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Cancer Patent Abstract
The present invention relates to novel compounds, pharmaceutical
compositions and methods for treating tumors, cancer and hyperproliferative
diseases including psoriasis, genital warts and hyperproliferative
cell growth diseases, including hyperproliferative keratinocyte
diseases such as hyperkeratosis, ichthyosis, keratoderma or lichen
planus. These compounds are described according to the chemical
structure: ##STR00001## where R.sup.1 is H, OH, F, Cl, Br, I, a
C.sub.1-C.sub.6 optionally substituted alkyl or alkenyl group, an
optionally substituted aryl group or a ##STR00002## group; R.sub.a
is a H, OH, C.sub.1-C.sub.10, optionally substituted alkyl or alkenyl
group, an optionally substituted O--(C.sub.1-C.sub.7 alkyl group)
or O-aryl group, an amine group which is optionally substituted
with at least one C.sub.1-C.sub.10 alkyl group which may be optionally
substituted, or a single optionally substituted aryl group, biphenyl
group, (C.sub.1-C.sub.6) alkylenearyl group, (C.sub.1-C.sub.6) alkylenebiphenyl
group, heteroaryl group, heterocyclic group, (C.sub.1-C.sub.6) alkylene
heteroaryl group or (C.sub.1-C.sub.6) alkylene heterocyclic group;
R.sup.2 is a ##STR00003## group; R.sub.b is a H, OH, C.sub.1-C.sub.10,
optionally substituted alkyl or alkenyl group, an optionally substituted
O--(C.sub.1-C.sub.7 alkyl group) or O-aryl group, an amine group
which is optionally substituted with at least one C.sub.1-C.sub.10
alkyl group which may be optionally substituted, or a single optionally
substituted aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl
group, (C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group,
heterocyclic group, (C.sub.1-C.sub.6) alkylene heteroaryl group
or (C.sub.1-C.sub.6) alkylene heterocyclic group; R.sup.3 and R.sup.6
are each independently selected from H, OH, F, Cl, Br, I, a C.sub.1-C.sub.6
optionally substituted alkyl or alkenyl group, an optionally substituted
aryl group, a carbamate, alkylene carbamate, urethane or alkylene
urethane; R.sup.4 is a ##STR00004## group, wherein R.sub.b is as
described above; and R.sup.5 is a ##STR00005## group, wherein R.sub.b
is as described above, with the proviso that at least one of R.sup.1
and R.sup.2 or R.sup.4 and R.sup.5 contains an R.sub.a or R.sub.b
group which is an amine group which is optionally substituted with
at least one C.sub.1-C.sub.10 alkyl group which may be optionally
substituted, or a single optionally substituted aryl group, biphenyl
group, (C.sub.1-C.sub.6) alkylenearyl group, (C.sub.1-C.sub.6) alkylenebiphenyl
group, heteroaryl group, heterocyclic group, (C.sub.1-C.sub.6) alkylene
heteroaryl group or (C.sub.1-C.sub.6) alkylene heterocyclic group;
or a stereoisomer, pharmaceutically acceptable salt, solvate, and
polymorph thereof.
Cancer Patent Claims
We claim:
1. A compound according to the structure: ##STR00061## where R.sup.1
is an optionally substituted ##STR00062## group; R.sub.a is a H,
OH, a C.sub.1-C.sub.10 optionally substituted alkyl or alkenyl group,
an optionally substituted O--(C.sub.1-C.sub.7 alkyl group) or O-aryl
group, an amine group which is optionally substituted with at least
one C.sub.1-C.sub.10 alkyl group which may be optionally substituted,
or a single optionally substituted aryl group, biphenyl group, (C.sub.1-C.sub.6)
alkylenearyl group, (C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl
group, heterocyclic group, (C.sub.1-C.sub.6) alkylene heteroaryl
group or (C.sub.1-C.sub.6) alkylene heterocyclic group; R.sup.2
is a ##STR00063## group; R.sub.b is a H, OH, C.sub.1-C.sub.10, optionally
substituted alkyl or alkenyl group, an optionally substituted O--(C.sub.1-C.sub.7
alkyl group) or O-aryl group, an amine group which is optionally
substituted with at least one C.sub.1-C.sub.10 alkyl group which
may be optionally substituted, or a single optionally substituted
aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl group,
(C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group, heterocyclic
group, (C.sub.1-C.sub.6) alkylene heteroaryl group or (C.sub.1-C.sub.6)
alkylene heterocyclic group; with the proviso that at least one
of R.sup.1 and R.sup.2 contains an R.sub.a or R.sub.b group which
is an amine group which is substituted with at least one C.sub.1-C.sub.10
alkyl group which is substituted, or a single optionally substituted
aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl group,
(C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group, heterocyclic
group, (C.sub.1-C.sub.6) alkylene heteroaryl group or (C.sub.1-C.sub.6)
alkylene heterocyclic group; or a stereoisomer, pharmaceutically
acceptable salt or solvate thereof.
2. The compound according to claim 1 wherein R.sub.a is OH or an
optionally substituted O--(C.sub.1-C.sub.7 alkyl group) or O-aryl
group; and R.sub.b is an amine group which is optionally substituted
with at least one C.sub.1-C.sub.10 alkyl group which may be optionally
substituted, or an optionally substituted aryl group, biphenyl group,
(C.sub.1-C.sub.6) alkylenearyl group, (C.sub.1-C.sub.6) alkylenebiphenyl
group, heteroaryl group, heterocyclic group, (C.sub.1-C.sub.6) alkylene
heteroaryl group or (C.sub.1-C.sub.6) alkylene heterocyclic group.
3. The compound according to claim 1 wherein R.sub.a is OH.
4. The compound according to claim 1 wherein R.sub.a is an optionally
substituted O--(C.sub.1-C.sub.7 alkyl group) or O-aryl group.
5. The compound according to claim 2 wherein R.sub.a is an optionally
substituted O--(C.sub.1-C.sub.7 alkyl group) or O-aryl group.
6. The compound according to claim 2 wherein R.sub.a is an optionally
substituted O--(C.sub.1-C.sub.7 alkyl group).
7. The compound according to claim 1 wherein R.sub.b is an amine
group which is optionally substituted with at least one C.sub.1-C.sub.10
alkyl group which may be optionally substituted, or a single optionally
substituted aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl
group, (C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group,
heterocyclic group, (C.sub.1-C.sub.6) alkylene heteroaryl group
or (C.sub.1-C.sub.6) alkylene heterocyclic group.
8. The compound according to claim 2 wherein R.sub.b is an amine
group which is optionally substituted with at least one C.sub.1-C.sub.10
alkyl group which may be optionally substituted, or a single optionally
substituted aryl group, (C.sub.1-C.sub.6) alkylenearyl group, heteroaryl
group, heterocyclic group, (C.sub.1-C.sub.6) alkylene heteroaryl
group or (C.sub.1-C.sub.6) alkylene heterocyclic group.
9. The compound according to claim 4 wherein R.sub.b is an amine
group which is optionally substituted with at least one C.sub.1-C.sub.10
alkyl group which may be optionally substituted, or a single optionally
substituted aryl group, (C.sub.1-C.sub.6) alkylenearyl group, heteroaryl
group, heterocyclic group, (C.sub.1-C.sub.6) alkylene heteroaryl
group or (C.sub.1-C.sub.6) alkylene heterocyclic group.
10. The compound according to claim 1 wherein R.sub.a is an optionally
substituted O--(C.sub.1-C.sub.7 alkyl group) and R.sub.b is an amine
group which is optionally substituted with at least one C.sub.1-C.sub.10
alkyl group which may be optionally substituted, or a single optionally
substituted aryl group, (C.sub.1-C.sub.6) alkylenearyl group, heteroaryl
group, heterocyclic group, (C.sub.1-C.sub.6) alkylene heteroaryl
group or (C.sub.1-C.sub.6) alkylene heterocyclic group.
11. The compound according to claim 1 wherein R.sub.b is an amine
group which is optionally substituted with a single cyclohexyl group,
an optionally substituted phenyl group, or an optionally substituted
benzyl group and R.sub.a is a O--(C.sub.1-C.sub.3 alkyl) group or
an O-phenyl group.
12. The compound according to claim 2 wherein R.sub.b is an amine
group which is optionally substituted with a single cyclohexyl group,
an optionally substituted phenyl group, or an optionally substituted
benzyl group and R.sub.a is a O--(C.sub.1-C.sub.3 alkyl) group or
an O-phenyl group.
13. The compound according to claim 4 wherein R.sub.b is an amine
group which is optionally substituted with a single cyclohexyl group,
an optionally substituted phenyl group, or an optionally substituted
benzyl group and R.sub.a is a O--(C.sub.1-C.sub.3 alkyl) group or
an O-phenyl group.
14. A pharmaceutical composition comprising an effective amount
of a compound according to claim 1 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
15. A pharmaceutical composition comprising an effective amount
of a compound according to claim 2 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
16. A pharmaceutical composition comprising an effective amount
of a compound according to claim 3 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
17. A pharmaceutical composition comprising an effective amount
of a compound according to claim 4 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
18. A pharmaceutical composition comprising an effective amount
of a compound according to claim 5 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
19. A pharmaceutical composition comprising an effective amount
of a compound according to claim 6 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
20. A pharmaceutical composition comprising an effective amount
of a compound according to claim 7 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
21. A pharmaceutical composition comprising an effective amount
of a compound according to claim 8 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
22. A pharmaceutical composition comprising an effective amount
of a compound according to claim 9 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
23. A pharmaceutical composition comprising an effective amount
of a compound according to claim 10 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
24. A pharmaceutical composition comprising an effective amount
of a compound according to claim 11 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
25. A pharmaceutical composition comprising an effective amount
of a compound according to claim 12 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
26. A pharmaceutical composition comprising an effective amount
of a compound according to claim 13 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
27. A compound according to the chemical structure: ##STR00064##
28. A pharmaceutical composition comprising an effective amount
of a compound according to claim 27 in combination with a pharmaceutically
acceptable carrier, additive or excipient.
Cancer Patent Description
FIELD OF THE INVENTION
The present invention relates to novel compounds, pharmaceutical
compositions and methods for treating tumors, cancer and hyperproliferative
diseases including psoriasis, genital warts and hyperproliferative
cell growth diseases, including hyperproliferative keratinocyte
diseases such as hyperkeratosis, ichthyosis, keratoderma or lichen
planus.
BACKGROUND OF THE INVENTION
Chronic myelogenous leukaemia (CML) is a hematological stem cell
disorder that is associated with a specific chromosomal abnormality
whereby the Abelson (c-abl) proto-oncogene, translocated from chromosome
9, is fused to the breakpoint cluster region (bcr) gene on chromosome
22 as shown in slide 2. The bcr-abl fusion gene codes for a tyrosine
kinase that is activated constitutively and is thus able to transform
cells and cause malignancy: white blood cells divide constantly
leading to a blast crisis. Recently, a selective inhibitor of p210-Bcr-Abl
tyrosine kinase, STI-571, was designed by Druker and co-workers.
