U.S. patent application number 10/950890 was filed with the patent office on 2005-02-24 for water-soluble shps as novel alkylating agents.
This patent application is currently assigned to VION PHARMACEUTICALS, INC.. Invention is credited to Doyle, Terrence W., King, Ivan, Lin, Xu.
Application Number | 20050043244 10/950890 |
Document ID | / |
Family ID | 33511222 |
Filed Date | 2005-02-24 |
United States Patent
Application |
20050043244 |
Kind Code |
A1 |
Lin, Xu ; et al. |
February 24, 2005 |
Water-soluble SHPs as novel alkylating agents
Abstract
The present invention relates to compounds according to the
structure (I): 1 Where R is --CH.sub.3 or --CH.sub.2CH.sub.2Cl; R'
is C.sub.1-C.sub.7 alkyl or --CH.sub.2CH.sub.2Cl; R.sub.2 or
R.sub.4 is OP0.sub.3H.sub.2, N0.sub.2, OCO(Glu-OH), NHCO(Glu-OH),
NHR.sub.7 and unassigned groups of R.sub.2, R.sub.3, R.sub.4,
R.sub.5 and R.sub.6 are, independently, H, F, Cl, Br, I, OH,
OP0.sub.3H.sub.2, OCH.sub.3, CF.sub.3, OCF.sub.3, NO.sub.2, CN,
SO.sub.2CH.sub.3, SO.sub.2CF.sub.3, COCH.sub.3, COOCH.sub.3,
SCH.sub.3, SFs, NH.sub.2, NHR.sub.7, N(CH.sub.3).sub.2,
OPO.sub.3H.sub.2, or a C1-C7 alkyl group with the proviso that when
any two of unassigned groups of R.sub.2, R.sub.3, R.sub.4, R.sub.5
or R.sub.6 are other than H, the other two of unassigned groups of
R.sub.2, R.sub.3, R.sub.4, R.sub.5 or R.sub.6 are H. R.sub.7 is H
or polyglutamyl as described. Phosphoric acid and glutamic acid can
be a free acid or pharmaceutically acceptable salt thereof.
Inventors: |
Lin, Xu; (Branford, CT)
; Doyle, Terrence W.; (Killingworth, CT) ; King,
Ivan; (North Haven, CT) |
Correspondence
Address: |
Henry D. Coleman
714 Colorado Avenue
Bridgeport
CT
06605-1601
US
|
Assignee: |
VION PHARMACEUTICALS, INC.
New Haven
CT
|
Family ID: |
33511222 |
Appl. No.: |
10/950890 |
Filed: |
September 27, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10950890 |
Sep 27, 2004 |
|
|
|
10461282 |
Jun 13, 2003 |
|
|
|
Current U.S.
Class: |
514/19.6 ;
514/114; 514/19.3; 514/524; 514/562; 514/590 |
Current CPC
Class: |
A61P 1/16 20180101; C07C
311/55 20130101; A61P 35/02 20180101; A61P 13/12 20180101; A61P
25/00 20180101; A61P 15/00 20180101; A61K 38/16 20130101; C07F 9/12
20130101; A61P 11/00 20180101; A61P 13/10 20180101; A61K 38/04
20130101; A61P 35/00 20180101; A61P 13/08 20180101; A61P 1/04
20180101; A61P 17/00 20180101; A61P 1/18 20180101; A61P 43/00
20180101 |
Class at
Publication: |
514/012 ;
514/013; 514/014; 514/015; 514/016; 514/017; 514/018; 514/019;
514/114; 514/524; 514/562; 514/590 |
International
Class: |
A61K 038/16; A61K
038/10; A61K 038/08; A61K 038/06; A61K 038/05; A61K 038/04; A61K
031/277; A61K 031/175; A61K 031/195 |
Claims
1-22. (cancelled)
23. A pharmaceutical composition comprising an effective amount for
treating non-tumorous cancer of a compound or its pharmaceutically
acceptable salt according to the structure: 5Where R is --CH.sub.3
or --CH.sub.2CH.sub.2Cl; R' is C.sub.1-C.sub.7 alkyl or
--CH.sub.2CH.sub.2Cl; one of R.sub.2 or R.sub.4, but not both, is
selected from OPO.sub.3H.sub.2, NO.sub.2, OCO(Glu), NHCO(Glu) and
NHR.sub.7 and the other of R.sub.2 or R.sub.4 which is unassigned,
and R.sub.3, R.sub.5 and R.sub.6, are, independently selected from
H, F, Cl, Br, I, OH, OPO.sub.3H.sub.2, OCH.sub.3, CF.sub.3,
OCF.sub.3, NO.sub.2, CN, SO.sub.2CH.sub.3, SO.sub.2CF.sub.3,
COCH.sub.3, COOCH.sub.3, SCH.sub.3, SF.sub.5, NHR.sub.8,
N(R.sub.9).sub.2 and C.sub.1-C.sub.7 alkyl, with the proviso that
at least two of R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are
H; R.sub.7 is H, glutamyl or a polyglutamic acid polypeptide
residue (--COCH(NHR.sub.7a)CH.sub.2CH.sub.2CO.sub.2H where R.sub.7a
is glutamyl or a polyglutamic acid polypeptide residue having from
1 to 50 peptide linkages; R.sub.8 is H or C.sub.1-C.sub.7 alkyl;
and R.sub.9 is CH.sub.3 or CH.sub.2CH.sub.3; optionally, in
combination with a pharmaceutically acceptable additive, carrier,
or excipient.
24. The composition according to claim 23 wherein R is
--CH.sub.2CH.sub.2Cl.
25. The composition according to claim 23 wherein said R' is
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,
n-pentyl, isopentyl, n-hexyl, isohexyl or substituted hexyl.
26. The composition according to claim 25 wherein R' is methyl.
27. The composition according to claim 23 wherein R.sub.2 is
OPO.sub.3H.sub.2 or its pharmaceutically acceptable salt.
28. The composition according to claim 23 wherein R.sub.4 is F, Cl
or OCH.sub.3 when R.sub.3, R.sub.5 and R.sub.6 are each H.
29. The composition according to claim 23 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.
30. The composition according to claim 23 two of R.sub.3, R.sub.4,
R.sub.5 or R.sub.6 are independently F or Cl.
31. The composition according to claim 30 wherein R.sub.4 and
R.sub.5 are independently F or Cl.
32. The composition according to claim 30 wherein R.sub.5 and
R.sub.6 are independently F or Cl.
33. The composition according to claim 31 wherein R.sub.4 and
R.sub.5 are Cl.
34. The composition according to claim 32 wherein R.sub.5 and
R.sub.6 are Cl.
35. The composition according to claim 23 wherein R.sub.5 is
OPO.sub.3H.sub.2 or its pharmaceutically acceptable salt.
36. The composition according to claim 23 wherein R.sub.2 is
NO.sub.2 when R.sub.3, R.sub.4 and R.sub.6 are each H.
37. The composition according to claim 23 wherein R.sub.4 is
NO.sub.2 and R.sub.2, R.sub.3 and R.sub.6 are each H.
38. The composition according to claim 23 wherein R.sub.4 is
OCO(Glu) and R.sub.2, R.sub.3, R.sub.5 and R.sub.6 are each H.
39. The composition according to claim 38 wherein Glu is in the
form of a pharmaceutically acceptable salt.
40. The composition according to claim 23 wherein R.sub.4 is
NHCO(Glu) and R.sub.2, R.sub.3, R.sub.5 and R.sub.6 are each H.
41. The composition according to claim 40 for wherein Glu is in the
form of a pharmaceutically acceptable salt.
42. The composition according to claim 23 wherein R.sub.4 is
NHR.sub.7 and R.sub.2, R.sub.3, R.sub.5 and R.sub.6 are each H.
43. The composition according to claim 42 for wherein R.sub.7 is
.alpha.-glutamyl or a pharmaceutically acceptable salt thereof.
44. The composition according to claim 43 wherein R.sub.7 is H,
.alpha.-glutamyl or a pharmaceutically acceptable salt thereof or a
polyglutamic acid polypeptide residue or a pharmaceutically
acceptable salt thereof.
45. A method of treating non-tumorous cancer in a patient in need
of therapy comprising administering to said patient an effective
amount of a compound or its pharmaceutically acceptable salt
according to the structure: 6Where R is --CH.sub.3 or
--CH.sub.2CH.sub.2Cl; R' is C.sub.1-C.sub.7 alkyl or
--CH.sub.2CH.sub.2Cl; one of R.sub.2 or R.sub.4, but not both, is
selected from OPO.sub.3H.sub.2, NO.sub.2, OCO(Glu), NHCO(Glu) and
NHR.sub.7 and the other of R.sub.2 or R.sub.4 which is unassigned,
and R.sub.3, R.sub.5 and R.sub.6, are, independently selected from
H, F, Cl, Br, I, OH, OPO.sub.3H.sub.2, OCH.sub.3, CF.sub.3,
OCF.sub.3, NO.sub.2, CN, SO.sub.2CH.sub.3, SO.sub.2CF.sub.3,
COCH.sub.3, COOCH.sub.3, SCH.sub.3, SF.sub.5, NHR.sub.8,
N(R.sub.9).sub.2 and C.sub.1-C.sub.7 alkyl, with the proviso that
at least two of R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are
H; R.sub.7 is H, glutamyl or a polyglutamic acid polypeptide
residue --COCH(NHR.sub.7a)CH.sub.2CH.sub.2C- O.sub.2H where
R.sub.7a is glutamyl or a polyglutamic acid polypeptide residue
having from 1 to 50 peptide linkages; R.sub.8 is H or
C.sub.1-C.sub.7 alkyl; and R.sub.9 is CH.sub.3 or CH.sub.2CH.sub.3;
optionally, in combination with a pharmaceutically acceptable
additive, carrier, or excipient.
46. The method according to claim 45 wherein R is
--CH.sub.2CH.sub.2Cl.
47. The method according to claim 45 wherein said R' is methyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl,
isopentyl, n-hexyl, isohexyl or substituted hexyl.
48. The method according to claim 47 wherein R'--CH.sub.3.
49. The method according to claim 45 wherein R.sub.2 is
OPO.sub.3H.sub.2 or a pharmaceutically acceptable salt thereof.
50. The method according to claim 45 wherein R.sub.4 is F, Cl or
OCH.sub.3 and R.sub.3, R.sub.5 and R.sub.6 are each H.
51. The method according to claim 45 wherein R.sub.5 is F, Cl,
OCH.sub.3 or OCF.sub.3 and R.sub.3, R.sub.4 and R.sub.6 are each
H.
52. The method according to claim 45 wherein two of R.sub.3,
R.sub.4, R.sub.5 or R.sub.6 are independently F or Cl.
53. The method according to claim 52 wherein R.sub.4 and R.sub.5
are independently F or Cl.
54. The method according to claim 52 wherein R.sub.5 and R.sub.6
are independently F or Cl.
55. The method according to claim 53 wherein R.sub.4 and R.sub.5
are Cl.
56. The method according to claim 54 wherein R.sub.5 and R.sub.6
are Cl.
57. The method according to claim 45 wherein R.sub.5 is
OPO.sub.3H.sub.2 or a pharmaceutically acceptable salt thereof.
58. The method according to claim 45 wherein R.sub.2 is NO.sub.2
and R.sub.3, R.sub.4 and R.sub.6 are each H.
59. The method according to claim 45 wherein R.sub.4 is NO.sub.2
and R.sub.2, R.sub.3 and R.sub.6 are each H.
60. The method according to claim 45 wherein R.sub.4 is OCO(Glu)
and R.sub.2, R.sub.3, R.sub.5 and R.sub.6 are each H.
61. The method according to claim 60 wherein Glu is in the form of
a pharmaceutically acceptable salt.
62. The method according to claim 45 wherein R.sub.4 is NHCO(Glu)
and R.sub.2, R.sub.3, R.sub.5 and R.sub.6 are each H.
63. The method according to claim 62 wherein Glu is in the form of
a pharmaceutically acceptable salt.
64. The method according to claim 45 wherein R.sub.4 is NHR.sub.7
and R.sub.2, R.sub.3, R.sub.5 and R.sub.6 are each H.
65. The method according to claim 64 wherein R.sub.7 is a
.alpha.-glutamyl or a pharmaceutically acceptable salt thereof.
