m, 15H). 3113NMR (161 MHz, CDC13):
d
2.99 (s), 2.88 (s).
LC/MS = 609 (M+ H+).
Retention time: 2.21 min
LC: Thermo Electron Surveyor HPLC
MS: Finnigan LCQ Advantage MAX Mass Spectrometer
Column: Phenomenex Polar RP 30 mm X 4.6 mm
Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid
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Gradient: 0 min-0.1 min 5% ACN, 0.1 min-1.95 min 5%-100% ACN, 1.95 min-3.5
min 100% ACN, 3.5 min-3.55 mm 100%-5% ACN, 3.55 min-4 min 5% ACN.
The following PD-B compounds as examples are made by the general
procedures:
Compound PD-B-8j
NH2
0
HN¨P-0 0 N,
01
Compound PD-B-8k
NH2
= 0
\ N
0 HN¨PI-0 0 N, j
0
Compound PD-B-81
NH2
Cro)\9TN
0 HN¨P-0 0 N,
oI
0 F
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Compound PD-B-8m
NH2
0
0 HN-Fi'-0-v0 N,
0
Compound PD-B-8n
NH2
= 0
o
11 ________ \
HN-P-0 0 N,
0
, and
Compound PD-B-8o
NH2
\</ 0
o
HN-P-0 0 N,
0
6
=
General procedure for preparation of a nucleoside prodrug (Method E):
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Non-limiting examples of 3',5'-cyclic mono-phosphoramidate prodrugs
comprising the instant invention may be prepared according to general Scheme
3.
Scheme 3
R8 R9
,X1
,X1._____(
0 X2 N N N X2 \ \
CI 11 0, // N
P., , \
/ 0
/4b-"--t__1)---o\ N\ 1\
Ry/, HN N
R9 N
R9
R3 R1
\ --------- RY, Rxo'R6
R1
Rx.;./._0\ i =__ R-0 \ s:.
, 1 N-P-6 -:- 2
R HO R2 H ti R
0 0 o
PD-Al PD-C
RY, Rx '
FR- y-4'NH2 HCI Re
R8 .Xlri\N
.X1 0 X2\ \ \
X2 \ \ \
N 0 i
t N
µ, _---
N
.,-_-_-(,\
N , N--- \R9
3/.'..----R . "R6 R9 0/3;14'16
HO _____________ ...R1 ________________ ... \ .- .
HO-P¨e --
II R2
HO 1.2 0
40 Compound 41
Scheme 3 illustrates chemical processes that may be useful for preparation of
compound PD-C. Accordingly, PD-Al is converted to PD-C in the presence of a
base when Ar is substituted with an electron withdrawing group such as p-nitro
or p-
chloro group (European Journal of Medicinal Chemistry, 2009, 44, 3769).
Alternatively; compound 40 is converted to Compound 41 according to Bioorganic
and Medicinal Chemistry Letters, 2007, 17, 2452, which is then coupled with a
amino
acid ester salt to form PD-C.
Compound PD-C-8q
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NH2
____________________ 0
HN-P-0 0 N, .dN
Ha
NH2
CI
PD-A-8 p
N
0 N.
r, 0
H
0 0
PD-C-8q
A solution of PD-A-8p in DMS0 is treated at room temperature with
potassium t-butoxide (-1 eq.) and the resulting mixture is stirred for about
10 min. to
about 2 h. The mixture is then cooled to 0 C and neutralized with 1N HCl to ¨
pH 6.
The mixture is purified by HPLC to afford compound PD-C--8q.
Additionally, the following PD-C compounds as examples are made by the
general procedures:
Compound PD-C-8r
NH2
NJ
H'
0 0
Compound PD-C-8s
2NH
NJ
N
H
0 0 ,and
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Compound PD-C-8t
NH2
0 N,
E-1 0 =
Compound PD-D-8u
N
NH2 H2
N. -)
0 N 1) POCI3, P0(0Me)3
0
2) KOH(aq), ACN -"-..
O
Compound 8 Compound 41-1
Compound 8 is dissolved in P0(0Me)3 (0.1 - 0.5 M solution) and cooled to
0 C under argon. To this stirring solution is added P0C13 (1.0 - 5.0 eq.)
dropwise, and
the reaction mixture is allowed to warm to room temperature for about 2 -16 h.
The
resulting solution is added dropwise to a rapidly stirring solution of
acetonitrile and
0.05 - 0.5 M aqueous KOH. When addition is complete, the solvents are removed
under reduced pressure. The resulting residue is dissolved in water and
purified by
HPLC to give Compound 41-1.
NH2 NH2
N NN
1) DCM, P0(0Me)3, DMF
o 0 N oxalyl-CI
HO- P 2) IPA ,=0==
o o Fli
Compound 41-1 Compound PD-D-8u
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A solution of Compound 41-1 in DCM and P0(0Me)3 is prepared and cooled
to 0 C. To this solution is added oxalyl chloride (1.0 - 5.0 eq.) followed by
a catalytic
amount of DMF. The mixture is allowed to stir for about 10 min. to about lh.
When
activation is complete, a large volume of 2-propanol is added to the reaction
mixture
and allowed to stir and warm to room temperature. The solvents arc removed
under
reduced pressure, and the resulting crude material is purified by preparative
HPLC to
give Compound PD-D-8u.
Compound PD-E-8v
NH 2 NH2
1) DCM, PO(OMe)3, DMF
oxalyl-CI NyJk-N
N 2) DCM, TEA
F NH2 H u -F
0 0
Compound 41-1 Compound PD-
E-8v
Compound PD-E-8v is prepared from Compound 41-1 in a matter similar to
that of Compound PD-D-8u substituting 2-aminopropane for 2-propanol.
Compound PD-F-8w
HO
NH2
_________________________________ 0
0
NH
HO F
Compound PD-F-8w
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Compound PD-F-8w is prepared in a matter similar to that of Compound 20
substituting Compound 8 for Compound 18.
