U.S. patent application number 11/093648 was filed with the patent office on 2005-11-24 for antiviral agents and methods of treating viral infections.
This patent application is currently assigned to Vion Pharmaceuticals, Inc.. Invention is credited to Cheng, Yung-chi, Doyle, Terrance W., King, Ivan C., Sartorelli, Alan C., Sznol, Mario.
Application Number | 20050261251 11/093648 |
Document ID | / |
Family ID | 23094766 |
Filed Date | 2005-11-24 |
United States Patent
Application |
20050261251 |
Kind Code |
A1 |
King, Ivan C. ; et
al. |
November 24, 2005 |
Antiviral agents and methods of treating viral infections
Abstract
The present invention relates to methods of treating viral or
fungal infections using 3-aminopyridine-2-carboxyaldehyde
thiosemicarbazone (3-AP) and
3-amino-4-methylpyridine-2-carboxaldehyde thiosemicarbazone (3-AMP)
and its prodrug forms and to pharmaceutical compositions comprising
these compounds.
Inventors: |
King, Ivan C.; (North Haven,
CT) ; Doyle, Terrance W.; (Killingworth, CT) ;
Sznol, Mario; (Woodbridge, CT) ; Sartorelli, Alan
C.; (Woodbridge, CT) ; Cheng, Yung-chi;
(Woodbridge, CT) |
Correspondence
Address: |
Henry D. Coleman
714 Colorado Avenue
Bridgeport
CT
06605-1601
US
|
Assignee: |
Vion Pharmaceuticals, Inc.
New Haven
CT
Yale University
New Haven
CT
|
Family ID: |
23094766 |
Appl. No.: |
11/093648 |
Filed: |
March 30, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11093648 |
Mar 30, 2005 |
|
|
|
10126050 |
Apr 18, 2002 |
|
|
|
6911460 |
|
|
|
|
60285559 |
Apr 20, 2001 |
|
|
|
Current U.S.
Class: |
514/89 ;
514/352 |
Current CPC
Class: |
A61K 31/44 20130101;
Y02A 50/387 20180101; Y02A 50/393 20180101; Y02A 50/30 20180101;
Y02A 50/389 20180101; Y02A 50/385 20180101 |
Class at
Publication: |
514/089 ;
514/352 |
International
Class: |
A61K 031/675; A61K
031/44 |
Claims
1. A method of treating a viral infection in a patient comprising
administering to said patient a pharmaceutical composition
comprising an anti-viral effective amount of at least one compound
according to the structure: 3Where R.sup.1 is H or a
C.sub.1-C.sub.3 alkyl group, preferably H or CH.sub.3; R.sup.2 is H
or CO.sub.2R.sup.3; R.sup.3 is CHRR' or 4where R is H, CH.sub.3,
CH.sub.2CH.sub.3, CH.sub.2CH.sub.2CH.sub.3, i-propyl; R' is a free
acid phosphate, phosphate salt or S--S--R" group; R" is
CH.sub.2CH.sub.2NHR.sup.4, CH.sub.2CH.sub.2OH, CH.sub.2COOR.sup.5,
ortho- or para-substituted C.sub.1-C.sub.3 alkylphenyl or ortho or
para nitrophenyl; R.sup.4 is H or a C.sub.1-C.sub.18 acyl group
(preferably, a C.sub.1-C.sub.4 group), benzoyl, or a substituted
benzoyl group; R.sup.5 is H, a C.sub.1-C.sub.18 alkyl group
(preferably, a C.sub.1-C.sub.3 group), phenyl, substituted phenyl,
benzyl, or a substituted benzyl group; R.sup.6, R.sup.7, R.sup.8,
R.sup.9 and R.sup.10 are independently selected from H, a free acid
phosphate, a phosphate salt, or an S--S--R" group, a
C.sub.1-C.sub.3 alkyl group, F, Cl, Br, I, OCH.sub.3, OCF.sub.3,
CF.sub.3, NO.sub.2, CN, SO.sub.2CF.sub.3, SO.sub.2CH.sub.3,
COOCH.sub.3, SF.sub.5, COCH.sub.3, NH.sub.2, N(CH.sub.3).sub.2,
SCH.sub.3 or OH; With the proviso that when any two of R.sup.6,
R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are other than H, the other
of R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are H, and with
the proviso that no more than one of R.sup.6, R.sup.7, R.sup.8,
R.sup.9 and R.sup.10 is a free acid phosphate, a phosphate salt or
S--S--R".
2-4. (canceled)
5. The method according to claim 1 wherein R.sup.1 and R.sup.2 are
H.
6. The method according to claim 1 wherein said viral infection is
caused by an agent selected from the group consisting of human
immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human T-cell
leukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytial
virus (RSV), human papilloma virus (HPV), adenovirus, hepatitis B
virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV),
varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex
viruses 1 and 2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8,
also known as Kaposi's sarcoma-associated virus) and flaviviruses,
including Yellow Fever virus, Dengue virus, Japanese Encephalitis
and West Nile viruses.
7. A method of treating a viral infection comprising administering
in combination, at least one compound according to the structure:
5Where R.sup.1 is H or a C.sub.1-C.sub.3 alkyl group, preferably H
or CH.sub.3; R.sup.2 is H or CO.sub.2R.sup.3; R.sup.3 is CHRR' or
6where R is H, CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, i-propyl; R' is a free acid phosphate,
phosphate salt or S--S--R" group; R" is CH.sub.2CH.sub.2NHR.sup.4,
CH.sub.2CH.sub.2OH, CH.sub.2COOR.sup.5, ortho- or para-substituted
C.sub.1-C.sub.3 alkylphenyl or ortho or para nitrophenyl; R.sup.4
is H or a C.sub.1-C.sub.18 acyl group (preferably, a
C.sub.1-C.sub.4 group), benzoyl, or a substituted benzoyl group;
R.sup.5 is H, a C.sub.1-C.sub.18 alkyl group (preferably, a
C.sub.1-C.sub.3 group), phenyl, substituted phenyl, benzyl, or a
substituted benzyl group; R.sup.6, R.sup.7, R.sup.8, R.sup.9 and
R.sup.10 are independently selected from H, a free acid phosphate,
a phosphate salt, or an S--S--R" group, a C.sub.1-C.sub.3 alkyl
group, F, Cl, Br, I, OCH.sub.3, OCF.sub.3, CF.sub.3, NO.sub.2, CN,
SO.sub.2CF.sub.3, SO.sub.2CH.sub.3, COOCH.sub.3, SF.sub.5,
COCH.sub.3, NH.sub.2, N(CH.sub.3).sub.2, SCH.sub.3 or OH; With the
proviso that when any two of R.sup.6, R.sup.7, R.sup.8, R.sup.9 and
R.sup.10 are other than H, the other of R.sup.6, R.sup.7, R.sup.8,
R.sup.9 and R.sup.10 are H, and with the proviso that no more than
one of R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is a free
acid phosphate, a phosphate salt or S--S--R" and at least one
anti-viral agent which inhibits the growth or replication of
viruses by a mechanism other than by inhibition of viral
ribonucleotide reductase.
8-10. (canceled)
11. The method according to claim 7 wherein R.sup.1 and R.sup.2 are
H.
12. The method according to claim 7 wherein said viral infection is
caused by an agent selected from the group consisting of human
immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human T-cell
leukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytial
virus (RSV), human papilloma virus (HPV), adenovirus, hepatitis B
virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV),
varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex
viruses 1 and 2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8,
also known as Kaposi's sarcoma-associated virus) and flaviviruses,
including Yellow Fever virus, Dengue virus, Japanese Encephalitis
and West Nile viruses.
13. The method according to claim 7 wherein said anti-viral agent
is selected from the group consisting of acyclic nucleosides,
interferons, reverse transcriptase inhibitors, nucleoside transport
inhibitors 2',3'-dideoxynucleosides, 3TC, AZT,
2',3'-dideoxycytidine, 2',3'-dideoxyadenosine,
2',3'-dideoxyinosine, 2',3'-dideoxythymidine,
2',3'-dideoxy-2',3'-didehydrothymidine and
2',3'-dideoxy-2',3'-didehydroc- ytidine, .beta.-LFddC,
.beta.-LFd4C, .beta.-Ld4C, tenofovir DF, adefovir, dipivoxil,
immunomodulators such as interleukin II (IL2) and granulocyte
macrophage colony stimulating factor (GM-CSF), erythropoetin,
ampligen, thymodulin, thymopentin, foscarnet, ribavirin and
inhibitors of HIV binding to CD4 receptors such as soluble CD4, CD4
fragments, CD4 hybrid molecules and glycosylation inhibitors such
as 2-deoxy-D-glucose, castanospermine and 1-deoxynojirimycin.
14. (canceled)
15. A pharmaceutical composition comprising an anti-viral effective
amount of at least one compound according to the structure: 7Where
R.sup.1 is H or a C.sub.1-C.sub.3 alkyl group, preferably H or
CH.sub.3; R.sup.2 is H or CO.sub.2R.sup.3; R.sup.3 is CHRR' or
8where R is H, CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, i-propyl; R' is a free acid phosphate,
phosphate salt or S--S--R" group; R" is CH.sub.2CH.sub.2NHR.sup.4,
CH.sub.2CH.sub.2OH, CH.sub.2COOR.sup.5, ortho- or para-substituted
C.sub.1-C.sub.3 alkylphenyl or ortho or para nitrophenyl; R.sup.4
is H or a C.sub.1-C.sub.18 acyl group (preferably, a
C.sub.1-C.sub.4 group), benzoyl, or a substituted benzoyl group;
R.sup.5 is H, a C.sub.1-C.sub.18 alkyl group (preferably, a
C.sub.1-C.sub.3 group), phenyl, substituted phenyl, benzyl, or a
substituted benzyl group; R.sup.6, R.sup.7, R.sup.8, R.sup.9 and
R.sup.10 are independently selected from H, a free acid phosphate,
a phosphate salt, or an S--S--R" group, a C.sub.1-C.sub.3 alkyl
group, F, Cl, Br, I, OCH.sub.3, OCF.sub.3, CF.sub.3, NO.sub.2, CN,
SO.sub.2CF.sub.3, SO.sub.2CH.sub.3, COOCH.sub.3, SF.sub.5,
COCH.sub.3, NH.sub.2, N(CH.sub.3).sub.2, SCH.sub.3 or OH; With the
proviso that when any two of R.sup.6, R.sup.7, R.sup.8, R.sup.9 and
R.sup.10 are other than H, the other of R.sup.6, R.sup.7, R.sup.8,
R.sup.9 and R.sup.10 are H, and with the proviso that no more than
one of R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 is a free
acid phosphate, a phosphate salt or S--S--R" and at least one other
anti-viral agent which inhibits the growth or replication of
viruses by a mechanism other than by inhibition of viral
ribonucleotide reductase.
16-18. (canceled)
19. The composition according to claim 15 wherein said composition
is used to treat a viral infection caused by an agent selected from
the group consisting of human immunodeficiency viruses 1 and 2
(HIV-1 and HIV-2), human T-cell leukemia viruses 1 and 2 (HTLV-1
and HTLV-2), respiratory syncytial virus (RSV), human papilloma
virus (HPV), adenovirus, hepatitis B virus (HBV), hepatitis C virus
(HCV), Epstein-Barr virus (EBV), varicella zoster virus (VZV),
cytomegalovirus (CMV), herpes simplex virus 1 and 2 (HSV-1 and
HSV-2), human herpes virus 8 (HHV-8, also known as Kaposi's
sarcoma-associated virus) and flaviviruses, including Yellow Fever
virus, Dengue virus, Japanese Encephalitis and West Nile
viruses.
20. The composition according to claim 15 wherein said other
anti-viral agent is selected from the group consisting of acyclic
nucleosides such as acyclovir or ganciclovir, interferons such as
alpha, beta or gamma-interferon, reverse transcriptase inhibitors
and nucleoside transport inhibitors such as dipyridamole,
2',3'-dideoxynucleosides, 3TC, AZT, 2',3'-dideoxycytidine,
2',3'-dideoxyadenosine, 2',3'-dideoxyinosine,
2',3'-dideoxythymidine, 2',3'-dideoxy-2',3'-didehydrothymidine and
2',3'-dideoxy-2',3'-didehydrocytidine, .beta.-LFddC, .beta.-LFd4C,
.beta.-Ld4C, tenofovir DF, adefovir, dipivoxil, immunomodulators
such as interleukin II (IL2) and granulocyte macrophage colony
stimulating factor (GM-CSF), erythropoetin, ampligen, thymodulin,
thymopentin, foscarnet, ribavirin and inhibitors of HIV binding to
CD4 receptors e.g. soluble CD4, CD4 fragments, CD4 hybrid
molecules, glycosylation inhibitors such as 2-deoxy-D-glucose,
castanospermine and 1-deoxynojirimycin.
21. The composition according to claim 15 wherein R.sup.1 and
R.sup.2 are H.
22. The composition according to claim 21 wherein said other
anti-viral agent is selected from the group consisting of 3TC, DDI,
D4T, P-LFd4C, .beta.-LFddC, AZT, acyclovir and gancyclovir.
23. The composition according to claim 21 wherein said other
anti-viral agent is selected from the group consisting of 3TC, DDI,
D4T and AZT.
24-35. (canceled)
36. A method of treating an HIV infection in a patient in need
thereof comprising administering to said patient in combination, an
effective amount of at least one compound according to the
structure: 9Where R.sup.1 and R.sup.2 are both H, with an effective
amount of at least one anti-HIV agent selected from the group
consisting of ddI and D4T, said administration producing a
synergistic effect in treating said patient.
37. The method according to claim 36 wherein said anti-HIV agent is
ddI.
38. The method according to claim 36 wherein said anti-HIV agent is
D4T.
39. A method of treating an HSV infection in a patient in need
thereof comprising administering in combination, an effective
amount of at least one compound according to the structure: 10Where
R.sup.1 and R.sup.2 are both H, with an effective amount of
acyclovir.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of provisional paten
application 60/285,559 filed Apr. 20, 2001.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of treating viral
infections using 3-aminopyridine-2-carboxyaldehyde
thiosemicarbazone (3-AP, Triapine.TM.) and
3-amino-4-methylpyridine-2-carboxaldehyde thiosemicarbazone (3-AMP)
and their prodrug forms and to pharmaceutical compositions
comprising these compounds. Combination therapy with other
antiviral agents, in particular, nucleoside antiviral agents,
represents another aspect of the present invention.