66. The method according to claim 64 wherein R.sub.7 is H, a
.alpha.-glutamyl or a pharmaceutically acceptable salt thereof or a
polyglutamic acid polypeptide residue or a pharmaceutically
acceptable salt thereof.
67. The method according to claim 45 wherein said cancer is
selected from the group consisting of acute lymphocytic leukemia,
acute myelogenous leukemia, or hairy cell leukemia.
68. (Cancelled)
69. A method of treating a drug-resistant non-tumorous cancer in a
patient in need thereof, said method comprising administering to
said patient an effective amount of a composition according to
claims 23 or 24.
70. A method of treating non-tumorous cancer in a patient in need
thereof said method comprising administering to said patient an
effective amount of a composition according to claims 23 or 24 in
combination with at least one additional anti-cancer agent.
71. A method of treating non-tumorous cancer in a patient in need
thereof said method comprising administering to said patient an
effective amount of a composition according to claims 23 or 24 in
combination with at least one additional anti-cancer agent selected
from the group consisting of antimetabolites, Ara C, etoposide,
doxorubicin, taxol, hydroxyurea, vincristine, cytoxan, mitomycin C,
adriamycin, topotecan, campothecin, irinotecan, gemcitabine, and
cis-platin.
72. A pharmaceutical composition according to any of claims 23-44
in combination with at least one additional anti-cancer agent.
73. A pharmaceutical composition according to claims 23 or 24 in
combination with at least one additional anti-cancer agent selected
from the group consisting of antimetabolites, Ara C, etoposide,
doxorubicin, taxol, hydroxyurea, vincristine, cytoxan, mitomycin C,
adriamycin, topotecan, campothecin, irinotecan, gemcitabine,
campothecin and cis-platin.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to metabolically activated
sulfonyl hydrazine prodrugs (SHPs) exhibiting anti-tumor activity
in mammals. Methods of treating neoplasia, especially including
cancer, using compounds according to the present invention, are
additional aspects of the present invention.
[0002] This invention was made with government support under grant
number 1 R.sub.43 CA92968-01 awarded by the Department of Health
and Human Services. As such, the government retains certain rights
in the invention.
BACKGROUND OF THE INVENTION
[0003] Alkylating agents are among the most effective therapeutic
agents currently available to treat different malignancies, and are
widely used in the clinic (Katzung, In Basic & Clinical
Pharmacology, 7th edition, 1998, Appleton & Lange, Stamford,
881). The high degree of cytotoxicity is attributed to the ability
to induce DNA interstrand cross-linking thereby inhibiting
replication (Rajski and Williams, Chem Reviews 1998, 98: 2723).
Among the alkylating agents, the CNU (chloroethylnitrosourea)
series have been widely used clinically to treat brain tumors,
colon cancer and lymphomas (DeVita, et al. Cancer Res. 1965, 25:
1876; and Nissen, et al. Cancer 1979, 43: 31), however, their
clinical usefulness is limited due to delayed and cumulative bone
marrow depression and hepatic toxicity (Panasci, et al. Cancer Res.
1977, 37: 2615; and Gibson and Hickman, Biochem Pharmacol. 1982,
31: 2795).
[0004] A series of 1,2-bis(sulfonyl)hydrazine prodrugs (SHPs) with
the ability to generate chloroethylating and carbamoylating
species, but lacking hydroxyethylating and vinylating species,
generated by the CNUs had been developed recently (Sartorelli, et
al. see U.S. Pat. No. 6,040,338; U.S. Pat. No. 5,637,619; U.S. Pat.
No. 5,256,820; U.S. Pat. No. 5,214,068; U.S. Pat. No. 5,101,072;
U.S. Pat. No. 4,849,563; and U.S. Pat. No. 4,684,747. The antitumor
activity has been suggested to result from chloroethylating and
subsequent cross-linking of DNA (Kohn, In Recent Results in Cancer
Research, Eds. Carter, et al., 1981, Springer, Berlin, vol. 76:
141; and Shealy, et al., J Med Chem. 1984, 27: 664). The
carbamoylating species (i.e., the isocyanate) can react with thiol
and amine functionalities on proteins and inhibit DNA polymerase
(Baril, et al. Cancer Res. 1975, 35: 1), the repair of DNA strand
breaks (Kann, et al. Cancer Res. 1974, 34: 398) and RNA synthesis
and processing (Kann, et al. Cancer Res. 1974, 34: 1982). However,
hydroxyethylation of DNA is a carcinogenic and/or mutagenic event
(Swenson, et al. J Natl Cancer Inst. 1979, 63: 1469).
[0005]
1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-(methylaminocarbonyl)hy-
drazine (VNP40101M), the current lead compound in the SHP series,
has lower toxicity to hosts and better anti-tumor activities
against the L1210 murine leukemia, L1210/BCNU, L1210/CTX, L1210/MEL
(1,3-bis(2-chloroethyl)-1-nitrosourea, cyclophosphamide and
melphalan resistant sublines), P388 leukemia, M109 lung carcinoma,
B16 melanoma, C26 colon carcinoma and U251 glioma than
chloroethylnitrosourea (CNU) derivatives and other SHP analogs
(Shyam, et al. J Med Chem. 1999, 42: 941). In addition, VNP4011M is
effective in crossing the blood brain barrier (BBB) and eradicating
leukemia cells implanted intracranially (>6.54 log cell kill),
rivaling the efficacy of BCNU (Finch, et al. Cancer Biochem
Biophys. 2001, 61: 3033). 2
[0006] The anti-tumor activity of VNP40101M is probably due to the
release of 90CE and methyl isocyanate. 90CE further fragments to
yield methyl 2-chloroethyldiazosulfone (1) FIG. 1, a relatively
specific O.sup.6-guanine chloroethylator, producing minimal
alkylation of the N.sup.7-position of guanine (Penketh, et al. J
Med Chem. 1994, 37: 2912; and Penketh, et al. Biochem Pharmacol.
2000, 59: 283). Methyl isocyanate released from VNP40101M has the
ability to inhibit various DNA repair enzymes including
O.sup.6-alkylguanine-DNA alkyltransferase leading to stabilization
of the O.sup.6-alkylguanine monoalkyl species in DNA, which leads
to a larger percentage of interstrand cross-links (Baril, et al.
Cancer Res. 1975, 35: 1).
[0007] VNP40101M is currently in clinical trials in patients with
solid tumors and hematologic malignancies. VNP40101M is not very
soluble in aqueous solution; polyethylene glycol (PEG) and ethanol
are included in the vehicle of the finished product to promote
solubility. Both PEG and ethanol are acceptable vehicles for human
use but may cause side effects such as hemolysis and phlebitis at
high concentrations, as indicated in animal studies. VNP40101M is
very well tolerated in humans and could be given at higher 110
doses, and could in theory produce a higher degree of efficacy, if
PEG and ethanol could be eliminated from the vehicle. Therefore,
our aim was to synthesize a series of SHPs that (a) were capable of
improving its water-solubility and stability in aqueous solution at
pH 3 to 9; (b) were capable of forming chloroethylating species;
(c) were devoid of hydroxyethylating activity; (d) were capable of
forming methyl isocyanate; and (e) were capable of improving
pharmacokinetic profiles (e.g., longer half-life in vivo).
[0008] The present inventors conceived that water-soluble
enzymatically-activated SHPs (I) might satisfy the above
conditions. An example of such an SHP would be the phosphate
containing derivatives shown in FIG. 2 for the following
reasons:
[0009] (a) In general, a phosphate-bearing analog, including its
salt form may have good water-solubility and stability at neutral
pH;
[0010] (b) The bioconversion of compounds of general structure I is
believed to proceed via alkaline phosphatase (AP) cleavage of the
oxygen-phosphorous bond to form the phenol intermediate, which may
subsequently undergo fragmentation resulting in the formation of
chloroethylating or methylating species and carbamoylating agent
without generating hydroxyethylating agent, as shown in FIGS. 1 and
2.
[0011] (c) The bioconversion of compounds I may also generate a
quinone methide which itself can cause damage to DNA and thereby
contribute to inhibition of cellular replication (Lin, et al. J Med
Chem. 1986, 29: 84).
[0012] (d) Compounds I may be considered as prodrugs of VNP40101M
that has been identified as an alkylating agent against a broad
anticancer spectrum of neoplastic disease states, including, for
example, numerous solid tumors. Thus, compounds I may generate the
same active species as VNP40101M.
[0013] Further examples of bioactivated prodrugs are shown in FIGS.
3 and 4. The nitro analogs shown in FIG. 3 are examples of
compounds that would be both water soluble and selectively
activated under conditions of hypoxia. Release of VNP40101M would
only occur on reduction of the nitro group under conditions of
hypoxia. Compounds II would be reduced by nitroreductase (NR) to
the corresponding amino analogs, which would be subsequently
fragmented into VNP40101M and a quinone-imine methide. NR, an
enzyme isolated from E. Coli or Bacillus spp., is widely used in
ADEPT (antibody-directed enzyme prodrug therapy) or GDEPT
(gene-directed enzyme prodrug therapy) for cancer therapy
(Anlezark, et al. WO93/08288, 1993).
[0014] FIG. 4 illustrates the use of peptidases to generate
VNP40101M intratumorally. Cleavage of compounds derived from
conjugation of glutamyl residues to appropriately substituted
phenols and aromatic amines by carboxypeptidases such as
carboxypeptidase G2 (CPG2) and carboxypeptidase A (CPA) has been
shown previously. CPG2, an enzyme isolated from Pseudomonas, is
capable of removing glutamate residues from folates and
methotrexate. It has been employed in the ADEPT or GDEPT system to
activate prodrugs containing glutamate residues (Bagshawe, et al.
WO88/07378, 1988; Springer, et al.
[0015] U.S. Pat. No. 6,025,340, 2000; Springer, et al. U.S. Pat.
No. 6,004,550, 1999). CPA from bovine pancreas readily cleaves drug
.alpha.-peptides (derivatives in which an amino acid is linked to
the .alpha.-carboxyl group of the glutamate moiety). Also, it has
been employed in the ADEPT (Wolfe, et al. Bioconjug Chem. 1999, 10:
38; Huennekens, Adv Enzyme Regul. 1997, 37: 77; and Vitols, et al.
Cancer Res. 1995, 55: 478). As shown in FIG. 4, Compounds III and
Compounds IV would be cleaved by the corresponding CPG2 or CPA,
which may either be introduced as an antibody conjugate or a
transgene (Pawelek, et al. U.S. Pat. No. 6,190,657, 2001), to form
VNP40101M and a quinone methide or quinone-imine methide.
OBJECTS OF THE INVENTION
[0016] In one aspect, an object of the present invention is to
provide compounds, pharmaceutical compositions and methods for the
treatments of neoplasia, including animal and human cancer.
[0017] In another aspect of the invention, an object of the present
invention is to provide methods of treating neoplasia utilizing
compositions that exhibit favorable and enhanced characteristics of
activity, pharmacokinetics, bioavailability and reduced
toxicity.
[0018] 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.
[0019] One or more of these and/or other objects of the invention
may be readily gleaned from the description of the invention that
follows.
BRIEF DESCRIPTION OF THE INVENTION
[0020] The present invention relates to compounds or their
pharmaceutically acceptable salts according to the structure (I):
3
[0021] Where R is --CH.sub.3 or --CH.sub.2CH.sub.2Cl;
[0022] R' is C.sub.1-C.sub.7 alkyl or --CH.sub.2CH.sub.2Cl; one of
R.sub.2 or R.sub.4, but not both, is selected from
OPO.sub.3H.sub.2, NO.sub.2, OCO(Glu), NHCO(Glu) and NHR.sub.7 and
the other of R.sub.2 or R.sub.4 hich is unassigned, and R.sub.3,
R.sub.5 and R.sub.6, are, independently selected from H, F, Cl, Br,
I, OH, OPO.sub.3H.sub.2, OCH.sub.3, CF.sub.3, OCF.sub.3, NO.sub.2,
CN, SO.sub.2CH.sub.3, SO.sub.2CF.sub.3, COCH.sub.3, COOCH.sub.3,
SCH.sub.3, SF.sub.5, NHR.sub.8, N(R.sub.9).sub.2, OPO.sub.3H.sub.2
and C.sub.1-C.sub.7 alkyl, with the proviso that at least two of
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are H;
[0023] R.sub.7 is H, glutamyl, preferably
.alpha.-glutamyl(--COCH(NH.sub.2- )CH.sub.2CH.sub.2CO.sub.2H) or a
polyglutamic acid polypeptide residue
(--COCH(NHR.sub.7a)CH.sub.2CH.sub.2CO.sub.2H where R.sub.7a is
glutamyl (preferably, .alpha.-glutamyl) or a polyglutamic acid
polypeptide residue) having from 1 to 50 peptide linkages,
preferably from 2 to 10 peptide linkages;
[0024] R.sub.8 is H or C.sub.1-C.sub.7 alkyl; and
[0025] R.sub.9 is CH.sub.3 or CH.sub.2CH.sub.3. The phosphoric acid
and/or glutamic acid substituents can be in the free acid form or a
pharmaceutically acceptable salt thereof.