Compound PD-G-8x
NH2
_______________________________ 0
N
0 HN¨F1)-0¨VO
0
Ha
Compound PD-G-8x
About 90 mM Compound 8 in THF is cooled to about -78 C and about 2.2 to
about 5 equivalents of t-butylmagnesium chloride (about 1 M in THF) is added.
The
mixture is warmed to about 0 C for about 30 min and is again cooled to about -
78 C.
A solution of (2S)-2- [chloro(1-phenoxy)phosphoryl]amino} ethyl isobutyrate
(W02008085508) (1 M in THF, about 2 equivalents) is added dropwise. The
cooling
is removed and the reaction is stirred for about one to about 24 hours. The
reaction is
quenched with water and the mixture is extracted with ethyl acetate. The
extracts are
dried and evaporated and the residue purified by chromatography to give
Compound
PD-G-8x.
Compound 6
NH NH2
\ N N \i \iN
HO
HO N CH3OH 0 0
OH
AcOH
HO F HO F
4 6
Compound 4 (about 0.04 mmol) and anhydrous Me0H (about 5 mL) is
treated with acetic acid (about 5 mL) and the reaction is stin-cd overnight at
room
temperature. Saturated Nal IC03 is added to neutralize the reaction mixture
and the
crude material is purified using a HPLC system (acetonitrile-H20) to give 6.
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Compound 7
NH NH2
CNN , N
Bz0-vN' 1. Al(CH3)3 HO-y
2. NH3
E3Zo 1 HO F
3b 7
To a dry, argon purged round bottom flask (50 mL) is added compound 3b
(about 0.39 mmol) and anhydrous dichloromethane (about 10 mL). The flask is
placed into a dry ice/acetone bath (¨ -78 C) and the solution is stirred for
about 10
min. BF3-Et20 (about 0.10 mL) is added dropwise and the reaction is stirred
for
about 10 mm. A1Me3 (about 1.16 mmol, 2.0 M in toluene) is then added. After a
few
minutes, the dry ice/acetone bath is removed and the reaction mixture is
stirred at
room temperature to about 45 C over about 4 h to about 4 d. A solution of
pyridine
(about 2 mL) in Me0H (about 10 mL) is added and the solvent is removed under
reduced pressure. The crude material is purified by chromatography and is
treated
with ammonium hydroxide in methanol for about 16 h at about room temperature.
The mixture is concentrated and the residue is purified by HPLC to give 7.
Compound 8
NH2
NH2
NNT-4
N,
Bz
/OA r0
Br
, Bz N
OH
xo c5 ,
Bz CI' Bz
BuLi THF
1 a 8b
To a suspension of 7-bromoimidazo[1,2-f][1,2,4]triazin-4-amine (obtained
according to ACS Medicinal Chemistry Letters, 2010, 1, 286; 375 mg, 1.75 mmol)
in
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THF (4.0 mL) under an atmosphere of argon was added 1,2-bis-
Rehlorodimethyl)silanyliethane (452 mg, 2.10 mmol). After 60 min, the reaction
was
cooled to -78 C and BuLi (1.6 M in THF, 3.8 mL, 6.10 mmol) was added. After 10
min at -78 C, a solution of la (obtained according to WO 200631725, 782 mg,
2.10
mmol) in THF ( 1.0 mL) was added dropwise. The resulting mixture was stirred
at -
78 C for 1 hour. Saturated aqueous ammonium chloride was added and allowed to
want' to 0 C. Water was added until all solids became soluble. The mixture was
extracted with ethyl acetate. The organic extract was dried with sodium
sulfate,
filtered and concentrated in vacuo. The residue was purified by silica gel
chromatography (ethyl acetate / hexanes), affording 8b (606 mg, 59%) as a
yellow
solid.
LC/MS = 508 (M+
Retention time: 2.17-2.26 min
LC: Thermo Electron Surveyor HPLC
MS: Finnigan LCQ Advantage MAX Mass Spectrometer
Column: Phenomenex Polar RP 30 mm X 4.6 mm
Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid
Gradient: 0 min-0.1 mm 5% ACN, 0.1 min-1.95 min 5%-100% ACN, 1.95 min-3.5
mm 100% ACN, 3.5 min-3.55 min 100%-5% ACN, 3.55 min-4 min 5% ACN.
NH2 NH2
Bz 0 /0 _
Et3SiH Bz N
OH
/1:5 BF3-Et20 /a -E
Bz Bz
8b Bc
To a solution of compound 8b (510 mg, 1.39 mmol) in diehloroethane (10.0
mL) at 0 C under an atmosphere of argon, was added triethyl silane (1.77 mL,
11.09
mmol) and then BF3=Et20 (1.41 mL, 11.09 mmol). The reaction mixture was
stirred
at 55 C for 16h. The reaction was cooled to 0 C and quenched with saturated
NaHCO3 (aq). The reaction was extracted with DCM and then Et0Ac. The
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combined organic phase was dried over sodium sulfate, filtered and
concentrated.
The residue was purified by silica gel chromatography (ethyl acetate/hexanes),
affording 8c (453 mg, 64%). 1H NMR (400 MHz, CDCI3): d 8.10-7.94 (m, 5H), 7.6-
7.33 (m, 7H), 5.91 (dd, 1H), 5.78 (d, J=24.6 Hz, 1H), 4.87 (dd, 1H), 4.70 (m,
1H),
4.58 (dd, 1H), 1.31 (d, J=22.4 Hz, 3H).
LC/MS = 491 (M-1).
Retention time: 2.36 min.
LC: Thermo Electron Surveyor HPLC
MS: Finnigan LCQ Advantage MAX Mass Spectrometer
Column: Phenomenex Polar RP 30 mm X 4.6 mm
Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid
Gradient: 0 min-0.1 min 5% ACN, 0.1 min-1.95 min 5%400% ACN, 1.95 min-3.5
min 100% ACN, 3.5 min-3.55 min 100%-5% ACN, 3.55 min-4 min 5% ACN.