See Drucker, et al., Nature Med., 2, 561-566 (1996) and Schindler,
et al., Science, 289, 1938-1941 (2000). STI-571 (tradename: Gleevec.RTM.)
is the first kinase inhibitor approved by the FDA and blocks the
ATP-binding site on Abl and Bcr-Abl kinases, resulting in both inhibition
of proliferation and induction of apoptosis in Bcr-Abl positive
cell lines. While STI-571 leads to a complete hematological response
in 96% of the patients treated for more than four weeks, patients
with advanced disease often relapse, their tumor cells become resistant
to the drug and these eventually grow out of control. One of the
possible causes of resistance of cancerous cells to STI-571 is a
mutation that replaces a single amino acid in the active site of
the kinase, preventing binding of the drug to the kinase. See Gorre,
et al., Science, 293, 876-880 (2001).
##STR00006##
The present invention relates to the goal of preparing novel tyrosine
kinase inhibitors that would be given alone or in combination with
STI-571 to cancer patients since the cancerous cells should be less
able to become resistant to all the drugs at once. In addition,
novel active substances that would hit alternative targets and work
alone or in synergy with STI-571 are also sought after targets.
OBJECTS OF THE INVENTION
It is an object of the invention to provide novel compounds which
can be used to treat one or more of tumors, cancer and proliferative
diseases as otherwise described herein.
It is an additional object of the invention to provide pharmaceutical
compounds based upon the compounds disclosed herein.
It is yet another object of the invention to provide methods to
treat patients for one or more of tumors, cancer, hyperproliferative
diseases including psoriasis, genital warts and hyperproliferative
cell growth diseases, including hyperproliferative keratinocyte
diseases such as hyperkeratosis, ichthyosis, keratoderma or lichen
planus.
It is still another object of the present invention to use the
present compounds alone or synergistically with other anti-tumor/anti-cancer
agents for the treatment of tumors and/or cancer in patients.
It is yet another object of the present invention to provide pharmaceutical
compositions which may be advantageously used in combination with
other anti-tumor/anti-cancer agents in the interest of providing
synergistic therapy to patients in need of such therapy.
One or more of these and/or other objects of the invention will
be readily apparent from a review of the disclosure of the present
invention herein.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts a number of representative compounds according to
the present invention.
FIG. 2 depicts a chemical scheme directed to certain furan compounds
according to the present invention.
FIG. 3 depicts a chemical scheme directed to alternative furan
compounds according to the present invention.
FIG. 4 shows a simple chemical scheme directed to the synthesis
of acetylene compounds according to the present invention.
FIGS. 5-12 show the effect of varying the chemical structure of
a number compounds according to the present invention on biological
activity.
FIG. 13 shows the effect of compounds on cell growth.
FIG. 14 shows the effect of compounds on the number of viable cells.
SUMMARY OF THE INVENTION
The present invention relates to compounds according to either
of the structures set forth below:
##STR00007## where R.sup.1 is H, OH, F, Cl, Br, I, a C.sub.1-C.sub.6
optionally substituted alkyl or alkenyl group, an optionally substituted
aryl group or a
##STR00008## group; R.sub.a is a H, OH, C.sub.1-C.sub.10, optionally
substituted alkyl or alkenyl group, an optionally substituted O--(C.sub.1-C.sub.7
alkyl group) or O-aryl group, an amine group which is optionally
substituted with at least one C.sub.1-C.sub.10 alkyl group which
may be optionally substituted, or a single optionally substituted
aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl group,
(C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group, heterocyclic
group, (C.sub.1-C.sub.6) alkylene heteroaryl group or (C.sub.1-C.sub.6)
alkylene heterocyclic group; R.sup.2 is a
##STR00009## group; R.sub.b is a H, OH, C.sub.1-C.sub.10, optionally
substituted alkyl or alkenyl group, an optionally substituted O--(C.sub.1-C.sub.7
alkyl group) or O-aryl group, an amine group which is optionally
substituted with at least one C.sub.1-C.sub.10 alkyl group which
may be optionally substituted, or a single optionally substituted
aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl group,
(C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group, heterocyclic
group, (C.sub.1-C.sub.6) alkylene heteroaryl group or (C.sub.1-C.sub.6)
alkylene heterocyclic group; R.sup.3 and R.sup.6 are each independently
selected from H, OH, F, Cl, Br, I, a C.sub.1-C.sub.6 optionally
substituted alkyl or alkenyl group, an optionally substituted aryl
group, a carbamate, alkylene carbamate, urethane or alkylene urethane;
R.sup.4 is a
##STR00010## group, wherein R.sub.a is as described above; and
R.sup.5 is a
##STR00011## group, wherein R.sub.b is as described above, with
the proviso that at least one of R.sup.1 and R.sup.2 or R.sup.4
and R.sup.5 contains an R.sub.a or R.sub.b group which is an amine
group which is optionally substituted with at least one C.sub.1-C.sub.10
alkyl group which may be optionally substituted, or a single optionally
substituted aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl
group, (C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group,
heterocyclic group, (C.sub.1-C.sub.6) alkylene heteroaryl group
or (C.sub.1-C.sub.6) alkylene heterocyclic group; or a stereoisomer,
pharmaceutically acceptable salt, solvate, and polymorph thereof.
In preferred aspects of the present invention, the compounds according
to the present invention contain at least one ester group and at
least one amide group, preferably with each such group bonded directly
to the acetylene or furan moieties, such that R.sup.1 forms an ester
group with the acetylenic group and R.sup.2 forms an amide group
with the acetylenic moiety. In the case of the furan compounds,
R.sup.4 preferably forms an amide group such that R.sub.b is preferably
an amine or substituted amine group and R.sup.5 preferably forms
an ester group with the furan moiety, such that R.sub.a is preferably
an O-alkyl or O-aryl group as otherwise defined hereinabove. These
compounds are presented below.
##STR00012##
Pharmaceutical compositions based upon the above-described compounds
comprise an effective amount of compound in combination with a pharmaceutically
acceptable carrier, additive or excipient.
Other aspects of the present invention are directed to methods
of treating tumors, cancer, hyperproliferative diseases including
psoriasis, genital warts and hyperproliferative cell growth diseases,
including hyperproliferative keratinocyte diseases such as hyperkeratosis,
ichthyosis, keratoderma or lichen planus, the method comprising
administering to a patient in need thereof an effective amount of
a compound according to the formula:
##STR00013## where R.sup.1 is H, F, Cl, Br, I, a C.sub.1-C.sub.6
optionally substituted alkyl or alkenyl group, an optionally substituted
aryl group or a
##STR00014## group; R.sub.a is a C.sub.1-C.sub.10, optionally substituted
alkyl or alkenyl group, an optionally substituted O--(C.sub.1-C.sub.7
alkyl group) or O-aryl group, an amine group which is optionally
substituted with at least one C.sub.1-C.sub.10 alkyl group which
may be optionally substituted, or a single optionally substituted
aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl group,
(C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group, heterocyclic
group, (C.sub.1-C.sub.6) alkylene heteroaryl group or (C.sub.1-C.sub.6)
alkylene heterocyclic group; R.sup.2 is a
##STR00015## group; R.sub.b is a C.sub.1-C.sub.10, optionally substituted
alkyl or alkenyl group, an optionally substituted O--(C.sub.1-C.sub.7
alkyl group) or O-aryl group, an amine group which is optionally
substituted with at least one C.sub.1-C.sub.10 alkyl group which
may be optionally substituted, or a single optionally substituted
aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl group,
(C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group, heterocyclic
group, (C.sub.1-C.sub.6) alkylene heteroaryl group or (C.sub.1-C.sub.6)
alkylene heterocyclic group; R.sup.3 and R.sup.6 are each independently
selected from H, F, Cl, Br, I, a C.sub.1-C.sub.6 optionally substituted
alkyl or alkenyl group, an optionally substituted aryl group, a
carbamate, alkylene carbamate, urethane or alkylene urethane; R.sup.4
is a
##STR00016## group, wherein R.sub.b is as described above, and
R.sup.5 is a
##STR00017## group, wherein R.sub.a is as described above; with
the proviso that at least one of R.sup.1 and R.sup.2 or R.sup.4
and R.sup.5 contains an R.sub.a or R.sub.b group which is an amine
group which is optionally substituted with at least one C.sub.1-C.sub.10
alkyl group which may be optionally substituted, or a single optionally
substituted aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl
group, (C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group,
heterocyclic group, (C.sub.1-C.sub.6) alkylene heteroaryl group
or (C.sub.1-C.sub.6) alkylene heterocyclic group; or a stereoisomer,
pharmaceutically acceptable salt, solvate, and polymorph thereof
in combination with a pharmaceutically acceptable carrier, additive
or excipient.
DETAILED DESCRIPTION OF THE INVENTION
The following terms are used throughout the specification to describe
the present invention:
"Patient" or "subject" is used throughout the
specification to describe an animal, generally a mammalian animal,
including a human, to whom treatment or use with the compounds or
compositions according to the present invention is provided. For
treatment or use with/or of those conditions or disease states which
are specific for a specific animal (especially, for example, a human
subject or patient), the term patient or subject refers to that
particular animal.
"Alkyl" refers to a fully saturated monovalent hydrocarbon
radical containing carbon and hydrogen which may be a straight chain,
branched, or cyclic group. Examples of alkyl groups are methyl,
ethyl, n-butyl, n-heptyl, isopropyl, 2-methylpropyl, cyclopropyl,
cyclopropylmethyl, cyclobutyl, cyclopentyl, cyclopentylethyl and
cyclohexyl. "Cycloalkyl" groups refer to cyclic alkyl
groups such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
C.sub.1-C.sub.7 alkyl groups are preferably used in certain aspects
of the present invention, although the alkyl group may be larger,
in certain advantageous instances. The term alkyl also refers to
monocyclic, bicyclic, tricyclic and tetracyclic alkyl (i.e., hydrocarbon)
groups. Alkyl groups according to the present invention may be substituted
or unsubstituted.
The term "alkenyl" refers to an alkyl group with at least
one double bond between adjacent carbon atoms within the chemical
group. The term "alkylene" refers to an optionally substituted
group having the general formula --(CH.sub.2).sub.n-- where n is
a positive integer from 1 to 12, preferably from 1 to 6, more preferably
from 1 to 3.