[0026] In certain preferred aspects of the present invention,
preferred agents in the class of Compounds I are
ortho-phosphate-bearing series where R is --CH.sub.2CH.sub.2Cl; R'
is --CH.sub.3; R.sub.2 is a phosphate group which can be the free
acid or its pharmaceutically acceptable salt (preferably Na). In
particularly preferred aspects of the ortho-phosphate-bearing SHPs,
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 of the
ortho-phosphate-bearing SHPs, R.sub.5 is Cl, F or Br (preferably
Cl, F) when R.sub.3, R.sub.4 and R.sub.6 are H. Still in other
preferred aspects of the ortho-phosphate-bearing SHPs, two of
R.sub.3, R.sub.4, R.sub.5 and 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 and R.sub.6 are H.
[0027] Preferred agents in the class of Compounds II are
meta-phosphate-bearing nitro-containing analogs of SHPs. The
phosphate group can be the free acid or its pharmaceutically
acceptable salt (preferably Na). In a particularly preferred
aspects of the nitro-containing SHPs, R.sub.2 is NO.sub.2 when
R.sub.4 is H. In other preferred aspects of the nitro-containing
SHPs, R.sub.4 is NO.sub.2 when R.sub.2 is H.
[0028] Preferred agents in the classes of Compounds III and IV are
glutamyl residue-conjugated analogs of SHPs. In particularly
preferred aspects of both of these SHPs, the acid-terminal can be
the free acid or its pharmaceutically acceptable salt (preferably,
Na). In still other preferred aspects of Compounds IV, R.sub.7 can
be H or a polyglutamic acid polypeptide residue.
[0029] 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. They also exhibit at least one or more
improvements such as an enhanced anti-neoplasia activity, a reduced
toxicity, a higher water-solubility, or a more favorable
pharmacokinetic profile compared to VNP40101M. Thus, preferred
compounds according to the present invention could have a higher
therapeutic index (i.e., a better benefit/risk ratio), than
VNP40101M.
[0030] Compounds according to the present invention may be used in
pharmaceutical compositions for the treatment of cancer, as well as
a number of other conditions and/or disease states. Examples
according to the present invention may be as intermediates in the
synthesis of other compounds exhibiting biological activity as well
as standards for determining the biological activity of the present
compounds. In some applications, the present compounds may be used
for treating microbial infections, especially including viral,
bacterial, and fungal infections. These compounds 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.
[0031] A further aspect of the present invention relates to the
treatment of 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 treating neoplasia in mammals comprising
administering an effective amount of a compound as described
hereinabove to a patient suffering from cancer. The treatment of
solid malignant tumors, leukemia, and lymphomas comprising
administering to a patient an anti-tumor effective amount of one or
more these agents is a preferred embodiment of the present
invention. The treatment of various other related disease states
may also be effected using the compounds of the present invention.
This method may also be used 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.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
[0032] FIG. 1 is a pictorial representation of a suggested
mechanism of activation of VNP40101M.
[0033] FIGS. 2, 3 and 4 are pictorial representations of the
certain chemical embodiments and their proposed mechanisms of
activation according to the present invention.
[0034] FIGS. 5-9 are pictorial representations of chemical schemes
for synthesizing compounds according to the present invention.
[0035] FIGS. 10-15 are pictorial representations of experimental
results which are presented in the present application related to
the efficacy and toxicity of certain preferred embodiments
according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The following terms shall be used throughout the
specification to describe the present invention.
[0037] 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 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. Preferably, in most aspects
of the present invention, patients are human patients.
[0038] 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 an
intended result within its use in context, generally, 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 tumor, cancerous tissue, or the like, depending
upon the disease or condition treated.
[0039] The term "neoplasia" is used throughout the specification 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 initiated the new growth
cease. Neoplasia could be a distinct mass of tissue that may be
benign (benign tumor) or malignant (carcinoma). As used herein, the
term neoplasia is used to describe all cancerous disease states and
embraces or encompasses the pathological process associated with
malignant hematogenous, ascitic, and solid tumors.
[0040] The term "cancer" and the term "tumor" used in this
application is interchangeable with the term "neoplasia".
[0041] Cancer 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,
Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma,
melanoma, acute lymphocytic leukemia, acute myelogenous leukemia,
Ewing's sarcoma, small cell lung cancer, choriocarcinoma,
rhabdomyosarcoma, Wilms' Tumor, neuroblastoma, hairy cell leukemia,
mouth/pharynx, oesophagus, larynx, kidney, lymphoma, among others.
The treatment of tumors comprising administering to a patient an
anti-tumor effective amount of one or more these agents is a
preferred embodiment of the present invention.
[0042] The term "alkyl" is used throughout the specification to
describe a fully saturated hydrocarbon radical containing between
one to seven carbon units. Alkyl groups for use in the present
invention include linear, branched-chain or cyclic groups, such as
preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl,
tert-butyl, pentyl, isopentyl, hexyl, cyclohexyl, methylcyclopropyl
and methylcyclohexyl.
[0043] The term "salt" is used throughout the specification to
describe 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
cancer, the term "salt" shall mean a pharmaceutically acceptable
salt, consistent with the use of the compounds as pharmaceutical
agents.
[0044] The term "glu" or ("Glu)" used in chemical formulas
according to the present invention refers to a glutamic acid
residue or derivative within context which may be bonded on its
amino group with another carboxyl group (e.g., RCOOH) or on one of
its carboxyl groups with a hydroxyl group or an amino group.
Glutamic acid (HOOCCH(NH.sub.2)CH.sub.2- CH.sub.2COOH) has two
reactive carboxylic acid groups and a reactive amino group, which
may be used to form glutamyl residues in compounds according to the
present invention. As indicated by the chemical structure, glutamic
acid may be bonded on one of the two carboxyl groups with another
amino group (RNH.sub.2) or a hydroxyl group (ROH) to form the
corresponding .alpha.-glutamyl or .gamma.-glutamyl amide or ester
compound.
[0045] Alternatively, glutamate may be bonded on its amino group
with a carboxyl group forming an N-glutamyl amide derivative. In
the present compounds, a single glutamyl residue may be present, as
well as a polyglutamyl polypeptide residue, which is a polypeptide
formed from two or more glutamate amino acids. The term OCO(Glu) is
representative of glutamyl residue (.alpha.- or .gamma.-) which is
bonded to a free hydroxyl group to form the ester with the .alpha.-
or .gamma.-carboxylic group (preferably, the .alpha.carboxylic acid
group of glutamic acid). The term NHCO(Glu) is representative of a
glutamyl residue (.alpha.- or .gamma.-) which is bonded to a free
amine group to form the amide with the .alpha.- or
.gamma.-carboxylic group (preferably, the .alpha.-carboxylic acid
group of glutamic acid). The term "glutamyl" refers to the
glutamate amino acid which has been derivatized as an ester or
amide. .alpha.-glutamyl refers to a glutamate derivative formed at
the .alpha.-carboxyl group of glutamic acid (ester or amide).
.gamma.-glutamyl refers to a glutamate derivative formed at the
.gamma.-carboxyl group of glutamic acid (ester or amide).
N-glutamyl refers to a glutamate derivative formed at the amino
group of glutamic acid (amide). Polyglutamic acid polypeptide
residue refers to a polypeptide residue which contains more than
one glutamic acid and preferably contains exclusively glutamic
acid.
[0046] The present invention relates to compounds according to the
structure (I): 4
[0047] Where R is --CH.sub.3 or --CH.sub.2CH.sub.2Cl;
[0048] R' is C.sub.1-C.sub.7 alkyl or --CH.sub.2CH.sub.2Cl; one of
R.sub.2 or R.sub.4, but not both, is selected from
OPO.sub.3H.sub.2, NO.sub.2, OCO(Glu), NHCO(Glu) and NHR.sub.7 and
the other of R.sub.2 or R.sub.4 which is unassigned, and R.sub.3,
R.sub.5 and R.sub.6, are, independently selected from H, F, Cl, Br,
I, OH, OPO.sub.3H.sub.2, OCH.sub.3, CF.sub.3, OCF.sub.3, NO.sub.2,
CN, SO.sub.2CH.sub.3, SO.sub.2CF.sub.3, COCH.sub.3, COOCH.sub.3,
SCH.sub.3, SF.sub.5, NHR.sub.8, N(R.sub.9).sub.2, OPO.sub.3H.sub.2
and C.sub.1-C.sub.7 alkyl, with the proviso that at least two of
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are H;
[0049] R.sub.7 is H, glutamyl, preferably,
.alpha.-glutamyl(--COCH(NH.sub.- 2)CH.sub.2CH.sub.2CO.sub.2H) or a
polyglutamic acid polypeptide residue
--COCH(NHR.sub.7a)CH.sub.2CH.sub.2CO.sub.2H where R.sub.7a is
glutamyl (preferably, .alpha.-glutamyl) or a polyglutamic acid
polypeptide residue having from 1 to 50 peptide linkages,
preferably from 2 to 10 peptide linkages;
[0050] R.sub.8 is H or C.sub.1-C.sub.7 alkyl; and
[0051] R.sub.9 is CH.sub.3 or CH.sub.2CH.sub.3. Phosphoric acid and
glutamic acid (glutamyl) can be a free acid or a pharmaceutically
acceptable salt thereof.
[0052] The present compounds represent prodrug forms of
intermediates that are believed to exhibit their activity through
chloroethylation, methylation, and/or carbamoylation mechanisms, as
illustrated in FIGS. 1 to 4. The rationale for the new prodrug
design was that enzyme-activated prodrugs could be converted into
active alkylating species 1 and methyl isocyanate via a sequence of
enzyme activation and prompt fragmentation. For Compounds I,
dephosphorylation can be accomplished by the AP enzyme activation
to give intermediate 2 or 3 and subsequent benzyl group
fragmentation generated the alkylating and carbamoylating species,
as shown in FIG. 2. For Compounds II, AP-activated
dephosphorylation and NR-activated reduction can afford
intermediate 6 or 7, which can generate the alkylating and
carbamoylating species via prompt fragmentation, as shown in FIG.
3. For Compounds III and IV, cleavage of the compound can be
catalyzed by the CPG2 or CPA enzyme to appropriately substituted
phenol 10 and aromatic amine 11, and prompt fragmentation then
produces the alkylating and carbamoylating species, as illustrated
in FIG. 4.
[0053] 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 the AP enzyme. The subsequent fragmentation step is usually
rapid. It is possible that phosphate-linked prodrugs with longer
half-lives in circulation, allowing them to act as an active
alkylating species depot; or prodrugs with a different distribution
than VNP40101M, may hawe desirable properties. One approach to this
goal is to slow down the dephosphorylation step, the rate-limiting
step in the bio-activation of the phosphate-bearing SHPs 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 the P--O bond cleavage.
In addition, the subsequent fragmentation step also may be affected
by substitution at the phenyl ring with electron-releasing or
electron-withdrawing groups. Based upon these considerations, a
number of phosphate-bearing SHPs were synthesized readily in good
quantities and evaluated. The disodium salts of these prodrugs were
very soluble in water.
[0054] The compounds according to the present invention are
primarily useful for their anti-neoplastic activity, including
their activity against solid tumors. In addition, these
compositions may also find use as intermediates in the chemical
synthesis of other useful anti-neoplastic agents that are, in turn,
useful as therapeutic agents or for other purposes.
