U.S. patent application number 11/473839 was filed with the patent office on 2007-01-11 for novel class of cytodifferentiating agents and histone deacetylase inhibitors, and methods of use thereof.
Invention is credited to Sandro Belvedere, Ronald Breslow, Leland Gershell, Paul A. Marks, Thomas A. Miller, Victoria M. Richon, Richard A. Rifkind.
Application Number | 20070010536 11/473839 |
Document ID | / |
Family ID | 26849835 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070010536 |
Kind Code |
A1 |
Breslow; Ronald ; et
al. |
January 11, 2007 |
Novel class of cytodifferentiating agents and histone deacetylase
inhibitors, and methods of use thereof
Abstract
The present invention provides the compound having the formula:
##STR1## wherein each of R.sup.1 and R.sub.2 is, substituted or
unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha,
pyridineamino, piperidino, t-butyl, aryloxy, arylalkyloxy, or
pyridine group; wherein A is an amido moiety, --O--, --S--, --NH--,
or --CH.sub.2--; and wherein n is an integer from 3 to 8. The
present invention also provides a method of selectively inducing
growth arrest, terminal differentiation and/or apoptosis of
neoplastic cells and thereby inhibiting proliferation of such
cells. Moreover, the present invention provides a method of
treating a patient having a tumor characterized by proliferation of
neoplastic cells. Lastly, the present invention provides a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and a therapeutically acceptable amount of the compound
above.
Inventors: |
Breslow; Ronald; (Englewood,
NJ) ; Belvedere; Sandro; (New York, NY) ;
Gershell; Leland; (New York, NY) ; Miller; Thomas
A.; (New York, NY) ; Marks; Paul A.;
(Washington, CT) ; Richon; Victoria M.; (New York,
NY) ; Rifkind; Richard A.; (New York, NY) |
Correspondence
Address: |
MINTZ LEVIN COHN FERRIS GLOVSKY & POPEO
666 THIRD AVENUE
NEW YORK
NY
10017
US
|
Family ID: |
26849835 |
Appl. No.: |
11/473839 |
Filed: |
June 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10281875 |
Oct 25, 2002 |
7126001 |
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11473839 |
Jun 22, 2006 |
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09645430 |
Aug 24, 2000 |
6511990 |
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10281875 |
Oct 25, 2002 |
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60208688 |
Jun 1, 2000 |
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60152755 |
Sep 8, 1999 |
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Current U.S.
Class: |
514/263.4 ;
514/317; 514/357; 514/370; 514/575; 544/277; 546/226; 546/304;
562/621; 562/623 |
Current CPC
Class: |
C07D 231/40 20130101;
C07C 235/74 20130101; C07F 9/4465 20130101; C07C 2601/14 20170501;
C07D 215/38 20130101; C07D 333/38 20130101; A61P 43/00 20180101;
C07C 311/03 20130101; C07D 215/40 20130101; C07C 311/06 20130101;
C07D 215/54 20130101; C07D 277/46 20130101; C07D 233/64 20130101;
A61P 35/00 20180101; C07D 249/08 20130101; C07C 327/32 20130101;
C07F 9/2458 20130101; C07C 233/07 20130101; C07C 259/06 20130101;
C07C 311/42 20130101; C07C 233/13 20130101; C07C 271/22 20130101;
C07C 237/22 20130101; C07D 233/56 20130101; C07C 237/12 20130101;
C07D 213/82 20130101; C07D 231/12 20130101; C07D 213/75
20130101 |
Class at
Publication: |
514/263.4 ;
514/317; 514/370; 514/357; 514/575; 544/277; 546/226; 546/304;
562/621; 562/623 |
International
Class: |
A61K 31/52 20060101
A61K031/52; A61K 31/445 20060101 A61K031/445; A61K 31/44 20060101
A61K031/44; C07C 259/04 20060101 C07C259/04 |
Claims
1-5. (canceled)
6. A compound having the formula: ##STR141## wherein each of
R.sub.1, and R.sub.2 is, substituted or unsubstituted, aryl,
cycloalkyl, cycloalkylamino, naphthyl, pyridineamino, piperidino,
9-purine-6-amino, thiazoleamino, hydroxyl, branched or unbranched
alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, or pyridine group;
wherein R.sub.3 is a hydroxyl, group; wherein R.sub.4, is hydrogen,
a halogen, a phenyl, or a cycloalkyl moiety; wherein A may be the
same or different and represents an amide moiety, --O--, --S--,
--NR.sub.5--, or --CH.sub.2--, where R.sub.5 is a substituted or
unsubstituted C.sub.1-C.sub.5, alkyl; and wherein n is an integer
from 3 to 10, or an enantiomer or pharmaceutically acceptable salt
thereof.
7-54. (canceled)
55. A compound having the formula: ##STR142## wherein each of
R.sub.7 is substituted or unsubstituted aryl, substituted or
unsubstituted cycloalkyl, cycloalkylamino, naphthyl, pyridineamino,
piperidino, 9-purine-6-amino, thiazoleamino, hydroxyl, branched or
unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, or
pyridinyl group; wherein R.sub.2 is -sulfonamide-R.sub.8, or
-amide-R.sub.8, wherein R.sub.8 is substituted or unsubstituted
aryl, substituted or unsubstituted cycloalkyl, cycloalkylamino,
naphthyl, pyridineamino, piperidino, 9-purine-6-amino,
thiazoleamino, hydroxyl, branched or unbranched alkyl, alkenyl,
alkyloxy, aryloxy, arylalkyloxy, or pyridinyl group; and wherein
R.sub.5 is C(O)--R.sub.6, wherein R.sub.6 is hydroxyl; and n is an
integer from 3 to 10, or an enantiomer or a pharmaceutically
acceptable salt thereof.
56. The compound of claim 55, wherein R.sub.2 is --NH--C(O)--Y or
--NH--SO.sub.2--Y, and wherein Y is selected from the group
consisting of: ##STR143##
57. The compound of claim 55, wherein R.sub.7 is selected from the
group consisting of: ##STR144##
58. A pharmaceutical composition comprising the compound of any one
of claims 6 and 55-57 and a pharmaceutically acceptable
carrier.
59. A pharmaceutically acceptable salt of the compound of claim
55.
60. The compound of claim 55, wherein --NH--R.sub.7 is selected
from the group consisting of: ##STR145##
61. The compound of claim 55, wherein R.sub.7 is a phenyl and
R.sub.2 is -amide-R.sub.8, wherein R.sub.8 is quinoline.
62. The compound of claim 6 having the formula: ##STR146## or a
pharmaceutically acceptable salt or enantiomer thereof.
63. The compound of claim 62, wherein n=5.
64. The compound of claim 6 having the formula: ##STR147## or a
pharmaceutically acceptable salt or enantiomer thereof.
65. The compound of claim 64, wherein n=5.
66. The compound of claim 6 having the formula: ##STR148## or a
pharmaceutically acceptable salt or enantiomer thereof.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/645,430, filed Aug. 24, 2000 which claims the benefit of
U.S. Provisional Application 60/208,688 filed Jun. 1, 2000 and U.S.
Provisional Application 60/152,755; filed Sep. 8, 1999.
[0002] Throughout this application various publications are
referenced by Arabic numerals within parentheses. Full citation for
these publications may be found at the end of the specification
immediately preceding the claims. The disclosures of these
publications in their entireties are hereby incorporated by
reference into this application in order to more fully describe the
state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
[0003] Cancer is a disorder in which a population of cells has
become, in varying degrees, unresponsive to the control mechanisms
which normally govern proliferation and differentiation. A recent
approach to cancer therapy has been to attempt induction of
terminal differentiation of the neoplastic cells (1). In cell
culture models differentiation has been reported by exposure of
cells to a variety of stimuli, including: cyclic AMP and retinoic
acid (2, 3), aclarubicin and other anthracylcines (4).
[0004] There is abundant evidence that neoplastic transfonnation
does not necessarily destroy the potential of cancer cells to
differentiate (1, 5, 6). There are many examples of tumor cells
which do not respond to the normal regulators of proliferation and
appear to be blocked in the expression of their differentiation
program, and yet can be induced to differentiate and cease
replicating. A variety of agents, including some relatively simple
polar compounds (5, 7-9), derivatives of vitamin D and retinoic
acid (10-12), steroid hormones (13); growth factors (6, 14),
proteases (15, 16), tumor promoters (17,18), and inhibitors of DNA
or RNA synthesis (4, 19-24), can induce various transformed cell
lines and primary human tumor explants to express more
differentiated characteristics.
[0005] Early studies by the some of present inventors identified a
series of polar compounds that were effective inducers of
differentiation in a number of transformed cell lines (8,9). One
such effective inducer was the hybrid polar/apolar compound
N,N'-hexamethylene bisacetamide (HMBA) (9), another was
suberoylanilide hydroxamic acid (SAHA) (39, 50). The use of these
compounds to induce murine erythroleukemia (MEL) cells to undergo
erythroid differentiation with suppression of oncogenicity has
proved a useful model to study inducer-mediated differentiation of
transformed cells (5, 7-9).
[0006] HMBA-induced MEL cell terminal erythroid differentiation is
a multistep process. Upon addition of HMBA to MEL cells (745A-DS19)
in culture, there is a latent period of 10 to 12 hours before
commitment to terminal differentiation is detected. Commitment is
defined as the capacity of cells to express terminal
differentiation despite removal of inducer (25). Upon continued
exposure to HMBA there is progressive recruitment of cells to
differentiate. The present inventors have reported that MEL cell
lines made resistant to relatively low levels of vincristine become
markedly more sensitive to the inducing action of HMBA and can be
induced to differentiate with little or no latent period (26).
[0007] HMBA is capable of inducing phenotypic changes consistent
with differentiation in a broad variety of cells lines (5). The
characteristics of the drug induced effect have been most
extensively studied in the murine erythroleukemia cell system (5,
25, 27, 28). MEL cell induction of differentiation is both time and
concentration dependent. The minimum concentration required to
demonstrate an effect in vitro in most strains is 2 to 3 mM; the
minimum duration of continuous exposure generally required to
induce differentiation in a substantial portion (>20%) of the
population without continuing drug exposure is about 36 hours.
[0008] There is evidence that protein kinase C is involved in the
pathway of inducer-mediated differentiation (29). The in vitro
studies provided a basis for evaluating the potential of HMBA as a
cytodifferentiation agent in the treatment of human cancers (30).
Several phase I clinical trials with HMBA have been completed
(31-36). Clinical trials have shown that this compound can induce a
therapeutic response in patients with cancer (35, 36). However,
these phase I clinical trials also have demonstrated that the
potential efficacy of HMBA is limited, in part, by dose-related
toxicity which prevents achieving optimal blood levels and by the
need for intravenous administration of large quantities of the
agent, over prolonged periods. Thus, some of the present inventors
have turned to synthesizing compounds that are more potent and
possibly less toxic than HMBA (37).
[0009] Recently, a class of compounds that induce differentiation,
have been shown to inhibit histone deacetylases. Several
experimental antitumor compounds, such as trichostatin A (TSA),
trapoxin, suberoylanilide hydroxamic acid (SAHA), and
phenylbutyrate have been shown to act, at least in part, by
inhibiting histone deacetylases (38, 39, 42). Additionally, diallyl
sulfide and related molecules (43), oxamflatin, (44), MS-27-275, a
synthetic benzamide derivative, (45) butyrate derivatives (46),
FR901228 (47), depudecin (48), and m-carboxycinnamic acid
bishydroxamide (39) have been shown to inhibit histone
deacetylases. In vitro, these compounds can inhibit the growth of
fibroblast cells by causing cell cycle arrest in the G1 and G2
phases (49-52), and can lead to the terminal differentiation and
loss of transforming potential of a variety of transformed cell
lines (49-51). In vivo, phenylbutyrate is effective in the
treatment of acute promyelocytic leukemia in conjunction with
retinoic acid (53). SAHA is effective in-preventing the formation
of mammary tumors in rats, and lung tumors in mice (54, 55).
[0010] U.S. Pat. No. 5,369,108 (41) issued to some of the present
inventors discloses compounds useful for selectively inducing
terminal differentiation of neoplastic cells, which compounds have
two polar end groups separated by a flexible chain of methylene
groups, wherein one or both of the polar end groups is a large
hydrophobic group. Such compounds are stated to be more active than
HMBA and HMBA related compounds.
[0011] However, U.S. Pat. No. 5,369,108 does not disclose that an
additional large hydrophobic group at the same end of the molecule
as the first hydrophobic group would further increase
differentiation activity about 100 fold in an enzymatic assay and
about 50 fold in a cell differentiation assay.
[0012] This new class of compounds of the present invention may be
useful for selectively inducing terminal differentiation of
neoplastic cells and therefore aid in treatment of tumors in
patients.
SUMMARY OF THE INVENTION
[0013] The subject invention provides a compound having the
formula: ##STR2## wherein R.sub.1 and R.sub.2 are the same or
different and are each a hydrophobic moiety; wherein R.sub.3 is
hydroxamic acid, hydroxylamino, hydroxyl, amino, alkylamino, or
alkyloxy group; and n is an integer from 3 to 10, or a
pharmaceutically acceptable salt thereof.
[0014] The subject invention also provides A compound having the
formula: ##STR3## wherein each of R.sub.1 and R.sub.2 is,
substituted or unsubstituted, aryl, cycloalkyl, cycloalkylamino,
naphtha, pyridineamino, piperidino, 9-purino-6-amine, thiazoleamino
group, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy,
aryloxy, arylalkyloxy, or pyridine group; wherein R.sub.3 is
hydroxamic acid, hydroxylamino, hydroxyl, amino, alkylamino, or
alkyloxy group; wherein R.sub.4 is hydrogen, a halogen, a phenyl,
or a cycloalkyl moiety; wherein A may be the same or different and
represents an amide moiety, --O--, --S--, --NR.sub.5--, or
--CH.sub.2--, where R.sub.5 is a substituted or unsubstituted
C.sub.1-C.sub.5 alkyl; and wherein n is an integer from 3 to 10, or
a pharmaceutically acceptable salt thereof.
[0015] The subject invention also provides a method of selectively
inducing terminal differentiation of neoplastic cells and thereby
inhibiting proliferation of such cells which comprises contacting
the cells under suitable conditions with an effective amount of the
aforementioned compound.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1. The effect of Compound 1 according to the subject
invention on MEL cell differentiation.
[0017] FIG. 2. The effect of Compound 1 according to the subject
invention on Histone Deacetylase 1 activity.
[0018] FIG. 3. The effect of Compound 2 according to the subject
invention on MEL cell differentiation.
[0019] FIG. 4. The effect of Compound 3 according to the subject
invention on MEL cell differentiation.
[0020] FIG. 5. The effect of Compound 3 according to the subject
invention on Histone Deacetylase 1 activity.
[0021] FIG. 6. The effect of Compound 4 according to the subject
invention on MEL cell differentiation.
[0022] FIG. 7. The effect of Compound 4 according to the
subject-invention on Histone Deacetylase 1 activity.
[0023] FIG. 8. A photoaffinity label (3H-498) binds directly to
HDAC 1.
[0024] FIG. 9. SAHA causes accumulation of acetylated histones H3
and H4 in the CWR22 tumor xenograft in mice.
[0025] FIG. 10. SAHA causes accumulation of acetylation histones H3
and H4 in peripheral blood nonnuclear cells in patients. SAHA was
administered by IV infusion daily.times.3. Samples were isolated
before (Pre), following infusion (Post) and 2 hours
after-infusion.