NH2
NH2
Bz N' NH3 HO--N0FN",
-F Me0H
Bz HC-5
8c Compound 8
To a solution of 8c (500 mg, 01.02 mmol) in THF (5.0 mL) was added lithium
hydroxide (122 mg, 5.09 mmol) as a solution in H20 (5.0 triL) and was stirred
at room
temperature for I h. The reaction was cooled to 0 C and was neutralized with
IN HCI
in water (5.1 mL). The mixture was concentrated and the residue was purified
by RP
HPLC (water / acetonitrile), affording Compound 8 (185 mg, 64%). 1f1NMR (400
MHz, CD30D): d 7.97 (s, 1H), 7.63 (s, 1H), 5.54 (d, J=24.8 Hz, 1H), 4.03 (dd,
1H),
3.88 (m, 1H), 3.71 (dd, 1H), 1.80 (d, J-22.1 Hz, 3H).
LC/MS = 284 (M + H ).
Retention time: 1.06 min.
LC: Thermo Electron Surveyor HPLC
MS: Finnigan LCQ Advantage MAX Mass Spectrometer
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Column: Phenomenex Polar RP 30 mm X 4.6 mm
Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid
Gradient: 0 min-0.1 mm 5% ACN, 0.1 min-1.95 min 5%-100% ACN, 1.95 min-3.5
min 100% ACN, 3.5 min-3.55 min 100%-5% ACN, 3.55 min-4 min 5% ACN.
Alternative procedure for Compound 8
NH2 NH2
HO N
NaBH4 HONj
0
Ha HO F
le Compound 8
Compound le (crude obtained from the previous reaction step) was dissolved
in Et0H. Excess sodium borohydride was added in portions until the reaction
was
nearly complete. The mixture was neutralized with acetic acid. The mixture was
concentrated and the solid residue was purified by silica gel column
chromatography
(0-10% Me0H / dichloromethane), affording compound 27 (210 mg, 50% in two
steps).
Additional alternative procedure for Compound 8
NH2 NH2
HO-V2r¨NN N j\I
s----- Raney Ni
H b HO F
Id 8
Raney Ni (about 500 mg) was neutralized by washing with H70, and added to
a solution of 1d (about 100 mg) in ethanol (about 10 mL). The mixture was then
heated to 80 C until the reaction is complete. The catalyst was removed by
filtration
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and the solution was concentrated in vacuo. The mixture was concentrated and
the
residue was purified by HPLC to give 8.
Compound 9
NH2
0 N
H
HO¨P-0 N,
0
OH '"CN N
z
HO F
9
Into a flask containing Compound 3 (120 mg, 0.39 mmoL, 1 equiv.) was
added P0(0Me)3 (1.5 mL, 0.25 M) and cooled to 0 C before adding P0C13 (125 pL,
1.37 mmoL, 3.5 equiv.). The reaction mixture was allowed to stir for 5 hr
before the
reaction was quenched with water. It was directly purified by HPLC to furnish
the
monophosphate Compound 9.
LC MS m/z 387.95 [M +
Compound 10
0 NH2
\
0
0 N,
JO " CN N
0---' .
) HO -F
0
10
Into a flask containing Compound 9 (30 mg, 0.078 mmoL, 1 equiv.) was
added NMP (0.8 mL, 0.1 M) followed by addition of TEA (43 pL, 0.31 mmoL, 4
equiv.), tetrabutylammonium bromide (25 mg, 0.078 mmoL, 1 equiv.) before
adding
chloromethylisopropyl carbonate (60 pL, 0.38 mmoL, 5 equiv.). The reaction
mixture was heated to 50 C and allowed to stir overnight. It was purified
directly by
HPLC, affording Compound 10.
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1H NMR (400 MHz, CDC13) d 7.98 (s, 1 H), 7.01 (d, J = 4.7 Hz, 1 H), 6.72 (d, J
= 4.7
Hz, 1 H), 6.04 (bs, 2 I-1), 5.74 ¨5.61 (m, 4 H), 4.91 (ddt, J= 12.6, 9.4, 6.3
Hz, 2 H),
4.64 --4.28 (in, 4 H), 1.37 - 1.19 (in, 15 H).
31P NMR (162 MHz, CDC13) d -4.06.
19F NMR (376 MHz, CDC13) d -76.58, -151.95 TFA salt.
LC MS m/z 620.03[M + H4].
Compound 11
NH2
0
-
N¨P-0
0 H
0
11
A solution of Compound B-2 in DMSO is treated with about 3 mole
equivalents of potassium t-butoxide for about 15 min to 24 hours. The reaction
is
quenched with 1N 1-IC1 and Compound 11 is isolated by reverse-phase HPLC.
Compound 12
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s' NH2
/0 0
Bz NH3 Bz
_________________ OH OH
,6 /0 F
Bz Bz
lb 12a
Raney Ni
NH2 NH2
1. TMSCN
TMSOTf
__________________________________________ 8z0
HO 0 _-,-__/ 0
N
_________________ 'ON 2. NH3 OH
HO
F Bz0 F
12 12b
Compound lb (about 1 mmol) is placed in a steel bomb reactor. The reactor
is charged with liquid ammonia (about 30 mL) and the mixture is stirred at
about 0 C
to 50 C for about 16 h. The ammonia is evaporated and the residue is purified
to give
12a. A solution of 12a (about 100 mg) in ethanol (about 10 mL) is treated with
Raney Ni (about 500 mg) that is neutralized by washing with H20. The mixture
is
then heated to about 35 to about 80 C until the reaction is complete. The
catalyst is
removed by filtration and the solution is concentrated in vacuo. The mixture
is
concentrated and the residue is purified by HPLC to give 12b. To a solution of
compound 12b (about 50 mg) and TMSCN (about 0.5 mmol) in acetonitile (about
2.0 mL) at about 0 C is added TMSOTf (about 0.5 mmol). The reaction mixture is
stirred at room temperature for about 1 h, then at 65 C for about 3 d. The
reaction is
quenched with saturated NaHCO3 at room temperature, and diluted with CH3CO2Et.