BACKGROUND
[0003] Triapine is a ribonucleotide reductase (RNR) inhibitor that
reduces the cellular pool of DNA precursors (dNTPs) by interfering
with their de novo synthesis (Cory et al., (1994) Biochem.
Pharmacol. 48, 335-44). Depletion of dNTPs resulted in inhibition
of DNA synthesis. Triapine was first developed as an anti-cancer
agent for its action against the growth of tumor cells both in
vitro and in vivo (Liu et al., (1992) J. Med. Chem. 35, 3672-77).
It has recently been shown that concentration of Triapine reached
1.0 micromolar in cancer patients receiving a 96-hour infusion of
Triapine at a dose of 96 mg/mm2 (Modiano et al., Proc. Am. Assoc.
Cancer Res., 42, 834, 2001). The mechanism of action of Triapine is
similar to that of another anti-cancer agent, hydroxyurea, which
has been approved for treatment of cancers in humans.
[0004] Recently, hydroxyurea, another known RNR inhibitor, was
shown to have synergistic effects against HIV-infected cells (human
immunodeficiency viruses, the causative agents of AIDS) when
combined with 2',3'-dideoxyinosine (DDI) (Gao et al., (1998)
Biochem. Pharmacol. 56, 105-12). The mechanism of action is
unknown, but may be due to the depletion of dNTPs in cells treated
with hydroxyurea.
[0005] 3-AP and 3-AMP, like other thiosemicarbazone analogs of this
class (Springarn and Sartorelli, J. Med. Chem. (1979) 22, 1314-6),
have very strong iron binding affinity and are capable of removing
iron from ferritin. Iron is required RNR activity and for normal
physiological function of organisms and iron deprivation inhibits
proliferation of protozoa (Merali et al., Antimicrob. Agents
Chemother. (1996) 40, 1298-1300), bacteria (Lowy et al.,
Antimicrob. Agents Chemother. (1984) 25, 375-6), fungi (Newman et
al., Antimicrob. Agents Chemother. (1995) 39, 1824-9; Shulman et
al., Arzneimittelforschung (1972) 22, 154-8; Kerbs et al.,
Sabouraudia (1979), 17, 241-50), and viruses (Dai et al., Virology
(1994) 205, 210-6; Cinatl et al., Antiviral Res. (1994) 25, 73-77;
Bayraktar et al., J. Viral Hepat. (1996) 3, 129-35; Martelius et
al, Transplantation (1999) 15, 1753-61; Georgiou et al, J. Infect.
Dis. (2000) 181, 484-90). In addition to depletion of intracellular
dNTP pools, that 3-AP inhibits viral dissemination could be
mediated through its iron chelating properties (Chouteau et al., J.
Hepatol. (2001) 34, 108-13; Georgiou et al, J. Infect. Dis. (2000)
181, 484-90; Bayraktar et al., J. Viral Hepat. (1996) 3, 129-35;
Conti et al., Boll. Ist. Sieroter Milan (1990) 69, 431-6).
[0006] 3-AP, because of its strong iron-chelating property, can be
used to remove excessive tissue iron in sickle cell disease
patients who require regular blood transfusion (Cohen and Martin,
Semin. Hematol. (2001) 38 (Suppl. 1), 69-72). As a potent inhibitor
of RNR, 3-AP could also be used for the treatment of psoriasis
(Smith, Clin. Exp. Dermatol. (1999) 24, 2-6).
[0007] Chronic HBV (hepatitis B virus) infection remains a
therapeutic challenge for clinicians. The recent development of
lamivudine has provided new hope in the therapy of chronic
hepatitis B. However, due to the slow kinetics of viral clearance
and the spontaneous genetic variability of HBV, lamivudine therapy
is associated with the selection of drug resistant mutants in up to
50% of patients after 3 years of therapy. It is therefore important
to continue research to develop new anti-HBV strategies using in
vitro and in vivo evaluation in experimental models of HBV
replication.
[0008] Herpes simplex virus (HSV) encodes a RNR which is similar to
the one encoded by mammalian cells. HSV replication does not
require the expression of viral RNR in exponentially growing cells
but is required for viral replication in quiescent cells (Goldstein
and Weller, (1988) Virol. 166, 1-51). Duan et al., Antimicrob.
Agents Chemother. (1998) 42, 1629-35, showed that the RNR inhibitor
BILD1633 SE in combination with acyclovir had activity against
acyclovir-resistant HSV strains.
[0009] Recently, flavivirus infections, including West Nile virus
infections, have become increasingly frequent in the United States.
The flaviviruses are agents of infectious disease which predominate
in East, Southeast and South Asia and Africa, although they may be
found in other parts of the world as well. Japanese encephalitis
virus is the causative agent of Japanese encephalitis (JE). The
mortality rate from JE is rather high and the disease brings heavy
sequelae. Although found in Japan, the disease has spread to other
parts of Asia and is now found predominantly outside of Japan,
primarily in South and Southeast Asia.
[0010] Dengue viruses are causative agents of dengue fever/dengue
hemorrhagic fever. Infection with dengue viruses is a major public
health problem in tropical countries, expecially in Southeast Asia
and the Western Pacific, but dengue viruses may also be found in
the Americas. As the dengue virus is transmitted to humans via the
Aedes aegypti mosquito, it is not unexpected that tropical and
subtropical countries, in particular, those in Southeast Asia, are
highly endemic for dengue.
[0011] Viral replication requires dNTPs, and depletion of
intracellular dNTPs by Triapine may prevent viruses from
multiplying. In addition, this new strategy may be used in
combination with other anti-viral agents to treat, or to prevent or
delay the development of drug resistant mutants.
OBJECTS OF THE INVENTION
[0012] It is an object of the invention to provide novel
combination pharmaceutical compositions for treating viral
infections in patients.
[0013] It is another object of the invention to provide novel
methods for reducing viral growth, elaboration and replication and
for treating viral infections in patients.
[0014] It is also an object of the invention to provide methods for
treating fungal infections in patients.
[0015] Any one or more of these and/or other objects of the present
invention may be readily gleaned from a review of the description
of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 is a representation of certain chemical embodiments
according to the present invention.
[0017] FIGS. 2-3 are representations of chemical schemes for
synthesizing compounds according to the present invention.
[0018] FIG. 4 depicts the antiviral activity of combinations of
Triapine with D4T, ddI and AZT.
SUMMARY OF THE INVENTION
[0019] The present invention relates to methods for inhibiting the
growth, replication or elaboration of a virus population or for
treating a variety of virus infections, including, for example,
human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human
T-cell leukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory
syncytial virus (RSV), human papilloma virus (HPV), adenovirus,
hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr
virus (EBV), varicella zoster virus (VZV), cytomegalovirus (CMV),
herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), human herpes
virus 8 (HHV-8, also known as Kaposi's sarcoma-associated virus)
and flaviviruses, including Yellow Fever virus, Dengue virus,
Japanese Encephalitis and West Nile viruses, said method comprising
administering an anti-viral effective amount of a composition
according to the present invention to a patient in need thereof to
treat, prevent or reduce the likelihood of contracting a viral
infection.
[0020] The present invention therefore relates to a method of
treating inhibiting the growth, replication and/or the elaboration
of a viral population or a viral infection in a patient, comprising
administration to said patient an effective amount of a compound
according to the structure: 1
[0021] Where R.sup.1 is H or a C.sub.1-C.sub.3 alkyl group,
preferably H or CH.sub.3;
[0022] R.sup.2 is H or CO.sub.2R.sup.3;
[0023] R.sup.3 is CHRR' or 2
[0024] where R is H, CH.sub.3, CH.sub.2CH.sub.3,
CH.sub.2CH.sub.2CH.sub.3, i-propyl;
[0025] R' is a free acid phosphate, phosphate salt or S--S--R"
group;
[0026] R" is CH.sub.2CH.sub.2NHR.sup.4, CH.sub.2CH.sub.2OH,
CH.sub.2COOR.sup.5, ortho- or para-substituted C.sub.1-C.sub.3
alkylphenyl or ortho or para nitrophenyl;
[0027] R.sup.4 is H or a C.sub.1-C.sub.18 acyl group (preferably, a
C.sub.1-C.sub.4 group), benzoyl, or a substituted benzoyl
group;
[0028] R.sup.5 is H, a C.sub.1-C.sub.18 alkyl group (preferably, a
C.sub.1-C.sub.3 group), phenyl, substituted phenyl, benzyl, or a
substituted benzyl group;
[0029] R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are
independently selected from H, a free acid phosphate, a phosphate
salt, or an S--S--R" group, a C.sub.1-C.sub.3 alkyl group, F, Cl,
Br, I, OCH.sub.3, OCF.sub.3, CF.sub.3, NO.sub.2, CN,
SO.sub.2CF.sub.3, SO.sub.2CH.sub.3, COOCH.sub.3, SF.sub.5,
COCH.sub.3, NH.sub.2, N(CH.sub.3).sub.2, SCH.sub.3 or OH;
[0030] With the proviso that when any two of R.sup.6, R.sup.7,
R.sup.8, R.sup.9 and R.sup.10 are other than H, the other of
R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are H, and with the
proviso that no more than one of R.sup.6, R.sup.7, R.sup.8, R.sup.9
and R.sup.10 is a free acid phosphate, a phosphate salt or
S--S--R".
[0031] In preferred aspects of the present invention, R.sup.6,
R.sup.8 or R.sup.10 is a free acid phosphate or a phosphate salt.
In other preferred aspects of the present invention, R.sup.6 is a
free acid phosphate or phosphate salt and the other of R.sup.7,
R.sup.8, R.sup.9 and R.sup.10 are H. In still other aspects of the
present invention R.sup.1 and R.sup.2 are both H.
[0032] The invention provides methods of use relating to treatment
of infections caused by viruses, including, for example, human
immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human T-cell
leukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytial
virus (RSV), human papilloma virus (HPV), adenovirus, hepatitis B
virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV),
varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex
viruses 1 and 2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8,
also known as Kaposi's sarcoma-associated virus) and flaviviruses,
including Yellow Fever virus, Dengue virus, Japanese Encephalitis
and West Nile viruses. The method includes the use of prodrug forms
of 3-AP and 3-AMP as otherwise described herein for the treatment
of viral infections.
[0033] In alternative embodiments, compounds according to the
present invention may be used to treat fungal infections including,
for example, infections caused by Piedraia hortae, Trichosporon
beigelii, Malassezia furfur, Epidermophyton spp., Microsporum spp.,
Trichophyton spp., Blastomyces dermatitidis, Coccidioides immitis,
Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus spp.
and Candida albicans. Methods of treating fungal infections
comprising administering an anti-fungal effective amount of one or
more of 3-AP, 3-AMP or one of its prodrugs as otherwise described
herein.
[0034] Compositions according to the present invention may be used
alone or in combination with reverse transcriptase inhibitors,
protease inhibitors, other immunomodulators, all for the treatment
of HIV infections, as well as other viral infections. The invention
also provides methods of use for the treatment of HBV infections by
combining one or more compounds according to the present invention
along with reverse trancriptase inhibitors, or interferon, or both.
The invention also provides a method for the treatment of HSV
infection by combining an effective amount of one or more of the
compositions as described above with one or more anti-HSV agents
such as acyclovir, DDI and D4T in amounts effective to treat the
viral infection. Without being limited by way of theory, it is
believed that the use of the presently described compounds exhibit
their anti-viral activity primarily through inhibition of cellular
and viral RNR and/or chelation of divalent metal ions such as iron,
among others. Use of compositions in combination therapy which
inhibit viruses in a manner other than through RNR are particularly
preferred as they often provide synergistic anti-viral activity in
combination with 3-AP (R.sup.1 and R.sup.2 are H), 3-AMP (R.sup.1
is CH.sub.3 and R.sup.2 is H) and prodrug compositions according to
the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The following terms shall be used throughout the
specification to describe the present invention.
[0036] The term "patient" is used throughout the specification to
describe an animal, preferably a human, to whom treatment,
including prophylactic treatment, with the compositions according
to the present invention is provided. For treatment of those
infections, conditions or disease states which are specific for a
specific animal such as a human patient, the term patient refers to
that specific animal.
[0037] The term "virus" shall be used to describe all types of
viruses, the growth or replication of which may be inhibited or
disease states of which may be treated using one or more methods
according to the present invention. Viruses which may be treated
according to the present invention include, for example, human
immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human T-cell
leukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytial
virus (RSV), human papilloma virus (HPV), adenovirus, hepatitis B
virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV),
varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex
viruses 1 and 2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8,
also known as Kaposi's sarcoma-associated virus) and flaviviruses,
including Yellow Fever virus, Dengue virus, Japanese Encephalitis
and West Nile viruses, among numerous others.
[0038] The term "human immunodeficiency virus" shall be used to
describe human immunodeficiency virus (HIV) and its infections,
which term shall be used to embrace both human immunodeficieny
virus 1 (HIV-1) and human immunodeficiency virus 2 (HIV-2).
[0039] The term "human T-cell leukemia virus" shall be used to
describe human T-cell leukemia virus and its infections, which term
shall be used to embrace both human T-cell leukemia virus 1
(HTLV-1) and human T-cell leukemia virus 2 (HTLV-2).
[0040] The term "Hepatitis B Virus" (HBV) is used to describe the
virus (serum hepatitis virus) which produces viral heptatis type B
in humans. This is a viral disease with a long incubation period
(about 50 to 160 days) in contrast to Hepatitis A virus (infectious
hepatitis virus) which has a short incubation period. The virus is
usually tramsmitted by injection of infected blood or blood
derivatives or merely by use of contaminated needles, lancets or
other instruments. Clinically and pathologically, the disease is
similar to viral hepatitis type A; however, there is no
cross-protective immunity. Viral antigen (HBAg) is found in the
serum after infection.
[0041] The term "Hepatitis C Virus" or (HCV) is used throughout the
specification to describe the hepatitis virus which is the
causative agent of non-A, non-B hepatitis. The disease in the acute
stage is, in general, milder than hepatitis B, but a greater
proportion of such infections become chronic.
[0042] The term "Epstein-Barr virus" (EBV) is used throughout the
specification to describe a herpetovirus found in cell cultures of
Burkitt's lymphoma. EBV is the causative agent in infectious
mononucleosis, as well as in a number of other related
conditions/disease states, including EBV-associated lymphomas.