[0026] FIGS. 11a-11f. Show the effect of selected compounds on
affinity purified human epitope-tagged (Flag) HDAC 1.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The subject invention provides a compound having the
formula: ##STR4## wherein R.sub.1 and R.sub.2 are the same or
different and are each a hydrophobic moiety; wherein R.sub.3 is
hydroxamic acid, hydroxylamino, hydroxyl, amino, alkylamino, or
alkyloxy group; and n is an integer from 3 to 10; or a
pharmaceutically acceptable salt of the compound.
[0028] In the foregoing compound each of R.sub.1 and R.sub.2 is
directly attached or through a linker, and is, substituted or
unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha,
pyridineamino, piperidino, 9-purine-6-amine, thiazoleamino group,
hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy,
arylalkyloxy, or pyridine group.
[0029] Where a linker is used, the linker may be an amide moiety,
--O--, --NH--, or --CH.sub.2--.
[0030] According to this invention, n may be 3-10, preferably 3-8,
more preferably 3-7, yet more preferably 4, 5 or 6, and most
preferably 5.
[0031] In another embodiment of the invention, the compound has the
formula: ##STR5## wherein each of R.sub.1 is, substituted or
unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha,
pyridineamino, piperidino, 9-purine-6-amine, thiazoleamino group,
hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy,
arylalkyloxy, or pyridine group. R.sub.2 may be -amide-R.sub.5,
wherein R.sub.5 is, substituted or unsubstituted, aryl, cycloalkyl,
cycloalkylamino, naptha, pyridineamino, piperidino,
9-purine-6-amine, thiazoleamino group, hydroxyl, branched or
unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, or
pyridine group.
[0032] In a further embodiment of the invention the compound has
the formula: ##STR6## wherein each of R.sub.1 and R.sub.2 is,
substituted or unsubstituted, aryl, cycloalkyl, cycloalkylamino,
naphtha, pyridineamino, piperidino, 9-purine-6-amine, thiazoleamino
group, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy,
aryloxy, arylalkyloxy, or pyridine group; wherein R.sub.3 is
hydroxamic acid, hydroxylamino, hydroxyl, amino, alkylamino, or
alkyloxy group; wherein R.sub.4 is hydrogen, a halogen, a phenyl,
or a cycloalkyl moiety; wherein A may be the same or different and
represents an amide moiety, --O--, --S--, --NR.sub.5--, or
--CH.sub.2--, where R.sub.5 is a substituted or unsubstituted
C.sub.1-C.sub.5 alkyl; and wherein n is an integer from 3 to 10, or
a pharmaceutically acceptable salt thereof.
[0033] In another embodiment the compound has the formula:
##STR7##
[0034] In yet another embodiment, the compound has the formula:
##STR8##
[0035] In a further embodiment, the compound has the formula:
##STR9## wherein each of R.sub.1 and R.sub.2 is, substituted or
unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha,
pyridineamino, piperidino, t-butyl, aryloxy, arylalkyloxy, or
pyridine group; and wherein n is an integer from 3 to 8.
[0036] The aryl or cycloalkyl group may be substituted with a
methyl, cyano, nitro, trifluoromethyl, amino, aminocarbonyl,
methylcyano, chloro, fluoro, bromo, iodo, 2,3-difluoro,
2,4-difluoro, 2,5-difluoro, 3,4-difluoro, 3,5-difluoro,
2,6-difluoro, 1,2,3-trifluoro, 2,3,6-trifluoro, 2,4,6-trifluoro,
3,4,5-trifluoro, 2,3,5,6-tetrafluoro, 2,3,4,5,6-pentafluoro, azido,
hexyl, t-butyl, phenyl, carboxyl, hydroxyl, methoxy, phenyloxy,
benzyloxy, phenylaminooxy, phenylaminocarbonyl, methyoxycarbonyl,
methylaminocarbonyl, dimethylamino, dimethylaminocarbonyl, or
hydroxylaminocarbonyl group.
[0037] In a further embodiment, the compound has the formula:
##STR10## or an enantiomer thereof.
[0038] In a yet further embodiment, the compound has the formula:
##STR11## or an enantiomer thereof.
[0039] In a further embodiment, the compound has the formula:
##STR12## or an enantiomer thereof.
[0040] In a yet further embodiment, the compound has the formula:
##STR13## or an enantiomer thereof.
[0041] In a further embodiment, the compound has the formula:
##STR14## or an enantiomer thereof.
[0042] In a yet further embodiment, the compound has the formula:
##STR15## or an enantiomer thereof.
[0043] In a yet further embodiment, the compound has the formula:
##STR16## or an enantiomer thereof.
[0044] In a further embodiment, the compound has the formula:
##STR17## or an enantiomer thereof.
[0045] In a further embodiment, the compound has the formula:
##STR18## or an enantiomer thereof.
[0046] In a yet further embodiment, the compound has the formula:
##STR19## or an enantiomer thereof.
[0047] In a further embodiment, the compound has the formula:
##STR20## or an enantiomer thereof.
[0048] This invention is also intended to encompass enantiomers and
salts of the compounds listed above.
[0049] In a further embodiment, the compound has the formula:
##STR21## wherein R.sub.1 and R.sub.2 are the same or different and
are each a hydrophobic moiety: wherein R.sub.5' is --C(O)--NHOH
(hydroxamic acid), --C(O)--CF.sub.3 (trifluoroacetyl),
--NH--P(O))H--CH.sub.3, --SO.sub.2NH.sub.2 (sulfonamide),
--SH(thiol), --(O)--R.sub.6, wherein R.sub.6 is hydroxyl, amino,
alkylamino, or alkyloxy group; and n is an integer from 3 to 10, or
a pharmaceutically acceptable salt thereof.
[0050] In the foregoing compound, each of R.sub.1 and R.sub.2 may
be directly attached or through a linker, and is, substituted or
unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha,
pyridineamino, piperidino, 9-purine-6-amine, thiazoleamino group,
hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy,
arylalkyloxy, or pyridine group.
[0051] The linker may be an amide moiety, --O--, --S--, --NH--, or
--CH.sub.2--.
[0052] In another embodiment, the compound has the formula:
##STR22## wherein each of R.sub.7 is, substituted or unsubstituted,
aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino,
piperidino, 9-purine-6-amine, thiazoleamino group, hydroxyl,
branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy,
arylalkyloxy, or pyridine group.
[0053] In the foregoing compound, R.sub.2 may be
-sulfonamide-R.sub.6, or -amide-R.sub.8 wherein R.sub.8 is,
substituted or unsubstituted, aryl, cycloalkyl, cycloalkylamino,
naphtha, pyridineamino, piperidino, 9-purine-6-amine, thiazoleamino
group, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy,
aryloxy, arylalkyloxy, or pyridine group.
[0054] The R.sub.2 may be --NH--C--(O)--Y, --NH--SO.sub.2--Y,
wherein Y is selected from the group consisting of: ##STR23##
[0055] The R.sub.7 may be selected from the group consisting of the
following and designated R.sub.7: ##STR24##
[0056] In yet another embodiment, the compound has the formula:
##STR25## wherein R.sub.1 and R.sub.2 are the same or different and
are each a hydrophobic moiety: wherein R.sub.5' is --C(O)--NHOH
(hydroxamic acid), --C(O)--CF.sub.3 (trifluoroacetyl),
--NH--P(O))H--CH.sub.3, --SO.sub.2NH.sub.2 (sulfonamide),
--SH(thiol), --C(O)--R.sub.6, wherein R.sub.6 is hydroxyl, amino,
alkylamino, or alkyloxy group; and wherein L is a linker consisting
of --(CH.sub.2)--, --C(O)--, --S--, --O--, --(CH.dbd.CH)--,
-phenyl-, or -cycloalkyl-, or any combination thereof, or a
pharmaceutically acceptable salt thereof.
[0057] L may also be a linker consisting of --(CH.sub.2).sub.n--,
--C(O)--, --S--, --O--, --(CH.dbd.CH).sub.m--, -phenyl-, or
-cycloalkyl-, or any combination thereof, wherein n is an integer
from 3 to 10, and m is an integer from 0 to 10,
[0058] In the foregoing compound, n may be from 4-7, and m is from
0-7. Preferably n is 5 or 6, most preferably n is 6. Preferably m
is from 1-6, more preferably m is 2-5, most preferably m is 3 or
4,
[0059] In the compound, each of R.sub.1 and R.sub.2 may be directly
attached or through a linker, and is, substituted or unsubstituted,
aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino,
piperidino, 9-purine-6-amine, thiazoleamino group, hydroxyl,
branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy,
arylalkyloxy, pyridine group.
[0060] The linker may be an amide moiety, --O--, --S--, --NH--, or
--CH.sub.2--.
[0061] This invention is also intended to encompass enantiomers,
salts and pro-drugs of the compounds disclosed herein.
[0062] In another embodiment the compound may have the formula:
##STR26## wherein L is a linker selected from the group consisting
of --(CH.sub.2)--, --(CH.dbd.CH)--, -phenyl-, -cycloalkyl-, or any
combination thereof; and wherein each of R.sub.1 and R.sub.2 are
independently substituted or unsubstituted, aryl, cycloalkyl,
cycloalkylamino, naphtha, pyridineamino, piperidino,
9-purine-6-amine, thiazoleamino group, hydroxyl, branched or
unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, or
pyridine group.
[0063] In a preferred embodiment, the linker L comprises the moiety
##STR27##
[0064] In another preferred embodiment, the compound has the
formula: ##STR28##
[0065] Any of the disclosed compounds can be formed into a
pharmaceutical composition together with a pharmaceutically
acceptable carrier.
[0066] Any of the compounds can also be formed into a
pharmaceutically acceptable salt of the compound using well known
pharmacological techniques.
[0067] A prodrug of any of the compounds can also be made using
well known pharmacological techniques.
[0068] Any of the compounds can be used in a method of inducing
differentiation of tumor cells in a tumor comprising contacting the
cells with an effective amount of the compound so as to thereby
differentiate the tumor cells.
[0069] Any of the compounds can also be used in a method of
inhibiting the activity of histone deacetylase comprising
contacting the histone deacetylase with an effective amount of the
compound so as to thereby inhibit the activity of histone
deacetylase.
[0070] This invention, in addition to the above listed compounds,
is further intended to encompass the use of homologs and analogs of
such compounds. In this context, homologs are molecules having
substantial structural similarities to the above-described
compounds and analogs are molecules having substantial biological
similarities regardless of structural similarities.
[0071] In a further embodiment, the subject invention provides a
pharmaceutical composition comprising a pharmaceutically effective
amount of any one of the aforementioned compounds and a
pharmaceutically acceptable carrier.
[0072] In a yet further embodiment, the subject invention provides
a method of selectively inducing growth arrest, terminal
differentiation and/or apoptosis of neoplastic cells and thereby
inhibiting proliferation of such cells which comprises contacting
the cells under suitable conditions with an effective amount of any
one of the aforementioned compounds.
[0073] The contacting should be performed continuously for a
prolonged period of time, i.e. for at least 48 hours, preferably
for about 4-5 days or longer.
[0074] The method may be practiced in vivo or in vitro. If the
method is practiced in vitro, contacting may be effected by
incubating the cells with the compound. The concentration of the
compound in contact with the cells should be from about 1 nM to
about 25 mM, preferably from about 20 nM to about 25 mM, more
preferably from about 40 nM to 100 .mu.M, yet more preferably from
about 40 nM to about 200 nM. The concentration depends upon the
individual compound and the state of the neoplastic cells.
[0075] The method may also comprise initially treating the cells
with an antitumor agent so as to render them resistant to an
antitumor agent and subsequently contacting the resulting resistant
cells under suitable conditions with an effective amount of any of
the compounds above, effective to selectively induce terminal
differentiation of such cells.
[0076] The present invention also provides a method of treating a
patient having a tumor characterized by proliferation of neoplastic
cells which comprises administering to the patient an effective
amount of any of the compounds above, effective to selectively
induce growth arrest, terminal differentiation and/or apoptosis of
such neoplastic cells and thereby inhibit their proliferation.
[0077] The method of the present invention is intended for the
treatment of human patients with tumors. However, it is also likely
that the method would be effective in the treatment of tumors in
other mammals. The term tumor is intended to include any cancer
caused by the proliferation of neoplastic cells, such as prostate
cancer, lung cancer, acute leukemia, multiple myeloma, bladder
carcinoma, renal carcinoma, breast carcinoma, colorectal carcinoma,
neuroblastoma or melanoma.
[0078] Routes of administration for the compound of the present
invention include any conventional and physiologically acceptable
route, such as, for example, oral, pulmonary, parenteral
(intramuscular, intraperitoneal, intravenous (IV) or subcutaneous
injection), inhalation (via .a fine powder formulation or a fine
mist), transdermal, nasal, vaginal, rectal, or sublingual routes of
administration and can be formulated in dosage forms appropriate
for each route of administration.
[0079] The present invention also provides a pharmaceutical
composition comprising a pharmaceutically acceptable carrier, such
as sterile pyrogen-free water, and a therapeutically acceptable
amount of any of the compounds above. Preferably, the effective
amount is an amount effective to selectively induce terminal
differentiation of suitable neoplastic cells and less than an
amount which causes toxicity in a patient.
[0080] The present invention provides the pharmaceutical
composition above in combination with an antitumor agent, a
hormone, a steroid, or a retinoid.
[0081] The antitumor agent may be one of numerous chemotherapy
agents such as an alkylating agent, an antimetabolite, a hormonal
agent, an antibiotic, colchicine, a vinca alkaloid, L-asparaginase,
procarbazine, hydroxyurea, mitotane, nitrosoureas or an imidazole
carboxamide. Suitable agents are those agents which promote
depolarization of tubulin. Preferably the antitumor agent is
colchicine or a vinca alkaloid; especially preferred are
vinblastine and vincristine.
[0082] In embodiments where the antitumor agent is vincristine, an
amount is administered to render the cells are resistant to
vincristine at a concentration of about 5 mg/ml. The administration
of the agent is performed essentially as described above for the
administration of any of the compounds. Preferably, the
administration of the agent is for a period of at least 3-5 days.
The administration of any of the compounds above is performed as
described previously.
[0083] The pharmaceutical composition may be administered daily in
2-6 hour infusions for a period of 3-21 days, for example, daily in
a 4 hour infusion for a period of 5 days.
[0084] This invention will be better understood from the
Experimental Details which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims which follow thereafter.
Experimental Details
[0085] Examples 1-5 show the synthesis of substituted
L-.alpha.-aminosuberic hydroxamic acids according to the subject
invention, and Examples 6 and 7 show the effects of compounds 1-5
on MEL cell differentiation and Histone Deacetylase activity.
EXAMPLE 1
Synthesis of Compound 1
[0086] N-Boc-.omega.-methyl-(L)-.alpha.-aminosuberate, Boc-Asu
(OMe) was prepared according to a published procedure (40).
("Boc"=t-butoxycarbonyl; "Asu"=.alpha.-aminosuberate (or
.alpha.-aminosuberic acid))
[0087] N-Cbz-.omega.-t-butyl-(L)-.alpha.-aminosuberate,
dicyclohexylamine salt was purchased from Research Plus, Bayonne,
N.J.
