U.S. patent application number 09/977659 was filed with the patent office on 2002-08-29 for modified prodrug forms of ap/amp.
Invention is credited to Doyle, Terrence W., Karra, Srinivasa, Li, Zujin, Lin, Xu, Mao, John, Qiao, Qi, Xu, Yang.
Application Number | 20020119955 09/977659 |
Document ID | / |
Family ID | 22906907 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119955 |
Kind Code |
A1 |
Doyle, Terrence W. ; et
al. |
August 29, 2002 |
Modified prodrug forms of AP/AMP
Abstract
The present invention relates to compounds according to the
structure: 1 Where R is H or CH.sub.3; R.sub.2 is phosphate which
can be free acid or salt; R.sub.3 is H, F, Cl, Br, I, OCH.sub.3,
OCF.sub.3, CF.sub.3 or a C.sub.1-C.sub.3 alkyl group; R.sub.4 is H,
F, Cl, Br, I, OCH.sub.3, OCF.sub.3 or CF.sub.3; and R.sub.5 and
R.sub.6 are each independently H, F, Cl, Br, I, OCH.sub.3,
OCF.sub.3 or CF.sub.3, with the proviso that when any two of
R.sub.3, R.sub.4, R.sub.5 or R.sub.6 are other than H, the other
two of R.sub.3, R.sub.4, R.sub.5 or R.sub.6 are H which may be used
to treat neoplasia, including cancer.
Inventors: |
Doyle, Terrence W.;
(Killingworth, CT) ; Karra, Srinivasa; (Hamden,
CT) ; Li, Zujin; (Orange, CT) ; Lin, Xu;
(Branford, CT) ; Mao, John; (Guilford, CT)
; Qiao, Qi; (Nutley, NJ) ; Xu, Yang;
(Cheshire, CT) |
Correspondence
Address: |
Henry D. Coleman
Coleman Sudol Sapone, P.C.
714 Colorado Avenue
Bridgeport
CT
06605-1601
US
|
Family ID: |
22906907 |
Appl. No.: |
09/977659 |
Filed: |
October 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60240529 |
Oct 13, 2000 |
|
|
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Current U.S.
Class: |
514/89 ;
546/22 |
Current CPC
Class: |
A61K 31/407 20130101;
A61P 43/00 20180101; A61K 31/70 20130101; C07D 213/75 20130101;
A61K 31/44 20130101; C07F 9/58 20130101; A61K 45/06 20130101; A61K
31/704 20130101; A61P 35/00 20180101; A61K 31/7068 20130101; A61K
31/47 20130101; A61K 31/675 20130101; A61K 47/52 20170801; A61K
33/243 20190101; A61K 31/44 20130101; A61K 31/195 20130101; A61K
31/47 20130101; A61K 31/44 20130101; A61K 31/704 20130101; A61K
31/44 20130101; A61K 31/7068 20130101; A61K 31/44 20130101; A61K
33/24 20130101; A61K 31/44 20130101; A61K 31/407 20130101; A61K
2300/00 20130101; A61K 31/44 20130101; A61K 2300/00 20130101; A61K
31/47 20130101; A61K 2300/00 20130101; A61K 31/675 20130101; A61K
2300/00 20130101; A61K 31/704 20130101; A61K 2300/00 20130101; A61K
31/7068 20130101; A61K 2300/00 20130101; A61K 33/24 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/89 ;
546/22 |
International
Class: |
A61K 031/675; C07F
009/58 |
Claims
1. A compound according to the structure: 6Where R is H or
CH.sub.3; R.sub.2 is phosphate which can be free acid or salt;
R.sub.3 is H, F, Cl, Br, I, OCH.sub.3, OCF.sub.3, CF.sub.3 or a
C.sub.1-C.sub.3 alkyl group; R.sub.4is H, F, Cl, Br, I, OCH.sub.3,
OCF.sub.3 or CF.sub.3; and R.sub.5 and R.sub.6 are each
independently H, F, Cl, Br, I, OCH.sub.3, OCF.sub.3 or CF.sub.3,
with the proviso that at least one of R.sub.3, R.sub.4, R.sub.5 or
R.sub.6 are other than H and when any two of R.sub.3, R.sub.4,
R.sub.5 or R.sub.6 are other than H, the other two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are H.
2. The compound according to claim 1 wherein R.sub.4 is Cl, F or Br
when R.sub.3, R.sub.5 and R.sub.6 are each H.
3. The compound according to claim 2 wherein R.sub.4 is Cl.
4. The compound according to claim 1 wherein R.sub.5 is F, Cl,
OCH.sub.3 or OCF.sub.3 when R.sub.3, R.sub.4 and R.sub.6 are each
H.
5. The compound according to claim 4 wherein R.sub.5 is F or
Cl.
6. The compound according to claim 5 wherein R.sub.5 is F.
7. The compound according to claim 5 wherein R.sub.5 is Cl.
8. The compound according to claim 1 wherein two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are other than H and are selected from
F, Cl, Br or I.
9. The compound according to claim 8 wherein two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are each F or Cl.
10. The compound according to claim 8 wherein R.sub.4 and R.sub.5
are F or Cl.
11. The compound according to claim 8 wherein or R.sub.5 and
R.sub.6 are F or Cl.
12. The compound according to claim 10 wherein R.sub.4 and R.sub.5
are Cl.
13. The compound according to claim 11 wherein R.sub.5 and R.sub.6
are Cl.
14. A pharmaceutical composition comprising an effective amount of
a compound for treating neoplasia according to the structure:
7Where R is H or CH.sub.3; R.sub.2 is free acid phosphate or
phosphate salt; R.sub.3 is H, F, Cl, Br, I, OCH.sub.3, OCF.sub.3,
CF.sub.3 or a C.sub.1-C.sub.3 alkyl group; R.sub.4 is H, F, Cl, Br,
I, OCH.sub.3, OCF.sub.3 or CF.sub.3; and R.sub.5 and R.sub.6 are
each independently H, F, Cl, Br, I, OCH.sub.3, OCF.sub.3 or
CF.sub.3, with the proviso that at least one of R.sub.3, R.sub.4,
R.sub.5 or R.sub.6 is other than H and when any two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are other than H, the other two of
R.sub.3, R.sub.4, R.sub.5 or R.sub.6 are H, optionally, in
combination with a pharmaceutically acceptable additive, carrier or
excipient.
15. The composition according to claim 14 wherein R.sub.4 is Cl, F
or Br when R.sub.3, R.sub.5 and R.sub.6 are each H.
16. The composition according to claim 15 wherein R.sub.4 is
Cl.
17. The composition according to claim 14 wherein R.sub.5 is F, Cl,
OCH.sub.3 or OCF.sub.3 when R.sub.3, R.sub.4 and R.sub.6 are each
H.
18. The composition according to claim 17 wherein R.sub.5 is F or
Cl.
19. The composition according to claim 18 wherein R.sub.5 is F.
20. The composition according to claim 18 wherein R.sub.5 is
Cl.
21. The composition according to claim 14 wherein two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are other than H and are selected from
F, Cl, Br or I.
22. The composition according to claim 21 wherein two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are each F or Cl.
23. The composition according to claim 21 wherein R.sub.4 and
R.sub.5 are F or Cl.
24. The composition according to claim 21 wherein or R.sub.5 and
R.sub.6 are F or Cl.
25. The composition according to claim 23 wherein R.sub.4 and
R.sub.5 are Cl.
26. The composition according to claim 24 wherein R.sub.5 and
R.sub.6 are Cl.
27. The composition according to claim 14 wherein said neoplasia is
cancer.
28. A method of treating neoplasia in a patient in need of therapy
comprising administering to said patient an effective amount of a
compound according to the structure: 8Where R is H or CH.sub.3;
R.sub.2 is phosphate which can be free acid or phosphate salt;
R.sub.3 is H, F, Cl, Br, I, OCH.sub.3, OCF.sub.3, CF.sub.3 or a
C.sub.1-C.sub.3 alkyl group; R.sub.4 is H, F, Cl, Br, I, OCH.sub.3,
OCF.sub.3 or CF.sub.3; and R.sub.5 and R.sub.6 are each
independently H, F, Cl, Br, I, OCH.sub.3, OCF.sub.3 or CF.sub.3,
with the proviso that at least one of R.sub.3, R.sub.4, R.sub.5 or
R.sub.6 is other than H and when any two of R.sub.3, R.sub.4,
R.sub.5 or R.sub.6 are other than H, the other two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are H, optionally, in combination with
a pharmaceutically acceptable additive, carrier or excipient.
29. The method according to claim 28 wherein R.sub.4 is Cl, F or Br
when R.sub.3, R.sub.5 and R.sub.6 are each H.
30. The method according to claim 29 wherein R.sub.4 is Cl.
31. The method according to claim 28 wherein R.sub.5 is F, Cl,
OCH.sub.3 or OCF.sub.3 when R.sub.3, R.sub.4 and R.sub.6 are each
H.
32. The method according to claim 31 wherein R.sub.5 is F or
Cl.
33. The method according to claim 32 wherein R.sub.5 is F.
34. The method according to claim 32 wherein R.sub.5 is Cl.
35. The method according to claim 28 wherein two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are other than H and are selected from
F, Cl, Br or I.
36. The method according to claim 35 wherein two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are each F or Cl.
37. The method according to claim 35 wherein R.sub.4 and R.sub.5
are F or Cl.
38. The method according to claim 35 wherein or R.sub.5 and R.sub.6
are F or Cl.
39. The method according to claim 37 wherein R.sub.4 and R.sub.5
are Cl.
40. The method according to claim 38 wherein R.sub.5 and R.sub.6
are Cl.
41. The method according to claim 28 wherein said neoplasia is
cancer.
42. The method according to claim 41 wherein said cancer is stomach
cancer, colon cancer, rectal cancer, liver cancer, pancreatic
cancer, lung cancer, breast cancer, cervix uteri cancer, corpus
uteri cancer, ovary cancer, prostate cancer, testis cancer, bladder
cancer, renal cancer, brain/cns cancer, head and neck cancer,
throat cancer, Hodgkins disease, non-Hodgkins leukemia, multiple
myeloma leukemias, melanoma, acute lymphocytic leukemia, acute
mylogenous leukemia, Ewings Sarcoma, small cell lung cancer,
choriocarcinoma, rhabdomyosarcoma, Wilms Tumor, neuroblastoma,
hairy cell leukemia, mouth/pharynx, oesophagus, larynx, melanoma,
kidney or lymphoma,
43. The method according to claim 41 wherein said cancer is lung
cancer, breast cancer or prostate cancer.
44. A method of treating neoplasia in a patient in need of therapy
comprising administering to said patient in combination an
effective amount of a compound according to the structure: 9Where R
is H or CH.sub.3; R.sub.2 is phosphate which can be free acid or
salt; R.sub.3 is H, F, Cl, Br, I, OCH.sub.3, OCF.sub.3, CF.sub.3 or
a C.sub.1-C.sub.3 alkyl group; R.sub.4is H, F, Cl, Br, I,
OCH.sub.3, OCF.sub.3 or CF.sub.3; and R.sub.5 and R.sub.6 are each
independently H, F, Cl, Br, I, OCH.sub.3, OCF.sub.3 or CF.sub.3,
with the proviso that when any two of R.sub.3, R.sub.4, R.sub.5 or
R.sub.6 are other than H, the other two of R.sub.3, R.sub.4,
R.sub.5 or R.sub.6 are H, optionally, in combination with a
pharmaceutically acceptable additive, carrier or excipient and an
effective amount of at least one anti-cancer agent which acts to
damage DNA.
45. The method according to claim 44 wherein R.sub.4 is Cl, F or Br
when R.sub.3, R.sub.5 and R.sub.6 are each H.
46. The method according to claim 45 wherein R.sub.4 is Cl.
47. The method according to claim 44 wherein R.sub.5 is F, Cl,
OCH.sub.3 or OCF.sub.3 when R.sub.3, R.sub.4 and R.sub.6 are each
H.
48. The method according to claim 47 wherein R.sub.5 is F or
Cl.
49. The method according to claim 48 wherein R.sub.5 is F.
50. The method according to claim 48 wherein R.sub.5 is Cl.
51. The method according to claim 44 wherein two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are other than H and are selected from
F, Cl, Br or I.
52. The method according to claim 51 wherein two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are each F or Cl.
53. The method according to claim 52 wherein R.sub.4 and R.sub.5
are Cl.
54. The method according to claim 52 wherein or R.sub.5 and R.sub.6
are Cl.