[0043] The term "Varicella-Zoster virus" (VZV) is used to describe
Herpesvirus varicellae, also known as chicken pox or herpes zoster.
Varicella results from a primary infection with the virus; herpes
zoster results from secondary invasion by the same or by
reactivation of infection which in many instances may have been
latent for a number of years. Both the primary and secondary
infections of VZV may be treated using compositions according to
the present invention.
[0044] The term "respiratory syncytial virus" (RSV) is used
throughout the specification to describe an RNA-containing virus of
the genus Pneumovirus that causes minor respiratory infection with
rhinitis and cough in adults, but is capable of causing bronchitis
and bronchopneumonia in young children. The virus is named for the
tendency to form syncytia in tissue culture.
[0045] The term "adenovirus" is used throughout the specification
to describe a virus of the family adenoviridae which are
doublestranded DNA-containing viruses, which infect mammals and
birds. The virion is 70 to 90 nm in diameter and is naked (has no
envelope). The virus develops in nuclei of infected cells;
isolation requires tissue cultures since laboratory animals are not
susceptible to apparent infection. The family includes two genera,
Mastadenovirus and Acviadenovirus.
[0046] The term "Human Herpes Virus 8" (HHV-8) is used throughout
the specification to describe a herpetovirus which is believed to
be the causative agent of Kaposi's sarcoma in AIDS patients.
[0047] The term "Human Papilloma Virus" (HPV) is used throughout
the specification to describe a virus which causes genital warts.
Also known as infectious warts virus, HPV is a universal, common,
often recurrent viral infection with a large number of serotypes.
HPV infection can lead to the formation of genital warts which can,
in turn, lead to genital and/or cervical cancer. Genital warts
caused by HPV types 1, 2, 6, 11, 16 and 18 are generally
transmitted sexually and are often associated with cervical and/or
genital cancer. HPV may mature to produce a papillary tumor or
wart, which is a circumscribed benign epithelial tumor projecting
from the surrounding surface. It is generally a benign epithelial
neoplasm consisting of villous or arborescent outgrowths of
fibrovascular stroma covered by neoplastic cells.
[0048] The term "flavivirus" is used throughout the specification
to describe viruses belonging to the genus Flavivirus of the family
Togaviridae. According to virus taxonomy, about 50 viruses
including Hepatitis C virus (HCV), Yellow Fever virus, Dengue
Virus, Japanese Encephalitis virus, West Nile virus and related
flaviviruses are members of this genus. The viruses belonging to
the genus Flavivirus are simply called flaviviruses. These viruses
were formerly classified as group B arboviruses. The flaviviruses
are agents of infectious disease and predominate in East, Southeast
and South Asia and Africa, although they may be found in other
parts of the world as well.
[0049] The term "Yellow Fever virus" is used to describe the
flavivirus which is the causative agent of yellow fever. Yellow
fever is a tropical mosquito-borne viral hepatitis, due to Yellow
Fever virus (YFV), with an urban form transmitted by Aedes aegypti,
and a rural, jungle or sylvatic form from tree-dwelling mammals by
various mosquitos of the Haemagogus species complex. Yellow fever
is characterized clinically by fever, slow pulse, albuminuria,
jaundice, congesion of the face and hemorrhages, especially
hematemesis ("black vomit"). It is fatal in about 5-10% of the
cases.
[0050] The term "Dengue virus" is used throughout the specification
to descibe the flavivirus which is the causative agent(s) of dengue
fever/dengue hemorrhagic fever. Dengue is a disease of tropical and
subtropical regions occurring epidemically and caused by Dengue
virus, one of a group of arboviruses which causes the hemorrhagic
fever syndrome. Four grades of severity are recognized: grade I:
fever and constitutional symptoms, grade II: grade I plus
spontaneous bleeding (of skin, gums or gastrointestinal tract),
grade III: grade II plus agitation and circulatory failure and
grade IV: profound shock. The disease is transmitted by a mosquito
of the genus Aedes (generally A. aegyptiI, but frequently, A.
albopictus). Also called Aden, bouquet, breakbone, dandy, date,
dengue (hemorrhagic) or polka, solar fever, stiffneck fever,
scarlatina rheumatica or exanthesis arthorosia. "Hemorrhagic
dengue" is a more pathogenic epidemic form of dengue which has
erupted in a number of epidemic outbreaks in the Pacific region in
recent years.
[0051] The term `West Nile virus" is used to describe the
flavivirus which is the causative agent of West Nile fever, a
disease characterized by headache, fever, masculopapular rash,
myalgia, lymphadenopathy and leukopenia. The virus is spread by
Culex mosquitoes from a reservoir in birds. Although in the past,
West Nile virus infections had been considered virtually
nonexistent in the United States, recent developments have
suggested that West Nile and other flavivirus infections will
appear with greater regulatory in the future in the United
States.
[0052] The term fungus shall mean "fungus" as that term is
generally known in the art. Fungal infections which may be treated
using 3-AP, 3-AMP and their prodrug forms alone or in combination
with other anti-fungal agents including, for example infections
caused by Piedraia hortae, Trichosporon beigelii, Malassezia
furfur, Epidermophyton spp., Microsporum spp., Trichophyton spp.,
Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus
neoformans, Histoplasma capsulatum, Aspergillus spp., Candida
albicans.
[0053] The term "anti-fungal agent" shall used to describe a
compound which may be used to treat a fungus infection other than
3-AP, 3-AMP or prodrugs of 3-AP and 3-AMP according to the present
invention. Anti-fungal agents according to the present invention
include, for example, terbinafine, fluconazole, itraconazole,
posaconazole, clotrimazole, griseofulvin, nystatin, tolnaftate,
caspofungin, amphotericin B, liposomal amphotericin B, and
amphotericin B lipid complex.
[0054] The term "pharmaceutically acceptable salt" is used
throughout the specification to describe a salt form of one or more
of the compositions (and in particularly preferred aspects
according to the present invention, phosphate salts) herein which
are presented to increase the solubility of the compound in saline
for parenteral delivery or in the gastric juices of the patient's
gastrointestinal tract in order to promote dissolution and the
bioavailability of the compounds. Pharmaceutically acceptable salts
include those derived from pharmaceutically acceptable inorganic or
organic bases and acids. Suitable salts include those derived from
alkali metals such as potassium and sodium, alkaline earth metals
such as calcium, magnesium and ammonium salts, among numerous other
acids well known in the pharmaceutical art. Sodium and potassium
salts are particularly preferred as neutralization salts of
carboxylic acids and free acid phosphate containing compositions
according to the present invention.
[0055] The term "inhibitory effective concentration" or "inhibitory
effective amount" is used throughout the specification to describe
concentrations or amounts of compounds according to the present
invention which substantially or significantly inhibit the growth
or replication of susceptible viruses, especially including human
immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human T-cell
leukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytial
virus (RSV), human papilloma virus (HPV), adenovirus, hepatitis B
virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV),
varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex
viruses 1 and 2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8,
also known as Kaposi's sarcoma-associated virus) and flaviviruses,
including Yellow Fever virus, Dengue virus, Japanese Encephalitis
and West Nile viruses, among numerous others. This term also refers
to amounts of concentrations of compounds (whether 3-AP, 3-AMP or
its prodrugs or more traditional anti-fungal agents as described in
the present specification) which inhibit the growth of fungi.
[0056] The term "therapeutic effective amount" or "therapeutically
effective amount" is used throughout the specification to describe
concentrations or amounts of compounds according to the present
invention which are therapeutically effective in treating viruses
or fungi according to the present invention.
[0057] The term "preventing effective amount" is used throughout
the specification to describe concentrations or amounts of
compounds according to the present invention which are
prophylactically effective in preventing, reducing the likelihood
of infection or delaying the onset of infections in patients caused
by human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human
T-cell leukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory
syncytial virus (RSV), human papilloma virus (HPV), adenovirus,
hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr
virus (EBV), varicella zoster virus (VZV), cytomegalovirus (CMV),
herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), human herpes
virus 8 (HHV-8, also known as Kaposi's sarcoma-associated virus)
and flaviviruses, including Yellow Fever virus, Dengue virus,
Japanese Encephalitis and West Nile viruses, among numerous others.
This term shall also be used to describe amounts or concentrations
of anti-fungal agents which are prophylactically effective in
preventing, reducing the likelihood or delaying the onset of a
fungal infection.
[0058] The term "effective amount" shall mean an amount or
concentration of a compound according to the present invention
which is effective within the context of its administration or use,
including, for example, the treatment or prevention of viral and/or
fungal infections.
[0059] The term "coadministration" or "combination therapy" is used
to describe a therapy in which at least two active compounds in
effective amounts are used to treat a viral or fungal infection at
the same time. Although the term coadministration preferably
includes the administration of two active compounds to the patient
at the same time, it is not necessary that the compounds be
administered to the patient at the same time, although effective
amounts of the individual compounds will be present in the patient
at the same time.
[0060] The term "alkyl" is used throughout the specification to
describe a hydrocarbon radical containing between one and three
carbon units. Alkyl groups for use in the present invention include
linear or branched-chain groups, such as propyl and isopropyl.
[0061] The present invention relates to the use of 3-AP and 3-AMP,
including their prodrug forms, as otherwise described herein for
the inhibition of viral infections and the treatment of viral
diseases in animals, including humans. Viruses which can be treated
by the present invention include, for example, human
immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), human T-cell
leukemia viruses 1 and 2 (HTLV-1 and HTLV-2), respiratory syncytial
virus (RSV), human papilloma virus (HPV), adenovirus, hepatitis B
virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV),
varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex
viruses 1 and 2 (HSV-1 and HSV-2), human herpes virus 8 (HHV-8,
also known as Kaposi's sarcoma-associated virus) and flaviviruses,
including Yellow Fever virus, Dengue virus, Japanese Encephalitis
and West Nile viruses, among numerous others.
[0062] The present invention also describes the use of 3-AP and
3-AMP and its prodrug forms in combination with other agents which
may be used to treat viral infections and to prevent a viral
infection or reduce the likelihood that an exposure of an animal to
a virus will result in a viral infection in that animal. Another
aspect of the present invention relates to combination therapy with
at least one compound according to the present invention, in
combination with at least one additional anti-viral agent which
inhibits viruses by a mechanism other than by inhibition of viral
RNR, which combination therapy produces synergistic inhibition of
viral infections.
[0063] The present invention may be used in the treatment of HIV
infections. For example, compositions according to the present
invention may be combined with other anti-HIV agents, especially
including, for example, D4T, DDI, AZT, lamivudine,
Beta-L-5-Fluoro-2',3'-dideoxydidehydr- ocytidine (.beta.-LFd4C),
Beta-L-5-Fluoro-2',3'-dideoxycytidine (.beta.-LFddC),
Beta-L-2',3'-dideoxydidehydrocytidine (.beta.-Ld4C) or other
nucleoside anti-HIV agents for the treatment of AIDS related
conditions such as AIDS-related complex (ARC) and AIDS-related
neurological conditions. The present invention is also useful in
the prevention or the reduction of the likelihood of progression to
clinical illness of individuals who are anti-HIV antibody or
HIV-antigen positive and in prophylaxis following exposure to
HIV.
[0064] The present invention may also be used in the treatment of
HBV infections, including the use in combination with other
anti-HBV agents for the treatment of acute or chronic HBV
infections, especially including for example, lamivudine,
Beta-L-5-Fluoro-2',3'-dideoxydidehydro- cytidine (.beta.-LFd4C),
Beta-L-5-Fluoro-2',3'-dideoxycytidine (.beta.-LFddC) or other
nucleoside anti-HBV agents. The invention is also useful in the
prevention or the reduction of the likelihood of progression to
clinical illness of individuals who are anti-HBV antibody or
HBV-antigen positive and in prophylaxis following potential
exposure to HBV such as after a liver transplant.
[0065] The present invention is also directed to the use of one or
more compounds as otherwise described herein in combination with
other anti-HSV agents for the treatment of HSV infections,
especially for example, acyclovir (ACV). The invention is also
useful in the prevention or the reduction of the likelihood of
progression to clinical illness of individuals who are infected
with HSV or in prophylaxis following potential exposure to HSV.
[0066] The present invention also relates to compositions and
methods for use in treating fungal infections either alone or using
combinations of agents. Suitable therapeutic agents for use in such
combination include 3-AP, 3-AMP or one or more of its prodrug forms
as described in detail hereinabove, alone or in combination with
another anti-fungal agents such as terbinafine, fluconazole,
itraconazole, posaconazole, clotrimazole, griseofulvin, nystatin,
tolnaftate, caspofungin, amphotericin B, liposomal amphotericin B,
and amphotericin B lipid complex.
[0067] In another aspect, the present invention is directed to the
use of one or more compounds according to the present invention in
a pharmaceutically acceptable carrier in combination with at least
one other anti-viral agent at a suitable dose ranging from about 1
to about 100 mg/kg of body weight per day, preferably within the
range of about 2 to 50 mg/kg/day, most preferably in the range of 3
to 20 mg/kg/day. The desired dose may conveniently be presented in
a single dose or as divided doses administered at appropriate
intervals, for example as two, three, four or more sub-doses per
day.
[0068] Ideally, the active ingredient should be administered to
achieve peak plasma concentrations of the active compound of from
about 0.05 to about 5 uM, preferably about 0.1 to 2 uM, most
preferably about 0.2 to about 1 uM. This may be achieved, for
example, by the intravenous injection of about a 0.1 to 10%
solution of the active ingredient, optionally in saline, or orally
administered as a bolus containing about 0.1 to about 5 g of the
active ingredient. Desirable blood levels may be maintained by a
continuous infusion to preferably provide about 0.01 to about 2.0
mg/kg/hour or by intermittent infusions containing about 0.4 to
about 15 mg/kg of the active ingredient. Oral dosages, where
applicable, will depend on the pharmacokinetics of the compounds to
be administered. While it is possible that, for use in therapy, a
compound of the invention may be administered as the raw chemical
it is preferable to present the active ingredient as a
pharmaceutical formulation.
[0069] Pharmaceutical formulations include those suitable for oral,
rectal, nasal, topical (including buccal and sub-lingual), vaginal
or parenteral (including intramuscular, sub-cutaneous and
intravenous) administration. Compositions according to the present
invention may also be presented as a bolus, electruary or paste.