The term "substituted" refers to a chemical group or
moiety which occurs on (is bonded to) another group and may include
one or more functional groups such an alkyl containing from 1 to
6 carbon atoms, preferably a lower alkyl containing 1-3 carbon atoms,
aryl, substituted aryl, acyl, ester, halogen (i.e., alkyl halos,
e.g., CF.sub.3), hydroxy, alkoxy, carboxy, alkoxyalkyl, amino, alkyl
and dialkyl amino, acylamino, acyloxy, aryloxy, aryloxyalkyl, carboxyalkyl,
carboxamido, thio, thioethers, both saturated and unsaturated cyclic
hydrocarbons, heterocycles and the like. Alkyl and ester groups,
for example,
##STR00018## groups are preferred substituents in certain aspects
of the present invention. Substituted, for example, as in "substituted
alkyl" or "substituted alkenyl", means that in the
hydrocarbyl, hydrocarbylene, alkyl, alkenyl or other moiety, at
least one hydrogen atom bound to a carbon atom is replaced with
one or more substituents that are functional groups such as hydroxyl,
alkoxy, thio, amino, halo, and the like, as described above. When
the term "substituted" appears prior to a list of possible
substituted groups, it is intended that the term apply to every
member of that group.
The term "aryl" refers to a substituted or unsubstituted
monovalent aromatic radical having a single ring (e.g., phenyl)
or multiple condensed rings (e.g., naphthyl). Other examples include
heterocyclic aromatic ring groups having one or more nitrogen, oxygen,
or sulfur atoms in the ring, such as imidazolyl, furyl, pyrrolyl,
pyridyl, thienyl and indolyl. The term "heteroaryl" refers
to an aryl group which contains at least one atom selected from
O, N and S.
"Halo" and "halogen" are used in the conventional
sense to refer to a chloro, bromo, fluoro or iodo substituent. The
terms "haloalkyl," "haloalkenyl" or "haloalkynyl"
(or "halogenated alkyl," "halogenated alkenyl,"
or "halogenated alkynyl") refers to an alkyl, alkenyl
or alkynyl group, respectively, in which at least one of the hydrogen
atoms in the group has been replaced with a halogen atom.
"Heterocycle" or "heterocyclic" refers to a
carbocylic ring wherein one or more carbon atoms have been replaced
with one or more heteroatoms such as nitrogen, oxygen or sulfur.
A substitutable nitrogen on an aromatic or non-aromatic heterocyclic
ring may be optionally substituted. The heteroatoms N or S may also
exist in oxidized form such as NO, SO and SO.sub.2. Examples of
heterocycles include, but are not limited to, piperidine, pyrrolidine,
morpholine, thiomorpholine, piperazine, tetrahydrofuran, tetrahydropyran,
2-pyrrolidinone, .delta.-velerolactam, .delta.-velerolactone and
2-ketopiperazine, among numerous others.
The term "biphenyl" refers to a group which contains
two optionally substituted aryl groups, preferably phenyl groups,
which are linked together at a single carbon atom on each phenyl
group.
The terms "carbamate", "alkylene carbamate",
"urethane" or "alkylene urethane" refers to
a substituent or moiety which may be represented by the structure
##STR00019## where R is a optionally substituted C.sub.1-C.sub.8
alkyl group or an aryl group, X is O (carbamate) or N (urethane)
and y is from 0 to 6. Compounds according to the present invention
based upon a furan skeleton may contain carbamates, alkylene carbamates,
urethanes or alkylene urethanes as indicated at R.sup.3 and R.sup.6
of the furan ring.
The term "compound" is used herein to refer to any specific
chemical compound disclosed herein. Within its use in context, the
term generally refers to a single compound, but in certain instances
may also refer to stereoisomers and/or optical isomers (including
racemic mixtures) of disclosed compounds.
The term "effective amount" refers to the amount of a
selected compound according to the present invention which is used
in an amount to produce an intended effect within the context of
its use and in particular, the treatment method to be used. The
precise amount of a compound according to the present invention
used in a given context will vary depending upon the particular
compound selected and its intended use, the disease or condition
to be treated, the method of delivery, the age and weight of the
subject, route of administration, and so forth, but may be easily
determined by routine experimentation. In the case of the treatment
of a condition or disease state, an effective amount is that amount
which is used to effectively treat the particular condition or disease
state.
The term "pharmaceutically acceptable" refers to a carrier,
additive or excipient which is not unacceptably toxic to the subject
to which it is administered.
The term "neoplasia" or "cancer" is used throughout
the specification to refer to the pathological process that results
in the formation and growth of a cancerous or malignant neoplasm,
i.e., abnormal tissue that grows by cellular proliferation, often
more rapidly than normal and continues to grow after the stimuli
that initiated the new growth cease. Malignant neoplasms show partial
or complete lack of structural organization and functional coordination
with the normal tissue and most invade surrounding tissues, metastasize
to several sites, and are likely to recur after attempted removal
and to cause the death of the patient unless adequately treated.
As used herein, the term neoplasia is used to describe all cancerous
disease states and embraces or encompasses the pathological process
associated with malignant hematogenous, ascitic and solid tumors.
Representative cancers include, for example, stomach, colon, rectal,
liver, pancreatic, lung, breast, cervix uteri, corpus uteri, ovary,
prostate, testis, bladder, renal, brain/CNS, head and neck, throat,
Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, leukemia,
melanoma, acute lymphocytic leukemia, acute myelogenous leukemia,
Ewing's sarcoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma,
Wilms' tumor, neuroblastoma, hairy cell leukemia, mouth/pharynx,
oesophagus, larynx, kidney cancer and lymphoma, among others, which
may be treated by one or more compounds according to the present
invention.
The term "tumor" is used to describe a malignant or benign
growth or tumefacent.
The term "hyperproliferative disease state" refers to
a disease state in which cells are growing in an uncontrolled manner,
whether that growth is cancerous or not. Such a disease state may
be reflected in psoriasis or genital warts or other hyperproliferative
cell growth diseases, including hyperproliferative keratinocyte
diseases including hyperkeratosis, ichthyosis, keratoderma or lichen
planus, all of which disease states may be treated using compounds
according to the present invention.
The present invention includes the compositions comprising the
pharmaceutically acceptable acid addition salts of compounds of
the present invention. The acids which are used to prepare the pharmaceutically
acceptable acid addition salts of the aforementioned base compounds
useful in this invention are those which form non-toxic acid addition
salts, i.e., salts containing pharmacologically acceptable anions,
such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate,
bisulfate, phosphate, acid phosphate, acetate, lactate, citrate,
acid citrate, tartrate, bitartrate, succinate, maleate, fumarate,
gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate,
benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1,1'-methylene-bis-(2-hydroxy-3
naphthoate)] salts, among others.
The invention also includes compositions comprising base addition
salts of the present compounds. The chemical bases that may be used
as reagents to prepare pharmaceutically acceptable base salts of
the present compounds that are acidic in nature are those that form
non-toxic base salts with such compounds. Such non-toxic base salts
include, but are not limited to those derived from such pharmacologically
acceptable cations such as alkali metal cations (e.g., potassium
and sodium) and alkaline earth metal cations (e, calcium and magnesium),
ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine),
and the lower alkanolammonium and other base salts of pharmaceutically
acceptable organic amines, among others.
The compounds of this invention include all stereoisomers where
relevant (i.e., cis and trans isomers) and all optical isomers of
the present compounds (e.g., R and S enantiomers), as well as racemic,
diastereomeric and other mixtures of such isomers, as well as all
polymorphs of the compounds.
The compositions of the present invention may be formulated in
a conventional manner using one or more pharmaceutically acceptable
carriers and may also be administered in controlled-release formulations.
Pharmaceutically acceptable carriers that may be used in these pharmaceutical
compositions include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic acid,
potassium sorbate, partial glyceride mixtures of saturated vegetable
fatty acids, water, salts or electrolytes, such as prolamine sulfate,
disodium hydrogen phosphate, potassium hydrogen phosphate, sodium
chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol, sodium
carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block
polymers, polyethylene glycol and wool fat.
The compositions of the present invention may be administered orally,
parenterally, by inhalation spray, topically, rectally, nasally,
buccally, vaginally or via an implanted reservoir. The term "parenteral"
as used herein includes subcutaneous, intravenous, intramuscular,
intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques.
Preferably, the compositions are administered orally, intraperitoneally
or intravenously.
Sterile injectable forms of the compositions of this invention
may be aqueous or oleaginous suspension. These suspensions may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents and suspending agents. The sterile
injectable preparation may also be a sterile injectable solution
or suspension in a non-toxic parenterally-acceptable diluent or
solvent, for example as a solution in 1,3-butanediol. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil may be
employed including synthetic mono- or di-glycerides. Fatty acids,
such as oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural pharmaceutically-acceptable
oils, such as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil solutions or suspensions may also contain a
long-chain alcohol diluent or dispersant, such as Ph. He1v or similar
alcohol.
The pharmaceutical compositions of this invention may be orally
administered in any orally acceptable dosage form including, but
not limited to, capsules, tablets, aqueous suspensions or solutions.
In the case of tablets for oral use, carriers which are commonly
used include lactose and corn starch. Lubricating agents, such as
magnesium stearate, are also typically added. For oral administration
in a capsule form, useful diluents include lactose and dried corn
starch. When aqueous suspensions are required for oral use, the
active ingredient is combined with emulsifying and suspending agents.
If desired, certain sweetening, flavoring or coloring agents may
also be added.
Alternatively, the pharmaceutical compositions of this invention
may be administered in the form of suppositories for rectal administration.
These can be prepared by mixing the agent with a suitable non-irritating
excipient which is solid at room temperature but liquid at rectal
temperature and therefore will melt in the rectum to release the
drug. Such materials include cocoa butter, beeswax and polyethylene
glycols.
The pharmaceutical compositions of this invention may also be administered
topically, especially to treat skin cancers, psoriasis or other
diseases which occur in or on the skin. Suitable topical formulations
are readily prepared for each of these areas or organs. Topical
application for the lower intestinal tract can be effected in a
rectal suppository formulation (see above) or in a suitable enema
formulation. Topically-acceptable transdermal patches may also be
used.
For topical applications, the pharmaceutical compositions may be
formulated in a suitable ointment containing the active component
suspended or dissolved in one or more carriers. Carriers for topical
administration of the compounds of this invention include, but are
not limited to, mineral oil, liquid petrolatum, white petrolatum,
propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying
wax and water. Alternatively, the pharmaceutical compositions can
be formulated in a suitable lotion or cream containing the active
components suspended or dissolved in one or more pharmaceutically
acceptable carriers. Suitable carriers include, but are not limited
to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters
wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
For ophthalmic use, the pharmaceutical compositions may be formulated
as micronized suspensions in isotonic, pH adjusted sterile saline,
or, preferably, as solutions in isotonic, pH adjusted sterile saline,
either with our without a preservative such as benzylalkonium chloride.
Alternatively, for ophthalmic uses, the pharmaceutical compositions
may be formulated in an ointment such as petrolatum.
The pharmaceutical compositions of this invention may also be administered
by nasal aerosol or inhalation. Such compositions are prepared according
to techniques well-known in the art of pharmaceutical formulation
and may be prepared as solutions in saline, employing benzyl alcohol
or other suitable preservatives, absorption promoters to enhance
bioavailability, fluorocarbons, and/or other conventional solubilizing
or dispersing agents.