N-Boc-.omega.-methyl-(L)-.alpha.-aminosuberateanilide,
Boc-Asu(OMe)-NHPh
[0088] ##STR29##
[0089] N-Boc-.omega.-methyl-(L)-.alpha.-aminosuberate (493 mg, 1.63
mmoles) was dissolved under Ar in 7 mL of dry CH.sub.2Cl.sub.2. EDC
(470 mg, 2.45 mmoles) was added, followed by aniline (230 .mu.L,
2.52 mmoles). The solution was stirred at room temperature for 2 h
30 min, then washed with dilute HCl (pH 2.4, 2.times.5 mL), sat.
NaHCO.sub.3 (10 mL), and H.sub.2O (2.times.10 mL). The product was
purified by column chromatography (Silica gel, Hexanes:AcOEt
3.5:1). The isolated yield was 366 mg (60%).
[0090] .sup.1H-NMR and Mass Spectroscopy were consistent with the
product.
N-Benzoyl-.omega.-methyl-(L)-.alpha.-aminosuberateanilide,
PhCOHN-Asu(OMe)-NHPh
[0091] ##STR30##
[0092] 90 mg of
N-Bloc-.omega.-methyl-(L)-.alpha.-aminosuberateanilide (0.238
mmoles) were treated with 3.2 mL of 25% trifluoroacetic acid (TFA)
CH.sub.2Cl.sub.2 for 30 min. The solvent was removed and the
residue left under high vacuum for 12 h. It was dissolved under Ar
in 3 mL of dry CH.sub.2Cl.sub.2 and
benzotriazole-1-yloxy-tris-pyrrolidinophosphonium
hexafluorophosphate (PyBOP) (149 mg, 0.286 mmoles), benzoic acid
(44 mg, 0.357 mmoles) and diisopropylethylamine (114 .mu.L, 0.655
mmoles). The solution was stirred at room temperature for 1 h. The
product was purified by column chromatography (Silica gel,
Hexanes:AcOEt 3:1-2:1) as a white solid: 75 mg, 82%.
[0093] .sup.1H-NMR and Mass Spectroscopy were consistent with the
product.
[0094] The foregoing coupling reaction was also successfully
accomplished using 1-(3 dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDC) as a reagent.
N-Benzoyl-(L)-.alpha.-aminosuberoylanilide,
PhCONH-Asu(OH)--NHPh
[0095] ##STR31##
[0096] 75 mg (0.196 mmoles) of N-benzoyl-aminosuberateanilide were
stirred for 6 h at 0.degree. C. in 1M NaOH:THF:MeOH 1:1:1. After
complete disappearance of the starting material, the solution was
neutralized (1M HCl) and extracted with AcOEt. The organic phase
was collected and dried. Solvent removal yielded the product as a
white solid: 67 mg, 93%.
[0097] .sup.1H-NMR and Mass Spectroscopy were consistent with the
product.
N-Benzoyl-(L)-.alpha.-aminosuberoylanilide-.omega.-hydroxamic acid,
PhCONH-Asu (NHOH)--NHPh
[0098] ##STR32##
[0099] To a suspension of 26 mg of
N-benzoyl-.omega.-methyl-(L)-.omega.-aminosuberateanilide (I2) in 1
mL of dry CH.sub.2Cl.sub.2 was added 58 mg of H.sub.2NOTBDPS
(H.sub.2NO-t-butyldiphenylsilyl) followed by 22 mg of EDC. The
reactionwas stirred at room temperature for 4 h. The intermediate
protected hydroxamic acid was purified by column chromatography
(silica gel, CH.sub.2Cl.sub.2:MeOH 100:0-98-2). It was deprotected
by treatment with 5% TFA in CH.sub.2Cl.sub.2 for 1 h 30 min. The
product was precipitated from acetone-pentane.
[0100] .sup.1H-NMR (d.sub.6-DMSO, 500 MHz) .delta.=10.29 (s, 1H),
8.53 (d, 1H), 7.90 (d, 2H), 7.60 (d, 2H), 7.53 (m, 1H), 7.46 (t,
2H), 7.28 (t, 2H), 7.03 (t, 2H), 4.53 (q, 1H), 1.92 (t, 2H), 1.78
(m, 2H), 1.50-1.25 (m, 6H).
[0101] ESI-MS: 384 (M+1), 406 (M+Na), 422 (M+K)
EXAMPLE 2
Synthesis of Compound 2
N-Nicotinoyl-(L)-.alpha.-aminosuberoylanilide-.omega.-hydroxamic
acid, C.sub.5H.sub.4NCO-Asu (NHOH)--NHPh
[0102] ##STR33##
[0103] It was prepared from
N-Boc-.omega.-methyl-L-.alpha.-aminosuberate following the same
procedure used for the benzoyl analog. Yields and chromatographic
behaviour were comparable.
[0104] .sup.1H-NMR (d.sub.6-DMSO, 500MHz) .delta.=10.30 (s, 1H),
10.10 (s, 1H), 9.05 (m, 1H), 8.80 (m, 1H), 8.71 (m, 1H), 8.24 (m,
1H), 7.60 (m, 2H), 7.30 (m, 2H), 7.04 (m, 1H), 4.56 (m, 1H), 1.93
(t, 2H), 1.79 (m, 2H), 1.55-1.30 (m, 6H). ESI-MS: 385 (M+1), 407
(M+Na)
EXAMPLE 3
Synthesis of Compound 3
N-benzyloxycarbonyl-.omega.-t-butyl-(L)-aminosuberic acid,
N-Cbz-(L)-Asu (OtBu)-OH
[0105] ##STR34##
[0106] N-Cbz-(L)-Asu (OtBu)-OH, dicyclohexylamine salt (100 mg,
0.178 mmol) was partitioned between 1 M HCl (5 mL) and EtOAc (10
mL). The organic layer was removed, and the aqueous portion washed
with EtOAc (3.times.3 mL). The organic fractions were combined,
washed with brine (1.times.2 mL), and dried (MgSO.sub.4). The
mixture was filtered and concentrated to a colorless film (67 mg,
0.176 mmol, 99%). This compound was used immediately in the next
step.
N-benzyloxycarbonyl-.omega.-t-butyl-(L)-.alpha.-aminosuberateanilide,
N-Cbz-(L)-Asu (OtBu)-NHPh
[0107] ##STR35##
[0108] N-Cbz-(L)-Asu (OtBu)-OH (67 mg, 0.176 mmol) was dissolved in
dry CH.sub.2Cl.sub.2 (2.5 mL). Aniline (17 .mu.L, 0.187 mmol),
PyBOP (97 mg, 0.187 mmol), and iPr.sub.2NEt (46 .mu.L, 0.266 mmol)
were added and the mixture stirred for 2 h. The reaction was
complete as indicated by TLC. The mixture was diluted with EtOAc (5
mL) and water (5 mL), and the layers separated. The aqueous portion
was washed with EtOAc (3.times.3 mL) and the organic fractions
combined. This solution was washed with 1 M HCl (1.times., 2 mL)
and brine (1.times.2 mL), dried (MgSO.sub.4), filtered, and
concentrated to a crude oil. This was passed through a plug of
silica gel (30% EtOAc/hexanes) to remove baseline impurities,
affording the compound (76 mg, 0.167 mmol, 94%).
[0109] .sup.1H NMR (CDCl.sub.3, 400 MHz, no TMS) .delta. 8.20 (br
s, 1H), 7.47 (d, 2H), 7.32 (m, 5H), 7.28 (t, 2H), 7.08 (t, 1H),
5.39 (d, 1H), 5.10 (m, 2H), 4.26 (m, 1H), 2.18 (t, 2H), 1.93 (m,
1H), 1.67 (m, 1H), 1.55 (m, 3H), 1.42 (s, 9H), 1.36 (m, 3H).
N-benzyloxycarbonyl-(L)-.alpha.-aminosuberateanilide, N-Cbz-(L)-ASU
(OH)--NHPh
[0110] ##STR36##
[0111] N-Cbz-(L)-ASU (OtBu)-anilide (76 mg, 0.167 mmol) was
dissolved in dry CH.sub.2Cl.sub.2 (5 mL) and TFA (0.5 mL) added
dropwise. The reaction was complete by TLC after 3 h. The mixture
was concentrated in vacuo to give the title compound (80 mg,
crude). This compound was taken on without purification to the next
step.
[0112] .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 11.93 (br s,
1H), 9.99 (br s, 1H), 7.57 (m, 3H), 7.34 (m, 5H), 7.29 (t, 2H),
7.03 (t, 1H), 5.02 (m, 2H), 4.11 (m, 1H), 2.17 (t, 2H), 1.46 (m,
2H).
N-benzyloxycarbonyl-(L)-.alpha.-aminosuberateanilide-.omega.-hydroxamic
acid, N-Cbz-(L)-Asu (NH--OH)--NHPh
[0113] ##STR37##
[0114] N-Cbz-(L)-Asu (OH)-anilide (80 mg, crude) and
O-t-butyldiphenylsilyl-hydroxylamine (60 mg, 0.221 mmol) were
dissolved in CH.sub.2Cl.sub.2 (4 mL). To this was added PyBOP (125
mg, 0.241 mmol) and iPr.sub.2NEt (52 .mu.L, 0.302 mmol) and stirred
overnight. TLC indicated reaction completion. The mixture was
concentrated in vacuo and then passed through a plug of silica gel
(50% EtOAc/hexanes) to remove baseline impurities. Evaporation of
volatiles afforded 107 mg of material which was then dissolved in
dry CH.sub.2Cl.sub.2 (5 mL) and TFA (0.25 mL) was added. Monitoring
by TLC indicated completion after 1.5 h. Concentrated in vacuo to
remove all volatiles. The residue was taken up in EtOAC (3 mL), and
then hexanes was added slowly to result in the precipitation of a
white gel. The supernatant was removed, and the precipitate washed
with hexanes (3.times.2 mL). This material was taken to dryness
under reduced pressure, to afford the title compound (40 mg, 0.097
mmol, 59%).
[0115] .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 10.32 (s, 1H),
10.00 (s, 1H), 8.64 (br s, 1H), 7.57 (m, 3H), 7.37 (m, 5H), 7.30
(t, 2H), 7.04 (t, 1H), 5.02 (m, 2H), 4.12 (m, 1H), 1.93 (t, 2H),
1.62 (m, 2H), 1.45 (m, 2H), 1.29 (m, 4H); ESI-MS 414 (M+1).
EXAMPLE 4
Synthesis of Compound 4
N-benzyloxycarbonyl-(L)-.alpha.-aminosuberoyl-8-quinolinamide-.omega.-hydr-
oxamic acid
[0116] ##STR38##
[0117] Prepared in similar manner to compound 3.
[0118] .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 10.45 (s, 1H),
10.31 (s, 1H), 8.85 (dd, 1H), 8.63 (dd, 1H), 8.42 (dd, 1H), 8.13
(dd, 1H), 8.68 (m, 2H), 7.60 (t, 1H), 7.37 (m, 2H), 7.28 (m, 2H),
5.10 (m, 2H), 4.24 (m, 1H), 1.93 (t, 2H), 1.85 (m, 1H), 1.70 (m,
1H), 1.50 (m, 2H), 1.42 (m, 2H), 1.30 (m, 2H); ESI-MS 465
(M+1).
EXAMPLE 5
Synthesis of Compound 5
N-Benzoyl-(L)-.alpha.-aminosuberoyl-8-quinolinamide-.omega.-hydroxamic
acid
[0119] ##STR39##
[0120] A sample of the N-Cbz-.omega.-t-butyl
L-.alpha.-aminosuberoyl-8-quinolinamide (90 mg, 0.178 mmoles) was
obtained from the previous synthesis. The Cbz group was removed by
hydrogenation in MeOH on 5% Pd on C. The resulting free amine was
coupled with benzoic acid using EDC in dry CH.sub.2Cl.sub.2 (69%
over the two steps). After TFA deprotection of the t-butyl ester,
the usual coupling with H.sub.2NOTBDPS followed by deprotection
afforded the desired hydroxamic acid.
[0121] .sup.1H-NMR (d.sub.6-DMSO, 500MHz) .delta.=10.55 (s, 1H),
10.30 (s, 1H), 9.03 (m, 1H), 8.78 (m, 1H), 8.62 (m, 1H), 8.40 (m,
1H), 7.97 (m, 2H), 7.67-7.46 (m, 6H), 4.66 (m, 1H), 1.94 (t, 2H),
1.87 (m, 1H), 1.80-1.20 (m, 7H). ESI-MS : 435 (M+1).
EXAMPLE 6
Synthesis of Compound with Inverted Amide Group
[0122] A compound having the following formula: ##STR40## is
synthesized by treating a malonic ester: ##STR41## with a base, and
then adding: ##STR42## where X is a halogen, to form: ##STR43##
from which R is removed by reaction with an amine and a
carbodiimide reagent to form: ##STR44## from which R' is removed
and converted to hydroxamic acid (NHOH) as in the previous
examples.
[0123] In the foregoing scheme, R may be t-butyl, removed with
trifluoroacetic acid; R' may be methyl, removed with a base or LiI;
and each R'' may be the same or different, depending on the reagent
used.
EXAMPLE 7
Effect of Compound 1
(N-Benzoyl-(L)-.alpha.-aminosuberoylanilide-.omega.-hydroxamic
acid, PhCONH-Asu (NHOH)--NHPh) on MEL Cell Differentiation and
Histone Deacetylase Activity
[0124] Murine Erythroleukemia (MEL) Cell Differentiation.
[0125] The MEL cell differentiation assay was used to assess the
ability of Compound 1 to induce terminal differentiation. MEL cells
(logarithmically dividing) were cultured with the indicated
concentrations of Compound 1. Following a 5-day culture period,
cell growth was determined using a Coulter Counter and
differentiation was determined microscopically using the benzidine
assay to determine hemoglobin protein accumulation on a per cell
basis.
[0126] It was observed, as shown in FIG. 1, that Compound 1 (200
nM) is able to induce MEL cell differentiation.
[0127] Histone Deacetylase (HDAC) Enzymatic Activity.
[0128] The effect of Compound 1 on affinity purified human
epitope-tagged (Flag) HDAC 1 was assayed by incubating the enzyme
preparation in the absence of substrate on ice for 20 min with the
indicated amounts of Compound 1. Substrate ([.sup.3H]acetyl-labeled
murine erythroleukemia cell-derived histone) was added and the
samples were incubated for 20 min at 37.degree. C. in a total
volume of 30 .mu.l. The reaction were then stopped and released
acetate was extracted and the amount of radioactivity released
determined by scintillation counting.
[0129] It was observed, as shown in Eigure 2, that, Compound 1 is a
potent inhibitor of HDAC1 enzymatic activity (ID.sub.50=1 nM).
EXAMPLE 8
Effect of Compound 2
(N-Nicotinoyl-(L)-.alpha.-aminosuberoylanilide-.omega.-hydroxamic
acid, C5H4NCO-Asu (NHOH)--NHPh) on MEL Cell Differentiation
[0130] Murine Exythroleukemia (MEL) Cell Differentiation:
[0131] The MEL cell differentiation assay was used to assess the
ability of Compound 2 to induce terminal differentiation. MEL cells
(logarithmically dividing) were cultured with the indicated
concentrations of Compound 2. Following a 5-day culture period
differentiation was determined microscopically using the benzidine
assay to determine hemoglobin protein accumulation on a per cell
basis.