[0055] In preferred Compounds I according to the present invention,
R is --CH.sub.2CH.sub.2Cl; R' is --CH.sub.3; R.sub.2 is a phosphate
group which can be free acid or salt (preferably Na). In
particularly preferred aspects of the ortho-phosphate-bearing SHPs,
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 of the
ortho-phosphate-bearing SHPs, R.sub.5 is Cl, F or Br (preferably
Cl, F) when R.sub.3, R.sub.4 and R.sub.6 are H. Still in other
preferred aspects of the ortho-phosphate-bearing SHPs, two of
R.sub.3, R.sub.4, R.sub.5 and 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 and R.sub.6 are H.
[0056] In preferred Compounds II according to the present
invention, they are meta-phosphate-bearing nitro-containing analogs
of SHPs. The phosphate group can be free acid or salt (preferably
Na). In particularly preferred aspects of the nitro-containing
SHPs, R.sub.2 is NO.sub.2 when R.sub.4 is H. In other preferred
aspects of the nitro-containing SHPs, R.sub.4 is NO.sub.2 when
R.sub.2 is H.
[0057] In preferred Compounds III and IV according to the present
invention, they are glutamyl residue-conjugated analogs of SHPs. In
particularly preferred aspects of the both SHPs, the acid-terminal
can be free acid or salt (preferably Na). Still in preferred
aspects of Compounds IV, R.sub.7 can be H or polyglutamyl.
[0058] Compounds according to the present invention are synthesized
by the adaptation of techniques that are well known in the art and
are derived from 90CE. The synthesis of 90CE is shown in FIG. 5
(see Sartorelli, et al. U.S. Pat. No. 4,684,747, 1987, relevant
portions of which are incorporated by reference herein).
[0059] As demonstrated in FIG. 6,
2-aminocarbonyl-1,2-bis(methylsulfonyl)-- 1-(substituted)hydrazines
of Compounds 1 (19 and 20, R.ident.CH.sub.2CH.sub.2Cl) are
synthesized respectively by reacting 90CE with phosgene or its
equivalents, such as triphosgene or trichloromethyl chloroformate
(see, Majer, et al. J. Org. Chem. 1994, 59: 1937; and Pridgen, et
al. J. Org. Chem. 1989, 54: 3231), and a further condensation in
situ with an appropriate N-alkyl-N-benzylamine (15 or 16, where R'
is --CH.sub.3; R.sub.2 or R.sub.4 is a phosphate group, such as
diethyl phosphonooxy group; and unassigned groups of R.sub.2,
R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are each independently of the
indicated structure or a related alkyl group, with the proviso that
when any two of unassigned groups of R.sub.2, R.sub.3, R.sub.4,
R.sub.5 or R.sub.6 are other than H, the other two of unassigned
groups of R.sub.2, R.sub.3, R.sub.4, R.sub.5 or R.sub.6 are H).
This coupling reaction can be achieved in high yield while using
N,N,-diisopropylethylamine (DIEA) as a base and keeping the
reaction at 0.degree. C. in dry acetonitrile-dichloromethane
solvent overnight. Following deprotection of 17 or 18 with
trimethylsilyl bromide (TMSBr) (Matulic-Adamic, et al. J Org Chem.
1995, 60: 2563), the phosphate free acid form 19 or 20 is treated
with saturated sodium bicarbonate (NaHCO.sub.3) solution to afford
the corresponding disodium salt 21 or 22, respectively. Reversed
phase column chromatography may be employed for purification of the
above water-soluble SHPs (19-22).
[0060] As shown in FIG. 7, the N-benzyl-N-methylamines (15 and 16)
can be prepared from the corresponding salicylaldehydes, salicylic
acids, or 4-substituted phenols. Commercially available
salicylaldehydes react with diethyl chlorophosphate to give their
corresponding diethylphosphonoxy-benzaldehydes (23 or 24) under
mild conditions. Using sodium borohydride as a reducing agent, the
reductive amination of a respective 23 or 24 with methylamine
affords the corresponding N-benzylmethylamine (15 or 16).
Commercially available salicylic acids are first reduced to the
corresponding salicyl alcohols (25) using lithium aluminum hydride
(LAH) as a reducing agent at reflux. Selective phosphorylation
(Silverberg, et al. Tetrahedron Lett. 1996, 37: 771) of the phenol
of 25 was acheived with diethyl phosphite, carbon tetrachloride,
DIEA and catalytic amounts of 4-dimethylaminopyridine (DMAP) to
provide the benzyl alcohols (26). Pyridinium chlorochromate
(PCC)-oxidation (Kasmai, et al. J. Org. Chem. 1995, 60: 2267) is
used for the transformation of the benzyl alcohols (26) to the
benzaldehydes (24). Under Duff formylation conditions using
hexamethylene-tetraamine (HMTA) in trifluoroacetic acid (TFA) at
reflux (Lindoy, et al., Synthesis 1998, 1029), the corresponding
salicylaldehydes (27 or 28) were obtained from commercially
available 4-subtituted phenols, and then were converted to 24 and
16 via similar phosphorylation and reductive amination steps
described above. The synthesis of the appropriate
N-alkyl-N-benzylamine derivatives for use in these reaction schemes
is well known in the art and uses standard chemical techniques.
[0061] A similar synthetic strategy can also be employed for
nitro-containing SHPs of Compounds II, as shown in FIG. 8. Using
commercially available nitrobenzaldehydes as the starting material,
reaction with diethyl chlorophosphate can give their corresponding
diethylphosphonoxy-benzaldehydes (29 or 30) under mild conditions.
Using sodium borohydride as a reducing agent, the reductive
amination of a respective 29 or 30 with methylamine affords the
corresponding nitro-containing N-benzylmethylamine (31 or 32). This
coupling reaction of 31 or 32 with 90CE can be achieved in high
yield while using phosgene or its equivalents as a carbonyl
coupling agent and DIEA as a base. Following TMSBr-deprotection of
33 or 34, the phosphate free acid form 35 or 36 is treated with
saturated NaHCO.sub.3 solution to afford the corresponding disodium
salt 37 or 38, respectively. Reversed phase column chromatography
may be employed for purification of the above water-soluble
Compounds II (35-38).
[0062] Preparation of the glutamic acid substituted phenols of
Compounds III is outlined in FIG. 9. Glutamic acid di-tert-butyl
ester (39) reacted with phosgene or its equivalents, followed by
condensation in situ with 4-hydroxy-benzaldehyde at 0.degree. C.
overnight to give the glutamate-bearing benzaldehyde 40 in good
yield. Reductive amination of 40 provided the secondary amine 41 in
fair yield. Reaction of compound 41 with phosgene at 0.degree. C.,
followed by condensation in situ with 90CE gave 42 successfully.
Following a published procedure (Mann, et al. Tetrahedron 1990, 46:
5377), deprotection of 42 was easily accomplished by treatment with
formic acid to afford the free acid 43. Further treatment of 43
with saturated NaHCO.sub.3 solution or an appropriate amine can
provide a respective water-soluble glutamate 44 such as disodium
salt, triethanolamine salt, triethylamine salt, or lutidine
salt.
[0063] The synthesis of glutamyl substituted aromatic amino analogs
(Compounds IV) is illustrated in FIG. 10. Following a literature
procedure (Jones, et al. Bio-org Med Chem Lett. 2000, 10: 1987),
commercially available N-Boc-glutamic acid 5-tert-butyl ester (45)
can react with 4-aminobenzyl alcohol to form the amide 46 in high
yield, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC)
and 1-hydroxybenzotriazole (HOBT) as promoters under mild
conditions. As an alternative to prepare a N-benzyl-N-methylamine
by PCC-oxidation and reductive amination, a published procedure for
the one-pot conversion of the benzyl alcohol 46 into the secondary
amine 47 is employed which utilizes manganese dioxide in the
presence of sodium borohydride (Kanno, et al. Tetrahedron Lett.
2002, 43: 7337). Reaction of the amine 47 with phosgene at
0.degree. C., followed by condensation in situ with 90CE can
generate 48 successfully, using DIEA as promoter. Deprotection of
48 can be easily accomplished by treatment with a dilute
hydrochloric acid to afford the free acid 49.
[0064] Finally, a pharmaceutically acceptable salt, such as sodium
salt 50, can be formed after further treatment with saturated
NaHCO.sub.3 solution.
[0065] After synthesis, the crude product generally is purified by
reversed phase column chromatography and lyophylization. The
synthesis evidences that the SHPs of the present invention may be
readily converted to their corresponding phosphate salts.
Modification of the disclosed chemical synthetic methods may be
readily made by those of ordinary skill in the art in order to
provide alternative synthetic pathways to the present
compounds.
[0066] Pharmaceutical compositions based upon the present novel
chemical compounds comprise the above-described compounds in a
therapeutically effective amount for the treatment of a condition
or disease such as cancer, optionally in combination with a
pharmaceutically acceptable additive, carrier or excipient.
[0067] Certain of the compounds, in pharmaceutical dosage form, may
be used as prophylactic agents for preventing a disease or
condition from manifesting itself.
[0068] The present compounds or their derivatives can be provided
in the form of pharmaceutically acceptable salts. As used therein,
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.
[0069] Nonlimiting examples of such salts include the sodium and
potassium salts of phosphate and glutamate, among others such as
triethanolamine salt, triethylamine salt, lutidine salt, or other
pharmaceutically acceptable salts known in the art. Modifications
of the active compound can affect the solubility, pharmacokinetic
parameters 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.
[0070] 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.
Paranteral administration and in particular, intravenous or
intramuscular administration is preferred.
[0071] Pharmaceutical compositions based upon these novel chemical
compounds comprise the above-described compounds in a
therapeutically effective amount for treating 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 regiment to be employed, the pharmacokinetics of the
agent used, as well as the patient (animal or human) treated.
[0072] 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.
[0073] The routineer will take advantage of favorable
pharmacokinetic parameters of the prodrug 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 effects of the compound.
[0074] 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, intravenous, intramuscular, subcutaneous, transdermal
(which may include a penetration enhancement agent), buccal and
suppository administration, among other routes of administration,
including, in certain instances, oral administration.
[0075] 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 may comprise sterile water or aqueous
sodium chloride solution in combination with other ingredients that
aid dispersion, such as ethanol and other pharmaceutically
acceptable solvents, including DMSO, among others. Of course, where
solutions are 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.
[0076] Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can be included 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; chelafing 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).
[0077] In preparing pharmaceutical compositions in oral dosage
form, any one or more of the usual pharmaceutical media may be
used. Thus, for liquid oral preparations such as suspensions,
elixirs and solutions, suitable carriers and additives including
water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring agents and the like may be used. For solid oral
preparations such as powders, tablets, capsules, and for solid
preparations such as suppositories, suitable carriers and additives
including starches, sugar carriers, such as dextrose, mannitol,
lactose and related carriers, diluents, granulating agents,
lubricants, binders, disintegrating agents and the like may be
used. If desired, the tablets or capsules may be enteric coated or
sustained release by standard techniques.
[0078] In one embodiment, the active compounds may be prepared with
carrier that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery system. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters and
polyactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art.
[0079] 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 is 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 be used in
this aspect of the present invention.
[0080] 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 described 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. In a preferred aspect, the
compositions according to the present invention may also be used to
treat drug-resistant forms of tumors or cancer, especially those
tumors or cancers which are resistant to traditional cancer
drugs.
[0081] 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.
[0082] The present compounds are prodrug forms of reactive
intermediates. In certain pharmaceutical dosage forms, the present
compounds may be modified to other prodrug forms to take advantage
of a particular route of administration of the active compounds.
One of ordinary skill in the art will recognize how to readily
modify the present compounds to alternative prodrug forms to
facilitate delivery of active compounds to a targeted site within
the patient. The individual of ordinary skill also will take
advantage of favorable pharmacokinetic parameters of the prodrug
forms, where applicable, in delivering the present compounds to a
targeted site within the patient to maximize the intended
anti-neoplastic effect of the compound.
