U.S. patent application number 12/776691 was filed with the patent office on 2010-12-09 for novel tricyclic nucleosides or nucleotides as therapeutic agents.
This patent application is currently assigned to BIOTA SCIENTIFIC MANAGEMENT PTY LTD. Invention is credited to Phillip Dan Cook, Deborah K. Ewing, Gregory Ewing, Yi Jin, John Lambert, Marija Prhavc, Vasanthankumar Rajappan, Vivek K. Rajwanshi, Kandasamy Sakthivel.
Application Number | 20100311684 12/776691 |
Document ID | / |
Family ID | 34272671 |
Filed Date | 2010-12-09 |
United States Patent
Application |
20100311684 |
Kind Code |
A1 |
Cook; Phillip Dan ; et
al. |
December 9, 2010 |
NOVEL TRICYCLIC NUCLEOSIDES OR NUCLEOTIDES AS THERAPEUTIC
AGENTS
Abstract
Nucleosides and nucleotides containing a tricyclic base portion
thereof are useful for treating infectious diseases and
proliferative disorders, such as viral infections or cancer
respectively.
Inventors: |
Cook; Phillip Dan;
(Fallbrook, CA) ; Ewing; Gregory; (Oceanside,
CA) ; Ewing; Deborah K.; (Oceanside, CA) ;
Jin; Yi; (Carlsbad, CA) ; Lambert; John;
(Blackburn South, AU) ; Prhavc; Marija;
(Encinitas, CA) ; Rajappan; Vasanthankumar;
(Carlsbad, CA) ; Rajwanshi; Vivek K.; (Vista,
CA) ; Sakthivel; Kandasamy; (Karur, IN) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE, SUITE 5400
SEATTLE
WA
98104
US
|
Assignee: |
BIOTA SCIENTIFIC MANAGEMENT PTY
LTD
Notting Hill
AU
|
Family ID: |
34272671 |
Appl. No.: |
12/776691 |
Filed: |
May 10, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10568917 |
Nov 29, 2006 |
7713941 |
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PCT/US2004/027819 |
Aug 27, 2004 |
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12776691 |
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60498425 |
Aug 27, 2003 |
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Current U.S.
Class: |
514/49 ; 514/23;
536/26.26; 536/27.1; 536/29.2 |
Current CPC
Class: |
C07H 19/04 20130101;
A61P 31/04 20180101; A61P 35/00 20180101; A61P 43/00 20180101; A61P
31/14 20180101; A61P 31/18 20180101; A61P 31/20 20180101; C07H
19/23 20130101; A61P 31/12 20180101 |
Class at
Publication: |
514/49 ;
536/27.1; 536/29.2; 514/23; 536/26.26 |
International
Class: |
A61K 31/7064 20060101
A61K031/7064; C07H 19/23 20060101 C07H019/23; C07H 7/06 20060101
C07H007/06; A61P 31/12 20060101 A61P031/12 |
Claims
1. A compound of the formula (I) which may be a D- or L-nucleotide
or nucleoside: ##STR00077## wherein A is O, S, CH.sub.2, NH, CHF,
CF.sub.2 or protected N; R.sup.1, R.sup.2, R.sup.2', R.sup.3,
R.sup.3', and R.sup.4 are independently H, F, Cl, Br, I, OH, SH,
NH.sub.2, NHOH, NHNH.sub.2, N.sub.3, COOH, CN, CONH.sub.2,
C(S)NH.sub.2, COOR, R, OR, SR, SSR, NHR, or NR.sub.2, wherein
R.sup.2 and R.sup.2' are not both hydrogen; L is O, S, NH, NR,
CY.sub.2O, CY.sub.2S, CY.sub.2NH, CY.sub.2, CY.sub.2CY.sub.2,
CY.sub.2OCY.sub.2, CY.sub.2SCY.sub.2, or CY.sub.2NHCY.sub.2,
wherein Y is H, F, Cl, Br, alkyl, alkenyl, or alkynyl, and wherein
alkyl, alkenyl, and alkynyl may each optionally contain one or more
heteroatoms; R.sup.5 is OH, monophosphate, diphosphate, or
triphosphate, or a phosphonate, phosphoamidate, or phosphoester
thereof; B is a base selected from formula (II): ##STR00078##
wherein dashed lines (---) indicate an optional .pi. bond; X is N,
NH, or NR; Z is C-G, O, >C.dbd.O, >C.dbd.S, or CH-G, wherein
if Z is a participant in a .pi. bond then Z is C-G, and when Z is
not a participant in a .pi. bond then Z is O, >C.dbd.O,
>C.dbd.S, or CH-G; Z.sup.1 is C-G or CH-G, wherein if Z' is a
participant in a .pi. bond then Z' is C-G, and when Z.sup.1 is not
a participant in a .pi. bond then Z' is CH-G; Z.sup.2 is C-G or
CH-G, wherein if Z.sup.2 is a participant in a .pi. bond then
Z.sup.2 is C-G, and when Z.sup.2 is not a participant in a .pi.
bond then Z.sup.2 is CH-G; Z.sup.3 is CH or N; Z.sup.4 is C-G,
>C.dbd.O, >C.dbd.S, >C.dbd.NH or >C.dbd.NR, wherein if
Z.sup.4 is a participant in a .pi. bond then Z.sup.4 is C-G, and if
Z.sup.4 is not a participant in a .pi. bond then Z.sup.4 is
>C.dbd.O, >C.dbd.S, >C.dbd.NH or >C.dbd.NR; each G is
independently H, F, Cl, Br, I, OH, SH, NH.sub.2, NHOH, N.sub.3,
COOH, CN, CONH.sub.2, C(S)NH.sub.2, C(.dbd.NH)NH.sub.2, R, OR, SR,
NHR, or NR.sub.2; and each R is independently alkyl, alkenyl,
alkynyl, aryl, acyl, or aralkyl, optionally containing one or more
heteroatoms; or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1 wherein Z.sup.4 is a participant in a
.pi. bond, and base B is: ##STR00079##
3. The compound of claim 2 wherein G is H, F, Cl, Br, I, R, OR, SR,
NH.sub.2, NHR or NR.sub.2, and R is alkyl.
4. The compound of claim 2 wherein X is NH or NR, and base B is
selected from: ##STR00080##
5. The compound of claim 2 wherein X is N and base B is:
##STR00081##
6. The compound of claim 1 wherein Z.sup.4 is not a participant in
a .pi. bond, and base B is selected from: ##STR00082##
7. The compound of claim 6 wherein X is NH or NR, and base B is
selected from: ##STR00083##
8. The compound of claim 7 wherein X is NH, and base B is selected
from: ##STR00084##
9. The compound of claim 6 wherein X is NH or NR, and base B is
selected from: ##STR00085##
10. The compound of claim 6 wherein X is N, and base B is selected
from: ##STR00086##
11. The compound of claim 1 wherein the base B is selected from one
of the following structures: ##STR00087## ##STR00088##
12. The compound of claims 1 wherein the nucleotide or nucleoside
has the structure: ##STR00089##
13. The compound of claim 12 wherein R.sup.1, R.sup.3 and R.sup.4
are hydrogen.
14. The compound of claim 13 wherein R.sup.2, R.sup.2' and R.sup.3'
are independently H, F, Cl, Br, I, OH, N.sub.3, CN, R or OR, and R
is alkyl.
15. The compound of claim 13 wherein R.sup.2' and R.sup.3' are
OH.
16. The compound of claim 15 wherein R.sup.2 is H or methyl.
17. The compound of claim 1 wherein R.sup.5 is OH, monophosphate,
or a monophosphonate thereof.
18. A method for treating a viral infection comprising
administering a therapeutically effective amount of a compound of
claim 1 to a mammal in need thereof.
19. The method of claim 18, wherein the compound is administered in
combination with one or more active anti-viral agents.
Description
RELATED APPLICATION
[0001] This application is related to provisional application Ser.
No. 60/498,425 filed Aug. 27, 2003 which is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel tricyclic nucleosides
and nucleotides, their preparation, and their use for the treatment
of infectious disease, including viral infections, and of
proliferative disorders, including cancer.
BACKGROUND OF THE INVENTION
[0003] Viral infections are a major threat to human health and
account for many serious infectious diseases. Hepatitis C virus
(HCV), a major cause of viral hepatitis, has infected more than 200
million people worldwide. Current treatment for HCV infection is
restricted to immunotherapy with interferon-.alpha. alone or in
combination with ribavirin, a nucleoside analog. This treatment is
effective in only about half the patient population. Therefore,
there is an urgent need for new HCV drugs. Hepatitis C virus
comprises a positive-strand RNA genome enclosed in a nucleocapsid
and lipid envelope and consists of approximately 9600
ribonucleotides, which encodes a polyprotein of about 3000 amino
acids (Dymock et al. Antiviral Chemistry & Chemotherapy 2000,
11, 79). A HCV protein, NS5B, released from the polyprotein,
possesses polymerase activity and is involved in the synthesis of
double-stranded RNA from the single-stranded viral RNA genome that
serves as the template. The reproduction of HCV virus may be
prevented through the manipulation of NS5B's polymerase activity.
The competitive inhibition of NS5B protein would suppress or
prevent the formation of the double-stranded HCV RNA.
Alternatively, a nucleoside analog also may be incorporated into
the extending RNA strand and act as a chain-terminator.
Furthermore, a deteriorating nucleoside analog also may be
incorporated into the extending RNA, which may cause mutagenic
damage to the viral genome. Recently, several PCT patent
applications (WO 99/43691, WO 01/32153, WO 01/60315, WO 01/79246,
WO 01/90121, WO 01/92282, WO 02/18404, WO 02/057287, WO 02/057425)
have described nucleoside analogs as anti-HCV agents in in vitro
assays.
[0004] Hepatitis B virus (HBV) has acutely infected almost a third
of the world's human population, and about 5% of the infected are
chronic carriers of the virus (Delaney I V et al. Antiviral
Chemistry & Chemotherapy 2001, 12, 1-35). Chronic HBV infection
causes liver damage that frequently progresses to cirrhosis and/or
liver cancer later in the life. Despite the availability and
widespread use of effective vaccines and chemotherapy, the number
of chronic carriers approaches 400 million worldwide. Therefore,
more effective anti-HBV drugs need to be developed. Human
immunodeficiency virus (HIV) causes progressive degeneration of the
immune system, leading to the development of AIDS. A number of
drugs have been used clinically, including reverse transcriptase
inhibitors and protease inhibitors. Currently, combination
therapies are used widely for the treatment of AIDS in order to
reduce the drug resistance. Despite the progress in the development
of anti-HIV drugs, AIDS is still one of the leading epidemic
diseases. Certain acute viral infections also impose a great threat
to human life, including the newly-discovered West Nile virus and
SARS virus.
[0005] Bacterial infections long have been the sources of many
infectious diseases. The widespread use of antibiotics produces
many new strains of life-threatening bacteria. Fungal infections
are another type of infectious diseases, some of which also can be
life-threatening. There is an increasing demand for the treatment
of bacterial and fungal infections. Antimicrobial drugs based on
new mechanisms of action are especially important.
[0006] Proliferative disorders are one of the major
life-threatening diseases and have been intensively investigated
for decades. Cancer now is the second leading cause of death in the
United States, and over 500,000 people die annually from this
proliferative disorder. All of the various cells types of the body
can be transformed into benign or malignant tumor cells.
Transformation of normal cells into cancer cells is a complex
process and thus far is not fully understood. The treatment of
cancer consists of surgery, radiation, and chemotherapy. While
chemotherapy can be used to treat all types of cancer, surgery and
radiation therapy are limited to certain cancer at certain sites of
the body. There are a number of anticancer drugs widely used
clinically. Among them are alkylating agent such as cisplatin,
antimetabolites, such as 5-fluorouracil, and gemcitabine. Although
surgery, radiation, and chemotherapies are available to treat
cancer patients, there is no cure for cancer at the present time.
Cancer research is still one of the most important tasks in medical
and pharmaceutical organizations.
[0007] Nucleoside drugs have been used clinically for the treatment
of viral infections and proliferative disorders for decades. Most
of the nucleoside drugs are classified as antimetabolites. After
they enter cells, nucleoside analogs are phosphorylated
successively to nucleoside 5'-monophosphates, 5'-diphosphates, and
5'-triphosphates. In most cases, nucleoside triphosphates, e.g.,
3'-azido-3'-deoxythymidine (AZT, an anti-HIV drug) triphosphate and
arabinosylcytosine (cytarabine, an anticancer drug) triphosphate,
are the active chemical entities that inhibit DNA or RNA synthesis,
through a competitive inhibition of polymerases and subsequent
incorporation of modified nucleotides into DNA or RNA sequences. In
a few cases, nucleoside analogs exert effects at lower phosphate
levels. For instance, 5-fluoro-2'-deoxyuridine (an anticancer drug)
5'-monophosphate and 2',2'-difluoro-2'-deoxycytidine (an anticancer
drug) 5'-diphosphate have been shown to inhibit thymidylate
synthase and ribonucleotide reductase, respectively. Although
nucleoside analogs themselves may act at the nonphosphate level
such as the inhibitors of adenosine kinases and the ligands of
adenosine receptors, currently, clinically-useful nucleoside drugs
primarily depend on cellular activation by nucleoside kinases and
nucleotide kinases.
[0008] At least, two criteria are pertinent for nucleoside
antiviral drugs: 1. nucleoside analogs should anabolize to
nucleotides in cells; and 2. the anabolized nucleotides should
target selectively viral enzymes. In order to be phosphorylated in
cells and selectively to target preferred enzymes, nucleoside
analogs should have favorable modifications on their sugar and base
moieties. To obtain such favorable nucleoside analogs, a general
approach is to generate diverse nucleoside analogs by modifying the
base or the sugar, or by modifying both base and sugar moieties.
Numerous examples exist in the literature for the synthesis of a
variety of modified nucleosides (Chemistry of Nucleosides and
Nucleotides Vol. 1 (1988), Vol. 2 (1991), Vol. 3 (1994), edited by
Leroy B. Townsend, Plenum Press).
[0009] However, there are certain classes of nucleoside compounds
that were not explored intensively for their antiviral and
anti-proliferative activities before the present invention. A class
of such compounds is tricyclic nucleosides. Disclosures on
tricyclic nucleosides are very limited considering the existence of
various tricyclic heterocycles. A well-known tricyclic nucleoside
is triciribine (TCN), having potent cytotoxicity against cancer
cells (Porcari et al. J. Med. Chem. 2000, 43, 2438-2448). A number
of its modified derivatives were prepared and screened against
viruses and cancer (Porcari et al. Nucleosides Nucleotides 1999,
18, 2475-2497; J. Med. Chem. 2000, 43, 2457-2463). Another known
tricyclic nucleoside is
2-(2-deoxy-.beta.-D-erythro-pentofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraa-
zabenz[cd]azulen-7-one, but its biological activity was not
reported (Helv. Chim. Acta, 2000, 83, 911-927). The PCT publication
WO 03/061385 describes tricyclic nucleoside libraries. The present
invention discloses novel tricyclic nucleosides and nucleotides and
their use for the treatment of infectious disease, including viral
infections, and of proliferative disorders, including cancer.
SUMMARY OF THE INVENTION
[0010] The present invention relates to novel tricyclic nucleosides
and derivatives thereof, their preparation, and their use for the
treatment of viral infections and proliferative disorders.
[0011] In one embodiment, a compound of the formula (I) which may
be a D- or L-nucleoside is provided
##STR00001##
[0012] wherein
[0013] A is O, S, CH.sub.2, CHF, or CF.sub.2;
[0014] R.sup.1, R.sup.2, R.sup.2', R.sup.3, R.sup.3', and R.sup.4
are independently selected from the group consisting of H, F, Cl,
Br, I, OH, SH, NH.sub.2, NHOH, NHNH.sub.2, N.sub.3, COOH, CN,
CONH.sub.2, C(S)NH.sub.2, COOR, R, OR, SR, SSR, NHR, and
NR.sub.2;
[0015] R.sup.4' is -L-R.sup.5;
[0016] L is selected from the group consisting of O, S, NH, NR,
CY.sub.2O, CY.sub.2S, CY.sub.2NH, CY.sub.2, CY.sub.2CY.sub.2,
CY.sub.2OCY.sub.2, CY.sub.2SCY.sub.2, and CY.sub.2NHCY.sub.2,
wherein Y is selected from the group consisting of H, F, Cl, Br,
alkyl, alkenyl, and alkynyl, wherein alkyl, alkenyl, and alkynyl
may each optionally contain one or more heteroatoms;
[0017] R.sup.5 is OH, monophosphate, diphosphate, or triphosphate,
optionally masked with prodrug moieties, or a mono di or
triphosphate mimic;
[0018] B is a base selected from the group of heterocycles
consisting of
##STR00002##
[0019] each Z is independently selected from the group consisting
of N,N--.sup.-BH.sub.2GM.sup.+, C-G, O, S, NR, >C.dbd.O,
>C.dbd.S, >C.dbd.NH, >C.dbd.NR, >S.dbd.O,
>S(O).sub.2 and CH-G;
[0020] wherein if Z is a participant in a .pi. bond (double bond),
Z is independently N or C-G;
[0021] wherein if Z is not a participant in a .pi. bond, Z is
independently N--.sup.-BH.sub.2GM.sup.+, O, S, NR, >C.dbd.O,
>C.dbd.S, >C.dbd.NH, >C.dbd.NR, >S.dbd.O,
>S(O).sub.2 and CH-G;
[0022] X is O, S, SO, SO.sub.2, Se, SeO, SeO.sub.2, NH, or NR;
[0023] W is C, CH or N;
[0024] wherein if W is a participant in one .pi. bond, W is C;
[0025] wherein if W is not a participant in a .pi. bond, W is CH or
N; and
[0026] .sup.-BH.sub.2GM.sup.+ is an ion pair and lye is a
cation;
[0027] G is selected from the group consisting of H, F, Cl, Br, I,
OH, SH, NH.sub.2, NHOH, N.sub.3, COOH, CN, CONH.sub.2,
C(S)NH.sub.2, C(.dbd.NH)NH.sub.2, R, OR, SR, NHR, and NR.sub.2,
when two or more G groups are present on a molecule, they may be
same as or different from one another;
[0028] R is selected from the group consisting of alkyl, alkenyl,
allynyl, aryl, acyl, and aralkyl, optionally containing one or more
heteroatoms;
[0029] dashed lines (---) indicate a possible .pi. or double
bond.
[0030] Thus, structures of formulae II and III may have one or more
ring double bonds and, in some instances, may have two or more ring
double bonds.
[0031] In one preferred embodiment L is CH.sub.2.
[0032] Preferably W is C. Preferably X is NH.
[0033] In another embodiment, the seven-membered ring portion of
the base contains one or two and preferably one N in the backbone
of the ring.
[0034] In another embodiment, the Z in the five-membered ring of
the base is C.
[0035] In yet another embodiment, each Z in the seven-membered ring
portion of the base is preferably C-G, >C.dbd.O, >C.dbd.S.
Preferably CH-G is CH.sub.2, CH-halo, and C-G is CH, C-alkyl
preferably CCH.sub.2, C--OR, preferably, C--O alkyl, more
preferably COCH.sub.3.
[0036] In one embodiment, at least one of R.sup.2 or R.sup.2' is
not H. In another preferred embodiment, the sugar
##STR00003##
[0037] Thus, compounds of the invention may have the formula
##STR00004##
[0038] Some embodiments, in compounds of the invention of formula
(I), B is a base selected from the group of heterocycles consisting
of
##STR00005##
[0039] Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.7 and Z.sup.8 are
independently N or C-G; and
[0040] Z.sup.5, Z.sup.6, and Z.sup.9 are independently selected
from the group consisting of N--.sup.-BH.sub.2GM.sup.+, O, S, NR,
>C.dbd.O, >C.dbd.S, >C.dbd.NH, >C.dbd.NR, >S.dbd.O,
>S(O).sub.2 and CH-G.
[0041] In a preferred embodiment, the base is
##STR00006## ##STR00007##
[0042] Preferably the compound of the invention is not
##STR00008##
[0043] In another aspect the component is not
##STR00009##
[0044] In another embodiment, is provided a method for the
treatment of a viral or bacterial infection, or proliferative
disorder comprising administering an effective amount of a compound
of the formula (I), or a pharmaceutically acceptable salt or a
prodrug thereof, optionally in combination with one or more
antiviral, antibacterial, or antiproliferative agents. In one
aspect, the viral infection is caused by an RNA virus, such as HCV
or a DNA or retrovirus such as HBV or HIV.
[0045] The invention is also directed to a process of making
compounds of the invention. For example, a compound having the
formula
##STR00010##
may be made by cyclising a compound having the formula
##STR00011##
[0046] wherein each of Z.sup.1-Z.sup.4 is independently Z; and
wherein each of W.sup.1-W.sup.2 is independently W. Preferably, in
this process A is O, CH.sub.2 or optionally protected N;
[0047] W.sup.1 is C (if p bond) or N (if no p bond);
[0048] W.sup.2 is C, CH or N;
[0049] W.sup.4 is H or trialkyltin;
[0050] Z.sup.1 and Z.sup.2 are each independently CH, C-halogen,
C-alkyl, C-aryl, C--O-alkyl or
[0051] C--S-alkyl;
[0052] Z.sup.3 is CH, C-alkyl, C-halogen, N, CNHR, CNH.sub.2,
CNR.sub.2, C.dbd.O, or C.dbd.S;
[0053] Z.sup.4 is CH, C-halogen, C-alkyl, C-aryl, C--O-alkyl,
C--S-alkyl, C--OH, C--NH.sub.2, C--NHR or C--NR.sub.2;
[0054] R.sup.1, R.sup.2, R.sup.2', R.sup.3, R.sup.3', R.sup.4 are
each independently H, halogen, alkyl, O-alkyl, OH, optionally
protected O, methyl, H or F; and
[0055] R.sup.5 is an optionally protected OH or NH.sub.2. This
process may further comprise reacting
##STR00012##
with
##STR00013##
to form
##STR00014##
[0056] wherein Y is a halogen; and
[0057] wherein W.sup.4 is H or a metal-containing compound capable
of metal-mediated cross coupling. The OH and NH groups may be
optionally deprotected.
[0058] In another embodiment, a process to make
##STR00015##
[0059] comprises cyclising a compound having the formula
##STR00016##
[0060] wherein each of Z.sup.1-Z.sup.4 and Z.sup.6 is independently
Z;
[0061] each of W.sup.1 and W.sup.2 is independently W;
[0062] wherein Y is halogen; and
[0063] Nu is a nucleophile. Preferably, A is O, CH.sub.2 or
optionally protected N;
[0064] X is optionally protected N, O, or S;
[0065] Nu is an alcohol, an alkylthiol, or an alkylamine;
[0066] W.sup.1 is C (if p bond) or N (if no p bond);
[0067] W.sup.2 is C, CH or N;
[0068] Z.sup.1 and Z.sup.2 are each independently CH, C-halogen,
C-alkyl, C-aryl, C--O-alkyl, or
[0069] C--S-alkyl;
[0070] Z.sup.3 is CH, C-alkyl, C-halogen, N, CNIIR, CNH.sub.2,
CNR.sub.2, C.dbd.O, or C.dbd.S;
[0071] Z.sup.4 is CH or C-halogen, C-alkyl, C-aryl, C--O-alkyl,
C--S-alkyl, C--OH, C--NH.sub.2, C--NHR or
[0072] C--NR.sub.2;
[0073] R.sup.1, R.sup.2, R.sup.2', R.sup.3, R.sup.3', R.sup.4 are
each independently H, halogen, alkyl, O-alkyl, OH, optionally
protected O, methyl, or F;
[0074] R.sup.5 is an optionally protected OH or NH.sub.2; and
[0075] Z.sup.6, is CH.sub.2, O, NH, NR or S. This process further
comprises reacting
##STR00017##
with
##STR00018##
to form
##STR00019##
[0076] wherein W.sup.4 is H or a metal-containing compound capable
of cross coupling.
[0077] In addition, the compound of the invention may be further
modified, for example, to add various functional groups. In one
embodiment, a compound having the formula
##STR00020##
may be modified using a nucleophile and/or electrophile to form a
compound selected from the group consisting of:
##STR00021## ##STR00022##
[0078] wherein each of Z.sup.1-Z.sup.4 are independently Z;
[0079] wherein each of W.sup.1 and W.sup.2 are independently W;
and
[0080] wherein Nu is a nucleophile. Preferably, in this process
[0081] A is O, CH.sub.2 or optionally protected N;
[0082] Nu is an alcohol, an alkylthiol, or an alkylamine;
[0083] W.sup.1 is C (if p bond) or N (if no p bond);
[0084] W.sup.2 is C, CH or N;
[0085] Z.sup.1 and Z.sup.2 are each independently CH, C-halogen,
C-alkyl, C-aryl, C--O-alkyl, or C--S-alkyl;
[0086] Z.sup.3 is CH, C-alkyl, C-halogen, N, CNHR, CNH.sub.2,
CNR.sub.2, C.dbd.O, or C.dbd.S;
[0087] Z.sup.4 is CH or C-halogen, C-alkyl, C-aryl, C--O-alkyl,
C--S-alkyl, C--OH, C--NH.sub.2, C--NHR or C--NR.sub.2;
[0088] R.sup.2, R.sup.21, R.sup.3, R.sup.3', R.sup.4 are each
independently H, halogen, alkyl, O-alkyl, OH, optionally protected
O, methyl, or F;
[0089] R.sup.5 is an optionally protected OH or NH.sub.2;
[0090] Q is O, NR, NH or S; and
[0091] R.sup.6 is alkyl, aryl, alkenyl or alkynyl.