[0132] It was observed, as shown in FIG. 3, that Compound 2 (800
nM) is able to induce MEL cell differentiation.
EXAMPLE 9
Effect of Compound 3
(N-benzyloxycarbonyl-(L)-.alpha.-aminosuberateanilide
.omega.-hydroxamic acid, N-Cbz-(L)-Asu (NHOH)--NHPh) on MEL Cell
Differentiation and Histone Deacetylase Activity
[0133] Murine Erythroleukemia (MEL) Cell Differentiation:
[0134] The MEL cell differentiation assay was used to assess the
ability of Compound 3 to induce terminal differentiation. MEL cells
(logarithmically dividing) were cultured with the indicated
concentrations of Compound 3. Following a 5-day culture period
differentiation was determined microsdopically using the benzidine
assay to determine hemoglobin protein accumulation on a per cell
basis.
[0135] It was observed, as shown in FIG. 4, that Compound 3 (400
nM) is able to induce MEL cell differentiation.
[0136] Histone Deacetylase (HDAC) Enzymatic Activity:
[0137] The effect of Compound 3 on affinity purified human epitope
tagged (Flag) HDAC1 was assayed by incubating the enzyme
preparation in the absence of substrate on ice for 20 min with the
indicated amounts of HPC. Substrate ([.sup.3H]acetyl-labelled
murine erythroleukemia cell-derived histone) was added and the
samples were incubated for 20 min at 37.degree. C. in a total
volume of 30 .mu.l. The reactions were then stopped and relaesed
acetate was extracted and the amount of radioactivity released
determined by scintillation counting.
[0138] It was observed, as shown in FIG. 5, that Compound 3 is a
potent inhibitor of HDAC1 enzymatic activity (ID.sub.50-100
nM).
EXAMPLE 10
Effect of Compound 4
(N-benzyloxycarbonyl-(L)-.alpha.-aminosuberoyl-8-quinolinamide-.omega.-hy-
droxamc acid) on MEL Cell Differentiation and Histone Deacetylase
Activity
[0139] Murine Erythroleukemia (MEL) Cell Differentiation:
[0140] The MEL cell differentiation assay was used to assess the
ability of Compound 4 to induce terminal differentiation. MEL cells
(logarithmically dividing) were cultured with the indicated
concentrations of Compound 4. Following a 5-day culture period
differentiation was determined microscopically using the benzidine
assay to determine hemoglobin protein accumulation on a per cell
basis.
[0141] It was observed, as shown in FIG. 6, that Compound 4 (40 nM)
is able to induce MEL cell differentiation.
[0142] Histone Deacetylase (HDAC) Enzymatic Activity:
[0143] The effect of Compound 4 on affinity purified human
epitopetagged (Flag) HDAC1 was assayed by incubating the enzyme
preparation in the absence of substrate on ice for 20 min with
indicated amounts of HPC. Substrate ([.sup.3H]acetyl-labelled
murine erythroleukemia cell-derived histone) was added and the
samples were incubated for 20 min at 37.degree. C. in a total
volume of 30 .mu.l. The reactions were then stopped and released
acetate was extracted and the amount of radioactivity released
determined by scintillation counting.
[0144] It was observed, as shown in FIG. 7, that Compound 4 is a
potent inhibitor of HDAC1 enzymatic activity (ID.sub.50<10
nM).
[0145] SAHA Inhibits the Activity of Affinity Purified HDAC1 and
HDAC3 (39).
[0146] Crystallographic studies with SAHA and a HDAC related
protein reveal that SAHA inhibits HDAC by a direct interaction with
the catalytic site (66). Additional studies demonstrate that a
tritium labeled photoaffinity SAHA analog (.sup.3H-498) that
contains an azide moiety (67) binds directly to HDAC1 (FIG. 8).
These results indicate that this class of hydroxamic acid based
compound inhibits HDAC activity through a direct interaction with
the HDAC protein.
[0147] SAHA causes the accumulation of acetylated histones H3 and
H4 in vivo. The in vivo effect of SAHA has been studied using the
CWR.sub.22 human prostate xenograft in mice (68). SAHA (50
mg/kg/day) caused a 97% reduction in mean final tumor volume
compared to controls with no apparent toxicity. SAHA administration
at this dose caused an increase in acetylated histones H3 and H4 in
the tumor xenograft (FIG. 9).
[0148] SAHA is currently in Phase I Clinical Trials in patients
with solid tumors. SAHA causes an accumulation of acetylated
histones H3 and H4 in the peripheral blood mononuclear cells
isolated from patients undergoing treatment (FIG. 10).
[0149] Table I shows a summary of the results of the Examples 7-10,
testing compounds 1-4, and also compares the results to the results
obtained from using SAHA. TABLE-US-00001 TABLE 1 Summary of Test
results of compounds 1-4, and comparison to SAHA results. MEL
Differentiation HDAC Inhibition Compound Range Opt. % B+ Range
ID.sub.50 1 0.1 to 50 .mu.M 200 nM 44% 0.0001 to 100 .mu.M 1 nM 2
0.2 to 12.5 .mu.M 800 nM 27% TBT 3 0.1 to 50 .mu.M 400 nM 16% 0.01
to 100 .mu.M 100 nM 4 0.01 to 50 .mu.M 40 nM 8% 0.01 to 100 .mu.M
<10 nM SAHA 2500 nM 68% 0.01 to 100 .mu.M 200 nM
EXAMPLE 12
Modified Inhibitors of HDAC
[0150] In additional studies we found that compounds 6 and 7 shown
below were very effective inhibitors of the enzyme HDAC. Compound 6
had ID.sub.50 of 2.5 nM, and compound 7 had ID.sub.50 of 50 nM.
This contrasts with an ID.sub.50 for SAHA of 1 .mu.M, much higher.
Note that the 1 .mu.M ID.sub.50 for SAHA as an inhibitor of HDAC is
of the same general magnitude as its 2.5 .mu.M optimal dose for the
cytodifferentiation of MEL cells, but this close similarity is not
true for all the compounds examined. In some cases very effective
HDAC inhibitors are less effective as cytodifferentiaters, probably
because the drugs are metabolized in the cell assays. Also, all
cell types are not the same, and some compounds are much better
against human tumor cells such as HT-29 than they are against MEL
cells. Thus, inhibition of HDAC cells is a preliminary indicator.
##STR45##
EXAMPLE 13
Evolution of Compounds Without a Hydroxamic Acid Portion
[0151] Of the above compounds which are hydroxamic acids, we have
found that they undergo enzymatic hydrolysis rather rapidly to the
carboxylic acids, so their biological lifetimes are short. We were
interested in evolving compounds which might be more stable in
vivo. Thus we have developed inhibitors of HDAC that are not
hydroxamic acids, and that can be used as cytodifferentiating
agents with longer biological lifetimes. Furthermore, we found that
the newly .evolved compounds have better selectivity to HDAC than,
e.g. SAHA.
[0152] We have evolved compounds that have double bonds, similarly
to Trichostatin A (TSA) to see if the resulting compounds have even
greater efficacy. Also, the chain in TSA is only five carbons, not
the six of SAHA. In Oxamflatin there is a chain of four carbons
containing a double bond and an ethinyl link between the hydroxamic
acid and the first phenyl ring, and Oxamflatin has been claimed to
be an effective inhibitor of HDAC. We incorporate some of these
features in our compounds, including those compounds that are not
hydroxamic acids.
[0153] Also disclosed are simple combinatorial methods for
screening a variety of such compounds for efficacy and selectivity
with respect to HDAC inhibition.
[0154] Furthermore, since there are many important enzymes that
contain Zn(II), hydroxamic. acids, and perhaps some of the other
metal coordinating groups can also bind to Zn(II) and other metals.
##STR46##
[0155] Since the target for HDAC is an acetyllysine sidechain of
histone, we make compounds in which transition state analogs of the
substrate are present. For example, we synthesize compounds like
SAHA in which the hydroxamic acid group --CO--NHOH, is replaced by
a trifluoroacetyl group, --CO--CF.sub.3. The resulting 8 will
easily form a hydrate, and thus bind to the Zn(II) of HDAC in a
mimic 9 of the transition state 10 for deacetylation. This is
related to the work published by Lipscomb [56] on the binding to
carboxypeptidase A of a substrate analog 11 containing a
CF.sub.3--CO--CH.sub.2 group in place of the normal amide. The
hydrate of the ketone coordinated to the Zn(II) as a mimic of the
transition state for catalyzed hydrolysis of an amide substrate.
Our synthesis of a particular example 12 in the fluoroketone series
is shown in Scheme below: ##STR47##
[0156] After the malonic ester alkylation, the aldehyde is prepared
and then converted to the trifluoromethylcarbinol with Rupperts
reagent [57, 58]. The malonic bis-anilides are prepared, and the
carbinol oxidized to the ketone 12 with the Dess-Martin reagent
[59]. Other approaches were tried unsuccessfully. In particular,
attempts to convert a carboxylic acid derivative directly to a
trifluoromethyl ketone did not work.
[0157] Compound 12 has been tested with HDAC and found to be an
inhibitor of the enzyme. Thus, we also adapt this synthesis to the
preparation of analogs of 12 with unsaturation, etc., in the chain,
and other groups at the left end of the molecule.
EXAMPLE 14
Evolution of Compounds where the Hydroxamic Acid Group is Replaced
bv NH--P(O)OH--CH.sub.3
[0158] An analog of SAHA in which the CH.sub.2--CO--NHOH group is
replaced by NH--P--(O)OH--CH.sub.3 may be synthesized by the
general scheme shown below. The resulting compound, 13, binds to
the Zn(II) of HDAC the way a related group binds to the Zn(II) of
carboxypeptidase in analogs such as that prepared by Bartlett [60].
##STR48##
[0159] A classic inhibitor of the Zn(II) enzyme carbonic anhydrase
is a sulfonamide, whose anion binds to the Zn(II) [61]. Thus
compound 14, an analog of SAHA with a sulfonamide group, is
synthesized as shown below. In the last step we react a carboxylic
sulfonic bis-chloride with aniline and ammonia. Since the
carboxylic acid chloride reacts faster, we use the sequence of
aniline, then ammonia, but the sequence may be reversed, or the
mixture may be separated if the two are of similar reactivity.
[0160] In the course of the synthesis of 14, we use a thiol 15
easily made from the corresponding haloacid. Thiols are also
inhibitors of Zn(II) enzymes such as carboxypeptidase A and related
peptidases such as Angiotensin Converting Enzyme (ACE), so we
convert 15 to 16 as an inhibitor of HDAC. A similar synthesis can
be used to attach the NH--P(O)OH--CH.sub.3 group to other
compounds, in particular compounds 6 and 7. ##STR49##
EXAMPLE 15
Varying the Linker Between the Zn(II) Binding Group and the
Hydrophobic Binding Groups
[0161] Based on the results with Oxamflatin, it seems that a phenyl
ring can be of the chain between the Zn(II) binding group and the
left hand section of the molecule as drawn, particularly when the
phenyl ring is meta substituted. Thus, we provide a synthesis t o
incorporate such meta substituted chains into other of our
compounds. We construct compounds 17 and 18. The simple syntheses,
not shown in detail, only require that instead of the hydroxamic
acid attached to the phenyl ring we make the aryl amides of 17 and
18. ##STR50##
[0162] Additional compounds may be synthesized, such as 19 and 20
to incorporate the trifluoromethyl ketone group of 12 that we know
is effective as a Zn (II) binder in HDAC. The syntheses involve
preparing compounds 21 and 22 and then adding CF.sub.3 to form the
carbinol, followed by oxidation as in the synthesis of 12. A simple
synthesis involves Heck coupling of compounds 23 and 24 with ethyl
acrylate, and conversion of the ester to aldehydes 21 and 22 by
reduction to the carbinol and then reoxidation. All the chains
shown so far contain only carbon atoms, but thioether links may be
acceptable and even useful, and they add synthetic ease. Thus,
sulfonamides such as 25 and 26, related to 19 and 20, from the
corresponding thiophenol and bromomethylsulfonamide. A related
synthesis may be used to make the corresponding phosphonamidates 27
and 28, if this class proves to be useful HDAC inhibitors and
cytodifferentiators. In this case, (N-protected) m-aminobenzoic
acid is used to acylate the arylamines, then phosphorylate the
anilino group. ##STR51##
EXAMPLE 16
Varying the Left Hand of the Molecule, Carrying the Hydrophobic
Groups
[0163] To vary the hydrophobic groups, we synthesized compound 29,
as an intermediate that can be treated with various amines to make
the compounds 30. Then deprotection of the hydroxamic=cid group
will generate the general class 31. The synthesis is shown in the
scheme below. ##STR52##
[0164] In the synthesis the O-protected hydroxylamine is acylated
with bromohexanoic acid, and the compound then alkylates the
bis-pentafluoro ester of malonic acid. The resulting 29 then reacts
with various amines, and the protecting group is removed with
acid.
[0165] With this compound as the starting material, we synthesize
related libraries carrying the other Zn(II) binding groups. For
example, alkylation of the malonate with compound 32 lets us make a
phosphonamidate library, and compound 33 will let us make a
CF.sub.3--CO library. In a similar way, a sulfonamide library can
be made if the work described earlier indicates that this is a
promising Zn(II) binding group for HDAC. Of course after malonate
alkylation and aminolysis the compound from 32 will be
demethylated, while that from 33 will be oxidized. ##STR53##
[0166] This also allows to expand on the structure of compound 6,
the derivative of aminosuberic acid. As described, this was one of
the most effective HDAC inhibitor we have examined. We prepared
this compound using an enzymatic hydrolysis to achieve optical
resolution and selectivity among the two carbomethoxy groups of 34
so that we could convert one of them to the aminoquinoline amide of
6 while protecting the nitrogen as a carbobenzoxy group. At the end
of the synthesis we converted the remote carbomethoxy group to a
hydroxamate. However, 6 is an intermediate that can be used to
prepare other derivatives. The carbobenzoxy group from 6 can be
removed and the amine 35 can be acetylated with a variety of
carboxylic acids to prepare library 36, or sulfonic acid chlorides
to prepare the corresponding sulfonamides. ##STR54## ##STR55##
[0167] Also, we synthesize a different library of amides 37 related
to 6, and then expand it with a library of other amides 38 by
acylating the amino group after deprotection. We also we synthesize
a group of compounds 39 in which after the carbobenzoxy group of 37
is removed we make a library of sulfonamides using various sulfonyl
chlorides. In all this, it the hydroxamic acid group may be
protected. ##STR56##
[0168] The foregoing synthesis schemes can be used to generate
compounds having a large number of variation. Some substituent
groups that are likely to result in compounds having potential good
affinity to HDAC or having got differentiating activity are as
follows:
[0169] Some Aminies that can be incorporated in place of the
aniline in SAHA, or as the X group in compounds 37 and 38:
##STR57##
[0170] Some carboxylic and sulfonic acids that can be incorporated
as group Y--CO in compound 38 or 39: ##STR58##
EXAMPLE 17
Synthesis Using the Foregoing Schemes
[0171] Reagents and starting materials were obtained from
commercial suppliers and used without further purification unless
otherwise indicated. For moisture-sensitive reactions, solvents
were freshly distilled prior to use: tetrahydrofuran was distilled
under argon from sodium metal utilizing benzophenone as an
indicator; dichloromethane and acetonitrile were distilled from
powdered calcium hydride. Anhydrous benzene, anhydrous DIEA, and
anhydrous pyridine were drawn by syringe from a sealed bottle
purchased from Aldrich. Tert-Butanol was dried over 4 .ANG.
molecular sieves before use. Sodium hydride was purchased as a 60%
dispersion in mineral oil. Aniline, diisopropylamine,
N-methylaniline, and benzyl alcohol were freshly distilled before
use. Deuterated solvents were obtained from Cambridge Isotope
Laboratories. Air- and/or moisture-sensitive reactions were carried
out under an atmosphere of dry argon in oven- or flame-dried
glassware equipped with a tightly-fitting rubber septum. Syringes
and needles were oven-dried before use. Reactions at 0.degree. C.
were carried out in an ice/water bath. Reactions at -78.degree. C.
were carried out in a dry ice/acetone bath.