[0083] The amount of compound included within the therapeutically
active formulations according to the present invention is an
effective amount for treating cancer. In general, a therapeutically
effective amount of the compound according to the present invention
in dosage form usually ranges from less than about 0.05 mg/kg to
about 500 mg/kg of body weight of the patient to be treated, or
considerably more, depending upon the compound used, the tumor type
to be treated, the ability of the active compound to localize in
the tissue to be treated, the route of administration and the
pharmacokinetics of the compound in the patient. In the case of
treating cancer, the compound is preferably administered in amounts
ranging from about 0.05 mg/kg to about 250 mg/kg or more at one
time. This dosage range generally produces effective blood level
concentrations of active compound ranging from about 0.01 to about
500 micrograms per ml of blood in the patient to be treated. The
duration of treatment may be for one or more days or may last for
several months or considerably longer (years) depending upon the
disease state treated. In a more preferred embodiment, the compound
is given to the patient at doses of 0.1 mg/kg to 100 mg/kg, twice
per day to once per 14 days, for the duration of 1 week to 52
weeks.
[0084] The concentration of active compound in the patient 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 given to the patient will
be also vary with the severity of the condition to be alleviated.
It is to be further understood that for any particular patient,
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.
[0085] The active compound according to the present invention can
be 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, antiflammatories, or antiviral compounds, among other
agents.
[0086] Compounds according to the present invention may be
administered alone or in combination with other agents, especially
including other compounds of the present invention. 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 lease one
additional anti-neoplastic/anticancer agent such as
antimetabolites, Ara C, etoposide, doxorubicin, taxol, hydroxyurea,
vincristine, cytoxan (cyclophosphamide) or mitomycin C, among
numerous others, including topoisomerase I and topoisomerase II
inhibitors, such as adriamycin, topotecan, campothecin and
irinotecan, other agent such as gemcitabine and agents based upon
campothecin and cis-platin. In theory, the present compounds, which
act by a mechanism to damage DNA, will act synergistically with
compounds that act by a mechanism to reduce or prevent DNA repair.
Thus, the present compounds may be advantageously combined with any
compound which acts by a mechanism to reduce or prevent DNA repair,
especially including inhibitors of enzymes catalyzed DNA repair,
such as inhibitors of ribonucleotide reductase (RR) and inhibitors
of O.sup.6-alkylguanine-DNA alkyltransferase (AGT). 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
anticancer agents produces a synergistic (i.e., more than additive)
result which is unexpected. In another embodiment, the compounds
according to the present invention are given either simultaneously
or sequentially with antibodies (conjugated or unconjugated),
viruses, or bacteria. The antibodies, viruses, or bacteria could
carry enzymes or gene encoding enzymes that activate the compounds
described in the present invention. The enzymes include but not
limit to NR, CPG2 and CPA.
[0087] In another aspect of the present invention, the present
compositions may be used to treat tumors and/or cancer which are
resistant to one or more traditional anti-tumor/anti-cancer agents,
such as those which have been described hereinabove. In this aspect
of the invention, an effective amount of a composition is
administered to a patient suffering from a drug-resistant tumor or
cancer in order to treat the tumor or cancer. In this aspect of the
present invention, the present compositions may be administered
alone or in combination with other effective anti-tumor/anti-cancer
agents.
[0088] While not being limited by way of theory, it is believed
that the compounds according to the present invention primarily
induce their therapeutic effect in treating malignant tumors by
functional as combined chloroethylating and carbamoylating
agents.
[0089] Having generally described the invention, reference is now
made to the following specific examples that 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 that 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
[0090] 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 Bruker 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 reserved
phase column chromatography (RPCC) was packed with CAT gel (Water,
preparative C-18, 125 .ANG., 55-105 .mu.m) eluting with milli-Q
de-ionized water. Electrospray mass (ESMS) analyses were conducted
on Q-Tof manufactured by Micromass (Manchester, UK) at Keck
Laboratory of Yale University and the mass accuracy could be
<0.02%.
Examples 1-3
Preparation of Salicylaldehydes (27a, 27b, 28b) via Formylation of
4-Substituted Phenols (Duff Formylation)
[0091] General procedure. To a solution of the appropriately
substituted phenol (10.0 g) in TFA (100 mL) was added HMTA (1.1
equivalent) in small portions. The reaction solution was heated at
reflux overnight. After cooling, the solution was treated with 50%
H.sub.2SO.sub.4 solution (40 mL) for 4 h at room temperature, and
then was extracted with ether (3.times.100 mL). The combined ether
phases were washed with 5 M HCl solution and water, and then dried
over anhydrous MgSO.sub.4. After filtration, the filtrate was
evaporated and purified.
[0092] 2-Hydroxy-5-trifluoromethyl-benzaldehyde (27a). Following
the general procedure and FCC purification with 30% ethyl
acetate-hexane, 4-trifluoromethyl phenol (10.0 g, 61.7 mmol) gave
2-hydroxy-5-trifluorome- thyl-benzaldehyde 27 (3.9 g, 34%) as a
pink solid.
[0093] Rf (20% ethyl acetate-hexane): 0.47.
[0094] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 11.31 (s, 1H, OH),
9.96 (s, 1H, CHO), 7.87 (d, J=1.6 Hz, 1H, C6-H (Ph)), 7.76 (dd,
J=2.0 and 8.5 Hz, 1H, C4-H (Ph)) and 7.11 (d, J=8.8 Hz, 1H, C3-H
(Ph)).
[0095] .sup.13CNMR (75 MHz, CDCl.sub.3) .delta. 195.8, 163.8, 133.4
(d), 131.0 (d), 125.3, 122.1 (m), 119.8 and 118.6.
[0096] 4,5-Dichloro-2-hydroxy-benzaldehyde (27b) and
5,6-dichloro-2-hydroxy-benzaldehyde (28). Following the general
procedure and FCC purification with 5-10% ethyl acetate-hexane,
3,4-dichlorophenol (10.0 g, 61.1 mmol) gave
5,6-dichloro-2-hydroxy-benzaldehyde 28 (2.2 g, 19%) as a light
yellow solid.
[0097] Rf (40% ethyl acetate-hexane): 0.63.
[0098] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 11.98 (s, 1H, OH),
10.44 (s, 1H, CHO), 7.55 (d, J=9.4 Hz, 1H, C4-H (Ph)) and 6.89 (d,
J=9.3 Hz, 1H, C3-H (Ph)).
[0099] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 195.4, 162.4,
137.8 (2C), 135.6, 123.8 and 118.1. From the same reaction
continuous FCC purification with 10% ethyl acetate-hexane gave
4,5-dichloro-2-hydroxy-be- nzaldehyde 27b (1.8 g, 15%) as a light
yellow solid.
[0100] Rf (40% ethyl acetate-hexane): 0.47.
[0101] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.97 (s, 1H, OH),
9.84 (s, 1H, CHO), 7.64 (s, 1H, C3-H (Ph)) and 7.15 (s, 1H, C6-H
(Ph)).
[0102] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 194.7, 160.0,
149.9, 141.5, 134.0, 123.6 and 119.9.
Examples 4-6
Preparation of Diethylphosphonoxy-benzaldehydes (23, 24a-b) from
Phenolic Aldehydes
[0103] General procedure. To a stirred ice-cold solution of the
appropriately substituted aldehyde (10.0 g) in acetonitrile (120
mL) was added diethyl chlorophosphate (1.1 equivalent) and TEA (I
0.1 equivalent). The reaction mixture was kept at room temperature
overnight. After removal of the precipitate by filtration, the
filtrate was evaporated and dried. The crude
diethylphosphonoxy-benzaldehyde could be used without further
purification.
[0104] 4-Diethylphosphonoxy-benzaldehyde (23). Following the
general procedure, 4-hydroxybenzaldehyde (9.0 g, 73.8 mmol) was
converted to 4-diethylphosphonoxy-benzaldehyde 23 (18.9 g, 99%)
isolated as a light yellow oil.
[0105] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 9.98 (s, 1H, CHO),
7.91 (d, J=9.0 Hz, 2H, C3-H (Ph)), 7.39 (d, J=8.4 Hz, 2H, C2-H
(Ph)), 4.26 (m, 4H, CH.sub.2) and 1.38 (t, J=6.9 Hz, 6H,
CH.sub.3).
[0106] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 190.5, 155.2 (d),
133.0, 131.4, 120.3 (d), 64.7 (d) and 15.8 (d).
[0107] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 4.6.
[0108] TOF ESMS calculated for (M+H)=259.07, observed 259.10.
[0109] 2-Diethylphosphonoxy-benzaldehyde (24a). Following the
general procedure, salicylaldehyde (9.0 g, 73.8 mmol) gave 24a
(18.9 g, 99%) as a colorless oil.
[0110] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.42 (s, 1H,
CHO), 7.90 (d, J=7.7 Hz, 1H, C3-H (Ph)), 7.61 (t, J=8.0 Hz, 1H,
C5-H (Ph)), 7.48 (d, J=8.7 Hz, 1H, C6-H (Ph)), 7.31 (t, J=7.1 Hz,
1H, C4-H (Ph)), 4.27 (m, 4H, CH.sub.2) and 1.37 (t, J=7.1 Hz, 6H,
CH.sub.3).
[0111] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 188.2, 152.5 (d),
135.5, 128.5, 127.1 (d), 125.2, 120.9 (d), 64.9 (d) and 15.8
(d).
[0112] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.0.
[0113] TOF ESMS calculated for (M+H)=259.07, observed 259.07.
[0114] 5-Chloro-2-diethylphosphonoxy-benzaldehyde (24b). Following
the general procedure, 5-chlorosalicylaldehyde (10.0 g, 73.8 mmol)
gave 24b (19.4 g, 90%) as a colorless oil.
[0115] .sup.1H NMR (300 MHz, CDCl.sub.3) 610.34 (s, 1H, CHO), 7.84
(d, J=2.5 Hz, 1H, C3-H (Ph)), 7.55 (dd, J=8.8 and 2.5 Hz, 1H, C5-H
(Ph)), 7.45 (d, J=8.8 Hz, 1H, C6-H (Ph)), 4.27 (m, 4H, CH.sub.2)
and 1.38 (t, J=6.8 Hz, 6H, CH.sub.3).
[0116] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 187.0, 151.1 (d),
135.2, 131.2, 128.2 (d), 128.1, 122.6 (d), 65.2 (d) and 16.0
(d).
[0117] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.1.
[0118] TOF ESMS calculated for (M+H)=293.03, observed 293.04.
Examples 7-9
Preparation of 4-Chloro-2-diethylphosphonoxy-benzaldehyde (24c)
from 4-Chlorosalicylic Acid
[0119] 5-Chloro-2-hydroxymethyl-phenol (25). A solution of 4-chloro
salicylic acid (10.0 g, 58.0 mmol) in THF (150 mL) was treated with
LAH (1.5 equivalent) at reflux for 2 h. After cooling to ambient
temperature, the reaction solution was quenched by 1 N NaHSO.sub.4
solution (200 mL), and then extracted with ether (300 mL). After
separation, the organic layer was dried over anhydrous MgSO.sub.4,
filtered and concentrated. The dried crude product 25 (7.5 g, 81%)
was obtained as a gray solid, and was pure enough for use without
further purification.
[0120] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 9.85 (s, 1H,
Ph-OH), 7.27 (d, J=8.2 Hz, 1H, C3-H (Ph)), 6.81 (d, J=8.2 Hz, 1H,
C4-H (Ph)), 6.78 (s, 1H, C6-H (Ph)), 5.03 (m, 1H, OH) and 4.41 (d,
J=4.1 Hz, 2H, PhCH.sub.2).
[0121] .sup.13C NMR (75 MHz, DMSO-d.sub.6) 8155.0, 131.0, 128.6,
128.0, 118.5, 114.2 and 57.7.
[0122] 4-Chloro-2-diethylphosphonoxy-benzyl alcohol (26). A
solution of 25 (8.6 g, 54.8 mmol), DIEA (2.1 equivalent) and DMAP
(0.1 equivalent) in acetonitrile (200 mL) was placed in -20.degree.
C. bath. To the above cold solution was added CC14 (5.0 equivalent)
and diethyl phosphite (1.1 equivalent). The reaction solution was
kept for 2 h at room temperature. The solvent was rotary
evaporated, and the crude oil was purified by FCC with 60% ethyl
acetate-hexane to obtain 26 (10.9 g, 68%) as a light yellow
oil.
[0123] Rf (80% ethyl acetate-hexane): 0.34.