55. The method according to claim 44 wherein said neoplasia is
cancer.
56. The method according to claim 55 wherein said cancer is stomach
cancer, colon cancer, rectal cancer, liver cancer, pancreatic
cancer, lung cancer, breast cancer, cervix uteri cancer, corpus
uteri cancer, ovary cancer, prostate cancer, testis cancer, bladder
cancer, renal cancer, brain/cns cancer, head and neck cancer,
throat cancer, Hodgkins disease, non-Hodgkins leukemia, multiple
myeloma leukemias, skin melanoma, acute lymphocytic leukemia, acute
mylogenous leukemia, Ewings Sarcoma, small cell lung cancer,
choriocarcinoma, rhabdomyosarcoma, Wilms Tumor, neuroblastoma,
hairy cell leukemia, mouth/pharynx, oesophagus, larynx, melanoma,
kidney or lymphoma,
57. The method according to claim 56 wherein said anti-cancer agent
is selected from the group consisting of cytoxan, mitomycin C,
Etoposide, adriamycin, topotecan, irinotecan, gemcitabine,
campothecin, cis-platin, chlorambucil, melphalan and mixtures
thereof.
58. The method according to claim 56 wherein R.sub.3, R.sub.4 and
R.sub.6 are H, R.sub.5 is F or Cl and said anti-cancer agent is
cytoxan or mitomycin C.
59. The method according to claim 58 wherein said cancer is lung
cancer, prostate cancer, colon cancer, melanoma or breast cancer.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority from U.S.
provisional application serial No. 60/240,529 of same title filed
Oct. 13, 2000.
BACKGROUND OF THE INVENTION
[0002] The reductive conversion of ribonucleotides to
deoxyribonucleotides by the enzyme Ribonucleotide Reductase (RR) is
a crucial, rate-controlling step in the pathway leading to the
biosynthesis of DNA. (Cory, J. G. In "Inhibitors of Ribonucleotide
Diphosphate Reductase Activity", International Encyclopedia of
Pharmacology and Therapeutics, Cory, J. G.; Cory, A. H, Eds.;
Pergamon Press: New York, (1989); Section 128, pp 1-16). Since
deoxyribonucleotides are present in extremely low levels in
mammalian cells, an excellent correlation exists between tumor
growth rate and specific activity of ribonucleotide reductase
(Elford, et al., J. Biol. Chem. (1970), 245, 5228). Mammalian
Ribonucleotide Reductase is composed of two dissimilar proteins,
often referred to as R1, which binds the ribonucleotide substrate,
and R2, which contains non-heme iron and a free tyrosyl radical
(Reichard, P.; Ehrenberg, A. Science, (1983), 221, 514). Both R1
and R2 contribute to the activity of the enzyme.
[0003] Currently, there are two broad classes of RR inhibitors. The
first class includes nucleoside analogs whose mechanism of action
involves binding to the R1 subunit of the enzyme; several of these
are in clinical development. Among these,
2',2'-difluoro-2'-deoxycytidine (Gemcitabine, Trade name: Gemzar,
Eli Lilly) was recently approved by the FDA for the treatment of
pancreatic cancer (Baker, et al., J. Med. Chem. (1991), 34, 1879),
and 2'-fluoromethylene-2'-deoxycytidine is being evaluated in
clinical trials for the treatment of various tumors (McCarthy, J.
R. and Sunkara, P. S. In Design, Synthesis, and Antitumor Activity
of An Inhibitor of Ribonucleotide Reductase, Weiner, D. B.;
Williams, W. V. Eds.; CRC Press:Boca Raton, (1994), 68 1364). The
second class of RR inhibitors includes N-hydroxyurea (Reichard
& Ehrenberg, Science, (1983), 221, 514 and Wright, et al., Cell
Biol. (1990), 68, 1364) and HCTs [N-Heterocyclic Carboxaldehyde
Thiosemicarbazones], which act by destroying the free radical of
the R2 subunit. HCTs have been demonstrated to be the most potent
inhibitors of ribonucleotide reductase, being 80-5000 fold more
effective than N-hydroxyurea in vitro (See, Liu, et al., J. Med.
Chem. (1992), 35, 3672 and J. Med. Chem. (1995), 38, 4234).
[0004] It is also broadly accepted that HCTs exert their enzyme
inhibitory effect through their high binding affinity for iron on
the R2 subunit, since iron is an essential element at the active
site of ribonucleotide reductase. Several years ago, a phase I
clinical evaluation of the lead compound in this series, 5-HP
(DeConti, et al., Cancer Res. (1972), 32, 1455 and Moore, et al.,
Cancer Res. (1971), 31, 235) demonstrated that, while the compound
gave good activity in animal models it was inactive in patients
with solid tumors presumably due to its rapid metabolism in humans.
Modification of 5-HP through the introduction of steric hindrance
and replacement of the hydroxy group with an amino moiety has
resulted in a series of 3-amino-bearing compounds (e.g., 1A (3-AP)
and 1B (3-AMP) (See Below)). Among these agents, 3-AP possesses
excellent antitumor activity (Liu, et al., J. Med. Chem. (1992),
35, 3672) and drastically reduced clearance rates. It is currently
in Phase 1 clinical trials. A single dose clinical trial was halted
once the drug reached a pharmacokinetic endpoint without displaying
any drug related toxicities. Additional Phase 1 studies of extended
dosing schedules (daily times 5 and 96 hour continuous infusion)
are in progress. 2
[0005] Despite the in vivo activity displayed by 3-AP, the
therapeutic potential of this compound may be limited by its poor
water-solubility. Therefore, to improve its water solubility and
therapeutic index, the synthesis of two phosphate-bearing
water-soluble prodrugs 2 (para 3-AP prodrug) and 3 (ortho 3-AP
prodrug) was developed. The phosphate-bearing prodrugs were
designed to give good water-solubility at neutral pH and increased
bioavailability.
[0006] Preliminary in vitro evaluation of the 3-AP prodrugs showed
that they were rapidly converted to 3-AP by alkaline phosphatase
enzyme. In contrast the in vivo PK studies in Beagle dogs showed
that 3-AP released from ortho phosphate-bearing prodrug 3 with a
half-life of 14.2 h, whereas para prodrug 2 has a half-life of 1.5
h. Prodrugs 2 and 3 were also evaluated in the M-109 solid tumor
bearing mice in vivo against 3-AP and cytoxan. The results from
these experiments showed that the ortho prodrug 3 has better
efficacy with reduced toxicity than the parent 3 -AP and has
comparable activity to that of cytoxan. With the aim to further
improve the biological and pharmaceutical profiles and to maximize
the therapeutic utility of the 3-AP prodrugs, a series of ortho
phosphate-bearing prodrugs were designed. 3
OBJECTS OF THE INVENTION
[0007] In one aspect of the invention, an object of the present
invention is to provide compounds, pharmaceutical compositions and
methods for the treatment of neoplasia, including cancer, in
patients.
[0008] In another aspect of the invention, an object of the present
invention is to provide methods of treating neoplasia utilizing
compositions which exhibit favorable and enhanced characteristics
of activity, pharmacokinetics, bioavailability and reduced
toxicity.
[0009] It is yet another object of the invention to provide
compositions and methods for the treatment of cancers which are
resistant to treatment with traditional chemotherapeutic
agents.
[0010] One or more of these and/or other objects of the invention
may be readily gleaned from the description of the invention which
follows.
BRIEF DESCRIPTION OF THE INVENTION
[0011] The present invention relates to compounds according to the
structure: 4
[0012] Where R is H or CH.sub.3;
[0013] R.sub.2 is phosphate which can be the free acid or salt;
[0014] R.sub.3 is H, F, Cl, Br, I, OCH.sub.3, OCF.sub.3, CF.sub.3
or a C.sub.1-C.sub.3 alkyl group;
[0015] R.sub.4 is H, F, Cl, Br, I, OCH.sub.3, OCF.sub.3, CF.sub.3,
NO.sub.2, CN, SO.sub.2CF.sub.3, COOCH.sub.3, SF.sub.5,
SO.sub.2CH.sub.3, COCH.sub.3,
[0016] NH.sub.2, N(CH.sub.3).sub.2, SCH.sub.3, OH; and
[0017] R.sub.5 and R.sub.6 are each independently H, F, Cl, Br, I,
OCH.sub.3, OCF.sub.3, CF.sub.3, NO.sub.2, CN, SO.sub.2CF.sub.3,
COOCH.sub.3, SF.sub.5, SO.sub.2CH.sub.3, COCH.sub.3, NH.sub.2,
N(CH.sub.3).sub.2, SCH.sub.3 or OH,
[0018] with the proviso that when any two of R.sub.3, R.sub.4,
R.sub.5 or R.sub.6 are other than H, the other two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are H.
[0019] In particularly preferred aspects of compounds according to
the present invention, R.sub.4 is Cl, F or Br (preferably, Cl) when
R.sub.3, R.sub.5 and R.sub.6 are H. In other preferred aspects
according to the present invention, R.sub.5 is F, Cl, OCH.sub.3 or
OCF.sub.3 (preferably F) when R.sub.3, R.sub.4 and R.sub.6 are H.
Still in other preferred aspects according to the present invention
when two of R.sub.3, R.sub.4, R.sub.5 or R.sub.6 are selected from
F, Cl, Br or I, (preferably, both substituents are the same and
more preferably, both substituents are Cl), the other two of
R.sub.3, R.sub.4, R.sub.5 or R.sub.6 are H. In still other
preferred aspects of the present invention, when R.sub.4 and
R.sub.5 or R.sub.5 and R.sub.6 are both F or Cl (preferably, both
are Cl), then the other of R.sub.3 and R.sub.6 or R.sub.3 and
R.sub.4 are both H. Compounds according to the present invention
and especially the preferred compositions according to the present
invention, as set forth above, are extremely effective compounds
for the treatment of neoplasia, including cancer, and exhibit at
least one or more of significantly enhanced anti-neoplasia
activity, enhanced higher maximum tolerated doses (MTD) with
reduced toxicity and prolonged half-life consistent with favorable
pharmacokinetics compared to para or ortho 3-AP prodrugs 2 and 3.
This represents an unexpected result. Thus, preferred compounds
according to the present invention may be used at much higher
doses, to greater effect against neoplasia, including cancer and
with enhanced half-life in the blood stream and reduced
toxicity.
[0020] Compounds according to the present invention may be used in
pharmaceutical compositions having biological/pharmacological
activity for the treatment of, for example, neoplasia, including
cancer, as well as a number of other conditions and/or disease
states, as intermediates in the synthesis of compounds exhibiting
biological activity as well as standards for determining the
biological activity of the present compounds as well as other
biologically active compounds. In some applications, the present
compounds may be used for treating microbial infections, especially
including viral infections. These compositions comprise an
effective amount of any one or more of the compounds disclosed
hereinabove, optionally in combination with a pharmaceutically
acceptable additive, carrier or excipient.
[0021] A further aspect of the present invention relates to the
treatment of neoplasia, including cancer, comprising administering
to a patient in need thereof an effective amount of a compound as
described hereinabove, optionally in combination with a
pharmaceutically acceptable additive, carrier or excipient. The
present invention also relates to methods for inhibiting the growth
of neoplasia, including a malignant tumor or cancer comprising
exposing the neoplasia to an inhibitory or therapeutically
effective amount or concentration of at least one of the disclosed
compounds. This method may be used therapeutically, in the
treatment of neoplasia, including cancer or in comparison tests
such as assays for determining the activities of related analogs as
well as for determining the susceptibility of a patient's cancer to
one or more of the compounds according to the present invention.
Primary utility resides in the treatment of neoplasia, including
cancer, especially including lung cancer, breast cancer and
prostate cancer, among others.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 is a representation of certain chemical embodiments
according to the present invention.
[0023] FIGS. 2-3 are representations of chemical schemes for
synthesizing compounds according to the present invention.
[0024] FIGS. 4-15 are representations of experimental results which
are presented in the present application related to the efficacy,
pharmacokinetics, bioavailability, combination chemotherapy, and
toxicity of certain preferred embodiments according to the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The following terms shall be used throughout the
specification to describe the present invention.
[0026] The term "patient" is used throughout the specification to
describe an animal, including a mammal and 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.
[0027] The term "effective amount" is used throughout the
specification to describe concentrations or amounts of compounds
according to the present invention which may be used to produce a
favorable change in the disease or condition treated, whether that
change is a remission, a decrease in growth or size of cancer or a
tumor, a favorable physiological result, a reduction in the growth
or elaboration of a microbe, or the like, depending upon the
disease or condition treated.