[0172] Chromatography
[0173] Analytical thin-layer chromatography (TLC) was conducted on
glass plates precoated with silica gel 60 F-254, 0.25 mm thickness,
manufactured by EM Science, Germany. Eluted compounds were
visualized by one or more of the following: short-wave ultraviolet
light, I.sub.2 vapor, KMnO.sub.4 stain, or FeCl.sub.3 stain.
Preparative TLC was carried out on Whatman precoated plates of
either 500 .mu.m or 1000 .mu.m silica gel thickness. Flash column
chromatography was performed on Merck Kieselgel 60, 230-400
mesh.
[0174] Instrumentation
[0175] NMR spectra were measured on Bruker DPX300 and DRX400
spectrometers; .sup.1H was observed at 300 and 400 MHz, and
.sup.19F at 376 MHz. Chemical shifts are reported as .delta. values
in ppm relative to the solvent residual peak. Mass spectra were
obtained on a Nermag R-10-1 instrument for chemical ionization (CI)
or, electron impact ionization (EI) spectra, and on a Jeol JMS
LCmate for electrospray ionization (ESI+) spectra. CI spectra were
run with either ammonia (NH.sub.3) or methane (CH.sub.4) as the
ionization gas.
(E,E)-7-t-Butoxycarbonyl-octa-2,4-dienedioic acid 8-t-butyl ester
1-methyl ester (40)
[0176] ##STR59##
[0177] To a stirred solution of NaH (60% disp., 234 mg, 5.85 mmol)
in THF (35 mL) at 0.degree. C. was added di-t-butyl malonate (1.20
mL, 5.37 mmol) dropwise. Gas evolution was observed, and the
solution was allowed to warm to ambient temperature and stirred for
6 h. A solution of methyl 6-bromo-2,4-hexadienoate (62) (1.00 g,
4.88 mmol) in THF (20 mL) was prepared in a separate flask and
stirred in a water bath. To this was cannulated dropwise the
malonate mixture, and the reaction allowed to proceed overnight.
The reaction was quenched with sat. NH.sub.4Cl (5 mL), then
H.sub.2O (10 mL) was added and the mixture extracted with Et.sub.2O
(3.times.15 mL). The organic fractions were combined and washed
with H.sub.2O (1.times.10 mL ), then with brine, dried over
MgSO.sub.4, and filtered. Evaporation under reduced pressure
followed by flash chromatography (0-20% EtOAc/hexanes) gave 40 as a
clear colorless oil (850 mg, 2.49 mmol, 51%). TLC R.sub.f 0.66 (20%
EtOAc/hexanes); .sup.1H-NMR (CDCl.sub.3, 400 MHz) .delta. 7.26 (dd,
1H), 6.26 (dd, 1H), 6.10 (m, 1H), 5.82 (d, 1H), 3.78 (s, 3H), 3.12
(t, 1H), 2.64 (t, 2H), 1.41 (s, 18H).
(E,E)-7-Carboxy-octa-2,4-dienedioic acid 1-methyl ester (41)
[0178] ##STR60##
[0179] To a stirred solution of 40 (200 mg, 0.59 mmol) in
CH.sub.2Cl.sub.2 (10 mL) was added TFA (1 mL). The reaction was
allowed to proceed overnight. Volatiles were removed under reduced
pressure to leave 41 as a white solid (112 mg, 0.49 mmol, 83%).
.sup.1H-NMR (CD.sub.3OD, 400 MHz) .delta. 7.11 (dd, 1H), 6.33 (dd,
1H), 6.16 (m, 1H), 5.81 (d, 1H), 3.76 (s, 3H), 3.15 (t, 1H), 2.70
(t, 2H).
4-Pentenoic acid phenylamide (42)
[0180] ##STR61##
[0181] To a stirred solution of oxalyl chloride (2.0 M in
CH.sub.2Cl.sub.2, 11.5 mL, 23.1 mmol) in CH.sub.2Cl.sub.2 (100 mL)
and DMF (1 drop) at 0.degree. C. was added 4-pentenoic acid (2.25
mL, 22.0 mmol). This was allowed to warm to ambient temperature.
Upon cessation of gas evolution, the mixture was returned to
0.degree. C. and a solution of aniline (2.00 mL, 22.0 mmol) and TEA
(6.72 mL, 26.3 mmol) in CH.sub.2Cl.sub.2 (5 mL) was added dropwise.
After warming to ambient temperature, the reaction was allowed to
proceed for 3 h. The mixture was concentrated under reduced
pressure, and then partitioned between HCl (1 N, 10 mL) and EtOAc
(30 mL) and the layers separated. The aqueous portion was extracted
with EtOAc (3.times.15 mL) and the organic layers combined, washed
with brine, dried over MgSO.sub.4, and filtered. Concentration
under reduced pressure gave a yellowish solid, which was
recrystallized with toluene to obtain 42 as white crystals (1.97 g,
11.24 mmol, 51%). TLC R.sub.f 0.68 (50% EtOAc/hexanes); .sup.1H-NMR
(300 MHz, CDCl.sub.3) .delta. 7.49 (d, 2H), 7.29 (t, 2H), 7.08 (t,
1H), 5.88 (m, 1H), 5.10 (dd, 2 H), 4.42 (br s, 4 H).
(E,E)-Octa-2,4-dienedioic acid 8-t-butyl ester 1-methyl ester
(43)
[0182] ##STR62##
[0183] To a stirred solution of diisopropylamine (2.06 mL, 14.7
mmol) in THF (25 mL) at -78.degree. C. was added n-BuLi (2.0 M in
hexanes, 6.2 mL, 12.4 mmol) and allowed to stir 20 min at this
temperature. A solution of phosphonate 43a (63) (2.66 g, 11.3 mmol)
in THF (4 mL) was then added dropwise, giving a deep yellow color
upon addition. After 20 min at -78.degree. C., the mixture was
warmed to 0.degree. C. and a solution of aldehyde 43b (64) (1.78 g,
11.3 mmol) in THF (4 mL) was added dropwise. After addition the
solution was allowed to warm to ambient temperature and stirred
overnight. It was diluted with Et.sub.2O (30 mL) and washed with
H.sub.2O (3.times.10 mL). The aqueous washings were combined and
extracted with Et.sub.2O (2.times.10 mL), and the organic portions
combined, washed with brine, dried over MgSO.sub.4, and filtered.
Evaporation under reduced pressure followed by flash chromatography
(10-20% EtOAc/hexanes) gave 43 as a clear oil (1.54 g, 57%). TLC
R.sub.f 0.56 (20% EtOAc/hexanes); .sup.1H-NMR (400 MHz, CDCl.sub.3)
.delta. 7.22 (dd, 1H), 6.19 (dd, 1H), 6.08 (m, 1H), 5.77 (d, 1H),
2.42 (m, 2H), 2.32 (t, 2H), 1.42 (s, 9H).
(E,E)-7-Phenylcarbamoyl-hepta-2,4-dienoic acid methyl ester
(44)
[0184] ##STR63##
[0185] To a stirred solution of diester 43 (1.00 g, 4.61 mmol) in
CH.sub.2Cl.sub.2 (40 mL) was added TFA (4.0 mL) and let react for 6
h. The mixture was concentrated under reduced pressure to remove
volatiles. A white solid consisting of the crude acid (710 mg, 3.85
mmol) remained. This acid (400 mg, 2.17 mmol) was dissolved in
CH.sub.2Cl.sub.2 (20 mL) and to this stirred solution were added
DMAP (13 mg), aniline (218 .mu.L, 2.39 mmol), and EDC (500 mg, 2.61
mmol). After 1.5 h, the mixture was diluted with EtOAc and washed
with H.sub.2O. The layers were separated, and the aqueous extracted
with EtOAc (3.times.15 mL).
[0186] The organic portions were combined and washed with HCl (1 N,
1.times.5 mL) and brine, dried over MgSO.sub.4, and filtered.
Concentration under reduced pressure left a brown solid. This was
dissolved in a minimum of CH.sub.2Cl.sub.2, then passed through a
plug of silica gel (20-30% EtOAc/hexanes, 200 mL) to remove
baseline impurities. The eluent was concentrated to a light brown
oil which was taken up in a small amount of CH.sub.2Cl.sub.2 and
from which crystals were precipitated upon the addition of
hexanes/diethyl ether. The mother liquor was drawn off, the
crystals rinsed with ether, and the liquid fraction concentrated
and this procedure repeated several times to ultimately give 44 as
off-white crystals (324 mg, 1.25 mmol, 58%). TLC R.sub.f 0.44 (50%
EtOAc/hexanes); .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.47 (d,
1H), 7.30 (t, 2H), 7.24 (m, 1H), 7.09 (t, 1H), 6.24 (dd, 1H), 6.14
(m, 1H), 5.81 (d, 1H), 3.72 (s, 3H), 2.60 (m, 2H), 2.47 (t,
2H).
(E,E)-7-(Methyl-phenyl-carbamoyl)-hepta-2,4-dienoic acid methyl
ester (45)
[0187] ##STR64##
[0188] The crude acid intermediate from the first step of the
preparation of 44 (200 mg, 1.09 mmol) and N-methylaniline (130
.mu.L, 1.19 mmol) were dissolved in CH.sub.2Cl.sub.2 (10 mL) and
stirred. EDC (271 mg, 1.41 mmol) and DMAP (5 mg) were then added
and the reaction run overnight. The mixture was partitioned between
H.sub.2O and EtOAc and the layers separated. The aqueous layer was
extracted with EtOAc (3.times.10 mL), the,organic portions combined
and washed with HCl (1 N, 1.times.5 mL), then brine, dried over
MgSO.sub.4, and filtered. Evaporation under reduced pressure left
pure 45 as a brown oil (286 mg, 1.05 mmol, 96%). TLC R.sub.f 0.81
(5% MeOH/CH.sub.2Cl.sub.2); .sup.1H-NMR (300 MHz, CDCl.sub.3)
.delta. 7.40 (t, 2H), 7.35 (t, 1H), 7.20 (d, 2H), 7.15 (dd, 1H),
6.20 (m, 2H), 5.76 (d, 1H), 3.70 (s, 3H), 3.24 (s, 3H), 2.42 (m,
2H), 2.18 (t, 2H).
(E,E )-7-Phenylcarbamoyl-hepta-2,4-dienoic acid (46)
[0189] ##STR65##
[0190] Ester 45 (260 mg, 0.95 mmol) was dissolved in MeOH (7.5 mL).
A solution of LiOH.H.sub.2O (200 mg, 4.76 mmol) in H.sub.2O (2.5
mL) was then added and the mixture stirred for 6 h. The reaction
was acidified with HCl (1 N) until pH 2 and then extracted with
EtOAc (3.times.10 mL). The organic fractions were combined and
washed with H.sub.2O and brine, dried over MgSO.sub.4, and
filtered. Evaporation under reduced pressure left the product pure
46 as a brown solid (200 mg, 0.77 mmol, 81%). TLC R.sub.f 0.13 (40%
EtOAc/hexanes); .sup.1H-NMR (300 MHz, CD.sub.3OD) .delta. 7.47 (t,
2H), 7.41 (d, 1H), 7.28 (d, 2H), 7.19 (dd, 1H), 6.18 (dd, 1H), 6.05
(m, 1H), 3.27 (s, 3H), 3.40 (m, 2H), 2.22 (t, 2H).
(E,E)-Octa-2,4-dienedioic acid 1-hydroxyamide 8-phenylamide
(47)
[0191] ##STR66##
[0192] Acid 46 (200 mg, 0.77 mmol) and TBDPSO--NH, (220 mg, 0.81
mmol) were dissolved in CH.sub.2Cl.sub.2 (8 mL). To this stirred
solution were added EDC (178 mg, 0.93 mmol) and DMAP (5 mg) and the
reaction allowed to proceed overnight. The mixture was concentrated
and then passed through a plug of silica gel (EtOAc). Evaporation
under reduced pressure left a light brown oil (383 mg, 0.75 mmol,
97%). The protected hydroxamate (270 mg, 0.53 mmol) was dissolved
in CH.sub.2Cl.sub.2 (10 mL) and TFA was added (0.5 mL). The
solution was stirred for 2 h, and a new spot on TLC was observed
which stained with FeCl.sub.3. The solution was concentrated under
reduced pressure and diethyl ether added, giving a residue which
adhered to the flask. The liquid phase was drawn off, the residue
was triturated with EtOAc, the liquid removed, and evaporation of
all volatiles from the residue gave 47 as a brown gum (23 mg, 0.084
mmol, 16%). TLC R.sub.f 0.22 (5% MeOH/CH.sub.2Cl.sub.2);
.sup.1H-NMR (400 MHz, CD.sub.3OD) .delta. 7.50 (t, 2H), 7.40 (t,
1H), 2.27 (d, 2H), 7.08 (m, 1H), 6.11 (m, 1H), 5.97 (m, 1H), 5.80
(m, 1H), 3.23 (s, 3H), 3.39 (m, 2H), 2.21 (t, 2H).
Octanedioic acid hydroxyamide phenylamide (48)
[0193] ##STR67##
[0194] The title compound 48 was obtained as a brown gum (9 mg) by
a series of steps analogous to the preparation of 47. TLC R.sub.f
0.20 (5% MeOH/CH.sub.2Cl.sub.2); .sup.1H-NMR (400 MHz, CD.sub.3OD)
.delta. 7.51 (t, 2H), 7.41 (t, 1H), 7.30 (d, 2H), 3.29 (s, 3H),
2.11 (m, 4H), 1.58 (m, 4H), 1.22 (m, 4H).
Octanedioic acid benzylamide (49)
[0195] ##STR68##
[0196] To a stirred solution of suberoyl chloride (1.00 mL, 5.55
mmol) in THF (40 mL) at 0.degree. C. was added a solution of
benzylamine, (0.61 mL, 5.55 mmol) and DIEA (1.45 mL, 8.33 mmol) in
THF (10 mL) dropwise. The mixture was allowed to warm to ambient
temperature and stirred for 1 h. Then, HCl (10 mL, 1 N) was added
and the mixture stirred for 0.5 h. The contents were diluted with
EtOAc (30 mL) and the layers separated. The aqueous portion was
extracted with EtOAc (3.times.10 mL), the organics combined, washed
with brine (5 mL), and dried over MgSO.sub.4. Filtration and
concentration under reduced pressure left 49 as an off-white solid.
.sup.1H-NMR. (300 MHz, DMSO-d.sub.6) .delta. 11.98 (br s, 1H), 9.80
(t, 1H), 7.32 (m, 2H), 7.23 (m, 3H), 4.25 (d, 2H), 2.19 (t, 2H),
2.12 (t, 2 h), 1.50 (m, 4H), 1.25 (m, 4H).