[0092] In another embodiment, a pharmaceutical composition is
provided comprising a therapeutically effective amount of a
compound of the formula (I) or a pharmaceutically acceptable salt
or a prodrug thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0093] The tricyclic nucleosides of the invention also include
derivatives such as nucleotide mimics and/or prodrugs thereof.
[0094] For example, in some embodiments, nucleotide mimics of the
compounds of the Invention of formula (I) discussed above
include:
[0095] a compound in which R.sup.5 is a monophosphate mimic of
having formula (X) or (XI):
##STR00023##
[0096] where X.sup.1', X.sup.4', and X.sup.6' independently are O,
S, NH, or NR; X.sup.2', X.sup.3', and X.sup.5' are selected
independently from the group consisting of H, F, OH, SH, NH.sub.2,
NHOH, N.sub.3, CN, .sup.-BH.sub.2GM.sup.+, .sup.-BH.sub.3M.sup.+,
R, OR, SR, NHR, and NR.sub.2. The substituents
.sup.-BH.sub.2GM.sup.+ and .sup.-BH.sub.3M.sup.+ are ion pairs,
which are linked to phosphorus through the negatively charged
boron. M.sup.4 is a cation.
[0097] In some embodiments, nucleotide mimics of the compounds of
the Invention of formula (I) discussed above include di- and
tri-phosphate mimics including:
[0098] a compound in which R.sup.5 is a di- or tri-phosphate moiety
of formula (XII):
##STR00024##
[0099] X.sup.2, X.sup.3, and X.sup.4 are selected independently
from the group consisting of O, S, Se, NH and NR;
[0100] X.sup.5 and X.sup.6 are selected independently from the
group consisting of O, S, Se, O.sub.2, CY.sub.2CO, CHOH,
C(OH).sub.2, CH.sub.2O, CH.sub.2CH.sub.2, CH.sub.2CHNH.sub.2,
CH.sub.2CH.sub.2CHNH.sub.2, CY.sub.2OCY.sub.2, CY.sub.2, CRY,
CY.sub.2CY.sub.2, CHR, CC, HC.dbd.CH, NH, NR, NOH, NOR, NNH.sub.2,
and NNHR;
[0101] Y is selected from the group consisting of H, F, Cl, Br,
alkyl, alkenyl, and alkynyl, wherein alkyl, alkenyl, and alkynyl
may each optionally contain one or more heteroatoms;
[0102] R is selected from the group consisting of alkyl, alkenyl,
alkynyl, aryl, acyl, and aralkyl, each optionally containing one or
more heteroatoms;
[0103] X.sup.7, X.sup.8, X.sup.9, and X.sup.10 are selected
independently from the group consisting of H, F, OH, SH, NH.sub.2,
NHOH, NHOR, NHNH.sub.2, NHNHR, CN, N.sub.3, .sup.-BH.sub.3M.sup.+,
.sup.-BH.sub.2GM.sup.+, R, OR, SR, SeH, SeR, NHR, and NR.sub.2.
[0104] wherein n is 0 or 1. The substituents .sup.-BH.sub.2GM.sup.+
and .sup.-BH.sub.3M.sup.+ are ion pairs, which are is linked to
phosphorus through the negatively charged boron. M.sup.+ is a
cation.
[0105] Additional nucleotide phosphate mimics and methods of making
the phosphate mimics appropriate for the compounds of the invention
are described in PCT Publications WO 2003/072757 and WO
2003/073989, filed Feb. 28, 2003, which are incorporated herein by
reference in their entirety. Many nucleotide mimics of the present
invention can be prepared by similar approaches as published or by
using well-known knowledge of organophosphorous chemistry.
Generally, phosphate mimics of the nucleosides and nucleotides of
the invention can inhibit enzyme function without phosphorylation
and/or have enhanced nuclease stability relative to nucleotides
with unmodified phosphate.
[0106] The term phosphate mimic, unless otherwise specified, refers
to a phosphate analog, including, but not limited to, phosphonate,
phosphothioate, phosphoselenate, selenophosphate, thiophosphate,
P-boranophosphate, phosphoramidate, sulfamate, sulfonate, and
sulfonamide and/or a combination thereof. Preferred embodiments of
the phosphate mimics include phosphonate, phosphorothioate,
methylphosphonate, fluoromethylphosphonate,
difluoromethylphosphonate, vinylphosphonate, phenylphosphonate,
sulfonate, fluorophosphate, dithiophosphorothioate,
5'-methylenephosphonate, 5'-difluoromethylenephosphonate,
5'-deoxyphosphonate, 5'-aminophosphoramidate, and 5'-thiophosphate.
More preferred is phosphonate.
[0107] The terms diphosphate mimic and triphosphate mimic
specifically refer to a diphosphate analog and a triphosphate
analog, respectively, which comprises at least one of the phosphate
mimics, one of the modifications at the bridging site of
diphosphate and triphosphate, or replacements of non-bridging
phosphate oxygens. The modification at the bridging site, i.e., in
the X.sup.5 and X.sup.6 positions of formula (XII), includes the
replacement of 0 by other atoms or functions such as S, Se,
O.sub.2, NH, NHR, NR, CH.sub.2, CHF, CHOI, CHBr, CF.sub.2,
CCl.sub.2, CBr.sub.2, CHR, CYCO.sub.2, CH.sub.2O, CHOH,
C(OH).sub.2, CH.sub.2CH.sub.2, CC, CH.dbd.CH,
CH.sub.2CH.sub.2CHNR.sub.2, CH.sub.2CHNH.sub.2, CY.sub.2OCY.sub.2,
CY.sub.2, CY.sub.2CY.sub.2, and CR.sub.2 where R is selected from
the group consisting of alkyl, alkenyl, alkynyl, aryl, acyl, and
aralkyl each optionally containing one or more heteroatoms.
Non-bridging phosphate oxygens, e.g., in X.sup.7-X.sup.10 positions
of formula (XII) can be replaced by a variety of substituents
including H, F, OH, SH, NH.sub.2, NHOH, NHOR, NHNH.sub.2, NHNHR,
CN, N.sub.3, .sup.-BH.sub.3M.sup.+, R, OR, SR, Sell, SeR, NHR,
NR.sub.2, and R* where R is as defined herein, and wherein R* is a
prodrug substituent. M.sup.+ is a cation preferably a
pharmaceutically acceptable cation such as Ca.sup.2+, ammonium,
trialkylammonium or tertaalkylammonium, e.g., NH.sub.4.sup.+,
Et.sub.3NH.sup.+, Bu.sub.3NH.sup.+, and Bu.sub.4N.sup.4.
[0108] The .alpha.-P, .beta.-P, and .gamma.-P in the diphosphate
mimics and triphosphate mimics may independently adopt either R or
S configurations when they become a chiral phosphorus.
[0109] In some embodiments, the tricyclic nucleosides and
nucleotides of invention also include their prodrug derivatives. In
addition to those described herein, prodrug derivatives of
nucleosides, nucleotides and nucleotide phosphate mimics and
methods of making the prodrugs appropriate for use in the present
invention are described in PCT Publications WO 2003/072757 and WO
2003/073989. Such prodrug modification is to enhance drug
absorption and/or drug delivery into cells.
[0110] In one embodiment, such compounds of the invention include
prodrugs (e.g., one or more of an --OH group of a mono, di or
triphosphate, one or more of X.sup.2', X.sup.3' or X.sup.5', or
X.sup.7-X.sup.10 in (XII) is a prodrug substituent R*) of the
compounds of formula (I) discussed herein.
[0111] R* is a prodrug substituent which may be conjugated to one
or more X.sup.7-X.sup.10 positions. The term prodrug, unless
otherwise specified, refers to a masked (protected) form of a
nucleotide, such as a mimic of formula (X) or (XI) that is formed
when one or more of X.sup.2', X.sup.3' or X.sup.5' is V or to a
masked (protected) form of a nucleotide mimic of formula (XII) when
one or more of X.sup.7-X.sup.10 is R*. The prodrug of a nucleoside
5'-monophosphate mimic can mask the negative charges of the
phosphate mimic moiety entirely or partially, mask the negative
charges of the di-phosphate (X.sup.7, X.sup.8, X.sup.10) mimic or
tri-phosphate (X.sup.7-X.sup.10) mimic moiety or phosphate moiety,
entirely or partially, or mask a heteroatom substituted alkyl, aryl
or aryalkyl (W', see below) attached to a phosphate or phosphate
mimic moiety in order to enhance drug absorption and/or drug
delivery into cells. The prodrug can be activated either by
cellular enzymes such as lipases, esterases, reductases, oxidases,
nucleases or by chemical cleavage such as hydrolysis to release
(liberate) the nucleotide mimic after the prodrug enters cells.
Prodrugs are often referred to as cleavable prodrugs. Prodrugs
substituents include, but are not limited to: proteins; antibiotics
(and antibiotic fragments); D- and L-amino acids attached to a
phosphate moiety or a phosphate mimic moiety via a carbon atom
(phosphonates), a nitrogen atom (phosphoamidates), or an oxygen
atom (phosphoesters); peptides (up to 10 amino acids) attached to a
phosphate moiety or a phosphate mimic moiety via a carbon atom
(phosphonates), a nitrogen atom (phosphoamidates), or an oxygen
atom (phosphoesters); drug moieties attached to a phosphate moiety
or a phosphate mimic moiety via a carbon atom (phosphonates), a
nitrogen atom (phosphoamidates), or an oxygen atom (phosphoesters);
steroids; cholesterols; folic acids; vitamins; polyamines;
carbohydrates; polyethylene glycols (PEGS); cyclosaligenyls;
substituted 4 to 8-membered rings, with or without heteroatom
substitutions, 1,3-phosphoamidate attachments to a terminal
phosphate or phosphate mimic moiety (.gamma. or .beta.) or
connecting between an .alpha.,.beta. or .beta.,.gamma. phosphate
moiety or phosphate mimic moiety; acylthioethoxy, (SATE)
RCOSCH.sub.2CH.sub.2O--; RCOSCH.sub.2CH.sub.2O--W'--O--;
RCOSCH.sub.2CH.sub.2O--W'--S--; RCOSCH.sub.2CH.sub.2O--W'--NH--;
RCOSCH.sub.2CH.sub.2O--W'--; RCOSCH.sub.2CH.sub.2O--W'--CY.sub.2--;
acyloxymethoxy, RCOOCH.sub.2O--; RCOOCH.sub.2O--W'--O--;
RCOOCH.sub.2O--W'--S--; RCOOCH.sub.2O--W'--NH--;
RCOOCH.sub.2O--W'--; RCOOCH.sub.2O--W--CY.sub.2--;
alkoxycarbonyloxymethoxy, ROCOOCH.sub.2O--;
ROCOOCH.sub.2O--W'--O--; ROCOOCH.sub.2O--W'--S--;
ROCOOCH.sub.2O--W'--NH--; ROCOOCH.sub.2O--W'--;
ROCOOCH.sub.2O--W'--CY.sub.2--; acylthioethyldithioethoxy (DTE)
RCOSCH.sub.2CH.sub.2SSCH.sub.2CH.sub.2O--;
RCOSCH.sub.2CH.sub.2SSCH.sub.2CH.sub.2O--W'--;
RCOSCH.sub.2CH.sub.2SSCH.sub.2CH.sub.2O--W'--O--;
RCOSCH.sub.2CH.sub.2SSCH.sub.2CH.sub.2O--W'--S--;
RCOSCH.sub.2CH.sub.2SSCH.sub.2CH.sub.2O--W'--NH--;
RCOSCH.sub.2CH.sub.2SSCH.sub.2CH.sub.2O--CY.sub.2--;
acyloxymethylphenylmethoxy (PAOB)
RCO.sub.2--C.sub.6H.sub.4--CH.sub.2--O--;
RCO.sub.2--C.sub.6H.sub.4--CH.sub.2--O--W'--;
RCO.sub.2--C.sub.6H.sub.4--CH.sub.2--O--W'--O--;
RCO.sub.2--C.sub.6H.sub.4--CH.sub.2--O--W'--S--;
RCO.sub.2--C.sub.6H.sub.4--CH.sub.2--O--W'--NH--;
RCO.sub.2--C.sub.6H.sub.4--CH.sub.2--O--W'--CY.sub.2--;
1,2-O-diacyl-glyceryloxy, RCOO--CH.sub.2--CH(OCOR)--CH.sub.2O--;
1,2-O-dialkyl-glyceryloxy, RO--CH.sub.2--CH(OR)--CH.sub.2O--;
1,2-S-dialkyl-glyceryloxy, RS--CH.sub.2--CH(SR)--CH.sub.2O--;
1-O-alkyl-2-O-acyl-glyceryloxy,
RO--CH.sub.2--CH(OCOR)--CH.sub.2O--;
1-S-alkyl-2-O-acyl-glyceryloxy,
RS--CH.sub.2--CH(OCOR)--CH.sub.2O--; 1-O-acyl-2-O-alky-glyceryloxy,
RCOO--CH.sub.2--CH(OR)--CH.sub.2O--;
1-O-acyl-2-S-alky-kglyceryloxy,
RCOO--CH.sub.2--CH(SR)--CH.sub.2O--; any substituent attached via a
carbon, nitrogen or oxygen atom to a nucleoside di- or
tri-phosphate mimic that liberates the di- or tri-phosphate mimic
in vivo.
[0112] A combination of prodrug substituents may be attached
(conjugated) to one or more X.sup.2', X.sup.3' and X.sup.5'
positions on a nucleoside mono-phosphate mimic or to one or more
X.sup.7-X.sup.10 positions on a nucleoside di- or tri-phosphate
mimic. W' is alkyl, aryl, aralkyl as described above or a
heterocycle. Preferred prodrug substituents (R*) in positions
X.sup.2', X.sup.3' or X.sup.5' include 2,3-O-diacylglyeeryloxy,
2,3-O-dialkylglyeeryloxy, 1-O-alkyl-2-O-acylglyceryloxy,
1-O-acyl-2-O-alkylglyceryloxy,
1-S-alkyl-2-O-acyl-1-thioglyceryloxy, acyloxymethoxy,
S-acyl-2-thioethoxy, S-pivaloyl-2-thioethoxy, acyloxymethoxy,
pivaloyloxymethoxy, alkoxycarbonyloxymethoxy,
S-alkyldithio-S'-ethyoxy acyloxymethoxy, S-acyl-2-thioethoxy,
S-pivaloyl-2-thioethoxy, pivaloyloxymethoxy,
alkoxycarbonyloxymethoxy, and S-alkyldithio-S'-ethyoxy.
[0113] The term moiety, unless otherwise specified, refers to a
portion of a molecule. Moiety may be, but is not limited to, a
functional group, an acyclic chain, a prodrug masking group, an
aromatic ring, a carbohydrate, a carbocyclic ring, or a
heterocycle.
[0114] The term base, unless otherwise specified, refers to the
base moiety of a nucleoside or nucleotide. The base moiety is the
heterocycle portion of a nucleoside or nucleotide. The base moiety
of a nucleotide of formula (I) is a tricyclic heterocycle
represented by formulae II-III. Preferably, the base moiety of a
nucleotide of formula (I) may be a tricyclic heterocycle
represented by any one of formulae IV-IX. The nucleoside base is
attached to the sugar moiety of a nucleoside in such ways that both
.beta.-D- and .alpha.-L-nucleoside can be produced.
[0115] The term sugar refers to the ribofuranose portion of a
nucleoside. The sugar moiety of formula (I) nucleosides and
nucleotides mimics and/or prodrugs thereof may contain one or more
substituents at their C1-, C2-, C3- and C4-position of the
ribofuranose. Substituents may direct to either the .alpha.- or
.beta.-face of the ribofuranose. The nucleoside base that can be
considered as a substituent at the C-1 position of the ribofuranose
directs to the .beta.-face of the sugar. The .beta.-face is the
side of a ribofuranose on which a purine or pyrimidine base of
natural .beta.-D-nucleosides is present. The .alpha.-face is the
side of the sugar opposite to the .alpha.-face. The sugar moiety of
the present invention is not limited to a ribofuranose and its
derivatives, instead it may be a carbohydrate, a carbohydrate
analog, a carbocyclic ring, or other ribofuranose analogs.
[0116] The term sugar-modified nucleoside or nucleotide refers to a
nucleoside or nucleotide containing a modified sugar moiety.
[0117] The term base-modified nucleoside or nucleotide refers to a
nucleoside or nucleotide containing a modified base moiety.
[0118] The term alkyl, unless otherwise specified, refers to a
saturated straight, branched, or cyclic hydrocarbon of C1 to C18.
Alkyls may include, but are not limited to, methyl, ethyl,
n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, t-butyl,
cyclobutyl, n-pentyl, isopentyl, neopentyl, cyclopentyl, n-hexyl,
cyclohexyl, dodecyl, tetradecyl, hexadecyl, or octadecyl.
[0119] The term alkenyl, unless otherwise specified, refers to an
unsaturated hydrocarbon of C2 to C18 that contains at least one
carbon-carbon double bond and may be straight, branched or cyclic.
Alkenyls may include, but are not limited to, olefinic, propenyl,
allyl, 1-butenyl, 3-butenyl, 1-pentenyl, 4-pentenyl, 1-hexenyl, or
cyclohexenyl.
[0120] The term alkynyl, unless otherwise specified, refers to an
unsaturated hydrocarbon of C2 to C18 that contains at least one
carbon-carbon triple bond and may be straight, branched or cyclic.
Alkynyls may include, but are not limited to, ethynyl, 1-propynyl,
2-propynyl, 1-butynyl, or 3-butynyl.
[0121] The term aryl, unless otherwise specified, refers to an
aromatic moiety with or without one or more heteroatoms. Aryls may
include, but are not limited to, phenyl, biphenyl, naphthyl,
pyridinyl, pyrrolyl, and imidazolyl optionally containing one or
more substituents. The substituents may include, but are not
limited to, hydroxy, amino, thio, halogen, cyano, nitro, alkoxy,
alkylamino, alkylthio, hydroxycarbonyl, alkoxycaxbonyl, or
carbamoyl.
[0122] The term aralkyl, unless otherwise specified, refers to a
moiety that contains both an aryl and an alkyl, an alkenyl, or an
alkynyl. Aralkyls can be attached through either the aromatic
portion or the non-aromatic portion. Aralkyls may include, but are
not limited to, benzyl, phenethyl, phenylpropyl, methylphenyl,
ethylphenyl, propylphenyl, butylphenyl, phenylethenyl,
phenylpropenyl, phenylethynyl, or phenylpropynyl.
[0123] The term acyl, unless otherwise specified, refers to
alkylcarbonyl. Acyls may include, but are not limited to, formyl,
acetyl, fluoroacetyl, difluoroacetyl, trifluoroacetyl,
chloroacetyl, dichloroacetyl, trichloroacetyl, propionyl, benzoyl,
toluoyl, butyryl, isobutyryl, or pivaloyl.
[0124] The term heteroatom refers to oxygen, sulfur, nitrogen, or
halogen. When one or more heteroatoms are attached to alkyl,
alkeneyl, alkynyl, acyl, aryl, or arakyl, a new functional group
may be produced. For instance, when one or more heteroatoms are
attached to an alkyl, substituted alkyls may be produced,
including, but not limited to, fluoroalkyl, chloroalkyl,
bromoalkyl, iodoalkyl, alkoxy, hydroxyalkyl, alkylamino,
aminoalkyl, alkylthio, thioalkyl, azidoalkyl, cyanoalkyl,
nitroalkyl, carbamoylalkyl, carboxylalkyl, and acylalkyl.
[0125] Benzoazulenes, such as benzo[cd]azulene refer to a class of
tricyclic compounds having a fused, 5, 6, and 7-membered rings that
may contain one or more heteroatoms, preferably O or N, in the
backbone of the ring, and thus derivatives of benzoazulene is also
included in such term.
[0126] The term halogen or halo refers to fluorine, chlorine,
bromine, or iodine.
[0127] The term function refers to a substituent. Functions may
include, but are not limited to, hydroxy, amino, sulthydryl, azido,
cyano, halo, nitro, hydroxyamino, hydroxycarbonyl, alkoxycarbonyl,
or carboxyl either protected or unprotected.
[0128] R may be formula (I) is a univalent substituent and present
on the base, sugar and other moieties. R may be selected from the
group consisting of alkyl, alkenyl, alkynyl, aryl, acyl, and
aralkyl optionally containing one or more heteroatoms, which are as
defined above.
[0129] A "protecting group" for example for O, S, or N such as
hydroxy or NH.sub.2, includes acyl groups, silyl groups, and the
like. Suitable protecting groups are described by Greene, T. W., et
al., in Protecting Groups in Organic Synthesis. 2.sup.nd Ed., John
Wiley & Sons, Inc. (1991), incorporated herein by reference.
"Nucleophile" and "electrophile" have their ordinary meaning in the
art. Examples of preferred nucleophiles are alcohols, alkylthiols
or alkylamines, which may be optionally protected. "Nu" refers to
both the free nucleophiles and the nucleophiles as attached to the
tricyclic compound of the invention. Thus, a nucleophile may be,
for example, DMF or MeO--. Preferably, a nucleophile may be
optionally protected N, O or S.
[0130] In addition to using prodrug approaches, the delivery of the
nucleosides and nucleotides may be assisted by using a
therapeutically acceptable carrier such as liposomal suspensions,
cationic lipids, and polyimines. In compounds of formula (I) where
a chiral center is present, the invention encompasses enantiomers,
or stereoisomers and mixtures thereof, such as enantiomerically
enriched mixtures.
[0131] The term "infection" or "microbial infection" refers to the
infection caused by an infectious agent or microbe, such as
bacteria, parasite (including protozoan), virus or fungus
(including unicellular and multicellular). Examples of microbes
that cause such infection include: Acanthamoeba, African Sleeping
Sickness (Trypanosomiasis), amebiasis, American Trypanosomiasis
(Chagas Disease), Bilharzia (Schistosomiasis), cryptosporidiosis
(diarrheal disease, Cryptosporidium Parvum), Giardiasis (diarrheal
disease, Giardia lamblia), hepatitis A, B, C, D, E, leishmaniasis
(skin sores and visceral), malaria (Plasmodium falciparum),
Salmonella enteritides infection (stomach cramps, diarrhea and
fever), tuberculosis (mycobacterium tuberculosis), varicella
(chicken pox), yellow fever, pneumonias, urinary tract infections
(Chlamydia and Mycoplasma), meningitis and meningococcal
septicemia, skin and soft tissue infections (Staphylococcus
aureus), lower respiratory tract infections (bacterial pathogens or
hepatitis C).
[0132] Common infections caused by microbes are further outlined in
the following chart:
TABLE-US-00001 Infection Bacteria Fungus Protozoa Virus AIDS X
Athlete's Foot X Chicken Pox X Common Cold X Diarrheal Disease X X
X Flu X Genital Herpes X Malaria X X Meningitis X Pneumonia X X
Sinusitis X X Skin Disease X X X X Strep Throat X Tuberculosis X
Urinary Tract Infections X Vaginal Infections X X Viral Hepatitis
X
Chemical Synthesis
[0133] The novel nucleosides and nucleotides, and prodrugs thereof,
of the present invention can be prepared by those who are skillful
in synthetic organic and nucleoside chemistry using established
synthetic methodology (Chemistry of Nucleosides and Nucleotides
Vol. 1, 2, 3, edited by Townsend, Plenum Press; Handbook of
Nucleoside Synthesis by Vorbruggen Ruh-Pohlenz, John Wiley &
Sons, Inc., 2001; The Organic Chemistry of Nucleic Acids by
Yoshihisa Mizuno, Elsevier, 1986). The nucleosides of the present
invention can be converted to their corresponding monophosphate,
diphosphate, and triphosphate by established phosphorylation
procedures. Similarly, known methods in the art can be used to
synthesize the nucleotide prodrugs and phosphate mimics. The
following schemes and descriptions serve as representative
syntheses of the nucleosides of the present invention. As such,
other compounds such as those having -L-R.sup.4' groups other than
CH.sub.2R.sup.5 may similarly be made.
##STR00025##
[0134] Gycosyl benzo[cd]azulenes can be prepared by modification of
optionally protected and functionalized 7-deazapurine analogues
followed by Stine, Heck or other metal-mediated cross coupling
chemistry to introduce an .alpha.,.beta.-unsaturated ester or other
carbonyl group. Such process allows for stereoselective synthesis
of an intermediate capable of efficient cyclisation to the
inventive compound. Any compound capable of metal-mediated cross
coupling may be used, such as a tin derivative, such as
trialkyltin. More preferably tributyltin. Cyclisation and optional
deprotection of the product delivers the target nucleoside which
contains the benzo[cd]azulene, a key element of the invention.