The organic phase was separated, washed with brine, dried over Na2SO4,
filtered and
concentrated. The residue is purified by RP-HPLC then dissolved in methanol
(about
1 mL). Ammonium hydroxide (28% in water, about 0.8 mL) is added and the
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mixture is stirred at about room temperature for 16 h. The mixture is
concentrated
and the residue is purified by RP HPLC to give 12.
Compound 13
NH2
0
HO,
0,
T,
Hd
13
Compound 13 is prepared in the same manner as Compound 9 using
Compound 12 as a starting material.
Compound 14
NH2
0 N,/
-
HO¨P-0
0
14
Compound 14 is prepared by treating Compound 13 with about one to about
five equivalents of DCC in pyridine and heating the reaction to reflux for
about one to
about 24 hours. Compound 14 is isolated by conventional ion exchange and
reverse-
phase HPLC.
Compound 15
2NH
o
NI
0 "CN
OTh
0¨P-6
0
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A solution of about 0.4 mmol of Compound 14 in about 10 mL of DMF is
treated with about 0.8 mmol of DIPEA and about 0.8 mmol of chloromethyl
isopropyl
carbonate (W02007/027248). The reaction is heated to about 25 to about 80 C
for
about 15 mm to about 24 hours. The solvent is removed under vacuum and the
residue is purified by HPLC to give Compound 15.
Compound 16
NHDMTr
N
HOrNJ \
0
HO
16
Compound 3 (about 0.22 inmoL) is dissolved in anhydrous pyridine (about 2
mL) and chlorotrimethylsilane (about 0.17 mL) is added. The mixture is stirred
at
about 0 C to about 25 C for about one to about 24 hours. Additional
chlorotrimethylsilane (about 0.1 mL) is added and the reaction is stirred for
about one
to about 24 hours. 4.4'-Dimethoxytrityl chloride (about 0.66 mmol) and DMAP
(about 0.11 to about 0.22 mmol) is sequentially added. The mixture is stirred
for
about one to about 24 hours. A solution of TBAF (1.0 M, about 0.22 mL) in THF
is
added and the reaction is stirred for about one to about 24 hours. The mixture
is
partitioned between ethyl acetate and water. The ethyl acetate layer is dried
and
concentrated. The residue is purified chromatography to afford Compound 16.
Compound 17
TrO NHDMTr
_______________________________ 0 N
HC5
17
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A mixture of about 1.25 mmol of Compound 16 and about 1.9 mmol of
triethylammonium 2-(2,2-dimethy1-3-(trityloxy)propanoylthio)ethyl phosphinate
(W02008082601) is dissolved in anhydrous pyridine (about 19 mL). Pivaloyl
chloride (about 2.5 mmol) is added dropwise at about -30 to about 0 C and the
solution is stirred at for about 30 min to about 24 hours. The reaction is
diluted with
methylene chloride and is neutralized with aqueous ammonium chloride (about
0.5M). The methylene chloride phase is evaporated and the residue is dried and
is
purified by chromatography to give Compound 17.
Compound 18
TrO
NHDMTr
0
Ns
N
0
NH
'CN
Ho
18
To a solution of about 0.49 rnmol of Compound 17 in anhydrous carbon
tetrachloride (about 5 mL) is added dropwise benzylamine (about 2.45 mmol).
The
reaction mixture is stirred for about one to about 24 hours. The solvent is
evaporated
.. and the residue is purified by chromatography to give Compound 18.
Compound 20
HO
NH2
_________________________________ 0 N
0 O¨P-0 0 Ns
HO
25
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A solution of about 2 mmol of Compound 18 in methylene chloride (about 10
mL) is treated with an aqueous solution of trifluoroacetie acid (90%, about 10
mL).
The reaction mixture is stirred at about 25 to about 60 C for about one to
about 24
hours. The reaction mixture is diluted with ethanol, the volatiles are
evaporated and
the residue is purified by chromatography to give Compound 20,
Compound 21
0
F 9
ir NH
0 HN-P-0-07S-N, ,J
0 \ NH2
_
HO F
21
About 90 mM Compound 2 in THF is cooled to about -78 C and about 2.2 to
about 5 equivalents of t-butylmagnesium chloride (about I M in THF) is added.
The
mixture is warmed to about 0 C for about 30 min and is again cooled to about -
78 C.
A solution of (2S)-2- [chloro(1-phenoxy)phosphoryl]aminolpropyl pivalo ate
(W02008085508) (1 M in THF, about 2 equivalents) is added dropwise. The
cooling
is removed and the reaction is stirred for about one to about 24 hours. The
reaction is
quenched with water and the mixture is extracted with ethyl acetate. The
extracts are
dried and evaporated and the residue purified by chromatography to give
Compound
21.
Compound 22
\
0
N-P11- NO2
H '
0
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22a
Compound 22a was obtained in a procedure similar to that for preparation of
C-la.
11-1 NMR (400 MHz, CDC13) d 8.11 (d, J= 9.0 Hz, 2 H), 8.02 (s, 1 H), 7.48 (t,
J= 7.5
Hz, 2 H), 7.42- 7.25 (m, 4 H), 7.21 (dt, J= 14.9, 5.5 Hz, 2 H), 7.08 (t, J=
7.3 Hz, 2
H), 5.17- 5.03 (m, 2 H), 4.99 (dd, J= 16.5, 9.7 Hz, 2 H), 3.44 (s, 1H), 3.35 -
3.21
(m, 2 H), 3.19 (d, J 9.2 Hz, 1H), 3.00 - 2.80 (m, 2 H).
31P NMR (162 MHz, CDC13) d 4.27.