Octanedioic acid benzylamide hydroxyamide (50)
[0197] ##STR69##
[0198] This compound was prepared from 49 through its protected
hydroxamate as-described for earlier compounds. Obtained 50 as a
white solid. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta. 10.30 (s,
1H), 8.27 (t, 1H), 7.28 (m, 2H), 7.23 (m, 3H), 5.65 (d, 2H), 2.11
(t, 2H), 1.91 (t, 2H), 1.46 (m, 4H), 1.23 (m, 4H).
(7S)-7-Benzyloxycarbonylamino-7-phenylcarbamoyl-heptanoic acid
t-butyl ester (51)
[0199] ##STR70##
[0200] N-Cbz-L-2-aminosuberic acid 8-t-butyl ester,
dicyclohexylamine salt (100 mg, 0.18 mmol) was dissolved in HCl (5
mL; 1 N) and extracted with EtOAc (3.times.10 mL). The extracts
were combined, washed with brine, and dried over MgSO.sub.4.
Evaporation left the free acid as a white solid (68 mg, 0.179
mmol). This was dissolved in CH.sub.2Cl.sub.2 (2.5 mL), to which
were added aniline (17 .mu.L, 0.19 mmol), DIEA (46 .mu.L, 0.27
mmol), and finally Py.BOP (97 mg, 0.19 mmol). The solution was
stirred for 1 h, then concentrated, and the residue partitioned
between H.sub.2O (5 mL) and EtOAC (10 mL). The layers were
separated, and the aqueous portion extracted with EtOAc (3.times.10
mL). The extracts were pooled and washed with HCl (1 N), then
brine, dried over MgSO.sub.4, and filtered. Concentration under
reduced pressure gave a solid residue which was passed through a
plug of silica gel (30% EtOAc/hexanes). The collected eluent was
evaporated to give 51 as a white solid (76 mg, 0.167 mmol, 94%).
TLC R.sub.f 0.38 (30% EtOAc/hexanes) .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 8.21 (s, 1H), 7.48 (d, 2H), 7.32 (m, 5H), 7.28
(t, 2H), 7.08 (t, 1H ), 5.39 (br d, 1H), 5.10 (m, 2H), 4.26 (br dd,
1H), 2.07 (t, 2H), 1.92 (m, 1H), 1.66 (m, 1H), 1.55 (m, 2H), 1.42
(s, 9H), 1.38 (m, 4H).
(7S)-7-Benzyloxycarbonylamino-7-phenylcarbamoyl-heptanoic acid
(52)
[0201] ##STR71##
[0202] To a solution of ester 51 (76 mg, 0.167 mmol) i n
CH.sub.2Cl.sub.2 (5 mL) was added TFA (0.5 mL) and the reaction
solution stirred for 5 h. The solution was concentrated
under-reduced pressure to give crude 52 as a white solid (80 mg),
which was used in the next step without purification. TLC R.sub.f
0.32 (5% MeOH/CH.sub.2Cl.sub.2); .sup.1H-NMR (400 MHz,
DMSO-d.sub.6), 7.55 (d, 1H), 7.35 (m, 4H), 7.29 (t, 2H) 7.03 (t,
1H) 5.02 (m, 2H), 4.11 (br dd, 1H), 2.17 (t, 2H), 1.59 (m, 2H),
1.48 (m, 2H), 1.22, (m, 4H).
(1S)-(6-Hydroxycarbamoyl-1-phenylcarbamoyl-hexyl-carbamic acid
benzyl ester (53)
[0203] ##STR72##
[0204] To a solution of crude acid 52 (80 mg) and TBDPSO--NH.sub.2
(60 mg, 0.221 mmol) in CH.sub.2Cl.sub.2 were added DIEA (52 .mu.L,
0.302 mmol) followed by Py.BOP (125 mg, 0.241 mmol). The solution
was stirred for 3 h, then concentrated under reduced pressure. The
residue was passed through a plug of silica gel (50% EtOAc/hexanes
and the collected eluent evaporated. A white foam (107 mg, 0.164
mmol, 82%) was obtained, this was dissolved in CH.sub.2Cl.sub.2 (5
mL) and TFA (0.25 mL) was added and the solution stirred for 2 h. A
new spot that stained with FeCl.sub.3 was indicated by TLC
analysis. The mixture was concentrated under reduced pressure, and
the residue was solvated in a minimum of EtOAc and the product
precipitated with hexanes. The resulting white gel was rinsed with
hexanes and dried under vacuum, to give 53 as a white solid (40 mg,
0.097 mmol, 58% over three steps). .sup.1H-NMR (400 MHz,
DMSO-d.sub.6) .delta. 10.31 (s, 1H), 9.99 (s, 1H), 7.59 (d, 2H),
7.56 (d, 1H), 7.37 (m, 4H), 7.29 (t, 2H), 7.02 (t, 1H), 5.02 (m,
2H), 4.11 (dt, 1H), 1.90 (t, 2H), 1.61 (m, 2H), 1.47 (m, 2H), 1.30
(m, 4H). MS (ESI+) calcd for C.sub.22H.sub.27N.sub.3O.sub.5 413,
found 414 [M+H].sup.+.
(7S)-7-Benzyloxycarbonylamino-7-(quinolin-8-ylcarbamoyl)-heptanoic
acid t-butyl ester (54)
[0205] ##STR73##
[0206] The title compound was made from N-Cbz-L-2-aminosuberic acid
8-t-butyl ester, dicyclohexylamine salt in a manner similar to that
for 51. Flash chromatography (0-1% MeOH/CH.sub.2Cl.sub.2) gave 54
as a light brown solid (70 mg, 0.138 mmol, 82%). TLC R.sub.f 0.42
(2% MeOH/CH.sub.2Cl.sub.2); .sup.1H-NMR (400 MHz, CDCl.sub.3)
.delta. 10.19 (s, 1H), 8.77 (dd, 1H), 8.71 (dd, 1H), 8.15 (dd, 1H),
7.52 (m, 2H), 7.45 (m, 1H), 7.33 (m, 4H); 5.50 (br d, 1H), 5.15 (m,
2H), 4.51 (br dd, 1 H), 2.17 (t, 2H), 2.00 (m, 1H), 1.79 (m, 1H),
1.56 (m, 2H), 1.45 (m, 2H), 1.40 (s, 9H), 1.38 (m, 2H).
(7S)-7-Benzyloxycarbonylamino-7-(quinolin-8-ylcarbamoyl)-heptanoic
acid (55)
[0207] ##STR74##
[0208] Prepared from 54 in a manner similar to that for 52.
Obtained 55 as a brown solid (72 mg, 0.129 mmol). TLC R.sub.f 0.16
(50% EtOAc/hexanes); .sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta.
11.92 (br s, 1H), 10.46 (s, 1H), 8.49 (dd, 1H), 8.63 (dd, 1H), 8.42
(dd, 1H), 8.10 (d, 1H), 7.68 (dd, 1H), 7.58 (t, 1H), 7.36 (m, 2H),
7.28 (m, 2H), 5.09 (m, 2H), 4.22 (m, 1H), 2.19 (t, 2H), 1.83 (m,
1H), 1.67 (m, 1H), 1.48 (m, 2H), 1.39 (m, 2H), 1.28 (m, 2H).
(1S)-[6-Hydroxycarbamoyl-1-(quinolin-8-ylcarbamoyl)-hexyl]-carbamic
acid benzyl ester (56)
[0209] ##STR75##
[0210] Prepared from 55 in a manner similar to that for 53.
Obtained 56 as a white solid (15 mg, 0.032 mmol, 44%). .sup.1H-NMR
(400 MHz, DMSO-d.sub.6) .delta. 10.46 (s, 1H), 10.31 (s, 1H), 8.85
(dd, 1H), 8.63 (dd, 1H), 8.42 (dd, 1H), 8.12 (d, 1H), 8.66 (m, 2H),
7.58 (t, 1H), 7.37 (m, 2H), 7.28 (m, 2H), 7.20-6.90 (1H), 5.10 (m,
2H), 4.10 (m, 1H), 1.92 (t, 2H), 1.82 (m, 1H), 1.68 (m, 1H), 1.49
(m, 2H), 1.40 (m, 2H), 1.26 (m, 2H). MS (ESI+) calcd for
C.sub.25H.sub.28N.sub.4O.sub.5 464, found 465 [M+H].sup.+.
(7S)-(Cyclohexanecarbonyl-amino)-7-phenylcarbamoyl-heptanoic acid
methyl ester (57)
[0211] ##STR76##
[0212] To a solution of 5 (81 mg, 0.214 mmol) in CH.sub.2Cl.sub.2
(10 mL) was added TFA (0.5 mL) and the solution stirred for 2 h.
The mixture was concentrated under reduced pressure. To a solution
of this amine (62 mg, 0.223 mmol) and cyclohexane carboxylic acid (
3 .thrfore..mu.L, 0.245 mmol) in CH.sub.2Cl.sub.2 (4 mL) were added
Py.BOP (140 mg, 0.268 mmol) and DIEA (58 .mu.L, 0.335 mmol). The
solution was stirred for 2 h, concentrated under reduced pressure,
and the product purified by flash chromatography (40%
EtOAc/hexanes). Evaporation left crude 57 as a white solid (95 mg)
containing a small amount of unreacted cyclohexane acid impurity.
This material was used in the next step without further
purification. TLC R.sub.f 0.58 (50% EtOAc/hexanes); .sup.1H-NMR
(400 MHz, CDCl.sub.3) .delta. 8.58 (s, 1H), 7.50 (d, 2H), 7.28 (t,
2H), 7.07 (t, 1H), 6.14 (d, 1H), 4.56 (dt, 1H), 3.64 (s, 3 H), 2.28
(t, 2H), 2.13 (tt, 1H), 1.94 (m, 1H), 1.85 (m, 2H), 1.76 (m, 2H),
1.64 (m, 4H), 1.41 (m, 5H), 1.22 (m, 4H).
(7S)-(Cyclohexanecarbonyl-amino)-7-phenylcarbamoyl-heptanoic acid
(58)
[0213] ##STR77##
[0214] To a solution of ester 57 (95 mg) in MeOH (2.5 mL) at
0.degree. C. was added a solution of NaOH (1 M, 2.5 mL). A white
precipitate formed upon addition, which was re-dissolved by the
addition of THF (2.5 mL). Additional NaOH (1 M, 1.0 mL) was added
after 3 h and the temperature maintained at 0.degree. C. Upon
complete disappearance of starting material by TLC analysis, the
reaction contents were acidified with HCl (1 N) to obtain a white
precipitate. The supernatant was drawn off, and the solid filtered
under aspiration. The combined liquors were extracted with EtOAc
(3.times.5 mL), and the extracts combined, washed with brine, dried
over MgSO.sub.4, and filtered. Concentration under reduced pressuxe
left a white solid which was combined with the filter cake and
dried under vacuum to obtain the carboxylic acid 58 (75 mg, 0.200
mmol, 90%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta. 11.95 (s,
1H), 9.98 (s, 1H), 7.90 (d, 1H), 7.58 (d, 1H), 7.28 (t, 2H), 7.02
(t, 1H), 4.33 (dt, 1H), 2.22 (tt, 1H), 2.17 (t, 2H), 1.67 (m, 6H),
1.60 (m, 2H), 1.46 (m, 2H), 1.22 (m, 9H).
(2S)-2-(Cyclohexanecarbonyl-amino)-octanedioic acid 8-hydroxyamide
1-phenylamide (59)
[0215] ##STR78##
[0216] Acid 58 (70 mg, 0.187 mmol), TBDPSO--NH.sub.2 (61 mg, 0.224
mmol), and DMAP (5 mg) were dissolved in CH.sub.2Cl.sub.2 (4 mL)
and EDC (47 mg, 0.243 mmol) was added. The solution was stirred
overnight. After concentration under reduced pressure, the material
was purified by flash chromatography (50% EtOAc/hexanes).
Evaporation of the combined product fractions gave a white foam (80
mg, 0.131 mmol, 70%). To a solution of this protected hydroxamate
in CH.sub.2Cl.sub.2 (2 mL) and THF (3 mL) was added TFA (0.25 mL)
and stirred for 1.5 h. A new spot which stained immediately with
FeCl.sub.3 was observed on TLC. The solution was concentrated and
all volatiles removed under vacuum. The residue was triturated with
EtOAc and obtain a white gel precipitate which was transferred to a
plastic tube with EtOAc (5 mL). The tube was centrifuged to form a
pellet, the supernatant drained, and EtOAc (10 mL) added. The
pellet was resuspended with sonication, then centrifuged again, the
supernatant discarded, and the residue dried under vacuum. A white
solid 59 (18 mg, 0.046 mmol, 35%) was obtained. .sup.1H-NMR (400
MHz, DMSO-d.sub.6) .delta. 10.31 (s, 1H) 9.97 (s, 1H), 7.89 (d,
1H), 7.57 (d, 2H), 7.28 (t, 2H), 7.02 (t, 1H), 4.33 (dt, 1H), 2.22
(t, 2H), 1.91 (t, 2H), 1.61 (m, 6H), 1.68 (m, 2H), 1.45 (m, 2H),
1.21 (9H).
Octanedioic acid hydroxyamide quinolin-8-ylamide (60)
[0217] ##STR79##
[0218] This compound was prepared from suberic acid monomethyl
ester in similar fashion to 48, with the use of 8-aminoquinoline.
The crude residue obtained after TFA deprotection of the protected
hydroxamate was taken up in a small volume of EtOAc and
precipitated with hexanes to give 60 as a white solid (18 mg, 0.057
mmol, 21% from the carboxylic acid). .sup.1H-NMR (400 MHz,
DMSO-d.sub.6) .delta. 10.31 (s, 1H), 10.02 (s, 1H), 8.92 (dd, 1H),
8.61 (dd, 1H), 8.40 (dd, 1H), 7.65 (dd, 1H), 7.63 (dd, 1H), 7.56
(t, 1H), 2.56 (t, 1H), 1.93 (t, 1H), 1.63 (m, 2H), 1.49 (m, 2H),
1.28 (m, 4H). MS (ESI+) calcd for C.sub.17H.sub.21N.sub.3O.sub.3
315, found 316 [M+H].sup.+.
2-t-Butoxycarbonyl-octanedioic acid 1-t-butyl ester 8-ethyl ester
(61)
[0219] ##STR80##
[0220] To a stirred suspension of NaH (60% disp., 197 mg, 4.913
mmol) in THF (25 mL) at 0.degree. C. was added di-t-butyl malonate
(1.00 mL, 4.466 mmol) and the mixture allowed to warm to ambient
temperature. After 1 h, gas had ceased evolving and ethyl
6-bromohexanoate (0.88 mL, 4.913 mmol) was added dropwise. The
reaction was brought to reflux overnight. The reaction was
carefully quenched with H.sub.2O (10 mL) and diluted with EtOAc.
After separation of the layers, the aqueous portion was extracted
with EtOAc (3.times.10 mL). The extracts were pooled and washed
with H,O, then brine, dried over MgSO.sub.4, and filtered.
Concentration under reduced pressure gave a yellow oil which was
passed through a plug of silica gel (10% EtOAc/hexanes).
Evaporation left a light yellow syrup 61 (1.52 g, 4.24 mol, 95%).