Tablets and capsules for oral administration may contain
conventional excipients such as binding agents, fillers,
lubricants, disintegrants, or wetting agents. The tablets may be
coated according to methods well known in the art. Oral liquid
preparations may be in the form of, for example, aqueous or oily
suspensions, solutions, emulsions, syrups or elixirs, or may be
presented as a dry product for constitution with water or other
suitable vehicle before use. Such liquid preparations may contain
conventional additives such as suspending agents, emulsifying
agents, non-aqueous vehicles (which may include edible oils), or
preservatives.
[0070] When desired, the above described formulations may be
adapted to provide sustained release characteristics of the active
ingredient(s) in the composition.
[0071] In the pharmaceutical aspect according to the present
invention, the compound(s) according to the present invention is
formulated preferably in admixture with a pharmaceutically
acceptable carrier. In general, it is preferable to administer the
pharmaceutical composition parenterally and in particular, in
intravenously or intramuscular dosage form, but a number of
formulations may be administered via other parenteral routes, such
as transdermal, buccal, subcutaneous, suppository or other route,
including via an oral route of administration. Intravenous and
intramuscular formulations are preferably administered in sterile
saline. Of course, one of ordinary skill in the art may modify the
formulations within the teachings of the specification to provide
numerous formulations for a particular route of administration
without rendering the compositions of the present invention
unstable or compromising their therapeutic activity. In particular,
the modification of the present compounds to render them more
soluble in water or other vehicle, for example, may be easily
accomplished by minor modifications (such as salt formulation,
etc.) which are well within the ordinary skill in the art. It is
also well within the routineer's skill to modify the route of
administration and dosage regimen of a particular compound in order
to manage the pharmacokinetics of the present compounds for maximum
beneficial effect to the patient.
[0072] The routineer will take advantage of favorable
pharmacokinetic parameters of the pro-drug forms of the present
invention, where applicable, in delivering the present compounds to
a patient suffering from a viral infection to maximize the intended
effect of the compound.
[0073] The pharmaceutical compositions according to the invention
may also contain other active ingredients such as antimicrobial
agents, antiinfective agents, or preservatives.
[0074] The invention thus provides, in a further aspect, a
pharmaceutical composition combination comprising an effective
amount of 3-AP, 3-AMP or one or more of its prodrug forms as
otherwise described herein or a pharmaceutically acceptable
derivative thereof together with another therapeutically active
agent, and in particular, another antiviral agent. Effective
amounts or concentrations of each of the active compounds are to be
included within the pharmaceutical compositions according to the
present invention.
[0075] Pharmaceutical formulations comprising at least one of 3-AP,
3-AMP and prodrug forms presented in combination with an effective
amount of at least one additional anti-viral agent in further
combination with a pharmaceutically acceptable carrier, represent a
further aspect of the present invention.
[0076] The present invention also relates to compositions and
methods for use in treating viral infections using combinations of
agents. Suitable therapeutic agents for use in such combinations
with 3-AP, 3-AMP or one or more of its prodrug forms include
acyclic nucleosides such as acyclovir or ganciclovir, interferons
such as alpha., beta or gamma.-interferon, reverse transcriptase
inhibitors and nucleosides, transport inhibitors such as
dipyridamole, 2',3'-dideoxynucleosides (including .beta.-L-FddC),
2,3'-dideoxy-2',3'-didehydronucleosides (including .beta.-L-Fd4C),
3TC (lamivudine), AZT, 2',3'-dideoxycytidine (DDC),
2',3'-dideoxyadenosine, 2',3'-dideoxyinosine (DDI),
2',3'-dideoxythymidine (DDT),
2',3'-dideoxy-2',3'-didehydrothymidine (D4T) and
2',3'-dideoxy-2',3'-didehydrocytidine (D4C), tenofirir DF,
adefovir, dipivoxil, immunomodulators such as interleukin II (IL2)
and granulocyte macrophage-colony stimulating factor (GM-CSF),
erythropoetin, ampligen, thymodulin, thymopentin, foscarnet,
ribavirin and inhibitors of HIV binding to CD4 receptors e.g.
soluble CD4, CD4 fragments, CD4 hybrid molecules, glycosylation
inhibitors such as 2-deoxy-D-glucose, castanospermine and
1-deoxynojirimycin.
[0077] The individual components of such combinations maybe
administered either sequentially or simultaneously in separate or
combined pharmaceutical formulations.
[0078] When one or more of the compounds according to the present
invention is used in combination with a second therapeutic agent
active against the same virus or fungus the dose of each compound
may be either the same as or differ from that when the compound is
used alone. Appropriate doses will be readily appreciated by those
skilled in the art.
[0079] Chemical Synthesis
[0080] Compositions according to the present invention are
synthesized using the general and synthetic methods which are set
forth in U.S. Pat. No. 5,767,134, issued Jun. 16, 1998 and as
otherwise set forth herein. Additional prodrug forms of 3-AP and
3-AMP are synthesized by the methods which may be followed in the
'134 patent as well as methods which are described below.
[0081] A number of phosphate bearing prodrugs (as set forth in FIG.
1) were synthesized readily in good quantities and evaluated. The
disodium salts of these prodrugs were very soluble in water.
[0082] As set forth in attached FIG. 2, the 5-chloro prodrug
compound 6 was synthesized as shown. Thus, the acid 20 was prepared
from, for example, 2-chloro-3-nicotinic acid methyl ester 18 or a
related derivative in a two-step sequence consisting of a Heck
reaction (See, Jeffery, Tetrahedron (1996), 52, 10113 and Dieck and
Heck, J. Org. Chem. (1975), 40, 1083 and a NaOH promoted ester
hydrolysis. The chloro ortho-phosphate linker 21 was prepared via
an oxidative coupling between the bis-TMSE-phosphite (McCombie et
al., J. Chem. Soc. (1945), 381) and 2-hydroxybenzyl alcohol.
Initially, problems were encountered in the large-scale preparation
of linker 21 as it decomposed during purification giving low
yields. The conditions were standardized by using Et.sub.3N as
buffer to neutralize the acidity of silica gel to obtain the linker
in good quantities (88%). Heating a reaction mixture consisting of
the acid 20, the linker 21, triethylamine and diphenylphosphoryl
azide under Curtius rearrangement conditions (Shipps et al., J.
Bioorg. Med. Chem. (1996), 4, 655) provided the desired carbamate
22 (58%), which was converted sequentially to the aldehyde 23 (72%)
and its corresponding thiosemicarbazone 24 in 63% yield. The
removal of the 2-trimethylsilylethyl (TMSE) group in 24 was
effected cleanly with TFA (Chao et al., J. Org. Chem. (1994), 59,
6687) and provided the 3-AP prodrug free acid 6, which was in turn
converted to the disodium salt 25 upon treatment with saturated
sodium bicarbonate solution and reverse phase column
purification.
[0083] The other substituted ortho prodrugs were synthesized
essentially following the same route using appropriate
phosphate-bearing substituted benzyl linkers such as 21. Coupling
of these linkers to 25, followed by functional group manipulations
furnished the corresponding prodrugs (FIG. 3). The synthesis
evidences that the prodrugs of the present invention may be readily
converted to their corresponding phosphate salts. The water
solubility of these phosphate salt compounds is excellent and is
significantly greater than corresponding non-prodrug forms. The
solubility of parental 3-AP in aqueous solution is less than 0.1
mg/ml, where as that of the prodrugs is between 16 and 35
mg/ml.
[0084] Having generally described the invention, reference is now
made to the following specific examples which are intended to
illustrate preferred and other embodiments and comparisons. The
included examples are not to be construed as limiting the scope of
this invention as is more broadly set forth above and in the
appended claims.
[0085] All reagents were purchased at commercial quality and used
without further purification, and solvents were dried and/or
distilled before use where necessary. All NMR spectra (.sup.1H,
.sup.13C, and .sup.31P) were determined on a Brucker AC300
spectrometer. Chemical shifts are measured in parts per million
(ppm) relative to tetramethylsilane. Coupling constants are
reported in hertz (Hz). Flash column chromatography (FCC) was
performed with Merck silica gel 60 (230-400 mesh), and pre-treated
with triethylamine for all trimethylsilylethyl (TMSE) protected
compounds. Reversed phase column chromatography (RPCC) was packed
with CAT gel (Waters, preparative C18 125 .ANG., 55-105 .mu.m),
eluting with milli-Q de-ionized water.
EXAMPLES 1-3
General Procedures for Preparation of the Nicotinic Acid (20)
Example 1
Preparation of 2-chloronicotinic acid methyl ester (18)
[0086] To a mixture of 2-chloronicotinic acid (Aldrich, 100.0 g,
0.63 mol) in 1,4-dioxane (500 mL) was added thionyl chloride (70
mL, 0.96 mol). The suspension was heated under reflux for 22 h with
a gas trap to absorb hydrogen chloride gas. After evaporation of
the solvent, the residue was dissolved in methanol (300 mL). To the
solution was added dropwise triethylamine (TEA, 120 mL, 1.26 mol)
at 0.degree. C. over 2 h. The solvents were evaporated and the
residue was suspended in ethyl acetate. The precipitate was removed
by filtration. The filtrate was concentrated to afford the ester 18
(92.3 g, 86%) as an oil:
[0087] Rf(1:5 v/v ethyl acetate-hexane) 0.38.
[0088] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.53 (dd, 4.8 Hz,
1H), 8.19 (dd, 7.6 Hz, 1H), 7.37 (dd, 7.7 Hz, 1H) and 3.97 (s,
3H).
[0089] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 164.5, 151.6,
149.6, 140.0, 126.4, 121.9 and 52.5.
Example 2
Preparation of 2-styrylnicotinic acid methyl ester (19)
[0090] To a solution of the ester 18 (48.8 g, 0.28 mol) in DMF (450
mL) was added styrene (165 mL, 1.42 mol), palladium acetate (6.5 g,
30 mmol), sodium acetate (47 g, 0.57 mol) and triphenyl phosphine
(30 g, 0.11 mol). The mixture was heated under reflux for 22 h. The
palladium-catalyst was removed by filtration through a Celite pad.
The filtrate was concentrated under reduced pressure, and the
residue was dissolved in a minimum amount of ethyl acetate. To the
above solution was added hexane. After removal of the precipitate
by filtration, the filtrate was concentrated. The resulting crude
material was purified by FCC (1:1 v/v ethyl acetate-hexane) to
afford the ester 19 (55.0 g, 81%) as a light yellow oil:
[0091] Rf(1:5 v/v ethyl acetate-hexane) 0.41.
[0092] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.70 (dd, 1H),
8.10 (dd, 1H), 8.16 (d, 1H), 7.94 (d, 1H), 7.64 (d, 2H), 7.4-7.3
(m, 3H), 7.18 (dd, 1H) and 3.94 (s, 3H).
[0093] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 166.7, 155.3,
152.0, 138.6, 136.7, 135.9, 128.6, 128.5, 127.5, 124.8, 123.8,
121.3 and 52.4.
Example 3
Preparation of 2-styrylnicotinic acid (20)
[0094] A solution of the ester 19 (55.0 g, 0.23 mol) in THF (100
mL) was treated with a 3 N NaOH solution (110 mL, 0.25 mol) for 21
h at ambient temperature. After removal of solvents, the residue
was taken up in water and ethyl ether. The phases were separated,
and the aqueous phase was washed with ether (2.times.). The
resulting aqueous phase was neutralized with a 2 N HCl solution,
and the precipitate was then collected by filtration to afford the
acid 20 (50.2 g, 97%) as a cream solid:
[0095] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.72 (dd, 1H),
8.19 (dd, 1H), 8.10 (d, 1H), 7.86 (d, 1H), 7.62 (d, 2H) and 7.4-7.3
(m, 4H).
[0096] .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 167.9, 153.7,
151.8, 138.6, 136.4, 134.5, 128.9, 128.7, 127.2, 125.3 and
122.1.
EXAMPLES 4-5
General Procedures for Preparation of the Phosphate Linkers (21,
26a-k)
Example 4
Preparation of bis(2-trimethylsilylethyl)phosphite
(TMSE-phosphite)
[0097] To a solution of 2-(trimethylsilyl)ethanol (Aldrich, 25.0 g,
0.21 mol) in ethyl ether (200 mL) containing pyridine (11.4 mL,
0.14 mol) was added phosphorus trichloride (6.2 mL, 70 mmol) in one
portion at -78.degree. C. The reaction mixture was kept for 5 min
while stirring, and then diluted with ethyl ether (500 mL). After
warming to ambient temperature, the mixture was stirred for 18 h
continually. The precipitate was removed by filtration, and the
filtrate was then bubbled by ammonia gas for 10 min. The
precipitate was removed by filtration through a Celite pad, and the
filtrate was concentrated to afford TMSE-phosphite (20.7 g, 99%) as
a colorless oil:
[0098] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.76 (d, 1H), 4.13
(m, 4H), 1.07 (m, 4H) and 0.0 (s, 18H).
[0099] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 64.0 (d), 19.6 (d)
and -1.6 (d).
[0100] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 18.5.
Example 5
Preparation of 2-(TMSE-phosphonooxy)benzyl alcohols (21, 26a-k)
[0101] General Procedure. To a solution of the corresponding
2-hydroxybenzyl alcohol (10 mmol) in acetonitrile (40 mL) was added
N,N'-diisopropylethylamine (DIEA, 11 mmol), 4-dimethylaminopyridine
(DMAP, 1 mmol), and carbon tetrachloride (50 mmol). While stirring
at -30.degree. C., to the solution was added
bis(2-trimethylsilylethyl)phosp- hite (stored in refrigerator, 11
mmol) immediately. After warming to ambient temperature, the
reaction mixture was stirred for 3 h. The solvents were evaporated
under reduced pressure, and the residual product was purified by
FCC (1:1 v/v ethyl acetate-hexane) to afford the corresponding
TMSE-protected phosphate linker (21, 26a-k).
[0102] 2-Bis(2-trimethylsilylethyl)phosphonooxy-5-chlorobenzyl
alcohol (21).
[0103] Following the above procedure, 5-chloro-2-hydroxybenzyl
alcohol (5.0 g, 32 mmol) gave 21 (12.2 g, 88%) as an oil:
[0104] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.33 (d, 1H), 7.11
(m, 1H), 7.02 (m, 1H), 4.49 (s, 2H), 4.12 (m, 4H), 1.00 (m, 4H) and
-0.07 (s, 18H).