[0124] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.40 (d, J=8.2 Hz,
1H, C3-H (Ph)), 7.23 (s, 1H, C6-H (Ph)), 7.19 (d, J=8.5 Hz, 1H,
C4-H (Ph)), 4.62 (s, 2H, PhCH.sub.2), 4.24 (m, 4H, CH.sub.2) and
1.37 (t, J=7.4 Hz, 6H, CH.sub.3).
[0125] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 148.2 (d), 133.6
(d), 131.6 (d), 131.0, 125.8, 120.9 (d), 65.1 (d), 59.1 and 15.9
(d).
[0126] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 6.0.
[0127] 4-Chloro-2-diethylphosphonoxy-benzaldehyde (24c). To a
stirred solution of 26 (10.7 g, 36.3 mmol) in dichloromethane (600
mL) was added PCC in small portions over 30 min at room
temperature. The reaction was monitored by TLC. Then, the reaction
mixture was passed through a Celite Filter pad, and the filtrate
was rotary evaporated. The residual oil was purified by a silica
gel pad eluting with ethyl acetate to obtain 24d (9.5 g, 90%) as a
green oil.
[0128] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.34 (s, 1H,
CHO), 7.84 (s, 1H, C3-H (Ph)), 7.52 (s, 1H, C6-H (Ph)), 7.29 (s,
1H, C4-H (Ph)), 4.29 (in, 4H, CH.sub.2) and 1.39 (s, 6H,
CH.sub.3).
[0129] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 186.8, 152.4 (d),
140.9, 129.3, 125.4 (d), 125.3, 121.1, 64.9 (d) and 15.6 (d).
[0130] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 4.8.
[0131] TOF ESMS calculated for (M+H)=293.03, observed 293.06.
Examples 10-13
Preparation of N-(Diethylphosphonoxybenzyl)-N-methylamines (15,
16a-c)
[0132] General Procedure. To a solution of the corresponding
diethylphosphonoxy-benzaldehyde (23 or 24a-c, 10 mmol) in
dichloromethane (10 mL) was added methylamine (2 N in THF, 2.0
equivalent). The reaction solution was kept at room temperature
overnight, filtered through a silica gel pad, the filtrate was
rotary evaporated and dried in vacuum. The resulting crude oil was
dissolved in methanol (50 mL). To the above solution was added
NaBH.sub.4 (2.0 equivalent) in small portions at 0.degree. C., and
the solution was kept stirring continuously for 4 h. After
evaporation, the residue was distributed in water (50 mL) and
dichloromethane (50 mL). The aqueous phase was separated and
extracted with dichloromethane (50 mL) once. The combined organic
phases were dried over anhydrous MgSO.sub.4, filtered and
evaporated. The crude N-(diethylphosphonoxybenzyl)-N-methylamines
(15 or 16a-c) was pure enough for use without further
purification.
[0133] N-(4-Diethylpho sphonoxybenzyl)-N-methyl amine (15).
Following the general procedure, 23 (29.9 g, 116 mmol) gave 15
(22.3 g, 71%) as a yellow oil.
[0134] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.31 (d, J=8.0 Hz,
2H, C3-H (Ph)), 7.17 (d, J=8.5 Hz, 2H, C2-H (Ph)), 4.21 (m, 4H,
CH.sub.2), 3.73 (s, 2H, PhCH.sub.2), 2.42 (s, 3H, NCH.sub.3) and
1.34 (t, J=6.9 Hz, 6H, CH.sub.3).
[0135] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 149.4 (d), 135.6,
129.3, 119.5 (d), 64.2 (d), 54.5, 35.1 and 15.7 (d).
[0136] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.3.
[0137] TOF ESMS calculated for (M+H)=274.11, observed 274.11.
[0138] N-(2-Diethylphosphonoxybenzyl)-N-methyl amine (16a).
Following the general procedure, 24a (19.0 g, 73.6 mmol) gave 16a
(16.4 g, 82%) as a light yellow oil.
[0139] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.37 (d, J=7.4 Hz,
1H, C3-H (Ph)), 7.33 (d, J=7.9 Hz, 1H, C6-H (Ph)), 7.24 (t, J=7.2
Hz, 1H, C5-H (Ph)), 7.14 (t, J=7.3 Hz, 1H, C4-H (Ph)), 4.22 (m, 4H,
CH.sub.2), 3.82 (s, 2H, PhCH.sub.2), 2.44 (s, 3H, NCH.sub.3) and
1.35 (t, J=7.0 Hz, 6H, CH.sub.3).
[0140] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 148.6 (d), 130.5
(d), 130.2, 128.1, 124.8, 119.7, 64.4 (d), 49.9, 35.5 and 15.8
(d).
[0141] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.6.
[0142] TOF ESMS calculated for (M+H)=274.11, observed 274.13.
[0143] N-(4-Chloro-2-diethylphosphonoxybenzyl)-N-methyl amine
(16b). Following the general procedure, 24b (21.9 g, 74.9 mmol)
gave 16b (18.3 g, 80%) as a light yellow oil.
[0144] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.39 (d, J=2.2 Hz,
1H, C3-H (Ph)), 7.27 (d, J=7.9 Hz, 1H, C6-H (Ph)), 7.20 (dd, J=8.3
and 2.6 Hz, 1H, C5-H (Ph)), 4.23 (m, 4H, CH.sub.2), 3.78 (s, 2H,
PhCH.sub.2), 2.45 (s, 3H, NCH.sub.3) and 1.36 (t, J=7.0 Hz, 6H,
CH.sub.3).
[0145] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 147.1 (d), 132.8
(d), 130.1 (d), 129.7, 127.7, 121.1, 64.6 (d), 49.6, 35.6 and 15.9
(d).
[0146] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.6.
[0147] TOF ESMS calculated for (M+H)=308.07, observed 308.08.
[0148] N-(5-Chloro-2-diethylphosphonoxybenzyl)-N-methyl amine
(16c). Following the general procedure, 24c (23.9 g, 81.7 mmol)
gave 16c (18.9 g, 76%) as a light yellow oil.
[0149] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.35 (s, 1H, C6-H
(Ph)), 7.30 (d, J=10.4 Hz, 1H, C3-H (Ph)), 7.14 (d, J=8.4 Hz, 1H,
C4-H (Ph)), 4.24 (m, 4H, CH.sub.2), 3.78 (s, 2H, PhCH.sub.2), 2.43
(s, 3H, NCH.sub.3) and 1.37 (t, J=6.8 Hz, 6H, CH.sub.3).
[0150] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 140.9 (d), 133.0,
131.0, 129.4 (d), 125.1, 120.3 (d), 64.7 (d), 49.4, 35.5 and 15.9
(d).
[0151] .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 5.4.
[0152] TOF ESMS calculated for (M+H)=308.07, observed 308.08.
Examples 14-15
Preparation of Free Phosphoiiic Acids (19, 20a-c)
[0153] General Procedure. To a cold stirred solution of 90CE (10
mmol) in acetonitrile (40 mL) was added phosgene (20% in toluene,
1.0 equivalent) and DIEA (1.0 equivalent). The reaction solution
was kept at 0.degree. C. for 20 min. Then, to the above solution
was added a solution of the corresponding
N-(diethylphosphonoxy-benzyl)-N-methylamine (15 or 16a-c, 10 mmol)
in dichloromethane (5 mL) and DIEA (another 1.0 equivalent). The
final reaction solution was kept at 5.degree. C. overnight. After
evaporation, the residue was distributed in water (80 mL) and
dichloromethane (80 mL). The aqueous phase was separated and
extracted with dichloromethane (80 mL) twice. The combined organic
phases were dried over anhydrous MgSO.sub.4, filtered and
evaporated. The crude protected phosphates (17 or 18a-c) were
obtained as oils.
[0154] A solution of the respective diethyl-protected phosphate (17
or 18a-c, 10 mmol) in dichloromethane (60 mL) was treated with
excess TMSBr (40 mL) at 5.degree. C. overnight. After evaporation
and drying in vacuum, the crude free phosphoric acid (19 or 20a-c)
was obtained as a glassy solid.
[0155] To the crude compound (19 or 20a, 10 mmol) was added water
(about 30 mL). The suspension was stirred for 2 h at ambient
temperature, and then a minimum amount of water was added to
complete dissolution. The aqueous solution was purified by RPCC
with de-ionized water. The fractions were monitored by .sup.31P NMR
and combined. After lyophylization, the purified free phosphoric
acid (19 or 20a) was obtained as a white powder.
[0156] 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-hydrazine (90CE).
Following a published procedure (Shyam, et al. J Med Chem. 1987,
30: 2157), the reaction of 2-hydroxyethyl-hydrazine and
methanesulfonyl chloride in the presence of pyridine as a base
provided the mesylate, which subsequently reacted with lithium
chloride to result in 90CE. 90CE was obtained as a white solid
after purification by FCC with 5% methanol in dichloromethane.
[0157] Rf (50% ethyl acetate-hexane): 0.30.
[0158] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.82 (s, 1H, NH),
3.99 (t, J=6.8 Hz, 2H, ClCH.sub.2), 3.86 (t, J=5.6 Hz, 2H,
NCH.sub.2), 3.19 (s, 3H, SCH.sub.3) and 3.13 (s, 3H,
SCH.sub.3).
[0159] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 54.0, 41.0, 40.2
and 38.4.
[0160] Phosphoric acid
mono-{4-{N-[1,2-bis(methylsulfonyl)-2-(2-chloroethy-
l)-hydrazinylcarbonyl]-N-methylaminomethyl}-phenyl} ester (19).
Following the general procedure, 15 (15.4 g, 56.8 mmol) and 90CE
(14.2 g, 1.0 equivalent) gave phosphoric acid
4-{N-[1,2-bis(methylsulfonyl)-2-(2-chlor-
oethyl)-hydrazinylcarbonyl]-N-methylaminomethyl-phenyl ester
diethyl ester (17, 25.2 g, 80%). Compound 17 (11.0 g, 20.1 mmol)
was converted to 19 (3.8 g, 38%) as a white powder.
[0161] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.11 (d, J=8.1 Hz,
2H, C3-H (Ph)), 6.95 (d, J=7.5 Hz, 2H, C2-H (Ph)), 4.36 (m, 2H,
PhCH.sub.2), 3.89 (m, 2H, ClCH.sub.2), 3.67 (m, 2H, NCH.sub.2),
3.23 (s, 3H, NCH.sub.3), 2.92 (s, 3H, SCH.sub.3) and 2.87 (s, 3H,
SCH.sub.3).
[0162] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 156.6, 153.8 (d),
133.4, 132.0, 123.0 (d), 57.3, 55.7, 43.6, 42.0, 40.4 and 39.1.
[0163] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 9.
[0164] TOF ESMS calculated for (M+H)=494.01, observed 493.98.
[0165] Phosphoric acid
mono-{2-{N-[1,2-bis(methylsulfonyl)-2-(2-chloroethy-
l)-hydrazinylcarbonyl]-N-methylaminomethyl}-phenyl} ester (20a).
Following the general procedure, 16a (16.4 g, 60.5 mmol) and 90CE
(15.1 g, 1.0 equivalent) gave phosphoric acid
2-{N-[1,2-bis(methylsulfonyl)-2-(2-chlor-
oethyl)-hydrazinylcarbonyl]-N-methylaminomethyl}-phenyl ester
diethyl ester (18a, 29.7 g, 89%). Compound 18a (10.5 g, 19.2 mmol)
gave 20a (2.6 g, 27%) as a white powder.
[0166] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.1-7.2 (m, 3H,
C3-H, C5-H and C6-H (Ph)), 6.94 (m, 1H, C4-H (Ph)), 4.45 (m, 2H,
PhCH.sub.2), 3.84 (m, 2H, ClCH.sub.2), 3.64 (m, 2H, NCH.sub.2),
3.20 (s, 31H, NCH.sub.3), 2.93 (s, 3H, SCH.sub.3) and 2.92 (s, 3H,
SCH.sub.3).
[0167] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 156.8, 152.5 (d),
131.8, 131.7, 128.7 (d), 126.7, 122.5, 57.2, 51.4, 43.5, 42.1, 40.5
and 39.9.
[0168] .sup.31P NMR (121 MHz, D.sub.2O) .delta. 9.8.
[0169] TOF ESMS calculated for (M+H)=494.01, observed 494.00.