[0135] In Scheme 1, preferably A is O, CH.sub.2 or optionally
protected N; Y is halogen; W.sup.1 is C (if p bond) or N (if no p
bond); W.sup.2 is C, CH or N; W.sup.4 is H or trialkyltin; Z.sup.1
and Z.sup.2 are each independently CH, C-halogen, C-alkyl, C-aryl,
C--O-alkyl or C--S-alkyl; Z.sup.3 is CH, C-alkyl, N, CNHR,
CNH.sub.2, CNR.sub.2, C.dbd.O, or C.dbd.S; Z.sup.4 is CH,
C-halogen, C-alkyl, C-aryl, C--O-alkyl, C--S-alkyl, C--OH,
C--NH.sub.2, C--NHR or C--NR.sub.2; R.sup.1, R.sup.2, R.sup.2',
R.sup.3, R.sup.3', R.sup.4 are each independently H, halogen,
alkyl, O-alkyl, OH, optionally protected O, methyl, H or F; R.sup.5
is an optionally protected OH or NH.sub.2.
##STR00026##
[0136] In Scheme 2, preferably A is O, CH.sub.2 or optionally
protected N; Y is halogen; W.sup.1 is C (if p bond) or N (if no p
bond); W.sup.2 is C, CH or N; W.sup.4 is H or trialkyltin; Z.sup.1
and Z.sup.2 are each independently CH, C-halogen, C-alkyl, C-aryl,
C--O-alkyl or C--S-alkyl; Z.sup.3 is CH, C-alkyl, C-halogen, N,
CNHR, CNH.sub.2, CNR.sub.2, C.dbd.O, or C.dbd.S; Z.sup.4 is CH,
C-halogen, C-alkyl, C-aryl, C--O-alkyl, C--S-alkyl, C--OH,
C--NH.sub.2, C--NHR or C--NR.sub.2; R.sup.1, R.sup.2, R.sup.2',
R.sup.3, R.sup.3', R.sup.4 are each independently H, halogen,
alkyl, O-alkyl, OH, optionally protected O, methyl, or F; R.sup.5
is an optionally protected OH or NH.sub.2.
##STR00027##
[0137] In Scheme 3, preferably A is O, CH.sub.2 or optionally
protected N; Y is halogen; X is optionally protected N, O or S;
W.sup.1 is C (if p bond) or N (if no p bond); W.sup.2 is C, CH or
N; W.sup.4 is H or trialkyltin; Z.sup.1 and Z.sup.2 are each
independently CH, C-halogen, C-alkyl, C-aryl, C--O-alkyl or
C--S-alkyl; Z.sup.3 is CH, C-alkyl, C-halogen, N, CNHR, CNH.sub.2,
CNR.sub.2, C.dbd.O, or C.dbd.S; Z.sup.4 is CH or C-halogen,
C-alkyl, C-aryl, C--O-alkyl, C--S-alkyl, C--NH.sub.2, C--NHR or
C--NR.sub.2; Z.sup.6 is CH.sub.2, O, NH or NR; R.sup.1, R.sup.2,
R.sup.2', R.sup.3, R.sup.3', R.sup.4 are each independently H,
halogen, alkyl, O-alkyl, OH, optionally protected O, methyl, or F;
R.sup.5 is an optionally protected OH or NH.sub.2; Nu is optionally
protected N, O, or S.
##STR00028##
[0138] In Scheme 4, preferably A is O, CH.sub.2 or optionally
protected N; Y is halogen; X is optionally protected N, O or S;
W.sup.1 is C (if p bond) or N (if no p bond); W.sup.2 is C, CH or
N; W.sup.4 is H or trialkyltin; Z.sup.3 is CH, C-alkyl, C-halogen,
N, CNHR, CNH.sub.2, CNR.sub.2, C.dbd.O, or C.dbd.S; Z.sup.4 is CH
or C-halogen, C-alkyl, C-aryl, C--O-alkyl, C--S-alkyl, C--NH.sub.2,
C--NHR or C--NR.sub.2; Z.sup.5, Z.sup.6, Z.sup.9 are each
independently CH.sub.2, O, NH or NR; R.sup.1, R.sup.2, R.sup.2',
R.sup.3, R.sup.3', R.sup.4 are each independently H, halogen,
alkyl, O-alkyl, OH, optionally protected O, methyl, or F; R.sup.5
is an optionally protected OH or NH.sub.2; Nu is a nucleophile such
as optionally protected N, O, or S; Lv is Leaving group.
[0139] Gycosyl benzo[cd]azulenes can alternatively be prepared by
glycosylation of intact benzo[cd]azulenes as shown in Scheme 2.
Conditions used for such glycosidations are well known to
practitioners of the art and can be found in the references cited
above.
##STR00029##
[0140] In Scheme 5, preferably A is O, CH.sub.2 or optionally
protected N; Z.sup.1 and Z.sup.2 are each independently CH,
C-halogen, C-alkyl, C-aryl, C--O-alkyl or C--S-alkyl; Z.sup.3 is
CH, C-alkyl, C-halogen; N, CNHR, CNH.sub.2, CNR.sub.2, C.dbd.O, or
C.dbd.S; Z.sup.4 is CH, C-halogen, C-alkyl, C-aryl, C--O-alkyl,
C--S-alkyl, C--OH, C--NH.sub.2, C--NHR or C--NR.sub.2; R.sup.2,
R.sup.2', R.sup.3, R.sup.3), R.sup.4 are each independently H,
halogen, alkyl, O-alkyl, OH, optionally protected O, methyl, or F;
R.sup.5 is an optionally protected OH or NH.sub.2; Lv is Leaving
group.
[0141] Certain gycosyl benzo[cd]azulene-7-ones can be modified by
well known functional group interconversions (FGIs). For example,
the 8,9-double bond can be manipulated by hydrogenation, addition
or addition-elimination processes. The 7-carbonyl function can also
be converted into a 7-thiocarbonyl or undergo O-alkylation.
##STR00030##
[0142] When equipped with an appropriate leaving group such as a
chlorine atom that the 4-position of the gycosyl benzo[cd]azulene
framework, a range of nucleophiles can engage in nucleophilic
substitutions at this position. Suitable nucleophiles include
alcohols, alkyl thiols and alkylamines.
[0143] In Scheme 6, preferably A is O, CH.sub.2 or optionally
protected N; Nu is nucleophile such as optionally protected N, O,
or S; W.sup.1 is C (if p bond) or N (if no p bond); W.sup.2 is C,
CH or N; Z.sup.1 and Z.sup.2 are each independently CH, C-halogen,
C-alkyl, C-aryl, C--O-alkyl, or C--S-alkyl; Z.sup.3 is CH, C-alkyl,
C-halogen, N, CNHR, CNH.sub.2, CNR.sub.2, C.dbd.O, or C.dbd.S;
Z.sup.4 is CH or C-halogen, C-alkyl, C-aryl, C--O-alkyl,
C--S-alkyl, C--OH, C--NH.sub.2, C--NHR or C--NR.sub.2; R.sup.1,
R.sup.2, R.sup.21, R.sup.3, R.sup.3', R.sup.4 are each
independently H, halogen, alkyl, O-alkyl, OH, optionally protected
O, methyl, or F; R.sup.5 is an optionally protected OH or NH.sub.2;
X is O or S; R.sup.6 is alkyl, aryl, alkenyl or alkynyl.
##STR00031##
[0144] When equipped with a 4-amino group the gycosyl
benzo[cd]azulene framework can be modified by interception of a
derived diazonium ion using standard techniques.
[0145] In Scheme 7, preferably A is O, CH.sub.2 or optionally
protected N; W.sup.1 is C (if .pi. bond) or N (if no .pi. bond);
W.sup.2 is C, CH, or N; Z.sup.1 and Z.sup.2 are each independently
CH, C-halogen, C-alkyl, C-aryl, C--O-alkyl or C--S-alkyl; Z.sup.3
is CH, C-alkyl, C-halogen, N, CNHR, CNH.sub.2, CNR.sub.2, C.dbd.O,
or C.dbd.S; Y is halogen; R.sup.1, R.sup.2, R.sup.2', R.sup.3,
R.sup.31, R.sup.4 are each independently H, halogen, alkyl,
O-alkyl, OH, optionally protected O, methyl, or F; R.sup.5 is OH,
NH.sub.2 or optional protecting group; R.sup.6, R.sup.7, and
R.sup.8 are each independently alkyl, aryl, alkenyl or alkynyl.
##STR00032##
[0146] In Scheme 8, preferably A is O, CH.sub.2 or optionally
protected N; W.sup.1 is C (if .pi. bond) or N (if no .pi. bond);
W.sup.2 is C, CH or N; Z.sup.1 and Z.sup.2 are each independently
CH, C-halogen, C-alkyl, C-aryl, C--O-alkyl or C--S-alkyl; Z.sup.3
is CH, C-alkyl, C-halogen, N, CNHR, CNH.sub.2, CNR.sub.2, C.dbd.O,
or C.dbd.S; Y is halogen; R.sup.1, R.sup.2, R.sup.2', R.sup.3,
R.sup.3, R.sup.4 are each independently H, halogen, alkyl, O-alkyl,
OH, optionally protected O, methyl, or F; R.sup.5 is optionally
protected OH or NH.sub.2.
[0147] The compounds described here can be converted into their
corresponding mono-, di- and triphosphates using well established
methods. Furthermore, prodrugs of mono-, di- and triphosphates can
be prepared in order to optimise the biological efficacy of these
phosphorylated compounds. Methods for preparing such prodrugs are
well known in the art (see Wagner, C. R., et al. Med. Res. Rev.,
2000, 20, 417-451).
##STR00033##
[0148] In Scheme 9, preferably A is O, CH.sub.2 or optionally
protected N; R.sup.1, R.sup.2, R.sup.2', R.sup.3, R.sup.3', R.sup.4
are each independently H, halogen, alkyl, O-alkyl, OH, optionally
protected O, methyl or F, Base is as described herein.
[0149] An alternative to the use of phosphates and prodrugs of
these is the use of phosphate mimics and their prodrugs (for
prodrugs, see Wagner, C. R., et al. Med. Res. Rev., 2000, 20,
417-451). One such phosphate mimic is shown below and this can be
prepared using appropriately protected nucleosides and known
conditions.
##STR00034##
[0150] Methods of preparing tri-, di, and mono-phosphate mimics
useful for making compounds of the invention is found in WO
2003/072757 and WO 2003/073989 filed Feb. 28, 2003. A
representative scheme is described above.
[0151] In Scheme 10, preferably A is O, CH.sub.2 or optionally
protected N; R.sup.1, R.sup.2, R.sup.2', R.sup.3, R.sup.3', R.sup.4
are each independently H, halogen, alkyl, O-alkyl, OH, optionally
protected O, methyl or F; X' is O, S, NH, CF.sub.2, CHF, CClH,
CBr.sub.2 or CHBr; Base is as described herein.
Biological Assays
[0152] Antiviral assays are conducted according to published,
widely used protocols. In order to obtain the therapeutic index,
compound-induced cytotoxicity to host cells is also measured in
parallel with antiviral activities. To determine the mode of action
of antiviral nucleosides the corresponding nucleoside triphosphates
are subject to enzyme-based assays for the inhibition of viral
polymerases according to known protocols (Ranjith-Kumar et al. J.
Virol. 2001, 75, 8615; Dhanak et al. J. Biol. Chem. 2002, 277,
38322-38327). Some compounds of the present invention showed
K.sub.i values of less than 1 .mu.M against HCV NS5B.
[0153] Since the replicon RNA replication mimics the replication of
HCV RNA in infected hepatocytes, compounds that have the inhibitory
effects in replicon assays are potentially useful as anti-HCV
drugs. The HCV replicon-containing cell lines (Randall and Rice,
Current Opinion in Infectious Diseases 2001, 14, 743) are used for
the identification of potential anti-HCV compounds. Among them is a
widely used subgenomic replicon system developed by Lohmann et al.
(Science 1999, 285, 110; J. General Virol. 2000, 81, 1631; J.
Virol. 2001, 75, 1437, 2002, 76, 4008). Some compounds of the
present invention showed potent anti-HCV activity with EC.sub.50
values of low .mu.M.
[0154] Widely used protocols developed by Korba et al. (Antiviral
Res. 1992, 19, 55), and Pai et al. (Antimicrobial Agents Chemother.
1996, 40, 380) are useful for the determination of in vitro
anti-HBV activity.
[0155] Anti-HIV assays can be conducted according to the protocols
developed by Schinazi et al. (Antimiromobial Agents Chemother.
1990, 34, 1061; 1992, 36, 2423; 1993, 37, 875) or other widely used
protocols (Kimpton et al. J. Virol. 1992, 66, 2232; Chan et al. J.
Med. Chem. 2001, 44, 1866).
Biological Applications and Administration
[0156] The nucleosides, nucleotide mimics and/or their prodrugs of
the present invention may be useful for the inhibition of a variety
of enzymes including, but not limited to, DNA or RNA polymerases,
helicases, ribonucleotide reductases, protein kinases, and
telomerases and for the modulation of G-proteins, P2 purinergic
receptors and the allosteric sites of a variety of enzymes.
[0157] The nucleosides, nucleotide mimics and/or their prodrugs of
the present invention are useful as human therapeutics for the
treatment of infectious diseases caused by viruses including, but
not limited to, HIV, HBV, HCV, HDV, HSV, HCMV, small pox, West Nile
virus, SARS virus, influenza viruses, measles, rhinovirus, RSV,
VZV, EBV, vaccinia virus, and papilloma virus.
[0158] The nucleosides, nucleotide mimics and/or their prodrugs of
the present invention are useful for the treatment of infectious
diseases caused by infectious agents such as parasites, bacteria
and fungi.
[0159] Those nucleosides, nucleotide mimics and/or their prodrugs
that have potent cytotoxicities to fast-dividing cancerous cells
are useful for the treatment of proliferative disorders, including,
but not limited to, lung cancer, liver cancer, prostate cancer,
colon cancer, breast cancer, ovarian cancer, melanoma, and
leukemia.
[0160] As the ligands of P2 receptors and O-proteins as well as the
inhibitors of protein kinases, the nucleosides, nucleotide mimics
and/or their prodrugs of the present invention are useful for the
treatment of a wide range of other diseases and disorders such as
inflammatory diseases, autoimmune diseases, Type 2 diabetes, and
cardiovascular diseases.
[0161] In order to overcome drug resistance, combination therapies
are widely used in the treatment of infectious diseases and
proliferative disorders. The nucleosides, nucleotide mimics and/or
their pro drugs of the present invention may be therapeutically
administered as a single drug, or alternatively may be administered
in combination with one or more other active chemical entities to
form a combination therapy. The other active chemical entities may
be a small molecule, a polypeptide, or a polynucleotide.
[0162] The pharmaceutical composition of the present invention
comprises at least one of the compounds represented by the formulas
herein or pharmaceutically acceptable salts, esters or prodrugs
thereof as active ingredients. The compositions include those
suitable for oral, topical, intravenous, subcutaneous, nasal,
ocular, pulmonary, and rectal administration. The compounds of the
invention can be administered to mammalian individuals, including
humans, as therapeutic agents.
[0163] Accordingly, the compounds of the invention are useful as
anti-microbial infection agents. The present invention provides a
method for the treatment of a patient afflicted with an infection
comprising administering to the patient a therapeutically effective
anti-microbial amount of a compound of the invention. The term
"microbe infection" as used herein refers to an abnormal state or
condition characterized by microbial transformation of cells,
microbial replication and/or microbial proliferation. Microbial
infections for which treatment with a compound of the invention
will be particularly useful include the microbes mentioned
above.
[0164] The term "treat" as in "to treat a disease" is intended to
include any means of treating a disease in a mammal, including (1)
preventing the disease, i.e., avoiding any clinical symptoms of the
disease, (2) inhibiting the disease, that is, arresting the
development or progression of clinical symptoms, and/or (3)
relieving the disease, i.e., causing regression of clinical
symptoms.
[0165] For example, the compounds of the invention are useful as
antiviral agents. The present invention provides a method for the
treatment of a patient afflicted with a viral infection comprising
administering to the patient a therapeutically effective antiviral
amount of a compound of the invention. The term "viral infection"
as used herein refers to an abnormal state or condition
characterized by viral transformation of cells, viral replication
and proliferation. Viral infections for which treatment with a
compound of the invention will be particularly useful include the
viruses mentioned above.
[0166] A "therapeutically effective amount" of a compound of the
invention refers to an amount which is effective, upon single or
multiple dose administration to the patient, in controlling the
growth of e.g., the microbe or tumor or in prolonging the
survivability of the patient beyond that expected in the absence of
such treatment. As used herein, "controlling the growth" refers to
slowing, interrupting, arresting or stopping the microbial or
proliferative transformation of cells or the replication and
proliferation of the microbe and does not necessarily indicate a
total elimination of e.g., the microbe or tumor.
[0167] Accordingly, the present invention includes pharmaceutical
compositions comprising, as an active ingredient, at least one of
the compounds of the invention in association with a pharmaceutical
carrier. The compounds of this invention can be administered by
oral, parenteral (intramuscular, intraperitoneal, intravenous (IV)
or subcutaneous injection), topical, transdermal (either passively
or using iontophoresis or electroporation), transmucosal (e.g.,
nasal, vaginal, rectal, or sublingual) or pulmonary (e.g., via dry
powder inhalation) routes of administration or using bioerodible
inserts and can be formulated in dosage forms appropriate for each
route of administration.
[0168] Solid dosage forms for brat administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is admixed with at least one inert
pharmaceutically acceptable carrier such as sucrose, lactose, or
starch. Such dosage forms can also comprise, as is normal practice,
additional substances other than inert diluents, e.g., lubricating,
agents such as magnesium stearate. In the case of capsules,
tablets, and pills, the dosage forms may also comprise buffering
agents. Tablets and pills can additionally be prepared with enteric
coatings.
[0169] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, with the elixirs containing inert diluents commonly used in
the art, such as water. Besides such inert diluents, compositions
can also include adjuvants, such as wetting agents, emulsifying and
suspending agents, and sweetening, flavoring, and perfuming
agents.
[0170] Preparations according to this invention for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or
vehicles are propylene glycol polyethylene glycol, vegetable oils,
such as olive oil and corn oil, gelatin, and injectable organic
esters such as ethyl oleate. Such dosage forms may also contain
adjuvants such as preserving, wetting, emulsifying, and dispersing
agents. They may be sterilized by, for example, filtration through
a bacteria retaining filter, by incorporating sterilizing agents
into the compositions, by irradiating the compositions, or by
heating the compositions. They can also be manufactured using
sterile water, or some other sterile injectable medium, immediately
before use.
[0171] Compositions for rectal or vaginal administration are
preferably suppositories which may contain, in addition to the
active substance, excipients such as cocoa butter or a suppository
wax. Compositions for nasal or sublingual administration are also
prepared with standard excipients well known in the art.
[0172] Topical formulations will generally comprise ointments,
creams, lotions, gels or solutions. Ointments will contain a
conventional ointment base selected from the four recognized
clasks: oleaginous bases; emulsifiable bases; emulsion bases; and
water-soluble bases. Lotions are preparations to be applied to the
skin or mucosal surface without friction, and are typically liquid
or semiliquid preparations in which solid particles, including the
active agent, are present in a water or alcohol base. Lotions are
usually suspensions of solids, and preferably, for the present
purpose, comprise a liquid oily emulsion of the oil-in-water type.
Creams, as known in the art, are viscous liquid or semisolid
emulsions, either oil-in-water or water-in-oil. Topical
formulations may also be in the form of a gel, i.e., a semisolid,
suspension-type system, or in the form of a solution.
[0173] Finally, formulations of these drugs in dry powder form for
delivery by a dry powder inhaler offer yet another means of
administration. This overcomes many of the disadvantages of the
oral and intravenous routes.
[0174] The dosage of active ingredient in the compositions of this
invention may be varied; however, it is necessary that the amount
of the active ingredient shall be such that a suitable dosage form
is obtained. The selected dosage depends upon the desired
therapeutic effect, on the route of administration, and on the
duration of the treatment desired. Generally, dosage levels of
between 0.001 to 10 mg/kg of body weight daily are administered to
mammals.
[0175] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to prepare and use the compounds disclosed and
claimed herein.
[0176] In order to overcome drug resistance, combination therapies
are widely used in the treatment of viral infections. The
nucleoside analogs, corresponding 5'-monophosphate, 5'-diphosphate,
5'-triphosphate, and prodrugs thereof of the present invention may
be therapeutically administered as a single drug, or alternatively
they may be administered in combination with one or more other
active chemical entities to form a combination therapy. The other
active chemical entities may be a small molecule, a polypeptide, or
a polynucleotide.
[0177] All references mentioned herein are incorporated herein by
reference in their entirety.
[0178] The following Examples are offered to illustrate but not to
limit the invention.
EXAMPLES
Example 1
2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-1,2,3,5,6-pentaazaben-
zo[cd]azulene-7-one (1.7)
##STR00035##
[0180] For the preparation of tricyclic nucleoside 1.7,
4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidine 1.2 was prepared by the
iodination of 4-amino-1H-pyrazolo[3,4-d]pyrimidine 1.1 using
N-iodosuccinimide in DMF at 80.degree. C. The glycosylation of 1.2
with commercially available 2-C-methyl-tetrabenzoyl ribofuranose
1.3 in boiling nitromethane in the presence of boron trifluoride
etherate gave 2'C-methyl nucleoside 1.4 in 65% yield after
purification. Removal of ester blocking groups with ammonia in MeOH
provided the free nucleoside 13 in 83% isolated yield. The use of
methyl acrylate in the Pd[0] catalyzed cross coupling reaction of
1.5 afforded the 7-alkenyl nucleoside 1.6. The treatment of
compound 1.6 with NaOMe/MeOH resulted in an intramolecular
cyclization, yielding the target tricyclic nucleoside 1.7.
Example 1
Step-A: 4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidine (1.2)
[0181] 4-Amino-1H-pyrazolo[3,4-d]pyrimidine was prepared according
to the published method (J. Med. Chem. 1993, 36, 3424-3430).
Example 1
Step-B:
4-Amino-3-iodo-1-(2-C-methyl-2,3,5-tri-O-benzoyl-.beta.-D-ribofura-
nosyl)pyrazolo[3,4-d]-pyrimidine (1.4)
[0182] 1-O-Acetyl-2-C-methyl-2,3,5-tri-O-benzoyl-D-ribofuranose
(1.0 g, 1.72 mmol) was dissolved in anhydrous nitromethane (10.0
mL) and to this solution compound 1.2 (312 mg, 1.21 mmol) was
added. The resulting suspension was brought to reflux and
borontrifluoride etherate (0.23 mL, 1.78 mmol) was added. The
suspension became a clear solution, which was heated at reflux for
2 hr. The mixture was cooled, the solvents were evaporated and the
off-white foamy residue was dissolved in ethyl acetate and then
poured with stirring into aq. sat. NaHCO.sub.3. The aqueous layer
was extracted twice with ethyl acetate and the combined organic
layers were dried (Na.sub.2SO.sub.4), filtered and evaporated to
give an off-white solid. This material was purified by flash column
chromatography on silica gel using CH.sub.2Cl.sub.2 to 2-3% MeOH in
dichoromethane as eluent to give the desired compound 1.4 (811 mg)
as a yellow foam.
Example 1
Step-C:
4-Amino-3-iodo-1-(2-C-methyl-.beta.-D-ribofuranosyl)-1H-pyrazolo[3-
,4-d]pyrimidine (1.5)
[0183] A solution of compound 1.4 (540 mg, 0.75 mmol) in MeOHic
NH.sub.3 (120 mL, saturated at 0.degree. C.) was stirred in a bomb
at 45.degree. C. for 16 hr. The mixture was evaporated to dryness
and then the residue co-evaporated with additional MeOH.
Purification by silica gel column chromatography using 6-14% MeOH
in dichloromethane as eluent gave the desired compound 1.5 (244 mg)
as an off-white solid.
Example 1
Step-D:
4-Amino-1-(2-C-methyl-.beta.-D-ribofuranosyl)-3-[2-methoxycarbonyl-
)ethenyl]-1H-pyrazolo[3,4-d]pyrimidine (1.6)
[0184] To a solution of compound 1.5 from Step-C (392 mg, 0.54
mmol) in DYE (10 mL) was added CuI (44 mg, 0.23 mmol), methyl
acrylate (2.0 mL, 23.23 mmol), triethylamine (0.332 mL, 2.36 mmol)
and tetrakis(triphenylphosphine)palladium[0] (133 mg, 0.12 mmol),
heated at 70.degree. C. under Ar. The reaction mixture was heated
at 70.degree. C. for 36 hr. After this time, further CuI (44 mg,
0.23 mmol), methyl acrylate (2.0 mL, 23.23 mmol), triethylamine
(0.332 mL, 2.36 mmol) and tetrakis(triphenylphosphine)palladium[0]
(133 mg, 0.12 mmol) were added and the mixture was heated at
70.degree. C. for a further 6 hr. Then the reaction mixture was
cooled to room temperature and 8 mL of 1/1 MeOH/CH.sub.2Cl.sub.2
was added. 100 mg of Dowex 1.times.2-100 Bicarb form was added, the
suspension stirred at room temperature for 45 min then filtered.
The resin was washed with 5.times.10 mL MeOH/CH.sub.2Cl.sub.2:1/1,
the solvents were evaporated and the residual DMF was finally
evaporated by azeotropic evaporation with toluene (2.times.10 mL).