[0105] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 146.5 (d), 134.7
(d), 130.8, 130.2, 128.6, 122.0 (d), 67.7 (d), 59.4, 19.5 (d) and
-1.6.
[0106] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.1.
[0107] 2-Bis(2-trimethylsilylethyl)phosphonooxy-5-fluorobenzyl
alcohol (26a).
[0108] Following the above procedure, 5-fluoro-2-hydroxybenzyl
alcohol (17.0 g, 119 mmol) gave 26a (31.7 g, 62%) as an oil:
[0109] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.2-7.1 (m, 1H),
7.0-6.9 (m, 1H), 4.63 (s, 1H), 4.3-4.1 (m, 4H), 1.2-1.1 (m, 4H) and
0.0 (s, 18H).
[0110] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 159.8 (d), 143.7
(dd), 135.2 (dd), 121.8 (dd), 116.4 (d), 115.0 (d), 67.6 (d), 59.4,
19.5 (d) and -1.6.
[0111] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.4.
[0112] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-59.8.
[0113] 2-Bis(2-trimethylsilylethyl)phosphonooxy-5-nitrobenzyl
alcohol (26b).
[0114] Following the above procedure, 2-hydroxy-5-nitrobenzyl
alcohol (4.5 g, 27 mmol) gave 26b (6.4 g, 53%) as an oil:
[0115] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.14 (m, 1H), 7.81
(m, 1H), 7.14 (m, 1H), 4.48 (s, 2H), 4.06 (m, 4H), 0.90 (m, 4H) and
-0.20 (m, 18H).
[0116] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 152.0 (d), 144.6,
134.7 (d), 123.7, 123.4, 119.8, 67.9 (d), 58.4, 19.3 (d) and
-1.6.
[0117] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 4.4.
[0118] 2-Bis(2-trimethylsilylethyl)phosphonooxy-5-methoxybenzyl
alcohol (26c).
[0119] Following the above procedure, 2-hydroxy-5-methoxybenzyl
alcohol (11.0 g, 25 mmol) gave 26c (7.7 g, 70%) as an oil:
[0120] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.06 (dd, 1H),
6.94 (d, 1H), 6.74 (dd, 1H), 4.57 (s, 2H), 4.3-4.1 (m, 4H), 3.74
(s, 3H), 1.1-1.0 (m, 4H) and 0.0 (m, 18H).
[0121] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 157.1, 141.7 (d),
134.0 (d), 121.9 (d), 125.3, 114.5, 67.5 (d), 60.2, 55.6, 19.6 (d)
and -1.6.
[0122] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.9.
[0123]
2-Bis(2-trimethylsilylethyl)phosphonooxy-5-trifluoromethoxybenzyl
alcohol (26d).
[0124] Following the above procedure,
2-hydroxy-5-trifluoromethoxybenzyl alcohol (1.9 g, 9.1 mmol) gave
26d (3.3 g, 62%) as an oil:
[0125] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.20 (d, 1H), 7.19
(dd, 1H), 7.09 (dd, 1H), 4.61 (s, 2H), 4.24 (m, 4H), 1.08 (m, 4H)
and 0.0 (s, 18H).
[0126] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 146.4 (dd), 135.0
(d), 123.0, 122.1, 121.4, 67.8 (d), 59.6, 19.6 (d) and -1.6.
[0127] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.2.
[0128] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-58.7.
[0129]
2-Bis(2-trimethylsilylethyl)phosphonooxy-5-trifluoromethylbenzyl
alcohol (26e).
[0130] Following the above procedure,
2-hydroxy-5-trifluoromethylbenzyl alcohol (4.1 g, 22 mmol) gave 26e
(7.9 g, 77%) as an oil:
[0131] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.72 (br s, 1H),
7.51 (dd, 1H), 7.29 (d, 1H), 4.66 (s, 2H), 4.23 (m, 4H), 1.09 (m,
4H) and 0.0 (s, 18H).
[0132] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 150.6 (d), 133.8
(d), 127.7 (d), 126.1, 121.3 (d), 68.0 (d), 59.6, 19.6 (d) and
-1.6.
[0133] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.8.
[0134] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-62.9.
[0135] 2-Bis(2-trimethylsilylethyl)phosphonooxy-3,5-dichlorobenzyl
alcohol (26f).
[0136] Following the above procedure, 3,5-dichloro-2-hydroxybenzyl
alcohol (4.6 g, 24 mmol) gave 26f (7.2 g, 63%) as an oil:
[0137] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.34 (d, 1H), 7.32
(dd, 1H), 4.56 (s, 2H), 4.25 (m, 4H), 1.08 (m, 4H) and 0.0 (s,
18H).
[0138] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 149.9, 143.3 (d),
136.9 (d), 131.2 (d), 129.9, 129.5, 127.6 (d), 68.3 (d), 59.8, 19.5
(d) and -1.6.
[0139] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.7.
[0140] 2-Bis(2-trimethylsilylethyl)phosphonooxy-4,5-dichlorobenzyl
alcohol (26g).
[0141] Following the above procedure, 4,5-dichloro-2-hydroxybenzyl
alcohol (3.6 g, 18 mmol) gave 26g (5.2 g, 59%) as an oil:
[0142] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.53 (s, 1H), 7.28
(s, 1H), 4.55 (s, 2H), 4.21 (m, 4H), 1.08 (m, 4H) and 0.0 (s,
18H).
[0143] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 146.6 (d), 133.5
(d), 131.9 (d), 131.6, 129.5 (d), 123.0 (d), 68.1 (d), 59.1, 19.6
(d) and -1.5.
[0144] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.0.
[0145] 2-Bis(2-trimethylsilylethyl)phosphonooxy-5,6-dichlorobenzyl
alcohol (26h).
[0146] Following the above procedure, 5,6-dichloro-2-hydroxybenzyl
alcohol (4.8 g, 25 mmol) gave 26h (8.6 g, 73%) as an oil:
[0147] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.35 (d, 1H), 7.04
(dd, 1H), 4.76 (s, 2H), 4.22 (m, 4H), 1.08 (m, 4H) and 0.0 (s,
18H).
[0148] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 147.6 (d), 134.7,
133.5 (d), 130.6 (d), 129.9, 120.8 (d), 68.1 (d), 57.3, 19.6 (d)
and -1.6.
[0149] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.4;
[0150] 2-Bis(2-trimethylsilylethyl)phosphonooxy-3-methylbenzyl
alcohol (26i).
[0151] Following the above procedure, 2-hydroxy-3-methylbenzyl
alcohol (2.0 g, 14 mmol) gave 26i (1.7 g, 88%) as an oil:
[0152] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.16 (m, 1H),
7.0-6.9 (m, 2H), 4.48 (s, 2H), 4.13 (m, 4H), 2.22 (s, 3H), 0.97 (m,
4H) and -0.09 (s, 18H).
[0153] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 146.9 (d), 133.5
(d), 131.0, 130.4 (d), 129.4, 125.6 (d), 67.6 (d), 60.1, 19.5 (d),
16.8 and -1.6.
[0154] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.9.
[0155] 2-Bis(2-trimethylsilylethyl)phosphonooxy-4-chlorobenzyl
alcohol (26j).
[0156] Following the above procedure, 4-chloro-2-hydroxybenzyl
alcohol (4.2 g, 26 mmol) gave 26j (9.6 g, 84%) as an oil:
[0157] R.sub.f (4:1 v/v ethyl acetate-hexane) 0.67.
[0158] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.27 (d, 1H),
7.2-7.1 (m, 2H), 4.55 (s, 2H), 4.21 (m, 4H), 1.07 (m, 4H) and 0.0
(s, 18H).
[0159] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 148.5 (d), 133.9,
131.7 (d), 131.5, 126.0, 121.4 (d), 67.9 (d), 59.4, 19.5 (d) and
-1.6.
[0160] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.8.
[0161] 2-Bis(2-trimethylsilylethyl)phosphonooxy-4-methoxybenzyl
alcohol (26k).
[0162] Following the above procedure, 2-hydroxy-4-methoxybenzyl
alcohol (2.7 g, 17 mmol) gave 26k (2.5 g, 33%) as an oil:
[0163] Rf (4:1 v/v ethyl acetate-hexane) 0.70.
[0164] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.29 (d, 1H),
6.8-6.7 (m, 2H), 4.53 (s, 2H), 4.22 (m, 4H), 3.75 (s, 3H), 1.09 (m,
4H) and 0.0 (s, 18H).
[0165] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 160.1 (d), 149.1
(d), 131.9, 125.3 (d), 111.2, 107.3 (d), 67.6 (d), 59.7, 55.5, 19.6
(d) and -1.6.
[0166] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.4.
EXAMPLES 6-10
General Procedures for Preparation of the 3-AP Prodrugs (25,
30a-k)
Example 6
Preparation of (2-styrylpyridin-3-yl)carbamic acid
2-(TMSE-phosphonooxy)be- nzyl esters (22, 27a-k) (Curtius
Rearrangement)
[0167] General Procedure. To a solution of 2-styrylnicotinic acid
(20, 20 mmol) in benzene (100 mL) containing triethylamine (TEA, 32
mmol) was added diphenylphosphorylazide (32 mmol). The solution was
heated at reflux for 10 min, and the corresponding TMSE-protected
phosphate linker (21 or 26a-k, 20 mmol) was then added. The
reaction mixture was kept under reflux for 3 h. Next, the solvents
were evaporated under reduced pressure. The residual product was
purified by FCC (1:4 v/v ethyl acetate-hexane) to afford the
corresponding carbamate (22 or 27a-k).
[0168] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-chlorobenzyl ester
(22).
[0169] Following the above procedure, 21 (9.4 g, 21 mmol) gave 22
(10.6 g, 58%) as an oil:
[0170] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.23 (d, 1H), 7.92
(br s, 1H), 7.56 (d, 1H), 7.4-7.0 (m, 11H), 5.11 (s, 2H), 4.10 (m,
4H), 0.94 (m, 4H) and -0.14 (s, 18H).
[0171] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 153.7, 151.9,
147.3 (d), 146.8, 145.2, 136.5, 134.8, 131.4, 130.2, 129.5, 129.3,
129.2, 128.5, 128.3, 127.2, 122.3, 121.3, 121.2, 67.4 (d), 61.6,
19.4 (d) and -1.7.
[0172] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.1.
[0173] Mass Calcd. For C.sub.31H.sub.42ClN.sub.2O.sub.6PSi.sub.2:
661.277; Found: 661.2
[0174] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-fluorobenzyl ester
(27a).
[0175] Following the above procedure, the cruse carbamate 27a
obtained from 26a (31.0 g, 73 mmol) was directly used for the
further reaction without purification.
[0176] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-nitrobenzyl ester
(27b).
[0177] Following the above procedure, 26b (4.3 g, 9.6 mmol) gave
27b (2.3 g, 35%) as an oil:
[0178] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.3-7.0 (m, 14H),
5.21 (s, 2H), 4.16 (m, 4H), 0.99 (m, 4H) and -0.12 (m, 18H).
[0179] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 153.5, 153.2 (d),
149.9, 145.6, 144.3, 136.4, 135.1, 131.1, 129.3, 129.0 (d), 128.5,
128.4, 127.2, 124.9, 124.8, 122.4, 120.9, 120.3, 68.0 (d), 62.1,
19.5 (d), 17.1 and -1.6.
[0180] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 4.5.
[0181] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-methoxybenzyl ester
(27c).
[0182] Following the above procedure, the cruse carbamate 27c
obtained from 26c (6.0 g, 26 mmol) was directly used for the
further reaction without purification.
[0183] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-trifluoromethoxybenzyl
ester (27d).
[0184] Following the above procedure, 26d (1.9 g, 8.5 mmol) gave
27d (3.4 g, 83%) as an oil:
[0185] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.39 (dd, 1H),
8.13 (br s, 1H), 7.74 (d, 1H), 7.60 (m, 2H), 7.4 1 (dd, 1H),
7.4-7.1 (m, 7H), 5.30 (s, 2H), 4.27 (m, 4H), 1.10 (m, 4H) and 0.0
(s, 18H).
[0186] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 153.4, 147.3 (d),
145.7, 145.4, 136.6, 135.4, 131.2, 129.8, 129.7, 129.2 (d), 128.6,
128.5, 127.4, 127.0, 125.2, 122.7, 122.5, 122.2, 121.5, 120.8,
120.0 (d), 118.6, 67.6 (d), 62.0, 19.6 (d) and -1.6.
[0187] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.3.
[0188] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-58.7.
[0189] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-trifluoromethylbenzyl
ester (27e).
[0190] Following the above procedure, 26e (5.1 g, 11 mmol) gave 27e
(5.3 g, 71%) as an oil:
[0191] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.40 (dd, 1H),
8.14 (br s, 1H), 7.74 (d, 2H), 7.6-7.5 (m, 4H), 7.4-7.1 (m, 8H),
5.29 (s, 2H), 4.29 (m, 4H), 1.11 (m, 4H) and 0.0 (s, 18H).
[0192] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 153.4, 145.4 (m),
136.5, 135.4, 131.1, 129.7, 128.6, 128.5, 128.2, 128.1, 127.4,
127.1 (d), 122.5, 120.8, 120.5, 120.0 (d), 67.7 (d), 61.9, 19.6 (d)
and -1.6.
[0193] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.0.
[0194] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-62.7.
[0195] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-3,5-dichlorobenzyl ester
(27f).
[0196] Following the above procedure, 26f (6.3 g, 13 mmol) gave 27f
(7.1 g, 76%) as an oil:
[0197] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.39 (dd, 1H),
8.11 (br s, 1H), 7.74 (d, 1H), 7.59 (br d, 2H), 7.4-7.2 (m, 8H),
7.18 (dd, 2H), 5.35 (s, 2H), 4.29 (m, 4H), 1.11 (m, 4H) and 0.0 (s,
18H).
[0198] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 153.3, 145.4 (d),
136.5, 135.4, 131.9 (d), 131.1, 131.0, 130.2, 129.8, 129.7, 128.7,
128.6, 128.3, 128.0 (d), 127.4, 122.5, 120.8, 67.9 (d), 62.2, 19.5
(d) and -1.6.
[0199] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.0.
[0200] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4,5-dichlorobenzyl ester
(27g).