The amount of compound in a pharmaceutical composition of the instant
invention that may be combined with the carrier materials to produce
a single dosage form will vary depending upon the host and disease
treated, the particular mode of administration. Preferably, the
compositions should be formulated to contain between about 0.5 milligram
to about 750 milligrams, more preferably about 1 milligram to about
600 milligrams, and even more preferably about 10 milligrams to
about 500 milligrams of active ingredient.
It should also be understood that a specific dosage and treatment
regimen for any particular patient will depend upon a variety of
factors, including the activity of the specific compound employed,
the age, body weight, general health, sex, diet, time of administration,
rate of excretion, drug combination, and the judgment of the treating
physician and the severity of the particular disease or condition
being treated.
Other aspects of the present invention are directed to methods
of treating tumors, cancer, hyperproliferative diseases including
psoriasis, genital warts and hyperproliferative cell growth diseases,
including hyperproliferative keratinocyte diseases such as hyperkeratosis,
ichthyosis, keratoderma or lichen planus, the method comprising
administering to a patient in need thereof an effective amount of
a compound according to the formula:
##STR00020## where R.sup.1 is H, OH, F, Cl, Br, I, a C.sub.1-C.sub.6
optionally substituted alkyl or alkenyl group, an optionally substituted
aryl group or a
##STR00021## group; R.sub.a is a H, OH, C.sub.1-C.sub.10, optionally
substituted alkyl or alkenyl group, an optionally substituted O--(C.sub.1-C.sub.7
alkyl group) or O-aryl group, an amine group which is optionally
substituted with at least one C.sub.1-C.sub.10 alkyl group which
may be optionally substituted, or a single optionally substituted
aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl group,
(C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group, heterocyclic
group, (C.sub.1-C.sub.6) alkylene heteroaryl group or (C.sub.1-C.sub.6)
alkylene heterocyclic group; R.sup.2 is a
##STR00022## group; R.sub.b is a H, OH, C.sub.1-C.sub.10, optionally
substituted alkyl or alkenyl group, an optionally substituted O--(C.sub.1-C.sub.7
alkyl group) or O-aryl group, an amine group which is optionally
substituted with at least one C.sub.1-C.sub.10 alkyl group which
may be optionally substituted, or a single optionally substituted
aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl group,
(C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group, heterocyclic
group, (C.sub.1-C.sub.6) alkylene heteroaryl group or (C.sub.1-C.sub.6)
alkylene heterocyclic group; R.sup.3 and R.sup.6 are each independently
selected from H, OH, F, Cl, Br, I, a C.sub.1-C.sub.6 optionally
substituted alkyl or alkenyl group, or an optionally substituted
aryl group; R.sup.4 is a
##STR00023## group, wherein R.sub.b is as described above; and
R.sup.5 is a
##STR00024## group, wherein R.sub.b is as described above, with
the proviso that at least one of R.sup.1 and R.sup.2 or R.sup.4
and R.sup.5 contains an R.sub.a or R.sub.b group which is an amine
group which is optionally substituted with at least one C.sub.1-C.sub.10
alkyl group which may be optionally substituted, or a single optionally
substituted aryl group, biphenyl group, (C.sub.1-C.sub.6) alkylenearyl
group, (C.sub.1-C.sub.6) alkylenebiphenyl group, heteroaryl group,
heterocyclic group, (C.sub.1-C.sub.6) alkylene heteroaryl group
or (C.sub.1-C.sub.6) alkylene heterocyclic group; or a stereoisomer,
pharmaceutically acceptable salt, solvate, and polymorph thereof,
optionally in combination with a pharmaceutically acceptable additive,
carrier or excipient.
Representative compounds of the present invention can be readily
synthesized in accordance with the general synthetic methods described
below and are illustrated more particularly in the schemes that
follow. Since the schemes are illustrative, the invention should
not be construed as being limited by the chemical reactions and
conditions expressed. The preparation of the various starting materials
used in the schemes is well within the skill of persons versed in
the art. Compounds not specifically mentioned may be readily synthesized
by analogy following techniques and methods well known to those
of skill in the art.
Unless specified to the contrary, reactions herein occur at approximately
atmospheric pressure and at a temperature of between about 0.degree.
C. and the boiling point of any organic solvent used in the reaction.
Inert organic solvents such as dichloromethane, diethyl ether, dimethylformamide,
chloroform or tetrahydrofuran are preferred solvents in the reactions
disclosed herein, although other solvents may be used where appropriate
or indicated. Reaction times can range from about one hour to about
forty-eight hours, and reactants optionally are stirred, shaken,
or agitated. Reactions can be done in one pot or in steps, unless
specified to the contrary.
General Chemistry and Structure Activity
Two scaffolds described hereafter preferably have been used: a
planar scaffold, represented by compounds containing a furan core
with various groups displayed at C-2, C-3, C-4 and C-5 of the furan
and an acetylene core (structure index).
##STR00025##
##STR00026## Some furans (R1=H or CH.sub.3, R2=CONHCy or CO.sub.2CH.sub.3,
R3=CO.sub.2CH.sub.3 or CONHCy, R4=CH.sub.3 or H) were prepared by
Diels-Alder cycloaddition of an acetylene bearing an amide and an
ester group (R5 and R6) on each side of the triple bond, followed
by regioselective reduction of the unsubstituted double bond and
retro Diels-Alder reaction (FIG. 2). The other furans (R1=H, R2=CONHCy,
R3=CO.sub.2CH.sub.3, R4=CH.sub.2CH.sub.2CH.sub.2NHCO.sub.2R with
R.dbd.CH.sub.3, (CH.sub.2).sub.3CH.sub.3, CH.sub.2CH(CH.sub.2CH.sub.3).sub.2,
CH.sub.2CH(NHBoc)CH(CH.sub.3).sub.2, CH.sub.2Cy, CH.sub.2Ph) were
prepared from pyrrolidinone after conversion to its corresponding
imide, diazotransfer, deacylation, cycloaddition with an acetylene
bearing an amide and an ester groups on each side of the triple
bond, cycloreversion and heating with an alcohol (FIG. 3).
The acetylenes were prepared by deprotonation of a propiolate (either
methyl or tert-butyl) with n-butyllithium and subsequent reaction
with an isocyanate (FIG. 4). Modifications may be readily made following
the aforementioned schemes. The furan and acetylene libraries were
both tested for activity in a cell-based assay where the death of
engineered murine myeloid cells (32D-bcr-abl), the survival of which
depends on the activity of bcr-abl tyrosine kinase, was sought.
The control cell line that enables the determination of the selectivity
of the compounds is also a murine cell line, which does not depend
on bcr-abl kinase activity to survive.
The biological activity of these compounds was evaluated in two
ways, a murine cell line differential proliferation assay and a
NCI 60 human carcinoma cell-line growth inhibition and cell death
assay. The murine cell death assays were performed using a p210bcrabl
transformed 32D murine cell line. This is a differential assay,
measuring the ability of the molecule to selectively inhibit the
transformed cells (32 Dbcrabl) over the non-transformed, growth
factor dependent parental cell line (32D). The NCl assay was performed
in the Developmental Therapeutics Program at the National Cancer
Institute. This assay measures the inhibition of cell growth and
cell death from sixty different human carcinomas. Both the acetylene
and furan structural cores represent novel chemical entities with
no prior reports of biological activity. Biological data collected
thus far show that both the ester and amide moiety provide the greatest
activity and are preferred. In addition, subtle changes in R1 confer
major differences in biological activity, suggesting a specific
cellular target for these molecules. The molecules K1P, AC19, AC22,
AN7A and AN7B (FIG. 1) showed good activity against a number of
carcinoma and model carcinoma cell lines. At present, the use of
these compounds is to be preferred in the treatment of tumors and
cancer.
Biological and Structure Activity
The furan and acetylene libraries were both tested for activity
in a cell-based assay where the death of engineered murine myeloid
cells (32D-bcr-abl), the survival of which is depends on the activity
of bcr-abl tyrosine kinase, was sought. The control cell line that
enables the determination of the selectivity of the compounds is
also a murine cell line, which does not depend on bcr-abl kinase
activity to survive.
We have found that a carbonyl on either side of the triple bond
was clearly preferred for activity since compounds AC2, AC4, AC5
and AC11 showed little or no activity (see structure index in FIG.
1). Investigation of the importance of the ester on activity, compounds
K1P and AC1 were compared. As seen in FIG. 5, the two compounds
performed equally well in the assay in terms of activity and selectivity.
At 100 nM, the compounds are still active with 50% of leukemic cells
surviving and 100% of the control cells surviving. The optimization
was therefore continued at the amide site. The ester can be later
modified to improve the pharmacokinetic properties of the future
drug. Investigation of the scaffold right side was then carried
out as shown in FIG. 6. Changing the amide group from cyclohexylamide
(K1P) to phenylamide (AC6) increased both selectivity and activity
while adding an electron donating group at the para position of
the aromatic ring (AC3) decreased activity and selectivity dramatically.
The position of substitution on the ring was further tested with
compounds AC15, AC16, and AC17. It was found that activity was regained
at the 1 uM level in the case of the ortho, meta and di-meta substitution
while selectivity remained low (FIG. 7). FIG. 8 shows the effect
of the nature of the substituent on the phenyl ring and revealed
that two electron withdrawing groups such as fluorine at the ortho
position (AC9) rendered the molecule inactive while two bulky inductive
group such as isopropyl groups (AC10) had moderate activity but
no selectivity. Adding an extra methylene unit between the phenyl
ring and the amide nitrogen gave an active benzylamide compound
(AC13) with little selectivity. These cell assays were usually carried
out using 50 cell/well in average and FIG. 8 shows the result of
an assay that used a varying number of cells per well, while keeping
the concentration of the compound constant at 1 uM. Complete selectivity
and activity was observed at 2000 cells per well. Using the phenylamide
(AC6) as a lead, acetylenes AC19, AC21 and AC22 were designed and
prepared to explore the space available for binding on the amide
side. As seen FIG. 9, these 3 new compounds all exhibit excellent
activity at 100 nM but only AC19 shows selectivity. Next a combination
of STI-571 and compounds AC22 and AC19 was tested. FIG. 10 shows
that STI-571 is moderately active (30% of the myeloid engineered
murine cells are killed but the all the control cells are still
alive) and selective at 10 nM in our assay while AC22 is very active
but not selective at 100 nM and not active at 10 nM. As a combination,
however, AC22 and STI-571 show complete activity and 70% selectivity,
while at 10 nM the activity remains and the selectivity is enhanced
to 100%. Neither of these compounds, on its own, displays such activity
and selectivity, therefore it seems that a combination of AC22 and
STI-571 act in synergy to combat leukemic cells while being non
toxic to control cells. A different result is obtained with AC19,
which, in combination with STI-571 does not increase the activity
or selectivity compared to STI-571 alone. This suggests that AC19
and STI-571 might be acting with the same target while AC22 and
STI-571 might bind to different sites thereby showing a multiplicative
effect of either compound (FIG. 11).