Examples 16-19
Preparation of the Disodium Salts (21, 22a-c)
[0170] General Procedure. The corresponding crude phosphoric acid
(19 or 20a-c, 10 mmol) was neutralized with an aqueous saturated
sodium bicarbonate (NaHCO.sub.3) solution (100 mL). The suspension
was stirred for 2 h at ambient temperature, and then added to a
minimum amount of water to make homogenous. The aqueous solution
was purified by RPCC with de-ionized water. The fractions were
monitored by 31P NMR and combined. After lyophylization, the
corresponding disodium salt (21 or 22a-c) was obtained as a white
powder.
[0171] Phosphoric acid
4-{N-11,2-bis(methylsulfonyl)-2-(2-chloroethyl)-hyd-
razinylcarbonyl]-N-methylaminomethyl}-phenyl ester disodium salt
(21). Following the general procedure, crude 19 (9.5 g, 19.3 mmol)
gave 21 (5.6 g, 54%) as a white powder.
[0172] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.08 (d, J=8.3 Hz,
2H, C3-H (Ph)), 6.97 (d, J=8.1 Hz, 2H, C2-H (Ph)), 4.38 (m, 2H,
PhCH.sub.2), 3.92 (m, 2H, ClCH.sub.2), 3.71 (m, 2H, NCH.sub.2),
3.26 (s, 3H, NCH.sub.3), 2.95 (s, 3H, SCH.sub.3) and 2.89 (s, 3H,
SCH.sub.3).
[0173] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 156.5, 156.1 (d),
131.7, 131.4, 122.9 (d), 57.4, 55.8, 43.7, 42.0, 40.5 and 39.0.
.sup.31P NMR (121 MHz, D.sub.2O) .delta. 14.2.
[0174] TOF ESMS calculated for (M-H)=492.01, observed 492.10.
[0175] Phosphoric acid
2-{N-[1,2-bis(methylsulfonyl)-2-(2-chloroethyl)-hyd-
razinylcarbonyl]-N-methylaminomethyl}-phenyl ester disodium salt
(22a). Following the general procedure, crude 20a (8.8 g, 17.8
mmol) gave 22a (5.7 g, 59%) as a white powder.
[0176] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.21 (d, J=8.4 Hz,
1H, C3-H (Ph)), 7.0-7.2 (m, 2H, C5-H and C6-H (Ph)), 6.86 (t, J=7.2
Hz, 1H, C4-H (Ph)), 4.52 (m, 2H, PhCH.sub.2), 3.90 (m, 2H,
ClCH.sub.2), 3.68 (m, 2H, NCH.sub.2), 3.27 (s, 3H, NCH.sub.3), 2.97
(s, 3H, SCH.sub.3) and 2.93 (s, 3H, SCH.sub.3).
[0177] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 156.7, 154.6 (d),
131.3, 130.9, 128.3 (d), 124.7, 122.4, 57.3, 51.4, 43.6, 42.0, 40.5
and 39.9.
[0178] .sup.31P NMR (121 MHz, D.sub.2O) 614.1.
[0179] TOF ESMS calculated for (M-H)=492.01, observed 492.05.
[0180] Phosphoric acid
2-{N-[1,2-bis(methylsulfonyl)-2-(2-chloroethyl)-hyd-
razinylcarbonyl]-N-methylaminomethyl}-4-chloro-phenyl ester
disodium salt (22b). Following the general procedure, crude 20b
(14.5 g, 27.6 mmol) gave 22b (8.3 g, 53%) as a white powder.
[0181] .sup.1H NMR (300 MHz, D.sub.2O) .delta.7.26 (s, 1H, C3-H
(Ph)), 7.17 (m, 1H, C5-H (Ph)), 7.08 (d, J=7.2 Hz, 1H, C6-H (Ph)),
4.48 (m, 2H, PhCH.sub.2), 3.93 (m, 2H, ClCH.sub.2), 3.72 (m, 2H,
NCH.sub.2), 3.30 (s, 3H, NCH.sub.3), 3.03 (s, 3H, SCH.sub.3) and
3.01 (s, 3H, SCH.sub.3).
[0182] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 156.8, 153.3 (d),
134.6, 130.8, 128.9, 126.7, 123.6, 57.3, 51.3, 43.6, 42.1, 40.6 and
40.1.
[0183] .sup.31P NMR (121 MHz, D.sub.2O) 614.2.
[0184] TOF ESMS calculated for (M-H)=525.96, observed 525.93.
[0185] Phosphoric acid
2-{N-[1,2-bis(methylsulfonyl)-2-(2-chloroethyl)-hyd-
razinyl-carbonyl]-N-methylaminomethyl}-5-chloro-phenyl ester
disodium salt (22c). Following the general procedure, crude 20c
(16.2 g, 30.8 mmol) gave 22c (8.9 g, 51%) as a white powder.
[0186] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.28 (s, 1H, C6-H
(Ph)), 7.03 (d, J=8.4 Hz, 1H, C3-H (Ph)), 6.87 (d, J=8.2 Hz, 1H,
C4-H (Ph)), 4.47 (m, 2H, PhCH.sub.2), 3.91 (m, 2H, ClCH.sub.2),
3.70 (m, 2H, NCH.sub.2), 3.26 (s, 3H, NCH.sub.3), 3.00 (s, 3H,
SCH.sub.3) and 2.96 (s, 3H, SCH.sub.3).
[0187] .sup.13C NMR (75 MHz, D.sub.2O) 6156.7, 155.3 (d), 135.6,
132.0, 127.0 (d), 124.5, 122.4, 57.3, 51.0, 43.7, 42.0, 40.5 and
40.0.
[0188] .sup.31P NMR (121 MHz, D.sub.2O) 614.1.
[0189] TOF ESMS calculated for (M-H)=525.96, observed 526.01.
Example 20
Preparation of the Carbamate (40)
[0190] 4-Formylphenyloxycarbonyl-glutamic acid di-tert-butyl ester
(40). To a cold stirred solution of 4-hydroxybenzyl alcohol (2.0 g,
16.9 mmol) in acetonitrile (50 mL) and dichloromethane (50 mL) was
added phosgene (20% in toluene, 1.0 equivalent) and DIEA (1.0
equivalent). The reaction solution was kept at 0.degree. C. for 30
min. Next, to the above solution was added a solution of glutamic
acid di-tert-butyl ester 39 (1.0 equivalent) in dichloromethane (50
mL) including DIEA (2.0 equivalent). The reaction mixture was kept
at 0.degree. C. overnight. Then, the mixture was treated with 0.5 N
KHSO4 solution (50 mL). After separation, the organic phase was
washed with brine (80 mL), dried over anhydrous MgSO.sub.4, rotary
evaporated and dried in vacuum. The crude carbamate 40 (6.7 g, 97%)
was obtained as a light yellow semi-solid.
[0191] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 9.97 (s, 1H, CHO),
7.89 (d, J=8.1 Hz, 2H, C3-H (Ph)), 7.32 (d, J=8.7 Hz, 2H, C2-H
(Ph)), 5.91 (d, J=7.8 Hz, 1H, NH), 4.32 (m, 1H, C.sup.1H), 2.35 (m,
2H, C.sup.2H), 2.00 (m, 2H, C.sup.3H), 1.50 and 1.46 (s,
2.times.9H, CH.sub.3).
[0192] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 191.0, 172.1,
170.6, 155.6, 153.1, 133.4, 131.1, 121.9, 82.7, 80.9, 54.1, 31.4,
28.0, 27.9 and 27.5.
[0193] TOF ESMS calculated for (M+H)=408.20, observed 408.19.
Example 21
Preparation of the N-Benzyl-N-methylamine (41)
[0194] 4-(Methyaminomethyl)phenyloxycarbonyl-glutamic acid
di-tert-butyl ester (41). A stirred solution of 40 (6.1 g, 14.9
mmol) in dichloromethane (50 mL) was treated with 2 N
methylamine-THF solution (10 mL) at 0.degree. C. overnight. After
removal of solvents, the residual oil was dissolved in methanol (80
mL) and placed in an ice-bath. To the above solution was added
sodium borohydride in small portions over 30 min. The reaction
solution was kept at 0.degree. C. for 1 hour, and solvent was then
evaporated. The residue was worked up with brine and
dichloromethane. After separation, the organic phase was dried over
anhydrous MgSO.sub.4, rotary evaporated and dried in vacuum. The
crude amine 41 (5.4 g, 78%) was obtained as a light yellow glassy
solid.
[0195] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.04 (d, J=8.3 Hz,
2H, C3-H (Ph)), 6.78 (d, J=8.1 Hz, 2H, C2-H (Ph)), 5.36 (d, J=7.4
Hz, 1H, CONH), 4.39 (m, 1H, C.sup.1H), 2.86 (d, J=4.6 Hz, 3H,
NCH.sub.3), 2.74 (d, J=4.8 Hz, 2H, NCH.sub.2), 2.34 (m, 2H,
C.sup.2H), 1.92 (m, 2H, C.sup.3H), 1.49 and 1.42 (s, 2.times.9H,
CH.sub.3).
[0196] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 172.6, 172.4,
157.9, 156.1, 128.6, 128.3, 115.6, 82.0, 80.7, 53.9, 51.5, 33.8,
31.6, 28.0, 27.9 and 27.6.
[0197] TOF ESMS calculated for (M+H)=423.25, observed 423.24.
Example 22
Preparation of the N-Benzyl-N-methylaminocarbonyl-hydrazine
(42)
[0198]
4-{N-(1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazin-2-yl-carbon-
ayl)-N-methyaminomethyl}phenyloxycarbonyl-glutamic acid
di-tert-butyl ester (42). To a cold stirred solution of 90CE (1.5
g, 5.9 mmol) in acetonitrile (30 mL) was added phosgene (20% in
toluene, 1.0 equivalent) and DIEA (1.0 equivalent). The reaction
solution was kept at 0.degree. C. for 30 min. Then, to the above
solution was added a solution of 41 (1.0 equivalent) in
acetonitrile (30 mL) including DIEA (1.0 equivalent). The reaction
mixture was kept at 0.degree. C. overnight. After evaporation of
solvent, the resulting residue was worked up with water and
dichloromethane. After separation, the organic phase was dried over
anhydrous MgSO.sub.4, rotary evaporated and dried in vacuum. The
crude N-benzyl-N-methylaminocarbonyl-hydrazine 42 (3.6 g, 87%) was
obtained as a light yellow glassy solid.
[0199] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.31 (d, J=8.0 Hz,
2H, C3-H (Ph)), 7.21 (d, J=8.2 Hz, 2H, C2-H (Ph)), 5.38 (d, J=7.5
Hz, 1H, CONH), 4.52 (bs, 1H, C.sup.1H), 3.82 (m, 2H, ClCH.sub.2),
3.67 (m, 2H, NCH.sub.2), 3.55 (s, 3H, NCH.sub.3), 3.23 and 3.14 (s,
2.times.3H, SO.sub.2CH.sub.3), 2.87 (s, 2H, NCH.sub.2Ph), 2.35 (m,
2H, C.sup.2H), 1.98 (m, 2H, C.sup.3H), 1.47 and 1.43 (s,
2.times.9H, CH.sub.3).
[0200] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 172.7, 172.1,
157.6, 148.8, 137.0, 128.9, 128.7, 121.0, 81.7, 80.5, 53.8, 53.4,
51.2, 41.9, 41.7, 41.3, 40.4, 31.5, 27.9, 27.8 and 27.4.
[0201] TOF ESMS calculated for (M+H)=699.21, observed 699.18.
Example 23
Preparation of the Glutamic Acid (43) and Its disodium Salt
(44)
[0202]
4-{N-(1,2-Bis(metliylsulfonyl)-1-(2-chloroethyl)hydrazin-2-yl-carbo-
nayl)-N-methyaminomethyl}plienyloxycarbonyl-glutaniic acid (42) and
its disodium salt (44). The crude glassy solid 42 (4.4 g, 6.4 mmol)
was treated with formic acid (200 mL) at 5.degree. C. overnight.
After frozen at -78.degree. C., the desired glutamic acid 43 was
obtained by lyophylization as sticky white solid.
[0203] Without further purification, crude 43 was treated with
saturated NaHCO.sub.3 solution (200 mL) at room temperature for 2
hours. The resulting milky mixture was purified by RPFCC with
de-ionized water. The fractions were monitored by HPLC and
combined. After lyophylization, the disodium salt 44 (0.78 g, 20%)
was obtained as a white powder.