[0201] Following the above procedure, 26g (11.3 g, 50 mmol) gave
27g (17.4 g, 75%) as an oil:
[0202] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.38 (dd, 1H),
8.12 (br s, 1H), 7.74 (d, 1H), 7.60 (dd, 2H), 7.53 (s, 1H), 7.51
(d, 1H), 7.4-7.2 (m, 6H), 7.17 (dd, 2H), 5.23 (s, 2H), 4.27 (m,
4H), 1.10 (m, 4H) and 0.0 (s, 18H).
[0203] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 153.4, 147.7 (d),
145.4, 136.6, 135.4, 133.1, 131.3, 131.2, 128.9, 128.6, 128.5,
127.7 (d), 127.4, 122.5, 120.8, 67.8 (d), 61.5, 19.6 (d) and
-1.6.
[0204] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.2.
[0205] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5,6-dichlorobenzyl ester
(27h).
[0206] Following the above procedure, 26h (6.2 g, 28 mmol) gave 27h
(9.6 g, 75%) as an oil:
[0207] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.38 (dd, 1H),
8.20 (br s, 1H), 7.73 (d, 1H), 7.60 (br d, 2H), 7.48 (d, 1H),
7.4-7.2 (m, 7H), 7.18 (dd, 2H), 5.48 (s, 2H), 4.28 (m, 4H), 1.10
(m, 4H) and 0.0 (s, 18H).
[0208] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 153.4, 149.2 (d),
145.2, 136.5, 135.4, 134.7, 131.3, 131.0, 129.8, 129.4, 128.6,
128.5, 127.4, 127.3 (d), 122.5, 120.7, 119.6 (d), 67.7 (d), 59.9,
19.6 (d) and -1.6.
[0209] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.0.
[0210] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-3-methylbenzyl ester
(27i).
[0211] Following the above procedure, 26i (1.4 g, 6.2 mmol) gave
27i (1.5 g, 53%) as an orange oil:
[0212] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.23 (dd, 1H),
7.97 (br s, 1H), 7.66 (m, 1H), 7.58 (d, 1H), 7.4-6.9 (m, 10H), 5.27
(s, 2H), 4.11 (m, 4H), 2.27 (s, 3H), 0.94 (m, 4H) and -0.11 (s,
18H).
[0213] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 153.9, 147.6 (d),
146.4, 145.1, 136.6, 134.7, 131.7, 131.6, 131.0, 130.8 (d), 128.5,
128.4 (d), 128.3, 127.6, 127.3, 125.3, 122.3, 121.2, 67.1 (d),
62.9, 19.5 (d), 17.1 and -1.6.
[0214] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.6.
[0215] Mass Calcd. For C.sub.32H.sub.45 N.sub.2O.sub.6PSi.sub.2:
640.859; Found: 640.2
[0216] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4-chlorobenzyl ester
(27j).
[0217] Following the above procedure, 26j (9.3 g, 21 mmol) gave 27j
(12.6 g, 87%) as an oil:
[0218] Rf (1:1 v/v ethyl acetate-hexane) 0.82.
[0219] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.28 (dd, 1H),
8.15 (br s, 1H), 7.72 (d, 1H), 7.60 (d, 2H), 7.4-7.3 (m, 7H),
7.2-7.1 (m, 2H), 5.26 (s, 2H), 4.28 (m, 4H), 1.11 (m, 4H) and 0.0
(s, 18H).
[0220] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 153.6, 149.7 (d),
145.2, 136.6, 135.3, 135.1, 131.4, 131.3, 128.6, 128.5, 127.4,
125.9 (d), 125.4, 122.4, 120.9 (d), 120.8, 67.6 (d), 62.1, 19.5 (d)
and -1.6.
[0221] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 65.1.
[0222] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4-methoxybenzyl ester
(27k).
[0223] Following the above procedure, 26k (2.8 g, 6.5 mmol) gave
27k (3.7 g, 86%) as an oil:
[0224] Rf(1:1 v/v ethyl acetate-hexane) 0.50.
[0225] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.36 (dd, 1H),
8.18 (br s, 1H), 7.72 (d, 1H), 7.4-7.3 (m, 6H), 7.62 (d, 2H),
7.2-7.1 (m, 1H), 6.97 (m, 1H), 6.71 (dd, 1H), 5.24 (s, 2H), 4.29
(m, 4H), 3.80 (s, 3H), 1.11 (m, 4H) and 0.0 (s, 18H).
[0226] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 160.5, 153.9,
150.0 (d), 145.0, 136.7, 135.1, 131.9, 131.6, 130.0, 128.6, 128.4,
127.4, 122.4, 120.9, 119.2 (d), 110.4,67.3 (d), 62.4, 55.5, 19.6
(d) and -1.6.
[0227] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.2.
Example 7
Preparation of (2-formylpyridin-3-yl)carbamic acid
2-(TMSE-phosphonooxy)be- nzyl esters (23, 28a-k) (ozonolysis)
[0228] General Procedure. The corresponding 2-styrylpyridine (22 or
27a-k, 10 mmol) was dissolved in dichloromethane (50 mL) and
ethanol (40 mL). The light yellow solution was ozonized at
-50.degree. C. till the solution turned to light blue. Nitrogen gas
was bubbled through the solution for 30 min to expel excess ozone.
To the solution was then added dimethyl sulfide (5 mL), and the
mixture was stirred for 2 h at room temperature. The solvent was
evaporated under reduced pressure, and the residual product was
purified by FCC (1:9 v/v ethyl acetate-hexane) to afford the
corresponding pyridine-2-carboxaldehyde (23 or 28a-k).
[0229] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-chlorobenzyl ester
(23).
[0230] Following the above procedure, 22 (2.4 g, 3.7 mmol) gave 23
(1.6 g, 72%) as an oil:
[0231] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.38 (br s, 1H),
9.90 (s, 1H), 8.67 (d, 1H), 8.28 (dd, 1H), 7.4-7.3 (m, 2H), 7.23
(dd, 1H), 7.13 (dd, 1H), 5.14 (s, 2H), 4.12 (m, 4H), 0.97 (m, 4H)
and -0.14 (m, 18H).
[0232] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 197.0, 152.9,
147.3 (d), 143.7, 138.3, 136.7, 130.1, 129.6, 129.5, 128.6, 128.5,
126.2, 121.3, 67.4 (d), 61.6, 19.4 (d) and -1.7.
[0233] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.1.
[0234] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-fluorobenzyl ester
(28a).
[0235] Following the above procedure, the crude 27a (31.0 g, 73
mmol) gave 28a (26.9 g, 64%) as an oil:
[0236] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.58 (s, 1H),
10.10 (s, 1H), 8.86 (d, 1H), 8.48 (dd, 1H), 7.52 (m, 1H), 7.4-7.3
(m, 1H), 7.21 (dd, 1H), 7.1-6.9 (m, 1H), 5.30 (s, 2H), 4.4-4.2 (m,
4H), 1.2-1.0 (m, 4H) and 0.0 (s, 18H).
[0237] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 197.1, 159.3 (d),
152.9, 144.5 (dd), 143.7, 143.5, 138.4, 136.8, 121.4 (dd), 116.2
(d), 115.9 (d), 67.3 (d), 61.8, 19.5 (d) and -1.6.
[0238] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.5.
[0239] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-59.3.
[0240] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-nitrobenzyl ester
(28b).
[0241] Following the above procedure, 27b (4.2 g, 9.4 mmol) gave
28b (2.8 g, 50%) as an oil:
[0242] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.42 (br s, 1H),
9.89 (s, 1H), 8.64 (d, 1H), 8.28 (dd, 1H), 8.21 (d, 1H), 8.05 (dd,
1H), 7.47 (d, 1H), 7.33 (dd, 1H), 5.21 (s, 2H), 4.17 (m, 4H), 0.98
(m, 4H) and -0.13 (m, 18H).
[0243] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 197.0, 153.5 (d),
152.7, 144.3, 143.9, 138.1, 136.8, 128.6, 128.3 (d), 126.2, 125.4,
125.2, 120.3, 67.9 (d), 61.4, 19.5 (d) and -1.7.
[0244] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 4.5.
[0245] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-methoxybenzyl ester
(28c).
[0246] Following the above procedure, the crude 27c (7.5 g, 17
mmol) gave 28c (9.8 g, 73%) as an oil:
[0247] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.37 (s, 1H),
9.93 (s, 1H), 8.71 (d, 1H), 8.30 (d, 1H), 7.34 (dd, 1H), 7.19 (d,
1H), 6.85 (d, 1H), 6.70 (d, 1H), 5.18 (s, 2H), 4.2-4.0 (m, 4H),
3.66 (s, 3H), 1.1-0.9 (m, 4H) and 0.0 (s, 18H).
[0248] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 196.9, 156.4,
153.1, 143.6, 142.4 (d), 138.5, 136.7, 128.6, 127.7 (d), 126.2,
120.9, 115.1, 114.3, 67.1 (d), 62.3, 55.5, 19.4 (d) and -1.7.
[0249] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.8.
[0250] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-trifluoromethoxybenzyl
ester (28d).
[0251] Following the above procedure, 27d (4.7 g, 6.6 mmol) gave
28d (3.2 g, 75%) as an oil:
[0252] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.54 (br s, 1H),
10.06 (s, 1H), 8.82 (d, 1H), 8.44 (dd, 1H), 7.48 (dd, 1H), 7.44
(dd, 1H), 7.32 (d, 1H), 7.25 (s, 1H), 7.2-7.1 (m, 1H), 5.30 (s,
2H), 4.26 (m, 4H), 1.10 (m, 4H) and 0.0 (s, 18H).
[0253] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 197.2, 153.0,
147.1 (d), 145.7, 143.9, 138.4, 136.9, 129.8, 128.8, 128.7, 126.4,
122.6, 122.2, 121.3, 120.0, 119.9, 67.6 (d), 61.8, 19.5 (d) and
-1.6.
[0254] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.3.
[0255] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-58.8.
[0256] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-trifluoromethylbenzyl
ester (28e).
[0257] Following the above procedure, 27e (12.2 g, 18 mmol) gave
28e (6.9 g, 63%) as an oil:
[0258] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.54 (br s, 1H),
10.06 (d, 1H), 8.82 (br d, 1H), 8.44 (dd, 1H), 7.72 (br s, 1H),
7.6-7.5 (m, 2H), 7.48 (dd, 1H), 7.3-7.1 (m, 2H), 5.33 (s, 2H), 4.27
(m, 4H), 1.10 (m, 4H) and 0.0 (s, 18H).
[0259] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 197.1, 153.0,
151.5 (d), 143.9, 138.4, 136.9, 129.8, 129.7, 128.7, 127.7, 127.6,
127.3 (d), 127.1, 127.0, 126.4, 126.3, 125.2, 120.3 (d), 120.0 (d),
67.7 (d), 61.8, 19.6 (d) and -1.6.
[0260] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 4.9.
[0261] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-62.7.
[0262] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-3,5-dichlorobenzyl ester
(28f).
[0263] Following the above procedure, 27f (8.0 g, 12 mmol) gave 28f
(5.1 g, 71%) as an oil:
[0264] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.54 (br s, 1H),
10.05 (s, 1H), 8.79 (d, 1H), 8.42 (dd, 1H), 7.46 (dd, 1H), 7.35
(dd, 2H), 7.24 (s, 1H), 5.38 (s, 2H), 4.27 (m, 4H), 1.12 (m, 4H)
and 0.0 (m, 18H).
[0265] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 197.2, 152.8,
149.9, 143.9, 143.6 (d), 138.4, 136.8, 131.8 (d), 131.0 (d), 130.1,
128.7, 128.0 (d), 127.8 (d), 126.3, 120.0 (d), 67.8 (d), 62.0, 19.6
(d) and -1.6.
[0266] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.1.
[0267] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4,5-dichlorobenzyl ester
(28g).
[0268] Following the above procedure, 27g (17.4 g, 25 mmol) gave
28g (13.4 g, 86%) as an oil:
[0269] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.51 (br s, 1H),
10.05 (s, 1H), 8.79 (d, 1H), 8.42 (dd, 1H), 7.53 (dd, 1H), 7.5-7.4
(m, 1H), 7.24 (s, 1H), 6.96 (dd, 1H), 5.23 (s, 2H), 4.28 (m, 4H),
1.10 (m, 4H) and 0.0 (m, 18H).
[0270] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 197.2, 152.9,
147.4 (d), 144.0, 138.3, 136.9, 133.1, 131.0, 128.8 (d), 128.7,
127.2 (d), 126.3, 122.2, 122.0, 67.8 (d), 61.3, 19.6 (d) and
-1.6.
[0271] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.1.
[0272] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5,6-dichlorobenzyl ester
(28h).
[0273] Following the above procedure, 27h (9.5 g, 14 mmol) gave 28h
(6.5 g, 77%) as an oil:
[0274] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.44 (br s, 1H),
10.05 (s, 1H), 8.86 (d, 1H), 8.44 (dd, 1H), 7.50 (d, 1H), 7.47 (d,
1H), 7.41 (d, 1H), 7.38 (m, 1H), 5.46 (s, 2H), 4.25 (m, 4H), 1.10
(m, 4H) and 0.0 (m, 18H).
[0275] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 197.0, 153.0,
149.9, 149.2 (d), 143.8, 138.5, 136.8, 135.0, 131.2, 129.8, 129.6,
128.7, 126.6 (d), 126.3, 119.5 (d), 67.7 (d), 59.9, 19.5 (d) and
-1.6.
[0276] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 4.9.
[0277] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-3-methylbenzyl ester
(28i).
[0278] Following the above procedure, 27i (1.5 g, 2.2 mmol) gave
28i (1.1 g, 86%) as an oil:
[0279] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.35 (br s, 1H),
9.92 (s, 1H), 8.71 (d, 1H), 8.28 (dd, 1H), 7.32 (dd, 1H), 7.16 (d,
1H), 7.05 (d, 1H), 6.96 (dd, 1H), 5.31 (s, 2H), 4.12 (m, 4H), 2.28
(s, 3H), 0.97 (m, 4H) and -0.12 (m, 18H).
[0280] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 196.9, 153.2,
147.2 (d), 143.6, 138.6, 136.6, 131.6, 130.9 (d), 128.6, 128.0 (d),
127.4, 126.2, 125.3, 67.1 (d), 62.9, 19.4 (d), 17.0 and -1.7.