LC MS m/z 452.09 [M + H ].
NH2
H
0
= - N
H0 -F
22b
Compound 22b was obtained in a procedure similar to that for preparation of
C-1 using Compound 3 and 22a.
1H NMR (400 MHz, CD30D) d 7.76 (d, J= 6.3 Hz, 1H), 7.38 (t, J= 8.2 Hz, 1 H),
7.27- 7.12 (m, 4 H), 7.06- 6.81 (m, 3 H), 6.74 (dd, J= 4.6, 3.5 Hz, 1 H), 4.95
-4.79
(m, 1 H), 4.35 -3.90 (m, 4 H), 3.23 (dt, J= 3.2, 1.6 Hz, 3H), 3.18 - 3.05 (in,
2 H),
2.82 (dt, J= 14.7, 7.3 Hz, 2 H), 1.15 (d, J= 22.4 Hz, 3 H).
31P NMR (162 MHz, CD30D) d 10.76, 10.71.
LC MS m/z 620.05 [M +
NH2
H
OH
N
Hd
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Compound 22
Into a flask containing the 22b (50 mg, 0.08 mmoL, 1 equiv.) was added
ethanol (4 mL) followed by Pd(OH)2 ( 56 mg, 0.08 mmoL, 1 equiv.) and ammonium
formate (42 mg, 0.64 mmoL, 8 equiv.). The reaction was heated to 80 C for
about an
hour. The solid was filtered off and the material purified by HPLC.
1H NMR (400 MHz, DMSO-d6) d 10.72 (s, 1H), 7.91 (s, 1 H), 7.95 - 7.89 (bs, 2
H),
7.41 (d, J= 7.7 Hz, 1 H), 7.26 (d, = 8.1 Hz, 1 H), 7.19 ¨ 6.66 (m. 3 H), 4.20
¨ 3.75
(m, 3 H), 2.99 (dd, J= 16.5, 9.6 Hz, 2 H), 2.89 ¨2.70 (m, 2 H), 2.48 ¨ 2.58
(m, 8 H),
1.10 (d, J= 22.3 Hz, 3 H).
31P NMR (162 MHz, DMSO-d6) d 7.49.
19F NMR (376 MHz, DMS0-(16) d -154.89.
LC MS m/z 530.21 [M +
Compound 23
NH2 NH2
N
131
'"CNN 1) POCI3, PO(OMe)3
N
HO
0,1 \ ____ .'/PN
2) K01-1(aq), ACN
HO F Hid u
Compound 3 Compound 23
Compound 3 (250mg, 0.82mmo1) was dissolved in P0(0Me)3 (5 mL, 0.16M)
and cooled to 0 C under argon. To this stirring solution was added POC13 (0.32
mL,
4.1 mmol) slowly dropwise, and the reaction mixture allowed to warm to room
temperature for 16 h. The resulting solution was added dropwise to a rapidly
stirring
solution of acetonitrile (400 mL) and 0.08M aqueous KOH (300 mL). When
addition
was complete, the reaction progress was checked by LCMS. When the reaction was
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complete, solvents were removed under reduced pressure. The resulting solid
residue
was dissolved in water and purified by HPLC to give 140mg of Compound 23
(yield;
47%).
1H-NMR (400MHz; CD30D) : d 8.15 (s, 1H), 7.40 (d, 1H; J = 4.8Hz), 7.09 (d, 1H;
J
= 4.8Hz), 4.64 (dd, 1H; J = 24Hz, 7.2Hz ), 4.50-4.36 (m, 3H), 1.32 (d, 3H;
J=22Hz).
19F-NMR (376MHz; CD30D) : d -153.11.
31P-NMR (162MHz; CD30D) : d -2.20.
MS [M + H4] = 370.2.
Compound 24
NH 2 NH2
N N
N, N,
0 N 1) DCM, 0 'CN FO(OMe)3, DMF
0,1 0,1
HO' U F 2) IPA )_c5
Compound 23 Compound 24
A solution of Compound 23 (7mg, 0.02 mmol) in DCM (2 mL) and
P0(0Me)3 (1 mL) was prepared and cooled to 0 C. To this solution was added
oxalyl-C1 (10 !IL) followed by DMF (2 p.L). The mixture was allowed to stir
for 1
min before an aliquot was taken out and quenched in Me0H and then checked by
LCMS for activation. Successive amounts of oxalyl-C1 (10 L) and DMF (2 gL)
were added until activation was complete. At this point, a large volume of 2-
propanol
(5 mL) was added to the reaction mixture and allowed to stir and warm to room
temperature. Once the reaction was complete, the solvents were removed under
reduced pressure, and the resulting crude material was purified by preparative
HPLC
to give 5.5 mg of Compound 24 (yield 70%).
11-1-NMR (400MHz; DMSO-do) : d 8.26 (hr, 1H), 8.15 (br, 1H), 7.97 (s, 1H),
7.00 (d,
11-1; J = 4.4Hz), 6.88 (d, 11-I; J = 4.4Hz), 4.59-4.51 (in, 2H), 4.37-4.25
(in, 2H), 1.23
(d, 3H; J=22.8Hz).
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19F-NMR (376MHz; CD30D) : d-151.72.
31P-NMR (162MHz; CD30D) : d-5.69.
MS [M + H4] -= 412Ø
Compound 25
NH2 NH2
1) DCM, P0(0Me)3, DMF
N exalyl-CI
\NJ _________________________________________ NJ
0 N 2) DCM, TEA 0 'CNN
0
7 '
d
NH3CI 0
Compound 23 0 Compound 25
Compound 25 was prepared from Compound 23 in a matter similar to that of
Compound 24 substituting the heptyl ester of alanine for 2-propanol (yield
5.3%).