[0028] 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 methyl, ethyl, propyl and
isopropyl.
[0029] The term "salt" shall mean any salt consistent with the use
of the compounds according to the present invention. In the case
where the compounds are used in pharmaceutical indications,
including the treatment of neoplasia, including cancer, the term
"salt" shall mean a pharmaceutically acceptable salt, consistent
with the use of the compounds as pharmaceutical agents.
[0030] The term "neoplasia" is used to describe the pathological
process that results in the formation and growth of a neoplasm,
i.e., an abnormal tissue that grows by cellular proliferation more
rapidly than normal tissue and continues to grow after the stimuli
that initated the new growth cease. Neoplasia exhibits partial or
complete lack of structural organization and functional
coordination with the normal tissue, and usually forms a distinct
mass of tissue which may be benign (benign tumor) or malignant
(carcinoma). The term "cancer" is used as a general term to
describe any of various types of malignant neoplasms, most of which
invade surrounding tissues, may metastasize to several sites and
are likely to recur after attempted removal and to cause death of
the patient unless adequately treated. As used herein, the term
cancer is subsumed under the term neoplasia.
[0031] A preferred therapeutic aspect according to the present
invention relates to methods for treating neoplasia, including
benign and malignant tumors and cancer in animal or human patients,
and in preferred embodiments, cancers which have developed drug
resistance, such as multiple drug resistant breast cancer
comprising administering therapeutically effective amounts or
concentrations of one or more of the compounds according to the
present invention to inhibit the growth or spread of or to actually
shrink the neoplasia in the animal or human patient being
treated.
[0032] Cancers which may be treated using compositions according to
the present invention include, for example, stomach, colon, rectal,
liver, pancreatic, lung, breast, cervix uteri, corpus uteri, ovary,
prostate, testis, bladder, renal, brain/cns, head and neck, throat,
Hodgkins disease, non-Hodgkins leukemia, multiple myeloma
leukemias, skin melanoma, acute lymphocytic leukemia, acute
mylogenous leukemia, Ewings Sarcoma, small cell lung cancer,
choriocarcinoma, rhabdomyosarcoma, Wilms Tumor, neuroblastoma,
hairy cell leukemia, mouth/pharynx, oesophagus, larynx, melanoma,
kidney and lymphoma, among others. Compounds according to the
present invention are particularly useful in the treatment of lung
cancer, breast cancer and prostate cancer.
[0033] In the present methods, in certain preferred embodiments, it
has been found advantageous to coadminister at least one additional
anti-neoplastia agent for the treatment of neoplasia, including
cancer. In these aspects according to the present invention, an
effective amount of one or more of the compounds according to the
present invention is co-administered along with an effective amount
of at least one additional anti-neoplastia/anti-cancer agent such
as, for example, cytoxan (cylophosphamide), mitomycin C, and
Etoposide, among numerous others, including topo I and topo II
agents, such as adriamycin, topotecan and irinotecan, other agents
such as gemcitabine, campothecin and agents based upon campothecin
and cis-platin, among other alkylating agents, including
chlorambucil and melphalan. It has unexpectedly been found that the
present compounds (as well the compound where R.sub.3, R.sub.4,
R.sub.5 and R.sub.6 are H), which act by a mechanism to reduce or
prevent DNA repair, will act synergistically with compounds which
act by damaging DNA. Thus, the present compounds may be
advantageously combined with any compound which acts by damaging
DNA, especially including alkylating agents and platinating agents.
By "co-administer" it is meant that the present compounds are
administered to a patient such that the present compounds as well
as the co-administered compound may be found in the patient's
bloodstream at the same time, regardless when the compounds are
actually administered, including simultaneously. In many instances,
the co-administration of the present compounds with traditional
anti-cancer agents produces a synergistic (i.e., more than
additive) result which is unexpected.
[0034] Pharmaceutical compositions based upon the present novel
chemical compounds comprise the above-described compounds in a
therapeutically effective amounts for the treatment of a condition
or disease such as neoplasia, including cancer, optionally in
combination with a pharmaceutically acceptable additive, carrier or
excipient.
[0035] Certain of the compounds, in pharmaceutical dosage form, may
be used as prophylactic agents for preventing a disease or
condition from manifesting itself.
[0036] The present compounds or their derivatives can be provided
in the form of pharmaceutically acceptable salts. As used herein,
the term pharmaceutically acceptable salts or complexes refers to
appropriate salts or complexes of the active compounds according to
the present invention which retain the desired biological activity
of the parent compound and exhibit limited toxicological effects to
normal cells. Nonlimiting examples of such salts inlcude the sodium
and potassium salts of phosphate, among others. Modifications of
the active compound can affect the solubility, bioavailability and
rate of metabolism of the active species, thus providing control
over the delivery of the active species. Further, the modifications
can affect the anticancer activity of the compound, in some cases
increasing the activity over the parent compound. This can easily
be assessed by preparing the derivatives and testing the anticancer
activity according to known methods well within the routineer's
skill in the art.
[0037] The compounds of this invention may be incorporated into
formulations for all routes of administration including for
example, oral and parenteral, including intravenous, intramuscular,
intraperitoneal, intrabuccal, transdermal and in suppository form.
Parenteral administration and in particular, intravenous or
intramuscular administration is preferred.
[0038] Pharmaceutical compositions based upon these novel chemical
compounds comprise the above-described compounds in a
therapeutically effective amount for treating neoplasia, cancer and
other diseases and conditions which have been described herein,
optionally in combination with a pharmaceutically acceptable
additive, carrier and/or excipient. One of ordinary skill in the
art will recognize that a therapeutically effective amount of one
of more compounds according to the present invention will vary with
the infection or condition to be treated, its severity, the
treatment regimen to be employed, the pharmacokinetics of the agent
used, as well as the patient (animal or human) treated.
[0039] In the pharmaceutical aspect according to the present
invention, the compound 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.
[0040] 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 targeted site within the host organism or patient to maximize the
intended effect of the compound.
[0041] The amount of compound included within therapeutically
active formulations according to the present invention is an
effective amount for treating the infection or condition. In its
most preferred embodiment, the present compounds, and in
particular, compounds where R.sub.4 is Cl or R.sub.5 is F, Cl,
OCH.sub.3 or OCF.sub.3 and the remaining substituents on the
benzene ring (other than the phosphate and urethane moiety) are H,
preferably are used for treating neoplasia, and in particular,
cancer, including, in certain instances, drug resistant cancers. In
general, a therapeutically effective amount of the present
preferred compound in dosage form usually ranges from slightly less
than about 0.025 mg./kg. to about 2.5 g./kg., preferably about
2.5-5 mg/kg to about 100 mg/kg of the patient or considerably more,
even more preferably about 20-50 mg/kg, more preferably about 25
mg/kg, depending upon the compound used, the condition or infection
treated and the route of administration, although exceptions to
this dosage range may be contemplated by the present invention. In
the case of the preferred compositions according to the present
invention as described above where R.sub.4 is Cl or R.sub.5 is F,
Cl, OCH.sub.3 or OCF.sub.3 and the remaining substituents on the
benzene (other than the phosphate and urethane moiety) are H,
because the compounds exhibit enhanced anti-cancer activity,
combined with reduced overall toxicity non-cancerous host cells and
the bioavailability of the compounds is also high, these compounds
may be administered at levels 3-10 fold higher than triapine 1A
with significantly less toxicity. At these doses, the AUC (area
under the curve) of triapine delivered from the prodrug form is
about 5 to 25 times greater than that achieveable by the
administration of triapine in non-prodrug form. The compounds
according to the present invention, therefore, represent an
unexpected result and are exceptional agents for the treatment of
neoplasia, especially cancer. The dosage range chosen for these
agents as set forth above is effective to generally produce
effective blood level concentrations of active compound, which may
range from less than about 0.04 to about 400 micrograms/cc or more
of blood in the patient. The more favorable bioavailablility
characteristics, coupled with the reduced toxicity and greater
activity of the present compounds on a molar basis compared to the
prior art Triapine.TM., evidences the present compounds as
unexpectedly favorable compounds for use in the treatment of
neoplasia, including cancer.
[0042] Administration of the active compound may range from
continuous (intravenous drip), including bolus administration,
intravenously or intramuscularly even less frequently than once a
day to several administrations per day and may include topical,
parenteral, intramuscular, intravenous, sub-cutaneous, transdermal
(which may include a penetration enhancement agent), buccal and
suppository administration, among other routes of administration,
including, in certain instances, oral administration.
[0043] To prepare the pharmaceutical compositions according to the
present invention, a therapeutically effective amount of one or
more of the compounds according to the present invention is
preferably intimately admixed with a pharmaceutically acceptable
carrier according to conventional pharmaceutical compounding
techniques to produce a dose. A carrier may take a wide variety of
forms depending on the form of preparation desired for
administration, e.g., intravenous or intramuscular. In preparing
pharmaceutical compositions in the appropriate dosage form, any of
the usual pharmaceutical media may be used. For parenteral
formulations, the carrier will usually comprise sterile water or
aqueous sodium chloride solution, though other ingredients
including those which aid dispersion may be included. Of course,
where sterile water is to be used and maintained as sterile, the
compositions and carriers must also be sterilized. Injectable
suspensions may also be prepared, in which case appropriate liquid
carriers, suspending agents and the like may be employed.
[0044] The present compounds may be used to treat animals, and in
particular, mammals, including humans, as patients. Thus, humans,
equines, canines, bovines and other animals, and in particular,
mammals, suffering from tumors, and in particular, cancer, or other
diseases as disclosed herein, can be treated by administering to
the patient an effective amount of one or more of the compounds
according to the present invention or its derivative or a
pharmaceutically acceptable salt thereof optionally in a
pharmaceutically acceptable carrier or diluent, either alone, or in
combination with other known pharmaceutical agents, depending upon
the disease to be treated). This treatment can also be administered
in conjunction with other conventional cancer therapies, such as
radiation treatment or surgery.
[0045] The active compound is included in the pharmaceutically
acceptable carrier or diluent in an amount sufficient to deliver to
a patient a therapeutically effective amount for the desired
indication, without causing serious toxic effects in the patient
treated.
[0046] The compound is conveniently administered in any suitable
unit dosage form, including but not limited to one containing 1 to
3000 mg, preferably 5 to 500 mg of active ingredient per unit
dosage form.
[0047] The concentration of active compound in the drug composition
will depend on absorption, distribution, inactivation, and
excretion rates of the drug as well as other factors known to those
of skill in the art. It is to be noted that dosage values will also
vary with the severity of the condition to be alleviated. It is to
be further understood that for any particular subject, specific
dosage regimens should be adjusted over time according to the
individual need and the professional judgment of the person
administering or supervising the administration of the
compositions, and that the concentration ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed composition. The active ingredient may be
administered at once, or may be divided into a number of smaller
doses to be administered at varying intervals of time.
[0048] The active compound according to the present invention can
also be mixed with other active materials that do not impair the
desired action, or with materials that supplement the desired
action, such as other anticancer agents, and in certain instances
depending upon the desired therapy or target, antibiotics,
antifungals, antinflammatories, or antiviral compounds, among
others agents.
[0049] Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,
citrates or phosphates and agents for the adjustment of tonicity
such as sodium chloride or dextrose. The parental preparation can
be enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic. If administered intravenously, preferred
carriers include, for example, physiological saline or phosphate
buffered saline (PBS).
[0050] In one embodiment, the active compounds may be prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art.
[0051] Liposomal suspensions may also be pharmaceutically
acceptable carriers. These may be prepared according to methods
known to those skilled in the art. For example, liposome
formulations may be prepared by dissolving appropriate lipid(s) in
an inorganic solvent that is then evaporated, leaving behind a thin
film of dried lipid on the surface of the container. An aqueous
solution of the active compound are then introduced into the
container. The container is then swirled by hand to free lipid
material from the sides of the container and to disperse lipid
aggregates, thereby forming the liposomal suspension. Other methods
of preparation well known by those of ordinary skill may also be
used in this aspect of the present invention.
[0052] A wide variety of biological assays have been used and are
accepted by those skilled in the art to assess anti-cancer activity
of compounds. Any of these methods can be used to evaluate the
activity of the compounds disclosed herein.
[0053] One common method of assessing activity is through the use
of test panels of cancer cell lines. These tests evaluate the in
vitro anti-cancer activity of particular compounds in cancer cell
lines, and provide predictive data with respect to the use of
tested compounds in vivo. Other assays include in vivo evaluations
of the compound's effect on human or in an appropriate animal
model, for example, using mouse tumor cells implanted into or
grafted onto mice or in other appropriate animal models.