[0281] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.8.
[0282] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4-chlorobenzyl ester
(28j).
[0283] Following the above procedure, 27j (12.2 g, 19 mmol) gave
28j (8.9 g, 80%) as an oil:
[0284] Rf (1:1 v/v ethyl acetate-hexane) 0.66.
[0285] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.49 (br s, 1H),
10.04 (s, 1H), 8.80 (d, 1H), 8.42 (dd, 1H), 7.5-7.4 (m, 3H), 7.16
(dd, 1H), 5.26 (s, 2H), 4.27 (m, 4H), 1.11 (m, 4H) and 0.0 (s,
18H).
[0286] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 197.1, 153.1,
149.3 (d), 143.8, 138.5, 136.8, 135.0, 131.0, 128.7, 126.3, 125.3
(d), 125.2, 120.5 (d), 67.5 (d), 61.9, 19.5 (d) and -1.6.
[0287] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.0.
[0288] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4-methoxybenzyl ester
(28k).
[0289] Following the above procedure, 27k (5.1 g, 8.0 mmol) gave
28k (2.7 g, 58%) as an oil:
[0290] Rf (1:1 v/v ethyl acetate-hexane) 0.44.
[0291] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.50 (br s, 1H),
10.05 (s, 1H), 8.84 (d, 1H), 8.42 (dd, 1H), 7.46 (dd, 1H), 7.36
(dd, 1H), 7.01 (s, 1H), 6.71 (dd, 1H), 5.24 (s, 2H), 4.27 (m, 4H),
3.80 (s, 3H), 1.10 (m, 4H) and 0.0 (s, 18H).
[0292] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 197.0, 160.9,
153.4, 150.2 (d), 143.6, 138.7, 136.7, 131.7, 128.7, 126.3, 118.5
(d), 110.6, 106.1 (d), 67.3 (d), 62.4, 55.5, 19.5 (d) and -1.6.
[0293] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.0.
Example 8
Preparation of pyridine-2-carboxaldehyde thiosemicarbazones (24,
29a-k)
[0294] General Procedure. The corresponding pyridine-2-formaldehyde
(23 or 28a-k, 10 mmol) was dissolved in ethanol-water (2:1 v/v, 150
mL). To the solution was added thiosemicarbazide (11 mmol). The
solution was stirred for 30 min at ambient temperature. After
addition of water (50 mL), the reaction mixture was stirred
vigorously for 2 h at room temperature. The yellow precipitate was
collected by filtration, washed with ethanol-water (1:4 v/v) and
dried in vacuum to afford the corresponding
pyridine-2-carboxaldehyde thiosemicarbazone (24, 29a-k).
[0295] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethyl silyl ethyl) phosphonooxy-5-chlorobenzyl ester
(24).
[0296] Following the above procedure, 23 (6.9 g, 12 mmol) gave 24
(4.9 g, 63%) as a yellow solid:
[0297] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.77 (br s,
1H), 10.03 (br s, 1H), 8.40 (dd, 1H), 8.28 (br s, 1H), 8.26 (s,
1H), 7.94 (br s, 1H), 7.5-7.4 (m, 4H), 5.20 (s, 2H), 4.06 (m, 4H),
0.98 (m, 4H) and -0.03 (m, 18H).
[0298] .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 178.5, 153.4,
148.0 (d), 144.2, 142.9, 141.0, 133.9, 129.5 (d), 128.9, 128.4,
128.0, 124.4, 121.6, 65.1 (d), 61.2, 18.9 (d) and -1.5.
[0299] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.8.
[0300] Mass Calcd. For C.sub.25H.sub.39ClN.sub.5O.sub.6PSSi.sub.2:
660.267; Found: 660.2
[0301] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-5-fluorobenzyl ester
(29a).
[0302] Following the above procedure, 28a (14.3 g, 25 mmol) gave
29a (12.9 g, 80%) as a yellow solid:
[0303] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.85 (s, 1H),
10.13 (s, 1H), 8.45 (d, 1H), 8.26 (s, 1H), 7.5-7.1 (m, 5H), 5.21
(s, 2H), 4.2-4.0 (m, 4H), 1.0-0.9 (m, 4H) and 0.0 (s, 18H).
[0304] .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 178.6, 159.8,
156.0, 153.4, 145.1 (d), 143.5, 141.5, 140.4, 134.2, 129.5, 124.5,
121.4 (d), 115.5, 64.4 (d), 61.3, 18.9 (d) and -1.6.
[0305] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 10.5.
[0306] .sup.19F NMR (282 MHz, DMSO-d.sub.6) .delta.-62.5.
[0307] Mass Calcd. For C.sub.25H.sub.39FN.sub.5O.sub.6PSSi.sub.2:
643.813 Found: 644.2
[0308] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-5-nitrobenzyl ester
(29b).
[0309] Following the above procedure, 28b (1.6 g, 2.7 mmol) gave
29b (1.3 g, 77%) as a yellow solid:
[0310] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.86 (br s,
1H), 10.14 (br s, 1H), 8.4-8.2 (m, 4H), 7.87 (br s, 2H), 7.64 (m,
1H), 7.52 (m, 1H), 5.26 (s, 2H), 4.05 (m, 4H), 0.97 (m, 4H) and
-0.03 (m, 18H).
[0311] .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 178.6, 155.3
(d), 153.4, 143.7, 142.7, 140.7, 134.1, 128.1 (d), 125.2, 124.6,
124.5, 120.1, 64.8 (d), 61.2, 18.9 (d) and -1.5.
[0312] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.2.
[0313] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-5-methoxybenzyl ester
(29c).
[0314] Following the above procedure, 28c (5.0 g, 8.8 mmol) gave
29c (4.4 g, 77%) as a yellow solid:
[0315] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.85 (s, 1H),
10.08 (s, 1H), 8.41 (d, 1H), 8.35 (d, 1H), 8.30 (s, 1H), 8.03 (s,
2H), 7.51 (dd, 1H), 7.26 (d, 1H), 7.01 (d, 1H), 6.90 (dd, 1H), 5.26
(s, 2H), 4.2-4.0 (m, 4H), 3.75 (s, 3H), 1.1-0.9 (m, 4H) and 0.0 (s,
18H).
[0316] .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 178.7, 155.7,
153.6, 143.9, 142.6(d), 134.2, 129.4, 128.4 (d), 124.5, 121.1,
114.1, 113.9, 64.9 (d), 61.9, 55.6, 19.1 (d) and -1.4.
[0317] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 10.3.
[0318] Mass Calcd. For C.sub.26H.sub.42 N.sub.5O.sub.7PSSi.sub.2:
655.848 Found: 656.2
[0319] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-5-trifluoromethoxybenzyl
ester (29d).
[0320] Following the above procedure, 28d (2.5 g, 3.9 mmol) gave
29d (1.9 g, 68%) as a yellow solid:
[0321] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.81 (s, 1H),
10.04 (br s, 1H), 8.42 (d, 1H), 8.26 (s, 1H), 7.95 (br s, 1H),
7.5-7.2 (m, 4H), 5.24 (s, 2H), 4.07 (m, 4H), 0.96 (m, 4H) and -0.04
(m, 18H).
[0322] .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 178.4, 153.4,
147.8 (d), 144.1, 144.0, 133.9, 129.4 (d), 124.4, 121.9, 121.7,
121.6, 121.4, 118.3, 65.1 (d), 61.2, 18.9 (d) and -1.6.
[0323] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.8.
[0324] .sup.19F NMR (282 MHz, DMSO-d.sub.6) .delta.-53.0.
[0325] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-5-trifluoromethylbenzyl
ester (29e).
[0326] Following the above procedure, 28e (5.3 g, 8.5 mmol) gave
29e (3.6 g, 61%) as a yellow solid:
[0327] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.81 (s, 1H),
10.03 (br s, 1H), 8.42 (d, 1H), 8.4-8.3 (m, 2H), 8.26 (s, 1H), 7.89
(br s, 1H), 7.79 (s, 1H), 7.75 (d, 2H), 7.56 (d, 2H), 7.49 (dd,
1H), 5.27 (s, 2H), 4.06 (m, 4H), 0.97 (m, 4H) and -0.04 (m,
18H).
[0328] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.5.
[0329] .sup.19F NMR (282 MHz, DMSO-d.sub.6) .delta.-56.2.
[0330] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-3,5-dichlorobenzyl ester
(29f).
[0331] Following the above procedure, 28f (4.8 g, 7.7 mmol) gave
29f (4.6 g, 85%) as a yellow solid:
[0332] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.75 (s, 1H),
10.04 (s, 1H), 8.39 (d, 1H), 8.30 (d, 1H), 8.26 (s, 1H), 7.91 (br
s, 1H), 7.69 (dd, 1H), 7.50 (d, 1H), 7.43 (dd, 1H), 5.29 (s, 2H),
4.24 (m, 4H), 1.03 (m, 4H) and -0.01 (m, 18H).
[0333] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 10.3.
[0334] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-4,5-dichlorobenzyl ester
(29g).
[0335] Following the above procedure, 28g (2.0 g, 3.2 mmol) gave
29g (1.5 g, 70%) as a yellow solid:
[0336] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.85 (s, 1H),
10.08 (s, 1H), 8.42 (d, 1H), 8.31 (d, 1H), 8.26 (s, 1H), 7.89 (m,
1H), 7.70 (s, 1H), 7.59 (s, 1H), 7.5-7.2 (m, 2H), 5.18 (s, 2H),
4.02 (m, 4H), 0.95 (m, 4H) and 0.0 (m, 18H).
[0337] .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 178.6, 153.3,
148.8, 143.8, 142.4, 140.6, 134.0, 130.8, 130.2 (d), 129.5, 128.3
(d), 125.8 (d), 124.4, 121.5, 119.9, 64.9 (d), 60.8, 18.9 (d) and
-1.5.
[0338] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.7.
[0339] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-5,6-dichlorobenzyl ester
(29h).
[0340] Following the above procedure, 28h (5.9 g, 9.5 mmol) gave
29h (3.6 g, 55%) as a yellow solid:
[0341] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.82 (s, 1H),
9.97 (br s, 1H), 8.50 (m, 1H), 8.41 (d, 1H), 8.27 (d, 1H), 8.23 (s,
1H), 7.72 (m, 1H), 7.67 (d, 1H), 7.48 (m, 1H), 7.41 (d, 1H),
7.3-7.1 (m, 1H), 5.32 (s, 2H), 4.03 (m, 4H), 0.96 (m, 4H) and -0.06
(m, 18H).
[0342] .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 178.3, 153.3,
150.6 (d), 143.8, 140.8, 134.0, 133.8, 133.3., 131.2, 130.3, 129.4,
126.7 (d), 124.4, 120.1, 119.9 (d), 64.8 (d), 59.4, 18.9 (d) and
-1.6.
[0343] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.6.
[0344] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-3-methylbenzyl ester
(29i).
[0345] Following the above procedure, 28i (1.1 g, 1.9 mmol) gave
29i (0.7 g, 57%) as a yellow solid:
[0346] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.76 (br s,
1H), 9.99 (br s, 1H), 8.42 (br s, 1H), 8.37 (m, 1H), 8.27 (br s,
1H), 8.24 (m, 1H), 8.03 (br s, 1H), 7.45 (dd, 1H), 7.25 (d, 1H),
7.19 (d, 1H), 7.11 (dd, 1H), 5.30 (s, 2H), 4.08 (m, 4H), 2.29 (s,
3H), 1.01 (m, 4H) and -0.03 (m, 18H).
[0347] .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 178.7, 153.8,
147.5 (d), 144.4, 143.2, 141.2, 134.3, 131.1, 130.7 (d), 128.9 (d),
126.4, 124.9, 124.6, 65.4 (d), 62.4, 19.2 (d), 16.9 and -1.4.
[0348] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 10.4.
[0349] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-4-chlorobenzyl ester
(29j).
[0350] Following the above procedure, 28j (10.5 g, 18.5 mmol) gave
29j (11.8 g, 97%) as a yellow solid:
[0351] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.74 (s, 1H),
10.05 (br s, 1H), 8.4-8.3 (m, 3H), 7.85 (br s, 1H), 7.56 (d, 1H),
7.5-7.4 (m, 3H), 5.21 (s, 2H), 4.22 (m, 4H), 1.04 (m, 4H) and -0.01
(s, 18H).
[0352] .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 153.2, 148.5,
148.4, 144.8, 144.5, 133.8, 133.0, 130.8, 126.5, 126.4, 125.4,
124.1, 119.7, 67.2 (d), 60.8, 18.9 (d) and -1.6.
[0353] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.5.
[0354] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-4-methoxybenzyl ester
(29k).
[0355] Following the above procedure, 28k (2.6 g, 4.5 mmol) gave
29k (2.7 g, 91%) as a yellow solid:
[0356] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 11.74 (d, 1H),
9.98 (br s, 1H), 8.4-8.3 (m, 3H), 7.82 (br s, 1H), 7.44 (m, 1H),
6.87 (m, 3H), 5.16 (s, 2H), 4.19 (m, 4H), 3.77 (s, 3H), 1.03 (m,
4H) and -0.01 (s, 18H).
[0357] .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 178.4, 160.0,
153.2, 150.2, 148.4, 144.8, 144.5, 133.8, 133.0, 130.8, 124.1,
119.0, 110.4, 106.0, 66.8 (d), 61.4, 55.3, 18.9 (d) and -1.6.
[0358] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.6.
Example 9
Preparation of Free phosphonic acids (6-17)
[0359] General Procedure. To a solution of the corresponding
TMSE-protected phosphate (24 or 29a-k, 10 mmol) in dichloromethane
(300-500 mL) was added trifluoroacetic acid (TFA, 20-50 mL) at
0.degree. C. The reaction mixture was stirred vigorously for 2 h in
an ice bath. A precipitate was collected by filtration, washed with
cold dichloromethane, and then dried in vacuum. More commonly, the
solvents were evaporated, and the resulting residual mixture was
then dried in vacuum. The corresponding free phosphonic acid (6-17)
was obtained as a yellow solid or glassy solid.
Example 10
Preparation of disodium salt of phosphonic acid (25, 30a-k)
[0360] General Procedure. The corresponding free phosphonic acid
(6-17, 10 mmol) was neutralized with an aqueous saturated sodium
bicarbonate (NaHCO.sub.3) solution (50-100 mL). The suspension was
stirred for 2 h at ambient temperature, and then added a minimum
amount of water to make homogenous. The aqueous solution was
purified by reversed phase column chromatography with de-ionized
water. The fractions were monitored by .sup.31P NMR and combined.