1H-NMR (400MHz; CD30D) : d 7.91 (s, 1H), 6.98 (d, 11-1; J = 4.8Hz), 6.92 (d,
1H; J
= 4.8Hz), 5.29 (dd, 1H; J = 24.4Hz, 8.8Hz), 4.66-4.60 (m, 2H), 4.48-4.40 (m,
1H),
4.15-4.11(m, 3H), 3.92(dd, 1H; J = 9.6Hz, 7.2Hz), 1.67-1.64 (m. 3H), 1.40-1.27
(m,
15H), 0.91-0.87(m, 6H).
19F-NMR (376MHz; CD30D) : d -151.46.
31P-NMR (162MHz; CD30D) : d 7.36.
MS [M + = 539.4.
Compound 26
NH2
0 \N
It n
N¨P¨`-"Thly0
H
\
,s-- N
HO
0
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Compound 26
Compound 26 is prepared from compound 22 in a matter similar to that for
preparation of compound 10.
Antiviral Activity
Another aspect of the invention relates to methods of inhibiting viral
infections, comprising the step of treating a sample or subject suspected of
needing
such inhibition with a composition of the invention.
Within the context of the invention samples suspected of containing a virus
include natural or man-made materials such as living organisms; tissue or cell
cultures; biological samples such as biological material samples (blood,
serum, urine,
cerebrospinal fluid, tears, sputum, saliva, tissue samples, and the like);
laboratory
samples; food, water, or air samples; bioproduct samples such as extracts of
cells,
particularly recombinant cells synthesizing a desired glycoprotein; and the
like.
Typically the sample will be suspected of containing an organism which induces
a
viral infection, frequently a pathogenic organism such as a tumor virus.
Samples can
be contained in any medium including water and organic solvent\water mixtures.
Samples include living organisms such as humans, and man made materials such
as
cell cultures.
If desired, the anti-virus activity of a compound of the invention after
application of the composition can be observed by any method including direct
and
indirect methods of detecting such activity. Quantitative, qualitative, and
semiquantitative methods of determining such activity arc all contemplated.
Typically one of the screening methods described above are applied, however,
any
other method such as observation of the physiological properties of a living
organism
are also applicable.
The antiviral activity of a compound of the invention can be measured using
standard screening protocols that are known. For example, the antiviral
activity of a
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compound can be measured using the following general protocols.
Cell-based Flavivirus Immunodetection assay
BHK21 or A549 cells are trypsinized, counted and diluted to 2x105 cells/mL
in Hams F-12 media (A549 cells) or RPMI-1640 media (BHK21 cells) supplemented
with 2% fetal bovine serum (FBS) and 1% penicillin/streptomycin. 2x104 cells
are
dispensed in a clear 96-well tissue culture plates per well and placed at 37
C, 5% CO2
overnight. On the next day, the cells are infected with viruses at
multiplicity of
infection (1\40I) of 0.3 in the presence of varied concentrations of test
compounds for
1 hour at 37 C and 5% CO2 for another 48 hours. The cells are washed once with
PBS and fixed with cold methanol for 10 mm. After washing twice with PBS, the
fixed cells are blocked with PBS containing 1% FBS and 0.05% Tween-20 for 1
hour
at room temperature. The primary antibody solution (4G2) is then added at a
concentration of 1:20 to 1:100 in PBS containing 1% FBS and 0.05% Tween-20 for
3
hours. The cells are then washed three times with PBS followed by one hour
incubation with horseradish peroxidase(HRP)-conjugated anti-mouse IgG (Sigma,
1:2000 dilution). After washing three times with PBS, 50 microliters of
3,3',5,5'-
tetramethylbenzidine (TMB) substrate solution (Sigma) is added to each well
for two
minutes. The reaction is stopped by addition of 0.5 M sulfuric acid. The
plates are
read at 450 nm absorbance for viral load quantification. After measurement,
the cells
are washed three times with PBS followed by incubation with propidium iodide
for 5
mm. The plate is read in a Tecan SafireTM reader (excitation 537 nm, emission
617
nm) for cell number quantification. Dose response curves are plotted from the
mean
absorbance versus the log of the concentration of test compounds. The EC50 is
calculated by non-linear regression analysis. A positive control such as N-
nonyl-
deoxynojirimycin may be used.
Cell-based Flavivirus cytopathic effect assay
For testing against West Nile virus or Japanese encephalitis virus, BHK21
cells are trypsinized and diluted to a concentration of 4 x 105 cells/mL in
RPMI-1640
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media supplemented with 2% FBS and 1% penicillin/streptomycin. For testing
against dengue virus, Huh7 cells are trypsinized and diluted to a
concentration of 4 x
105 cells/mL in DMEM media supplemented with 5% FBS and 1%
penicillin/streptomycin. A 50 microliter of cell suspension (2 x 104 cells) is
dispensed
per well in a 96-well optical bottom PIT polymer-based plates (Nunc). Cells
are
grown overnight in culture medium at 37 C, 5% CO2, and then infected with West
Nile virus (e.g. B956 strain) or Japanese encephalitis virus (e.g. Nakayama
strain) at
MO1 = 0.3, or with dengue virus (e.g. DEN-2 NGC strain) at MO1 = 1, in thc
presence
of different concentrations of test compounds. The plates containing the virus
and the
compounds are further incubated at 37 C, 5% CO2 for 72 hours. At the end of
incubation, 100 microliters of CellTiter-GloTm reagent is added into each
well.
Contents are mixed for 2 minutes on an orbital shaker to induce cell lysis.
The plates
are incubated at room temperature for 10 minutes to stabilize luminescent
signal.
Luminescence reading is recorded using a plate reader. A positive control such
as N-
nonyl-deoxynojirimycin may be used.
Antiviral Activity in a Mouse Model of Dengue Infection.