The residue was redissolved in MeOH and pre-adsorbed on silica gel.
Chromatographic purification on silica gel using 4-4.5% MeOH in
CH.sub.2Cl.sub.2 as eluent gave desired ester 1.6 (216 mg, yield
52%).
Example 1
Step-E:
2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-1,2,3,5,6-pen-
taazabenzo[cd]azulene-7-one (1.7)
[0185] A solution of ester 1.6 (208.3 mg, 0.57 mmol) in 0.1M
NaOCH.sub.3 in MeOH was heated at reflux for 3 h, cooled to
0.degree. C. and Dowex 50.times.100 (acidic form) was added until
the solution pH became neutral. The reaction mixture was filtered
and the resin was washed with MeOH. The solvent was evaporated, and
the residue was purified using silica gel column chromatography
using 4 to 4.5% MeOH in CH.sub.2Cl.sub.2 as eluent to give the
desired compound 1.7 (26.5 mg) as an off-white solid.
[0186] .sup.1H NMR (DMSO-d.sub.6) .delta. 11.6 (s, NH, 1H), 8.56
(s, H-4, 1H), 7.31 (d, J 12 Hz, CH, 1H), 6.28 (d, J 12 Hz, CH, 1H),
6.15 (s, H-1', 1H), 5.28 (s, 2'-OH, 1H), 5.14 (d, J 6.9 Hz, 3'-OH,
1H), 4.65 (t, J 5.7 Hz, 5'-OH, 1H), 3.95-4.09 (m, H-3', H-4', 2H),
3.62-3.69 (m, H-5', 2H), 0.83 (s, CH.sub.3, 3H). MS m/z 332
(M-H).sup.+.
Example 2
2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6,8,9-tetrahydro-7-oxa-2,3,5,6-tet-
raazabenzo[cd]azulene (2.9)
##STR00036##
[0188] Bromination (NBS/THF) of starting pyrrolo pyrimidine 2.1
afforded bromopyrrolo pyrimidine 2.2 in 63% yield. Treatment of 2.2
with n-butyllithium in THF at -78.degree. C. resulted in a
selective lithio-bromo exchange to yield an intermediate which on
treatment with (2-bromoethoxy) tert-butyldimethylsilane
(-30.degree. C./5h) delivered pyrimidine 2.3 in 43% yield along
with recovered starting pyrimidine 2.2 (30%). When the reaction was
repeated on large scale (7.0 g of 2.2), significant improvement was
achieved (-20.degree. C./16 h) and pyrimidine 2.3 was isolated in
55% yield (5.5 g). Stereoselective glycosylation of 2.3 with bromo
sugar 2.4 (freshly prepared form commercially available
3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-1-O-methyl-.alpha.-D-ribo-
furanose using HBr in acetic acid/CH.sub.2Cl.sub.2) with 85%
KOH/TDA-[(tris[2-(2-methoxyethoxy)ethyl]amine afforded nucleoside
2.5 in 26% isolated yield. Removal of the TBDMS group of 2.5 with
tetrabutylammonium fluoride/THF gave diol 2.6. Compound 2.6
underwent Mitsunobu coupling with N-hydroxyphthalimide to give the
corresponding phthalimidooxyethyl nucleoside 2.7 in 88% yield. 2.7
was treated with anhydrous H.sub.2NNH.sub.2 to remove the phthaloyl
group and this free aminooxy intermediate (crude) 2.8 was heated in
EtOH to give 2.8. Removal of the dichlorobenzyl groups of 2.8 by
using BCl.sub.3 in CH.sub.2Cl.sub.2 delivered tricyclic nucleoside
2.9.
Example 2
Step-A: 5-Bromo-4-chloropyrrolo[2,3-d]pyrimidine (2.2)
[0189] To solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine was
prepared according to a literature procedure (Townsend, L. B. et
al., J. Med. Chem. 1988, 31, 2086-2092).
Example 2
Step-B:
5-[2-(tert-Butyldimethylsiloxy)ethyl]-4-chloro-7H-pyrrolo[2,3-d]py-
rimidine (2.3)
[0190] A suspension of compound 2.2 from Step A (2.0 g, 8.6 mmol)
in THF (40.0 mL) was cooled to -78.degree. C. under an argon
atmosphere. n-BuLi (1.6M in hexanes, 13.4 mmol) was then added over
1.0 hr via syringe. A suspension formed and
(2-bromoethoxy)-tert-butyldimethylsilane (7.4 mL, 34.4 mmol) was
added via syringe while maintaining the reaction mixture at
-78.degree. C. The reaction mixture was allowed to slowly reach
-30.degree. C. and stirred for 2 h, then -30 to -10.degree. C. for
1 h, and -10 to 0.degree. C. for 1 hr. The reaction mixture became
dark brown in color, was treated with NH.sub.4Cl, CH.sub.2Cl.sub.2
and water. The reaction mixture was extracted with CH.sub.2Cl.sub.2
and the extracts were dried over Na.sub.2SO.sub.4, filtered and
evaporated. The beige residue was purified by flash column
chromatography using 23% EtOAc in hexanes as eluent to give the
desired compound 2.3 (1.16 g, 43%) as a white solid. (Brown, D. M.
et al., J. Chem. Soc. PT-1, 3565-3570.)
Example 2
Step-C: 5-[2-tert-Butyldimeythylsilocy)ethyl]-4-chloro-7-[2-C-meth
1-3,5-bis-O-(2,4-dichlorophenylmethyl)-.beta.-D-ribofuranosyl]-7H-pyrrolo-
[2,3-d]pyrimidine (2.5)
[0191] Compound 2.3 (2.5 g, 8.00 mmol) was suspended in CH.sub.3CN
(50 mL), and powdered 85% KOH, (1.3 g, 19.73 mmol) followed by
TDA-1 (tris[2-(2-methoxyethoxy)ethyl]amine) (0.2 mL, 0.62 mmol)
were added. After stirring at room temperature for 10 min, a
freshly prepared solution of bromo sugar 2.4 (prepared from
commercially available
3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-1-O-methyl-.alpha.-D-ribo-
furanose; (i) Helv. Chim. Acta, 1995, 78, 486; (ii) WO 02/057287,
2002) (10.1 mmol) in anhydrous acetonitrile (50 mL) was added via
cannula, and the reaction stirred for 24 hr at room temperature
then cooled in an ice/water bath and treated with CH.sub.2Cl.sub.2
(100 mL) and water (80 mL). The aqueous material was extracted
three times with CH.sub.2Cl.sub.2, the combined organic layers were
dried over Na.sub.2SO.sub.4, filtered and evaporated. The crude
product was purified on a silica gel column using 20% ethyl
acetate/hexanes as eluent to give the desired nucleoside 2.5 (1.03
g, 13%). Further elution with 25% EtOAc/hexanes as eluent gave a
mixture of the desired nucleoside 2.5 and starting base 2.3 (350
mg).
Example 2
Step-D:
4-Chloro-7-[2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-.beta.-
-D-ribofuranosyl]-5-(2-hydroxyethyl)-7H-pyrrolo[2,3-d]pyrimidine
(2.6)
[0192] To a solution of compound 2.5 (1.14 g, 1.471=01) in THF (30
mL) was added a 1.0M solution of tetrabutylammonium fluoride in THF
(2.2 mmol) at room temperature. The colorless solution was stirred
for 5 hr at room temperature and then diluted by addition of 10 mL
of CH.sub.2Cl.sub.2 and 10 mL of brine. The aqueous layer was
extracted three times with CH.sub.2Cl.sub.2, dried over
Na.sub.2SO.sub.4, filtered and evaporated. The residue obtained was
purified by silica gel column chromatography using 1-1.5% MeOH in
CH.sub.2Cl.sub.2 as eluent to give the desired compound 2.6 (900
mg, 88%) as a white solid.
Example 2
Step-E:
4-Chloro-7-[2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-.beta.-
-D-ribofuranosyl]-5-(2-phthalimidooxyethyl)-7H-pyrrolo[2,3-d]pyrimidine
(2.7)
[0193] To a solution of the compound 2.6 (359.0 mg, 0.54 mmol) in
THF (10 mL) were added triphenylphosphine (215.0 mg, 0.82 mmol) and
N-hydroxyphthalimide (132.0 mg, 0.0.81 mmol) followed by
diethylazodicarboxylate (DEAD) (153 .mu.L, 0.88 mmol), and the
solution was stirred overnight at room temperature. The reaction
mixture was diluted by adding 10 mL of CH.sub.2Cl.sub.2 and 10 mL
of water. The aqueous layer was extracted three times with
CH.sub.2Cl.sub.2, dried over Na.sub.2SO.sub.4, filtered and
evaporated. The product was purified by flash column chromatography
using 15-20% EtOAc in hexanes as eluent gave the desired compound
2.7 (369 mg, 85%) as a white solid.
Example 2
Step-F:
2-(2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-.beta.-D-ribofu-
ranosyl)-2,6,8,9-tetrahydro-7-oxa-2,3,5,6-tetranzabenzo[cd]azulene
(2.8)
[0194] To a suspension of compound 2.7 (880.0 mg, 1.09 mmol) in
acetonitrile (60 mL) was added anhydrous hydrazine (38 .mu.l, 1.19
mmol), and the solution was stirred for 2 hr at room temperature.
No starting material left as judged by the tlc. The white
precipitate (phthalic hydrazide) was filtered off and washed with
anhydrous acetonitrile, and then solution was evaporated to
dryness. The crude reaction product was dried under high vacuum to
give 864 mg of a white solid. The resulting free aminooxy
intermediate was redissolved in anhydrous EtOH (50 mL) and the
solution was heated at reflux for 2 d. After evaporation, reaction
mixture was purified by silicagel column chromatography using
CH.sub.2Cl.sub.2 to 2% MeOH in CH.sub.2Cl.sub.2 to give desired
compound 2.8 (466 mg) as a white foam.
Example 2
Step-G:
2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6,8,9-tetrahydro-7-oxa-2,3-
,5,6-tetraazabenzo[cd]azulene (2.9)
[0195] To a solution of compound 2.8 (128.0 mg, 0.20 mmol) in
CH.sub.2Cl.sub.2 (25 mL) at -78.degree. C., was added a 1.0M
solution of BCl.sub.3 in CH.sub.2Cl.sub.2 (2.0 mL, 2.0 mmol)
dropwise via syringe. The mixture was stirred at -78.degree. C. for
1.5 h, then at -35.degree. C. to -40.degree. C. for 2.5 hr. The
reaction was quenched with MeOH (8.0 ml) and the solvents were
evaporated. The resulting crude product was purified by flash
column chromatography over silica gel using 5% MeOH in
CH.sub.2Cl.sub.2 as eluent to give the title compound 2.9 (59.2 mg)
as a white foam.
[0196] .sup.1H NMR (DMSO-d.sub.6) .delta. 10.62 (s, NH, 1H), 8.19
(s, H-2, 1H), 7.47 (s, H-6, 1H), 6.15 (s, H-1', 1H), 5.09 (br s,
2'-OH, 3'-OH, 5'-OH, 3H), 4.29 (br s, OCH.sub.2CH.sub.2, 2H),
3.63-3.96 (m, H-3', H-4', H5', 4H), 2.91-2.96 (m,
OCH.sub.2CH.sub.2, 2H), 0.70 (s, CH.sub.3, 3H). MS m/z 381
(M+CH.sub.3COO).sup.-.
Example 3
2-(.beta.-D-ribofuranosyl)-2,6-dihydro-7H-1,2,3,5,6-pentaazabenzo[cd]azule-
ne-7-one (3.3)
##STR00037##
[0197] Example 3
Step-A:
4-Amino-1-(.beta.-D-ribofuranosyl)-3-[2-methoxycarbonyl)ethenyl]-1-
H-pyrazolo[3,4-d]pyrimidine (3.2)
[0198] To a solution of compound 3.1* (300 mg, 0.76 mmol) in DMF
(10 mL) was added CuI (29 mg, 0.15 mmol), methylacrylate (1.3 mL,
15.1 mmol), triethylamine (1.3 mL, 3.09 mmol) and
tetrakis(triphenylphosphine)palladium[0] (88 mg, 0.08 mmol) and the
mixture heated at 70.degree. C. for 36 hr under argon. After this
time, additional CuI, methylacrylate, triethylamine and Pd catalyst
were added, and the dark brown reaction mixture was heated for a
further 6 hr. Then the reaction mixture was cooled to room
temperature and 8 mL of 1/1 MeOH/CH.sub.2Cl.sub.2 was added. 100 mg
Dowex 1.times.2-100 Bicarb form, was added and the mixture stirred
for 45 min at room temperature then filtered. The resin was washed
with 2.times.10 mL MeOH/CH.sub.2Cl.sub.2:1/1 and the solvents
evaporated. Chromatographic purification on silica gel using 6-7%
MeOH in CH.sub.2Cl.sub.2 gave the desired compound 3.2 (120 mg) as
a light yellow solid.
[0199] *J. Med. Chem. 1993, 36, 3424-3430.
Example 3
Step-B:
2-(.beta.-D-ribofuranosyl)-2,6-dihydro-7H-1,2,3,5,6-pentaazabenzo[-
cd]azulene-7-one (3.3)
[0200] A solution of compound 3.2 (110 mg, 0.31 mmol) in 0.1M
NaOCH.sub.3 in MeOH was heated at reflux for 3 h, cooled to
0.degree. C. and treated with Dowex 50.times.100 (acidic form)
until the pH of the mixture became neutral. The reaction contents
were filtered and the resin was washed with MeOH/CH.sub.2Cl.sub.2
(1:1). The solvents were evaporated and the residue obtained was
purified by flash column chromatography using 4-4.5% MeOH in
CH.sub.2Cl.sub.2 as eluent to give the title compound 3.3 (10.7 mg)
as a white solid. .sup.1H NMR (DMSO-d.sub.6) .delta. 11.6 (s, NH,
1H), 8.58 (s, H-4, 1H), 7.35 (d, J 12 Hz, CH, 1H), 6.31 (d, J 12
Hz, CH, 1H), 6.11 (d, J 4.5 Hz, H-1', 1H), 5.46 (d, J 5.7 Hz,
2'-OH, 1H), 5.21 (d, J 3.9 Hz, 3'-OH, 1H), 4.80 (t, J 5.1 Hz,
5'-OH, 1H), 4.62-4.64 (m, H-2', 1H), 3.92-3.94 (m, H-3', 1H),
3.44-3.61 (m, H5', 2H), 0.83 (s, CH.sub.3, 3H).
Example 4
2-(2-C-methyl-.beta.-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraa-
zabenzo[cd]azulene (4.6)
##STR00038##
[0202] Treatment of 2.2 with n-butyllithium in THF at -78.degree.
C. resulted in a selective halogen-metal exchange to yield an
intermediate which on treatment with (3-bromopropoxy)
tert-butyldimethylsilane (-30.degree. C./5 h) afforded pyrimidine
4.1 in 36% yield along with the recovery of starting pyrimidine
(30%). When the reaction was repeated on large scale (starting 7.0
g. of 2.2), significant improvement was achieved (-20.degree. C./16
h) and pyrimidine 4.1 was isolated in 55% yield. Stereoselective
glycosylation of 4.1 with bromo sugar 2.4 in the presence of
KOH/TDA-1 afforded nucleoside 4.2 in 17% isolated yield. Removal of
the TBDMS protecting group of 4.2 allowed Mitsunobu coupling with
phthalimide to give the corresponding phthalimido derivative 4.4 in
quantitative yield. Phthalimide cleavage using hydrazine in
different solvents failed to produce primary amine and it was found
that an alternative reaction condition using
ethylenediamine/ethanol resulted in phthalimide cleavage followed
by in situ cyclization of the free amino intermediate to give
protected tricyclic 4.5 in 56% yield. Deprotection of the
dichlorobenzyl groups of 4.5 using BCl.sub.3 in
CH.sub.2Cl.sub.2/-78.degree. C. to -30.degree. C. delivered the
target tricyclic nucleoside 4.6 in 84% isolated yield after
purification by silica gel column chromatography.
Example 4
Step-A:
5-[2-(tert-Butyldimethylsiloxy)propyl]-4-chloro-7H-pyrrolo[2,3-d]p-
yrimidine (4.1)
[0203] A suspension of compound 2.2 (10.0 g, 43.0 mmol) in THF
(140.0 mL) was cooled to -78 0.degree. C. under an argon atmosphere
and n-BuLi (1.6M in hexanes, 67.3 mmol) was then added via syringe
over 1.5 hr. The resulting suspension was stirred for the next 30
min at -78.degree. C. then
(3-bromopropoxy)-tert-butyldimethylsilane (21.4 mL, 172.0 mmol) was
slowly added via syringe at -78.degree. C. over 1 hr. The reaction
mixture was allowed to slowly reach -30.degree. C. and stirred for
the next 2 h, and -30 to 10.degree. C. for 1 h, and -10 to
0.degree. C. for 1 hr. The reaction mixture became dark brown in
color, and was kept at 4.degree. C. overnight. The reaction was
quenched by adding aqueous NH.sub.4Cl (100 mL), and diluted with
CH.sub.2Cl.sub.2 (600 mL) and water (120 mL) then extracted with
CH.sub.2Cl.sub.2. The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered and evaporated. The beige colored
residue was purified by flash column chromatography using 25% EtOAc
in hexanes as eluent to give the desired compound 4.1 (5.5 g) as a
white solid.
Example 4
Step-B:
5-[2-(tert-Butyldimethylsiloxy)propyl]-4-chloro-7-(2-C-methyl-3,5--
bis-O-(2,4-dichlorophenylmethyl)-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (4.2)
[0204] Compound 4.1 (5.24 g, 16.0 mmol) was suspended in CH.sub.3CN
(120 mL) and powdered 85% KOH ((2.63 g, 40.0 mmol) was added
followed by TDA-1 (tris[2-(2-methoxyethoxy)ethyl]amine (0.4 mL,
1.24 mmol). After stirring at room temperature for 30 min, a
freshly prepared solution of bromo sugar 2.4 (20.0 mmol) in
anhydrous acetonitrile (120 mL) was added via cannula, and the
mixture stirred for 2 days at room temperature, cooled in an ice
bath and treated with CH.sub.2Cl.sub.2 (200 mL) and water (100 mL).
The aqueous material was extracted three times with
CH.sub.2Cl.sub.2 and the combined organic extracts were dried over
Na.sub.2SO.sub.4, filtered and evaporated. The crude product was
purified on a silica gel column using 15% ethyl acetate/hexanes as
eluent to give desired compound 4.2 (2.6 g) as a light yellow
solid.
Example 4
Step-C:
4-Chloro-7-[2-C-methyl-3,5-bis-O-2,4-dichlorophenylmethyl-.beta.-D-
-ribofuranosyl]-5-(2-hydroxypropyl)-7H-pyrrolo[2,3-d]pyrimidine
(4.3)
[0205] To a solution of compound 4.2 (800 mg, 1.01 mmol) in THF (20
mL) was added a 1.0 M solution of tetrabutylammonium fluoride in
THF (1.5 mmol) at room temperature. The colorless solution was
stirred for 4 hr at room temperature and then diluted by addition
of 150 mL of CH.sub.2Cl.sub.2 and water (50 mL). The aqueous layers
were extracted three times with CH.sub.2Cl.sub.2, and then dried
over Na.sub.2SO.sub.4, filtered and evaporated. Purification by
silica gel column chromatography using 1-2% MeOH in
CH.sub.2Cl.sub.2 as eluent gave the desired compound 4.3 (460 mg,
67%) as a white solid.
Example 4
Step-D:
4-Chloro-7-[2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-.beta.-
-D-ribofuranosyl]-5-(2-phthalimidoproyl)-7H-pyrrolo[2,3-d]pyrimidine
(4.41
[0206] To a solution of compound 4.3 (1.28 g, 1.89 mmol) in THF (70
mL) were added triphenylphosphine (648 mg, 2.47 mmol) and
phthalimide (364.0 mg, 2.47 mmol) followed by DEAD (440 .mu.L, 2.53
mmol), and the solution was stirred for overnight at room
temperature. The reaction mixture was diluted by adding 100 mL of
CH.sub.2Cl.sub.2 and water (100 mL) and the aqueous layers
extracted three times with CH.sub.2Cl.sub.2, dried over
Na.sub.2SO.sub.4, filtered and evaporated. The residue obtained was
purified by flash column chromatography using 20-30% EtOAc in
hexanes as eluent to give the desired compound 4.4 (1.5 g, 100%) as
a white solid.
Example 4
Step-E:
2-[2-C-methyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-.beta.-D-ribofu-
ranosyl]-6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulene
(4.5)
[0207] To a solution of compound 4.4 (161 mg, 0.2 mmol) in absolute
EtOH (15 mL) was added ethylenediamine (24 .mu.L, 0.4 mmol) and the
mixture was stirred at 50.degree. C. for 2 days. The solvents were
evaporated and the residue was purified by column chromatography
using 2-3% MeOH in CH.sub.2Cl.sub.2 as eluent to give the desired
compound 4.5 (70.7 mg, 55%) as an off-white foam.
Example 4
Step-F:
2-(2-C-methyl-.beta.-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,5,-
6-tetraazabenzo[cd]azulene (4.6)
[0208] To a solution of compound 4.5 (68.0 mg, 0.1 mmol) in
CH.sub.2Cl.sub.2 (10 mL) at -78.degree. C. was added 1.0 M solution
of BCl.sub.3 in CH.sub.2Cl.sub.2 (1.07 mmol) dropwise via syringe.
The mixture was stirred at -78.degree. C. for 1.5 h, then for 3 hr
at -35.degree. C. to -40.degree. C. The reaction was quenched by
adding MeOH (6.0 mL), the solvents were evaporated and the
resulting residue was purified by flash column chromatography over
silica gel using 6-7% MeOH in CH.sub.2Cl.sub.2 as eluent to give
title compound 4.6 (31.8 mg) as a white foam.
[0209] .sup.1H NMR (DMSO-d.sub.6) .delta. 7.99 (s, 1H), 7.5 (s, NH,
1H), 7.23 (s, H-7, 1H), 6.12 (s, H-1', 1H), 3.82-3.94 (m, H-3',
H-4', H-5', 4H), 2.78 (m, NCH.sub.2CH.sub.2CH.sub.2, 2H), 1.9 (m,
NCH.sub.2CH.sub.2CH.sub.2, 4H), 0.70 (s, CH.sub.3, 3H). MS m/z 379
(M+CH.sub.3COO).sup.-
Example 5
2-(2-O-Methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo-
[cd]azulen-7-one (5.9)
##STR00039##
[0211] Selective 2'-O-methylation of tricyclic nucleosides was
sought by introduction of selective and simultaneous protection of
3' and S'-hydroxyl groups in 5.1 through a
3',5'-O-tetraisopropyldisiloxane bridge, followed by methylation
and removal of 3',5'-OH protection to afford 5.4.
[0212] Reaction of commercially available 6-chlorotubercidine 5.1
with TIPDSCl.sub.2/imidazole in DMF/room temperature/overnight gave
5',3'-0 TIPDS protected compound 5.2 in 75% yield. Methylation of
5.2 in DMF using NaH/MeI/4 h/0.degree. C. gave 2'OCH.sub.3 compound
5.3 in 35% yield (2 steps). It was found that by decreasing the
reaction time from 4 hr to 1 h, compound 5.3 could be isolated in
68% yield. Removal of the TIPDS of 5.3 was accomplished by using 4
equivalent of 1.0M tetrabutylammonium fluoride in THF at 0.degree.
C./1 hr to give 5.4. Intermediate 5.4 was acetylated using
Ac.sub.2O/pyridine to give 5.5 in quantitative yield. Iodination of
5.5 using ICl in CH.sub.2Cl.sub.2 gave the iodo compound 5.6 in 66%
yield and subsequent amination of 5.6 with methanolic ammonia
120.degree. C./16 h provided 5.7 in 89% isolated yield. Stifle
coupling of 5.7 using (Z)-methyl-3-(tributylstannyl)acrylate
provided 5.8 (Z isomer) in 27% yield and the Stille coupling
product 5.8, when subjected to cyclization using DBU/dioxane
afforded target tricyclic nucleoside 5.9 in 48% yield.
Example 5
Step-A:
4-Chloro-7-[3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-.beta.-D-rib-
ofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine (5.2)
[0213] To a solution of commercially available 6-chlorotubercidine
5.1 (3.0 g, 10.5 mmol) in DMF (140 mL) was added imidazole (3.6 g,
52.9 mmol), and TIPDSiCl.sub.2 (1.2 eq) (4.0 mL, 12.5 mmol) at room
temperature under argon. The reaction mixture was stirred for 16 h
at room temperature and then quenched by adding 20 mL of EtOH. The
solvents were evaporated, water (100 mL) was added and the white
suspension was extracted, with CH.sub.2Cl.sub.2. The organic
extracts were dried over Na.sub.2SO.sub.4, filtered, evaporated and
the residue purified by flash column chromatography using gradient
5-7% EtOAc in hexanes to give the desired compound 5.2 (4.14 g,
75%) as a glassy solid.