[0054] Chemical Synthesis
[0055] Preliminary in vitro evaluation of the 3-AP prodrugs showed
that they were rapidly converted to 3-AP by alkaline phosphatase
enzyme. In contrast, the in vivo PK studies in Beagle dogs showed
that 3-AP released from ortho phosphate-bearing prodrug with a
half-life of 14.2 h, whereas para prodrug has a half-life of 1.5 h.
Prodrugs 2 and 3 were also evaluated in the M-109 solid tumor
bearing mice in vivo against 3-AP and cytoxan. The results from
these experiments showed that the ortho prodrug 3 has better
efficacy with reduced toxicity than the parent 3-AP (1A) and has
comparable activity to that of cytoxan. With the aim to further
improve the biological and pharmaceutical profiles and to maximize
the therapeutic utility of the 3-AP prodrugs, a series of ortho
phosphate-bearing prodrugs were designed based on the following
rationale.
[0056] The rationale for the new prodrug design was that the 3-AP
phosphate-linked prodrugs can release 3-AP via a sequence of
dephosphorylation to give 4 and subsequent benzyl group
fragmentation to give quinone methide 5 which can act as a
biological alkylating agent. 5
[0057] While not being limited by way of theory, it is theorized
that the rate-determining step in this prodrug activation process
would appear to be the P--O bond cleavage step, which is catalyzed
by alkaline phosphatase. The subsequent fragmentation step is
usually rapid. It is possible that 3-AP prodrugs with longer
half-lives in circulation, allowing them to act as a 3-AP depot; or
prodrugs with a different distribution than that of the parent
drug, may have desirable properties. One approach to this goal is
to slow down the dephosphorylation step, the rate-limiting step in
the bioactivation of 3-AP phosphate-bearing prodrugs by introducing
bulky substituents at the position alpha to the phosphate group .
These alkyl groups may impose steric hindrance by the close
proximity to the P--O bond cleavage site, thereby slowing down the
enzymatic dephosphorylation event. Another approach is to introduce
electron-releasing or electron-withdrawing groups in the phenyl
ring which may effect the rate of P--O bond cleavage. Similarly,
the subsequent fragmentation step also may be effected by
substitution at other positions with electron-releasing and
electron-withdrawing groups.
[0058] Based on these considerations, a number of phosphate bearing
prodrugs (FIG. 1) were synthesized readily in good quantities and
evaluated. The disodium salts of these prodrugs were very soluble
in water.
[0059] The 5-chloro prodrug compound 6 was synthesized as shown in
FIG. 2. 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 thio-semicarbazone 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.
[0060] 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.
[0061] 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. Other compounds not specifically presented in the
examples section of this application may be readily synthesized
following analogous methodologies and/or facile syntheses which are
presented and known in the art. One of ordinary skill may readily
synthesize all compounds set forth and described without engaging
in undue experimentation by simply following the detailed synthetic
methodology directly or adapting/modifying such synthetic
methodology using techniques well known in the art.
EXAMPLES
[0062] 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)
[0063] 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:
[0064] Rf (1:5 v/v ethyl acetate-hexane) 0.38.
[0065] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 88.53 (dd, 4.8 Hz,
1H), 8.19 (dd, 7.6 Hz, 1H), 7.37 (dd, 7.7 Hz, 1H) and 3.97 (s,
3H).
[0066] .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)
[0067] 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:
[0068] Rf (1:5 v/v ethyl acetate-hexane) 0.41.
[0069] .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).
[0070] .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)
[0071] 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 x). 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:
[0072] .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).
[0073] .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)
[0074] 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:
[0075] .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, 1H).
[0076] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 64.0 (d), 19.6 (d)
and -1.6 (d).
[0077] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 18.5.
Example 5
Preparation of 2-(TMSE-phosphonooxy)benzyl alcohols (21, 26a-k)
[0078] 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).
[0079] 2-Bis(2-trimethylsilylethyl)phosphonooxybenzyl alcohol (5
position on phenyl group is H, 21a).
[0080] Following the above procedure, 2-hydroxybenzyl alcohol (15.0
g, 0.12 mmol) gave the ortho phosphate linker
2-Bis(2-trimethylsilylethyl)ph- osphonooxybenzyl alcohol (39.78 g,
81%) as an oil:
[0081] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 87.43 (d, J=8.5
Hz, 1H), 7.27-7.16 (m, 3H), 4.63 (s, 2H), 4.29-4.19 (m, 4H), 4.12
(m, 4H), 1.14-1.08 (m, 4H), and 0.00 (s, 18H);
[0082] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 148.4 (d, J=8.84
Hz), 133.0 (d, J=4.59 Hz), 130.9, 129.1, 25.8, 121.0 (d, J=4.47
Hz), 67.5 (d, J=6.93), 60.1, 9.5 (d, J=5.76 Hz) and -1.563.
[0083] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.4.
[0084] 2-Bis(2-trimethylsilylethyl)phosphonooxy-5-chlorobenzyl
alcohol (21).
[0085] Following the above procedure, 5-chloro-2-hydroxybenzyl
alcohol (5.0 g, 32 mmol) gave 21 (12.2 g, 88%) as an oil:
[0086] .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).
[0087] .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.
[0088] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.1.
[0089] 2-Bis(2-trimethylsilylethyl)phosphonooxy-5-fluorobenzyl
alcohol (26a).
[0090] Following the above procedure, 5-fluoro-2-hydroxybenzyl
alcohol (17.0 g, 119 mmol) gave 26a (31.7 g, 62%) as an oil:
[0091] .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).
[0092] .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. .sup.31P NMR (121 MHz, CDCl.sub.3) .delta.
6.4.
[0093] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-59.8.
[0094] 2-Bis(2-trimethylsilylethyl)phosphonooxy-5-nitrobenzyl
alcohol (26b).
[0095] Following the above procedure, 2-hydroxy-5-nitrobenzyl
alcohol (4.5 g, 27 mmol) gave 26b (6.4 g, 53%) as an oil:
[0096] .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).
[0097] .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.
[0098] .sup.13P NMR (121 MHz, CDCl.sub.3) .delta. 4.4.
[0099] 2-Bis(2-trimethylsilylethyl)phosphonooxy-5-methoxybenzyl
alcohol (26c).
[0100] Following the above procedure, 2-hydroxy-5-methoxybenzyl
alcohol (11.0 g, 25 mmol) gave 26c (7.7 g, 70%) as an oil:
[0101] .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).
[0102] .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.
[0103] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.9.
[0104]
2-Bis(2-trimethylsilylethyl)phosphonooxy-5-trifluoromethoxybenzyl
alcohol (26d).
[0105] Following the above procedure,
2-hydroxy-5-trifluoromethoxybeiizyl alcohol (1.9 g, 9.1 mmol) gave
26d (3.3 g, 62%) as an oil:
[0106] .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).
[0107] .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.
[0108] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.2.
[0109] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-58.7.
[0110]
2-Bis(2-trimethylsilylethyl)phosphonooxy-5-trifluoromethylbenzyl
alcohol (26e).
[0111] Following the above procedure,
2-hydroxy-5-trifluoromethylbenzyl alcohol (4.1 g, 22 mmol) gave 26e
(7.9 g, 77%) as an oil:
[0112] .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).
[0113] .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.
[0114] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.8.
[0115] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-62.9.
[0116] 2-Bis(2-trimethylsilylethyl)phosphonooxy-3,5-dichlorobenzyl
alcohol (26f).
[0117] Following the above procedure, 3,5-dichloro-2-hydroxybenzyl
alcohol (4.6 g, 24 mmol) gave 26f (7.2 g, 63%) as an oil:
[0118] .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).
[0119] .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.
[0120] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.7.
[0121] 2-Bis(2-trimethylsilylethyl)phosphonooxy-4,5-dichlorobenzyl
alcohol (26g).
[0122] Following the above procedure, 4,5-dichloro-2-hydroxybenzyl
alcohol (3.6 g, 18 mmol) gave 26g (5.2 g, 59%) as an oil:
[0123] .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).
[0124] .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.
[0125] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.0.
[0126] 2-Bis(2-trimethylsilylethyl)phosphonooxy-5,6-dichlorobenzyl
alcohol (26 h).
[0127] Following the above procedure, 5,6-dichloro-2-hydroxybenzyl
alcohol (4.8 g, 25 mmol) gave 26 h (8.6 g, 73%) as an oil:
[0128] .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).
[0129] .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.
[0130] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.4.
[0131] 2-Bis(2-trimethylsilylethyl)phosphonooxy-3-methylbenzyl
alcohol (26i).
[0132] Following the above procedure, 2-hydroxy-3-methylbenzyl
alcohol (2.0 g, 14 mmol) gave 26i (1.7 g, 88%) as an oil:
[0133] .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).
[0134] .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.
[0135] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.9.
[0136] 2-Bis(2-trimethylsilylethyl)phosphonooxy-4-chlorobenzyl
alcohol (26j).
[0137] Following the above procedure, 4-chloro-2-hydroxybenzyl
alcohol (4.2 g, 26 mmol) gave 26j (9.6 g, 84%) as an oil:
[0138] Rf (4:1 v/v ethyl acetate-hexane) 0.67.
[0139] .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).
[0140] .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.
[0141] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.8.
[0142] 2-Bis(2-trimethylsilylethyl)phosphonooxy-4-methoxybenzyl
alcohol (26k).
[0143] Following the above procedure, 2-hydroxy-4-methoxybenzyl
alcohol (2.7 g, 17 mmol) gave 26k (2.5 g, 33%) as an oil:
[0144] Rf (4:1 v/v ethyl acetate-hexane) 0.70.
[0145] .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).
[0146] .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.
[0147] .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)
[0148] 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), prepared as described
above, 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).
[0149] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-benzyl ester (22a).
Following the above procedure, the crude carbamate obtained from
2-Bis(2-trimethylsilylethyl)phosphonooxybenzyl alcohol (21a) (29.72
g, 0.073 mol) was directly used for the further reaction without
purification.
[0150] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-chlorobenzyl ester
(22).
[0151] Following the above procedure, 21 (9.4 g, 21 mmol) gave 22
(10.6 g, 58%) as an oil:
[0152] .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).
[0153] .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.
[0154] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.1.
[0155] Mass Calcd. For C.sub.31H.sub.42ClN.sub.2O.sub.6PSi.sub.2:
661.277; Found: 661.2
[0156] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-fluorobenzyl ester
(27a).
[0157] Following the above procedure, the crude carbamate 27a
obtained from 26a (31.0 g, 73 mmol) was directly used for the
further reaction without purification.
[0158] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-nitrobenzyl ester
(27b).
[0159] Following the above procedure, 26b (4.3 g, 9.6 mmol) gave
27b (2.3 g, 35%) as an oil:
[0160] .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).
[0161] .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.
[0162] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 4.5.
[0163] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-methoxybenzyl ester
(27c).
[0164] 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.
[0165] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-trifluoromethoxybenzyl
ester (27d).
[0166] Following the above procedure, 26d (1.9 g, 8.5 mmol) gave
27d (3.4 g, 83%) as an oil:
[0167] .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).
[0168] .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.
[0169] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.3.
[0170] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-58.7.
[0171] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-trifluoromethylbenzyl
ester (27e).
[0172] Following the above procedure, 26e (5.1 g, 11 mmol) gave 27e
(5.3 g, 71%) as an oil:
[0173] .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).
[0174] .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.
[0175] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.0.
[0176] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-62.7.
[0177] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-3,5-dichlorobenzyl ester
(27f).
[0178] Following the above procedure, 26f (6.3 g, 13 mmol) gave 27f
(7.1 g, 76%) as an oil:
[0179] .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).
[0180] .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.
[0181] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.0.
[0182] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4,5-dichlorobenzyl ester
(27g).
[0183] Following the above procedure, 26g (11.3 g, 50 mmol) gave
27g (17.4 g, 75%) as an oil:
[0184] .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).
[0185] .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.
[0186] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.2.
[0187] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5,6-dichlorobenzyl ester
(27 h).
[0188] Following the above procedure, 26 h (6.2 g, 28 mmol) gave 27
h (9.6 g, 75%) as an oil:
[0189] .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).
[0190] .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.
[0191] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.0.
[0192] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-3-methylbenzyl ester
(27i).