TLC R.sub.f 0.44 (10% EtOAc/hexanes); .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 4.10 (q, 2H), 3.08 (t, 1H), 2.26 (t, 2H), 1.76
(m, 2H), 1.60 (m, 2H), 1.43 (s, 18H), 1.32 (m, 4H), 1.23 (m,
3H).
2-Carboxy-octanedioic acid 8-ethyl ester (62)
[0221] ##STR81##
[0222] To a solution of triester 61 (500 mg, 1.395 mmol) in
CH.sub.2Cl.sub.2 (20 mL) was added TFA (2.0 mL) and the reaction
mixture stirred overnight. Volatile components were evaporated
under vacuum, and the residue repeatedly dissolved in
CH.sub.2Cl.sub.2 and evaporated to remove all traces of TFA. A
solid 62 (327 mg, 1.33 mmol) was obtained and used directly in the
next step without further purification. .sup.1H-NMR (400 MHz,
DMSO-d.sub.6) .delta. 12.62 (br s, 2H), 4.03 (q, 2H), 3.16 (t, 1H),
2.25 (t, 2H), 1.67 (m, 2H), 1.49 (m, 2H), 1.25 (m, 4H), 1.16 (t,
3H).
7,7-Bis-(quinolin-8-ylcarbamoyl)-heptanoic acid ethyl ester
(65)
[0223] ##STR82##
[0224] Diacid 62 (150 mg, 0.609 mmol), 8-aminoquinoline (211 mg,
1.462 mmol), and DMAP (5 mg) were dissolved in THF (6 mL). To this
solution was added EDC (350 mg, 1.827 mmol) and the reaction
allowed to proceed overnight. The mixture was concentrated under
reduced pressure and the product purified by flash chromatography
(40% EtOAc/hexanes). Evaporation of the combined product fractions
left 63 as a light brown solid (100 mg, 0.201 mmol, 14%).
.sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta. 10.85 (s, 2H), 8.92
(dd, 2H), 8.64 (dd, 2H), 8.40 (dd, 2H), 7.68 (dd, 2H), 7.62 (dd,
2H), 7.57 (t, 2H), 4.35 (t, 1H), 3.98 (q, 2H), 2.24 (t, 2H), 2.00
(m, 2H), 1.51 (m, 2H), 1.37 (m, 4H), 1.12 (t, 3H).
7,7-Bis-(quinolin-8-ylcarbamoyl)-heptanoic acid (64)
[0225] ##STR83##
[0226] To a solution of ester 63 (94 mg, 0.212 mol) in MeOH (3 mL)
and THF (1 mL) was added a solution of LiOH.H.sub.2O (44 mg, 1.062
mmol) in H.sub.2O (1 mL) and the mixture was stirred for 5 h. After
acidification with HCl (1 N) to pH 7, EtOAc (10 mL) was added and
the layers separated. The aqueous portion was extracted with EtOAc
(3.times.5 mL), and the extracts combined, washed with sat.
NH.sub.4Cl (3 mL), H.sub.2O (3 mL), then brine, dried over
MgSO.sub.4, and filtered. Concentration under reduced pressure left
64 as a white solid (94 mg, 0.200 mmol, 94%). TLC R.sub.f 0.21 (50%
EtOAc/hexanes); .sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta. 11.88
(s, 1H), 10.85 (s, 2H), 8.93 (dd, 2H), 8.65 (dd, 2H), 8.40 (dd,
2H), 7.69 (dd, 2H), 7.63 (dd, 2H), 7.58 (t, 2H), 4.35 (t, 1H), 2.16
(t, 2H), 2.00 (m, 2H), 1.49 (m, 2H), 1.38 (m, 4H).
2-(Quinolin-8-ylcarbamoyl)-octanedioic acid 8-hydroxyamide
1-quinolin-8-ylamide (65)
[0227] ##STR84##
[0228] Acid 64 (94 mg, 0.200 mmol), TBDPSO--NH.sub.2 (74 mg, 0.272
mmol), and DMAP (5 mg) were dissolved in CH.sub.2Cl.sub.2 (4 mL)
and EDC (57 mg, 0.295 mmol) was added. The solution was stirred
overnight, then concentrated under reduced pressure. Purification
by flash chromatography (30-50% EtOAc/hexanes) and evaporation of
the combined product fractions gave a white foam. To a solution of
this protected hydroxamate in CH.sub.2Cl.sub.2 (4 mL) was added TFA
(0.2 mL) and the solution stirred for 4 h. TLC indicated complete
consumption of starting material and a new spot that stained with
FeCl.sub.3. The solution was concentrated under reduced pressure,
and the residue dissolved in a minumum of EtOAc. Addition of
hexanes gave a white precipitate, from which the mother liquor was
removed. After rinsing with hexanes, the residue was dried under
vacuum to leave 65 as a white solid (30 mg, 0.061 mmol, 22% from
the carboxylic acid). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.
10.85 (s, 2H), 10.30 (s, 1H), 8.93 (dd, 2H), 8.65 (dd, 2H), 8.40
(dd, 2H), 7.69 (dd, 2H), 7.63 (dd, 2H), 7.58 (t, 2H), 4.35 (t, 1H),
1.99 (m, 2H), 1.92 (t. 2H), 1.48 (m, 2H), 1.35 (m, 4H). MS (ESI+)
calcd for C.sub.27H.sub.27N.sub.5O.sub.4 485, found 486
[M+H].sup.+.
2-(Quinolin-3-ylcarbamoyl)-octanedioic acid 8-hydroxy&de
1-quinolin-3-ylamide (68)
[0229] ##STR85##
[0230] The title compound was made from diacid 62 as analogous to
65. 1H-NMR (400 MHz, DMSO-d6) .delta. 10.60 (s, 1H), 10.34 (s, 1H),
8.95 (dd, 2H), 8.74 (s, 2H), 7.93 (dd, 2H), 7.64 (dd, 2H), 7.56
(dd, 2H), 3.71 (t, 1H), 1.96 (m, 4H), 1.51 (m, 2H), 1.34 (m,
4H).
6-Bromohexanoic acid phenylamide (76)
[0231] ##STR86##
[0232] To a solution of 6-bromohexanoyl chloride (1.00 mL, 6.53
mmol) in THF (35 mL) at 0.degree. C. was added dropwise a solution
of aniline (0.60 mL, 6.53 mmol) and TEA (1.09 mL, 7.84 mmol) in THF
(5 mL). The reaction mixture was allowed to warm to ambient
temperature and stirred for 2 h. The mixture was filtered, the
solids rinsed with EtOAc, and the filtrate reduced under vacuum.
The residue was partitioned between H.sub.2O (15 mL) and EtOAc (20
mL) and the layers separated. The aqueous portion was extracted
with EtOAc (3.times.10 mL) and the organic layers combined, washed
with HCl (1 N), brine, dried over MgSO.sub.4, and filtered.
Concentration under reduced pressure left a brown oil which was
passed through a plug of silica gel (30% EtOAc/hexanes) under
aspiration. Concentration under reduced pressure left 67 as a solid
(1.55 g, 5.74 mmol, 88%). TLC R.sub.f 0.36 (25% EtOAc/hexanes);
.sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta. 9.85 (s, 1H), 7.57 (d,
2H), 7.27 (t, 2H), 7.01 (t, 1H), 3.53 (t, 2H), 2.30 (t, 2H), 1.81
(t, 2H) 1.63 (m, 2H), 1.42 (m, 2H); MS (ESI+) calcd for
C.sub.12H.sub.16BrNO 268+270, found 269+271 [M+H].sup.+.
Thioacetic acid S-(5-phenylcarbamoyl-pentyl) ester (68)
[0233] ##STR87##
[0234] Bromide 67 (200 mg, 0. 74 mmol), potassium thioacetate (110
mg, 0.96 mmol), and sodium iodide (10 mg) were combined in THF (625
mL) and the vigorously stirred mixture brought to reflux overnight.
The reaction mixture was concentrated, the passed through a plug of
silica gel (20% EtOAc/hexanes, 200 mL) under aspiration.
Evaporation under reduced pressure left 68 as an orange crystalline
solid (190 mg, 0.72 mmol, 97%). TLC R.sub.f 0.22 (25%
EtOAc/hexanes); .sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta. 9.83
(s, 1H), 7.56 (d, 2H), 7.27 (t, 2H), 7.00 (t, 1H) 2.82 (t, 2H),
2.30 (s, 3H), 2.28 (t, 2H), 1.57 (m, 2H), 1.52 (m, 2H), 1.35 (m,
2H).
6-Methanesulfonylamino-hexanoic acid (69)
[0235] ##STR88##
[0236] 6-aminohexanoic acid (904 mg, 6.89 mmol) and NaOH (415 mg,
10.34 mmol) were dissolved in H.sub.2O (30 mL) and cooled to 0-5
.degree. C. Methanesulfonyl chloride (0.586 mL, 7.58 mmol) was
added dropwise and the reaction mixture stirred for 2 h, then
warmed to ambient temperature and stirred for an additional 2 h.
The mixture was acidified with HCl (1 N) and extracted with EtOAc
(3.times.15 mL). The extracts were combined, washed with H.sub.2O,
then brine, dried over MgSO.sub.4, and filtered. Evaporation under
reduced pressure gave 69 as a white crystalline solid (207 mg, 0.99
mmol, 14%). .sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta. 11.95 (s,
1H), 6.91 (t, 1H), 2.90 (dt, 2H), 2.87 (s, 3H), 2.20 (t, 2H), 2.48
(m, 2H), 2.43 (m, 2H), 1.27 (m, 2H).
6-Methanesulfonylamino-hexanoic acid phenylamide (70)
[0237] ##STR89##
[0238] To a solution of acid 69 (100 mg, 0.48 mmol), aniline (60
.mu.L, 0.66 mmol), and DMAP (5 mg) in THF (5 mL) was added EDC (119
mg, 0.57 mmol). The reaction mixture was stirred overnight, then
partitioned between H.sub.2O (10 mL) and EtOAc (15 mL). The layers
were separated, and the aqueous portion extracted with EtOAc
(3.times.10 mL). The organic fractions were combined, washed with
sat. NH.sub.4Cl (5 mL), then brine, dried over MgSO.sub.4, and
filtered. Concentration under reduced pressure gave 70 as a white
crystalline solid (130 mg, 0.46 mmol, 95%). .sup.1H-NMR (4.0.0M Hz,
DMSO-d.sub.6) .delta. 9.84 (s, 1H), 7.57 (d, 2H), 7.26 (t, 2H),
7.00 (t, 1H), 6.92 (t, 1H), 2.91 (dt, 2H) 2.85 (s, 3H), 1.58 (m,
2H), 1.47 (m, 2H), 1.31 (m, 2H).
9,9,9-trifluoro-8-oxononanoic acid methyl ester (71)
[0239] ##STR90##
[0240] To a solution of suberic acid monomethyl ester (1.00 g, 5.31
mmol) in THF (15 mL) was added oxalyl chloride (2 mL) followed by
DMF (1 drop). The solution was stirred for 2 h, then concentrated
under reduced pressure. Volatiles were removed under high vacuum
overnight, leaving a yellow oil (1.08 g, 5.22 mmol, 98%). This
crude acid chloride was then transformed into the trifluoromethyl
ketone by a literature method as follows (65). To a solution of the
acid chloride (1.08 g, 5.22 mmol) in CH.sub.2Cl.sub.2 (45 mL) at
0.degree. C. were added trifluoroacetic anhydride (4.64 mL, 32.81
mmol) and pyridine (3.54 mL, 43.74 mmol). The mixture was allowed
to warm to ambient temperature and stirred for 2 h. After returning
to 0.degree. C., ice-cold H.sub.2O (20 mL) was added carefully.
Additional H.sub.2O (100 mL) was added and the layers separated.
The aqueous phase was extracted with CH.sub.2Cl2 (2.times.30 mL)
and the organic layers combined, washed with brine, dried over
MgSO.sub.4, and filtered. Evaporation under reduced pressure left a
brown oil, which was purified by flash chromatography (2-4%
MeOH/CH.sub.2Cl.sub.2) to give 71 as a clear oil (641 mg, 2.67
mmol, 49%). TLC R.sub.f 0.24 (2% MeOH/CH.sub.2Cl.sub.2);
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 3.67 (s, 3H), 2.71 (t,
2H), 2.31 (t, 2H), 1.65 (m, 4H), 1.35 (m, 4H).
9,9,9-trifluoro-8-oxo-nonanoic acid phenylamide (72)
[0241] ##STR91##
[0242] To solution of ester 71 (300 mg, 1.25 mmol) in THF (18 mL)
was added a solution of LiOH.H.sub.2O (262 mg, 6.24 mmol) in
H.sub.2O (6 mL) and the suspension was stirred overnight. The
mixture was then acidified with HCl (1 N) to pH 2 and then
extracted with EtOAc (3.times.15 mL). The extracts were combined,
washed with brine, dried over MgSO.sub.4, and filtered.
Concentration under reduced pressure left a white solid (211 mg,
0.93 mmol, 75%). To a solution of this acid (109 mg, 0.48 mmol),
EDC (111 mg, 0.58 mmol), and DMAP (5 mg) in CH.sub.2Cl.sub.2 (5 mL)
was added aniline (49 .mu.L, 0.53 mmol) and the reaction allowed to
proceed overnight. The solution was partitioned between H.sub.2O (5
mL) and EtOAc (10 mL). The layers were separated, and the aqueous
phase extracted with EtOAc (3.times.5 mL). The organic portions
were combined, washed with brine, dried over MgSO.sub.4, and
filtered. Evaporation under reduced pressure left a solid which was
purified by preparative TLC (30% EtOAC/hexanes) with isolation of
the least polar band by EtOAc extraction. The extract was
concentrated to give 72 as a yellowish solid (92 mg, 0.31 mmol,
65%). TLC R.sub.f 0.48 (50% EtOAc/hexanes); .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 7.51 (d, 2H), 7.32 (t, 2H), 7.10 (t, 1H), 2.72
(t, 2H), 2.36 (t, 2H), 1.72 (m, 4H), 1.40 (m, 4H); .sup.19F-NMR (?
MHz, CDCl.sub.3) -78.40 (s, 3F); MS (APCI+) calcd for
C.sub.15H.sub.19F.sub.3NO.sub.2 301, found 325 [M+Na].sup.+.
(5-Phenylcarbamoyl-pentyl)-carbamic acid t-butyl ester (73)
[0243] ##STR92##
[0244] To a solution of N-Boc-6-aminohexanoic acid (2.50 g, 10.81
mmol), EDC (2.69 g, 14.05 nimol), and DMAP (20 mg) in
CH.sub.2Cl.sub.2 (100 mL) was added aniline (1.04 mL, 11.35 mmol)
and the mixture stirred overnight. The solution was evaporated
under reduced pressure to a small volume, then partitioned between
H.sub.2O (20 mL) and EtOAc (30 mL). The layers were separated, and
the aqueous phase extracted with EtOAc (3.times.15 mL). The organic
portions were combined, washed with sat. NH.sub.4Cl (5 mL), then
brine, dried over MgSO.sub.4, and filtered. Concentration under
reduced pressure left pure 73 as a white solid (3.14 g, 10.25 mmol,
95%). TLC R.sub.f 0.40 (50% EtOAc/hexanes); .sup.1H-NMR (400 MHz,
DMSO-d.sub.6) .delta. 9.81 (s, 1H), 7.56 (d, 2H), 7.26 (t, 2H),
7.00 (t, 1H), 6.74 (t, 1H), 2.89 (dt, 2H), 2.27 (t, 2H), 1.56 (m,
2H), 1.38 (m, 2H), 1.35 (s, 9H), 1.25 (m, 2H).