Furans AN7A and AN7B were also evaluated for activity and selectivity
in our assay and showed incredible activity and selectivity (both
100% at 10 nM); the graph shown in FIG. 12 corresponds to the cell
assay results for furan AN7B. In order to obtain additional information
about the behavior of our compounds in cancerous cells, 18 compounds
were submitted to and accepted by the NCl for testing on 60 human
carcinoma cell lines as set forth in the examples section. This
would confirm the activity seen in our cell-based assay and maybe
give an insight into the mechanism of action and biological target
of the acetylene/furan compounds. While the results were somewhat
disappointing (AC19 did not show activity below 10 uM and AC22 showed
mediocre growth inhibition between 2.6 and 7.3 uM in leukemia cell
lines only), K1P had an interesting pattern of activity whereby
it inhibited growth of leukemia cell lines (GI50 between 0.8 and
5 uM) and renal cell lines (GI50: 2.5 uM to 2.7 uM). As a result,
K1P was singled out by the NCl for further testing: MTD (Maximum
Tolerated Dose) and murine hollow fiber in vivo assay. These are
being carried out at the present time, along with testing of furans
AN7A and AN7B in the 60 human cancer cell lines.
The invention is described further in the following examples, which
are illustrative and in no way limiting.
EXAMPLES
In order to access a representative range of compounds using commercially
available starting materials, the general strategy for the preparation
of the acetylenes involved the deprotonation of methyl propiolate
with n-butyllithium (1.6M in hexanes) at -78.degree. C. followed
by addition of the desired isocyanate, source of the diversity at
the amide moiety. It was found that the use of 1.05 eq of n-Buli
gave a cleaner product than the use of 1.5 eq. Also, for compounds
of preparation type 2 (from AC14 onwards), it was found that stirring
for a reduced amount of time increased the yield.
The final compounds were obtained in moderate to high yields (23-84%).
Purification of these compounds was found difficult; they were generally
purified by careful column chromatography followed by recrystallisation.
In some case, HPLC purification was also required.
General Experimental Conditions
.sup.1H-- and .sup.13C-- nuclear magnetic spectra (NMR) were recorded
as solutions in deuteriated chloroform (CDCl.sub.3) unless stated
otherwise with tetramethylsilane as the internal reference, on a
DPX-400 MHz or 500 MHz Brucker Avance FT-NMR spectrometer. Chemical
shift values (.delta.) are given in part per million (ppm) and coupling
constants (J) are expressed in Hertz (Hz). Mass spectra were recorded
at the UIUC School of chemical Science and were obtained by electron
ionisation (EI) technique. Elemental analyses were performed by
Atlantic Microlab, Inc. Routine analytical thin layer chromatography
(TLC) was carried out on J. T. Baker Si250 F.sub.254 glass-backed
plates. The plates were developed with the appropriate solvent system
and visualized either by UV lamp or dipping into Hanessian stain
and heating with a heat gun. Column chromatography was carried out
using 230-400 mesh, 60 A, silica gel from Silicycle. Reagents were
obtained from Aldrich, Sigma or Fluka. High performance liquid chromatography
was performed on a Varian Chrompack Microsorb-MV 100-5 C-18 column
(250.times.4.6.times.1/4'' mm) using Varian Prostar 210-SDM pumps
with an isocratic mixture of methanol and water (80:20); UV detection
was at 254 nm with a Varian Prostar 320-U/VIS detector. Solvents
were Analar grade except for THF, which was puriss grade (H.sub.2O<0.005%)
and used without further purification. When mixed solvent systems
were used, the ratios are v/v. When ethyl acetate and hexanes are
used as a mixture, the percentage given is in ethyl acetate (i.e.
ethyl acetate-hexanes 5%).
Type 1 Procedure for the Preparation of Acetylenes
##STR00027##
A solution of methyl propiolate (0.445 ml, 5 mmol) in dry THF (25
ml) was cooled to -78.degree. C. under N.sub.2, before a solution
of n-butyllithium (1.6M in hexanes, 3.3 ml, 5.25 mmol) was added
dropwise. The mixture was stirred at that temperature for 40 min
before the isocyanate (6.5 mmol) was added dropwise. The resulting
reaction mixture was allowed to stir at -78.degree. C. for 4 h.
Trimethylsilyl chloride (2 ml) was added and the mixture was stirred
for a further 30 min prior to addition of aqueous hydrochloric acid
(1N, 6.5 ml) and the reaction mixture was allowed to warm to room
temperature. The resulting two phases were separated and the aqueous
layer was extracted with ethyl acetate (3.times.20 ml). The combined
organic extracts were dried over sodium sulfate, filtered and concentrated
in vacuo to give an oil which was purified by column chromatography.
Type 2 Procedure for the Preparation of Acetylenes
##STR00028##
A solution of methyl propiolate (0.445 ml, 5 mmol) in dry THF (25
ml) was cooled to -78.degree. C. under N.sub.2, before a solution
of n-butyllithium (1.6M in hexanes, 3.3 ml, 5.25 mmol) was added
dropwise. The mixture was stirred at that temperature for 30 min
before the isocyanate (5 mmol) was added dropwise. The resulting
reaction mixture was allowed to stir at -78.degree. C. for 30 min.
A saturated aqueous solution of ammonium chloride (20 ml) was added
and the mixture was allowed to warm to room temperature. The resulting
two phases were separated and the aqueous layer was extracted with
ethyl acetate (3.times.20 ml). The combined organic extracts were
washed with saturated aqueous sodium hydrogen carbonate (20 ml),
dried over magnesium sulfate, filtered and concentrated in vacuo
to give an oil which was purified by column chromatography. The
resulting crystalline compounds were recrystallised from hexanes
and ethyl acetate.
Characterization of Compounds Used for Comparison:
##STR00029##
76% yield; R.sub.f 0.3 (hex/EA 4:1); (Found C, 66.82; H, 8.49;
N, 5.60; O, 19.04; C.sub.14H.sub.21NO.sub.3 requires C, 66.91; H,
8.42; N, 5.57; O, 19.10); (retention time minutes, seconds); .sup.1H-NMR
(400 MHz, CDCl.sub.3) 5.66 (1H, br s), 3.85-3.81 (1H, m), 1.93 (2H,
dd, J 12.6, 3.5), 1.71-1.70 (2H, m), 1.54 (1H, m), 1.50 (9H, s),
1.37-1.35 (2H, m) and 1.18-1.15 (3H, m); .sup.13C-NMR (100 MHz,
CDCl.sub.3) 151.3 (C.sub.q), 150.1 (C.sub.q), 85.1 (C.sub.q), 75.4
(C.sub.q), 74.8 (C.sub.q), 49.1 (CH), 32.6 (2.times.CH.sub.2), 27.9
(3.times.CH.sub.3), 24.9 (CH.sub.2) and 24.6 (CH.sub.2); m/z (EI)
251.152206 (M.sup.+, C.sub.14H.sub.21NO.sub.3 requires 251.152144),
251 (M.sup.+, 5%), 152 (MH.sup.+-CO.sub.2.sup.tBu, 47), 114 (100),
98 (12) and 83 (30).
##STR00030##
48% yield; R.sub.f (hex/EA); (Found C, 66.24; H, 5.04; N, 6.44;
O, 21.98; C.sub.12H.sub.11NO.sub.3 requires C, 66.35; H, 5.10; N,
6.45; O, 22.10); (retention time 3 minutes, 57 seconds); .sup.1H-NMR
(400 MHz, CDCl.sub.3) 7.38-7.27 (5H, m), 6.2 (1H, br s), 4.62 (2H,
d, J 5.9) and 3.82 (3H, s); .sup.13C-NMR (100 MHz, CDCl.sub.3) 152.6
(C.sub.q), 150.5 (C.sub.q), 136.4 (C.sub.q), 129.0, 128.2, 128.0
and 127.3 (5.times.CH), 77.2 (C.sub.q), 73.9 (C.sub.q), 53.4 (CH.sub.3)
and 44.1 (CH.sub.2); m/z (EI) 217.074208 (M.sup.+, C.sub.12H.sub.11NO.sub.3
requires 217.073893), 217 (M.sup.+, 23%), 159 (MH.sup.+-CO.sub.2Me,
68) and 106 (PhNH.sup.+, 100).
##STR00031##
66% yield; R.sub.f 0.15 (hex/EA, 4:1); (Found C, 63.12; H, 7.13;
N, 6.64; O, 23.07; C.sub.11H.sub.15NO.sub.3 requires C, 63.14; H,
7.22; N, 6.70; O, 22.94); (retention time 2 minutes, 56 seconds);
.sup.1H-NMR (500 MHz, CDCl.sub.3) 5.98 (1H, br s), 3.86-3.84 (1H,
m), 3.83 (3H, s), 1.95-1.92 (2H, m), 1.74-1.70 (2H, m), 1.61-1.57
(1H, m), 1.37-1.35 (2H, m) and 1.19-1.17 (2H, m); .sup.13C-NMR (125
MHz, CDCl.sub.3) 152.8 (C.sub.q), 149.7 (C.sub.q), 77.9 (C.sub.q),
73.2 (C.sub.q), 53.3 (CH.sub.3), 49.3 (CH), 32.6, 25.3 and 24.6
(5.times.CH.sub.2); m/z (EI) 209.1045986 (M.sup.+, C.sub.11H.sub.15NO.sub.3
requires 209.105194), 209 (M.sup.+, 6%), 166 (44) and 128 (100).
##STR00032##
68% yield; R.sub.f 0.16 (hex/EA, 4:1); (Found C, 56.59; H, 6.53;
N, 8.16; O, 28.64; C.sub.8H.sub.11NO.sub.3 requires C, 56.80; H,
6.55; N, 8.28; O, 28.37); (retention time 3 minutes, 5 seconds);
.sup.1H-NMR (400 MHz, CDCl.sub.3) 5.84 (1H, br s), 4.18-4.07 (1H,
m), 3.83 (3H, s) and 1.20 (6H, d, J 6.5); .sup.13C-NMR (100 MHz,
CDCl.sub.3) 152.8 (C.sub.q), 149.7 (C.sub.q), 77.7 (C.sub.q), 73.1
(C.sub.q), 53.4 (CH.sub.3), 45.5 (CH) and 22.3 (2.times.CH.sub.3);
m/z (EI) 169.073537 (M.sup.+, C.sub.8H.sub.11NO.sub.3 requires 169.073893),
169 (M.sup.+, 17%), 154 (M.sup.+-Me, 100), 138 (M.sup.+-OMe, 16)
and 111 (MH.sup.+-CO.sub.2Me, 61).