[0193] Following the above procedure, 26i (1.4 g, 6.2 mmol) gave
27i (1.5 g, 53%) as an orange oil:
[0194] .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), 7.46.9 (m, 10H), 5.27
(s, 2H), 4.11 (m, 4H), 2.27 (s, 3H), 0.94 (m, 4H) and -0.11 (s,
18H).
[0195] .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.
[0196] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.6.
[0197] Mass Calcd. For C.sub.32H.sub.45 N.sub.2O.sub.6PSi.sub.2:
640.859; Found: 640.2
[0198] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4-chlorobenzyl ester
(27j).
[0199] Following the above procedure, 26j (9.3 g, 21 mmol) gave 27j
(12.6 g, 87%) as an oil:
[0200] Rf (1:1 v/v ethyl acetate-hexane) 0.82.
[0201] .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).
[0202] .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.
[0203] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.1.
[0204] (2-Styrylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4-methoxybenzyl ester
(27k).
[0205] Following the above procedure, 26k (2.8 g, 6.5 mmol) gave
27k (3.7 g, 86%) as an oil:
[0206] Rf(1:l v/v ethyl acetate-hexane) 0.50.
[0207] .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).
[0208] .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.
[0209] .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)
[0210] 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).
[0211] (2-Formylpyridin-3-yl)carbamic acid
2-(trimethylsilylethylphosphono- oxy)benzyl ester (23a).
[0212] Following the above procedure, the crude 22a, prepared
above, gave 23a (29.81 g, 73%) as an oil:
[0213] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.49 (s, 1H),
10.06 (s, 1H), 8.84 (d, J=8.36 Hz, 1H), 8.43 (d, J=5.36 Hz, 1H),
7.49-7.16 (m, 5H), 5.34 (s, 2H), 4.32-4.24 (m, 4H), 1.11 (dd,
J=8.59 Hz, 6.57 Hz, 4H) and 0.0 (18H).
[0214] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 197.0, 153.2,
149.1 (d, J=6.45 Hz), 143.7, 138.5, 136.7, 130.1, 129.8, 128.6 (d,
J=6.68 Hz), 126.3, 124.9, 120.0, 67.2 (d, J=5.39 Hz, 2C), 62.5,
19.5 (d, J=6.58 Hz, 2C), -1.6.
[0215] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.2.
[0216] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-chlorobenzyl ester
(23).
[0217] Following the above procedure, 22 (2.4 g, 3.7 mmol) gave 23
(1.6 g, 72%) as an oil:
[0218] .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).
[0219] .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.
[0220] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.1.
[0221] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-fluorobenzyl ester
(28a).
[0222] Following the above procedure, the crude 27a (31.0 g, 73
mmol) gave 28a (26.9 g, 64%) as an oil:
[0223] .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).
[0224] .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.
[0225] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.5.
[0226] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-59.3.
[0227] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-nitrobenzyl ester
(28b).
[0228] Following the above procedure, 27b (4.2 g, 9.4 mmol) gave
28b (2.8 g, 50%) as an oil:
[0229] .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).
[0230] .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.
[0231] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 4.5.
[0232] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-methoxybenzyl ester
(28c).
[0233] Following the above procedure, the crude 27c (7.5 g, 17
mmol) gave 28c (9.8 g, 73%) as an oil:
[0234] .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).
[0235] .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.
[0236] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.8.
[0237] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-trifluoromethoxybenzyl
ester (28d).
[0238] Following the above procedure, 27d (4.7 g, 6.6 mmol) gave
28d (3.2 g, 75%) as an oil: .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).
[0239] .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.
[0240] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.3.
[0241] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-58.8.
[0242] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5-trifluoromethylbenzyl
ester (28e).
[0243] Following the above procedure, 27e (12.2 g, 18 mmol) gave
28e (6.9 g, 63%) as an oil:
[0244] .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).
[0245] .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.
[0246] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 4.9.
[0247] .sup.19F NMR (282 MHz, CDCl.sub.3) .delta.-62.7.
[0248] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-3,5-dichlorobenzyl ester
(28f).
[0249] Following the above procedure, 27f (8.0 g, 12 mmol) gave 28f
(5.1 g, 71%) as an oil:
[0250] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 810.54 (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).
[0251] .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.
[0252] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.1.
[0253] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4,5-dichlorobenzyl ester
(28g).
[0254] Following the above procedure, 27g (17.4 g, 25 mmol) gave
28g (13.4 g, 86%) as an oil:
[0255] .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).
[0256] .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.
[0257] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.1.
[0258] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-5,6-dichlorobenzyl ester
(28 h).
[0259] Following the above procedure, 27 h (9.5 g, 14 mmol) gave 28
h (6.5 g, 77%) as an oil:
[0260] .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).
[0261] .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.
[0262] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 4.9.
[0263] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-3-methylbenzyl ester
(28i).
[0264] Following the above procedure, 27i (1.5 g, 2.2 mmol) gave
28i (1.1 g, 86%) as an oil:
[0265] .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).
[0266] .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.
[0267] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.8.
[0268] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4-chlorobenzyl ester
(28j).
[0269] Following the above procedure, 27j (12.2 g, 19 mmol) gave
28j (8.9 g, 80%) as an oil:
[0270] Rf (1:1 v/v ethyl acetate-hexane) 0.66.
[0271] .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).
[0272] .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.
[0273] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.0.
[0274] (2-Formylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl)pho- sphonooxy-4-methoxybenzyl ester
(28k).
[0275] Following the above procedure, 27k (5.1 g, 8.0 mmol) gave
28k (2.7 g, 58%) as an oil:
[0276] Rf (1 :1 v/v ethyl acetate-hexane) 0.44.
[0277] .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).
[0278] .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.
[0279] .sup.31P NMR (121 MHz, CDCl.sub.3) .quadrature.5.0.
Example 8
Preparation of pyridine-2-carboxaldehyde thiosemicarbazones (24,
29a-k)
[0280] General Procedure. The corresponding pyridine-2-formaldehyde
(23, 23a 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, 24a, 29a-k).
[0281] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethyl silyl ethyl) phosphonooxy-5-chlorobenzyl ester
(24).
[0282] Following the above procedure, 23 (6.9 g, 12 mmol) gave 24
(4.9 g, 63%) as a yellow solid:
[0283] .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).
[0284] .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.
[0285] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.8.
[0286] Mass Calcd. For C.sub.25H.sub.39ClN.sub.5O.sub.6PSSi.sub.2:
660.267; Found: 660.2
[0287] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethyl silyl ethyl) phosphonooxy benzyl ester (24a).
Following the above procedure, 23a (29.74 g, 0.54 mol) gave 24a
(33.67 g, 90%)) as a yellow solid: .sup.1H NMR (DMSO-d.sub.6, 300
MHz): d 11.76 (s, 1H), 10.04 (s, 1H), 8.38 (d, J=4.32 Hz, 1H), 8.29
(s, 2H), 7.86-7.26 (m, 5H), 5.27 (s, 2H), 4.26-4.18 (m, 4H), 1.05
(dd, J=9.08 Hz, 7.64 Hz, 4H), 0.0 (s, 18H); .sup.13C NMR
(DMSO-d.sub.6, 75 MHz): d 178.9, 153.3, 148.0 (d, J=6.42 Hz),
144.6, 144.3, 140.6, 133.9, 129.5, 127.2 (d, J=5.54 Hz), 125.2,
124.1, 119.8 (d, J=4.32 Hz), 119.6, 66.7 (d, J=7.8 Hz, 2C), 61.3,
18.9 (d, J=6.5 Hz, 2C),-1.6; .sup.31P NMR (DMSO-d.sub.6, 121 MHz):
d 9.7
[0288] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-5-fluorobenzyl ester
(29a).
[0289] Following the above procedure, 28a (14.3 g, 25 mmol) gave
29a (12.9 g, 80%) as a yellow solid:
[0290] .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).
[0291] .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.
[0292] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 10.5.
[0293] .sup.19F NMR (282 MHz, DMSO-d.sub.6) .delta.-62.5.
[0294] Mass Calcd. For C.sub.25H.sub.39FN.sub.5O.sub.6PSSi.sub.2:
643.813 Found: 644.2
[0295] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-5-nitrobenzyl ester
(29b).
[0296] Following the above procedure, 28b (1.6 g, 2.7 mmol) gave
29b (1.3 g, 77%) as a yellow solid:
[0297] .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).
[0298] .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.
[0299] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.2.
[0300] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
[0301] 2-bis(2-trimethylsilylethyl) phosphonooxy-5-methoxybenzyl
ester (29c).
[0302] Following the above procedure, 28c (5.0 g, 8.8 mmol) gave
29c (4.4 g, 77%) as a yellow solid:
[0303] .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).
[0304] .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.
[0305] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 10.3.
[0306] Mass Calcd. For C.sub.26H.sub.42 N.sub.5O.sub.7PSSi.sub.2:
655.848 Found: 656.2
[0307] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-5-trifluoromethoxybenzyl
ester (29d).
[0308] Following the above procedure, 28d (2.5 g, 3.9 mmol) gave
29d (1.9 g, 68%) as a yellow solid:
[0309] .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).
[0310] .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.
[0311] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.8.
[0312] .sup.19F NMR (282 MHz, DMSO-d.sub.6) .delta.-53.0.
[0313] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-5-trifluoromethylbenzyl
ester (29e).
[0314] Following the above procedure, 28e (5.3 g, 8.5 mmol) gave
29e (3.6 g, 61%) as a yellow solid:
[0315] .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).
[0316] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.5.
[0317] .sup.19F NMR (282 MHz, DMSO-d.sub.6) .delta.-56.2.
[0318] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-3,5-dichlorobenzyl ester
(29f).
[0319] Following the above procedure, 28f (4.8 g, 7.7 mmol) gave
29f (4.6 g, 85%) as a yellow solid:
[0320] .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).
[0321] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 10.3.
[0322] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-4,5-dichlorobenzyl ester
(29g).
[0323] Following the above procedure, 28g (2.0 g, 3.2 mmol) gave
29g (1.5 g, 70%) as a yellow solid:
[0324] .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).
[0325] .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.
[0326] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.7.
[0327] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-5,6-dichlorobenzyl ester
(29 h).
[0328] Following the above procedure, 28 h (5.9 g, 9.5 mmol) gave
29 h (3.6 g, 55%) as a yellow solid:
[0329] .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).
[0330] .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.
[0331] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.6.
[0332] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-3-methylbenzyl ester
(29i).
[0333] Following the above procedure, 28i (1.1 g, 1.9 mmol) gave
29i (0.7 g, 57%) as a yellow solid:
[0334] .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).
[0335] .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.
[0336] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 10.4.
[0337] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-4-chlorobenzyl ester
(29j).
[0338] Following the above procedure, 28j (10.5 g, 18.5 mmol) gave
29j (11.8 g, 97%) as a yellow solid:
[0339] .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).
[0340] .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.
[0341] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.5.
[0342] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-bis(2-trimethylsilylethyl) phosphonooxy-4-methoxybenzyl ester
(29k).
[0343] Following the above procedure, 28k (2.6 g, 4.5 mmol) gave
29k (2.7 g, 91%) as a yellow solid:
[0344] .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).
[0345] .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.
[0346] .sup.31P NMR (121 MHz, DMSO-d.sub.6) .delta. 9.6.
Example 9
Preparation of Free Phosphonic Acids (6-17)
[0347] General Procedure. To a solution of the corresponding
TMSE-protected phosphate (24, 24a 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 and 6a not shown) was
obtained as a yellow solid or glassy solid.
Example 10
Preparation of Disodium Salt of Phosphonic Acid (25, 30a-k)
[0348] 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.
[0349] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-chlorobenzyl ester (25).
[0350] Following the above procedure, 24 (1.1 g, 1.7 mmol) gave 25
(0.4 g, 49%) as a pale yellow powder:
[0351] .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).
[0352] .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.
[0353] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.3.
[0354] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-fluorobenzyl ester (30a).
[0355] 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:
[0356] .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).
[0357] .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, 117.2 and 65.2.
[0358] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.5.
[0359] .sup.19F NMR (282 MHz, D.sub.2O) 6 -57.4.
[0360] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-nitrobenzyl ester (30b).
[0361] Following the above procedure, 29b (2.1 g, 3.0 mmol) gave
30b (1.0 g, 73%) as a dark yellow powder:
[0362] .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).
[0363] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 13.8.
[0364] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-methoxybenzyl ester (30c).