Example 5
Step-B:
4-Chloro-7-[2-O-methyl-3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-.-
beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine (5.3)
[0214] To a solution of compound 5.2 (2.0 g, 3.79 mmol) in DMF
(45.0 mL) at 0.degree. C., was added methyl iodide (1.88 mL, 15.23
mmol) followed by NaH (in one portion) (228 mg, 5.7 mmol, 60%
suspension). The resulting reaction mixture was stirred for 1 hr at
0.degree. C. and then quenched with anhydrous ethanol (20 mL) and
diluted with 100 mL of CH.sub.2Cl.sub.2. The diluted reaction
mixture was washed with water and the organic phase was dried over
Na.sub.2SO.sub.4, filtered, evaporated and coevaporated three times
with toluene. The residue obtained was purified by flash column
chromatography using a gradient of 5-7% EtOAc in hexanes and
afforded the desired 2'-O-methyl nucleoside 5.3 (1.4 g, 68%)
Example 5
Step-C:
4-Chloro-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidine (5.4)
[0215] To a solution of compound 5.3 (3.62 g, 6.66 mmol) in THF
(100 mL), was added a 1.0 M solution of tetrabutylammonium fluoride
in THF (26.0 mmol) at room temperature. The colorless solution was
stirred for 1 hr at room temperature and then diluted by adding 150
mL of CH.sub.2Cl.sub.2 and water 50 mL. The aqueous portion was
extracted three times with CH.sub.2Cl.sub.2, dried over
Na.sub.2SO.sub.4, filtered and evaporated. Purification by silica
gel column chromatography using pure CH.sub.2Cl.sub.2 to 2% MeOH in
CH.sub.2Cl.sub.2 as eluent gave the desired compound 5.4 (1.24 g,
59%) as a colorless oil.
Example 5
Step-D:
4-Chloro-7-(2-O-methyl-3,5-di-O-acetyl-.beta.-D-ribofuranosyl)-7H--
pyrrolo[2,3-d]pyrimidine (5.5)
[0216] To a solution of compound 5.4 (2.44 mmol) pyridine (40 mL),
was added acetic anhydride (1.23 mL, 8.44 mmol) via syringe and the
solution stirred overnight at room temperature. The solvents were
evaporated in vacuo, the residue was dissolved in CH.sub.2Cl.sub.2
and the solution was washed with water then dried over
Na.sub.2SO.sub.4, filtered, evaporated and co-evaporated with
toluene three times. The residue obtained was purified by flash
column chromatography using a gradient of pure CH.sub.2Cl.sub.2 to
2.5% MeOH in CH.sub.2Cl.sub.2 to give the desired compound 5.5
(1.26 g) as a colorless oil.
Example 5
Step-E:
4-Chloro-5-iodo-7-(2-O-methyl-3,5-di-O-acetyl-.beta.-D-ribofuranos-
yl)-7H-pyrrolo[2,3-d]pyrimidine (5.6)
[0217] To a solution of compound 5.5 (1.25 g, 3.26 mmol) in
CH.sub.2Cl.sub.2 (80 mL), a 1.0 M solution of ICl in
CH.sub.2Cl.sub.2 (8.12 mmol) was added at room temperature and the
resulting dark brown solution was stirred for 4 hr at room
temperature. The solvents were evaporated in vacuo at 25.degree.
C.-35.degree. C. and the residue was dried under high vacuum for 30
min. A light brown sticky material was obtained which was purified
by flash column chromatography using gradient of pure
CH.sub.2Cl.sub.2 to 1.5% MeOH in CH.sub.2Cl.sub.2 to afford the
desired compound 5.6 (1.1 g, 66%) as a white solid.
Example 5
Step-F:
4-Amino-5-iodo-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,-
3-d]pyrimidine (5.7)
[0218] A solution of compound 5.6 (28.8 mg, 0.057 mmol) in MeOH (3
mL) was transferred to a steel bomb, cooled to -50 to -60.degree.
C., and treated with a saturated solution of ammonia in MeOH (10
mL). The reactor was sealed and heated at 118.degree. C. overnight.
The reaction vessel was cooled (0-5.degree. C.), opened carefully
and the reaction mixture was evaporated to dryness. The crude
product was dissolved in MeOH, adsorbed onto silica gel and
purified by flash column chromatography on silica gel using pure
CH.sub.2Cl.sub.2 to 4% MeOH in CH.sub.2Cl.sub.2 as eluents to give
the desired compound 5.7 (20.7 mg) as an off white solid.
Example 5
Step-G:
4-Amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-3-[2-methoxycarbonyl-
)ethenyl]-7H-pyrrolo[2,3-d]pyrimidine (5.8)
[0219] To a solution of compound 5.7 (156 mg, 0.38 mmol) in DMF
(12.0 mL), were added (Z)-methyl-3-(tributylstannyl)acrylate (J.
Am. Chem. Soc., 1993, 115, 1619) (0.29 mL, 0.77 mmol) and CuI (14.6
mg, 0.08 mmol). The mixture was stirred for 10 min at room
temperature and then Pd(PPh.sub.3).sub.2Cl.sub.2 was added, and
reaction the mixture was heated for 3.5 h at 70.degree. C. under
argon. The reaction mixture was cooled to room temperature and
filtered through a celite pad. The celite pad was washed with 8 mL
of 1/1 MeOH/CH.sub.2Cl.sub.2. After filtration, the washings were
combined and the solvents were evaporated in vacuo. The residue was
redissolved in MeOH and adsorbed onto silica gel and purified by
flash column chromatography using 2.5% MeOH in CH.sub.2Cl.sub.2 as
eluent to give the desired compound 5.8 (38 mg, 27%) as a yellow
solid.
Example 5
Step-H:
2-(2-O-Methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetra-
azabenz[cd]azulen-7-one (5.9)
[0220] To a solution of compound 5.8 (36 mg, 0.1 mmol) in
1,4-dioxane (6.0 mL), were added 3 A molecular sieves followed by
DBU (38 .mu.L, 0.25 mmol). The reaction mixture was stirred for 3.5
h at 110.degree. C. and then cooled to room temperature and
filtered, the solvents were evaporated and the residue purified by
flash column chromatography using CH.sub.2Cl.sub.2 to 2% MeOH in
CH.sub.2Cl.sub.2 as eluent to give title compound 5.9 (16 mg) as an
off white solid.
[0221] .sup.1H NMR (DMSO-d.sub.6) .delta. 10.69 (s, NH, 1H), 8.33
(s, 1H), 7.8 (s, H-1, 1H), 7.04 (d, J 11.7 Hz, CH, 1H), 6.13 (d, J
6.0 Hz, H-4, 1H), 5.67 (d, J 11.7 Hz, CH, 1H), 5.25 (d, J 5.4 Hz,
3'-OH, 1H), 5.12 (t, J 5.7 Hz, 5'-OH, 1H), 4.24-4.27 (m, H-4', 1H),
4.12-4.15 (m, H-3', 1H), 3.93 (m, H-2', 1H), 3.55-3.61 (m, H-5',
2H), 3.28 (s, OCH.sub.3, 3H). MS m/z 331 (M-H).sup.+
Example 6
2-(2-C-Methyl-.beta.-D-ribofuranosyl]-2,6-dihydro-7H-2,3,5,6-tetraazabenzo-
[cd]azulen-7-one (6.6)
##STR00040## ##STR00041##
[0223] The tricyclic nucleoside 6.7 was synthesized starting from
4-chloro-1H-pyrrolo[2,3-d]pyrimidine 2.1. The nucleobase 2.1 was
treated with N-iodosuccinimide in THF at room temperature for 4 hr
to provide 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine 6.1. Then
6.1 was converted to the corresponding sodium salt with sodium
hydride in acetonitrile and reacted with bromo-sugar 2.4 ((i) Helv.
Chico. Acta. 1995, 78, 486; (ii) WO 02/057287, 2002), to give
nucleoside 6.2. which was directly converted to
4-amino-5-iodo-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-.bet-
a.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine 6.3. A Stile
coupling reaction between nucleoside 6.3 and
Z-3-tributylstannylacrylate provided compound 6.4. The cyclization
of compound 6.4 was accomplished by heating in DBU/dioxane
overnight to afford the protected tricyclic nucleoside 6.5, which
was then treated with boron trichloride in CH.sub.2Cl.sub.2 to
produce nucleoside 6.6.
4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (6.1)
[0224] Compound 6.1 was prepared according to a published procedure
(Townsend, L. B. et al., 1990, 33 1982-1992.
Example 6
Step A:
4-Chloro-5-iodo-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-
-.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine (6.2)
[0225] Compound 2.4 was prepared according to the published
procedure. ((i). Helv. Chico. Acta. 1995, 78, 486; (ii). WO
02/057287, 2002).
[0226] A solution of compound 2.4 (25 mmol) in anhydrous
acetonitrile (90 mL) was added to a solution of the sodium salt of
4-chloro-5-iodo-1H-pyrrolo[2,3-d]pyrimidine [generated in situ from
4-chloro-5-iodo-1H-pyrrolo[2,3-d]pyrimidine 6.1 (6.99 g, 25 mmol)
in anhydrous acetonitrile (250 mL), and NaH (60% in mineral oil,
1.0 g, 25 mmol), after 4 hr of vigorous stirring at room
temperature]. The combined mixture was stirred at room temperature
for 24 h, and then evaporated to dryness. The residue was suspended
in water (250 mL) and extracted with CH.sub.2Cl.sub.2 (2.times.500
mL). The combined extracts were washed with brine (300 mL), dried
over Na.sub.2SO.sub.4, filtered and evaporated. The crude product
was purified on a silica gel column using ethyl acetate/hexanes
(1/4-1/2) as the eluent. Fractions containing the product were
combined and evaporated in vacuo to give the desired product 6.2
(6.26 g, yield 34%) as light yellow foam.
Example 6
Step B:
4-amino-5-iodo-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl--
.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine (6.3)
[0227] To a compound 6.2 (5 g, 6.7 mmol) in dioxane (100 mL) was
added conc. NH.sub.4OH (100 mL). The mixture was heated in a
stainless steel autoclave at 100.degree. C. for 3 h, then cooled
and evaporated in vacuo. The crude mixture was dissolved in 100 mL
of CH.sub.2Cl.sub.2 and washed with water and brine, dried over
MgSO.sub.4, filtered and concentrated to provide crude product. The
crude product was then purified on a silica gel column with 5% MeOH
in CH.sub.2Cl.sub.2 as eluent to give 4.32 g of 6.3 as white foam
(yield 88%).
Example 6
Step C:
4-Amino-5-[2-(methoxycarbonyl)ethenyl]-7-[3,5-bis-O-(2,4-dichlorop-
henylmethyl)-2-C-methyl-.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidin-
e (6.4)
[0228] To a solution of compound 6.3 (1.25 g, 1.726 mmol) in 10 mL
of anhydrous DMF was added Z-3-tributylstannylacrylate (1.1 mL, 2
eq.) (J. Am. Chem. Soc., 1993, 115, 1619), CuI (66 mg, 0.2 eq.) and
PdCl.sub.2(PPh.sub.3).sub.2 (121 mg, 0.1 eq.) at room temperature
under the argon atmosphere. The reaction mixture was heated at
70.degree. C. for 8 hr. Then the reaction mixture was cooled to
room temperature and filtered through a celite pad. The filtrate
was concentrated in vacuo to provide an orange-red oil as the crude
product which was purified on a silica gel column with 10-30% THF
in CH.sub.2Cl.sub.2 as eluent to give 995 mg 6.4 as yellow foam
(yield 81%).
Example 6
Step D:
2-[3,5-Bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-.beta.-D-ribofu-
ranosyl]-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one
(6.5)
[0229] To a solution of the compound 6.4 (1.15 g, 1.69 mmol) in 150
mL of anhydrous dioxane under an argon atmosphere at room
temperature was added DBU (630 .mu.L, 2.5 eq.) and 1 g of 4 A
molecular sieves. The reaction mixture was heated at reflux for 16
hr then cooled to room temperature and evaporated to dryness in
vacuo. The residue was purified by silica gel chromatography with
1% MeOH in CH.sub.2Cl.sub.2 as eluent to provide 772 mg of compound
6.5 as a yellow solid (yield: 71%).
Example 6
Step E:
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetra-
azabenzo[cd] azulen-7-one (6.6)
[0230] To a solution of the compound 6.5 (0.71 g, 1.1 mmol) in 30
mL of anhydrous CH.sub.2Cl.sub.2 at -78.degree. C. was added boron
trichloride (1M solution in CH.sub.2Cl.sub.2, 11 mL, 11 mmol)
dropwise. The mixture was stirred at -78.degree. C. for 2.5 h, then
at -30.degree. C. to -20.degree. C. for 3 hr. The reaction was
quenched by addition of methanolic/CH.sub.2Cl.sub.2 (1:1) (5 mL)
and the resulting mixture stirred at -15.degree. C. for 30 min.,
then neutralized with aqueous ammonia at 0.degree. C. and stirred
at room temperature for 15 min. The solid was filtered and washed
with CH.sub.2Cl.sub.2/MeOH (1/1, 3.times.30 mL). Chromatography
over silica gel using 5% MeOH in CH.sub.2Cl.sub.2 as eluent
furnished the 282 mg of the desired compound 6.6 as a yellow solid
(yield: 77.8%). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 10.67
(br s 1H, NH), 8.32 (s, 1H, H-4), 7.86 (s, 1H, H-1), 7.00 (d, J
12.6 Hz, H-8), 6.09 (s, 1H, H-1'), 5.63 (d, J 12.6 Hz, H-9), 5.16
(m, 31-1, 3.times.OH), 3.95 (m, 1H, H-3'), 3.88-3.56 (m, 3H, H-4',
2.times.H-5'), 0.73 (s, 3H, CH.sub.3); ES MS: 391.5
(M+CH.sub.3COO).sup.-.
Example 7
2-(2-C-methyl-.beta.-D-ribofuranosyl)-8,9-dihydro-3,5,6,9a-tetraazabenzo[c-
d]azulene (7.14)
##STR00042## ##STR00043## ##STR00044##
[0231] Example 7
Step A: 2-C-Methyl-D-ribofuranose (7.2)
[0232] To a suspension of
2-C-methyl-1,2,3,5-tetra-O-benzoyl-.beta.-D-ribofuranose 1.3, (50
g) in anhydrous MeOH (1000 mL) was added KCN (150 mg) and the
mixture was allowed to stir at room temperature under argon for 15
hr during which time all the material dissolved in the solvent and
the solution became clear. The solvent was evaporated and the
residue was dried under vacuum to deliver 14.9 g of product
7.2.
Example 7
Step B: 2,3-O-Isopropylidene-2-C-methyl-D-ribofuranose (7.3)
[0233] The material from Step A (7.2) (14.9 g, 86 mmol) was
dissolved in dry acetone (1000 mL) and 1 mL of conc.
H.sub.2SO.sub.4 was added. The mixture was stirred at room
temperature overnight and then carefully neutralized with saturated
aqueous NaHCO.sub.3 and the solvent evaporated. The residue was
dissolved in 500 mL ethyl acetate and washed with water (100 mL)
and brine (100 mL). The solution was dried over Na.sub.2SO.sub.4,
filtered and evaporated. The residue was purified on a silica gel
column using 2:1 hexanes:EtOAc. Evaporation of the solvent afforded
14 g of the product 7.3.
Example 7
Step C:
2,3-O-Isopropylidene-2-C-methyl-5-O-(triphenylmethyl)-D-ribofurano-
se (7.4).
[0234] To a solution of compound 7.3 (13.7 g, 67.4 mmol) in
pyridine was added chlorotriphenylmethane (23.5 g, 84.2 mmol) and
the mixture was heated at 60.degree. C. for 15 hr under an argon
atmosphere. The solvent was evaporated and the residue was
dissolved in ethyl acetate (200 mL) and washed with water (150 mL),
brine (150 mL) and dried over sodium sulphate. After filtration and
evaporation, the residue was loaded on a silica gel column and
eluted with 10:1 followed by 5:1 hexane:ethyl acetate. Evaporation
of solvent under reduced pressure afforded 15 g of 7.4 as a
colorless syrup.
Example 7
Step D:
3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-4-C-methyl-7-O-(triphenyl-
methyl)-D-allo- and D-altro-septononitrile (7.5).
[0235] To a suspension of NaH (95%, 1.23 g, 48.2 mmol) in dry DME
(250 mL), diethyl cyanomethylenephosphonate (10.06 mL, 62 mmol) was
added dropwise at 0.degree. C. over 15 minutes. After evolution of
hydrogen ceased, compound 7.4 (15 g, 33.5 mmol), in 250 mL dry DME
was added to the resulting solution over 30 minutes and then the
mixture was stirred at room temperature for 2 hr. The reaction
mixture was partitioned between ether (1000 mL) and water (1000 mL)
and the aqueous layer was extracted with 1000 mL ether. The
combined ether extracts were washed with water, dried
(Na.sub.2SO.sub.4) and filtered. The solvent was evaporated under
reduced pressure, and the residue was purified by silica gel column
chromatography using 4:1 hexane:ethyl acetate as eluent. The
solvent was evaporated to give 7.5 as an off-white foam (14.8
g).
Example 7
Step E:
(2E)-3,6-Anhydro-2-deoxy-2-C--[(N,N-dimethylamino)methylidene]-4,5-
-O-isopropylidene-4-C-methyl-7-O-(triphenylmethyl)-D-allo- and
D-altro-septononitrile (7.6).
[0236] To a solution of compound 7.5 (13.0 g, 27.68 mmol) in
anhydrous CH.sub.2Cl.sub.2 (60 mL) was added
bis(dimethylamino)-tert-butoxymethane (22.87 mL, 110.74 mmol)
followed by dry dimethylformamide (2.3 mL). The mixture was stirred
at room temperature for 20 hr. After removal of solvents the
mixture was chromatographed on a silica gel column pre-treated with
triethylamine to deliver 11.4 g of 7.6 as a viscous oil.
Example 7
Step F:
(2E)-3,6-Anhydro-2-deoxy-2-C-(hydroxymethylidene)-4,5-O-isopropyli-
dene-4-C-methyl-7-O-(triphenylmethyl)-D-allo- and
D-altro-septononitrile (7.7).
[0237] To a solution of compound 7.6 (6 g, 11.44 mmol) in
CHCl.sub.3 (120 mL) was added a solution of TFA (3 mL) in water
(200 mL) and the mixture was stirred vigorously at room temperature
for 16 hr. The organic layer was separated and washed with water
and dried (Na.sub.2SO.sub.4) then filtered. Removal of solvent
afforded 2-formyl nitrile (7.7) (2.5 g) in a form that was used as
such in the next step.
Example 7
Step G:
(2E)-3,6-Anhydro-2-deoxy-2-C-[(cyanomethylamino)methylidene]-4,5-O-
-isopropylidene-4-C-methyl-7-O-(triphenylmethyl)-D-allo- and
D-altro-septononitrile (7.8).
[0238] Crude 7.7 was dissolved in MeOH (25 mL) and 1.6 mL water was
added followed by aminoacetonitrile hydrochloride (0.78 g, 8.77
mmol) and sodium acetate trihydrate (1.3 g, 9.55 mmol). The mixture
was stirred at room temperature for 16 hr. After evaporation of the
solvent, the residue was dissolved in a minimum volume of
CH.sub.2Cl.sub.2 and loaded on a silica gel column which was eluted
with 30:1 CH.sub.2Cl.sub.2:MeOH. Evaporation of solvent under
reduced pressure gave 2.2 g of product 7.8 as an anomeric
mixture.
Example 7
Step H:
3-Amino-2-cyano-4-(2,3-O-isopropylidene-2-C-methyl-5-O-triphenylme-
thyl-.beta.-D-ribofuranosyl)-1H-pyrrole (7.9).
[0239] To a solution of compound 7.8 (8 g, 14.94 mmol) in
CH.sub.2Cl.sub.2 (100 mL) at 0.degree. C. was added
1,5-diaza[4.3.0]non-5-ene (DBN) (2.95 mL, 23.89 mmol) followed by
ethyl chloroformate (2.35 mL, 23.89 mmol). The mixture was kept at
0-4.degree. C. for 16 hr. Additional 2 mL of DBN was added and the
mixture was stirred at room temperature for 24 hr. After
evaporation of the solvent the residue was purified on silica gel
column using 4:1 hexanes:EtOAc followed by 3:1 hexanes:EtOAc to
obtain a major fraction as mixture of anomers. To this mixture of
anomers (6.42 g, 10.56 mmol) in MeOH (100 mL) was added sodium
carbonate (3 g) and stirred at room temperature for 1 hr. The
insoluble residue was filtered off and the solvent was removed
under vacuum. The residue was purified on a silica gel column using
3:1 hexanes:EtOAc to obtain 5 g of product 7.9 as 13 anomer.
Example 7
Step I
Part A: 3-(tert-Butyldiphenylsilyloxy)propyl
4-methylbenzenesulfonate
[0240] The title compound was prepared according to the procedure
described by Caprio et al. Tetrahedron, 2001, 57, 4023-4034.
Part B:
3-Amino-1-(3-tert-butyldiphenylsilyloxypropyl)-2-cyano-4-(2,3-O-is-
opropylidene-2-C-methyl-5-O-triphenylmethyl-.beta.-D-ribofuranosyl)-1H-pyr-
role (7.10)
[0241] To a solution of potassium t-butoxide (1M THF solution, 4.32
mL) in THF (30 mL) was added compound 7.9 (2 g, 3.73 mmol),
followed by catalytic amount of 18-crown-6. The mixture was stirred
under argon for 10 minutes during which time the solution turned
clear reddish brown. To this solution was added the compound
prepared according Part A (3.68 g, 7.47 mmol) dissolved in 1.5 in
of (anhydrous) dichloroethane. After 1 hour, a further equivalent
of tosylate was added and the mixture was stirred at room
temperature overnight. After evaporation of the solvent under
vacuum, the residue dissolved in minimum CH.sub.2Cl.sub.2 was
loaded on a silica gel column and eluted with 6:1 followed by 4:1
hexane:EtOAc. Evaporation of the solvent from the appropriate
fractions afforded 1.5 g of product 7.10.
Example 7
Step J:
4-Amino-5-(3-tert-butyldiphenylsilyloxypropyl)-7-(2,3-O-isopropyli-
dene-2-C-methyl-5-O-triphenylmethyl-.beta.-D-ribofuranosyl)-5H-pyrrolo-[3,-
2-d]pyrimidine (7.11).
[0242] Compound 7.10 (600 mg, 0.72 mmol) and formamidine acetate
(227.5 mg, 2.61 mmol) were mixed with 20 mL ethyl alcohol and
heated at reflux under argon atmosphere for 8 hr. The solvent was
evaporated and the residue was loaded on a silica gel column and
eluted with 20:1 MeOH:CH.sub.2Cl.sub.2 to afford 555 mg product
7.11.
Example 7
Step K:
4-Amino-5-(3-hydroxypropyl)-7-(2,3-O-isopropylidene-2-C-methyl-5-O-
-triphenylmethyl-.beta.-D-ribofuranosyl)-5H-pyrrolo[3,2-d]pyrimidine
(7.12).
[0243] Compound 7.11 (555 mg, 0.65 mmol) was dissolved in anhydrous
THF (10 mL) and 1.3 mL (1.3 mmol) of a 1M THF solution of
tetrabutylammonium fluoride was added. The mixture was stirred at
room temperature for 2 hr and the solvent was then evaporated under
reduced pressure and the residue was loaded on a silica gel column
and eluted with 15:1 CH.sub.2Cl.sub.2:MeOH to give 281 mg of
product 7.12.
Example 7
Step L:
2-(2,3-O-Isopropylidene-2-C-methyl-5-O-triphenylmethyl-.beta.-D-ri-
bofuranosyl)-8,9-dihydro-3,5,6,9a-tetraazabenzo c azulene
(7.13).
[0244] Compound 7.12 (160 mg, 0.26 mmol) was taken up in 2 mL
CH.sub.2Cl.sub.2 and kept at 0.degree. C. To this was added TEMPO
(2,2,6,6-tetramethyl-1-piperidinyloxy, free radical, 1 mg) followed
by an aqueous solution KBr (1 mg in 0.2 mL water) and
Aliquat.RTM.366 (6 .mu.L) and NaOCl (0.35 M, 0.92 mL). The mixture
was stirred at 0.degree. C. for 30 minutes. More CH.sub.2Cl.sub.2
was added and the reaction was washed with water (5 mL). The
organic layer was dried over Na.sub.2SO.sub.4, filtered and
evaporated under reduced pressure. The residue was used in the next
step without further purification.
Example 7
Step M:
2-(2-C-methyl-.beta.-D-ribofuranosyl)-8,9-dihydro-3,5,6,9a-tetraaz-
abenzo[cd]azulene (7.14).
[0245] Compound 7.13 was heated at 80.degree. C. in 90% acetic acid
for 12 hr. The solvent was evaporated and the crude product was
purified by reverse phase HPLC on a C18 column to afford 3 mg pure
product 7.14. .sup.1H NMR (DMSO-d.sub.6). .delta. 8.10 (s, 1H),
7.49 (s, 1H), 6.60 (s, 1H), 5.33 (s, 1H), 4.57 (m, 3H), 4.03 (m,
1H), 3.72 (m, 1H), 3.51 (an, 2H), 2.91 (m, 2H), 2.77 (m, 2H), 1.02
(s, 3H).