##STR00033##
84% yield; R.sub.f (hex/EA); (Found C, 65.59; H, 8.12; N, 6.66;
O, 22.93; C.sub.11H.sub.17NO.sub.3 requires C, 62.54; H, 8.11; N,
6.63; O, 22.72); (retention time 3 minutes, 39 seconds); .sup.1H-NMR
(400 MHz, CDCl.sub.3) 5.77 (1H, br s), 4.17-4.07 (1H, m), 1.50 (9H,
s) and 1.19 (6H, d, J 6.5); .sup.13C-NMR (100 MHz, CDCl.sub.3) 151.2
(C.sub.q), 150.2 (C.sub.q), 85.1 (C.sub.q), 75.3 (C.sub.q), 74.7
(C.sub.q), 42.4 (CH), 27.9 (3.times.CH.sub.3) and 22.3 (CH.sub.3);
m/z (EI) 211.121045 (M.sup.+, C.sub.11H.sub.17NO.sub.3 requires
211.120844), 211 (M.sup.+, 4%), 196 (M.sup.+-Me, 19), 155 (MH.sup.+-.sup.tBu,
44), 140 (M.sup.+-.sup.tBu-Me, 100) and 111 (MH.sup.+-.sup.tBu-.sup.iPr).
Characterization of Other Compounds:
##STR00034##
100%; R.sub.f 0.1 (hex/EA, 4:1); (Found C, 61.74; H, 4.87; N, 6.02;
O, 27.51; C.sub.12H.sub.11NO.sub.4 requires C, 61.80; H, 4.75; N,
6.01; O, 27.44); (retention time 3 minutes, 31 seconds); 1H-NMR
(400 MHz, CDCl.sub.3) 8.47 (1H, br s), 7.45 (2H, d, J 12.5), 6.88
(2H, d, J 12.5), 3.86 (3H, s) and 3.80 (3H, s); .sup.13C-NMR (100
MHz, CDCl.sub.3) 157.3 (C.sub.q), 152.7 (C.sub.q), 148.0 (C.sub.q),
129.5 (C.sub.q), 121.9 (CH), 114.3 (CH), 77.6 (C.sub.q), 74.1 (C.sub.q),
55.5 (CH.sub.3), 53.5 (CH.sub.3); m/z (EI) 233.069052 (M.sup.+,
C.sub.12H.sub.11NO.sub.4 requires 233.068808), 233 (M.sup.+, 7%),
122 (15) and 62 (100).
##STR00035##
71% yield; R.sub.f (hex/EA); (retention time 3 minutes, 32 seconds);
.sup.1H-NMR (400 MHz, CDCl.sub.3) 7.59 (1H, br s), 7.51 (2H, d,
J 7.7), 7.36-7.32 (2H, m), 7.18 (1H, m) and 3.86 (3H, s); .sup.13C-NMR
(100 MHz, CDCl.sub.3); m/z (EI) 203.058896 (M.sup.+, C.sub.11H.sub.9NO.sub.3
requires 203.058243), 203 (M.sup.+, 64%), 172 (M.sup.+-OMe, 35)
and 145 (MH.sup.+-CO.sub.2Me, 100).
##STR00036##
33% yield; R.sub.f (hex/EA); (retention time 2 minutes, 58 seconds);
dodgy .sup.1H-NMR (400 MHz, CDCl.sub.3) 7.32-7.25 (2H, m), 7.08-6.97
(2H, m), 3.88 (3H, s) and 3.80 (1H, br s); .sup.13C-NMR (100 MHz,
CDCl.sub.3) get peak labels on spectrum; m/z (EI) 239.039140 (M.sup.+,
C.sub.11H.sub.7F.sub.2NO.sub.3 requires 239.039400), 239 (M.sup.+,
100%), 220 (M.sup.+-F, 59) and 203 (M.sup.+-OMe, 62).
##STR00037##
80% yield; R.sub.f 0.26 (hex/EA, 4:1); (Found C, 70.88; H, 7.22;
N, 4.88; O, 16.49; C.sub.17H.sub.21NO.sub.3 requires C, 71.06; H,
7.37; N, 4.87; O, 16.70); (retention time 2 minutes, 13 seconds);
dodgy, doris' .sup.1H-NMR (300 MHz, CDCl.sub.3) 7.22-7.20 (3H, m),
3.90 (3H, s), 3.05 (2H, m) and 1.17 (12H, 2.times.d, J 7.0); .sup.13C-NMR
(75 MHz, CDCl.sub.3) 147.3 (C.sub.q.times.2), 146.3 (C.sub.q.times.2),
129.4 (C.sub.q), 124.0 (CH.times.3), 77.6 (C.sub.q), 67.6 (C.sub.q),
53.6 (CH.sub.3), 29.0 (CH.sub.3), 28.8 (CH.sub.3) and 23.7 (CH);
m/z (EI) 287.151570 (M.sup.+, C.sub.17H.sub.21NO.sub.3 requires
287.152144), 287 (M.sup.+, 52%), 255 (MH.sup.+-OMe, 57), 240 (100)
and 212 (92).
##STR00038##
% yield; R.sub.f (hex/EA); (retention time minutes, seconds); .sup.1H-NMR
(400 MHz, CDCl.sub.3); .sup.13C-NMR (100 MHz, CDCl.sub.3); m/z (EI)
(M.sup.+, CHNO requires).
##STR00039##
50% yield; R.sub.f 0.1 (hex/EA, 4:1); (retention time 3 minutes,
31 seconds); .sup.1H-NMR (400 MHz, CDCl.sub.3) 8.47 (1H, br s),
7.45 (2H, d, J 12.5), 6.88 (2H, d, J 12.5), 3.86 (3H, s) and 3.80
(3H, s); .sup.13C-NMR (100 MHz, CDCl.sub.3) 157.3 (C.sub.q), 152.7
(C.sub.q), 148.0 (C.sub.q), 129.5 (C.sub.q), 121.9 (CH), 114.3 (CH),
77.6 (C.sub.q), 74.1 (C.sub.q), 55.5 (CH.sub.3), 53.5 (CH.sub.3);
m/z (EI) 233.069052 (M.sup.+, C.sub.12H.sub.11NO.sub.4 requires
233.068808), 233 (M.sup.+, 7%), 122 (15) and 62 (100).
##STR00040##
56% yield; R.sub.f 0.44 (hex/EA, 1:1); (retention time 3 minutes,
27 seconds); .sup.1H-NMR (400 MHz, CDCl.sub.3) 8.28 (1H, dd, J 1.4,
8), 8.21 (1H, br s), 7.11 (1H, apparent dt, J 1.5, 7.7), 6.97 (1H,
dt, J 0.9, 8.7), 6.90 (1H, d, J 8.2), 3.91 (3H, s) and 3.87 (3H,
s); .sup.13C-NMR (100 MHz, CDCl.sub.3) 153.2 (C.sub.q), 148.1 (2.times.C.sub.q),
126.9 (C.sub.q), 125.7 (CH), 121.5 (CH), 120.9 (CH), 110.5 (CH),
78.1 (C.sub.q), 74.1 (C.sub.q), 56.2 (CH.sub.3) and 53.8 (CH.sub.3);
m/z (EI) 233.068297 (M.sup.+, C.sub.12H.sub.11NO.sub.4 requires
233.068808), 233 (M.sup.+, 100%), 218 (M.sup.+-Me, 3), 202 (M.sup.+-OMe,
25) and 175 (MH.sup.+-CO.sub.2Me, 40).
##STR00041##
30% yield; R.sub.f (hex/EA); (retention time 3 minutes, 42 seconds);
.sup.1H-NMR (500 MHz, CDCl.sub.3) 7.69 (1H, br s), 6.73 (2H, s),
6.30 (1H, s), 3.86 (3H, s) and 3.79 (6H, s); .sup.13C-NMR (125 MHz,
CDCl.sub.3) 161.2 (2.times.C.sub.q), 152.6 (C.sub.q), 148.1 (C.sub.q),
138.2 (C.sub.q), 95.5 (2.times.CH), 97.9 (CH), 77.4 (C.sub.q), 74.1
(C.sub.q), 55.5 (2.times.CH.sub.3) and 53.5 (CH.sub.3); m/z (EI)
263.079901 (M.sup.+, C.sub.13H.sub.13NO.sub.5 requires 263.079373),
263 (M.sup.+, 31%), 235 (45), 232 (M.sup.+--OMe, 35), 204 (M.sup.+-CO.sub.2Me,
100).
##STR00042##
81% yield; R.sub.f 0.14 (hex/EA, 4:1); (retention time 3 minutes,
26 seconds); .sup.1H-NMR (400 MHz, CDCl.sub.3) 8.21 (1H, s), 7.95-7.79
(3H, m), 7.70 (1H, br s), 7.51-7.42 (3H, m) and 3.92 (3H, s); .sup.13C-NMR
(100 MHz, CDCl.sub.3) 153.2 (C.sub.q), 148.8 (C.sub.q), 134.5 (C.sub.q),
133.9 (C.sub.q), 131.5 (C.sub.q), 129.5, 128.3, 128.0, 127.2, 126.2,
119.8 and 118.1 (7.times.CH), 78.1 (C.sub.q), 74.7 (C.sub.q), 53.9
(CH.sub.3); m/z (EI) 253.074299 (M.sup.+, C.sub.15H.sub.11NO.sub.3
requires 253.073893), 253 (M.sup.+, 67%), 222 (M.sup.+-OMe, 22),
195 (MH.sup.+-CO.sub.2Me, 28), 115 (100).
##STR00043##
48% yield; R.sub.f 0.11 (hex/EA, 4:1); (retention time 5 minutes,
53 seconds); .sup.1H-NMR (500 MHz, CDCl.sub.3) 7.63 (1H, br s),
7.41 (2H, d, J 8.5), 7.19 (2H, d, J 8.5), 3.86 (3H, s), 2.93-2.86
(1H, m) and 1.23 (6H, d, J 4.5); .sup.13C-NMR (125 MHz, CDCl.sub.3)
152.9 (C.sub.q), 148.2 (C.sub.q), 146.6 (C.sub.q), 134.4 (C.sub.q),
127.1 and 120.2 (4.times.CH), 77.2 (C.sub.q), 74.0 (C.sub.q), 53.5
(CH.sub.3), 33.7 (CH) and 23.9 (CH.sub.3.times.2); m/z (EI) (M.sup.+,
CHNO requires).
##STR00044##
26% yield; R.sub.f (hex/EA); (Found C, 73.14; H, 4.85; N, 5.05;
O, 17.00; C.sub.17H.sub.13NO.sub.3 requires C, 73.11; H, 4.69; N,
5.01; O, 17.19); .sup.1H-NMR (500 MHz, CDCl.sub.3) 7.67 (1H, br
s), 7.59-7.55 (6H, m), 7.46-7.42 (2H, m), 7.37-7.33 (1H, m) and
3.88 (3H, s); .sup.13C-NMR (125 MHz, CDCl.sub.3) 153.2 (C.sub.q),
148.6 (C.sub.q), 140.5 (C.sub.q), 138.9 (C.sub.q), 136.2 (C.sub.q),
129.3, 128.2, 127.8, 127.3 and 120.9 (9.times.CH), 77.9 (C.sub.q),
74.7 (C.sub.q) and 53.9 (CH.sub.3); m/z (EI) 279.088946 (M.sup.+,
C.sub.17H.sub.13NO.sub.3 requires 279.089543), 279 (M.sup.+, 100%),
193 (62), 168 (65) and 141 (70).