[0365] 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:
[0366] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.96 (br s, 1H),
7.70 (br s, 1H), 7.21 (br s, 1H), 7.0 8 (br S, 1H), 6.73 (s, 2H),
5.05 (s, 2H) and 3.65 (s, 3H).
[0367] .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.
[0368] .sup.31 P NMR (121 MHz, D.sub.2O) .delta. 14.6.
[0369] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-trifluoromethoxybenzyl ester (30d).
[0370] Following the above procedure, 29d (1.9 g, 2.6 mmol) gave
30d (0.5 g, 31%) as a pale yellow powder:
[0371] .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).
[0372] .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.
[0373] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.3.
[0374] .sup.19F NMR (282 MHz, D.sub.2O) .delta.-56.3.
[0375] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5-trifluoromethylbenzyl ester (30e).
[0376] Following the above procedure, 29e (3.6 g, 5.2 mmol) gave
30e (1.3 g, 45%) as a pale yellow powder:
[0377] .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).
[0378] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.0.
[0379] .sup.19F NMR (282 MHz, D.sub.2O) .delta.-59.4.
[0380] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-3,5-dichlorobenzyl ester (30f).
[0381] Following the above procedure, 29f (4.5 g, 6.5 mmol) gave
30f (0.8 g, 24%) as a pale yellow powder:
[0382] .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).
[0383] .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.
[0384] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.4.
[0385] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-4,5-dichlorobenzyl ester (30g).
[0386] Following the above procedure, 29g (2.5 g, 3.0 mmol) gave
30g (0.4 g, 23%) as a pale yellow powder:
[0387] .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).
[0388] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.3.
[0389] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-5,6-dichlorobenzyl ester (30 h).
[0390] Following the above procedure, 29 h (4.6 g, 6.6 mmol) gave
30 h (2.3 g, 64%) as a pale yellow powder:
[0391] .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).
[0392] .sup.1H NMR (121 MHz, D.sub.2O) .delta. 14.2.
[0393] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-3-methylbenzyl ester (30i).
[0394] Following the above procedure, 29i (1.2 g, 1.8 mmol) gave
30i (0.5 g, 57%) as a yellow powder:
[0395] .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).
[0396] .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.
[0397] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.2.
[0398] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-4-chlorobenzyl ester (30j).
[0399] Following the above procedure, 29j (4.2 g, 6.6 mmol) gave
30j (1.6 g, 48%) as a pale yellow powder:
[0400] .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).
[0401] .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.
[0402] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.3.
[0403] (2-Thiosemicarbazonomethylpyridin-3-yl)carbamic acid
2-(disodium phosphonooxy)-4-methoxybenzyl ester (30k).
[0404] Following the above procedure, 29k (2.9 g, 4.4 mmol) gave
30k (1.2 g, 54%) as a pale yellow powder:
[0405] .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).
[0406] .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.
[0407] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.3.
[0408] Biological Testing/Data
[0409] Bioconversion of prodrugs to 3-AP catalyzed by alkaline
phosphatase. The bioactivation of dimethyl para-prodrug and a
subset of the ortho phosphate prodrugs was studied using
4.65.times.10.sup.-5 unit of phosphatase enzyme solution (Type
VII-SA, from Bovine Intestinal Mucose, Sigma). Upon incubation with
phosphatase, all the prodrugs were converted cleanly to the parent
drug 3-AP. Under these experimental conditions, there was no
signficant increase in the half-life (T.sub.1/2) of bioactivation
of ortho phsophate prodrugs compared to that of unsubstituted ortho
prodrug (Table 1, below). Human serum stability studies were
conducted by incubating the prodrugs at 37.degree. C. in human
serum. This study showed that 4-chloro phosophate prodrug 16 is the
slowest releasing prodrug which has a half life of 1.5 times that
of ortho-prodrug.
1TABLE 1 Enzymatic Bioconversion and Serum Stability of 3-AP
Phosphate Prodrugs Half-life Alkaline Human Serum Buffered Saline
Prodrugs phosphatase 37.degree. C. 37.degree. C. pH 7.6, 37.degree.
C. Ortho-(2) 16.3 min 2.7 hr No hydrolysis Para-(3) 9.2 min 1.2 hr
5.5 hr 5-Cl-(6) 30.5 min 3.4 hr 162 hr 5-F-(7) Not Tested 3.8 hr No
hydrolysis 5-CH.sub.3O(9) 22.1 min 3.2 hr 151 hr 4-Cl-(16) 29.9 min
4.0 hr No hydrolysis 4-CH.sub.3O(17) Not Tested 13.3 hr 15.7 hr
[0410] In Vivo PK Studies of Ortho Phosphate Bearing 3-AP
Prodrugs
[0411] The pharmacokinetics of 3-AP prodrugs were characterized
following administration of a single intravenous dose of 7.2-8.5
mg/kg of 3-AP phosphate prodrugs (equivalent to 3 mg/kg of 3-AP) to
a beagle dog. The animal was dosed once with each prodrug weekly.
After each dose, a washout period of at least 6 days was maintained
before the next dose was administered. Concentrations of 3-AP
(Triapine) and prodrug in serum were determined by HPLC and used to
calculate various PK parameters. These PK parameters were compared
to those of 3-AP from equimolar doses from a separate study. Mean
serum drug concentration versus time data were analyzed by both
compartmental and non-compartmental models. AUC, total body
clearance (Cl), steady-state volume of distribution (Vd,ss),
terminal half-life (T1/2) , Cmax, Tmax were calculated for both
3-AP and the prodrugs.
[0412] Pharmacokinetic parameters of the prodrugs at equimolar i.v.
doses are presented in Table 2 below. Ortho-phosphate prodrug was
shown previously to undergo rapid bioconversion to 3-AP when
incubated in vitro with alkaline phosphatase. In vivo, the
conversion was considerably retarded suggesting that in vivo the
off-rate of the drug from the alkaline phosphatase is retarded. At
no time have we been able to detect the presence of the
intermediary phenol which is the expected cleavage product. The
serum half-lives of several of the ortho prodrugs were extended
relative to the half life of 3 -AP itself in the dog (.about.1.5
hrs) which is comparable to that seen in humans for Triapine.TM.
(3-AP). The 4-chloro (16), 5-methoxy (9) and 5-fluoro (7) analogs
were promising in this regard as reflected in their AUCs and
half-lives.
2TABLE 2 PK Values of Ortho Phosphate Bearing 3-AP Prodrugs in the
Dog C.sub.max AUC CI(mL/ Prodrugs (.mu.g/mL) (.mu.g .multidot.
min/L) T.sub.1/2 min/kg) V.sub.SS(L/kg) Ortho- (3) 125 63309 5.9 hr
0.11 0.14 5-Cl- (6) 136 24263 2.1 hr 0.32 0.08 139 27939 2.3 hr
0.28 0.09 5-F- (7) 114 44829 4.5 hr 0.47 0.11 5-NO.sub.2- (8) 126
3396 19 min 2.33 0.12 5-CH.sub.3O- (9) 126 51460 4.7 hr 0.15 0.12
5-CF.sub.3O- (10) 144 17584 1.4 hr 0.48 0.09 5-CF.sub.3- (11) 156
9579 43 min 0.86 0.08 3,5-Di-Cl- (12) 140 6939 34 min 1.2 0.08
4,5-Di-Cl- (13) 220 21499 1.1 hr 0.39 0.15 5,6-Di-Cl- (14) 202
34211 2.0 hr 0.24 0.12 3-CH.sub.3- (15) 147 66756 5.3 hr 0.11 0.08
4-Cl- (16) 120 46321 4.5 hr 0.17 0.10
[0413] Pharmacokinetic parameters of 3-AP itself following i.v.
administration of equimolar doses of the prodrugs are presented in
Table 3, below. The results of the study evidence that the prodrugs
ortho (3), 5-fluoro (7), and 4-chloro (16) appeared to provide an
extended release of the parent 3 -AP, resulting in sustained
concentrations of 3 -AP in serum compared to the other prodrugs in
the study. These compounds also exhibited increased stability in
aqueous solution.
3TABLE 3 PK Values of 3-AP in the Dog C.sub.max AUC T.sub.max
T.sub.1/2 V/F Prodrugs (.mu.g/mL) (.mu.g .multidot. min/L) (min)
(hour) (L/kg) Para- (2) 1.6 74.5 -- 1.5 2.80 Ortho- (3) 0.6 698 8.6
14.2 1.77 5-Cl- (6) 2.2 456 16.7 2.2 0.42 2.1 395 -- 2.2 3.67 5-F-
(7) 0.5 619 1.2 13.2 1.85 5-NO.sub.2- (8) 1.9 84 -- 0.5 4.27
5-CH.sub.3O- (9) 1.5 987 7.9 7.7 0.67 5-CF.sub.3O- (10) 9.2 709 --
0.9 0.92 5-CF.sub.3- (11) 3.0 280 -- 1.1 2.74 3,4-Di-Cl- (12) 2.1
163 -- 0.9 3.99 4,5-Di-Cl- (13) 2.7 392 -- 1.7 3.14 5,6-Di-Cl- (14)
3.6 1.6 2.32 3-CH.sub.3- (15) 1.5 998 7.8 7.8 5.18 4-Cl- (16) 0.8
888 9.0 12.4 1.21
[0414] Initially, both prodrugs were studied at a single dose of
7.5-7.7 mg/kg (equivalent to 3 mg/kg of Triapine). Based on the
findings, a rising dose pharmacokinetic and toxicokinetic study was
conducted for the 5-fluoro-prodrug at 20, 40, and 80 mg/kg, and the
4-chloro-prodrug at 20, and 30 mg/kg.
[0415] PK parameters of Triapine and the two prodrugs are presented
in Tables 4 and 5 and are compared to those obtained, in a separate
study, from administration of 3 mg/kg of Triapine (equivalent to
approximately 7.5 mg/kg of prodrugs). When compared to dogs treated
with Triapine, dogs receiving the 4-chloro- and 5-fluoro-prodrug
(at equimolar doses) showed an increased Triapine exposure
(expressed as AUC). The dose escalation study showed that the peak
serum concentrations and AUCs of Triapine were linearly related to
the dose of the prodrug.
4TABLE 4 Comparative Pharmacokinetics of Triapine Phosphate
Prodrugs in Dogs - PK Values of Triapine. Dose C.sub.max AUC
T.sub.max T.sub.1/2 V/F Prodrugs (mg/kg) (.mu.g/mL) (.mu.g
.multidot. min/L) (min) (hour) (L/kg) 5-Fluoro- 7.5 0.5 619 1.2
13.2 1.85 (7) 20 6.8 490 -- 0.8 2.90 40 12.4 1153 -- 1.1 3.22 80
32.0 2713 -- 1.0 2.50 4-Chloro- 7.7 0.8 888 9.0 12.4 1.21 (16) 20
13.0 2592 -- 2.3 1.53 30 31.9 5905 -- 2.1 0.94 Triapine 3 2.3 124
-- 1.8 3.57
[0416]
5TABLE 5 Comparative Pharmacokineties of Triapine Phosphate
Prodrugs in Dogs - PK Values of Pro drug. Cl Dose C.sub.max AUC
T.sub.1/2 (mL/ V.sub.SS Prodrugs (mg/kg) (.mu.g/mL) (mg .multidot.
min/L) (hour) min/kg) (L/kg) 5-Fluoro- 7.5 114 44829 4.5 hr 0.47
0.11 (7) 20 299.6 35877 1.4 hr 0.56 0.11 40 412.0 56679 1.6 hr 0.35
0.07 80 377.4 43863 1.3 hr 0.46 0.06 4-Chloro- 7.7 120 46321 4.5 hr
0.17 0.10 (16) 20 464 63080 1.6 hr 0.32 0.07 30 556 90291 1.9 hr
0.33 0.15 Triapine 3 2.3 124 -- 1.8 3.57
[0417] Serum prodrug peak concentrations and AUCs, on the other
hand, were not linear with dose, and appeared to be saturated at
the doses investigated. Serum concentration-time profiles of
prodrugs and Triapine are presented in attached FIGS. 4 and 5. This
data suggests that at IV doses.gtoreq.20 mg/kg, both prodrugs
exhibited an extended bioconversion to parent Triapine, which
persisted above 1 .mu.M (0.2 .mu.g/mL) for 24 hours. The sustained
serum Triapine levels were presumably attributable to the high
serum levels of prodrugs as well as their low total body clearance.
The pharmacokinetics of the 4-chloro Prodrug and of the Triapine
are shown in FIG. 6. High blood levels of Triapine (>1 .mu.lM)
were observed at 24 hours at doses that were well tolerated in the
dog.