Example 8
2-(2-C-Methyl-.beta.-D-ribofuranosyl]-2,6,8,9-tetrahydro-7H-2,3,54-tetraaz-
abenzo[cd]azulen-7-one (8.1)
##STR00045##
[0247] A mixture of tricyclic nucleoside 6.6 (20 mg, 0.06 mmol) and
10% Pd/C (12.8 mg, 0.2 eq.) in 20 mL of MeOH under H.sub.2 pressure
(32 psi) was shaken for 7 hr at room temperature. The mixture was
filtered through 0.45 .mu.m filter. The combined filtrates were
evaporated and purified on a silica gel column with 5% MeOH in
CH.sub.2Cl.sub.2. 12 mg of pure compound 8.1 was obtained (yield
60%). .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta.10.65 (br s, 1H,
NH), 8.37 (s, 1H, H-4), 7.48 (s, 1H, H-1), 6.12 (s, 1H, H-1'), 5.05
(m, 3H, 3.times.OH), 3.89-3.12 (m, 4H, H-3', H-4', 2.times.H-5%
2.86-2.74 (m, 4H, 2.times.H-8, 2.times.H-9), 0.64 (s, 3H,
CH.sub.3); LCMS: ES-MS 393.6 (M+CH.sub.3COO).
Example 9
2-(.beta.-D-ribofuranosyl-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[c]azulen-7--
one (9,4
##STR00046##
[0249] The nucleoside 9.2 was produced directly from nucleoside
9.1, which was prepared starting from D-ribose (Bheemarao G. et al;
J. Med. Chem. 2000, 43, 2883-2893), using liquid ammonia at
85.degree. C. overnight. The palladium[0] catalyzed cross-coupling
reaction of compound 9.2, followed by the cyclization of compound
9.3 in 0.1 N NaOMe in MeOH, delivered the desired tricyclic
nucleoside 9.4.
Example 9
Step A:
4-amino-5-iodo-7-.beta.-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidin-
e (9.2)
[0250] Compound 9.2 was prepared according to a published method
(Bergstrom, D. E., et al., J. Org. Chem., 1981, 46, 1423).
Example 9
Step B:
4-Amino-5-[2-(methoxycarbonyl)ethenyl]-7-(2-C-methyl-.beta.-D-ribo-
furanosyl)-7H-pyrrolo[2,3-d]pyrimidine (9.3)
[0251] To a solution of compound 9.2 (300 mg, 0.76 mmol) in 10 mL
of anhydrous DMF was added CuI (29 mg, 0.2 eq.), methyl acrylate
(1.37 mL, 20 eq.), triethylamine (212 .mu.L, 2 eq.) and
Pd(PPh.sub.3).sub.4 (88 mg, 0.1 eq.) at room temperature under an
argon atmosphere. The reaction mixture was heated at 70.degree. C.
for 24 hr. then cooled to room temperature and 20 mL of 1/1
MeOH/CH.sub.2Cl.sub.2 was added. Then, 1.0 g Dowex 1.times.2-100
Bicarb form was added and the suspension stirred at room
temperature for 45 min. then filtered. The resin was washed with
5.times.20 mL MeOH/CH.sub.2Cl.sub.2:1/1, DMF was finally evaporated
by co-evaporation with toluene (2.times.10 mL). Chromatographic
column purification on silica gel (eluent:
CH.sub.2Cl.sub.2/MeOH:90/10) gave 224 mg of final product 9.3
(yield 84%)
[0252] .sup.1H NMR (CD.sub.3OD) .delta. 8.11 (s, 1H, H-2), 8.00 (s,
1H, H-6), 7.96 (d, J 15.54 Hz, 1H), 6.35 (d, J 15.54 Hz, H2''),
6.52 (d, 1H, H-1'), 4.43 (t, 1H, H-3'), 4.34-4.26 (m, 2H, H-4',
H-2'), 3.85-3.64 (m, 2H, 2.times.H-5'), 3.79 (s, 3H,
OCH.sub.3).
Example 9
Step C:
2-(.beta.-D-Ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd-
]azulen-7-one (9.4)
[0253] A solution of compound 9.3 (140 mg, 0.4 mmoles) in 0.1 M
NaOMe in MeOH (80 mL) was heated at 70.degree. C. for 4 hr. The
solution was cooled and the solvent evaporated and the residue
purified by silica gel column using CH.sub.2Cl.sub.2/MeOH:90/10 as
eluent to give the final product 9.4.
[0254] .sup.1H NMR (CD.sub.3OD) .delta. 8.32 (s, 1H, H-2), 7.86 (s,
1H, H-6), 7.07 (d, J 12 Hz, H-8), 6.09 (s, 1H, H-1'), 5.70 (d, J 12
Hz, H-9), 4.57 (m, 1H, H-2'), 4.29 (m, 1H, H-3'), 4.12 (m, 1H,
H-4'), 3.88-3.72 (m, 2H, 2.times.H-5'); ES-MS: 377.4
(M+CH.sub.3COO).sup.-.
Example 10
2-(3-Deoxy-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetranzabenzo[cd-
]azulen-7-one (10.6)
##STR00047##
[0256] The nucleoside 5.1 was prepared starting from D-ribose ((i)
Journal of Heterocyclic Chemistry, 25(6), 1893-8, 1988; (ii)
Helvetica Chimica Acta, 71(6), 1573-85, 1988), then converted to
the 3'-deoxy nucleoside 10.2 as follows: 4.0 equiv of
a-acetoxyisobutyryl bromide was added to a suspension of nucleoside
5.1 in acetonitrile containing 1.1 equiv of H.sub.2O at room
temperature followed by treatment with DOWEX OH.sup.- resin in MeOH
to afford a crystalline sample of
4-methoxy-7-(2,3-anhydro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
ine 10.1. The epoxide was treated with 4.0 equiv of LiEt.sub.3BH in
THF at room temperature to give the 3'-deoxynucleoside 10.2 in 50%
combined yield for the two preceding steps. The 7-iodo group was
introduced by reacting the 3'-deoxynucleoside 10.2 with
N-iodosuccinimide in DMF to give compound 10.3 which, in turn, was
treated with anhydrous liquid ammonia to provide compound 10.4.
After the palladium[0] catalyzed cross-coupling reaction and
cyclization to form the tricyclic target, the desired nucleoside
10.6 was obtained.
Example 10
Step A:
4-methoxy-7-(2,3-anhydro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (10.1)
[0257] To a mixture of nucleoside 5.1 (250 mg, 0.875 mmol) in 12 mL
of acetonitrile were added H.sub.2O/acetonitrile (1/9) (157 .mu.L,
1 eq.) and .alpha.-acetoxyisobutyryl bromide (0.537 mL, 4 eq.).
After 2 hr stirring at room temperature, sat. NaHCO.sub.3 (aq.) was
added and the mixture was extracted with ethyl acetate. The
combined organic extracts were washed with brine, dried over
MgSO.sub.4 and evaporated. The foamy residue was suspended in MeOH
and stirred overnight with Dowex Off (previously washed with
anhydrous MeOH). The resin was filtered off, washed with MeOH and
the combined filtrates were evaporated to yield 225 mg of a pale
yellow foam 10.1, which was directly used in the next step without
further purification.
Example 10
Step B:
4-Methoxy-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine (10.2)
[0258] Superhydride LiEt.sub.3BH in 1M THF (8 mL, 10 eq.) was added
dropwise to an ice-cold deoxygenated (after 15 min purging with
argon) solution of anhydrous nucleoside 10.1 (218 mg, 0.8 mmol) in
anhydrous THF (10 mL) under argon. The resulting mixture was
stirred at 0.degree. C. for 2 h, then acidified cautiously and
finally purged with argon for 1 hr. The residue was purified on a
silica gel column with 5% MeOH in CH.sub.2Cl.sub.2 to yield 117 mg
of target compound 10.2 as off-white solid (yield 55%).
Example 10
Step C:
4-Methoxy-5-iodo-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidine (10.3)
[0259] To a solution of nucleoside 10.2 (350 mg, 1.32 mmol) in DMF
(10 mL) was added N-iodosuccinimide (327 mg, 1.1 eq.) at 0.degree.
C. The reaction mixture was stirred at 0.degree. C. under argon for
2 h, then warmed up to room temperature and stirred overnight. The
reaction was quenched by addition of 4 mL of MeOH. The solution was
evaporated to dryness, then redissolved in CHCl.sub.3, washed with
sat. aq. NaHCO.sub.3, Na.sub.2SO.sub.3 and water, then dried over
MgSO.sub.4. After evaporation, the residue was purified on a silica
gel column using 0-3% MeOH in CH.sub.2Cl.sub.2 to provide 353 mg of
pure compound 10.3 as white solid (yield: 68%).
Example 10
Step D:
4-Amino-5-iodo-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d-
]pyrimidine (10.4)
[0260] A mixture of compound 10.3 (50 mg, 0.128 mmol) and anhydrous
liquid ammonia (15 mL) was heated in a stainless steel autoclave at
120.degree. C. 2 days, then cooled and evaporated in vacuo. The
residue was purified on a silica gel column with 3% MeOH in
CH.sub.2Cl.sub.2 as eluent to give 30 mg of the compound 10.4 as a
white solid. (yield: 62%)
Example 10
Step E:
4-Amino-5-[2-methoxycarbonyl)ethenyl]-7-(3-deoxy-.beta.-D-ribofura-
nosyl)-7H-pyrrolo[2,3-d]-pyrimidine (10.5)
[0261] To a solution of compound 10.4 (50 mg, 0.132 mmol) in 2 mL
of anhydrous DMF were added CuI (5 mg, 0.2 eq.), methyl acrylate
(240 .mu.L, 20 eq.), triethylamine (37 uL, 2 eq.) and
Pd(PPh.sub.3).sub.4 (15 mg, 0.1 eq.) at room temperature under an
argon atmosphere. The reaction mixture was heated at 70.degree. C.
for 48 hr. then cooled to room temperature and 20 mL of 1/1
MeOH/CH.sub.2Cl.sub.2 was added. 100 mg Dowex 1.times.2-100 Bicarb
form was then added and the suspension was stirred at room
temperature for 45 min. then filtered. The resin was washed with
3.times.10 mL MeOH/CH.sub.2Cl.sub.2:1/1, and the solvent
evaporated. DMF was finally evaporated by azeotropic co-evaporation
with toluene (2.times.5 mL). The residue was purified by
chromatographic column purification on silica gel (eluent:
CH.sub.2Cl.sub.2/MeOH=95/5) to give 20 mg of final product 10.5
(yield 45%).
Example 10
Step F:
2-(3-Deoxy-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraaza-
benzo[cd]azulen-7-one (10.6)
[0262] A solution of compound 10.5 (20 mg, 0.06 mmoles) in 0.1 M
NaOMe in MeOH (12 mL) was heated at 70.degree. C. for 4 hr. The
solvent was evaporated and the residue purified by silica gel
column with CH.sub.2Cl.sub.2/MeOH=90/10) to give the final product
10.6.
[0263] .sup.1H NMR (300 MHz, CD.sub.3OD): .delta. 8.31 (s, 1H),
7.71 (s, 1H), 7.05 (d, J 12 Hz), 6.05 (s, 1H, H-1'), 5.73 (d, J 12
Hz, 1H), 3.87-3.66 (m, 4H, H-2', H-4' 2.times.H-5'), 2.31-2.09 (m,
1H, 2.times.H-3'); ES MS: 360.9 (M+CH.sub.3COO).
Example 11
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-9-methyl-2,6-dihydro-7H-2,3,5,6-tetr-
aazabenzo[cd]azulen-7-one (11.4)
##STR00048##
[0264] Example 11
Step A:
4-Chloro-5-iodo-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidine (11.1)
[0265] To a solution of the compound 6.3 (7.73 g, 10.39 mmoles) in
dichloromethane (200 mL) at -78.degree. C. was added boron
trichloride (1M in dichloromethane, 104 mL, 104 mmol) dropwise. The
mixture was stirred at -78.degree. C. for 2.5 h, then at
-30.degree. C. to -20.degree. C. for 3 hr. The reaction was
quenched by addition of methanolic/dichloromethane (1:1) (105 mL)
and the resulting mixture stirred at -15.degree. C. for 30 min.,
then neutralized with aqueous ammonia at 0.degree. C. and stirred
at room temperature for 15 min. The solid was filtered and washed
with CH.sub.2Cl.sub.2/MeOH (1/1, 250 mL). The chromatography over
silica gel using CH.sub.2Cl.sub.2 and CH.sub.2Cl.sub.2/MeOH (99/1
to 90/10) gradient as the eluent to furnish the desired compound
11.1 (2.24 g, yield 51%) as a colorless foam.
Example 11
Step B:
4-Amino-5-iodo-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,-
3-d]pyrimidine (11.2)
[0266] To the compound 11.1 (425 mg, 1 mmol) was added liquid
ammonia (20 mL). The mixture was heated in a stainless steel
autoclave at 85.degree. C. overnight, then cooled and evaporated in
vacuo. The crude mixture was purified on a silica gel column with
5% methanol in dichloromethane as eluent to give the product 11.2
as a light yellow foam (400 mg, 100% yield).
Example 11
Step C:
4-Amino-5-[1-methyl-2-(methoxycarbonyl)ethenyl]-7-(2-C-methyl-.bet-
a.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine (11.3)
[0267] To a solution of compound 11.2 (1 g, 2.46 mmol) in 20 mL of
anhydrous DMF were added CuI (94 mg, 0.2 eq.), methyl crotonate
(5.33 mL, 20 eq.), triethylamine (686 .mu.L, 2 eq.) and
Pd(PPh.sub.3).sub.4 (285 mg, 0.1 eq.) at room temperature under an
argon atmosphere. The reaction mixture was heated at 70.degree. C.
for 24 hr. then cooled to room temperature and 100 mL of 1/1
MeOH/CH.sub.2Cl.sub.2 was added. 2.0 g Dowex 1.times.2-100 Bicarb
form was then added and the suspension was stirred at room
temperature for 45 min., then filtered. The resin was washed With
3.times.50 mL MeOH/CH.sub.2Cl.sub.2:1/1.DMF was finally evaporated
by azeotropic co-evaporation with toluene (2.times.5 mL).
Chromatograph purification on silica gel (eluent:
CH.sub.2Cl.sub.2/MeOH: 95/5) gave 463 mg of product 11.2 (yield:
50%).
Example 11
Step D:
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-9-methyl-2,6-dihydro-7H-2,3,-
5,6-tetraazabenz[cd]azulen-7-one (11.4)
[0268] A solution of compound 11.3 (33 mg, 0.087 mmoles) in 0.1 M
NaOMe in MeOH (17 mL) was heated at 70.degree. C. for 4 hr. The
solution was evaporated and the residue purified by silica gel
chromatography using CH.sub.2Cl.sub.2/MeOH:95/5 as eluent to
provide 24 mg of final product 11.4 (yield 80%).
[0269] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 10.66 (br. 1H,
NH), 8.33 (s, 1H, H-4), 8.09 (s, 1H, H-1), 6.10 (s, 1H, H-1'), 5.67
(s, 1H, H-8), 5.16 (m, 3H, 3.times.OH), 4.05-3.65 (m, 4H, H-3',
H-4' 2.times.H-5'), 2.11 (s, 3H, CH.sub.3), 0.73 (s, 3H, CH.sub.3);
ESMS: 405.5 (M+CH.sub.3COO).
Example 12
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-8-methyl-2,6-dihydro-7H-2,3,5,6-tetr-
aazabenzo[cd]azulen-7-one (12.2)
##STR00049##
[0270] Example 12
Step A:
4-amino-5-[2-methyl-2-(methoxycarbonyl)ethenyl]-7-(2-C-methyl-.bet-
a.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (12.1)
[0271] To a solution of compound 11.2 (200 mg, 0.492 mmol) in 4 mL
of anhydrous DME was added CuI (19 mg, 0.2 eq.), a-methyl
methacrylate (1.06 mL, 20 eq.), triethylamine (137 .mu.L, 2 eq.)
and Pd(PPh.sub.3).sub.4 (57 mg, 0.1 eq.) at room temperature under
an argon atmosphere. The reaction mixture was heated at 70.degree.
C. for 24 hr. then cooled to room temperature and 100 mL of 1/1
MeOH/CH.sub.2Cl.sub.2 was added. 400 mg Dowex 1.times.2-100 Bicarb
form was then added the suspension stirred at room temperature for
45 min., then filtered. The resin was washed with 3.times.10 mL
MeOH/CH.sub.2Cl.sub.2:1/1. DMF was finally evaporated by azeotropic
co-evaporation with toluene (2.times.5 mL). Chromatograph
purification of the residue on silica gel (eluent:
CH.sub.2Cl.sub.2/MeOH:9515) gave 100 mg of ester 12.1 (yield:
54%).
Example 12
Step B:
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-8-methyl-2,6-dihydro-7H-2,3,-
5,6-tetraazabenzo[cd]azulen-7-one (12.2)
[0272] A solution of compound 12.1 (30 mg, 0.079 mmoles) in 0.1 M
NaOMe in MeOH (16 mL) was heated at 70.degree. C. for 4 hr. The
solution was evaporated and the residue purified by silica gel
column chromatography using CH.sub.2Cl.sub.2/MeOH:95/5) to provide
product 12.2.
[0273] .sup.1H NMR. (300 MHz, DMSO-d.sub.6) .delta. 10.60 (br s.
1H, NH), 8.28 (s, 1H, H-4), 7.73 (s, 1H, H-1), 7.09 (s, 1H, H-9),
6.07 (s, 1H, H-1'), 5.16 (m, 3H, 3.times.OH), 3.93-3.63 (m, 4H,
H-3', 4' 2.times.H-5'), 1.97 (s, 3H, CH.sub.3), 0.72 (s, 3H,
CH.sub.3); ES MS: 405.3 (M+CH.sub.3COO).
Example 13
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-9-methoxy-2,6-dihydro-7H-2,3,5,6-tet-
raazabenz[c,d]azulen-7-one (13.2)
##STR00050##
[0274] Example 13
Step A:
4-Amino-5-[1-methoxy-2-(methoxycarbonyl)ethenyl]-7-(2-C-methyl-.be-
ta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (13.1)
[0275] To a solution of compound 11.2 (200 mg, 0.492 mmol) in 5 mL
of anhydrous DMF were added CuI (19 mg, 0.2 eq.),
E-3-methoxymethacrylate (1.06 mL, 20 eq.), triethylamine (137
.mu.L, 2 eq.) and Pd(PPh.sub.3).sub.4 (57 mg, 0.1 eq.) at room
temperature under an argon atmosphere. The reaction mixture was
heated at 70.degree. C. for 24 hr. then cooled to room temperature
and 100 mL of 1/1 MeOH/CH.sub.2Cl.sub.2 was added. 400 mg Dowex
1.times.2-100 Bicarb form was then added and the suspension stirred
at room temperature for 45 min., then filtered. The resin was
washed with 3.times.10 mL MeOH/CH.sub.2Cl.sub.2:1/1. DMF was
finally evaporated by azeotropic co-evaporation with toluene
(2.times.5 mL). Chromatographic purification of the residue on
silica gel (eluent: CH.sub.2Cl.sub.2/MeOH:95/5) gave 87 mg of
product 13.1 (yield: 45%).
Example 13
Step B:
2-(2-C-Methoxy-.beta.-D-ribofuranosyl)-9-methoxy-2,6-dihydro-7H-2,-
3,5,6-tetronzabenzo[cd]azulen-7-one (13.2)
[0276] A solution of compound 13.1 (30 mg, 0.076 mmoles) in 0.1 M
NaOMe in MeOH (15 mL) was heated at 70.degree. C. for 4 hr. The
solution was evaporated and the residue purified by silica gel
column with CH.sub.2Cl.sub.2/MeOH:95/5) to provide nucleoside
13.2.
[0277] .sup.1H NMR. (300 MHz, DMSO-d.sub.6) .delta. 10.63 (br s 1H,
NH), 8.35 (s, 1H, H-4), 8.06 (s, 1H, H-1), 6.13 (s, 1H, H-1'), 5.30
(s, 1H, H-8), 5.22 (m, 3H, 3.times.OH), 3.32 (s, 3H, OCH.sub.3),
3.98-3.63 (m, 4H, H-3', H-4' 2.times.H-5'), 0.71 (s, 3H, CH.sub.3);
ES MS: 421.5 (M+CH.sub.3COO.sup.-).
Example 14
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-8-bromo-9-methoxy-2,6,8,9-tetrahydro-
-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one (14.1)
##STR00051##
[0279] To a stirred solution of nucleoside 6.6 (10 mg, 0.030 mmol)
in DMF (0.5 mL) was added N-bromosuccinimide (11.25 mg, 2.10 eq.)
at 0.degree. C. under argon. The reaction mixture was stirred at
0.degree. C. for 1 hr then quenched with MeOH (0.5 mL). The mixture
was evaporated to dryness and the residue was purified on a silica
gel column with 5% MeOH in CH.sub.2Cl.sub.2 to give compound 14.1
as a mixture of diastereoisomers (8 mg, 65%). The isolated compound
was characterized by .sup.1H NMR, COSY, NOESY and LCMS. .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta.11.29 (s, 1H, NH), 8.54 (s, 1H,
H-4), 8.00 (s, 1H, H-1), 6.24, 6.21 (2s, 1H, H-1'), 5.22-5.08 (m,
4H, 3.times.OH, H-8), 4.77-4.73 (m, 1H, H-9), 3.95-3.7 (m, 4H,
H-3', H-4' 2.times.H-5'), 3.24, 3.21 (2s, 3H, OCH.sub.3), 0.68,
0.66 (2s, 3H, CH.sub.3). ES MS: 501.7 (M+CH.sub.3COO.sup.-).
Example 15
4-Amino-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetra-
azabenzo[c,d]azulene-7-one (15.6)
##STR00052##
[0281] The sodium salt of
4-chloro-5-iodo-2-pivaloylamino-1H-pyrrolo[2,3-d]pyrimidine 15.1
(prepared in situ using sodium hydride) was reacted with protected
1-bromo-2-C-methyl-D-ribofuranose 2.4 (which was prepared with
HBr/AcOH in CH.sub.2Cl.sub.2 from the corresponding 1-O-methyl
analogue) to give the .beta.-anomer 15.2. Removal of
dichlorophenymethyl protecting groups was performed using boron
trichloride in CH.sub.2Cl.sub.2 to give the 4-chloro-nucleoside
15.3. Further ammonolysis and deprotection at elevated temperature
yielded 2,4-diamino nucleoside 15.4, which was converted under the
Heck coupling conditions with methylacrylate into the corresponding
5-methylpropenoate 15.5. This compound was converted into the
target tetraazabenzo[cd]azulene nucleoside 15.6 via sodium
methoxide mediated ring closure.
Example 15
Step A:
4-Chloro-5-iodo-2-pivaloylamino-7-[3,5-bis-O-(2,4-dichlorophenymet-
hyl)-2-C-methyl-.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
(15.2)
[0282] A solution of 2.4 (36.7 mmol) in anhydrous acetonitrile (50
mL) was added to a solution of sodium salt of
4-chloro-5-iodo-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine in
acetonitrile [generated in situ from
4-chloro-5-iodo-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine (Nucl.
Acid Res. 1998 (26), 3350-3357) (20.87 g, 55.1 mmol) in anhydrous
acetonitrile (1000 mL) and NaH (60% in mineral oil, 2.20 g, 55.1
mmol) after 4 hr of vigorous stirring at room temperature]. The
combined mixture was stirred at room temperature for 48 hr. The
solids were filtered then washed with acetonitrile (100 mL) and the
combined filtrate evaporated to provide a viscous oil. Purification
on a silica gel column, using hexanes/EtOAc gradient (15/1, 13/1,
11/1, 9/1, 7/1) as the eluent, yielded the target compound as a
colorless foam (7.02 g, 23%).
Example 15
Step B:
4-Chloro-5-iodo-2-pivaloylamino-7-(2-C-methyl-.beta.-D-ribofuranos-
yl)-7H-pyrrolo[2,3-d]pyrimidine (15.3)
[0283] To a solution of compound 15.2 (7.03 g, 8.43 mmol) in
CH.sub.2Cl.sub.2 (200 mL) at -75.degree. C. was added boron
trichloride (1M in CH.sub.2Cl.sub.2; 83.4 mL, 83.4 mmol). The
mixture was stirred at -75 to -70.degree. C. for 2 hr and then at
-30 to -20.degree. C. for 3 hr. The reaction was quenched by
addition of MeOH/CH.sub.2Cl.sub.2 (1/1, 9 mL) and the resulting
mixture stirred at -20 to -15.degree. C. for 30 min., then
neutralized with aq. ammonia (28%, 35 mL) at 0.degree. C. and
stirred at room temperature for 10 min. The solid which separated
was filtered and washed with MeOH/CH.sub.2Cl.sub.2 (1/1, 500 mL).
The combined filtrates were evaporated and the residue was purified
on a silica gel column using CH.sub.2Cl.sub.2/MeOH (50/1, 40/1) as
the eluents to furnish the target compound 15.3 as an off-white
solid (2.93 g, 80%).