##STR00045##
50% yield; R.sub.f (hex/EA); (Found C, 70.21; H, 4.32; N, 5.19;
C.sub.17H.sub.13NO.sub.4 requires C, 69.15; H, 4.44; N, 4.74; O,
21.67); (bad HPLC); .sup.1H-NMR (500 MHz, CDCl.sub.3) 7.53 (1H,
br s), 7.49 (2H, d, J 7.9), 7.35 (2H, app t, J 7.5), 7.11 (1H, app
t, J 7.5), 7.0 (4H, d J 7.5) and 3.87 (3H, s); .sup.13C-NMR (125
MHz, CDCl.sub.3) 157.0 (C.sub.q), 154.7 (C.sub.q), 152.7 (C.sub.q),
148.2 (C.sub.q), 131.8 (C.sub.q), 129.9, 129.8, 123.5, 121.9, 119.4,
119.0 and 118.8 (9.times.CH), 77.5 (C.sub.q), 74.3 (C.sub.q) and
53.5 (CH.sub.3); m/z (EI) 295.084774 (M.sup.+, C.sub.17H.sub.13NO.sub.4
requires 295.084458).
##STR00046##
7% yield; R.sub.f 0.15 (hex/EA, 4:1); (retention time 3 minutes,
27 seconds); .sup.1H-NMR (500 MHz, CDCl.sub.3) 7.32-7.25 (3H, m),
7.09 (2H, d, J 8.2), 6.66 (1H, d, J 1.4), 4.90 (1H, app dt, J 5.7,
7.8), 3.81 (3H, s), 3.74 (3H, s), 3.19 (1H, dd, J 5.7, 18.7) and
3.11 (1H, dd, J 5.7, 14); .sup.13C-NMR (125 MHz, CDCl.sub.3) 171.1
(C.sub.q), 153.0 (C.sub.q), 150.4 (C.sub.q), 135.4 (C.sub.q), 129.9,
129.6, 129.1, 128.0 and 127.8 (5.times.CH), 77.7 (C.sub.q), 74.5
(C.sub.q), 57.5 (CH.sub.3), 54.1 (CH.sub.3), 53.1 (CH) and 37.9
(CH.sub.2); m/z (EI) (M.sup.+, CHNO requires).
##STR00047##
27% yield; R.sub.f 0.44 (hex/EA, 1:1); (retention time 3 minutes,
26 seconds); .sup.1H-NMR (500 MHz, CDCl.sub.3) 7.20 (2H, d, J 8.6),
6.88 (2H, d, J 8.6), 6.23 (1H, br s), 4.42 (2H, d, J 5.8), 3.81
(3H, s) and 3.80 (3H, s); .sup.13C-NMR (125 MHz, CDCl.sub.3) 159.5
(C.sub.q), 152.7 (C.sub.q), 150.4 (C.sub.q), 129.4 (CH.times.2),
128.5 (C.sub.q), 114.3 (2.times.CH), 77.3 (C.sub.q), 73.8 (C.sub.q),
55.3 (CH.sub.3), 53.3 (CH.sub.3) and 43.7 (CH.sub.2); m/z (EI) 247.084683
(M.sup.+, C.sub.13H.sub.13NO.sub.4 requires 247.084458), 247 (M.sup.+,
56%), 217 (MH.sup.+-OMe, 40), 189 (MH.sup.+-CO.sub.2Me, 40), 136
(M.sup.+-CO-tp-CO.sub.2Me, 100), 121 (OMePhCH.sub.2.sup.+, 92).
Preparation of the Furan Library (K1 and K2):
##STR00048##
N-methylacetamide (1.00 g, 13.68 mmol) was dissolved in toluene
(75 ml) and stirred at room temperature before methyl malonyl chloride
(1.9 ml, 17.78 mmol) was added slowly. Nitrogen was bubbled through
the reaction mixture, which was heated to 85.degree. C. for 4 h.
Upon cooling, diethyl ether (20 ml) was added and the reaction mixture
was washed with sat. aq. sodium bicarbonate and sat. aq. sodium
chloride solutions. The organic phase was dried with sodium sulfate,
filtered and evaporated to dryness to afford a yellow liquid (1.72,
72%), which was used without further purification.
##STR00049##
Triethylamine (2.1 ml, 15 mmol) was added to a solution of imide
X (0.86 g, 5 mmol) in THF (20 ml) and methanesulfonyl azide (0.9
g, 7.5 mmol). The reaction mixture was left to stir at room temperature
overnight before concentration in vacuo. Purification by column
chromatography (gradient elution from 10% to 40% EA/hex) gave a
yellow oil (0.71 g, 71%).
##STR00050##
The diazoimide (132 mg, 0.66 mmol) was combined with the acetylene
(1.0 mmol) and rhodium perfluorobutyramidate (2.5 mg) in dry toluene
(4 ml). The reaction mixture was allowed to stir at room temperature
overnight then heated to 90.degree. C. for 8 h. Evaporation of the
solvent gave a yellow oil, which was purified by column chromatography
(10% up to 50% EA/hex).
##STR00051## less polar 16% yield; R.sub.f 0.52 (50% EA/hex); .sup.1H-NMR
(300 MHz, CDCl.sub.3) 7.04 (1H, br s), 3.94 (3H, s), 3.92 (3H, s),
3.88 (1H, m), 2.64 (3H, s), 1.94-1.92 (2H, m), 1.70-1.62 (3H, m),
1.44-1.31 (2H, m), 1.27-1.12 (3H, m); .sup.13C-NMR (75 MHz, CDCl.sub.3)
Doris' data. K2 (More Polar)
39% yield; R.sub.f 0.22 (50% EA/hex); .sup.1H-NMR (300 MHz, CDCl.sub.3)
5.95 (1H, br s), 3.90 (3H, s), 3.88 (3H, s), 3.83 (1H, m), 2.64
(3H, s), 2.03-2.00 (2H, m), 1.76-1.71 (2H, m), 1.61 (1H, m), 1.43-1.36
(2H, m), 1.27-1.22 (3H, m); .sup.13C-NMR (75 MHz, CDCl.sub.3) Doris'
data.
Preparation of the Furan Library with a 3-methylene Unit at the
5-Position
##STR00052##
A solution containing 2-pyrrolidinone (1.33 ml, 17.51 mmol) and
2,2,6-trimethyl-1,3-dioxen-4-one (2.75 ml, 21.1 mmol) in xylenes
(17.5 ml) was heated to reflux under a nitrogen atmosphere for 2
h. The solvent was removed under vacuum to give a brown oil, which
was purified by column chromatography (5:1 hex/EA) affording the
desired compound as a transparent oil that solidified in the freezer
(2.82 g, 96%).
##STR00053##
Triethylamine (3.0 ml, 21.3 mmol) was added to a solution of b-ketoimide
(1.8 g, 10.6 mmol) and methanesulfonyl azide (1.55 g, 12.8 mmol)
in acetonitrile (6 ml) at room temperature. The reaction mixture
was stirred overnight and the solvent was removed under reduced
pressure. The resulting residue was purified by column chromatography
(20% EA/hex) to give 1.99 g (96%) of the desired product as a yellow
oil.
##STR00054##
Pyrrolidine (2.14 ml, 25.64 mmol) was added dropwise to a solution
containing the diazoimide (1 g, 5.1 mmol) in dichloromethane (12
ml) at 0.degree. C. under nitrogen atmosphere. The resulting solution
was stirred at 0.degree. C. for 2 h. before the solvent was removed
under vacuum. The residue thereby obtained was purified by column
chromatography (1:2 EA/hex) to give the desired product in 80% yield
(632.4 g).
##STR00055##
A solution of acetylene (168 mg, 0.80 mmol) and rhodium perfluorobutyramidate
(cat) in toluene (1.5 ml) was heated to 95.degree. C. under a nitrogen
atmosphere. A solution of the diazoimide (112 mg, 0.73 mmol) in
toluene (1.5 ml) was then added dropwise over 25 min. The solution
was heated to reflux for 2 h. before it was cooled to room temperature
and the solvent was evaporated under reduced pressure. The residue,
typically yellow-brown was then dissolved in methanol (excess) and
heated under reflux for 30 min. Upon cooling, the alcohol evaporated
in vacuo and its corresponding residue was purified by column chromatography
to give a pale yellow oil.
Nucleophile=methanol: 13% yield;
Nucleophile=benzylalcohol: 26% yield;
##STR00056##
A solution of acetylene (168 mg, 0.80 mmol) and rhodium perfluorobutyramidate
(cat) in toluene (1.5 ml) was heated to 95.degree. C. under a nitrogen
atmosphere. A solution of the diazoimide (112 mg, 0.73 mmol) in
toluene (1s.5 ml) was then added dropwise over 25 min. The solution
was heated to reflux for 2 h. before it was cooled to room temperature
and a nucleophile (excess) and heated under reflux for 30 min. Upon
cooling, the solvent evaporated in vacuo and its corresponding residue
was purified by column chromatography to give a pale yellow oil.
Nucleophile=allylamine; 23% yield;
Nucleophile=2-ethyl-1-butanol; 13% yield;
Nucleophile=1-butanol; 19% yield;
Nucleophile=N-Boc L-valinol; 14% yield;
Nucleophile=cyclohexanol; 16% yield;
Nucleophile=N-Boc L-phenylalaminol; % yield;
Nucleophile=allylalcohol;
AN7A and AN7B
##STR00057##
2-Methylfuran (0.5 ml, 5.49 mmol) was added to a solution of the
acetylene (1 g, 4.77 mmol) in toluene (5 ml) and the resulting solution
was heated to reflux for 3 h before the solvent was evaporated under
vacuum. The resulting residue was purified by column chromatography
(40% EA/hex) to give the product as a yellow oil (1.1 g, 80%).
##STR00058##
The mixture of regioisomers (700 mg, 2.4 mmol) in n-hexane (7 ml)
was added to a stirred suspension of PdBaSO.sub.4 (cat) in n-hexane.
The mixture was degassed extensively and placed under hydrogen atmosphere
at 0.degree. C. The reaction was followed closely by analysis of
aliquots. After 30 min, the reaction was complete and the mixture
was filtered through a pad of celite.RTM. and evaporated to give
the desired compound as a transparent oil (700 mg, quant.).
##STR00059##
The mixture of regioisomers was heated to give, after column chromatography
purification (5% to 20% EA/hex), the two expected furans. The less
polar furan was obtained in 20%, the most polar furan in 32% and
a mixture of both in 25%. The furans could be further purified by
HPLC (80% MeOH/H2O, C18 column).