[0418] Clinical observations: For dogs which received the
4-chloro-prodrug, there were no early deaths. Treatment-related
clinical observations were recorded for both dogs. The male treated
at 20 mg/kg exhibited loose stool on days 2 and 3 (day 1 =day of
dosing). The female treated at 40 mg/kg exhibited emesis, diarrhea,
yellow mucous in the stool, reduced activity and was cyanotic (the
mouth was grey) on day 1 after dosing. These observations were not
present on day 2. The only adverse clinical sign on day 2 was the
absence of stool. The male dog treated at 30 mg/kg showed similar
clinical sign as the female at 40 mg/kg. The MTD for
4-chloro-prodrug was therefore established at 30-40 mg/kg.
[0419] For dogs which received 5-fluoro-prodrug, there were no
early deaths. There were no adverse observations from dogs treated
at 20 (female), and 40 mg/kg (male). Treatment-related clinical
observations were recorded for the female treated at 80 mg/kg. This
female exhibited emesis during the infusion and post infusion. In
addition, the dog was pale (pallor), diarrhea was noted, and had
yellow mucous in the stool. All observations were noted on day 1
and the dog was recovered on day 2. Based on the findings, the MTD
for 5-fluoro-prodrug was established at 80 mg/kg.
[0420] It should be noted that at MTDs (30 mg/kg for 4-chloro-, and
80 mg/kg for 5-fluoro-prodrug), both prodrugs achieved peak serum
levels of Triapine at approximately 32 .mu.g/mL, suggesting the
toxicity observed in dogs was due to Triapine, not the prodrug
itself.
[0421] In Vivo Anti-Tumor Efficacy
[0422] Twelve (12) 3-AP prodrugs have been evaluated for efficacy
and toxicity using the M109 murine lung carinoma model in Balb/c
mice. The procedure of the experiment is as follows: eight week
aged female Balb/c mice (about 20 grams) were subcutaneously
inoculated with 5.times.10.sup.5/mouse of M109 murine lung carinoma
cells at the right flank on day 0. The mice were then randomly
grouped and each group consisted of 8-10 mice. The treatment was
started on day 3 or 5 according to the schedule shown in Table 6,
below. 3-AP was used with the Triapine.TM. formulation while all
3-AP prodrugs were either dissolved or suspended in sterile
deionized water. The body weight of mice and the tumor volume were
measured twice weekly until the tumor in the control group became
necrotic or at least one animal was dead in the control group.
[0423] The results are summarized in Table 6, below. Based upon the
stability, pharmacokinetics and activity of these agents, it is
evident that the 5-fluoro and 4-chloro analogs (7 and 16,
respectively) exhibited the greatest activity as anti-cancer
agents. In addition, the 5-trifluoromethoxy derivative (10), the
4,5-dichloro derivative (13) and the 5,6-dichloro derivative (14)
also evidenced unexpectedly good activity.
6TABLE 6 Activity in the M109 Lung Carcinoma Model Inhibi- BW Dose
Schedule tion Loss Relative Prodrugs (mpk,ip) (day) (%) (%)
Activity 3-AP (1A) 5.5 (bid) 3-7,10-14 60 8.09 <CTX Ortho- (3)
48QD 5-9,12-16 70 12.6 <CTX 5-Cl- (6) 60QD 5-9,12-16 67 5.3 =CTX
5-Fl- (7) 60QD 5-9,12-16 75 4.7 =CTX 5-NO.sub.2- (8) 60QD 3-7, 33
9.7 <<CTX 10-14,17-21 5-CH.sub.3O- (9) 48QD 5-9,12-16 73 13.5
ND 5-CF.sub.3O- (10) 100QD 3-7, 81 8.7 =CTX 10-14,17-21 5-CF.sub.3-
(11) 100QD 3-7, 77 9.1 <CTX 10-14,17-21 3,5-Di-Cl- (12) 60QD
3-7, 58 9.0 <CTX 10-14,17-21 4,5-DiCl- (13) 60QD 3-7,10-14,17-
57 10.0 <CTX 21 5,6-Di-Cl- (14) 60QD 3-7,10-14,17- 59 7.0
<CTX 21 3-CH.sub.3- (15) 60QD 3-7,10-14 64 7.9 <CTX 4-Cl-
(16) 60QD 5-9,12-16 74 12.6 =CTX
[0424] CTX=Cytoxan
[0425] In general, the prodrugs of 3-AP could be administered at
doses as high as 8 times greater than the MTD of 3-AP on a molar
basis. These agents could also be given on a QD1-5 schedule weekly
for an extended period without excessive mortality in the mice.
Treatment with compounds 3, 6, 7, 9, 10 and 16 gave better efficacy
on M109 lung carcinoma compared with the 3-AP parent drug at the
MTD. Subsequent studies on M109 lung carcinoma were performed using
prodrugs 7 and 16 according to the schedule set forth in FIG. 7c
and evidenced effective inhibition against M109 lung carcinoma.
These agents (prodrugs 7 and 16) were also tested on other cancer
cell lines in mice such as HTB Human Lung Carcinoma, B16-F10
Melanoma, DLD-1 Colon Carcinoma, with results evidencing
effectiveness significantly greater than cytoxan (see FIGS. 7D, E
and F)
[0426] 5-Chloro (6), 5-Fluoro (7) and 4-Chloro (16), analogs were
engaged in further study such as the optimal dose and dosing
schedule, different dosing routes, as well as use in combination
chemotherapy. The results of these experiments are shown on the
appended graphs (see attached as FIGS. 4-10). The results which may
be readily obtained from the figures evidence that the prodrugs of
the present invention combine well with the DNA damaging agents
cytoxan and mitomycin C (FIGS. 8A-D and 9A-C). Similar results,
evidence that the present compounds also may be combined to great
effect with Etoposide (FIG. 9D) and cisplatin against human colon
carcinoma and human lovo colon carcinoma (FIGS. 10A and B).
[0427] Efficacy of 3-AP prodrugs on M109 Lung Carcinoma in Balb/c
Mice
[0428] Materials: M109 lung carinoma cells; BALB/c mice (female, 9
weeks, 18-20 g); cytoxan (Sigma); 3-AP prodrug (ortho 3-AP
prodrug(3), 5-methoxy 3-AP prodrug (9), 5-chloro 3-AP prodrug (6),
4-chloro 3-AP prodrug (16) and 5-fluoro 3-AP prodrug (7). 110
Balb/c mice were randomly divided into twelve groups:
7 Groups: Mice 1. Vehicle 0.2 ml Qd (day 5-9; 12-16; 19-23) 10 2.
200 mpk cytoxan I.p. 1/w 10 3. 48 mpk ortho 3-AP prodrug I.P., Qd,
(day 5-9; 12-16; 19-23) 10 4. 60 mpk ortho 3-AP prodrug I.P., Qd
(day 5-9; 12-16; 19-23) 10 5. 48 mpk 5-methoxy 3-AP prodrug I.P.,
Qd, (day 5-9; 12-16; 19-23) 10 6. 55 mpk 5-methoxy 3-AP prodrug
I.P., Qd (day 5-9; 12-16; 19-23) 10 7. 48 mpk 5-chloro 3-AP prodrug
I.P., Qd, (day 5-9); 12-16; 19-23) 10 8. 60 mpk 5-chloro 3-AP
prodrug I.P., Qd, (day 5-9); 12-16; 19-23) 10 9. 48 mpk 4-chloro
3-AP prodrug I.P., Qd, (day 5-9); 12-16; 19-23) 10 10. 60 mpk
4-chloro 3-AP prodrug I.P., Qd, (day 5-9); 12-16; 19-23) 10 11. 48
mpk 5-fluoro 3-AP prodrug I.P., Qd, (day 5-9); 12-16; 19-23) 7 12.
48 mpk 4-fluoro 3-AP prodrug I.P., Qd, (day 5-9); 12-16; 19-23)
8
[0429] 3-AP Prodrug Preparation: Each 3-AP prodrug stocking
solution (10.0 mg/ml) was made by dissolving the prodrug in
deionized sterile water before each injection. The following 3-AP
prodrug stock solutions were made for each prodrug by further
diluting the stocking solution with water.
8 Tube Stocking Water Conc. Of prodrug Volume 1. 1.8 ml 1.2 ml 6.0
mg/ml 3 ml 2. 1.65 ml 1.35 ml 5.5 mg/ml (Methoxy 3-AP) 3 ml 3. 1.44
ml 1.56 ml 4.8 mg/ml 3 ml
[0430] The M109 cells in log phase were removed by trypsinization,
washed with PBS, and reconstituted to 2.5.times.10.sup.6 cells/ml
PBS. The M109 suspensions were implanted into the animals
subcutaneously on day 0 (0.2 ml, 5.times.10.sup.5 cells/mouse) at
the right flank. The mice were randomly regrouped as per the above.
Drug treatment was started on day 5 according to the above
schedule. The mice were maintained in a clean temperature constant
laboratory. The bedding was changed at least twice a week. The mice
were provided enough food and drinking water. The drinking water
was autoclaved before use. The treatment with orthophosphate 3-AP
prodrug (3) and 5-methoxy 3-AP prodrug (9) was stopped before the
end of the experiment due to the severe toxic reactions or
mortality of the mice. The body weight and tumor were measured
twice per week until the end of the experiment. The mortality and
the appearance of mice were observed daily.
[0431] FIGS. 7A, 7B and 7C evidence the efficacy exhibited by
5-fluoro 3-AP prodrug (7)and the 4-chloro 3-AP prodrug (16)
relative to 200 mpk cytoxan in comparison to controls. The 3-AP
prodrugs evidenced exceptional tumor shrinking efficacy comparable
to cytoxan, with no mortality.
[0432] 3-AP Prodrug/Cytoxan Combination Chemotherapy on M109 Lung
Carcinoma in Balb/c Mice
[0433] Materials: M109 lung carinoma cells; BALB/c mice (female, 9
weeks, 19-21 g); cytoxan (Sigma); mitomycin C ; 4-chloro 3-AP
prodrug (16) and 5-fluoro 3-AP prodrug (7). 120 Balb/c mice were
randomly divided into fifteen groups, each group consisting of 8
mice:
9 Groups: Mice 1. Vehicle 0.2 ml Qd (day 3-7; 10-14) 8 2. 200 mpk
cytoxan I.P. 1/w X 3 (Start on Day 3) 8 3. 3 mpk mitomycin C, I.V.
QD (Day 3 & 17) 8 4. 45 mpk 5-chloro 3-AP prodrug I.P./ Qd (day
3-7; 10-14) 8 +100 mpk cytoxan, I.P. 1/W (Start on day 4) 5. 45 mpk
5-chloro 3-AP prodrug I.P./ Qd (day 3-7; 10-14) 8 +150 mpk cytoxan,
I.P. 1/W (Start on day 4) 6. 45 mpk 5-chloro 3-AP prodrug I.P./ Qd
(day 3-7; 10-14) 8 +200 mpk cytoxan, I.P. 1/W (Start on day 4) 7.
60 mpk 5-chloro 3-AP prodrug I.P./ Qd (day 3-7; 10-14) 8 +100 mpk
cytoxan, I.P. 1/W (Start on day 4) 8. 60 mpk 5-chloro 3-AP prodrug
I.P./ Qd (day 3-7; 10-14) 8 +150 mpk Cytoxan, I.P. 1/W (Start on
day 4) 9. 60 mpk 5-chloro 3-AP prodrug I.P./ Qd (day 3-7; 10-14) 8
+200 mpk Cytoxan, I.P. 1/W (Start on day 4) 10. 45 mpk 5-fluoro
3-AP prodrug I.P./ Qd (day 3-7; 10-14) 8 +100 mpk cytoxan, I.P. 1/W
(Start on day 4) 11. 45 mpk 5-fluoro 3-AP prodrug I.P./ Qd (day
3-7; 10-14) 8 +150 mpk cytoxan, I.P. 1/W (Start on day 4) 12. 45
mpk 5-fluoro 3-AP prodrug I.P./ Qd (day 3-7; 10-14) 8 +200 mpk
cytoxan, I.P. 1/W (Start on day 4) 13. 60 mpk 5-fluoro 3-AP prodrug
I.P./ Qd (day 3-7; 10-14) 8 +100 mpk cytoxan, I.P. 1/W (Start on
day 4) 14. 60 mpk 5-fluoro 3-AP prodrug I.P./ Qd (day 3-7; 10-14) 8
+150 mpk cytoxan, I.P. 1/W (Start on day 4) 15. 60 mpk 5-fluoro
3-AP prodrug I.P./ Qd (day 3-7; 10-14) 8 +200 mpk cytoxan, I.P. 1/W
(Start on day 4)
[0434] 3-AP prodrug preparation: Each 3-AP prodrug stocking
solution (10.0 mg/ml) was made by dissolving the prodrug in
deionized sterile water before each injection. The following 3-AP
prodrug stock solutions were made for each prodrug by further
diluting the stocking solution with water.