6-Aminohexanoic acid phenylamide, TFA salt (74)
[0245] ##STR93##
[0246] To a solution of carbamate 73 (300 rng, 0.98 mmol) in
CH.sub.2Cl.sub.2 (15 mL) was added TFA (0.75 mL) and the solution
stirred overnight. Complete consumption of starting material was
confirmed by TLC. The mixture was evaporated under reduced pressure
to remove all volatiles, leaving an off-white solid (295 mg, 0.92
mmol, 94%). Crude 74 was used without further purification.
N-(N-Phenylcarbamoyl-5-pentyl) phosphoramidic acid dimethyl ester
(75)
[0247] ##STR94##
[0248] To a stirred suspension of ammonium salt 74 (197 mg, 0.62
mmol) and DIEA (148 .mu.L, 0.85 mmol) in CH.sub.2Cl.sub.2 (7 mL) at
0.degree. C. was added dropwise dimethyl chlorophosphate (77 .mu.L,
0.72 mmol). The mixture was allowed to warm to ambient temperature
and stirred overnight. The solution was diluted with H.sub.2O (10
mL) and the layers separated. The aqueous phase was extracted with
CH.sub.2Cl.sub.2 (3.times.10 mL), the organic portions combined,
washed with sat. NH.sub.4Cl (5a), then brine, dried over
MgSO.sub.4, and filtered. After concentration, the residue was
purified by flash chromatography (2-5% MeOH/CH.sub.2Cl.sub.2), and
the fractions containing the more polar of the two UV-active bands
on TLC were combined and concentrated, giving 75 as a clear oil (40
mg, 0.13 mmol, 20%). TLC R.sub.f 0.23 (5% MeOH/CH.sub.2Cl.sub.2);
.sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta. 9.84 (s, 1H), 7.57 (d,
2H), 7.26 (t, 2H), 7.00 (t, 1H), 4.90 (dt, 1H), 3.51 (d, 6H), 2.71
(m, 2H), 2.28 (t, 2H), 1.56 (m, 2H), 1.40 (m, 2H), 1.29 (m,
2H).
Methyl N-(5-N-phenylcarbamoylpentyl) methylphosphonamidate (76)
[0249] ##STR95##
[0250] To a suspension of ammonium salt 74 (155 mg, 0.48 mmol) in
CH.sub.3CN (8 mL) were added DIEA (0.21 mL) and methyl
methylphosphonochloridate (77 mg, 0.600 mmol). The reaction mixture
was stirred overnight, during which time it clarified. The solution
was partitioned between H.sub.2O (10 mL) and EtOAc (15 mL) and the
layers separated. The aqueous portion was extracted with EtOAc
(3.times.10 mL) and the organics combined, washed with sat.
NH.sub.4Cl (1.times.5 mL), then brine, dried over MgSO.sub.4, and
filtered. The product was purified by flash chromatography (3-10%
MeOH/CH.sub.2Cl.sub.2), and the fractions containing the more polar
spot were combined and concentrated to give 76 as a clear oil (102
mg, 0.34 mmol, 71%). TLC R.sub.f 0.16 (58 MeOH/CH.sub.2Cl.sub.2);
.sup.1H-NMR (400 MHz, DMSO-d.sub.6) .delta. 9.85 (s, 1H), 7.57 (d,
2H), 7.26 (t, 2H), 7.00 (t, 1H), 4.52 (dt, 1H), 3.43 (d, 3H), 2.73
(m, 2H), 2.28 (t, 2H), 1.57 (m, 2H), 1.38 (m, 2H), 1.28 (m, 2H),
1.26 (d, 3H).
EXAMPLE 18
Synthesis of Compound 77
Diethyl 3-bromophenylmalonate
[0251] ##STR96##
[0252] Diethyl 3-bromophenyl malonate was prepared according to the
procedures of Cehnevert, R. and Desjardins, M. Can. J. Chem. 1994.
72, 3212-2317. .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 7.6 (s,
1H), 7.50 (d, 1H, J=7.9 Hz), 7.37 (d, 1H, J=7.9 Hz), 7.26 (t, 1H,
J=7.9 Hz), 4.58 (s, 1H), 4.22 (m, 4H), 1.29 (t, J=10 Hz).
3-bromophenyl malonyl di(phenylamide)
[0253] ##STR97##
[0254] Diethyl 3-bromophenyl malonate (1 g, 3.2 mmol) was added to
aniline (5 mL). The reaction mixture was purged with Ar (g) and
brought to reflux for 2 h. After cooling, the reaction mixture was
diluted with 10% HCl (20 mL) and ethyl acetate (50 mL). The organic
layer was separated and concentrated to afford 3-bromophenyl
malonyl di(phenylamide) as a white powder. (540 mg, 1.3 mmol, 42%).
.sup.1H NMR (d.sub.6-DMSO, 300 MHz) .delta. 10.3 (bs, 2H), 7.65 )
s, 1H), 7.60 (d, 4H, J=7.9 Hz), 7.54 (d, 1H, J=7.9 Hz), 7.46 (d,
1H, J=7.8 Hz), 7.35 (t, 1H, J=7.8Hz), 7.31 (t, 4H, J=7.8 Hz), 7.06
(t, 2H, J=7.6 Hz), 4.91 (9s, 1H).
3-(malonyl di(phenylamide))cinnamic acid
[0255] ##STR98##
[0256] 3-bromophenyl malonyl di(phenylamide) (500 mg, 1.22 mmol),
acrylic acid (115 mg, 1.6 mmol, 1.3 equiv.), Pd (OAc).sub.2 (2 mg),
tri-o-tolyl phosphone (20 mg), tributyl amine (0.6 mL) and xylenes
(5 mL) were heated to 120.degree. C. for 6 h in a sealed vessel.
After cooling, the reaction was diluted with 5% HCl (10 mL) and
ethyl acetate (50 mL). The organic layer was separated, filtered
and on standing 3-(malonyl di(phenyl amide)) cinnamic acid
precipitated as a white powder (450 mg, 1.12 mmol, 92%).
.sup.1H-NMR (d.sub.6-DMSO, 300MHz, .delta. 12.4 (bs, 1H), 10.3 (bs,
2H), 7.73 (s, 1H), 7.7-7.5 (m, 6H), 7.52 (d, 1H, J=7.7 Hz), 7.43
(t, 1H, J=7.6 Hz), 7.31 (t, 4H, J=7.5Hz), 7.06 (t, 2H, J=7.4 Hz),
6.52 (d, 1H, J=16 Hz), 4.95 (s, 1H). APCI-MS 401 (M+1).
3-(malonyl di(phenylamide))cinnamyl hydroxamic acid (77)
[0257] ##STR99##
[0258] 3-(malonyl di(phenylamide))cinnamic acid (200 mg, 0.5 mmol)
was dissolved in dry CH.sub.2Cl.sub.2 (10 mL).
Isobutylchlorofonmate (0.10 mL, 0.77 mmol) and triethylamine (0.20
mL) were added at 0.degree. C. with stirring. After 2 h at
25.degree. C., O-(t-butyldiphenyl silyl)hydroxylamine was added and
the mixture was stirred an additional 4 h. The crude reaction
mixture was applied directly to a pad a silica gel (15 g) and
elution with 20% ethyl acetate/hexanes afforded the corresponding
silyl protected hydroxamic acid (R.sub.f=0.58, 50% ethyl
acteate/hexanes) as a foam. This was treated directly with 10%
trifluoracetic acid in dichloromethane (10 mL) for 4 h. The
solvents were concentrated at 50.degree. C. by rotavap and the
residue was suspended in ethyl ether (10 mL). Filtration of the
resultant precipitate afforded compound 77 as a white powder (150
mg, 0.365 mmol, 73%). .sup.1H NMR (d.sub.6-DMSO, 300 MHz, .delta.
10.8 (bs, 0.5H), 10.2 (bs, 2H), 9.06 (bs, 0.5H), 7.7-7.55 (m, 5H),
7.53-7.38 (m, 4H), 7.31 (t, 4H, J=7.7 Hz), 7.06 (t, 2H, J=7.3 Hz),
6.50 (d, 1H, J=16Hz), 4.92 (s, 1H). APC.sub.1-MS 416 (M+1).
[0259] The effect of compound 77 on MEL cell differentiation and
Histone Deacetylase activity is shown in Table 2. Compound 77
corresponds to structure 683 in Table 2. As evident from Table 2,
compound 77 is expected to be a highly effective
cytodifferentiating agent.
[0260] Results
[0261] All the compounds which were prepared were tested. Table 2
below shows the results of testing of only a subgroup of compounds.
Table 2 is compiled from experiments similar to the experiments
described in Examples 7-10 above. The tested compounds were
assigned structure numbers as shown in Table 2. The structure
numbers were randomly assigned and do not correlate to the compound
numbers used elsewhere in this disclosure.
[0262] The results shown in Table 2 verify the generat accuracy of
the predictive principals for the design of compounds having cell
differentiation and HDAC inhibition activity discuss.ed above in
this disclosure. Based on the principals and synthesis schemes
disclosed, a number of additional compounds can readily be
designed, prepared and tested for cell differentiation and HDAC
inhibition activity.
[0263] FIGS. 11a-f show the effect of selected compounds on
affinity purified human epitope-tagged (Flag) HDAC1. The effect was
assayed by incubating the enzyme preparation in the absence of
substrate on ice for 20 minutes with the indicated amounts of
compound. Substrate([.sup.3H]acetyl-labeled murine erythroleukemia
cell-derived histones) was added and the samples were incubated for
20 minutes at 37.degree. C. in a total volume of 30 .mu.l. The
reactions were then stopped and released acetate was extracted and
the amount of radioactivity released determined by scintillation
counting. This is a modification of the HDAC Assay described in
Richon et al. 1998 (39). TABLE-US-00002 TABLE 2 Inhibition data of
selected compounds. MEL Diff Cells/ HDAC inh NO: Structure Range
Opt. % B+ ml x 10.sup.-5 Range ID.sub.50 SAHA (390) ##STR100## 0.5
to 50 .mu.M 2.5 .mu.M 68 3.6 0.001 to 100 .mu.M 200 nM 654
##STR101## 0.1 to 50 .mu.M 200 nM 44 9 0.0001 to 100 .mu.M 1 nM 655
##STR102## 0.1 to 50 .mu.M 400 nM 16 3.3 0.01 to 100 .mu.M 100 nM
656 ##STR103## 0.4 to 50 .mu.M 0 0.01 to 100 .mu.M >100 .mu.M
657 ##STR104## 0.4 to 50 .mu.M 0 0.01 to 100 .mu.M >100 .mu.M
658 ##STR105## 0.01 to 50 .mu.M 40 nM 8 13 0.0001 to 100 .mu.M 2.5
nM 659 ##STR106## 0.4 to 50 .mu.M 0 0.01 to 100 .mu.M 10 .mu.M 660
##STR107## 0.2 to 12.5 .mu.M 800 nM 27 0.01 to 100 .mu.M 50 nM 661
##STR108## 0.1 to 50 .mu.M 500 nM 7 0.01 to 100 .mu.M 20 nM 662
##STR109## 0.2 to 50 .mu.M 0 0.0001 to 100 .mu.M >100 .mu.M 663
##STR110## 0.2 to 50 .mu.M 200 nM 43 7 0.001 to 100 .mu.M 100 nM
664 ##STR111## 0.2 to 50 .mu.M 400 nM 33 22 0.001 to 100 .mu.M 50
nM 665 ##STR112## 0.1-50 .mu.M 150 nM 24 30 0.001 to 100 .mu.M 50
nM 666 ##STR113## 0.1-50 .mu.M 150 nM 31 28 0.001 to 100 .mu.M 100
nM 667 ##STR114## 0.02-10 .mu.M 80 nM 27 2 0.001 to 100 .mu.M 50 nM
668 ##STR115## 0.02-10 .mu.M 10 .mu.M 11 4.7 0.001 to 100 .mu.M 100
nM 669 ##STR116## 0.8 to 50 .mu.M 4 .mu.M 11 16.0 0.001 to 100
.mu.M 10 .mu.M 670 ##STR117## 0.4 to 50 .mu.M No effect up to 25
.mu.M -- 13.0 0.001 to 100 .mu.M >100 .mu.M 671 ##STR118## 0.4
to 50 .mu.M 3.1 .mu.M 35 12.5 0.001 to 100 .mu.M 200 nM 672
##STR119## 0.8 to 50 .mu.M 0 No inh 0.01 to 100 .mu.M 100 .mu.M 673
##STR120## 0.8 to 50 .mu.M 0 No inh 0.01 to 100 .mu.M 100 .mu.M 674
##STR121## 0.8 to 50 .mu.M 0 Dead at 25 .mu.M 0.01 to 100 .mu.M 50
.mu.M 675 ##STR122## 0.8 to 50 .mu.M 0 No inh 0.001 to 100 .mu.M
>100 .mu.M 676 ##STR123## 0.8 to 50 .mu.M 0 No inh 0.01 to 100
.mu.M 100 .mu.M 677 ##STR124## 0.05 to 25 .mu.M 1.6 .mu.M 23 4.5
0.001 to 100 .mu.M 5 nM 678 ##STR125## 0.8 to 50 .mu.M 0 No inh
0.001 to 100 .mu.M >100 .mu.M 679 ##STR126## 0.8 to 50 .mu.M 0
No inh 0.001 to 100 .mu.M >100 .mu.M 680 ##STR127## 0.01 to 100
.mu.M >100 .mu.M 681 ##STR128## 0.8 to 50 .mu.M 3 .mu.M 3 2.5
0.01 to 100 .mu.M 200 nM 682 ##STR129## 0.8 to 50 .mu.M 50 .mu.M 8
1.1 0.01 to 100 .mu.M 150 nM 683 ##STR130## 0.01 to 0.1 .mu.M 20 nM
9 9.0 0.001 to 100 .mu.M 1 nM 684 ##STR131## 0.4 to 50 .mu.M 0 No
inh 0.01 to 100 .mu.M 100 .mu.M 685 ##STR132## 0.125 to 5 .mu.M 1.0
.mu.M 20 1.0 0.01 to 100 .mu.M 150 nM 686 ##STR133## 0.4 to 50
.mu.M 0 No inh 0.01 to 100 .mu.M 100 .mu.M 687 ##STR134## 0.125 to
5 .mu.M 0 No inh 0.01 to 100 .mu.M 200 nM 688 ##STR135## 0.4 to 50
.mu.M 0 No inh 0.01 to 100 .mu.M >100 .mu.M 689 ##STR136## 5.0
to 40 .mu.M 35 .mu.M 48 0.01 to 100 .mu.M 200 nM 690 ##STR137## 5.0
to 40 .mu.M 10 .mu.M 38 0.01 to 100 .mu.M 150 nM 691 ##STR138## 1.0
to 25 .mu.M 0 No inh 0.01 to 100 .mu.M 100 nM 692 ##STR139## 0.03
to 5 .mu.M 1 .mu.M 27 0.01 to 100 .mu.M 1 nM 693 ##STR140## 0.4 to
50 .mu.M 0 No inh 0.01 to 100 .mu.M >100 .mu.M
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* * * * *