##STR00060## less polar: R.sub.f 0.5 (hex/EA 3:2); (Found C, 63.17;
H, 7.23; N, 5.24; C.sub.14H.sub.19NO.sub.4 requires C, 63.38; H,
7.22; N, 5.28); (retention time 7 minutes, 27 seconds); .sup.1H-NMR
(400 MHz, CDCl.sub.3) 9.23 (1H, br s), 7.90 (1H, s), 3.94-3.89 (1H,
m), 3.87 (3H, s), 2.68 (3H, s), 1.99-1.96 (2H, m), 1.77-1.73 (2H,
m), 1.55-1.54 (1H, m) and 1.43-1.31 (5H, m); .sup.13C-NMR (100 MHz,
CDCl.sub.3); m/z (EI); more polar R.sub.f 0.4 (hex/EA 3:2); (Found
C, 63.45; H, 7.24; N, 5.27; C.sub.14H.sub.19NO.sub.4 requires C,
63.38; H, 7.22; N, 5.28); (retention time 6 minutes, 14 seconds);
.sup.1H-NMR (400 MHz, CDCl.sub.3) 9.22 (1H, br s), 7.95 (1H, s),
3.96-3.92 (1H, m), 3.90 (3H, s), 2.57 (3H, s), 1.99-1.95 (2H, m),
1.76-1.72 (2H, m), 1.58 (1H, m) and 1.43-1.31 (5H, m); .sup.13C-NMR
(100 MHz, CDCl.sub.3); m/z (EI) Biological Testing
A number of compounds according to the present invention were tested
for activity using the following three systems: a low cell density
high throughput cell proliferation assay, a high cell density proliferation
assay and a cell free kinase inhibition assay as described below.
Activity was exhibited with several compounds according to the present
invention consistent with these compounds being used in the treatment
of tumors, cancer and other cell growth proliferation assays.
Low cell density high throughput cell proliferation assay Between
50 and 100 32D and 32DtetP210Bcr-Abl cells in 100 microliters of
medium without serum were exposed to the test compound for 15 minutes.
An equal volume of 20% serum supplemented tissue culture medium
was then added and the cells were inoculated in single well in 32
wells of a 96 well plate. The endpoint of the assay was the fraction
of the 32 wells in which a cell pellet was seen to develop which
was filled with live cells. This assay is subject to automated screening
by a plate reader for the cell pellet. We used this high throughput
assay to screen for compounds that suppress the growth of the 32DtetP210Bcr-Abl
cell line in the absence of IL-3, but allow the growth of the 32D
cell line in the presence of IL-3. Various concentrations of each
compound were tested. If a compound suppressed the growth of the
P210Bcr-Abl cell line without affecting the growth of the 32D cell
line, then the compound was considered to be selectively inhibitory
for the P210Bcr-Abl dependent growth.
As shown in FIG. 13, the compounds derived from the linear acetylene
compounds, AC22 and K1P, are inhibitory to the 32DtetP210bcrabl
cell line at 1 micromolar. AC22 also inhibits the 32Dtet cell line
at a similar concentration. The effect of these compounds were also
studied in combination with Imatinib (Gleevec or STI-571) to determine
whether the compound had an additive or synergistic effect with
Imatinib in inhibiting P210bcrabl dependent cell growth. As shown
above, the inhibition of the combination of AC19 and Imatinib (STI571)
at a concentration of 10 nanomolar of each drug (15% inhibition)
is less than the sum of the inhibitory effect of both drugs alone
(25%). When compound AC22 or K1P are added in combination with Imatinib
to the culture of the 32DtetP210bcrabl cell line at 10 nanomolar
concentration of both drugs, there is inhibition of the growth of
the P210bcrabl dependent proliferation (80%) This is greater than
the sum of the inhibition that is seen when the drugs (Imatinib
inhibits 25% and AC22 inhibits 10%) are used separately. A similar
synergism is seen for K1P and AC19: 45% inhibition together and
25% when used alone.
High Cell Density Cell Proliferation Assay (MTS)
This cell proliferation assay was performed using MTS tetrazolium
(Cell titer96 Aqueous; Promega, Madison, Wis.), which measure numbers
of viable cells. Between 2.times.10.sup.3 and 2.times.10.sup.4 STI-resistant
cells are washed twice in RF-10 and plated in quadruplicate in the
wells of a microtiter plate in 100 .mu.l of RF-10 medium supplemented
with various doses of test compounds (See FIG. 14). Controls using
the same concentration of Imatinib without cells were set up in
parallel. The plate is then incubated for 72 hours at 37 C in a
humidified 5% CO.sub.2 atmosphere. Twenty microliters of MTS were
then added to the wells and the plate was incubated for three hours.
Then the absorbance was recorded at 490-nm wavelength with a microplate
autoreader (Spectramax). Results are expressed as the mean optical
density of the 4-well set of each compound dose. All experiments
were repeated at least 3 times.
Compounds identified as being selectively inhibitor for P210BcrAbl
growth in the low density assay were also tested in a high cell
density assay in which the number of cells was closer to that present
in the blood stream in the presence of serum. This high cell density
assay was not used to screen drugs initially but was used for in
depth studies of compounds that were selected on the basis of the
low cell density assay.
Cell Free Kinase Inhibition Assay. A panel of 60 kinases (Upstate
Biotechnologies, Lake Placid, N.Y.) were analyzed in in vitro cell
free kinase assays. The results for those kinases with associated
inhibition are shown below.
TABLE-US-00001 % Inhibition at 10 .mu.M* Kinase K1P AC22 CaMKII
-- 40 CaMKIV -- 64 CDK2/cyclinA -- 50 CK1 46 (37) Fyn 49 (29) IKKBeta
-- 45 Lyn -- 98 PKCy -- 61 PKCbII (35) 48 *only % of inhibition
>45% is reported
Testing of Compounds According to the Present Invention by NCI
The following cells lines were used to test the activity of compounds
according to the present invention.
TABLE-US-00002 Cell Line Name Panel Name Cell Number Panel Number
Inoculation Density CCRF-CEM Leukemia 3 7 40000 HL-60(TB) Leukemia
8 7 15000 K-562 Leukemia 5 7 5000 MOLT-4 Leukemia 6 7 30000 RPMI-8226
Leukemia 10 7 30000 SR Leukemia 19 7 20000 A549/ATCC Non-Small Cell
Lung Cancer 4 1 7500 EKVX Non-Small Cell Lung Cancer 8 1 20000 HOP-18
Non-Small Cell Lung Cancer 27 1 20000 HOP-19 Non-Small Cell Lung
Cancer 28 1 20000 HOP-62 Non-Small Cell Lung Cancer 26 1 10000 HOP-92
Non-Small Cell Lung Cancer 29 1 20000 NCI-H226 Non-Small Cell Lung
Cancer 13 1 20000 NCI-H23 Non-Small Cell Lung Cancer 1 1 20000 NCI-H322M
Non-Small Cell Lung Cancer 17 1 20000 NCI-H460 Non-Small Cell Lung
Cancer 21 1 7500 NCI-H522 Non-Small Cell Lung Cancer 3 1 15000 LXFL
529 Non-Small Cell Lung Cancer 30 1 10000 DMS 114 Small Cell Lung
Cancer 9 2 20000 DMS 273 Small Cell Lung Cancer 11 2 5000 SHP-77
Small Cell Lung Cancer 13 2 40000 COLO 205 Colon Cancer 10 4 15000
DLD-1 Colon Cancer 11 4 5000 HCC-2998 Colon Cancer 2 4 10000 HCT-116
Colon Cancer 3 4 5000 HCT-15 Colon Cancer 15 4 10000 HT29 Colon
Cancer 1 4 5000 KM12 Colon Cancer 17 4 15000 KM20L2 Colon Cancer
18 4 10000 SW-620 Colon Cancer 9 4 10000 SF-268 CNS Cancer 14 12
15000 SF-295 CNS Cancer 15 12 10000 SF-539 CNS Cancer 16 12 15000
SNB-19 CNS Cancer 2 12 15000 SNB-75 CNS Cancer 5 12 20000 SNB-78
CNS Cancer 6 12 20000 TE671 CNS Cancer 10 12 20000 U251 CNS Cancer
9 12 7500 XF 498 CNS Cancer 17 12 20000 LOX IMVI Melanoma 1 10 7500
MALME-3M Melanoma 2 10 20000 M14 Melanoma 14 10 15000 RPMI-7951
Melanoma 3 10 20000 M19-MEL Melanoma 16 10 10000 SK-MEL-2 Melanoma
5 10 20000 SK-MEL-28 Melanoma 8 10 10000 SK-MEL-5 Melanoma 7 10
10000 UACC-257 Melanoma 21 10 20000 UACC-62 Melanoma 20 10 10000
IGROV1 Ovarian Cancer 10 6 10000 OVCAR-3 Ovarian Cancer 1 6 10000
OVCAR-4 Ovarian Cancer 2 6 15000 OVCAR-5 Ovarian Cancer 3 6 20000
OVCAR-8 Ovarian Cancer 5 6 10000 SK-OV-3 Ovarian Cancer 11 6 20000
786-0 Renal Cancer 18 9 5000 A498 Renal Cancer 13 9 20000 ACHN Renal
Cancer 23 9 10000 CAKI-1 Renal Cancer 15 9 10000 RXF 393 Renal Cancer
16 9 15000 RXF-631 Renal Cancer 17 9 10000 SN12C Renal Cancer 8
9 15000 SN12K1 Renal Cancer 10 9 10000 TK-10 Renal Cancer 24 9 15000
UO-31 Renal Cancer 4 9 15000 P388 Leukemia 1 7 5000 P388/ADR Leukemia
2 7 5000 PC-3 Prostate Cancer 1 11 7500 DU-145 Prostate Cancer 3
11 10000 MCF7 Breast Cancer 1 5 5000 NCI/ADR-RES Breast Cancer 2
5 15000 MDA-MB-231/ATCC Breast Cancer 5 5 20000 HS 578T Breast Cancer
6 5 20000 MDA-MB-435 Breast Cancer 11 5 15000 MDA-N Breast Cancer
12 5 15000 BT-549 Breast Cancer 13 5 20000 T-47D Breast Cancer 14
5 20000 MAXF 401 Breast Cancer 16 5 20000 MDA-MB-468 Breast Cancer
18 5 20000 SK-BR-3 Breast Cancer 10 5 20000
Results showed that the compound KIP exhibited enhanced activity
against certain leukemia cell lines, colon cancer cell lines, melanoma
cell lines, renal cancer cell lines and a breast cancer cell line,
thus showing the potential for broad activity of the compound K1P
as well as other compounds according to the present invention.
While the invention has been described with reference to specific
methods and embodiments, it will be appreciated that various modifications
may be made without departing from the invention. |