10 Tube Stocking Water Conc. Of prodrug Volume 1. 1.8 ml 1.2 ml 6.0
mg/ml 3 ml 2. 1.35 ml 1.65 ml 4.5 mg/ml 3 ml
[0435] The M109 cells in log phase were removed by trypsinization,
washed with PBS, and reconstituted to 2.5.times.10.sup.6 cells/ml
PBS. The M109 suspensions were implanted into the animals
subcutaneously on day 0 (0.2 ml, 5.times.10.sup.5 cells/mouse) at
the right flank. The mice were randomly regrouped as per the above.
Drug treatment was started on day 3 according to the above
schedule. The mice were maintained in a clean temperature constant
laboratory. The bedding was changed at least twice a week. The mice
were provided enough food and drinking water. The drinking water
was autoclaved before use. The body weight and tumors were measured
twice per week until the end of the experiment. The mortality and
the appearance of mice were observed daily. FIG. 7C depicts another
experiment with a comparison to cytoxan and prodrug (3).
[0436] FIGS. 7D-F, evidence the activity exhibited by 5-fluoro 3-AP
prodrug (7) and 4-chloro prodrug (16) under the indicated
conditions against HTB 177 Human Lung Carcinoma, B16-F10 Melanoma
and DLD-1 Colon Carcinoma, all carried in mice.
[0437] FIGS. 8A-D evidence the efficacy exhibited by 5-chloro 3-AP
prodrug (6) and the 5-fluoro 3-AP prodrug (7) in combination
chemotherapy (with cytoxan) relative to 200 mpk cytoxan against
M109 Lung Carcinoma sssin comparison to controls. The 3-AP prodrugs
in combination with cytoxan evidenced exceptional synergistic tumor
shrinking efficacy when compared to cytoxan alone. Note that no
mortality was exhibited during this experiment.
[0438] 5-Fluoro 3-AP Prodrug/Cytoxan or Mitomycin C Based
Combination Chemotherapy on M109 Lung Carcinoma in Balb/c Mice
[0439] Materials: M109 lung carinoma cells; BALB/c mice (female, 9
weeks, 19-21 g); cytoxan (Sigma); mitomycin C and 5-fluoro
ortho-3-AP prodrug (7).
[0440] 121 Balb/c mice were randomly divided into twelve groups,
each group consisting of 10 mice:
11 Groups: Mice 1. Vehicle 0.2 ml Qd (day 3-7; 10-14) 11 2. 200 mpk
cytoxan I.P. 1/w X 3 (Start on Day 3) 10 3. 3 mpk mitomycin C, I.V.
QD (Day 3 & 13) 10 4. 45 mpk 5-fluoro 3-AP prodrug I.P./ Qd
(day 3-7; 10-14) 10 +150 mpk cytoxan, I.P. 1/W (Start on day 4) 5.
45 mpk 5-fluoro 3-AP prodrug I.P./ Qd (day 3-7; 10-14) 10 +200 mpk
cytoxan, I.P. 1/W (Start on day 4) 6. 60 mpk 5-fluoro 3-AP prodrug
I.P./ Qd (day 3-7; 10-14) 10 +150 mpk cytoxan, I.P. 1/W (Start on
day 4) 7. 60 mpk 5-fluoro 3-AP prodrug I.P./ Qd (day 3-7; 10-14) 10
+200 mpk cytoxan, I.P. 1/W (Start on day 4) 8. 45 mpk 5-fluoro 3-AP
prodrug I.P./ Qd (day 3-7; 10-14) 10 +2 mpk mitomycin C, I.V. QD
(Day 3 & 13) 9. 60 mpk 5-fluoro 3-AP prodrug I.P./ Qd (day 3-7;
10-14) 10 +2 mpk mitomycin C, I.V. QD (Day 3 & 13) 10 10. 120
mpk 5-fluoro 3-AP, S.C. Qd (day 3-7)* 10 11. 150 mpk 5-fluoro 3-AP,
S.C. Qd (day 3-7; 10-14)** 10 12. 200 mpk 5-fluoro 3-AP, S.C. Qd
(day 3-6)*** 10 *Only one dosing schedule was given due to
mortality of mice; **Treatment was stopped after two dosing
schedules due to the body weight loss ***Treatment was stopped
after four days because of mortality of mice.
[0441] 3-AP Prodrug Preparation: 5-fluoro 3-AP prodrug (7) stocking
solution (20.0 mg/ml) was made by dissolving the prodrug in
deionized sterile water before each injection. The following 3 -AP
prodrug stock solutions were made for each prodrug by further
diluting the stocking solution with water.
12 Tube Stocking Water Conc. of prodrug Volume 1. 3.0 ml 0 ml 20.0
mg/ml 3 ml 2. 2.25 ml 0.75 ml 15.0 mg/ml 3 ml 3. 1.8 ml 1.2 ml 12.0
mg/ml 3 ml 4. 1.5 ml 1.5 ml 10.0 mg/ml 3 ml 2. 0.9 ml 2.1 ml 6.0
mg/ml 3 ml 2. 0.68 ml 2.32 ml 4.5 mg/ml 3 ml
[0442] Liquid nitrogen stored M109 lung and carcinoma cells
(1.times.10.sup.6 cels/ml.times.1 ml) were recovered by rapidly
thawing the cells at 37.degree. C. and cultured with 25 ml of DMEM
culture medium containing 10% FCS at 37.degree. C., in 5% CO.sub.2.
After passing two generations, the cells were washed twice with
PBS, pH 7.2, trypsinized and subcultured in the flasks containing
50 ml culture medium. Finally, M109 cells in log phase (about
90-95% saturation) were removed by trypsinization, washed with PBS,
and reconstituted to 5.times.10.sup.6 cells/ml PBS for tumor
implantation. The M109 suspensions were implanted into the animals
subcutaneously on day 0 (0.2 ml, 5.times.10.sup.5 cells/mouse) at
the right flank. The mice were randomly regrouped as per the above.
Drug treatment was started on day 3 according to the above
schedule. The mice were maintained in a clean temperature constant
laboratory. The bedding was changed at least twice a week. The mice
were provided enough food and drinking water. The drinking water
was autoclaved before use. The body weight and tumors were measured
twice per week until the end of the experiment. The mortality and
the appearance of mice were observed daily.
[0443] FIGS. 9A-C evidence the efficacy exhibited by 5-fluoro 3-AP
prodrug (7) and 5-chloro 3-AP prodrug (6) in combination
chemotherapy with mitomycin C relative to 200 mpk cytoxan and in
comparison to controls. These 3-AP prodrugs in combination with
cytoxan and mitomycin C evidenced exceptional synergistic tumor
shrinking efficacy when compared to cytoxan or mytomycin C alone.
In FIG. D, the results of an experiment comparing the efficacy of a
combination of 5-chloro 3-AP prodrug (6) with Etoposide compared
with Etoposide alone or control are presented. Synergistic activity
was evidenced by the drug combination in this experiment against
M109 lung carcinoma.
[0444] FIGS. 10A and 10B evidence the effect of combination therapy
utilizing 4-chloro 3-AP (16) and cisplatin against DLD-1 Human
Colon Carcinoma (FIG. 10A) and Human LoVo Colon Carcinoma (FIG.
10B). In both of these experiments, combination therapy evidenced
synergistic activity against the tumors tested.
[0445] LD 50 of 5-Fluoro 3-AP Prodrug in C57BL/6J Mice
[0446] Materials: C57BL/6J Mice (female, 8 weeks); 5-fluoro 3-AP
prodrug (7). 55 C57BL/6J mice were randomly divided into 11 groups,
each group consisting of 5 mice:
13 Groups: Mice 1. Vehicle 0.2 ml Sterilized deionized water, I.P.
QD 5 2. 100 mpk 5-fluoro 3-AP prodrug, I.P. Qd 5 3. 125 mpk
5-fluoro 3-AP prodrug, I.P. Qd 5 4. 150 mpk 5-fluoro 3-AP prodrug,
I.P. Qd 5 5. 175 mpk 5-fluoro 3-AP prodrug, I.P. Qd 5 6. 200 mpk
5-fluoro 3-AP prodrug, I.P. Qd 5 7. 175 mpk 5-fluoro 3-AP prodrug,
S.C.. Qd 5 8. 200 mpk 5-fluoro 3-AP prodrug, S.C.. Qd 5 9. 225 mpk
5-fluoro 3-AP prodrug, S.C.. Qd 5 10. 250 mpk 5-fluoro 3-AP
prodrug, S.C.. Qd 5 11. 300 mpk 5-fluoro 3-AP prodrug, S.C.. Qd
5
[0447] 3-AP prodrug preparation. 5-fluoro 3-AP prodrug (7) was
dissolved into sterile deionized water to make the stock solution
(30 mg/ml). The following 3-AP prodrug stock solutions were made
for each prodrug by further diluting the stocking solution with
water.
14 Tube Stocking Water Conc. of prodrug Volume 1. 3.0 ml 0 ml 30.0
mg/ml 3 ml 2. 2.5 ml 0.5 ml 25.0 mg/ml 3 ml 3. 2.25 ml 0.75 22.5
mg/ml 3 ml 4. 2 ml 1 ml 20 mg/ml 3 ml 5. 1.75 ml 1.25 ml 17.5 mg/ml
3 ml 6. 1.5 ml 1.5 ml 15 mg/ml 3 ml 7. 1.25 ml 1.75 ml 12.5 mg/ml 3
ml 8. 1 ml 2 ml 10 mg/ml 3 ml
[0448] Treatment was started on day 0 following the above
schedules. The mortality of the animals was recorded daily. The
body weight was measured twice per week and the appearance and
behavior of the mice were observed daily. FIG. 11 shows the
estimation of the LD 50 of 5-fluoro AP prodrug, which is
approximately 160 mpk.
[0449] Pharmacokinetic Study
[0450] The pharmacokinetics of 3-AP (1A) the orthophosphate prodrug
(2), and 5-F orthophosphate prodrug (7, 30a, FIG. 3) were
determined in beagle dogs (Canis familiaris). The dogs were dosed
at 20 mg/kg, 30 mg/kg and 40 mg/kg in the case of 3-AP and the
orthophosphate prodrug and at 20 mg/kg, 40 mg/kg and 80 mg/kg for
the 5-Fluoro orthophosphate prodrug. The dosing schedule for each
compound was based upon the Maximum Tolerated Dose for each
prodrug, which was significantly higher for the 5-Fluoro
orthophosphate produrg than for either 3-AP or the orthophosphate
prodrug (3). (It is noted here that even at doses of 80 mg/kg, the
5-Fluoro orthophosphate was not toxic to the animals, whereas in
the case of the 3-AP and the orthophosphate prodrug (3), the drug
was toxic at the 30 and 40 mg/kg level.
[0451] Drug levels were determined for the animals at the intervals
which are indicated in attached FIGS. 12-15. These figures evidence
that the orthophosphate prodrug had a significant impact on the
bioavailability of 3-AP and that the 5-fluorophosphate prodrug
provided a significantly greater bioavailabiity and high
concentrations of 3 -AP for long duration. The pharmacokinetic data
for 3-AP and the orthophosphate prodrug (3) is presented in FIG.
12.
[0452] In the case of the 5-fluorophosphate prodrug (7), FIGS.
13-15 set forth the data evidencing that the 5-fluorophosphate
derivative provided greater bioavailability of the prodrug compound
itself and greater bioavailability of 3-AP resulting from
degradation of the prodrug. In addition, the duration of the higher
levels of 3-AP was longer in the case of the 5-Fluoro phosphate
prodrug (see inset in FIG. 13) tan in the case of 3-AP or the
orthophosphate prodrug.
[0453] From the studies, it was shown that the 5-fluorophosphate
prodrug (7) was tolerated at higher levels (MTD) than either 3-AP
or the orthophosphate prodrug (3), and further, provided delivery
of 3-AP at higher blood concentrations initially and for a longer
duration than either the orthophosphate prodrug form or the 3-AP
drug itself.
[0454] 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.
* * * * *