Compounds are tested in vivo in a mouse model of dengue virus infection
(Schul et al. J. Infectious Dis. 2007; 195:665-74). Six to ten week old AG129
mice
(B&K Universal Ltd, H11, UK) are housed in individually ventilated cages. Mice
are
injected intraperitoneally with 0.4 mL TSV01 dengue virus 2 suspension. Blood
samples are taken by retro orbital puncture under isoflurane anesthesia. Blood
samples are collected in tubes containing sodium citrate to a final
concentration of
0.4%, and immediately centrifuged for 3 minutes at 6000g to obtain plasma.
Plasma
(20 microliters) is diluted in 780 microliters RPMI-1640 medium and snap
frozen in
liquid nitrogen for plaque assay analysis. The remaining plasma is reserved
for
cytokine and NS1 protein level determination. Mice develop dengue virernia
rising
over several days, peaking on day 3 post-infection.
For testing of antiviral activity, a compound of the invention is dissolved in
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vehicle fluid, e.g. 10% ethanol, 30% PEG 300 and 60% D5W (5% dextrose in
water;
or 6N HCl (1.5 eq):1N NaOH (pH adjusted to 3.5): 100 mM citrate buffer pH 3.5
(0.9% v/v:2.5% v/v: 96.6% v/v). Thirty six 6-10 week old AG129 mice are
divided
into six groups of six mice each. All mice are infected with dengue virus as
described
above (day 0). Group 1 is dosed by oral gavage of 200 mL/mouse with 0.2 mg/kg
of
a compound of the invention twice a day (once early in the morning and once
late in
the afternoon) for three consecutive days starting on day 0 (first dose just
before
dengue infection). Groups 2, 3 and 4 are dosed the same way with 1 mg/kg, 5
mg/kg
and 25 mg/kg of the compound, respectively. A positive control may be used,
such as
(2R,3R,4R,5R)-2-(2-amino-6-hydroxy-purin-9-y1)-5-hydroxymethy1-3-methyl-
tetrahydro-furan-3,4-diol, dosed by oral gavage of 200 microliters/mouse the
same
way as the previous groups. A further group is treated with only vehicle
fluid.
On day 3 post-infection approximately 100 microliter blood samples (anti-
coagulated with sodium citrate) are taken from the mice by retro-orbital
puncture
under isoflurane anesthesia. Plasma is obtained from each blood sample by
centrifugation and snap frozen in liquid nitrogen for plague assay analysis.
The
collected plasma samples are analyzed by plague assay as described in Schul et
al.
Cytokines are also analyzed as described by Schul. NS1 protein levels are
analyzed
using a PlateliaTM kit (BioRad Laboratories). An anti-viral effect is
indicated by a
reduction in cytokine levels and/or NS1 protein levels.
Typically, reductions in viremia of about 5-100 fold, more typically 10-60
fold, most typically 20-30 fold, are obtained with 5-50 mg/kg bid dosages of
the
compounds of the invention.
HEN' IC50 Determination
Assay Protocol: Either wild type or S282T (Migliaccio, et al, J. Biol. Chem.
2003, 49164-49170; Klumpp, et al., J. Biol. Chem. 2006, 3793-3799) mutant
polymerase enzyme was used in this assay. NS5b polyrnerase assay (40 41.,) was
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assembled by adding 28 j.LL polymerase mixture (final concentration: 50 mM
Tris-
HCI at pH 7.5, 10 mM KCL, 5 mM MgC12, 1 mM DTT, 10 m1v1 EDTA, 4 ng/[iL of
RNA template, and 75 nM HCV A21 NS5b polymerase) to assay plates followed by 4
pl of compound dilution. The polymerase and compound were pre-incubated at
35 C for 10 minute before the addition of 8 1_, of nucleotide substrate
mixture (33P-
a-labeled competing nucleotide at Km. and 0.5 mM of the remaining three
nucleotides). The assay plates were covered and incubated at 35 C for 90 min.
Reactions were then filtered through 96-well DEAE-81 filter plates via vacuum.
The
filter plates were then washed under vacuum with multiple volumes of 0.125 M
NaHPO4, water, and ethanol to remove unincorporated label. Plates were then
counted on TopCount to assess the level of product synthesis over background
controls. The IC50 value is determined using Prism fitting program.
Preferably, compounds described herein inhibited NS5b polymerase with an
IC50's below 1000 p.M, more preferably below 100 M, and most preferably below
10
M. For example, compound TP-1 has an IC50 of 0.15 iM against both wild type
HCV polymerase and the S282T mutant enzyme. Table II below shows the activity
of TP-1 and TP-2 against both wild type and the S282T mutant enzyme compared
to
the activities obtained with the triphosphate of 2'-methyl guanidine and the
triphosphate of (2R,3R,4R,5R)-2-(4-aminopyrrolo[1,2-11[1,2,4]triazin-7-y1)-3,4-
dihydroxy-5-(hydroxymethyl)-3-methyl-tetrahydrofuran-2-carbonitrile. This
demonstrates that replacing the 2' OH of the pyrrolo[1,2-f][1,2,41triazin-7-y1
nucleosides with a 2' F unexpectedly confers activity against resistant S282T
HCV
mutant strains of virus.