Example 15
Step C:
2,4-Diamino-5-iodo-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrol-
o[2,3-d]pyrimidine (15.4)
[0284] A mixture of the compound from Step B (2.92 g, 5.6 mmol) and
anhydrous liquid ammonia (50 mL) was heated in a stainless steel
autoclave at 110.degree. C. for 1 d, then cooled and the solvent
evaporated. The residue was treated with MeOH to yield 0.30 g of
15.4. The filtrate was evaporated and purified on silica gel column
with CH.sub.2Cl.sub.2/MeOH (20/1) to famish additional 1.52 g of
the target compound (total yield 77%).
Example 15
Step D:
2,4-Diamino-5-[(E)-1-(methoxycarbonyl)-2-ethenyl]-7-(2-C-methyl-.b-
eta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine (15.5)
[0285] To a solution of the compound from Step C (1.54 g, 3.66
mmol) in DMF (35 mL) were added copper iodide (139 mg, 0.73 mmol),
methyl acrylate (6.6 mL, 73.1 mmol), triethylamine (1.02 mL, 7.3
mmol), and tetrakis(triphenylphosphine)palladium[0] (422.5 mg, 0.37
mmol). The resulting mixture was stirred at 70.degree. C. for 10 h,
then cooled to room temperature and diluted with
MeOH/CH.sub.2Cl.sub.2 (1/1, 50 mL). Dowex HCO.sub.3.sup.- (3 g) was
added then and after 45 min of stirring, the resin was filtered
off, washed with CH.sub.2Cl.sub.2/MeOH (1/1, 150 mL) and the
combined filtrates concentrated. The residue was treated with MeOH
and the catalyst, which separated, was filtered off. The evaporated
filtrate was treated with MeOH again and the target compound, which
separated, was filtered off (627 mg). The filtrate was concentrated
in vacuo, and purified on a silica gel column using a
CH.sub.2Cl.sub.2/MeOH gradient (50/1, 30/1, 20/1 and 15/1) to
furnish an additional 175 mg of compound 15.5 (total yield
58%).
Example 15
Step E:
4-Amino-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5-
,6-tetraazabenzo[cd]azulene-7-one (15.6)
[0286] A solution of the compound from Step D (578 mg, 1.52 mmol)
in 0.1 N NaOMe/MeOH (250 mL) was heated at 60.degree. C. for 12 hr
and then neutralized at room temperature with Dowex H.sup.+. The
resin was filtered, washed with MeOH and the combined filtrates
concentrated in vacuo. Purification on a silica gel column with
CH.sub.2Cl.sub.2/MeOH (10/1 and 5/1) yielded the target compound
15.6 as a yellow solid (245 mg, 46%).
[0287] .sup.1H-NMR (DMSO-d.sub.6): .delta. 10.04 (br s, NH, 1H),
7.42 (s, 1H, H-1), 6.90 (d, H-9, J 11.7 Hz, 1H), 6.26 (br,
NH.sub.2, 2H), 5.91 (s, H-1', 1H), 5.56 (dd, J 11.7 Hz, J 1.6 Hz,
H-8, 1H), 5.21 (br s, 3'-OH, 1H), 5.06 (t, J 4.8 Hz, 5'-OH, 1H),
4.98 (s, 2'-OH, 1H), 3.75-3.88 (m, H-3', H-4', H-5', 3H), 3.62 (m,
H-5', 1H), 0.78 (s, Me, 3H). MS m/z=406.5
(M+CH.sub.3COO.sup.-).
Example 16
4-Fluoro-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetr-
aazabenzo[cd] azulene-7-one (16.2)
##STR00053##
[0289] Nucleoside 15.6 was converted with HF in pyridine and
tert-butylnitrite at low temperature into the corresponding
4-fluoro analogue 16.2, after 5'-O-tert-butyldimethylsilyl
derivatization with tert-butyldimethylsilyl chloride and imidazole
in DMF.
Example 16
Step A:
4-Amino-2-(5-O-tert-butyldimethylsilyl-2-C-methyl-.beta.-D-ribofur-
anosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-one
(16.1)
[0290] A mixture of
4-amino-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetr-
aazabenzo[cd]azulene-7-one (65 mg, 0.19 mmol) in DMF,
tert-butyldimethylsilyl chloride (70 mg, 0.45 mmol) and imidazole
(61 mg, 0.90 mmol) was stirred overnight at room temperature and
then concentrated in vacuo. The oily residue was dissolved in
CH.sub.2Cl.sub.2 (20 mL), washed with aq. HCl (0.1 N), sat. aq.
NaHCO.sub.3, water, brine and dried (Na.sub.2SO.sub.4). The
evaporated residue was purified on silica gel with hexanes/EtOAc
(1/1)+0.5% Et.sub.3N and EtOAc+0.5% Et.sub.3N to yield the target
compound 16.1 as colorless oil (38 mg, 44%).
Example 16
Step B:
4-Fluoro-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,-
5,6-tetraazabenzo[cd]azulene-7-one (16.2)
[0291] To a solution of HF/pyridine (1.5 mL) and pyridine (1 mL) at
-25.degree. C. was added a solution of 16.1 (30 mg, 0.07 mmol) in
pyridine (0.5 mL) followed by tert-butylnitrite (15 .mu.L, 0.13
mmol). Reaction mixture was allowed to warm to -5.degree. C.,
quenched with 6 N aq. NaOH and evaporated. The pale-yellow residue
was triturated with MeOH, filtered and thoroughly washed with MeOH
(100 mL combined filtrate). The evaporated filtrate was purified on
a silica gel coition with CH.sub.2Cl.sub.2/MeOH 20/1 to give the
target compound 16.2 as a yellow solid (5 mg, 22%).
[0292] .sup.1H-NMR (CD.sub.3OD): .delta. 7.05 (d, H-9,
J.sub.H8,H9=11.7 Hz, 1H), 7.81 (s, 1H, H-1), 6.09 (s, H-1', 1H),
5.78 (d, J.sub.H8,H911.7 Hz, H-8, 1H), 3.99-4.10 (m, H-4', H-5',
3H), 3.85 (dd, J.sub.gem, 12.9 Hz, J.sub.H5',H4' 3.5 Hz, H-5', 1H),
0.92 (s, Me, 3H). .sup.19F-NMR (CD.sub.3OD): .delta.-52.58. MS
m/z=409.6 (M+CH.sub.3COO.sup.-).
Example 17
7-Amino-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2H-2,3,5,6-tetraazabenzo[cd]-
azulene (17.2)
##STR00054##
[0294] Nucleoside 11.2 was reacted with acrylonitrile under
Heck-type coupling conditions. The nitrile 17.1 was converted into
the target 7-amino-tetraazabenzoazulen nucleoside 17.2 via sodium
methoxide mediated ring closure.
Example 17
Step A:
4-Amino-5-[(E/Z)-1-cyano-2-ethenyl]-7-(2-C-methyl-.beta.-D-ribofur-
anosyl)-7H-pyrrolo[2,3-d]pyrimidine (17.1)
[0295] To a solution of 11.2 (200 mg, 0.49 mmol) in DMF (5 mL) were
added copper iodide (19 mg, 0.1 mmol), acrylonitrile (0.65 mL, 9.8
mmol), triethylamine (0.137 mL, 0.99 mmol), and
tetrakis(triphenylphosphine)palladium[0] (57 mg, 0.05 mmol). The
resulting mixture was stirred at 70.degree. C. for 4 d., then
cooled to room temperature, diluted with MeOH/CH.sub.2Cl.sub.2
(1/1, 6 mL), and treated with Dowex HCO.sub.3.sup.- (0.5 g). After
1 hr stirring the resin was filtered off, washed with
CH.sub.2Cl.sub.2/MeOH (1/1, 50 mL) and combined filtrate
concentrated. The crude residue was purified on a silica gel column
with a CH.sub.2Cl.sub.2/MeOH gradient (50/1, 30/1, 10/1) to yield
the target stereoisomeric mixture (E/Z, 3/1) as yellow solid (75
mg, 46%).
Example 17
Step B:
7-Amino-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2H-2,3,5,6-tetraazab-
enzo[cd]azulene (17.2)
[0296] A mixture of the compound from Step A (75 mg, 0.23 mmol) in
0.1 N NaOMe/MeOH (18 mL) was heated at 60.degree. C. for 8 hr then
cooled to room temperature and evaporated in vacuo. Crude residue
was purified on a silica gel column with a CH.sub.2Cl.sub.2/MeOH
gradient (20/1, 10/1, 5/1) to yield the target compound 17.2 as
yellow solid (44 mg, 59%).
[0297] .sup.1H-NMR (DMSO-d.sub.6): .delta. 8.10 (s, H-4, 1H), 7.51
(s, 1H, H-1), 7.6-8.0 (2br, NH.sub.2, 2H), 6.85 (d, H-9,
J.sub.H8,H9=11.6 Hz, 1H), 6.01 (s, H-1', 1H), 5.52 (d, H-8,
J.sub.H8,H9 11.6 Hz, 1H), 5.11 (m, 2'-OH, 3'-OH, 5'-OH), 3.77-3.91
(m, H-3', H-4', H-5', 3H), 3.63 (m, H-5', 1H), 0.70 (s, Me, 3H). MS
m/z=390.8 (M+CH.sub.3COO.sup.-).
Example 18
7-Methoxy-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2H-2,3,5,6-tetraazabenzo[c-
d] azulene (18.3)
##STR00055##
[0299] Peracetylated nucleoside 18.1, prepared by treating the
compound 6.6 with acetic anhydride, triethylamine and DMAP in
acetonitrile and was reacted with trimethyloxonium
tetrafluoroborate in CH.sub.2Cl.sub.2 at ambient temperature to
furnish methoxy nucleoside 18.2. Removal of acetyl groups in MeOH
saturated with potassium carbonate yielded the target nucleoside
18.3.
Example 18
Step A:
2-(2,3,5-Tri-O-acetyl-2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihyd-
ro-7H-2,3,5,6-tetraazabenzo[cd]azulene-7-one (18.1).
[0300] To a solution of
2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenz-
o[cd]azulene-7-one 6.6 (136 mg, 0.41 mmol) in acetonitrile were
added acetic anhydride (0.71 mL, 7.5 mmol), triethylamine (1.05
mL), and DMAP (58 mg, 0.47 mmol). The mixture was stirred overnight
at room temperature, then evaporated and the residue partitioned
between water (75 mL) and CH.sub.2Cl.sub.2 (200 mL). The organic
layer was washed with brine and dried over Na.sub.2SO.sub.4. The
evaporated residue was treated with MeOH to yield the target
compound as yellow solid (150 mg, 80%).
Example 18
Step B:
7-Methoxy-2-(2,3,5-tri-O-acetyl-2-C-methyl-.beta.-D-ribofuranosyl)-
-2H-2,3,5,6-tetraazabenzo[cd]azulene-7-one (18.2).
[0301] A solution of the compound from Step A (50 mg, 0.11 mmol) in
CH.sub.2Cl.sub.2 (1 mL) and trimethoxyoxonium tetrafluoroborate (18
mg, 0.12 mmol) under argon was stirred at room temperature for 2 d.
At this point the reaction was quenched with sat. aq.
K.sub.2CO.sub.3 (1 mL) and the resulting mixture diluted with
CH.sub.2Cl.sub.2 (50 mL), washed with water, brine and dried over
Na.sub.2SO.sub.4. The evaporated residue was purified on a silica
gel column with CH.sub.2Cl.sub.2/MeOH (50/1) as the eluent to yield
the target compound 18.2 as yellow solid (29 mg, 56%).
Example 18
Step C:
7-Methoxy-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2H-2,3,5,6-tetraaz-
abenzo[cd]azulene (18.3).
[0302] A mixture of the compound from Step B (28 mg, 0.06 mmol) in
saturated methanolic K.sub.2CO.sub.3 (5 mL) was stirred at room
temperature for 30 min. and then concentrated in vacuo. The crude
evaporated residue was purified on a silica gel column with
CH.sub.2Cl.sub.2/MeOH (20/1) as the eluent to afford the target
compound 18.3 (15 mg, 72%) as a yellow solid.
[0303] .sup.1H-NMR (DMSO-d.sub.6): .delta. 8.30 (s, H-4, 1H), 7.68
(s, 1H, H-1), 6.83 (d, H-9, J.sub.H8,H9 11.6 Hz, 1H), 5.98 (s,
H-1', 1H), 5.84 (d, H-8, J.sub.H8,H9 11.4 Hz, 1H), 5.22 (s, 2'-OH,
1H), 5.17 (m, 3'-OH, 1H), 5.12 (t, 5'-OH, J.sub.5'OH,H5' 5.0 Hz,
1H), 3.78-3.88 (m, H-3', H-4', H-5', 3H), 3.65 (m, H-5', 1H), 3.49
(s, OMe, 3H), 0.72 (s, Me, 3H). MS m/z=405.9
(M+CH.sub.3COO.sup.-).
Example 19
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-2H-2,3,5,6-tetraazabenzo[cd]azulene--
4,7(3H,6H)-dione (19.1)
##STR00056##
[0305] To a solution
4-amino-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetr-
anzabenzo[cd]azulene-7-one 15.6 (35 mg, 0.1 mmol) in 50% aqueous
acetic acid (5 mL) was added sodium nitrite (42 mg, 0.6 mmol) and
the mixture stirred at room temperature for 4 hr. The mixture was
neutralized with 1M TEAB buffer and purified by reversed phase
ion-pairing HPLC on a Phenomenex Luna C18(2) 250.times.21 mm 10
.mu.m column. 100 mM triethylammonium acetate (TEAA), pH 7 was used
as the ion-pairing agent. A gradient of 20% to 55% MeOH over 40 min
was applied. The target compound eluted at 26 min followed by two
smaller peaks at 29 and 31 min. TEAA was removed by repeated
lyophilzation to yield the target compound as a fluffy yellow
material (28 mg, 80%).
[0306] .sup.1H-NMR (DMSO-d.sub.6): .delta. 11.0 (br s, 2NH, 2H),
7.53 (s, 1H, H-1), 6.95 (d, H-9, J.sub.H8,H9 11.7 Hz, 1H), 5.91 (s,
H-1', 1H), 5.62 (d, J.sub.H8,H9 11.7 Hz, H-8, 1H), 5.10 (br, 5'-OH,
3'-OH, 2'-OH, 3H), 3.77-3.89 (m, H-3', H-4', H-5', 3H), 3.63 (m,
H-5', 1H), 0.79 (s, Me, 3H). MS m/z 347.7 (M-1).
Example 20
4-Chloro-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetr-
aazabenzo[cd]azulen-7-One (20.8)
##STR00057##
[0308] The tricyclic nucleoside 20.8 was synthesized starting from
2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine 20.1. The nucleobase
20.1 was diazotized in the presence of copper chloride and the
resulting base 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine was treated
with N-iodosuccinimide in THF at room temperature to provide
2,4-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine 20.3. Then 20.3 was
converted to the corresponding sodium salt with sodium hydride in
acetonitrile and reacted with bromo-sugar 2.4, which was prepared
from
3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-1-O-methyl-.alpha.-D-ribo-
furanose ((i) Helv. Chim. Acta. 1995, 78, 486; (ii) WO 02/057287,
2002), to give nucleoside 20.4. The glycosylation product 20.4 was
allowed to react with ammonium hydroxide in dioxane at 100.degree.
C. provided 20.5. Methyl-cis-.beta.-(tributylstannyl)acrylate (J.
Am. Chem. Soc; 1993, 115, 1619) was coupled with compound 20.5
under Stille reaction conditions using PdCl.sub.2(PPh.sub.3).sub.2
and copper iodide to give Z-ester analog 20.6, which was further
reacted with DBU in dioxane to give protected tricycle 20.7.
Nucleoside 20.7 was treated with boron trichloride in
CH.sub.2Cl.sub.2 to afford tricyclic nucleoside 20.8.
Example 20
Step A: 2-Amino-4-chloro-5-iodo-1H-pyrrolo[2,3-d]pyrimidine
(20.2)
[0309] Compound 20.2 was prepared as described in Seela, F., at
al., Liebigs Ann. Chem., 1985, 312-320.
Example 20
Step B: 2,4-Dichloro-5-iodo-1H-pyrrolo[2,3-d]pyrimidine (20.3)
[0310] Compound 20.2 (3.80 g, 20.0 mmol) was dissolved in THF (200
mL) and cooled to -20.degree. C. for 20 min. N-Iodosuccinimide (7.0
g, 30.0 mmol) was slowly added and the resulting mixture was
stirred at room temperature. After 2 h, the mixture was evaporated
to dryness and the residue was re-dissolved in ethyl acetate,
washed with 5% sodium thiosulphate, saturated sodium chloride
solution and then dried over sodium sulfate and evaporated to
dryness. The crude product was purified by silica gel column
chromatography using 20% ethyl acetate in hexane to give 4.6 g of
compound 20.3 as a yellowish solid.
Example 20
Step C:
2,4-Dichloro-5-iodo-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-me-
thyl-.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine (20.4)
[0311] A solution of 2.4 ((i) Helv. Chim. Acta. 1995, 78, 486; (ii)
WO 02/057287, 2002) (8.8 g, 20.0 mmoles) in anhydrous acetonitrile
(300 mL) was added to a solution of the sodium salt of
4,6-dichloro-5-iodo-7H-pyrrolo[2,3-d].sub.p' yrimidine [generated
in situ from 4,6-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (3.1
g, 10.0 mmol) in anhydrous acetonitrile (100 mL), and NaH (60% in
mineral oil, 0.90 g, 37.0 mmol), after 4 hr of vigorous stirring at
room temperature]. The combined mixture was stirred at room
temperature for 40 hr, and then evaporated to dryness. The mixture
was filtered through a celite plug and the solid residue was
thoroughly washed with 500 mL of acetonitrile. The filtrates were
evaporated to dryness and the crude product was purified on a
silica gel column using 25% ethyl acetate in hexane to give 2.8 g
of the desired product 20.4 as a white foam.
Example 20
Step D:
4,2-Dichloro-5-iodo-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-me-
thyl-.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine (20.5)
[0312] The material from Step C (2.5 g, 2.5 mmol) in dioxane (50
mL) was placed in a pressure vessel and aqueous ammonium hydroxide
(50 mL) was added. The mixture was tightly sealed and heated to
100.degree. C. for 2 hr. After the reaction, the mixture was
evaporated to dryness and the crude product was purified using
silica gel column chromatography with 5-10% MeOH in
CH.sub.2Cl.sub.2 as eluent to give 2.10 g of the pure desired
product 20.5.
Example 20
Step E:
4-Amino-2-chloro-5-[1-(methoxycarbonyl)-2-ethenyl]-7-[3,5-bis-O-(2-
,4-dichlorophenylmethyl)-2-C-methyl-.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-
-d]pyrimidine (20.6).
[0313] To the material from Step D (2.1 g, 1.90 mmol) in 100 mL of
anhydrous DMF was added CuI (83.80 mg, 0.44 mmol),
methyl-cis-.beta.-(tributylstannyl)acrylate (1.5 mL, 4.4 mmol) and
PdCl.sub.2(PPh.sub.3).sub.2 (150.0 mg, 0.22 mmol) at room
temperature under an argon atmosphere. The reaction mixture was
heated at 70.degree. C. for 24 hr then cooled to room temperature
and filtered through a celite plug. The filtrate was evaporated to
dryness and the crude product purified on a silica gel column using
5-30% THF in CH.sub.2Cl.sub.2 as eluent to give 2.2 g of pure the
desired ester 20.6.
Example 20
Step F:
2-[3,5-Bis-O-(2,4-dichlorophenylmethyl)2-C-methyl-.beta.-D-ribofur-
anosyl]-4-chloro-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one
(20.7)
[0314] To a solution of compound 20.6 (1.9 g, 2.5 mmol) in dioxane
(40 mL) was added DBU (1.3 mL, 9.0 mmol). The mixture was heated at
reflux for 2 hr and then evaporated to dryness. The crude product
was purified by silica gel column chromatography using 5-10% MeOH
in CH.sub.2Cl.sub.2 as eluent to give 1.7 g of pure product
20.7.
Example 20
Step G:
4-Chloro-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,-
5,6-tetraazabenzo[cd]azulen-7-one (20.8)
[0315] To a solution of compound 20.7 obtained from Step F (1.7 mg,
2.4 mmol) in CH.sub.2Cl.sub.2 (200 mL) at -78.degree. C. was added
boron trichloride (1M in CH.sub.2Cl.sub.2, 25 mL, 25.0 mmol),
dropwise. The mixture was stirred at -78.degree. C. for 2.5 hr,
then at -30.degree. C. to -20.degree. C. for 3 hr. The reaction was
quenched by addition of MeOHic/CH.sub.2Cl.sub.2 (1:1) (50 mL) and
the resulting mixture stirred at -15.degree. C. for 30 minutes,
then neutralized with aqueous ammonia at 0.degree. C. and stirred
at room temperature for 15 minutes. The mixture was evaporated to
dryness and the residue was purified by silica gel column
chromatography using 5-20% ethanol in CH.sub.2Cl.sub.2 as eluent to
give 560 mg of pure yellowish tricyclic product 20.8.
[0316] .sup.1H NMR (DMSO-d.sub.6) .delta. 11.04 (d, J 1.5 Hz, 1H,
NH), 7.89 (s, 1H, H-6), 7.04 (d, J 11.7 Hz, H1''), 5.96 (s, 1H,
H-1'), 5.69 (dd, J 11.7, 1.5 Hz, H2''), 5.16 (m, 3H, 3.times.OH),
3.88-3.32 (m, 4H, H-3', H-4', 2.times.H-5'), 0.76 (s, 3H,
CH.sub.3).
[0317] Tricyclic nucleoside 20.8 was found to be a suitable
intermediate for the synthesis of C-4-functionalized tricyclic
nucleosides. Nucleoside 20.8 was reacted with sodium thiomethoxide
in DMF at elevated temperature. Two products were isolated in a
ratio of 1:1. These products were separated using reverse phase
HPLC and characterized as tricyclic nucleosides 21.1 and 21.2 using
.sup.1H NMR and LCMS analysis. Compound 20.8 was treated with
tert-butyldimethylsilyl chloride and imidazole in DMF at room
temperature to give 5'-TBDMS protected tricyclic nucleoside 21.3.
Nucleophilic displacement reaction of compound 21.3 using 2M
methylamine in TIM, followed by deprotection reaction using
tetrabutylammoniumfluoride (TBAF) afforded 4-methylamino derivative
21.4. In a similar fashion, 21.3 was reacted with sodium methoxide
in MeOH at reflux and the resulting product was deprotected with
TBAF to give 4-OMe analog 21.5.
Examples 21 and 22
2-(2-C-methyl-.beta.-D-ribofuranosyl)-4-methylthio-2,6-dihydro-7H-2,3,5,6--
tetraazabenzo[cd]azulen-7-one (21.1) and
2-(2-C-methyl-.beta.-D-ribofuranosyl)-4,9-di(methylthio)-2,6,8,9-tetrahyd-
ro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one (21.2)
##STR00058##
[0319] To a solution of the compound obtained from Example 20
(20.8) (100.0 mg, 0.30 mmol) in DMF (10 mL) was added sodium
thiomethoxide (105 mg, 1.5 mmol). The mixture was stirred at
120.degree. C. for 24 hr. After evaporation of the DMF under
reduced pressure, the residue was purified on a silica gel column
using 5-12% MeOH in CHCl.sub.3 to give a mixture of two products
(1:1). These two products were separated by reverse phase HPLC to
give 25 mg of 21.1 and 30 mg of 21.2.
[0320] For Example 21 (21.1): .sup.1H NMR (CD.sub.3OD) .delta. 7.70
(s, 1H, H-6), 7.00 (d, J 12.0 Hz, H1''), 6.19 (s, 1H, H-1'), 5.72
(d, J 12.0 Hz, H2''), 4.07-3.80 (m, 4H, H-3', H-4', 2.times.H-5'),
2.56 (s, 3H, SCH.sub.3), 0.91 (s, 3H, CH.sub.3).
[0321] For Example 22 (21.2): .sup.1H NMR (CD.sub.3OD) .delta. 7.54
(s, 1H, H-6), 6.29 (s, 1H, 2.times.H1'), 4.51-3.79 (m, 5H, H-3',
H-4', 2.times.H-5', CH), 3.21 (m, 2H, CH.sub.2), 2.59 (s, 3H,
SCH.sub.3), 2.14 (2.times.s, 3H, SCH.sub.3), 0.88 (2.times.s, 3H,
2.times.CH.sub.3).
Example 23
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-4-methylamino-2,6-dihydro-7H-2,3,5,6-
-tetraazabenzo[cd]azulen-7-one (21.4)
4-Chloro-2-(5-O-tert-butyldimethylsilyl-2-C-methyl-.beta.-D-ribofuranosyl)-
-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one (21.3)
[0322] To a solution of compound 20.8 (366 mg, 1.0 mmol) and
imidazole (68.0 mg, 1.0 mmol) in DMF (20 mL) was added
tert-butyldimethylsilylchloride (150.7 mg, 1.0 mmol). The mixture
was stirred at ambient temperature for 6 hr under inert atmosphere
and then treated with saturated sodium bicarbonate solution and
extracted with ethyl acetate, dried over sodium sulfate, and
evaporated to dryness. The crude product was purified using silica
gel column chromatography using 2-5% MeOH in CHCl.sub.3 to give 380
mg of the desired product 21.3.