[0204] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 7.15 (d, J=8.0 Hz,
2H, C3-H (Ph)), 7.08 (d, J=7.8 Hz, 2H, C2-H (Ph)), 4.31 (bs, 1H,
C.sup.1H), 3.83 (m, 2H, ClCH.sub.2), 3.67 (m, 2H, NCH.sub.2), 3.46
(s, 3H, NCH.sub.3), 3.16 (s, 3H, SO.sub.2CH.sub.3), 2.95 (m, 2H,
NCH.sub.2Ph), 2.70 (s, 3H, SO.sub.2CH.sub.3), 1.99 (m, 2H,
C.sup.2H) and 1.75 (m, 2H, C.sup.3H).
[0205] .sup.13C NMR (75 MHz, D.sub.2O) .delta. 185.0, 182.7, 162.0,
154.4, 150.8, 140.0, 131.0, 123.6, 59.1, 55.1, 53.5, 43.7, 42.9,
36.7, 36.4 and 31.2.
[0206] TOF ESMS calculated for (M+H)=631.05, observed 631.04, and
for (M+Na)=653.03, observed 653.03.
Example 24
[0207] Determination of Solubility and Stability in Aqueous
Solutions
[0208] The solubility of VNP40101M in water is 0.66 mg/mL at room
temperature (Krishna, et al. AAPS PharrnsciTech 2001, 2: article
14). The solubility of the newly synthesized SHPs (19, 20a, 21, and
22a) is much higher than that of VNP40101M, as shown in Table
1.
[0209] For the free phosphoric acid 19 and 20a, an excess amount of
the drug was placed in a glass vial containing 2.0 mL of water. The
vials were shaken in a Glas Col rotary apparatus at room
temperature for 24 hours. The suspension containing undissolved
drug was centrifuged; the supernatant was carefully separated and
analyzed by HPLC for drug concentration. The solubility of 19 and
-20a was found to be 293 and 46 mg/mL respectively. Aqueous
solutions of 19 and 20a were colorless. Similarly, the solubility
of the sodium salts 21 and 22a was determined visually by adding
incremental quantities of the drug to 2.0 mL of water in a glass
vial. The vials were shaken at room temperature in a Glas Col
rotary apparatus until the drug dissolved entirely. Additional
fixed quantities of drug were added and the vials shaken until
complete dissolution. This process was continued until no more drug
dissolved. Compounds 21 and 22a are highly water-soluble. Because
of limited drug supplies determination of the solubilities at
equilibrium could not be obtained. Solubilities of >0.98 and
>1.35 g/mL for 21 and 22a, respectively, were determined.
1TABLE 1 Water-solubility of the Projected SHPs at Room Temperature
Compound Water-solubility, mg/mL VNP40101M 0.66 PAP-101M (19) 293
OAP-101M (20a) 46 PAP-Na-101M (21) >980 OAP-Na-101M (22a)
>1350
[0210] The stabilities of PAP-101M (19), OAP-101M (20a), and
VNP40101M were investigated in potassium phosphate buffers (50 mM)
at pH 3, 5, 7 and 9 and at room temperature (22-25.degree. C.). One
sample was prepared at each pH for each drug; the initial drug
concentration in each sample was 50 .mu.g/mL. Each sample was
analyzed by HPLC repetitively, at various time points, to determine
the
2TABLE 2 Aqueous Stability of the Projected SHPs Half-life Compound
pH 3 pH 5 pH 7 pH 9 VNP40101M No 118 day 12.2 hr 6.9 min hydrolysis
PAP-101M (19) No No No No hydrolysis hydrolysis hydrolysis
hydrolysis OAP-101M (20a) No No No 171 day hydrolysis hydrolysis
hydrolysis
[0211] concentration of the respective drug. The first-order
kinetic half-lives of each drug were calculated. As demonstrated in
Table 2 below, the results indicate clearly that the
phosphate-bearing prodrugs (19 and 20a) were quite stable compared
to VNP40101M.
Example 25
Determination of In Vitro Bioconversion and Stability
[0212] Table 3 shows the bioconversion of compounds 19 and 20a in
the presence of AP (from bovine intestinal mucosa, Sigma) or human
plasma. Each drug, at a final concentration of approximately 50
.mu.g/mL, was incubated at 37.degree. C. in 50 mM Tris buffered
saline (pH 7.6) containing approximately 0.055 unit/mL of the
phosphatase enzyme.
3TABLE 3 In vitro Enzymatic Bioconversion and Human Plasma
Stability Half-life, min Alkaline Buffered saline phosphatase Human
plasma pH 7.6 Compound 37.degree. C. 37.degree. C. 37.degree. C.
VNP40101M Not tested 14.5 20.5 PAP-101M (19) 21.1 34.3 No
hydrolysis OAP-101M (20a) 29.0 53.3 No hydrolysis
[0213] A control sample of each drug at a final concentration of 50
.mu.g/mL in 50 mM Tris buffered saline (pH 7.6) without AP was also
incubated at 37.degree. C. Aliquots of each solution were taken
periodically; disappearance of the tested drug was determined by
HPLC.
[0214] The stability of compounds 19, 20a and VNP40101M was
evaluated in 100% human plasma (pooled mixed gender, BioChemed) at
a final concentration of 50 .mu.g/mL. Each drug (19 or 20a) was
incubated in human plasma at 37.degree. C. for a maximum of two
hours. Aliquots of the incubation mixture were taken at various
time points and extracted with acetonitrile. The extract was
separated by centrifugation and analyzed directly by HPLC. In a
similar manner, VNP40101M was incubated in human plasma at
37.degree. C. for a maximum of one hour. At various time points,
aliquots of the incubation mixture were removed and extracted with
0.5% H.sub.3PO.sub.4 in acetonitrile. The extract was separated by
centrifugation and analyzed directly by HPLC. For comparison, the
stability of each drug incubated in 50 mM Tris buffered saline (pH
7.6) instead of 100% human plasma was also determined.
[0215] It is clearly shown that (a) the projected prodrugs 19 and
20a were more stable in buffered saline and human plasma than
VNP40101M; and (b) they could be rapidly activated by alkaline
phosphatase. OAP-101M (20a) was shown to have a longer half-life
than PAP-101M (19).
Example 26
Pharmacokinetic Study in Rats
[0216] Preliminary investigations of the pharmacokinetic profiles
of prodrugs 19 and 20a were conducted in female Sprague-Dawley rats
(10 weeks old, 250 g, Charles River). Each prodrug was administered
as a single bolus intravenous (iv) injection via the jugular vein
at a dose of 50 mg per kg (mpk) of body weight. Blood samples were
collected on the day of dosing at the following time points:
pre-dose and approximately 2, 10, 30 min, 1, 2, and 24 hr, after
dosing. At each time point, approximately 0.2 mL of blood was
collected in a tube containing an anticoagulant (heparin), which
was immediately acidified by adding 0.005 mL of a 2.0 M citric acid
solution. Then, the tube was inverted 4 to 6 times and immediately
placed on ice. The blood samples were centrifuged within 30 min
after blood collection at 3,000 rpm for 10-20 min at 2-8.degree.
C., and the plasma fraction was transferred to a labeled Nunc
cryovial. The plasma samples were immediately frozen on dry ice and
stored at -20.degree. C. until HPLC-UV analysis. Animals were
euthanized with CO.sub.2 inhalation after experiments.
[0217] Bioanalytical methods were developed to quantify these
prodrugs in rat plasma using HPLC-UV at either 220 nm or 230 nm.
Each plasma sample (0.1 mL) was extracted with 0.2 mL of
acetonitrile. The extract was separated by centrifugation and
analyzed directly by HPLC-UV. HPLC calibration standards were
prepared in control rat plasma and processed as above. The standard
curve had a linear range of 1.0-50 .mu.g/mL.
[0218] Determination of 19 could not be done because of the fast
conversion or clearance of the compound from the circulation of
rats. As demonstrated in Table 4, pharmacokinetic parameters (area
under the concentration-time curve-AUC, total body clearance--Cl,
steady-state volume of distribution--Vss, maximum
concentration--Cmax, and terminal half-life-T.sub.1/2) were
calculated. Plasma half-life for 20a was approximately 14 min,
which was longer than that of 19. Comparison to VNP 101M (10 mpk of
radioactive VNP 101M was used in previous experiments) is difficult
because of the difference in doses used in two the studies.
4TABLE 4 Pharmacokinetic Parameters of 19 and 20a in Rats AUC Cl
(min*ug/ (mL/min/ Vss Cmax T.sub.1/2 Drug mL) kg) (mL/kg)
(.mu.g/mL) (min) 19 -- -- -- -- -- 20a 1312.0 37.8 145.6 238.6 14.0
VNP40101M* 325.8 .+-. 2.0 .+-. 0.91 .+-. 11.3 .+-. 20.9 .+-. 113.8
0.6 0.16 2.1 8.7 *The plasma VNP40101M level (.about.10 .mu.g/mL)
peaked at 2 min after administration of [.sup.14C]-VNP40101M. The
VNP40101M levels declined, with a half-life of .about.20 min. After
the first 2 hours, no VNP40101M could be detected in the plasma
(see, Almassian, et al. Proceedings AACR, 2001, 42: 326, article
1756).
Example 27
Evaluation of In Vivo Anti-tumor Activity
[0219] The anti-tumor effects of VNP40101M and prodrugs, including
PAP-101M (19), OAP-101M (20a), PAP-Na-101M (21), and OAP-Na-101M
(22a), were evaluated in both the B16-F10 murine melanoma and
HTB177 human lung carcinoma models. B16-FI0 melanoma cells were
implanted subcutaneously (5.times.10.sup.5 cells) into C57BU 6
mice, which were randomized into groups of ten immediately after
tumor cell implantation (Day 0). On Day 2, mice were injected
intraperitoneally with either a bolus injection of 0.1 mL PBS or
drug. The treatment was carried out weekly for four consecutive
weeks. Tumor measurement in three dimensions was determined once a
week with the formula L.times.H.times.W/2, where L, H, and W
represent length, height, and width, respectively. As shown in FIG.
11, B16-F10 tumors in the PBS control group grew exponentially,
reaching a size around 4,000 mm.sup.3 on Day 24. VNP40101M and
tested prodrugs effectively inhibited the growth of B16-F 10
melanoma. On Day 24, tumor growth was inhibited by 81% in mice
treated with 80 mg/kg of VNP40101M, and by 75 to 91% in mice
treated with equal molar doses of the prodrugs. Of all the
prodrugs, OAP-101M (20a) and OAP-Na-101M (22a) were the most
efficacious; tumor growth inhibitions were 91% and 89.5%,
respectively. The inhibitions were significantly (p<0.05) higher
than those in others groups. Tumor-bearing mice receiving OAP-101M
(20a) also survived longer than that received other drugs (FIG.
12). The toxicity of these drugs in mice was mild as determined by
body weight loss and animal appearance, as illustrated in FIG. 13.
The anti-tumor effects of these prodrugs had also been investigated
in HTB177 human lung carcinoma implanted in nu/nu CD-1 mice, as
demonstrated in FIGS. 14 and 15, and been shown that OAP-101M (20a)
and OAP-Na-101M (22a) holdout promise as well.
[0220] In short, we have shown that the water-soluble SHP prodrugs
OAP-101M (20a) and OAP-Na-101M (22a) have anti-tumor activity
against the B 16-F10 murine melanoma and HTB 177 human lung
carcinoma and the efficacy was as good as or better than VNP40
.mu.M.
SUMMARY
[0221] In summary, phosphate-bearing SHPs OAP-101M (20a) and
OAP-Na-101M (22a) possesses the following characteristics: (a)
highly water-soluble and stable in aqueous solution at pH 3 to 9;
(b) its conversion can be catalyzed by alkaline phosphatase (AP);
(c) has longer half-life in saline and in human plasma than
PAP-101M (19) and VNP40101M; (d) has better in vivo PK profiles
than PAP-101M; (e) has good anti-tumor activities against B16-F10
murine melanoma and HTB177 human lung carcinoma in mice as compared
to PAP-101M (19), PAP-Na-101M (21) and VNP40101M; and (f) its
sodium salt (22a) has even a higher water-solubility and similar
anti-tumor activity than the free acid 20a.
[0222] 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 not 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.
* * * * *