Table II
WT S282T
Triphosphate IC50(uM) IC50(uM) Note
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0
O 0 0
II II II / NH
,/
OH OH OH \NH2 0.1 20 from J. Bio. Chem.,
2003, 278, 49164
(200 fold shift)
Ho OH
2'-C-MeGTP
0
0 0 0
\ 'NH
HO('OPOF1'0 0 N
OH OH OH N---\ NH2 0.15 0.15 (1 fold shift)
Ho
TP-1
NH2
O 0 0 WO/2009/132135
II If II \ N \ N 0.525 111
HO-P-O-P-O-P-0-A _0 Ns (242 fold shift)
OH OH OH V N
''ONI
HO OH
NH2
O 0 0
HO-P-O-P-O-P-0-y Ns
OH OH OH N 0.24 1.60 (7 fold shift)
"ON
Ho
TP-2
NH2
O 0 0 N
ir
HO-P-O-P-O-P-0
OH OH OH 0.034
7-
HO F
TP-3
NH2
9 9 9
HO-P-O-P-O-P-0 0 N,
OH OH OH 0.30 1.6 (5.3 fold shift)
Ho
TP-8a
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IICV EC50 Determination
Replicon cells were seeded in 96-well plates at a density of 8 x 103 cells per
well in 100 tit of culture medium, excluding Geneticin. Compound was serially
diluted in 100% DMSO and then added to the cells at a 1:200 dilution,
achieving a
final concentration of 0.5% DMSO and a total volume of 200 L. Plates were
incubated at 37 C for 3 days, after which culture medium was removed and cells
were
lysed in lysis buffer provided by Promega's luciferase assay system. Following
the
manufacturer's instruction, 100 iL of luciferase substrate was added to the
lysed cells
and luciferase activity was measured in a TopCount luminometer. Preferably,
compounds described herein have EC50's below 1000 M, more preferably below
100 M, and most preferably below 10 M. The activities of representative
compounds of Formula I are shown in the Table III below.
Table III
Compound No. EC50, M
A-1 23
B-1 1.4-4.3
B-3 16-28
B-4 8.4-19
B-5 1 1.93-25.5
B-6 3.75-11.1
B-7 63-73
B-8 35-60
C-1 67-70
C-2 3.9-12
C-3 43-84
C-4 9.8-31
C-5 24-28
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C-6 11
10 6.5-8
22 31-45
23 39.4-40.3
24 40.3-70.5
25 9.7-10
___________________________________ _ ______
PD-A-8b 0.68
The cytotoxicity of a compound of the invention can be determined using the
following general protocol.
Metabolism Studies:
Applicants have observed that monophosphate prodings of nucleoside analogs
with a nitrogen at the X1 position can have enhanced activity over their
counterparts
with a carbon at the XI position. This difference in activity correlates to
the amount
of the active triphosphate analogs of the compounds in cells. This can be
quantified
by a metabolism study which quantifies the intracellular concentration of the
triphosphate analogs. The higher intracellular concentration of the
triphosphate
metabolite correlates to the prodrug with enhanced activity.
For example, comparison of the prodrug compound B-7 with prodrug
compound PD-A-8b shows increased activity when the XI position is nitorgen.
This
can be observed in Table III, where the HCV EC50 for the compound where the XI
position is nitrogen (compound PD-A-8b) is 0.68 tiM compared to 63-73 ).11VI
for
compound B-7. The activation of prodrug analog PD-A-8b (to its triphosphate
analog
TP-8a) was found to be more than two orders of magnitude more efficient than
that
observed for its prodrug counterpart where the X1 position is carbon, B-7 (to
its
triphosphate analog TP-3), as seen in Table IV.
Experimental:
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Huh-luc/neo replicon cells containing HCV genotype lb subgenomic replicons
were maintained in Dulbecco's modified eagle medium containing glutamax
supplemented with 10% heat inactivated fetal bovine scrum, penicillin-
streptomycin,
and G418 disulphate salt solution. Cells were transferred to twelve well
tissue culture
plates by trypsonization and grown to continency (0.88 X 106 cells/well).
Cells were
treated for 24 hours with 10 M nucleoside, or 10 M prodrug. After 24 hours,
cells
were washed 2 times with 2.0 mL ice cold 0.9% sodium chloride saline. Cells
were
then scraped into 0.5 mL 70% methanol (Me0H) and frozen overnight to
facilitate the
extraction of nucleotide metabolites. Extracted cell material in 70% Me0H was
transferred into tubes and dried. After drying, samples were resuspended in
1mM
Ammonium phosphate pFI 8.5 containing internal standard (100 nM ClATP).
Intracellular levels of the nucleoside triphosphates were quantified based on
authentic
standard curves by liquid chromatography coupled to tandem mass spectrometry.
Results
Table IV: Intracellular triphosphate analog concentrations formed in Huh-
luc/neo
replicon cells following 24 hour incubations with 10 M PD-A-8b and B-7.
Prodrug Triphosphate Intracellular Triphosphate Analog
Concentration
(pmol/million)
B-7 TP-3 <0.11a
PD-A-8b TP-8a 20.5b
a Intracellular concentrations were below the lower limit of quantification of
the assay.
Value is the average of results from 2 separate wells.
Cytotoxicity Cell Culture Assay (Determination of CC50):
The assay is based on the evaluation of cytotoxic effect of tested compounds
using
a metabolic substrate.
Assay protocol .for determination of CC50:
1. Maintain MT-2 cells in RPM1-1640 medium supplemented with 5% fetal bovine
serum and antibiotics.
186
2. Distribute the cells into a 96-well plate (20,000 cell in 100 41.., media
per well) and
add various concentrations of the tested compound in triplicate (1001AL/well).
Include untreated control.
3. Incubate the cells for 5 days at 37 C.
4. Prepare XTT solution (6 ml per assay plate) in dark at a concentration of
2mg/m1
in a phosphate-buffered saline pH 7.4. Heat the solution in a water-bath at 55
C
for 5 mm. Add 50 xL of N-methylphenazonium methasulfate (5 jAg/mL) per 6 mL
of XTT solution.
5. Remove 1004 media from each well on the assay plate and add 100 jiL of the
XTT substrate solution per well. Incubate at 37 C for 45 to 60 mm in a CO2
incubator.
6. Add 201AL of 2% Triton X-100 per well to stop the metabolic conversion of
XTT.
7. Read the absorbance at 450 nm with subtracting off the background at 650
nm.
8. Plot the percentage absorbance relative to untreated control and estimate
the CC50
value as drug concentration resulting in a 50% inhibition of the cell growth.
Consider the absorbance being directly proportional to the cell growth.
The invention has been described with reference to various specific and
preferred embodiments and techniques. However, one skilled in the art will
understand that many variations and modifications may be made while remaining
.. within the spirit and scope of the invention.
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