[0323] .sup.1H NMR (CDCl.sub.3) .delta. 7.68 (s, 1H, H-6), 7.24 (d,
J 11.4 Hz, H1''), 6.83 (s, 1H, H-1'), 5.85 (d, J 11.4 Hz, H2''),
4.14-3.72 (m, 4H, H-3', H-4', 2.times.H-5'), 0.99 (s, 12H,
CH.sub.3, (CH.sub.3).sub.3), 0.18 (s, 6H, 2.times.CH.sub.3)
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-4-methylamino-2,6-dihydro-7H-2,3,5,6-
-tetraazabenzo[cd]azulen-7-one (21.4)
[0324] Compound 21.3 (100 mg, 0.20 mmol) was added to 2M
methylamine solution in THF (25 mL) in a pressure vessel. The
vessel was tightly sealed and heated at 90.degree. C. for 8 hr.
After evaporation of the solvent and excess amine, the residue was
re-dissolved in THF (20 mL). To this solution, tetrabutylammonium
fluoride in THF (2 mL) was added and the solution stirred at room
temperature for 4 hr. After careful evaporation of the solvent, the
residue was purified on a silica gel column using 5-7% MeOH in
CHCl.sub.3 as eluent to give 46 mg of desired yellowish
product.
[0325] .sup.1H NMR (DMSO-d.sub.6) .delta. 10.05 (br s. 1H, NH),
7.42 (s, 1H, H-6), 6.89 (d, J 11.7 Hz, H1''), 6.65 (br s, 1H, NH),
5.93 (s, 1H, H-1'), 5.61 (d, J 11.7 Hz, H2''), 5.19-5.00 (m, 3H,
3.times.OH), 3.90-3.3.63 (m, 4H, H-3', H-4', 2.times.H-5'), 3.32,
(s, 3H, NCH.sub.3), 0.80 (s, 3H, CH.sub.3).
Example 24
4-Methoxy-2-(2-C-methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tet-
raazabenzo[cd]azulen-7-one (21.5)
[0326] To a solution of compound 21.3 (100.0 mg, 0.20 mmol) in
anhydrous MeOH (25 mL) was added freshly prepared sodium methoxide
(540.0 mg, 10.0 mmol). The resulting homogeneous solution was
heated to reflux for 24 hr and then neutralized with DOWEX resin
and filtered. The neutral methanolic solution was evaporated and
the residue was re-dissolved in THF. To this solution,
tetrabutylammonium fluoride in THF (2 mL) was added and the mixture
was stirred at room temperature for 4 hr. After careful evaporation
of the solvent, the residue was purified on a silica gel column to
give 38 mg of the desired yellowish product.
[0327] .sup.1H NMR (CD.sub.3OD) .delta. 7.64 (s, 1H, H-2), 7.00 (d,
J 11.7 Hz, H1''), 6.129 (s, 1H, H-1'), 5.73 (d, J 11.7 Hz, H2''),
3.81-4.13 (m, 7H, H-3', H-4', 2.times.H5', OCH.sub.3), 0.92 (s, 3H,
CH.sub.3).
Example 25
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo-
[cd]azulene-7-thione (23.2)
##STR00059##
[0328] Example 25
Step A:
2-(2-C-Methyl-2,3,5-tri-O-acetyl-.beta.-D-ribofuranosyl-2,6-dihydr-
o-7H-2,3,5,6-tetraazabenzo[cd] azulene-7-thione (23.1).
[0329] Compound 18.1 (250 mg, 0.54 mmol) was dissolved in dioxane
(5 mL) and pyridine (7 mL) was added, followed by phosphorus
pentasulfide (242 mg, 0.5 mmol). The mixture was heated at reflux
for 24 hr then the solvent was evaporated and the residue was
washed with pyridine (3.times.4 mL). The combined washings were
evaporated and the residue was dissolved in 50 mL CHCl.sub.3 and
washed with 30 mL 10% aqueous sodium bicarbonate followed by water.
The organic phase was dried over anhydrous sodium sulfate,
filtered, evaporated and the residue (180 mg) containing 23.1 was
used immediately in the next step.
Example 25
Step B:
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetra-
azabenzo[cd]azulene-7-thione (23.2).
[0330] To a suspension of compound 23.1 (180 mg, 0.38 mmol) in 4 mL
ethanol was added 0.22 mL of 1N sodium hydroxide solution in water.
The mixture was stirred at room temperature for 1.5 hr. after which
time the pH was brought to 6 with acetic acid and the solvent
evaporated. The residue was purified on silica gel column (10:1
CH.sub.2Cl.sub.2:MeOH) to afford 30 mg of product 23.2. .sup.1H NMR
(DMSO-d.sub.6) .delta. 10.49 (s, 1H), 8.52 (s, 1H), 7.50 (s, 1H),
7.00 (d, J 12 Hz, 1H), 6.08 (s, 1H), 5.65 (d, J 12 Hz, 1H), 5.18
(m, 3H), 3.82 (m, 3H), 3.68 (m, 1H), 0.73 (s, 3H).
Example 26
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-6,7-dihydro-2H-2,3,5,6-tetraazabenzo-
[cd]azulene (24.2)
##STR00060##
[0331] Example 27
Step A:
2-[3,5-Bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-.beta.-D-ribofu-
ranosyl]-6,7-dihydro-2H-2,3,5,6-tetraazabenzo[cd]azulene (24.1)
[0332] To compound 6.2 (372 mg, 0.5 mmol) and tri-n-butyltin
allylamine (prepared according to the literature procedure in
Corriu et. al. Journal of Organic Chemistry 1993, 58, 1443-1448) in
10 mL anhydrous toluene was added tetrakis(triphenylphosphine)
palladium[0] and the mixture was heated at reflux for 5 hr. The
solvent was evaporated and the residue dissolved in
CH.sub.2Cl.sub.2 and loaded on a silica gel column and eluted
successively with 75:1, 60:1, 40:1 CH.sub.2Cl.sub.2:MeOH. Pooling
and evaporation of the fractions afforded 160 mg of product
24.1.
Example 27
Step B:
2-(2-C-Methyl-.beta.-D-ribofuranosyl)-6,7-dihydro-2H-2,3,5,6-tetra-
azabenzo[cd]azulene (24.2).
[0333] To compound 24.1 (160 mg, 0.25 mmol) in CH.sub.2Cl.sub.2 (10
mL) at -78.degree. C. was added 1M solution of boron trichloride in
CH.sub.2Cl.sub.2 dropwise over 5 minutes and the solution stirred
at -78.degree. C. for 2.5 h and then at -25.degree. C. for 3 hr. To
this mixture was added 25 mL of 1:1 v/v CH.sub.2Cl.sub.2:MeOH and
the solution stirred at -15.degree. C. for 30 minutes. The mixture
was brought to room temperature and the solvent was evaporated
under reduced pressure. The residue was co-evaporated with MeOH
(5.times.10 mL) and a 10 mL MeOH solution was neutralized with
NH.sub.4OH and evaporated again. The residue was adsorbed on 2 g
silica gel and loaded on a silica column and eluted successively
with 50:1, 20:1 and 15:1 CH.sub.2Cl.sub.2:MeOH. Fractions eluting
at 20:1 and 15:1 were collected. Pooling of fraction and
evaporation gave 16 mg product 24.2. .sup.1H NMR (DMSO-d.sub.6)
.delta. 8.10 (s, 1H), 7.51 (s, 1H), 7.30 (t, 1H), 6.61 (d, J 9 Hz,
1H), 6.11 (s, 1H), 5.71 (dt, J 6, 12 Hz, 1H), 5.08 (m, 3H), 3.86
(m, 5H), 3.78 (m, 1H), 0.72 (s, 3H).
Example 27
9-Methoxy-2-(2-C-methyl-.beta.-D-ribofuranosyl)-6,7,8,9-tetrahydro-2H-2,3,-
5,6-tetraazabenzo[cd]azulene (25.1).
##STR00061##
[0335] The slower moving fraction from Step B in Example 27, Step B
was isolated to afford 8 mg of ether 25.1. .sup.1H NMR
(DMSO-d.sub.6) .delta. 8.00 (s, 1H), 7.53 (m, 3H), 6.11 (s, 1H),
5.11 (m, 4H), 4.43 (m, 1H), 3.97 (m, 1H), 3.84 (m, 3H), 3.66 (m,
2H), 3.26 (s, 3H), 0.68 (s, 3H).
Example 28
Nucleoside Monophosphates
[0336] To the compound appropriate nucleoside (0.156 mmol) (dried
over P.sub.2O.sub.5 in vacuo overnight) was added trimethyl
phosphate (1.5 mL). The mixture was stirred overnight in a sealed
container containing 4 A molecular sieves. It was then cooled to
0.degree. C. and phosphorous oxychloride (35.8 .mu.L, 2.5 eq.) was
added via syringe. The mixture was stirred for 3 hr at 0.degree.
C., then the reaction was quenched by addition of
tetraethylammonium bicarbonate (TEAB) (1M) (1.5 mL) and water (15
mL). The aqueous solution was washed with CHCl.sub.3 and ether then
lyophilized. The crude product was purified by HPLC using a C18
column with water and 5% acetonitrile in water to provide the
monophosphate as a triethylammonium salt after lyophilization.
Example 29
5'-p-Phenyl methoxyalaninylphosphate prodrugs
##STR00062##
[0338] To a solution of compound the appropriate nucleoside (0.6
mmol) in anhydrous THF (5 mL) was added phenyl
methoxyalaninylphosphorochloridate (40 mg, 5 eq.) (freshly prepared
following the literature procedure: J. Med. Chem. 1993, 36,
1048-1052 and Antiviral Research, 1999, 43, 37-53) and
1-methylimidazole (95 .mu.L, 10 eq.) at room temperature under
argon. The reaction was followed by TLC. After 36 hr, the reaction
mixture was evaporated and the residue was purified on silica gel
with 0-10% MeOH in CH.sub.2Cl.sub.2 as eluent to provide a 1:1
mixture of diastereomers.
Example 30
2-[5-O-Bis(pivaloyloxymethyl)phosphoryl-prodrugs
##STR00063##
[0340] To a solution of triethylammonium salt of compound
nucleoside monophosphate (0.024 mmol) in anhydrous MeOH (0.5 mL)
was added tributylstannyl methoxide (14 .mu.L, 2 eq.) at room
temperature under argon. The reaction mixture was stirred at room
temperature for 30 min then evaporated and co-evaporated with
acetonitrile three times. The residue was dissolved in anhydrous
acetonitrile (3 mL) and tetrabutylammonium bromide (15.5 mg, 2 eq.)
and iodomethyl piovalate (58 mg, 10 eq) were added. The reaction
mixture was heated at reflux for 1 hr cooled to room temperature
and the solvent was evaporated. The residue was purified on a
silica gel column with 1-5% MeOH in CH.sub.2Cl.sub.2 to provide the
prodrug.
Example 31
Nucleoside Diphosphates
##STR00064##
[0342] To a solution of the triethylammonium salt of
5'-monophosphate (0.031 mmol) [dried by coevaporation with
anhydrous DMF twice (2.times.1 mL)] in 0.5 mL of anhydrous DMF was
added N,N'-carbonyldiimidazole (25 mg, 5 eq.) at room temperature
under argon. The reaction mixture was stirred at room temperature
for 4 hr after which analytical TLC showed no starting material.
Then tributylammonium phosphate salt (1.5 n-Bu.sub.3N/phosphate,
which was prepared (see PCT, WO 88/03921) and further dried by
coevaporation with anhydrous DMF three times) was added to the
above solution. The reaction was followed by TLC and typically
after 3 days, LC-MS showed significant (>50%) conversion to
product. The reaction was quenched with 1 mL of triethylamine, 1 mL
of water, and stirred at room temperature for 40 min. The crude
product was purified by reverse phase HPLC to provide pure product
29.1.
Examples 32-42
Nucleoside 5'-Triphosphates
[0343] To an ice-cold mixture of nucleoside (0.1 mmol) in trimethyl
phosphate (1 mL, anhydrous) was added POCl.sub.3 (18.6 .mu.L, 0.2
mmol) and the mixture stirred at 0.degree. C. for 1 h. tributyl
amine (71.5 .mu.L, 0.3 mmol) was added, followed by acetonitrile
(0.1 mL, anhydrous) and tributylammonium pyrophosphate (182 mg, 0.4
mmol). After 30 min. the reaction was quenched with ice-cold 1M
triethylammonium bicarbonate buffer (5 mL, 1M, pH 8.5). The
products were purified by HPLC.
TABLE-US-00002 Calculated Observed Example Molecular m/z Number
Structure Weight [M - H].sup.- 32 ##STR00065## 572.251 571.6 33
##STR00066## 573.239 572.0 34 ##STR00067## 562.26 561.9 35
##STR00068## 560.284 559.9 36 ##STR00069## 572.251 571.8 37
##STR00070## 587.266 586.9 38 ##STR00071## 574.267 573.9 39
##STR00072## 542.225 541.1 40 ##STR00073## 590.241 589.8 41
##STR00074## 586.278 585.8 42 ##STR00075## 602.277 601.9
Example 43
Nucleoside-5'Triphosphate Mimic
2-(5-.alpha.-P.sub.I-borano-.beta.,.gamma.-difluoromethylene)triphosphono
2-C-methyl-.beta.-D-ribofuranosyl)-2,6,8,9-tetrahydro-7-oxa-2,3,5,6-tetra-
azabenzo[cd]azulene (43.2)
##STR00076##
[0344] Example 43
Step A
2-(2-C-methyl-2,30di-O-acetyl-.beta.-D-ribofuranosyl)-2,6,8,9-tetra-
hydro-7-oxa-2,3,5,6-tetraazabenzo[cd]azulene (43.1)
[0345] To a solution of compound 2.9 from example 2, step G (76 mg,
0.24 mmol) in pyridine (2.0 mL) was added
tert-butyldimethylsilylchloride (60 mg, 0.38 mmol). The mixture was
stirred at ambient temperature for 16 hr under inert atmosphere.
Added acetic anhydride (0.44 mL, 4.32 mmol), and stirred for 3 h at
room temperature. Added triethylamine (0.61 mL, 6.0 mmol), and DMAP
(35 mg, 0.29 mmol). The mixture was stirred overnight at room
temperature, then evaporated and the residue partitioned between
water (20 mL) and CH.sub.2Cl.sub.2 (60 mL). The organic layer was
dried over Na.sub.2SO.sub.4. Solvents were evaporated in vacuo. The
resulted crude product was dried on high vacuum for 2 h and
redissolved in THF (2.0 ml) and cooled to 0.degree. C. and added
1.0M solution of TBAF in THF (0.6 mmol) and stirred at 0.degree. C.
for 1 h. Reaction was quenched by adding absolute ethanol (2 ml),
and solvents were evaporated. The residue left was redissolved in
dichloromethane and washed with water. The organic portions were
dried over Na.sub.2SO.sub.4 and filtered, evaporated and crude
product was purified by silicagel column chromatography using 1-2%
methanol in dichloromethane to give 43.1 as light yellow foam (57
mg).
[0346] .sup.1H NMR (DMSO-d.sub.6) .delta. 8.57 (s, H-2, 1H), 7.70
(s, H-6, 1H), 6.62 (s, H-1', 1H), 5.39 (d J 5.7, H-3', 1H), 5.23
(t, J 5.7, 5'-OH, 1H), 4.52 (br s, OCH.sub.2CH.sub.2, 2H),
4.09-4.13 (m, H-4', 1H), 3.62-3.81 (m, H-5', 2H), 3.08-3.1 (m,
OCH.sub.2CH.sub.2, 2H), 2.51 (s, N--COCH.sub.3, 3H), 2.08, 2.04
(each s, 2.times.O--COCH.sub.3, 6H), 1.34 (s, CH.sub.3, 3H).
Example 43
Step B:
2-(5-.alpha.-P.sub.Iborano-.beta.,.gamma.-difluoromethylene)tripho-
sphono
2-C-methyl-.beta.-D-ribofuranosyl)-2,6,8,9-tetrahydro-7-oxa-2,3,5,6-
-tetraazabenzo[cd]azulene (43.2)
[0347] 2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one (29 mg, 0.15
mmol) was added to a stirred solution of 43.1 (43 mg, 0.1 mmol) in
anhydrous DMF (0.5 mL) and pyridine (0.1 mL) at 0.degree. C. under
argon. The reaction mixture was stirred at room temperature for 2
h, cooled with ice bath. Tributylamine (65 uL, 0.28 mmol) was
added, followed by addition of (difluoromethylene)diphosphonic acid
bis(tributylammonium) salt (89 mg, 0.15 mmol) in DMF (0.5 mL). The
reaction mixture was stirred at room temperature for 2 h and cooled
with ice. Borane-diisopropylethylamine complex (377 ul, 2.11 mmol)
was added, and the resulting mixture was stirred at room
temperature for 6 h, cooled with ice, and quenched by slow addition
of water (2 mL). The mixture was stirred at room temperature for 1
h, diluted with water (3 ml), extracted two times with chloroform
and aqueous portion was concentrated to about 2 ml. Aqueous ammonia
(33%) (2 ml) was added and stirred at room temperature for 10 h and
ammonia was evaporated and the remaining aqueous portion was
analysed by LCMS. LCMS showed the presence of two diastereoisomers
of 43.2. MS m/z 592.1 [M-H]
Example 44
HCV Replicon Assays
[0348] Doubling or 1/2-log dilutions of each compound were made in
DMSO, and aliquots were transferred to 96-well microplates to give
a final concentration range of 100-400 .mu.M downwards in the
presence of a constant concentration of 1% DMSO (ELISA and Reporter
methods) or 0.4% DMSO (hybridization method). The inhibitory
activity of these was assessed by three methods in Huh-7 cells
transfected with replicons coding for non-structural (NS) proteins
of HCV.
[0349] Replicon ELISA method: Huh-7 cells containing an HCV NS3
NS5b replicon were seeded into microplates containing compound
dilutions at a concentration of 20,000 cells per well. After 3 days
incubation the cell monolayers were washed and fixed with 1:1
acetone/MeOH. An ELISA was performed on the fixed cell sheets by
the sequential addition of HCV-specific monoclonal antibody,
horseradish peroxidase-conjugated secondary antibody and substrate
solution, with thorough washing between additions. The colour
development reaction was stopped with 12.5% sulphuric acid and the
plates read at 490 nm. The monolayers were then washed, dried and
stained with carbol fuchsin for microscopic assessment of
cytotoxicity.
[0350] Replicon reporter method: Huh-7 cells containing a replicon
expressing HCV NS3-NS5b plus a reporter gene were seeded into the
test microplates at a concentration of 15,000 cells per well. After
2 days incubation, the viability of the cells was assessed by the
addition of Resazurin (Sigma TOX-8) to all wells and reading the
plates at 595 nm 3 hours post addition. The signal from the
reporter gene product was measured immediately thereafter.
[0351] Replicon hybridization and cytotoxicity method: Huh-7 cells
containing an HCV NS3-NS5b replicon were seeded into microplates
containing compound dilutions at a concentration of 5,000 cells per
well. After 3 days incubation the media was replaced with MTS
solution and cytotoxicity was assessed by color development. After
reading the plates at 490 nm, the MTS solution was aspirated and
the cells were lysed and hybridized against HCV sequences using a
chemiluminescent readout.
[0352] Data analysis: The mean reading of duplicate wells at each
compound concentration was expressed as a percentage of the mean
value for compound-free control wells. Percentage inhibition was
plotted against concentration for each compound, and the 50%
inhibitory concentration (IC.sub.50) was calculated.
[0353] Compounds of Examples 1-27 were typically active in replicon
assays in the range of 5 to >1000 .mu.M
Example 45
[0354] Huh-7 and Vero Cells: The compounds were additionally
assessed for cytotoxicity in exponentially growing Huh-7 and Vero
cell cultures. Doubling dilutions of the compounds were made, as
described previously, and transferred to test microplates to give
final concentrations of 500 .mu.M downwards. Either Huh-7 cells or
Vero cells were added at concentrations of 12,000 and 3,000 cells
per well respectively. After incubation for 4 days at 37.degree.
C., MTT was added to all wells and the plates re-incubated for 2
hours. Acidified isopropanol was then added to all wells to lyse
the cells and dissolve any formazan that had been produced.
Absorbance was read at 570 nm, and the mean readings from duplicate
test wells were expressed as percentages of the mean readings from
compound free control wells. The 50% cytotoxic concentration
(CCID.sub.50) of each compound was calculated from the plot of
percentage cell survival against compound concentration.
[0355] HFF Cells: Cells were seeded into microtiter plates
containing IA-log dilutions of compounds at a concentration of
5,000 cells per well. After 3 days incubation the media was
replaced with MTS solution in media and cytotoxicity was assessed
by color development. Plates were read at 490 nm and CC.sub.50s
were calculated from percent inhibition as noted above.
[0356] Huh-7 and Vero Cells: The compounds were additionally
assessed for cytotoxicity in exponentially growing Huh-7 and Vero
cell cultures. Doubling dilutions of the compounds were made, as
described previously, and transferred to test microplates to give
final concentrations of 500 .mu.M downwards. Either Huh-7 cells or
Vero cells were added at concentrations of 12,000 and 3,000 cells
per well respectively. After incubation for 4 days at 37.degree.
C., MTT was added to all wells and the plates re-incubated for 2
hours. Acidified isopropanol was then added to all wells to lyse
the cells, and dissolve any formazan that had been produced.
Absorbance was read at 570 nm, and the mean readings from duplicate
test wells were expressed as percentages of the mean readings from
compound free control wells. The 50% cytotoxic concentration
(CCID.sub.50) of each compound was calculated from the plot of
percentage cell survival against compound concentration.
[0357] HFF Cells: Cells were seeded into microtiter plates
containing 1/2-log dilutions of compounds at a concentration of
5,000 cells per well. After 3 days incubation the media was
replaced with MTS solution in media and cytotoxicity was assessed
by color development. Plates were read at 490 nm and CC.sub.50s
were calculated from percent inhibition as noted above.
[0358] Huh-7 and Vero Cells: The compounds were additionally
assessed for cytotoxicity in exponentially growing Huh-7 and Vero
cell cultures. Doubling dilutions of the compounds were made, as
described previously, and transferred to test microplates to give
final concentrations of 500 .mu.M downwards. Either Huh-7 cells or
Vero cells were added at concentrations of 12,000 and 3,000 cells
per well respectively. After incubation for 4 days at 37.degree.
C., MTT was added to all wells and the plates re-incubated for 2
hours. Acidified isopropanol was then added to all wells to lyse
the cells and dissolve any formazan that had been produced.
Absorbance was read at 570 nm, and the mean readings from duplicate
test wells were expressed as percentages of the mean readings from
compound free control wells. The 50% cytotoxic concentration
(CCID.sub.50) of each compound was calculated from the plot of
percentage cell survival against compound concentration.
[0359] HFF Cells: Cells were seeded into microtiter plates
containing 1/2-log dilutions of compounds at a concentration of
5,000 cells per well. After 3 days incubation the media was
replaced with MTS solution in media and cytotoxicity was assessed
by color development. Plates were read at 490 nm and CC.sub.50s
were calculated from percent inhibition as noted above.
[0360] Compounds of Examples 1-27 were typically cytotoxic in the
range of 30 to >100 .mu.M.
Example 46
HCV Polymerase Inhibition Assay
[0361] The C-terminal his-tagged full-length HCV (Bartenschlager
1b) polymerase gene was cloned and expressed in Sf9 cells by
standard procedures. The enzyme was purified by nickel affinity
chromatography followed by S-Sepharose column chromatography.
Reactions contained 20 mM Tris HCl pH 7.0, 5 mM Hepes pH 7.0, 90 mM
NaCl, 12.5 mM MgCl.sub.2, 2% glycerol, 0.005% Triton X-100, 1.5 mM
DTT, 0.4 U/.mu.l RNasin, 20 .mu.g/ml RNA corresponding to 696
nucleotides of the 3' non-coding region of the HCV 1b genome, 2
.mu.M UTP (=K.sub.m), 0.02 .mu.Ci/.mu.l .sup.33P-labelled UTP, a
concentration equal to the K.sub.m of competing NTP (20 .mu.M ATP,
3 .mu.M GTP, or 0.5 .mu.M CTP), 500 .mu.M "non-competing" NTPs, and
100 nM HCV 1b polymerase (Bartenschlager, full length enzyme) in a
total volume of 25 .mu.l. Reactions were initiated with the
addition of enzyme and terminated after 2 hours with 5 .mu.l 0.5 M
EDTA. Stopped reactions were spotted onto either DEAF filter mats
or DEAF 96-well filter plates (Millipore). Unincorporated
nucleotides were washed from the filters. The filter mat was dried
and sealed in a bag together with 10 ml of OptiScint HiSafe
scintillation fluid. Filter plates were dried, and 75 ml OptiPhase
scintillation fluid was added to each well. The remaining
radioactivity was quantitated on a Wallac 1205 Betaplate counter or
Wallac 1240 MicroBeta plate counter.
[0362] Compounds of Examples 32-42 were typically inhibitory of
NS5B in the range of 100 to >1000 nM. Selected Examples were
more active and displayed IC.sub.50 values in the range of 30 to
100 nM.
* * * * *