After lyophylization, the corresponding disodium salt (25 or 30a-k)
was obtained as a pale yellow powder.
[0361] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-chlorobenzyl ester (25).
[0362] Following the above procedure, 24 (1.1 g, 1.7 mmol) gave 25
(0.4 g, 49%) as a pale yellow powder:
[0363] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.94 (br s, 2H),
7.72 (s, 1H), 7.2-7.0 (m, 3H) and 4.98 (s, 2H).
[0364] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 179.6, 157.0, 153.2,
147.2 (d), 145.2, 136.8, 131.4, 130.5, 128.8, 127.8, 123.6 and
65.4.
[0365] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.3.
[0366] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-fluorobenzyl ester (30a).
[0367] Following the above procedure, 29a (10.5 g, 16 mmol) gave 7
(6.2 g, 86%), which upon treatment with NaHCO.sub.3 gave 30a (4.0
g, 59%) as a pale yellow powder:
[0368] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 8.2 (br s, 1H), 7.8
(br m, 1H), 7.57 (br s, 1H), 7.15 (m, 1H), 6.93 (m, 1H), 6.81 (m,
1H), 6.78 (m, 1H) and 4.93 (s, 2H).
[0369] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 179.4, 161.5, 158.4,
156.5, 150.3, 147.3, 146.5, 136.7, 130.8, 130.4, 127.7, 123.5,
117.5, 117.2and 65.2.
[0370] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.5.
[0371] .sup.19F NMR (282 MHz, D.sub.2O) .delta.-57.4.
[0372] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-nitrobenzyl ester (30b).
[0373] Following the above procedure, 29b (2.1 g, 3.0 mmol) gave
30b (1.0 g, 73%) as a dark yellow powder:
[0374] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 8.0-7.8 (m, 4H),
7.40 (m, 1H), 7.17 (m, 1H) and 5.06 (s, 2H).
[0375] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 13.8.
[0376] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-methoxybenzyl ester (30c).
[0377] Following the above procedure, 29c (4.3 g, 16 mmol) gave 9
(2.9 g, 98%), which upon treatment with NaHCO.sub.3 gave 30c (1.6
g, 43%) as a pale yellow powder:
[0378] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.96 (br s, 1H),
7.70 (br s, 1H), 7.21 (br s, 1H), 7.08 (br s, 1H), 6.73 (s, 2H),
5.05 (s, 2H) and 3.65 (s, 3H).
[0379] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 174.5, 151.8, 151.1,
143.4, 142.1, 141.7, 135.9, 131.6, 126.4, 124.9, 122.6, 118.4,
111.7, 60.8 and 53.2.
[0380] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.6.
[0381] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-trifluoromethoxybenzyl ester (30d).
[0382] Following the above procedure, 29d (1.9 g, 2.6 mmol) gave
30d (0.5 g, 31%) as a pale yellow powder:
[0383] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.93 (br s, 1H),
7.86 (br d, 1H), 7.71 (s, 1H), 7.25 (d, 1H), 7.02 (m, 4H) and 5.01
(s, 2H).
[0384] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 179.5, 173.5, 157.1,
153.3, 147.1, 146.8, 145.5, 141.2 (m), 136.5, 132.4 (m), 130.2 (d),
127.7 (d), 124.5, 124.0, 123.1, 122.6, 121.0 and 65.4.
[0385] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.3.
[0386] .sup.19F NMR (282 MHz, D.sub.2O) .delta.-56.3.
[0387] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-trifluoromethylbenzyl ester (30e).
[0388] Following the above procedure, 29e (3.6 g, 5.2 mmol) gave
30e (1.3 g, 45%) as a pale yellow powder:
[0389] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.98 (br s, 1H),
7.89 (d, 1H), 7.77 (s, 1H), 7.4-7.3 (m, 3H), 7.08 (m, 1H) and 5.04
(s, 2H).
[0390] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.0.
[0391] .sup.19F NMR (282 MHz, D.sub.2O) .delta.-59.4.
[0392] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-3,5-dichlorobenzyl ester (30f).
[0393] Following the above procedure, 29f (4.5 g, 6.5 mmol) gave
30f (0.8 g, 24%) as a pale yellow powder:
[0394] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 8.31 (br s, 1H),
7.88 (br d, 2H), 7.6-7.5 (m, 2H), 7.2-6.8 (m, 5H) and 5.07 (s,
2H).
[0395] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 179.6, 156.6 (d),
149.4 (d), 147.4, 146.8 (d), 136.6, 134.2, 131.5, 131.1, 130.7 (d),
130.1, 128.5, 127.7 (d) and 65.6.
[0396] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.4.
[0397] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-4,5-dichlorobenzyl ester (30g).
[0398] Following the above procedure, 29g (2.5 g, 3.0 mmol) gave
30g (0.4 g, 23%) as a pale yellow powder:
[0399] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 8.07 (s, 1H), 7.99
(m, 1H), 7.85 (s, 1H), 7.39 (s, 1H), 7.19 (m, 2H) and 4.99 (s,
2H).
[0400] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.3.
[0401] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5,6-dichlorobenzyl ester (30h).
[0402] Following the above procedure, 29h (4.6 g, 6.6 mmol) gave
30h (2.3 g, 64%) as a pale yellow powder:
[0403] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 8.01 (s, 1H), 7.91
(br s, 1H), 7.73 (s, 1H), 7.23 (dd, 2H), 7.12 (m, 1H) and 5.18 (s,
2H).
[0404] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.2.
[0405] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-3-methylbenzyl ester (30i).
[0406] Following the above procedure, 29i (1.2 g, 1.8 mmol) gave
30i (0.5 g, 57%) as a yellow powder:
[0407] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 8.11 (br s, 2H),
7.91 (m, 2H), 7.71 (m, 1H), 7.00 (m, 2H), 6.84 (m, 1H), 5.22 (s,
2H) and 2.14 (s, 3H).
[0408] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 146.1, 134.6, 133.6,
131.1, 128.8, 127.9, 125.7, 66.6 and 19.1.
[0409] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.2.
[0410] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-4-chlorobenzyl ester (30j).
[0411] Following the above procedure, 29j (4.2 g, 6.6 mmol) gave
30j (1.6 g, 48%) as a pale yellow powder:
[0412] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.98 (s, 1H), 7.90
(m, 1H), 7.74 (s, 1H), 7.31 (s, 1H), 7.09 (m, 3H), 6.85 (m, 1H) and
5.00 (s, 2H).
[0413] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 180.0, 157.7, 155.9,
147.6, 147.4, 142.3, 137.1, 136.8, 133.2, 132.9, 128.3, 127.9,
124.9 and 65.9.
[0414] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.3.
[0415] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-4-methoxybenzyl ester (30k).
[0416] Following the above procedure, 29k (2.9 g, 4.4 mmol) gave
30k (1.2 g, 54%) as a pale yellow powder:
[0417] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 8.06 (s, 1H), 7.94
(s, 1H), 7.64 (s, 1H), 7.13 (m, 1H), 7.0-6.8 (m, 3H), 6.43 (m, 1H),
4.06 (s, 2H) and 3.58 (s, 3H).
[0418] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 161.5, 161.3, 155.1,
133.1, 127.3, 127.0, 111.4, 108.3 and 57.9.
[0419] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.3.
Example 11
Biological Data
Antiviral Aactivity of Triapine (R.sup.1 and R.sup.2 are Both H)
Plus Lamivudine Against HIV-1 Infection
[0420] MT-2 cells infected with strain IIIB of HIV (wildtype, 3TC
resistant, or AZT resistant) are used for determining antiviral
activities (Bridges et al., Biochem. Pharmacol. Vol. 51, 731-36,
1996). Cells are infected with virus at a multiplicity of infection
of 0.1 TCID.sub.50/ml and added to wells containing serial 2-fold
dilutions of drugs. MT-2 cells in RPMI 1640 medium supplemented
with 10% dialyzed fetal bovine serum and 100 .mu.g/mL Kanamycin are
infected with virus and immediately added to serial dilutions of
the drugs.
[0421] After 5 days, 20 .mu.L of MTT dye (2.5 mg/mL in PBS) is
added per well. At the end of the 4-h incubation period, 150 .mu.L
of acidified 2-propanol with 2% NP-40 nonionic detergent is added
per well. After the crystals of dye are dissolved (usually 1-2
days), the plates are read on a microplate reader. Using this
MTT-dye reduction method (Larder et al., (1990), Antimicrob. Agents
Chemother. 34, 436-41), the percentage of protection can be
calculated from the formula [(a-b)/(c-b).times.100] in which a is
the A.sub.595 of drug-treated virus-infected wells, b is the
A.sub.595 of no-drug infected cells, and c is the A.sub.595 of the
no-drug uninfected cells. The EC.sub.50 was calculated from linear
log 10 plots of the percentage protection verses inhibition
concentration. Concentrations of Triapine tested are ranging from
0.1 to 0.8 micromolar where concentration of lamivudine are from
0.02 to 2 micromolar.
Example 12
Antiviral Activity of Triapine (R.sup.1 and R.sup.2 are Both H)
Plus D4T, DDI and AZT Against HIV-1 Infection
[0422] Using the above-described experimental system, the ability
of Triapine to potentiate the anti-HIV activity of the
dideoxynucleoside analogs ddI (2',3'-dideoxyinosine), D4T
(2',3'-dideoxydidehydrothymidine) and AZT (3'-azidothymidine) was
examined in HIV-infected MT-2 cells (as described above).
Isobolograms, describing the interactions, were plotted according
to the method described in Bridges et al., Biochem. Pharmacol., 51,
731-736 (1996). The results, presented in FIG. 4, evidence that
triapine elicits synergistic anti-HIV activites when combined with
D4T and ddI and interacts in an additive fashion when combined with
AZT.
Example 13
Antiviral Activity of Triapine Plus Lamivudine Against HBV
Infection
[0423] The effects of drugs on HBV viral DNA replication are
assessed as described by Doong et al. (Proc. Natl. Acad. Sci. USA
88, 8495-99, 1991). A human hepatoma cell line carrying HBV
(designated 2.2.15) is used in this study (Price et al., (1989)
Proc. Natl. Acad. Sci. USA 86, 8541-44). Six-day-old cultures are
treated with various concentrations of the drug in culture medium
(minimal essential medium with Earle's salts and 10% fetal bovine
serum [MEME]). The drugs are left in the culture medium for 3 days,
and then the medium is aspirated and fresh medium containing the
same concentration of the drugs are added. At the end of the
subsequent 3-day period, the culture medium is harvested. An
aliquot of the culture medium (5 .mu.l) is used for the estimation
of the HBV surface antigen (HBVsAg) (as described below). The
culture medium is processed to obtain virions by polyethylene
glycol precipitation. The viral DNA recovered from the secreted
particles is subjected to Southern blot analysis (Sambrook et al.,
(1989) Molecular cloning laboratory manual, 2nd ed. Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.). Inhibition of viral
DNA replication is determined by comparison of the viral DNAs from
drug-treated and nontreated cultures. Southern blot analysis of the
DNA is performed, and the level of inhibition is determined by
hybridization of the blots to an HBV-specific probe followed by
autoradiography. Quantitation of the autoradiographs is performed
by densitometric scans on a densitometer. The percentage of
inhibition can be calculated from the formula
[1-(a-b)/(c-b).times.100] in which a is the densitometric unit of
drug-treated virus-infected wells, b is the densitometric unit of
no-drug infected cells, and c is the densitometric unit of the
no-drug uninfected cells. The ED.sub.50 was calculated from linear
log 10 plots of percentage inhibition verses the inhibition
concentration.
Example 14
Antiviral Activity of Triapine Plus Acyclovir Against HSV
Infection
[0424] To evaluate the antiviral activities of Triapine, acyclovir,
and combination thereof, HeLa S.sub.3 cells are seeded in
25-cm.sup.2 flasks and are used as host cells for virus infection
(laboratory strains of virus HSV-1, HSV-2, wild type (Darby et al.,
(1982) Nature, 289, 81-3; Cheng, et al., Antimicrob. Agents
Chemother. 18, 957-61, 1980). After a 1-hr adsorption period with
virus at 5-10 plaque-forming units per cell, the monolayers are
rinsed with phosphate-buffered saline, followed by the addition of
5 ml of growth medium containing various concentrations of drug.
Concentrations of Triapine tested range from 0.1 to 1.0 micromolar
where concentrations of acyclovir are from 1-50 micromolar. The
cells are incubated at 37.degree. C. for 48 hr and then are stored
frozen at -70.degree. C. until titration. The amounts of HSV
contained in cells are determined with Vero cells using the methods
described by Cheng et al. (Cheng, et al., Antimicrob. Agents
Chemother. 18, 957-61, 1980).
[0425] The results of the above-test are presented in the following
Table 1, below, which presents the antiviral activity of Triapine,
at low, non-toxic clinically achievable concentrations of 0.3, 0.6
and 1.0 .mu.M, in combination with acyclovir against HSV-1 (KOS)
and HSV-2 (333) strains of herpes simplex virus in a virus yield
assay. The experimental results evidence that in the presence of as
little as 0.3 and 0.6 .mu.M Triapine, the EC.sub.90 of ACV
decreased by 10- to greater than 24-fold for HSV-1. A greater than
4-fold decrease in the EC.sub.90 of ACV against HSV-2 is observed
with 0.6 .mu.M Triapine. These data evidence that Triapine may
markedly potentiate the anti-HSV activity of anti-HSV
nucleoside-type analogs such as ACV in treating HSV infections.
1TABLE 1 Virus Yield Assay in HSV-Infected Vero Cells EC.sub.90
(.mu.M) DRUG HSV-1 HSV-2 ACV 29.3 36.5 Triapine >1 >1 0.3 ACV
+ 0.3 .mu.M Triapine 2.9 4.7 0.6 ACV + 0.6 .mu.M Triapine 1.2 4.8
ACV + 1.0 .mu.M Triapine <0.005 2.2
[0426] It is to be understood by those skilled in the art that the
foregoing description and examples are illustrative of practicing
the present invention, but are in no way limiting. Variations of
the detail presented herein may be made without departing from the
spirit and scope of the present invention as defined by the
following claims.
* * * * *